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Oxidative Stress

Back Clinic Oxidative Stress Chiropractic and Functional Medicine Team. Oxidative stress is defined as a disturbance in the balance between the production of reactive oxygen (free radicals) and antioxidant defenses. In other words, it is an imbalance between the production of free radicals and the body’s ability to counteract or detoxify the harmful effects through neutralization by antioxidants. Oxidative stress leads to many pathophysiological conditions in the body. These include neurodegenerative diseases, i.e., Parkinson’s disease, Alzheimer’s disease, gene mutations, cancers, chronic fatigue syndrome, fragile X syndrome, heart and blood vessel disorders, atherosclerosis, heart failure, heart attack, and inflammatory diseases. Oxidation happens under a number of circumstances:

the cells use glucose to make energy
the immune system is fighting off bacteria and creating inflammation
the bodies detoxify pollutants, pesticides, and cigarette smoke
There are millions of processes taking place in our bodies at any given time that can result in oxidation. Here are a few symptoms:

Fatigue
Memory loss and or brain fog
Muscle and or joint pain
Wrinkles along with grey hair
Decreased eyesight
Headaches and sensitivity to noise
Susceptibility to infections
Choosing organic foods and avoiding toxins in your environment makes a big difference. This, along with reducing stress, can be beneficial in decreasing oxidation.


Dr. Alex Jimenez Presents: The Impact Of Stress (Part 2)

Dr. Alex Jimenez Presents: The Impact Of Stress (Part 2)


Introduction

Dr. Alex Jimenez, D.C., presents how chronic stress can impact the body and how it is correlated with inflammation in this 2-part series. Part 1 examined how stress correlates with various symptoms affecting the body’s gene levels. Part 2 looks at how inflammation and chronic stress correlate with the various factors that can lead to physical development. We refer our patients to certified medical providers who provide available treatments for many individuals suffering from chronic stress associated with the cardiovascular, endocrine, and immune systems affecting the body and developing inflammation. We encourage each of our patients by mentioning them to associated medical providers based on their analysis appropriately. We understand that education is a delightful way when asking our providers questions at the patient’s request and understanding. Dr. Jimenez, D.C., only uses this information as an educational service. Disclaimer

 

How Stress Can Impact Us?

Dr. Alex Jimenez, D.C., presents: Stress can create many emotions that can hugely impact many of us. Whether it is anger, frustration, or sadness, stress can make anyone reach a breaking point and cause underlying conditions that can develop into cardiovascular issues. So those people with the highest level of anger, when you look at the cardiovascular literature, have the least probability of survival. Anger is a bad player. Anger causes arrhythmia. This study looked at, now that we have people with ICDs and defibrillators, we can monitor these things. And we see that anger can trigger ventricular arrhythmias in patients. And it’s easy now to follow, with some of our technology.

 

Anger has been linked to episodes of atrial fibrillation. When you think about it, it’s adrenaline outpouring into the body and causing coronary constriction. It’s increasing the heart rate. All of these things can lead to arrhythmia. And it doesn’t have to be AFib. It can be APCs and VPCs. Now, some very interesting research has come out about telomerase and telomeres. Telomeres are little caps on the chromosomes, and telomerase is the enzyme linked to telomere formation. And now, we can understand through the language of science, and we’re starting to use technology and use science in a way that we could never do before to understand the impact of stress on telomeres and telomerase enzymes.

 

The Factors That Lead Up To Chronic Stress

Dr. Alex Jimenez, D.C., presents: So one of the key people to study this is the Nobel Prize-winning, Dr. Elizabeth Blackburn. And what she said is that this is a conclusion, and we’ll come back to some of her other studies. She tells us that the telomeres of babies from women in utero had a lot of stress or were even shorter in young adulthood compared to mothers who did not have the same stressful situations. Maternal psychological stress during pregnancy may exert a programming effect on the developing telomere biology system that is already apparent at birth as reflected by the setting of newborn leukocyte telemetry length. So children can come in imprinted, and even if they do, this can be transformed.

 

What about racial discrimination these boxes here show high racial discrimination leading to low telomere length, which most of us have ever thought about. So, shorter telomere length leads to an increased risk of cancer and overall mortality. Cancer incidence rates are 22.5 per 1000 person-years in the shortest telomere group, verse 14.2 in the middle group, and 5.1 in the longest telomere group. Shorter telomeres can lead to instability of the chromosome and result in cancer formation. So, now we understand, through the language of science, the impact of stress on the telomerase enzyme and the telomere length. According to Dr. Elizabeth Blackburn, 58 premenopausal women were caregivers of their chronically ill children verse women who had healthy children. The women were asked how they perceive stress in their lives and whether it impacts their health by affecting their cellular aging.

 

That was the question of the study as they looked at telomere length and telomerase enzyme, and this is what they found. Now, the keyword here is perceived. We are not to judge each other’s stress. Stress is personal, and some of our responses may be genetic. For example, someone who has homozygous comps with a sluggish gene may have much more anxiety than someone who doesn’t have this genetic polymorphism. Someone who has an MAOA in an MAOB may have more anxiety than someone who doesn’t have that genetic polymorphism. So there is a genetic component to our response, but what she found was perceived psychological stress. And the number of years caring for chronically ill children was associated with shorter telomere length and less telomerase activity, providing the first indication that stress can impact telomere maintenance and longevity.

 

How To Transform Our Stress Response?

Dr. Alex Jimenez, D.C., presents: That’s powerful, and many healthcare providers are under some form of stress. And the question is, what can we do to transform our response? Framingham also looked at depression and identified clinical depression as a bigger risk for cardiovascular events and poor outcomes than smoking, diabetes, high LDL, and low HDL, which is crazy because we spend all of our time on these things. Yet, we don’t spend much time dealing with the emotional aspects of vascular disease. This is affected depression, inventory, a simple screening test for depression, looking at people with high levels of depression versus low levels of depression. And you can see that as you go from the low to the highest level, as you work your way through, the chance of survival becomes less.

 

And many of us have our theories as to why this occurs. And is it because if we are depressed, we don’t say, “Oh, I’m going to eat some brussels sprouts, and I’m going to take those B vitamins, and I’m going to go out and exercise, and I’m going to do some meditation.” So post-MI independent risk factor for an event is depression. Our mindset regarding depression makes us incapable of functioning normally and can make our bodies develop issues that affect our vital organs, muscles, and joints. So, depression is a big player, as 75% of post-MI deaths are related to depression, right? So looking at patients, now, you have to ask the question: Is it the depression causing the problem, or is it the cytokine sickness that’s already led to the heart disease causing the depression? We have to factor all of this in.

 

And yet another study looked at over 4,000 people with no coronary disease at baseline. For every increase of five points on the depression scale, that increased risk by 15%. And those with the highest depression scores had a 40% higher coronary artery disease rate and a 60% higher death rate. So mostly everyone thinks it’s a cytokine sickness that leads to MI, vascular disease, and depression. And then, of course, when you have an event, and you come out with a whole host of issues around it, we know that people who are depressed have a twofold increase in mortality, a fivefold increase in death after a heart attack, and poor outcomes with surgery. It’s like this, what came first, the chicken or the egg?

 

How Depression Is Linked With Chronic Stress?

Dr. Alex Jimenez, D.C., presents: Every surgeon knows this. They don’t want to do surgery on depressed people. They know the outcome is not good, and of course, they are less likely to follow through on all of our great functional medicine recommendations. So what are some of the mechanisms of autonomic dysfunction have been evaluated heart rate variability and low levels of omega-3s, which have a profound effect on the brain, and low levels of vitamin D. There are those inflammatory cytokines we talked about not getting restorative sleep, and many of our heart patients do have apnea. And remember, don’t just think it’s the heavyset heart patients with thick short necks; it can be quite deceiving. And it’s really important to look at the structure of the face and, of course, social connection, which is the secret sauce. So is autonomic dysfunction a mechanism? One study looked at heart rate variability in people with a recent MI, and they looked at over 300 people with depression and those without depression. They found that four heart rate variability indices will lower in people with depression.

 

Gut Inflammation & Chronic Stress

Dr. Alex Jimenez, D.C., presents: So here are two groups of people having a heart attack and heart rate variability, rising to the top as a possible etiology. One of the many things that can also affect chronic stress in the body is how the gut microbiome plays its part in oxidative stress. The gut is everything, and many heart patients laugh because they would ask their cardiologists, “Why do you care about my gut microbiome? Why would this affect my heart?” Well, all that gut inflammation is causing cytokine sickness. And what a lot of us have forgotten since medical school is that many of our neurotransmitters come from the gut. So chronic inflammation and exposure to inflammatory cytokines appear to lead to alterations in dopamine function and the basal ganglia, reflected by depression, fatigue, and psychomotor slowing. So we can’t emphasize the role of inflammation and depression enough if we take a look at acute coronary syndrome and depression, which was associated with higher markers for inflammation, more elevated CRP, lower HS, lower heart rate variability, and something that never gets checked in the hospital, which is nutrition deficiencies.

 

And in this case, they looked at omega-3s and vitamin D levels, so at a minimum, an omega-3 check and a vitamin D level are warranted in all of our patients. And certainly, if you can get a full diagnosis for stress-induced inflammation. Another condition you must look at when it comes to stress-induced inflammation is osteoporosis in the joints. Many people with osteoporosis will have muscle loss, immune dysfunction, fat around the midline, and high blood sugar are associated with aging, and it can come from elevated cortisol levels in the body.

 

High cortisol heart disease risks are two times higher in people taking high doses of steroids. Small amounts of steroids don’t have the same risk, so it is not as big a deal. Of course, we try to get our patients off of steroids. But the point here is that cortisol is a stress hormone and is a stress hormone that raises blood pressure and puts weight on the midline, makes us diabetic, causes insulin resistance, and the list is endless. So, cortisol’s a big player, and when it comes to functional medicine, we have to look at the various tests that pertain to elevated levels of cortisol like food sensitivity, a 3-day stool valve, a nutra-valve, and an adrenal stress index test to look at what is going on with the patients. When there is a heightened sympathetic nervous system and high cortisol, we discussed everything from coagulopathy to decreased heart rate variability, central obesity, diabetes, and hypertension.

 

Parental Relationships & Chronic Stress

Dr. Alex Jimenez, D.C., presents: And turning on the renin-angiotensin system it’s all linked to stress. Let’s look at this study that looked at 126 Harvard Medical students, and they were followed for 35 years, a long research. And they said, what’s the incidence of significant illness, heart disease, cancer, hypertension? And they asked these students very simple questions, what was your relationship with your mom and your dad? Was it very close? Was it warm and friendly? Was it tolerant? Was it strained and cold? This is what they found. They found that if the students identified their relationship with their parents as strained 100% incidence of significant health risk. Thirty-five years later, if they said it was warm and close, the results cut that percentage in half. And it would help if you thought about what it is and what can explain this, and you’ll see how adverse childhood experiences make us sick in a few minutes and how we learn our coping skills from our parents.

 

Conclusion

Dr. Alex Jimenez, D.C., presents: Our spiritual tradition comes from our parents often. Our parents are the ones who frequently teach us how to get angry or how to resolve conflict. So our parents have had a profound effect on us. And when you think about that, our connection is also not very surprising. This is a 35-year follow-up study.

 

Chronic stress can lead to multiple issues that can correlate to illness and dysfunction in the muscles and joints. It can affect the gut system and lead to inflammation if it is not taken care of immediately. So when it comes to the impact of stress affecting our daily lives, it can be numerous factors, from chronic conditions to family history. Eating nutritious foods high in antioxidants, exercising, practicing mindfulness, and going to daily treatments can lower the effects of chronic stress and reduce the associated symptoms that overlap and cause pain to the body. We can continue with our health and wellness journey pain-free by utilizing various ways to lower chronic stress in our bodies.

 

Disclaimer

Dr. Alex Jimenez Presents: The Impact Of Stress (Part 2)

Dr. Alex Jimenez Presents: The Impact Of Stress


Introduction

Dr. Alex Jimenez, D.C., presents how stress can impact many individuals and correlate with many conditions in the body in this 2-part series. We refer our patients to certified medical providers who provide multiple available treatments for many people suffering from hypertension associated with the cardiovascular, endocrine, and immune systems affecting the body. We encourage each of our patients by mentioning them to associated medical providers based on their analysis appropriately. We understand that education is a delightful way when asking our providers questions at the patient’s request and understanding. Dr. Jimenez, D.C., only uses this information as an educational service. Disclaimer

 

How Stress Impacts the Body

Dr. Alex Jimenez, D.C., presents: Now everyone responds to changes in the environment differently. When it comes to many individuals doing everyday activities from working at their job, opening on the weekends, traffic jams, taking exams, or preparing for a big speech, the body goes through a constant state of hyperreactive to a stage of emotional, mental exhaustion that leaves the individual to be exhausted and stressed out. And the key is to recognize this before it happens, as we see this impact of stress on our patients and ourselves. And the first thing to realize is what the initiating event is causing this impact.

 

Whatever the initiating event, the most important part is our perception of the event. What does it mean to us? Is it our perception? When the body goes through this initiating event, it can cause the perception to lead to the response and the effect on our body. So perception is everything as we talk about stress and the stress response. Now, we have over 1400 chemical reactions that occur in the body. So for this talk’s purpose, we’ll discuss the three key ones: adrenaline and neuro-adrenaline, aldosterone, and of course, cortisol.

 

And why are these important? Because every one of these has a huge impact on cardiovascular disease. Now, in the 1990s, many doctors were starting to understand the effect of stress on the physical body. And what happens to people when their HPA-axis signals that they are under threat and start flooding their bodies with stress hormones? Well, we see enhanced coagulation. We see a shift in the renin and angiotensin system. It revs up. We see weight gain in people and insulin resistance. What a lot of people don’t realize is that lipids become abnormal with stress. Almost every one of our patients knows that tachycardia and arrhythmia occur when our adrenaline is flowing, and our blood pressure increases. Now, think about this through the language of medicine.

 

Around the 1990s, doctors were giving aspirin and Plavix at the time for coagulation. We continue to provide ACEs and ARBs to our patients. The impact of cortisol causes weight gain and insulin resistance. We give statins; we give metformin. We provide beta blockers for that, tachycardia, and calcium blockers for that high blood pressure. So every single hormone that gets turned on with stress, we have a drug that we’re using to balance that. And quite frankly, for years, we talked about how good beta blockers were for the heart. Well, when you think about that, beta blockers do block adrenaline. So when doctors look at this, they begin to think, “Well, maybe we need to medicate and meditate, right? We’re using all these drugs, but we may need to look at other ways to transform the stress response.”

 

What is Vasoconstriction?

Dr. Alex Jimenez, D.C., presents: We won’t read every one of these symptoms because there are so many, but it all comes down to the same thing. Stress. We have to think of someone who’s in an auto accident, for example, and that person is bleeding. So the body is beautiful in that it puts together a way to stop the individual from bleeding or vasoconstriction. Vasoconstriction is constructing these blood vessels and making the platelets sticky so they form a clot, and the blood can stop. This increases the cardiac output by raising the heart rate and increases aldosterone, which causes salt and water retention to raise the blood pressure. So for someone in a medical emergency, like an accident, bleeding, or losing volume, this is the beauty of the human body. But unfortunately, we see people living this way, literally 24/7. So we know the vasoconstriction and the platelet stickiness, and we see increases in markers for inflammation, homocysteine, CRP, and fibrinogen, all of which increase cardiovascular risk.

 

We see the impact of cortisol, not only raising blood pressure, not only causing diabetes and insulin resistance, but also depositing abdominal fat around the midline. And then, as you’ll see in a few minutes, there are links between stressful events and arrhythmias like atrial fibrillation and even ventricular fibrillation. For the first time in medicine, in cardiology, we have a syndrome called takosubo cardiomyopathy, which is affectionately called broken heart syndrome. And this is a syndrome in which the myocardium becomes acutely stunned to the point of causing severe left ventricular function or dysfunction. And usually, this is triggered by bad news and an emotionally stressful event. It looks like someone needs a heart transplant. So when we think about the old Framingham risk factors, we say, which of these are impacted by stress?

 

Symptoms of Stress

Dr. Alex Jimenez, D.C., presents: People have all sorts of maladaptive behaviors to stress, whether 20 friends in this pack of cigarettes, eating this Cinnabon because it makes me feel good right now, or all the cortisol will make me fat and diabetic. Lipids go up under stress; blood pressure goes up under stress. So every one of these risk factors is impacted by stress hormones. And, of course, we know that with the turning on of the RAS system or the renin-angiotensin system, we always see a worsening in heart failure. And this is very much described in the literature. And, for those of you who may work in the emergency room, ask your patients what they were doing before coming in with their episode of congestive heart failure or chest pain. And you’re going to hear stories like, I was watching a bad movie, or I was watching a war movie, or I got upset over the football game, or something like that.

 

We’ll talk about heart rate variability, which gets impacted by stress. And, of course, stress affects our ability to resist infections. And we know that people are under stress when they’re vaccinated. For example, Cleco lasers work but don’t produce antibodies to the vaccine when they’re under stress. And, of course, as you’ll see in a minute, severe stress can cause sudden cardiac death, MI, and so on. So it is a bad player that’s overlooked. And for many of our patients, stress drives the train. So when we’re talking about eating brussels sprouts and cauliflower and, you know, lots of green leafy vegetables, and someone is under so much stress that they’re trying to figure out, “How am I going to get through the day?” They’re not hearing any of the other things that we’re recommending.

 

So, chronic stress and affective disorders, whether depression, anxiety, or panic, put our foot on the accelerator and rev up the sympathetic nervous system. We know that the same things we see with aging, as you’ll see in a minute, are linked to increased levels of stress hormones, especially cortisol. So whether it’s osteoporosis, decreased bone density, endothelial dysfunction, platelet activation, hypertension, central obesity, or insulin resistance, this comes from a stress response. And we have to have a plan for our patients on how to handle this. American Institute of Stress says that 75 to 90% of all healthcare provider visits result from stress-related disorders. And that’s way too high, but by looking at the patients and where they were coming in with, they tell their stories to their doctors. The results are the same; it doesn’t matter whether it was headaches, muscle tension, angina, arrhythmia, or irritable bowel; it almost always had some stress trigger.

 

Acute & Chronic Stress

Dr. Alex Jimenez, D.C., presents: There’s a difference between acute and chronic stress with our perception and social connection. Even though we gain some strength from a higher power, stress can impact anyone, and most of us might not be able to handle it well. So a great study was done many years ago by Dr. Ray and Holmes that stated, 50 years ago, put together a method for quantifying life-changing events. So let’s look at some areas, such as life-changing events. How do life-changing events and how do they rank? Which are the big ones, and which are the little ones?

 

And how does that ranking lead to major medical problems like cancer, heart attack, and sudden death in the future? So they looked at 43 life-changing events, ranked them originally, and re-ranked them in the 1990s. And some of them remained the same. They gave an adjustment score to the event, and then they looked at numbers that would be linked to major illness. So, for example, a life-changing event. Number one, 100 life-changing units, is a death of a spouse. Anyone could relate to that. Divorce was number two, separation number three, and the end of a close family member. But also noticed that some things got ranked that are, you might not equate with, being a major life-changing event that can impact a stress response like marriage or retirement.

 

Conclusion

Dr. Alex Jimenez, D.C., presents: So it wasn’t the actual single event that made the difference. It was the adding up of events. And what they found after looking at 67 physicians was if you had a life-changing unit score of somewhere between zero and one 50, not a big deal, no real major illness, but once you hit that 300 mark, there was a 50% chance of major illness. So this timeline of events in the patient’s life. We want to know what was going on in their life when their symptoms started and then bring it back earlier to understand the environment in which this individual was living. The impact of stress can make many individuals develop chronic conditions and mask other symptoms that can lead to muscle and joint pain. In part 2, we will dive in more about how the impact of stress affects a person’s body and health.

 

Disclaimer

Dr. Alex Jimenez Presents: How Hypertension Is Explained

Dr. Alex Jimenez Presents: How Hypertension Is Explained


Introduction

Dr. Alex Jimenez, D.C., presents how hypertension affects the human body and some causes that can increase hypertension in many individuals in this 2-part series. We refer our patients to certified medical providers who provide multiple available treatments for many individuals suffering from hypertension associated with the cardiovascular and immune systems affecting the body. We encourage each of our patients by mentioning them to associated medical providers based on their analysis appropriately. We understand that education is a delightful way when asking our providers questions at the patient’s request and understanding. Dr. Jimenez, D.C., only makes use of this information as an educational service. Disclaimer

 

How To Look For Hypertension

Dr. Alex Jimenez, D.C., presents: Let’s go back to the decision tree so you can begin to think about how you will apply the go-to-it model in functional medicine to hypertension and how you will start better assessing somebody with hypertension rather than telling them that their blood pressure is elevated. Is the body influenced by inflammation, oxidative stress, or immune response? Is it affecting endothelial function or vascular smooth muscle from those three categories of reactions, inflammation, oxidative stress, or immune response? Do we choose a diuretic calcium channel blocker or an ACE inhibitor? And so to do that, it’s really important in our gather section. Taking the medical history and the timeline of their hypertension, you get a clue about the organ damage to the questionnaires. You’re looking at their anthropometrics.

 

This includes the following questions:

  • What are the inflammatory markers?
  • What are the biomarkers and clinical indicators?

 

Those are outlined through the clinical decision tree. And already just doing that, you’re going to expand and fine-tune your lens on what you might see in your hypertensive patient. Let’s add to the timeline when does hypertension begin? The timeframe of hypertension begins actually in prenatally. It’s important to ask your patient if they were early or large educational age. Was their mother stressed? Were they born early or premature? Was there nutritional stress in their pregnancy? If they know that, you can have two people with the same kidney size, but the person who didn’t have enough protein during pregnancy can have up to 40% less glomeruli. Knowing that will change how you adjust the medication decades later if you know they possibly have 40% less glomeruli.

 

The Timeline For Blood Pressure

Dr. Alex Jimenez, D.C., presents: So it’s important to take the timeline of their blood pressure. Then it’s also important to recognize what is happening when we begin to organize and collect data through the biomarkers; the basic biomarkers will give you clues about whether they have issues with insulin lipids, whether they have problems with vascular reactivity, autonomic nervous system balance, imbalance, coagulation, or immune toxin effects. So this is a reasonable thing to print off because, in your hypertensive patient, this is through just the biomarkers you can begin to get a clue as to what areas of dysfunction affect inflammation, oxidative stress, and immune response and how these biomarkers reflect that information for you. This is very reasonable to have in front of you to help change your thoughts about hypertension and also enables you to refine some of the characteristics of the person on the other side of your stethoscope in a more personalized, precise way.

 

But let’s start at the very beginning. Does your patient have high blood pressure? We know that depending on the end organ effects of their comorbidities, you may run someone a slightly higher blood pressure if you have a profusion issue in the brain and the kidneys or the heart, but some guidelines are there. Our 2017 American Heart Association guidelines for blood pressure categories are listed here. They’ve waxed and waned back and forth over the last couple of decades, but this is very clear. Having elevated blood pressure, anything above 120, really shifted how many people we start seeing or considering addressing the root causes of their blood pressure. So we will come back to this, especially in the case to help us look at how we categorize people with blood pressure issues.

 

The Criteria To Mesure Blood Pressure

Dr. Alex Jimenez, D.C., presents: What is the first step? It’s how do you have the blood pressure taken in your patient? Do they monitor it at home? Do they bring those numbers to you? How do you monitor blood pressure in your clinic? How do you get accurate readings in your clinic? Here are the criteria to accurately measure blood pressure and the questions to consider whether you’re doing all these. 

  • Do you ask your patient whether they’ve had caffeine in the last hour?
  • Whether they’ve smoked in the previous hour?
  • Were they exposed to smoke in the last hour? 
  • Is the place where you’re taking blood pressure warm and quiet?
  • Are they sitting with their back supported in a chair with their feet on the ground?
  • Do you use the roll-around side table to rest your arm at the heart level?
  • Are they sitting at the exam table with their feet dangling, and a nurse aide elevates their arm and puts in their axillary fold to hold their arm there?
  • Are their feet on the ground? 
  • Have they sat there for five minutes? 
  • Have they exercised in the previous 30 minutes? 

 

You may have systolic blood pressure if everything is in the criteria. Here’s the challenge. There are 10 to 15 millimeters of mercury higher when it comes to sitting and taking blood pressure. What about the cuff size? We know last century; most adults had an upper arm circumference of fewer than 33 centimeters. Over 61% of people now have an upper arm circumference greater than 33 centimeters. So the size of the cuff is different for around 60% of your adult patients, depending on your population. So you have to use a large cuff. So take a look at how blood pressure is collected in your office. Let’s say the blood pressure is elevated in your patients; then we have to ask, is it normal? Great.

 

The Different Types Of Hypertension

Dr. Alex Jimenez, D.C., presents: Is it elevated because of white-coat hypertension? Do they have normal blood pressure, elevated outside the clinic, or masked hypertension? Or do they just have sustained hypertension which is a challenge? We’ll talk about that. So when you interpret, it is also important to consider ambulatory blood pressure monitoring. So if you have somebody who’s hypertensive and don’t know whether the blood pressure goes down and you’re trying to figure out whether they have sustained hypertension, you can use 24-hour blood pressure monitoring. The mean daytime blood pressure above 130 over 80 is hypertensive the mean nighttime blood pressure above 110 over 65 is hypertensive. So why is this important? The average blood pressure dips to around 15% at night because of the issue with blood pressure dipping. Failure to have blood pressure drop while you sleep at night could develop problems that can affect a person throughout the day. 

 

If your patient sleeps at night, it should drop about 15% when they sleep. If they have non-dipping blood pressure, it is associated with comorbidities. What are some of those comorbidities in non-dipping blood pressure? Some of the conditions correlated with non-dipping blood pressure include:

  • Congestive Heart Disease
  • Cardiovascular Disease
  • Cerebrovascular Disease
  • Congestive Heart Failure
  • Chronic Renal Failure
  • Silent Cerebral Infractions

Co-morbidities Associated With Non-Blood Pressure

Dr. Alex Jimenez, D.C., presents: These are the comorbidities associated with non-blood pressure. All of us agree that elevated blood pressure is not necessarily good in all those conditions. So when you look at different people groups or other comorbidities, non-dipping blood pressure is most commonly associated with sodium-sensitive folks, people who have renal insufficiency, people who have diabetes, people who have left ventricular hypertrophy, people who have refractory hypertension or autonomic nervous system dysfunction and finally, sleep apnea. So, non-dipping blood pressure increases your association with subclinical cardiac damage. Okay, Reverse dipping means you are more hypertensive at night and is more ascent associated than during the day is more related to hemorrhagic stroke. And if you have somebody with nocturnal hypertension, you have to start thinking about things like the carotid arteries and increased carotid, internal medial thickness. You start thinking about left ventricular hypertrophy and may see it on EKG. Here’s what we know about nocturnal hypertension. Nocturnal hypertension is a nighttime blood pressure greater than 120 over 70. It is associated with greater predictability of cardiovascular morbidity and mortality.

 

If you have nocturnal hypertension, it increases your risk of mortality from cardiovascular disease by 29 to 38%. We must know what’s happening at night when we sleep, right? Well, what’s another refinement? Another refinement is recognizing that resting blood pressure is controlled by your renin-angiotensin system. Waking blood pressure is controlled by your sympathetic nervous system. So let’s talk about how their renal angiotensin system drives their nighttime hypertension, and you think about what medication they’re taking. You might change the medication dosing to nighttime. Well, studies have shown that if you have nighttime hypertension and are a non-dipper, it’s best to take your ACE inhibitors, ARBs, calcium channel blockers, and certain beta blockers at night before bed. But it makes sense that you wouldn’t move your diuretics to nighttime, or you will have a disruptive sleep.

 

Addressing Daytime & Nighttime Blood Pressure

Dr. Alex Jimenez, D.C., presents: So if we don’t address daytime and nighttime blood pressure, we have to consider the effect of blood pressure load. What is your average daytime blood pressure and your moderate sleeping blood pressure is. We know that blood pressure load in young adults is hypertensive only about 9% of the time. So meaning the systolic load is about 9% versus in the elderly, about 80% of the blood pressure load is systolic. And so when you have a higher systolic load, you have more complications and end-organ damage. So what we’re talking about is helping identify your patient with hypertension; what is their timeline? What is their phenotype? Are they only hypertensive during the day, or they’re hypertensive at night also? We have to look at what helps balance that.

 

Here’s the other point, only about 3.5% of people with hypertension do it have a genetic cause. Only 3.5% of people their genes cause hypertension. The power is at the bottom of the matrix and recognizing these patterns, right? So you look at exercise, sleep, diet, stress, and relationships. So we know that these four autonomic balances help determine blood pressure. We will examine the renal angiotensin system, plasma volume where they hold onto too much fluid, secondary salt load, and endothelial dysfunction. Abnormalities in any of these can lead to hypertension. We’ve been talking about another one that can lead to hypertension: the link between insulin resistance and hypertension.

 

This diagrammatically gives you an idea of the physiologic interactions between insulin resistance and hypertension. It affects increasing sympathetic tone and increasing renal-angiotensin system balance. So let’s spend a few minutes on the renin-angiotensin system pathway angiotensinogen down to angiotensin two. We take advantage of these enzymes by giving inhibitors to angiotensin-converting enzymes in our hypertensives patients. Elevated angiotensin two leads to cardiovascular hypertrophy, leads to sympathetic phase constriction, increased blood volume, sodium fluid, retention, and aldosterone release. Can you inquire about your patient biomarkers? Can you ask whether they have elevated renin levels?

 

Look For The Signs

Dr. Alex Jimenez, D.C., presents: Well, you can. You can check plasma renin activity and aldosterone levels. It’s important to do this if your patient is hypertensive and has never been on medication because this is where nitrous oxide is so important. This is where your endothelial nitric oxide synthase is present. This is where you have sheer and hemodynamic stress. This is where dietary intake of arginine or the environment that affects nitric oxide plays such a role in the health of this layer of endothelia. If you lay it all together somehow, miraculously, or at least in your mind’s eye, it’ll cover six tennis courts in the average adult. It’s a huge surface area. And the things that cause endothelial dysfunction are not new news to people in functional medicine. Increased oxidative stress and inflammation are two things we mentioned that play an effect.

 

And then, look at some of these other components, your ADMA being elevated and correlated with insulin resistance. It all begins to form together in a matrix that interacts. So you look at one comorbidity in cardiometabolic syndrome, and it affects another comorbidity. You suddenly see the interrelation between them or hyperhomocysteinemia, which is a one-carbon metabolism marker, meaning you’re looking at the adequacy of folate, b12, b6, riboflavin, and that activity of your one-carbon metabolism. So let’s look at some of these emerging risk markers to improve and track in patients with hypertension. Let’s reanalyze ADMA again. ADMA stands for asymmetric dimethyl arginine. Asymmetric, dimethyl arginine is a biomarker of endothelial dysfunction. That molecule inhibits nitric oxide synthase while impairing endothelial function, and in all of the comorbidities associated with cardiometabolic syndrome, ADMA can be elevated.

Conclusion

So, as a quick review, L-arginine is converted to nitric oxide via nitric oxide synthase, and nitric oxide adequacy leads to vasodilation. ADMA blocks this conversion. And if your ADMA levels are elevated and your nitric oxide levels are low, then you have decreased nitric oxide platelet aggregation increases in LDL oxidation. So many things reduce nitric oxide or are associated with lower nitric oxide levels, sleep apnea, low dietary arginine, protein, zinc insufficiency, and smoking.

 

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The Stressful Impact Of The Body’s Homeostasis

The Stressful Impact Of The Body’s Homeostasis

Introduction

Everybody deals with stress at some point in their lives. Whether it be a job interview, a huge deadline, a project, or even a test, stress is there to keep the body functioning in each scenario that the body is going through. Stress can help regulate the body’s immune system and help metabolize homeostasis as the body increases its energy throughout the day. When dealing with chronic stress can cause metabolic dysfunction in the body like gut disorders, inflammation, and an increase in blood glucose levels. Chronic stress can also affect a person’s mood and health, eating habits, and sleep quality. Today’s article will look at if stress is a good thing or a bad thing, how it affects the body, and the effects of what chronic stress does to the body. Refer patients to certified, skilled providers specializing in gut treatments for individuals that suffer from autonomic neuropathy. We guide our patients by referring to our associated medical providers based on their examination when it’s appropriate. We find that education is critical for asking insightful questions to our providers. Dr. Alex Jimenez DC provides this information as an educational service only. Disclaimer

 

Can my insurance cover it? Yes, it may. If you are uncertain, here is the link to all the insurance providers we cover. If you have any questions or concerns, please call Dr. Jimenez at 915-850-0900

02 LaValle Triad 1 Adrenal Thyroid Pancreas

Is Having Stress Good Or Bad?

 

Do you feel anxious all the time? How about feeling headaches that are constantly being a nuisance? Feeling overwhelmed and losing focus or motivation? All these signs are stressful situations that a person is going through. Research studies have defined stress or cortisol as the body’s hormone that provides a variety of effects on different functions in each system. Cortisol is the primary glucocorticoid that is from the adrenal cortex. At the same time, the HPA (hypothalamus-pituitary-adrenal) axis helps regulates the production and secretion of this hormone to the rest of the body. Now cortisol can be beneficial and harmful to the body, depending on the situation a person is in. Additional research studies have mentioned that cortisol begins and affects the brain and the rest of the body as stress in its acute form can cause the body to adapt and survive. The acute responses from cortisol allow neural, cardiovascular, immune, and metabolic function in the body. 

 

How Does It Affect The Body’s Metabolism?

Now cortisol affects the body’s metabolism when controlled in a slow, steady sleep cycle that decreases corticotropin‐releasing hormone (CRH) and increases growth hormone (GH). Research studies have shown that when the adrenal glands secrete cortisol, it starts to have a complex interaction with the hypothalamus and pituitary glands in the nervous and endocrine systems. This causes the adrenal and thyroid function in the body to be closely linked while under the control of the hypothalamus and tropic hormones. The thyroid competes with the adrenal organs for tyrosine. Research studies have found that tyrosine is used to produce cortisol under stress while preventing cognitive function decline that is responsive to physical stress. However, when the body can not produce enough tyrosine, it can cause hypothyroidism and cause the cortisol hormone to become chronic.


An Overview About Stress-Video

Have you experienced headaches that randomly show up out of nowhere? Have you constantly gained weight or lost weight? Do you feel anxious or stressed out always that it is affecting your sleep? These are all signs and symptoms of your cortisol levels turning into their chronic state. The video above shows what stress does to your body and how it can cause unwanted symptoms. When there is chronic stress in the body, the HPA axis (neuro‐endocrine) is imbalanced due to the stress‐mediated activators involved in autoimmune thyroid diseases (AITD). When there is chronic stress in the body, it can cause excessive production of inflammatory compounds in the body can generate IR. The inflammatory substances can damage or inactivate insulin receptors leading to insulin resistance. This then contributes to the breakdown of one or more factors needed to complete the glucose transport process in the body.


The Effects Of Chronic Cortisol In The Body

 

When there is chronic stress in the body and has not been treated or reduced right away, it can lead to something known as allostatic load. Allostatic load is defined as wear and tear of the body and brain due to chronic overactivity or inactivity of the body systems typically involved in environmental challenges and adaptation. Research studies have shown that allostatic load causes excess secretion of hormones like cortisol and catecholamine to respond to chronic stressors affecting the body. This causes the HPA axis to do one of two things: being overworked or failing to shut off after stressful events causing sleep disturbances. Other issues that chronic stress does to the body can include:

  • Increased insulin secretion and fat deposition
  • Altered immune function
  • Hypothyroidism (adrenal exhaustion)
  • Sodium and water retention
  • Loss of REM sleep
  • Mental and Emotional instability
  • Increase in cardiovascular risk factors

These symptoms cause the body to become dysfunctional, and research studies have pointed out that various stressors can damage the body. This can make it extremely difficult for a person to cope with stress and alleviate it.

Conclusion

Overall, stress or cortisol is a hormone the body needs to function correctly. Chronic stress in the body from various stressors can cause many metabolic dysfunctions like hypothyroidism, weight gain, insulin resistance, and metabolic syndrome, to name a few. Chronic stress can also cause sleep disorders since the HPA axis is wired up and can seem to calm down the slightest. When people start to find ways of dealing with these various stressors, they can reduce their stress levels back to normal and be stress-free.

 

References

Jones, Carol, and Christopher Gwenin. “Cortisol Level Dysregulation and Its Prevalence-Is It Nature’s Alarm Clock?” Physiological Reports, John Wiley and Sons Inc., Jan. 2021, www.ncbi.nlm.nih.gov/pmc/articles/PMC7749606/.

McEwen, Bruce S. “Central Effects of Stress Hormones in Health and Disease: Understanding the Protective and Damaging Effects of Stress and Stress Mediators.” European Journal of Pharmacology, U.S. National Library of Medicine, 7 Apr. 2008, www.ncbi.nlm.nih.gov/pmc/articles/PMC2474765/.

McEwen, Bruce S. “Stressed or Stressed out: What Is the Difference?” Journal of Psychiatry & Neuroscience : JPN, U.S. National Library of Medicine, Sept. 2005, www.ncbi.nlm.nih.gov/pmc/articles/PMC1197275/.

Rodriquez, Erik J, et al. “Allostatic Load: Importance, Markers, and Score Determination in Minority and Disparity Populations.” Journal of Urban Health : Bulletin of the New York Academy of Medicine, Springer US, Mar. 2019, www.ncbi.nlm.nih.gov/pmc/articles/PMC6430278/.

Thau, Lauren, et al. “Physiology, Cortisol – Statpearls – NCBI Bookshelf.” In: StatPearls [Internet]. Treasure Island (FL), StatPearls Publishing, 6 Sept. 2021, www.ncbi.nlm.nih.gov/books/NBK538239/.

Young, Simon N. “L-Tyrosine to Alleviate the Effects of Stress?” Journal of Psychiatry & Neuroscience : JPN, U.S. National Library of Medicine, May 2007, www.ncbi.nlm.nih.gov/pmc/articles/PMC1863555/.

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Diabetes & Stress Are Connected In The Body

Diabetes & Stress Are Connected In The Body

Introduction

As the world is in constant motion, many people have to endure stressful situations affecting their bodies and health. The body needs hormones like cortisol to keep functioning as it affects the immune, nervous, cardiovascular, and musculoskeletal systems, to name a few. Another essential function the body needs is glucose, which requires energy to be in constant motion. Situations that cause the cortisol levels and glucose levels to rise in the body can lead to chronic issues like diabetes and chronic stress. This causes the individual to be miserable and be in a serious situation if it is not controlled right away. Today’s article examines how cortisol and glucose affect the body and the interwoven connection between stress and diabetes. Refer patients to certified, skilled providers specializing in stress management and endocrine treatments for diabetic individuals. We guide our patients by referring to our associated medical providers based on their examination when it’s appropriate. We find that education is critical for asking insightful questions to our providers. Dr. Alex Jimenez DC provides this information as an educational service only. Disclaimer

 

Can my insurance cover it? Yes, it may. If you are uncertain, here is the link to all the insurance providers we cover. If you have any questions or concerns, please call Dr. Jimenez at 915-850-0900.

01 LaValle Advanced Diagnostics in DM & Stress

How Does Cortisol Affect The Body?

 

Have you been experiencing sleeping problems at night? What about frequent headaches that are a nuisance throughout the entire day? Or have you noticed excessive weight loss or weight gain around your midsection? Some of these symptoms are signs that your cortisol and glucose levels are high and can affect your body. Cortisol is a hormone produced in the endocrine system and can be beneficial or harmful to the body if it is not regularly checked. Research studies have defined cortisol as one of the prominent glucocorticoids secreted out due to the response of the body’s biochemicals, characterized by the HPA (hypothalamic‐pituitary‐adrenal) axis helps cognitive events. However, when the cortisol levels turn chronic in the body due to circumstances that cause the body to become dysfunctional, it can significantly impact a person and cause an imbalance in the HPA axis. Some of the symptoms that chronic cortisol leads to the body can include:

  • Hormonal imbalances
  • Insulin resistance
  • Weight gain
  • Increases in visceral “belly” fat
  • Increased cortisol output
  • Immune problems
    • Allergies and Asthma
    • Inflamed Joints
    • Poor exercise recovery

Additional information has provided that the presence of cortisol in the body can help increase blood glucose availability to the brain. With cortisol providing organ functionality, the blood glucose provides energy for the body.

 

How Cortisol & Glucose Work In The Body

Cortisol helps stimulate mass glucose mobilization in the liver, allowing block protein synthesis to push amino acids into sugar for the body. This is known as fatty acid liberation biotransformed into glucose. When this happens, it helps stimulate visceral fat storage if excess glucose is not utilized, thus causing weight gain. Research studies have shown that a lack of cortisol can cause a decrease in hepatic glucose production in the body. This will cause hypoglycemia, where the body doesn’t have enough glucose in its system. Additional research shows that cortisol responds to any stressor that affects a person with low glucose levels but can also become positive after a glucose load. Managing the body’s glucose and cortisol levels can help progress the development of diabetes.


How Cortisol Is Linked With Type 2 Diabetes- Video

Have you experienced stressful situations that cause your muscles to tense up? How about feeling your blood sugar either spiking up or down? Do you feel inflammatory effects all over your body that makes them ache? Stress can cause harmful effects to the body, activating inflammation, increasing sympathetic tone, and reducing glucocorticoid responsiveness. Stress can also be linked to diabetes, as the video above shows how the stress hormone cortisol is linked with type 2 diabetes. Research studies have mentioned that cortisol can become negatively associated with the mechanics of insulin resistance, increasing the beta-cell function and increasing the insulin released in the body. This can become dangerous for many individuals that have pre-existing diabetes and have been dealing with stress constantly. 


The Interwoven Connection Between Stress & Diabetes

 

The interwoven connection between stress and diabetes is shown as research studies have found that the pathophysiology of anxiety and diabetes has increased insulin resistance risk for the body. When a person is dealing with chronic stress, it can cause them to have many issues like:

  • Cold intolerance
  • Diminished cognition and mood
  • Food sensitivities
  • Low energy throughout the day

When this happens, the body is at a high risk of developing insulin resistance and type 2 diabetes. Research studies have mentioned that type 2 diabetes is characterized by insulin resistance and beta-cell dysfunction. The glucocorticoid in the body can become excessive to affect the cells, causing dysfunctionality. Additional research studies have shown that any perceived stress can become a vital risk factor that not only affects the body, like hypertension, BMI (body mass index), or diet quality but can cause a rise in type 2 diabetes. When individuals find ways to lower their chronic stress, it can help manage their glucose levels from reaching critical levels.

 

Conclusion

The body’s chronic stress can cause insulin resistance and cause diabetes to become pre-existing. The body needs cortisol and glucose to keep functioning and have the energy to move. When people start to suffer from chronic stress and diabetes, it can become challenging to manage; however, making minor changes to the body like finding ways to lower stress, eating healthy foods, and monitoring glucose levels can help the body reset the glucose and cortisol levels to normal. Doing this can relieve many individuals who want to continue their health journey being stress-free.

 

References

Adam, Tanja C, et al. “Cortisol Is Negatively Associated with Insulin Sensitivity in Overweight Latino Youth.” The Journal of Clinical Endocrinology and Metabolism, The Endocrine Society, Oct. 2010, www.ncbi.nlm.nih.gov/pmc/articles/PMC3050109/.

De Feo, P, et al. “Contribution of Cortisol to Glucose Counterregulation in Humans.” The American Journal of Physiology, U.S. National Library of Medicine, July 1989, pubmed.ncbi.nlm.nih.gov/2665516/.

Hucklebridge, F H, et al. “The Awakening Cortisol Response and Blood Glucose Levels.” Life Sciences, U.S. National Library of Medicine, 1999, pubmed.ncbi.nlm.nih.gov/10201642/.

Joseph, Joshua J, and Sherita H Golden. “Cortisol Dysregulation: The Bidirectional Link between Stress, Depression, and Type 2 Diabetes Mellitus.” Annals of the New York Academy of Sciences, U.S. National Library of Medicine, Mar. 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5334212/.

Kamba, Aya, et al. “Association between Higher Serum Cortisol Levels and Decreased Insulin Secretion in a General Population.” PloS One, Public Library of Science, 18 Nov. 2016, www.ncbi.nlm.nih.gov/pmc/articles/PMC5115704/.

Lee, Do Yup, et al. “Technical and Clinical Aspects of Cortisol as a Biochemical Marker of Chronic Stress.” BMB Reports, Korean Society for Biochemistry and Molecular Biology, Apr. 2015, www.ncbi.nlm.nih.gov/pmc/articles/PMC4436856/.

Thau, Lauren, et al. “Physiology, Cortisol.” In: StatPearls [Internet]. Treasure Island (FL), StatPearls Publishing, 6 Sept. 2021, www.ncbi.nlm.nih.gov/books/NBK538239.

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The Effects of Low Laser Therapy on Repairing The Calcaneal Tendon | El Paso, TX

The Effects of Low Laser Therapy on Repairing The Calcaneal Tendon | El Paso, TX

The body is a well-working machine that can endure anything that is thrown in its way. However, when it gets an injury, the body’s natural healing process will ensure that the body can get back to its daily activities. The healing process of an injured muscle varies throughout the body. Depending on how severe the damage is and how long the healing process will take, the body can recover to a mere few days to a few months. One of the most gruelly healing processes that the body has to endure is a ruptured calcaneal tendon.

The Calcaneal Tendon

The calcaneal tendon or the Achilles tendon is a thick tendon that is located in the back of the leg. This muscle-tendon is what makes the body move while walking, running, or even jumping. Not only that, the calcaneal tendon is the strongest tendon in the body, and it connects the gastrocnemius and soleus muscles at the heel bone. When the calcaneal tendon is ruptured, the healing process can last from weeks to months until it is fully healed. 

 

 

The Healing Effects of Low Laser Therapy

One of the ways that can help the damaged calcaneal tendons’ healing process is low laser therapy. Studies have shown that low laser therapy can speed up the damaged tendon repair after a partial lesion. Not only that but the combination of ultrasound and low laser therapy has been studied to be the physical agents for treating tendon injuries. The studies showed that the combination of low laser therapy and ultrasound has beneficial properties during the recovery process of treating calcaneal tendon injuries.

 

 

The study found that when patients are being treated for their calcaneal tendons, their hydroxyproline levels around the treated area are significantly increased with ultrasound and low laser therapy. The body’s natural biochemical and biomechanical structures on the injured tendon increase, thus affecting the healing process. Another study has shown that low laser therapy can help reduce fibrosis and prevent oxidative stress in the traumatized calcaneal tendon. The study even showed that after the calcaneal tendon is traumatized, inflammation, angiogenesis, vasodilation, and the extracellular matrix are formed in the affected area. So when patients are being treated with low laser therapy for about fourteen to twenty-one days, their histological abnormalities are alleviated, reducing collagen concentration and fibrosis; preventing oxidative stress from increasing in the body.

 

Conclusion

Overall, it is said that the effects of low laser therapy can help speed up the healing process of repairing the calcaneal tendon. The promising results have been proven since low laser therapy can help repair the damaged tendon, reducing oxidative stress and preventing fibrosis from escalating, causing more problems on the injured tendon. And with the combination of ultrasound, the calcaneal tendon can recover faster so the body can continue its everyday activities without any prolonged injuries.

 

References:

Demir, Huseyin, et al. “Comparison of the Effects of Laser, Ultrasound, and Combined Laser + Ultrasound Treatments in Experimental Tendon Healing.” Lasers in Surgery and Medicine, U.S. National Library of Medicine, 2004, pubmed.ncbi.nlm.nih.gov/15278933/.

Fillipin, Lidiane Isabel, et al. “Low-Level Laser Therapy (LLLT) Prevents Oxidative Stress and Reduces Fibrosis in Rat Traumatized Achilles Tendon.” Lasers in Surgery and Medicine, U.S. National Library of Medicine, Oct. 2005, pubmed.ncbi.nlm.nih.gov/16196040/.

Oliveira, Fla’via Schlittler, et al. Effect of Low Level Laser Therapy (830 Nm … – Medical Laser. 2009, medical.summuslaser.com/data/files/86/1585171501_uLg8u2FrJP7ZHcA.pdf.

Wood, Viviane T, et al. “Collagen Changes and Realignment Induced by Low-Level Laser Therapy and Low-Intensity Ultrasound in the Calcaneal Tendon.” Lasers in Surgery and Medicine, U.S. National Library of Medicine, 2010, pubmed.ncbi.nlm.nih.gov/20662033/.

Functional Endocrinology: Cortisol and Melatonin Circadian Rhythm

Functional Endocrinology: Cortisol and Melatonin Circadian Rhythm

Do you feel:

  • You cannot stay asleep at night?
  • You have a slow start in the morning?
  • Afternoon fatigue?
  • Waking up tired even after getting six or more hours of sleep?
  • Under a high amount of stress?

If you are experiencing any of these situations, then it might be due to your melatonin and cortisol levels affecting your body and circadian rhythm.

Across the world, millions of people have trouble sleeping. In the United States, there are roughly about 50-70 million people who have a poor quality of sleep. When a person has slept for less than eight hours, they become tired, and many problems can come to them, especially if their lives are hectic. With a hectic lifestyle and poor sleep, it can cause the body to have low energy to get any task done, the cortisol stress hormone will be raised, and diseases like high blood pressure and diabetes can cause problems that can be chronic if it is not treated.

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In functional endocrinology, melatonin and cortisol are hormones that the body produces naturally. The cortisol hormone or the stress hormone helps the body be in a state of “fight or flight” mode, which can be a good thing for anyone who is doing a project or going for a job interview. Although when cortisol hormone levels are high, it can lead the body to have complications like inflammation, chronic oxidative stress, and high blood pressure.

The Melatonin Circadian Rhythm

With the melatonin hormone, this hormone tells the body when it is time to sleep. Sometimes though, people do have a hard time sleeping, and taking melatonin supplements can actually relax the body and thus making the person fall asleep. Since the pineal gland produces melatonin from the brain, it can also be found in the eyes, the bone marrow, and the gut to relax the body and making the person fall asleep naturally. Some studies show that the circadian rhythm of the pineal gland that is producing melatonin. By doing this, the research shows that the administration of melatonin can:

  • One: induce sleep on individuals who have trouble falling asleep.
  • Two: inhibits the body to wake up naturally from the circadian pacemaker.
  • Three: shift the circadian biological clocks to increase sleep intake when a person is trying to sleep at an earlier time to get the full eight-hour benefits of sleep.

When a person is working at a 9 to 5 job, they are rising with their bodies and relaxing their bodies after a hard day at work. Studies found out that the melatonin and cortisol hormones help regulate the 24-hour pattern of the body’s function and responses tremendously. With the body’s hormone production cycle, it can be disturbed if the person is staying awake late at night or sleeping during the day. When this happens, the person can get disruptive disorders like mood swings, dizziness, be irritable and depressed, and have metabolic disorders. Not only that, but the body’s immune system and its endocrine system can also be damaged as well, causing the body to be a host to infections and diseases.

There have been more studies on the circadian rhythms in the body, as the studies show how people who work in the night shift have been associated with a vast number of adverse health problems that attack the cardiovascular and gastrointestinal system as well as disturbing the metabolic system. Anyone who has worked the night shift has to change their sleep schedule and adapt to the rapid reorientation in their sleep/wake schedule to go to work and do their job. Since everyone is working at a shift schedule, it can be stressful and can affect a worker’s body performance as well as affecting melatonin and cortisol secretion.

Ways To Support Cortisol and Melatonin

Surprisingly though, there are ways to lower cortisol levels and make sure that melatonin levels are working correctly for the body to function. For cortisol levels to be lowered, a person should do meditative practices, find an enjoyable hobby, and, most importantly, try deep breathing exercises to relax the body from unwanted stress. With deep breathing exercises, it can help the body to release any tension that a person is holding, and the muscles in the body began to relax, and the blood starts to flow. With the melatonin levels, they work together with the body’s circadian rhythm and make sure the body knows when it is time to wake up, sleep and eat. The melatonin hormone can also help regulate the body’s temperature, blood pressure, and hormone levels to make sure it is functioning correctly. When there are high levels of these systems, it can cause the body to develop chronic illnesses and harm the body in the process.

Research shows that melatonin hormones can bind to neurological receptors in the body, thus promoting relaxation. Since melatonin binds to neurological receptors, it can also reduce nerve activity and dopamine levels to make the eyes heavy, thus making the person fall asleep.

Conclusion

With the body being able to naturally produce melatonin and cortisol levels to make sure that the body does not get overly stressed throughout the entire day. Since melatonin is partnered with the body’s circadian rhythm, the body knows when to stay up and fall asleep. Since everyone has a hectic schedule, it is essential to take time and relax and get on a healthy sleep schedule so the body can be healthy and functioning. Some products are here to make sure that the endocrine system is functioning properly and supporting the adrenal glands and sugar metabolism.

The scope of our information is limited to chiropractic, musculoskeletal, and nervous health issues or functional medicine articles, topics, and discussions. We use functional health protocols to treat injuries or disorders of the musculoskeletal system. Our office has made a reasonable attempt to provide supportive citations and has identified the relevant research study or studies supporting our posts. We also make copies of supporting research studies available to the board and or the public upon request. To further discuss the subject matter above, please feel free to ask Dr. Alex Jimenez or contact us at 915-850-0900.


References:

Cajochen, C, et al. �Role of Melatonin in the Regulation of Human Circadian Rhythms and Sleep.� Journal of Neuroendocrinology, U.S. National Library of Medicine, Apr. 2003, www.ncbi.nlm.nih.gov/pubmed/12622846.

James, Francine O, et al. �Circadian Rhythms of Melatonin, Cortisol, and Clock Gene Expression during Simulated Night Shift Work.� Sleep, Associated Professional Sleep Societies, LLC, Nov. 2007, www.ncbi.nlm.nih.gov/pmc/articles/PMC2082093/.

Monteleone, P, et al. �Temporal Relationship between Melatonin and Cortisol Responses to Nighttime Physical Stress in Humans.� Psychoneuroendocrinology, U.S. National Library of Medicine, 1992, www.ncbi.nlm.nih.gov/pubmed/1609019.

Raman, Ryan. �How Melatonin Can Help You Sleep and Feel Better.� Healthline, Healthline Media, 3 Sept. 2017, www.healthline.com/nutrition/melatonin-and-sleep.

Zamanian, Zahra, et al. �Outline of Changes in Cortisol and Melatonin Circadian Rhythms in the Security Guards of Shiraz University of Medical Sciences.� International Journal of Preventive Medicine, Medknow Publications & Media Pvt Ltd, July 2013, www.ncbi.nlm.nih.gov/pmc/articles/PMC3775223/.


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By informing individuals about how the National University of Health Sciences provides knowledge for future generations who want to make a difference in the world. The University offers a wide variety of medical professions for functional and integrative medicine.

5 Ways You�re Hurting Your Gut El Paso, Texas

5 Ways You�re Hurting Your Gut El Paso, Texas

Ever wondered why you feel sluggish from a long day? Or feel sick to the stomach when you ate something bad or overindulged on your favorite food? Could it be that your gut is showing signs of stress and discomfort due to certain habits that you may encounter and didn�t even know about it?

In our previous article, we talked about the six types of food that our gut needs to be healthy. Since our gut contains trillions of microbiomes, both good and bad, these microbiomes play an important role in our overall health. A healthy microbiome improves our gut health, heart health, brain health, controls our weight and regulates our blood sugar. With the good bacteria in our gut, the bacteria benefit us with a good digestive system and destroys the harmful bacteria. But certain lifestyles and diet choices can actually increase the bad bacteria and lower the good bacteria and overall health.

 

 

11860 Vista Del Sol, Ste. 128 5 Ways You�re Hurting Your Gut El Paso, Texas

 

Here are five surprisingly lifestyle choices that are hurting your gut:

Not Eating a Wide Range of Foods

Our gut plays an important role in our overall health. When we eat good whole foods, our gut is happier; we have more energy to complete any task that is thrown at us and we are getting nutrients for our gut flora. However, during the past couple of decades, we have been leaning more into processed foods due to the economic pressures of increased food productions. FOA stated that �75 percent of the world�s food is generated from only 12 plants and five animal species� and that is very bad to our gut flora.

Here at Injury Medical & Chiropractic Clinic, we inform our patients about the importance of eating nutritious, whole foods to promote not only a healthy gut but a healthy mind. When the body gets introduced to a wide variety of whole foods (with a high fiber content), our gut starts to repair the damage of processed food that we may have consumed internally.

 

11860 Vista Del Sol, Ste. 128 5 Ways You�re Hurting Your Gut El Paso, Texas

Inadequate Prebiotic Consumption

Prebiotics are the fibers that don�t need to be digested and can pass through our gut. That may seem like a waste however, prebiotics encourages friendly bacteria to grow in our gut. Any high-fiber fruits like apples can actually help to grow helpful microbes like Bifidobacteria.

However, when you disregard prebiotics to your diet, you are harming your digestive health. Without prebiotics, our digestive system slows down the development and diversity for our gut flora. So in order to have a healthy microbiome development, you need to incorporate foods filled with both digestible and indigestible fibers to your diet. Some foods included in this category are oats, nuts, onions, garlic, leeks, asparagus, bananas, pears, chickpeas, and beans.

Sticking to a high fiber diet maybe challenging however, there is the option of taking prebiotic supplements. If you have a food allergen or food sensitivity to any high enriched fiber foods, taking prebiotic supplements can actually help grow Bifidobacterium and Faecalibacterium in your gut and be beneficial to your health without the discomfort.

 

Excessive Alcohol Consumption

Every adult enjoys alcohol once in a while. Yes, it�s one of those beverages that help you relax a bit after a long day, however, too much of it can lead to alcohol abuse and addiction. So, did you know that consuming that much alcohol is bad for your heart, liver, and brain; thus hurting your gut health and giving you dysbiosis?

One study stated, that the alcoholics with dysbiosis had a lower median abundance of Bacteroidetes and a high abundance of Proteobacteria. The ones that weren�t alcoholics were not affected by the study.

However; there is some good news on limiting yourself to alcoholism and that it can be beneficial to your gut bacteria. If you moderately consumed red wine responsibly, the polyphenols in the wine can help benefit your gut flora. So, enjoy a glass of wine once in a while as a small treat that should not be taken for granted.

Inadequate Sleep

In one of the previous articles, we talked about how to achieve a good night sleep through herbs. When we get little to no sleep through our hectic lives, it affects us through various health problems, including heart disease and obesity. In a 2016 study, researchers discovered the effect of short-term sleep deprivation on the gut microbiota after two days.

When our body doesn�t receive the recommended 8 hours of sleep, our gut takes a huge toll as we feel sluggish and exhausted. So, to make sure that our gut microbiome will be taken care of, we recommended to turn off your electronical devices at least 30 minutes before you get ready to settle down for the night. Turn off all the lights, and don�t drink any liquids at least two hours before bed, close your eyes and take a deep breath in a meditative state, and relax as you drift off into slumber town.

 

 

Inadequate Exercise

Through our fast-paced lifestyle and stressful jobs, it�s hard to find time to exercise. But when we actually do find time to exercise, not only do our minds feel good; but our body and gut feel good as well. However, things always come up when we are in an exercise routine and we have to skip exercising altogether. It happens to all of us and it�s hard to pick up where we left off when we tried to exercise.

When we don�t exercise at least a couple of times out of the week, our bodies take a huge toll on us as we gained weight, our stress is way too high, and we have a higher chance of getting a chronic disease. When this happens our gut flora is a huge disadvantage. Here at the clinic, we strive to inform our patients about the importance of exercising and that it not only changes their lives but also changes their mood entirely.

However, don�t just go into a hard exercise routine where you will injure yourself. Start off with a low-intensity workout then build it up as you go because your gut flora will thank you for it.

As a final say, we here at Injury Medical want to keep you informed on nutrition and ways to help you improve your ailments with these 5 surprises. But to also educate you on what may be hurting your gut. With these surprises and slight changes to your daily life, your gut will be thanking you for the long haul.

 


 

NCBI Resources

According to evidence from a 2016 research study, the gut�s immune system is fundamental towards preventing a variety of diseases and it may often contribute to metabolic disorders. However, it might also help provide a treatment goal when observing systemic inflammation in insulin resistance. Moreover, modified gut immunity has been linked with changes to the gut microbiota, intestinal barrier function, gut-residing immune cells, and resistance to antigens which enter the gastrointestinal, or GI, system. Although this has been previously believed to raise the danger of esophageal ailments including, pathogenic infections and chronic inflammation, which may ultimately lead to chronic health issues.

 

 

Multi-Dimensional Roles of Ketone Bodies

Multi-Dimensional Roles of Ketone Bodies

Ketone bodies are created by the liver and utilized as an energy source when glucose is not readily available in the human body. The two main ketone bodies are acetoacetate (AcAc) and 3-beta-hydroxybutyrate (3HB), while acetone is the third and least abundant, ketone body. Ketones are always present in the blood and their levels increase during fasting and prolonged exercise.�Ketogenesis is the biochemical process by which organisms produce ketone bodies through the breakdown of fatty acids and ketogenic amino acids.

Ketone bodies are mainly generated in the mitochondria of liver cells. Ketogenesis occurs when there are low glucose levels in the blood, particularly after other cellular carbohydrate stores, such as glycogen, have been exhausted. This mechanism can also occur when there is insufficient amounts of insulin. The production of ketone bodies is ultimately initiated to make available energy which is stored in the human body as fatty acids. Ketogenesis occurs in the mitochondria where it is independently regulated.

Abstract

Ketone body metabolism is a central node in physiological homeostasis. In this review, we discuss how ketones serve discrete fine-tuning metabolic roles that optimize organ and organism performance in varying nutrient remains and protect from inflammation and injury in multiple organ systems. Traditionally viewed as metabolic substrates enlisted only in carbohydrate restriction, recent observations underscore the importance of ketone bodies as vital metabolic and signaling mediators when carbohydrates are abundant. Complementing a repertoire of known therapeutic options for diseases of the nervous system, prospective roles for ketone bodies in cancer have arisen, as have intriguing protective roles in heart and liver, opening therapeutic options in obesity-related and cardiovascular disease. Controversies in ketone metabolism and signaling are discussed to reconcile classical dogma with contemporary observations.

Introduction

Ketone bodies are a vital alternative metabolic fuel source for all the domains of life, eukarya, bacteria, and archaea (Aneja et al., 2002; Cahill GF Jr, 2006; Krishnakumar et al., 2008). Ketone body metabolism in humans has been leveraged to fuel the brain during episodic periods of nutrient deprivation. Ketone bodies are interwoven with crucial mammalian metabolic pathways such as ?-oxidation (FAO), the tricarboxylic acid cycle (TCA), gluconeogenesis, de novo lipogenesis (DNL), and biosynthesis of sterols. In mammals, ketone bodies are produced predominantly in the liver from FAO-derived acetyl-CoA, and they are transported to extrahepatic tissues for terminal oxidation. This physiology provides an alternative fuel that is augmented by relatively brief periods of fasting, which increases fatty acid availability and diminishes carbohydrate availability (Cahill GF Jr, 2006; McGarry and Foster, 1980; Robinson and Williamson, 1980). Ketone body oxidation becomes a significant contributor to overall energy mammalian metabolism within extrahepatic tissues in a myriad of physiological states, including fasting, starvation, the neonatal period, post-exercise, pregnancy, and adherence to low carbohydrate diets. Circulating total ketone body concentrations in healthy adult humans normally exhibit circadian oscillations between approximately 100�250 �M, rise to ~1 mM after prolonged exercise or 24h of fasting, and can accumulate to as high as 20 mM in pathological states like diabetic ketoacidosis (Cahill GF Jr, 2006; Johnson et al., 1969b; Koeslag et al., 1980; Robinson and Williamson, 1980; Wildenhoff et al., 1974). The human liver produces up to 300 g of ketone bodies per day (Balasse and Fery, 1989), which contribute between 5�20% of total energy expenditure in fed, fasted, and starved states (Balasse et al., 1978; Cox et al., 2016).

Recent studies now highlight imperative roles for ketone bodies in mammalian cell metabolism, homeostasis, and signaling under a wide variety of physiological and pathological states. Apart from serving as energy fuels for extrahepatic tissues like brain, heart, or skeletal muscle, ketone bodies play pivotal roles as signaling mediators, drivers of protein post-translational modification (PTM), and modulators of inflammation and oxidative stress. In this review, we provide both classical and modern views of the pleiotropic roles of ketone bodies and their metabolism.

Overview of Ketone Body Metabolism

The rate of hepatic ketogenesis is governed by an orchestrated series of physiological and biochemical transformations of fat. Primary regulators include lipolysis of fatty acids from triacylglycerols, transport to and across the hepatocyte plasma membrane, transport into mitochondria via carnitine palmitoyltransferase 1 (CPT1), the ?-oxidation spiral, TCA cycle activity and intermediate concentrations, redox potential, and the hormonal regulators of these processes, predominantly glucagon and insulin [reviewed in (Arias et al., 1995; Ayte et al., 1993; Ehara et al., 2015; Ferre et al., 1983; Kahn et al., 2005; McGarry and Foster, 1980; Williamson et al., 1969)]. Classically ketogenesis is viewed as a spillover pathway, in which ?-oxidation-derived acetyl-CoA exceeds citrate synthase activity and/or oxaloacetate availability for condensation to form citrate. Three-carbon intermediates exhibit anti-ketogenic activity, presumably due to their ability to expand the oxaloacetate pool for acetyl-CoA consumption, but hepatic acetyl-CoA concentration alone does not determine ketogenic rate (Foster, 1967; Rawat and Menahan, 1975; Williamson et al., 1969). The regulation of ketogenesis by hormonal, transcriptional, and post-translational events together support the notion that the molecular mechanisms that fine-tune ketogenic rate remain incompletely understood (see Regulation of HMGCS2 and SCOT/OXCT1).

Ketogenesis occurs primarily in hepatic mitochondrial matrix at rates proportional to total fat oxidation. After transport of acyl chains across the mitochondrial membranes and ?-oxidation, the mitochondrial isoform of 3-hydroxymethylglutaryl-CoA synthase (HMGCS2) catalyzes the fate committing condensation of acetoacetyl-CoA (AcAc-CoA) and acetyl-CoA to generate HMG-CoA (Fig. 1A). HMG-CoA lyase (HMGCL) cleaves HMG-CoA to liberate acetyl-CoA and acetoacetate (AcAc), and the latter is reduced to d-?-hydroxybutyrate (d-?OHB) by phosphatidylcholine-dependent mitochondrial d-?OHB dehydrogenase (BDH1) in a NAD+/NADH-coupled near-equilibrium reaction (Bock and Fleischer, 1975; LEHNINGER et al., 1960). The BDH1 equilibrium constant favors d-?OHB production, but the ratio of AcAc/d-?OHB ketone bodies is directly proportional to mitochondrial NAD+/NADH ratio, and thus BDH1 oxidoreductase activity modulates mitochondrial redox potential (Krebs et al., 1969; Williamson et al., 1967). AcAc can also spontaneously decarboxylate to acetone (Pedersen, 1929), the source of sweet odor in humans suffering ketoacidosis (i.e., total serum ketone bodies > ~7 mM; AcAc pKa 3.6, ?OHB pKa 4.7). The mechanisms through which ketone bodies are transported across the mitochondrial inner membrane are not known, but AcAc/d-?OHB are released from cells via monocarboxylate transporters (in mammals, MCT 1 and 2, also known as solute carrier 16A family members 1 and 7) and transported in the circulation to extrahepatic tissues for terminal oxidation (Cotter et al., 2011; Halestrap and Wilson, 2012; Halestrap, 2012; Hugo et al., 2012). Concentrations of circulating ketone bodies are higher than those in the extrahepatic tissues (Harrison and Long, 1940) indicating ketone bodies are transported down a concentration gradient. Loss-of-function mutations in MCT1 are associated with spontaneous bouts of ketoacidosis, suggesting a critical role in ketone body import.

� With the exception of potential diversion of ketone bodies into non-oxidative fates (see Non-oxidative metabolic fates of ketone bodies), hepatocytes lack the ability to metabolize the ketone bodies they produce. Ketone bodies synthesized de novo by liver are (i) catabolized in mitochondria of extrahepatic tissues to acetyl-CoA, which is available to the TCA cycle for terminal oxidation (Fig. 1A), (ii) diverted to the lipogenesis or sterol synthesis pathways (Fig. 1B), or (iii) excreted in the urine. As an alternative energetic fuel, ketone bodies are avidly oxidized in heart, skeletal muscle, and brain (Balasse and Fery, 1989; Bentourkia et al., 2009; Owen et al., 1967; Reichard et al., 1974; Sultan, 1988). Extrahepatic mitochondrial BDH1 catalyzes the first reaction of ?OHB oxidation, converting it to back AcAc (LEHNINGER et al., 1960; Sandermann et al., 1986). A cytoplasmic d-?OHB-dehydrogenase (BDH2) with only 20% sequence identity to BDH1 has a high Km for ketone bodies, and also plays a role in iron homeostasis (Davuluri et al., 2016; Guo et al., 2006). In extrahepatic mitochondrial matrix, AcAc is activated to AcAc-CoA through exchange of a CoA-moiety from succinyl-CoA in a reaction catalyzed by a unique mammalian CoA transferase, succinyl-CoA:3-oxoacid-CoA transferase (SCOT, CoA transferase; encoded by OXCT1), through a near equilibrium reaction. The free energy released by hydrolysis of AcAc-CoA is greater than that of succinyl-CoA, favoring AcAc formation. Thus ketone body oxidative flux occurs due to mass action: an abundant supply of AcAc and rapid consumption of acetyl-CoA through citrate synthase favors AcAc-CoA (+ succinate) formation by SCOT. Notably, in contrast to glucose (hexokinase) and fatty acids (acyl-CoA synthetases), the activation of ketone bodies (SCOT) into an oxidizable form does not require the investment of ATP. A reversible AcAc-CoA thiolase reaction [catalyzed by any of the four mitochondrial thiolases encoded by either ACAA2 (encoding an enzyme known as T1 or CT), ACAT1 (encoding T2), HADHA, or HADHB] yields two molecules of acetyl-CoA, which enter the TCA cycle (Hersh and Jencks, 1967; Stern et al., 1956; Williamson et al., 1971). During ketotic states (i.e., total serum ketones > 500 �M), ketone bodies become significant contributors to energy expenditure�and are utilized in tissues rapidly until uptake or saturation of oxidation occurs (Balasse et al., 1978; Balasse and Fery, 1989; Edmond et al., 1987). A very small fraction of liver-derived ketone bodies can be readily measured in the urine, and utilization and reabsorption rates by the kidney are proportionate to circulating concentration (Goldstein, 1987; Robinson and Williamson, 1980). During highly ketotic states (> 1 mM in plasma), ketonuria serves as a semi-quantitative reporter of ketosis, although most clinical assays of urine ketone bodies detect AcAc but not ?OHB (Klocker et al., 2013).

Ketogenic Substrates and their Impact on Hepatocyte Metabolism

Ketogenic substrates include fatty acids and amino acids (Fig. 1B). The catabolism of amino acids, especially leucine, generates about 4% of ketone bodies in post-absorptive state (Thomas et al., 1982). Thus the acetyl-CoA substrate pool to generate ketone bodies mainly derives from fatty acids, because during states of diminished carbohydrate supply, pyruvate enters the hepatic TCA cycle primarily via anaplerosis, i.e., ATP-dependent carboxylation to oxaloacetate (OAA), or to malate (MAL), and not oxidative decarboxylation to acetyl-CoA (Jeoung et al., 2012; Magnusson et al., 1991; Merritt et al., 2011). In liver, glucose and pyruvate contribute negligibly to ketogenesis, even when pyruvate decarboxylation to acetyl-CoA is maximal (Jeoung et al., 2012).

Acetyl-CoA subsumes several roles integral to hepatic intermediary metabolism beyond ATP generation via terminal oxidation (also see The integration of ketone body metabolism, post-translational modification, and cell physiology). Acetyl-CoA allosterically activates (i) pyruvate carboxylase (PC), thereby activating a metabolic control mechanism that augments anaplerotic entry of metabolites into the TCA cycle (Owen et al., 2002; Scrutton and Utter, 1967) and (ii) pyruvate dehydrogenase kinase, which phosphorylates and inhibits pyruvate dehydrogenase (PDH) (Cooper et al., 1975), thereby further enhancing flow of pyruvate into the TCA cycle via anaplerosis. Furthermore, cytoplasmic acetyl-CoA, whose pool is augmented by mechanisms that convert mitochondrial acetyl-CoA to transportable metabolites, inhibits fatty acid oxidation: acetyl-CoA carboxylase (ACC) catalyzes the conversion of acetyl-CoA to malonyl-CoA, the lipogenic substrate and allosteric inhibitor of mitochondrial CPT1 [reviewed in (Kahn et al., 2005; McGarry and Foster, 1980)]. Thus, the mitochondrial acetyl-CoA pool both regulates and is regulated by the spillover pathway of ketogenesis, which orchestrates key aspects of hepatic intermediary metabolism.

Non-Oxidative Metabolic Fates of Ketone Bodies

The predominant fate of liver-derived ketones is SCOT-dependent extrahepatic oxidation. However, AcAc can be exported from mitochondria and utilized in anabolic pathways via conversion to AcAc-CoA by an ATP-dependent reaction catalyzed by cytoplasmic acetoacetyl-CoA synthetase (AACS, Fig. 1B). This pathway is active during brain development and in lactating mammary gland (Morris, 2005; Robinson and Williamson, 1978; Ohgami et al., 2003). AACS is also highly expressed in adipose tissue, and activated osteoclasts (Aguilo et al., 2010; Yamasaki et al., 2016). Cytoplasmic AcAc-CoA can be either directed by cytosolic HMGCS1 toward sterol biosynthesis, or cleaved by either of two cytoplasmic thiolases to acetyl-CoA (ACAA1 and ACAT2), carboxylated to malonyl-CoA, and contribute to the synthesis of fatty acids (Bergstrom et al., 1984; Edmond, 1974; Endemann et al., 1982; Geelen et al., 1983; Webber and Edmond, 1977).

While the physiological significance is yet to be established, ketones can serve as anabolic substrates even in the liver. In artificial experimental contexts, AcAc can contribute to as much as half of newly synthesized lipid, and up to 75% of new synthesized cholesterol (Endemann et al., 1982; Geelen et al., 1983; Freed et al., 1988). Because AcAc is derived from incomplete hepatic fat oxidation, the ability of AcAc to contribute to lipogenesis in vivo would imply hepatic futile cycling, where fat-derived ketones can be utilized for lipid production, a notion whose physiological significance requires experimental validation, but could serve adaptive or maladaptive roles (Solinas et al., 2015). AcAc avidly supplies cholesterogenesis, with a low AACS Km-AcAc (~50 �M) favoring AcAc activation even in the fed state (Bergstrom et al., 1984). The dynamic role of cytoplasmic ketone metabolism has been suggested in primary mouse embryonic neurons and in 3T3-L1 derived-adipocytes, as AACS knockdown impaired differentiation of each cell type (Hasegawa et al., 2012a; Hasegawa et al., 2012b). Knockdown of AACS in mice in vivo decreased serum cholesterol (Hasegawa et al., 2012c). SREBP-2, a master transcriptional regulator of cholesterol biosynthesis, and peroxisome proliferator activated receptor (PPAR)-? are AACS transcriptional activators, and regulate its transcription during neurite development and in the liver (Aguilo et al., 2010; Hasegawa et al., 2012c). Taken together, cytoplasmic ketone body metabolism may be important in select conditions or disease natural histories, but are inadequate to dispose of liver-derived ketone bodies, as massive hyperketonemia occurs in the setting of selective impairment of the primary oxidative fate via loss of function mutations to SCOT (Berry et al., 2001; Cotter et al., 2011).

Regulation of HMGCS2 and SCOT/OXCT1

The divergence of a mitochondrial from the gene encoding cytosolic HMGCS occurred early in vertebrate evolution due to the need to support hepatic ketogenesis in species with higher brain to body weight ratios (Boukaftane et al., 1994; Cunnane and Crawford, 2003). Naturally occurring loss-of-function HMGCS2 mutations in humans cause bouts of hypoketotic hypoglycemia (Pitt et al., 2015; Thompson et al., 1997). Robust HMGCS2 expression is restricted to hepatocytes and colonic epithelium, and its expression and enzymatic activity are coordinated through diverse mechanisms (Mascaro et al., 1995; McGarry and Foster, 1980; Robinson and Williamson, 1980). While the full scope of physiological states that influence HMGCS2 requires further elucidation, its expression and/or activity is regulated during the early postnatal period, aging, diabetes, starvation or ingestion of ketogenic diet (Balasse and Fery, 1989; Cahill GF Jr, 2006; Girard et al., 1992; Hegardt, 1999; Satapati et al., 2012; Sengupta et al., 2010). In the fetus, methylation of 5� flanking region of Hmgcs2 gene inversely correlates with its transcription, and is partially reversed after birth (Arias et al., 1995; Ayte et al., 1993; Ehara et al., 2015; Ferre et al., 1983). Similarly, hepatic Bdh1 exhibits a developmental expression pattern, increasing from birth to weaning, and is also induced by ketogenic diet in a fibroblast growth factor (FGF)-21-dependent manner (Badman et al., 2007; Zhang et al., 1989). Ketogenesis in mammals is highly responsive to both insulin and glucagon, being suppressed and stimulated, respectively (McGarry and Foster, 1977). Insulin suppresses adipose tissue lipolysis, thus depriving ketogenesis of its substrate, while glucagon increases ketogenic flux through a direct effect on the liver (Hegardt, 1999). Hmgcs2 transcription is stimulated by forkhead transcriptional factor FOXA2, which is inhibited via insulin-phosphatidylinositol-3-kinase/Akt, and is induced by glucagon-cAMP-p300 signaling (Arias et al., 1995; Hegardt, 1999; Quant et al., 1990; Thumelin et al., 1993; von Meyenn et al., 2013; Wolfrum et al., 2004; Wolfrum et al., 2003). PPAR? (Rodriguez et al., 1994) together with its target, FGF21 (Badman et al., 2007) also induce Hmgcs2 transcription in the liver during starvation or administration of ketogenic diet (Badman et al., 2007; Inagaki et al., 2007). Induction of PPAR? may occur before the transition from fetal to neonatal physiology, while FGF21 activation may be favored in the early neonatal period via ?OHB-mediated inhibition of histone deacetylase (HDAC)-3 (Rando et al., 2016). mTORC1 (mammalian target of rapamycin complex 1) dependent inhibition of PPAR? transcriptional activity is also a key regulator of Hmgcs2 gene expression (Sengupta et al., 2010), and liver PER2, a master circadian oscillator, indirectly regulates Hmgcs2 expression (Chavan et al., 2016). Recent observations indicate that extrahepatic tumor-induced interleukin-6 impairs ketogenesis via PPAR? suppression (Flint et al., 2016). Despite these observations, it is important to note that physiological shifts in Hmgcs2 gene expression have not been mechanistically linked to HMGCS2 protein abundance or to variations of ketogenic rate.

HMGCS2 enzyme activity is regulated through multiple PTMs. HMGCS2 serine phosphorylation enhanced its activity in vitro (Grimsrud et al., 2012). HMGCS2 activity is allosterically inhibited by succinyl-CoA and lysine residue succinylation (Arias et al., 1995; Hegardt, 1999; Lowe and Tubbs, 1985; Quant et al., 1990; Rardin et al., 2013; Reed et al., 1975; Thumelin et al., 1993). Succinylation of HMGCS2, HMGCL, and BDH1 lysine residues in hepatic mitochondria are targets of the NAD+ dependent deacylase sirtuin 5 (SIRT5) (Rardin et al., 2013). HMGCS2 activity is also enhanced by SIRT3 lysine deacetylation, and it is possible that crosstalk between acetylation and succinylation regulates HMGCS2 activity (Rardin et al., 2013; Shimazu et al., 2013). Despite the ability of these PTMs to regulate HMGCS2 Km and Vmax, fluctuations of these PTMs have not yet been carefully mapped and have not been confirmed as mechanistic drivers of ketogenesis in vivo.

SCOT is expressed in all mammalian cells that harbor mitochondria, except those of hepatocytes. The importance of SCOT activity and ketolysis was demonstrated in SCOT-KO mice, which exhibited uniform lethality due to hyperketonemic hypoglycemia within 48h after birth (Cotter et al., 2011). Tissue-specific loss of SCOT in neurons or skeletal myocytes induces metabolic abnormalities during starvation but is not lethal (Cotter et al., 2013b). In humans, SCOT deficiency presents early in life with severe ketoacidosis, causing lethargy, vomiting, and coma (Berry et al., 2001; Fukao et al., 2000; Kassovska-Bratinova et al., 1996; Niezen-Koning et al., 1997; Saudubray et al., 1987; Snyderman et al., 1998; Tildon and Cornblath, 1972). Relatively little is known at the cellular level about SCOT gene and protein expression regulators. Oxct1 mRNA expression and SCOT protein and activity are diminished in ketotic states, possibly through PPAR-dependent mechanisms (Fenselau and Wallis, 1974; Fenselau and Wallis, 1976; Grinblat et al., 1986; Okuda et al., 1991; Turko et al., 2001; Wentz et al., 2010). In diabetic ketoacidosis, the mismatch between hepatic ketogenesis and extrahepatic oxidation becomes exacerbated by impairment of SCOT activity. Overexpression of insulin-independent glucose transporter (GLUT1/SLC2A1) in cardiomyocytes also inhibits Oxct1 gene expression and downregulates ketones terminal oxidation in a non-ketotic state (Yan et al., 2009). In liver, Oxct1 mRNA abundance is suppressed by microRNA-122 and histone methylation H3K27me3 that are evident during the transition from fetal to the neonatal period (Thorrez et al., 2011). However, suppression of hepatic Oxct1 expression in the postnatal period is primarily attributable to the evacuation of Oxct1-expressing hematopoietic progenitors from the liver, rather than a loss of previously existing Oxct1 expression in terminally differentiated hepatocytes. In fact, expression of Oxct1 mRNA and SCOT protein in differentiated hepatocytes are extremely low (Orii et al., 2008).

SCOT is also regulated by PTMs. The enzyme is hyper-acetylated in brains of SIRT3 KO mice, which also exhibit diminished AcAc dependent acetyl-CoA production (Dittenhafer-Reed et al., 2015). Non-enzymatic nitration of tyrosine residues of SCOT also attenuates its activity, which has been reported in hearts of various diabetic mice models (Marcondes et al., 2001; Turko et al., 2001; Wang et al., 2010a). In contrast, tryptophan residue nitration augments SCOT activity (Br�g�re et al., 2010; Rebrin et al., 2007). Molecular mechanisms of residue-specific nitration or de-nitration designed to modulate SCOT activity may exist and require elucidation.

Controversies in Extrahepatic Ketogenesis

In mammals the primary ketogenic organ is liver, and only hepatocytes and gut epithelial cells abundantly express the mitochondrial isoform of HMGCS2 (Cotter et al., 2013a; Cotter et al., 2014; McGarry and Foster, 1980; Robinson and Williamson, 1980). Anaerobic bacterial fermentation of complex polysaccharides yields butyrate, which is absorbed by colonocytes in mammalians for terminal oxidation or ketogenesis (Cherbuy et al., 1995), which may play a role in colonocyte differentiation (Wang et al., 2016). Excluding gut epithelial cells and hepatocytes, HMGCS2 is nearly absent in almost all other mammalian cells, but the prospect of extrahepatic ketogenesis has been raised in tumor cells, astrocytes of the central nervous system, the kidney, pancreatic ? cells, retinal pigment epithelium (RPE), and even in skeletal muscle (Adijanto et al., 2014; Avogaro et al., 1992; El Azzouny et al., 2016; Grabacka et al., 2016; Kang et al., 2015; Le Foll et al., 2014; Nonaka et al., 2016; Takagi et al., 2016a; Thevenet et al., 2016; Zhang et al., 2011). Ectopic HMGCS2 has been observed in tissues that lack net ketogenic capacity (Cook et al., 2016; Wentz et al., 2010), and HMGCS2 exhibits prospective ketogenesis-independent �moonlighting� activities, including within the cell nucleus (Chen et al., 2016; Kostiuk et al., 2010; Meertens et al., 1998).

Any extrahepatic tissue that oxidizes ketone bodies also has the potential to accumulate ketone bodies via HMGCS2 independent mechanisms (Fig. 2A). However, there is no extrahepatic tissue in which a steady state ketone body concentration exceeds that in the circulation (Cotter et al., 2011; Cotter et al., 2013b; Harrison and Long, 1940), underscoring that ketone bodies are transported down a concentration gradient via MCT1/2-dependent mechanisms. One mechanism of apparent extrahepatic ketogenesis may actually reflect relative impairment of ketone oxidation. Additional potential explanations fall within the realm of ketone body formation. First, de novo ketogenesis may occur via reversible enzymatic activity of thiolase and SCOT (Weidemann and Krebs, 1969). When the concentration of acetyl-CoA is relatively high, reactions normally responsible for AcAc oxidation operate in the reverse direction (GOLDMAN, 1954). A second mechanism occurs when ?-oxidation-derived intermediates accumulate due to a TCA cycle bottleneck, AcAc-CoA is converted to l-?OHB-CoA through a reaction catalyzed by mitochondrial 3-hydroxyacyl-CoA dehydrogenase, and further by 3-hydroxybutyryl CoA deacylase to l-?OHB, which is indistinguishable by mass spectrometry or resonance spectroscopy from the physiological enantiomer d-?OHB (Reed and Ozand, 1980). l-?OHB can be chromatographically or enzymatically distinguished from d-?OHB, and is present in extrahepatic tissues, but not in liver or blood (Hsu et al., 2011). Hepatic ketogenesis produces only d-?OHB, the only enantiomer that is a BDH substrate (Ito et al., 1984; Lincoln et al., 1987; Reed and Ozand, 1980; Scofield et al., 1982; Scofield et al., 1982). A third HMGCS2-independent mechanism generates d-?OHB through amino acid catabolism, particularly that of leucine and lysine. A fourth mechanism is only apparent because it is due to a labeling artifact and is thus termed pseudoketogenesis. This phenomenon is attributable to the reversibility of the SCOT and thiolase reactions, and can cause overestimation of ketone body turnover due to the isotopic dilution of ketone body tracer in extrahepatic tissue (Des Rosiers et al., 1990; Fink et al., 1988). Nonetheless, pseudoketogenesis may be negligible in most contexts (Bailey et al., 1990; Keller et al., 1978). A schematic (Fig. 2A) indicates a useful approach to apply while considering elevated tissue steady state concentration of ketones.

� Kidney has recently received attention as a potentially ketogenic organ. In the vast majority of states, the kidney is a net consumer of liver-derived ketone bodies, excreting or reabsorbing ketone bodies from the bloodstream, and kidney is generally not a net ketone body generator or concentrator (Robinson and Williamson, 1980). The authors of a classical study concluded that minimal renal ketogenesis quantified in an artificial experimental system was not physiologically relevant (Weidemann and Krebs, 1969). Recently, renal ketogenesis has been inferred in diabetic and autophagy deficient mouse models, but it is more likely that multi-organ shifts in metabolic homeostasis alter integrative ketone metabolism through inputs on multiple organs (Takagi et al., 2016a; Takagi et al., 2016b; Zhang et al., 2011). One recent publication suggested renal ketogenesis as a protective mechanism against ischemia-reperfusion injury in the kidney (Tran et al., 2016). Absolute steady state concentrations of ?OHB from extracts of mice renal tissue were reported at ~4�12 mM. To test whether this was tenable, we quantified ?OHB concentrations in renal extracts from fed and 24h fasted mice. Serum ?OHB concentrations increased from ~100 �M to 2 mM with 24h fasting (Fig. 2B), while renal steady state ?OHB concentrations approximate 100 �M in the fed state, and only 1 mM in the 24h fasted state (Fig. 2C�E), observations that are consistent with concentrations quantified over 45 years ago (Hems and Brosnan, 1970). It remains possible that in ketotic states, liver-derived ketone bodies could be renoprotective, but evidence for renal ketogenesis requires further substantiation. Compelling evidence that supports true extrahepatic ketogenesis was presented in RPE (Adijanto et al., 2014). This intriguing metabolic transformation was suggested to potentially allow RPE-derived ketones to flow to photoreceptor or M�ller glia cells, which could aid in the regeneration of photoreceptor outer segment.

?OHB as a Signaling Mediator

Although they are energetically rich, ketone bodies exert provocative �non-canonical� signaling roles in cellular homeostasis (Fig. 3) (Newman and Verdin, 2014; Rojas-Morales et al., 2016). For example, ?OHB inhibits Class I HDACs, which increases histone acetylation and thereby induces the expression of genes that curtail oxidative stress (Shimazu et al., 2013). ?OHB itself is a histone covalent modifier at lysine residues in livers of fasted or streptozotocin induced diabetic mice (Xie et al., 2016) (also see below, The integration of ketone body metabolism, post-translational modification, and cell physiology, and Ketone bodies, oxidative stress, and neuroprotection).

?OHB is also an effector via G-protein coupled receptors. Through unclear molecular mechanisms, it suppresses sympathetic nervous system activity and reduces total energy expenditure and heart rate by inhibiting short chain fatty acid signaling through G protein coupled receptor 41 (GPR41) (Kimura et al., 2011). One of the most studied signaling effects of ?OHB proceeds through GPR109A (also known as HCAR2), a member of the hydrocarboxylic acid GPCR sub-family expressed in adipose tissues (white and brown) (Tunaru et al., 2003), and in immune cells (Ahmed et al., 2009). ?OHB is the only known endogenous ligand of GPR109A receptor (EC50 ~770 �M) activated by d-?OHB, l-?OHB, and butyrate, but not AcAc (Taggart et al., 2005). The high concentration threshold for GPR109A activation is achieved through adherence to a ketogenic diet, starvation, or during ketoacidosis, leading to inhibition of adipose tissue lipolysis. The anti-lipolytic effect of GPR109A proceeds through inhibition of adenylyl cyclase and decreased cAMP, inhibiting hormone sensitive triglyceride lipase (Ahmed et al., 2009; Tunaru et al., 2003). This creates a negative feedback loop in which ketosis places a modulatory brake on ketogenesis by diminishing the release of non-esterified fatty acids from adipocytes (Ahmed et al., 2009; Taggart et al., 2005), an effect that can be counterbalanced by the sympathetic drive that stimulates lipolysis. Niacin (vitamin B3, nicotinic acid) is a potent (EC50 ~ 0.1 �M) ligand for GRP109A, effectively employed for decades for dyslipidemias (Benyo et al., 2005; Benyo et al., 2006; Fabbrini et al., 2010a; Lukasova et al., 2011; Tunaru et al., 2003). While niacin enhances reverse cholesterol transport in macrophages and reduces atherosclerotic lesions (Lukasova et al., 2011), the effects of ?OHB on atherosclerotic lesions remain unknown. Although GPR109A receptor exerts protective roles, and intriguing connections exist between ketogenic diet use in stroke and neurodegenerative diseases (Fu et al., 2015; Rahman et al., 2014), a protective role of ?OHB via GPR109A has not been demonstrated in vivo.

Finally, ?OHB may influence appetite and satiety. A meta-analysis of studies that measured the effects of ketogenic and very low energy diets concluded that participants consuming these diets exhibit higher satiety, compared to control diets (Gibson et al., 2015). However, a plausible explanation for this effect is the additional metabolic or hormonal elements that might modulate appetite. For example, mice maintained on a rodent ketogenic diet exhibited increased energy expenditure compared to chow control-fed mice, despite similar caloric intake, and circulating leptin or genes of peptides regulating feeding behavior were not changed (Kennedy et al., 2007). Among proposed mechanisms that suggest appetite suppression by ?OHB includes both signaling and oxidation (Laeger et al., 2010). Hepatocyte specific deletion of circadian rhythm gene (Per2)�and chromatin immunoprecipitation studies revealed that PER2 directly activates the Cpt1a gene, and indirectly regulates Hmgcs2, leading to impaired ketosis in Per2 knockout mice (Chavan et al., 2016). These mice exhibited impaired food anticipation, which was partially restored by systemic ?OHB administration. Future studies will be needed to confirm the central nervous system as a direct ?OHB target, and whether ketone oxidation is required for the observed effects, or whether another signaling mechanism is involved. Other investigators have invoked the possibility of local astrocyte-derived ketogenesis within the ventromedial hypothalamus as a regulator of food intake, but these preliminary observations also will benefit from genetic and flux-based assessments (Le Foll et al., 2014). The relationship between ketosis and nutrient deprivation remains of interest because hunger and satiety are important elements in failed weight loss attempts.

Integration of Ketone Body Metabolism, Post-Translational Modification, and Cell Physiology

Ketone bodies contribute to compartmentalized pools of acetyl-CoA, a key intermediate that exhibits prominent roles in cellular metabolism (Pietrocola et al., 2015). One role of acetyl-CoA is to serve as a substrate for acetylation, an enzymatically-catalyzed histone covalent modification (Choudhary et al., 2014; Dutta et al., 2016; Fan et al., 2015; Menzies et al., 2016). A large number of dynamically acetylated mitochondrial proteins, many of which may occur through non-enzymatic mechanisms, have also emerged from computational proteomics studies (Dittenhafer-Reed et al., 2015; Hebert et al., 2013; Rardin et al., 2013; Shimazu et al., 2010). Lysine deacetylases use a zinc cofactor (e.g., nucleocytosolic HDACs) or NAD+ as co-substrate (sirtuins, SIRTs) (Choudhary et al., 2014; Menzies et al., 2016). The acetylproteome serves as both sensor and effector of the total cellular acetyl-CoA pool, as physiological and genetic manipulations each result in non-enzymatic global variations of acetylation (Weinert et al., 2014). As intracellular metabolites serve as modulators of lysine residue acetylation, it is important to consider the role of ketone bodies, whose abundance is highly dynamic.

?OHB is an epigenetic modifier through at least two mechanisms. Increased ?OHB levels induced by fasting, caloric restriction, direct administration or prolonged exercise provoke HDAC inhibition or histone acetyltransferase activation (Marosi et al., 2016; Sleiman et al., 2016) or to oxidative stress (Shimazu et al., 2013). ?OHB inhibition of HDAC3 could regulate newborn metabolic physiology (Rando et al., 2016). Independently, ?OHB itself directly modifies histone lysine residues (Xie et al., 2016). Prolonged fasting, or steptozotocin-induced diabetic ketoacidosis increased histone ?-hydroxybutyrylation. Although the number of lysine ?-hydroxybutyrylation and acetylation sites was comparable, stoichiometrically greater histone ?-hydroxybutyrylation than acetylation was observed. Distinct genes were impacted by histone lysine ?-hydroxybutyrylation, versus acetylation or methylation, suggesting distinct cellular functions. Whether ?-hydroxybutyrylation is spontaneous or enzymatic is not known, but expands the range of mechanisms through ketone bodies dynamically influence transcription.

Essential cell reprogramming events during caloric restriction and nutrient deprivation may be mediated in SIRT3- and SIRT5-dependent mitochondrial deacetylation and desuccinylation, respectively, regulating ketogenic and ketolytic proteins at post-translational level in liver and extrahepatic tissues (Dittenhafer-Reed et al., 2015; Hebert et al., 2013; Rardin et al., 2013; Shimazu et al., 2010). Even though stoichiometric comparison of occupied sites does not necessarily link directly to shifts in metabolic flux, mitochondrial acetylation is dynamic and may be driven by acetyl-CoA concentration or mitochondrial pH, rather than enzymatic acetyltransferases (Wagner and Payne, 2013). That SIRT3 and SIRT5 modulate activities of ketone body metabolizing enzymes provokes the question of the reciprocal role of ketones in sculpting the acetylproteome, succinylproteome, and other dynamic cellular targets. Indeed, as variations of ketogenesis reflect NAD+ concentrations, ketone production and abundance could regulate sirtuin activity, thereby influencing total acetyl-CoA/succinyl-CoA pools, the acylproteome, and thus mitochondrial and cell physiology. ?-hydroxybutyrylation of enzyme lysine residues could add another layer to cellular reprogramming. In extrahepatic tissues, ketone body oxidation may stimulate analogous changes in cell homeostasis. While compartmentation of acetyl-CoA pools is highly regulated and coordinates a broad spectrum of cellular changes, the ability of ketone bodies to directly shape both mitochondrial and cytoplasmic acetyl-CoA concentrations requires elucidation (Chen et al., 2012; Corbet et al., 2016; Pougovkina et al., 2014; Schwer et al., 2009; Wellen and Thompson, 2012). Because acetyl-CoA concentrations are tightly regulated, and acetyl-CoA is membrane impermeant, it is crucial to consider the driver mechanisms coordinating acetyl-CoA homeostasis, including the rates of production and terminal oxidation in the TCA cycle, conversion into ketone bodies, mitochondrial efflux via carnitine acetyltransferase (CrAT), or acetyl-CoA export to cytosol after conversion to citrate and release by ATP citrate lyase (ACLY). The key roles of these latter mechanisms in cell acetylproteome and homeostasis require matched understanding of the roles of ketogenesis and ketone oxidation (Das et al., 2015; McDonnell et al., 2016; Moussaieff et al., 2015; Overmyer et al., 2015; Seiler et al., 2014; Seiler et al., 2015; Wellen et al., 2009; Wellen and Thompson, 2012). Convergent technologies in metabolomics and acylproteomics in the setting of genetically manipulated models will be required to specify targets and outcomes.

Anti- and Pro-Inflammatory Responses to Ketone Bodies

Ketosis and ketone bodies modulate inflammation and immune cell function, but varied and even discrepant mechanisms have been proposed. Prolonged nutrient deprivation reduces inflammation (Youm et al., 2015), but the chronic ketosis of type 1 diabetes is a pro-inflammatory state (Jain et al., 2002; Kanikarla-Marie and Jain, 2015; Kurepa et al., 2012). Mechanism-based signaling roles for ?OHB in inflammation emerge because many immune system cells, including macrophages or monocytes, abundantly express GPR109A. While ?OHB exerts a predominantly anti-inflammatory response (Fu et al., 2014; Gambhir et al., 2012; Rahman et al., 2014; Youm et al., 2015), high concentrations of ketone bodies, particularly AcAc, may trigger a pro-inflammatory response (Jain et al., 2002; Kanikarla-Marie and Jain, 2015; Kurepa et al., 2012).

Anti-inflammatory roles of GPR109A ligands in atherosclerosis, obesity, inflammatory bowel disease, neurological disease, and cancer have been reviewed (Graff et al., 2016). GPR109A expression is augmented in RPE cells of diabetic models, human diabetic patients (Gambhir et al., 2012), and in microglia during neurodegeneration (Fu et al., 2014). Anti-inflammatory effects of ?OHB are enhanced by GPR109A overexpression in RPE cells, and abrogated by pharmacological inhibition or genetic knockout of GPR109A (Gambhir et al., 2012). ?OHB and exogenous nicotinic acid (Taggart et al., 2005), both confer anti-inflammatory effects in TNF? or LPS-induced inflammation by decreasing the levels of pro-inflammatory proteins (iNOS, COX-2), or secreted cytokines (TNF?, IL-1?, IL-6, CCL2/MCP-1), in part through inhibiting NF-?B translocation (Fu et al., 2014; Gambhir et al., 2012). ?OHB decreases ER stress and the NLRP3 inflammasome, activating the antioxidative stress response (Bae et al., 2016; Youm et al., 2015). However, in neurodegenerative inflammation, GPR109A-dependent ?OHB-mediated protection does not involve inflammatory mediators like MAPK pathway signaling (e.g., ERK, JNK, p38) (Fu et al., 2014), but may require COX-1-dependent PGD2 production (Rahman et al., 2014). It is intriguing that macrophage GPR109A is required to exert a neuroprotective effect in an ischemic stroke model (Rahman et al., 2014), but the ability of ?OHB to inhibit the NLRP3 inflammasome in bone marrow derived macrophages is GPR109A independent (Youm et al., 2015). Although most studies link ?OHB to anti-inflammatory effects, ?OHB may be pro-inflammatory and increase markers of lipid peroxidation in calf hepatocytes (Shi et al., 2014). Anti- versus pro-inflammatory effects of ?OHB may thus depend on cell type, ?OHB concentration, exposure duration, and the presence or absence of co-modulators.

Unlike ?OHB, AcAc may activate pro-inflammatory signaling. Elevated AcAc, especially with a high glucose concentration, intensifies endothelial cell injury through an NADPH oxidase/oxidative stress dependent mechanism (Kanikarla-Marie and Jain, 2015). High AcAc concentrations in umbilical cord of diabetic mothers were correlated with higher protein oxidation rate and MCP-1 concentration (Kurepa et al., 2012). High AcAc in diabetic patients was correlated with TNF? expression (Jain et al., 2002), and AcAc, but not ?OHB, induced TNF?, MCP-1 expression, ROS accumulation, and diminished cAMP level in U937 human monocyte cells (Jain et al., 2002; Kurepa et al., 2012).

Ketone body dependent signaling phenomena are frequently triggered only with high ketone body concentrations (> 5 mM), and in the case of many studies linking ketones to pro- or anti-inflammatory effects, through unclear mechanisms. In addition, due to the contradictory effects of ?OHB versus AcAc on inflammation, and the ability of AcAc/?OHB ratio to influence mitochondrial redox potential, the best experiments assessing the roles of ketone bodies on cellular phenotypes compare the effects of AcAc and ?OHB in varying ratios, and at varying cumulative concentrations [e.g., (Saito et al., 2016)]. Finally, AcAc can be purchased commercially only as a lithium salt or as an ethyl ester that requires base hydrolysis before use. Lithium cation independently induces signal transduction cascades (Manji et al., 1995), and AcAc anion is labile. Finally, studies using racemic d/l-?OHB can be confounded, as only the d-?OHB stereoisomer can be oxidized to AcAc, but d-?OHB and l-?OHB can each signal through GPR109A, inhibit the NLRP3 inflammasome, and serve as lipogenic substrates.

Ketone Bodies, Oxidative Stress, and Neuroprotection

Oxidative stress is typically defined as a state in which ROS are presented in excess, due to excessive production and/or impaired elimination. Antioxidant and oxidative stress mitigating roles of ketone bodies have been widely described both in vitro and in vivo, particularly in the context of neuroprotection. As most neurons do not effectively generate high-energy phosphates from fatty acids�but do oxidize ketone bodies when carbohydrates are in short supply, neuroprotective effects of ketone bodies are especially important (Cahill GF Jr, 2006; Edmond et al., 1987; Yang et al., 1987). In oxidative stress models, BDH1 induction and SCOT suppression suggest that ketone body metabolism can be reprogrammed to sustain diverse cell signaling, redox potential, or metabolic requirements (Nagao et al., 2016; Tieu et al., 2003).

Ketone bodies decrease the grades of cellular damage, injury, death and lower apoptosis in neurons and cardiomyocytes (Haces et al., 2008; Maalouf et al., 2007; Nagao et al., 2016; Tieu et al., 2003). Invoked mechanisms are varied and not always linearly related to concentration. Low millimolar concentrations of (d or l)-?OHB scavenge ROS (hydroxyl anion), while AcAc scavenges numerous ROS species, but only at concentrations that exceed the physiological range (IC50 20�67 mM) (Haces et al., 2008). Conversely, a beneficial influence over the electron transport chain�s redox potential is a mechanism commonly linked to d-?OHB. While all three ketone bodies (d/l-?OHB and AcAc) reduced neuronal cell death and ROS accumulation triggered by chemical inhibition of glycolysis, only d-?OHB and AcAc prevented neuronal ATP decline. Conversely, in a hypoglycemic in vivo model, (d or l)-?OHB, but not AcAc prevented hippocampal lipid peroxidation (Haces et al., 2008; Maalouf et al., 2007; Marosi et al., 2016; Murphy, 2009; Tieu et al., 2003). In vivo studies of mice fed a ketogenic diet (87% kcal fat and 13% protein) exhibited neuroanatomical variation of antioxidant capacity (Ziegler et al., 2003), where the most profound changes were observed in hippocampus, with increase glutathione peroxidase and total antioxidant capacities.

Ketogenic diet, ketone esters (also see Therapeutic use of ketogenic diet and exogenous ketone bodies), or ?OHB administration exert neuroprotection in models of ischemic stroke (Rahman et al., 2014); Parkinson�s disease (Tieu et al., 2003); central nervous system oxygen toxicity seizure (D’Agostino et al., 2013); epileptic spasms (Yum et al., 2015); mitochondrial encephalomyopathy, lactic acidosis and stroke-like (MELAS) episodes syndrome (Frey et al., 2016) and Alzheimer�s disease (Cunnane and Crawford, 2003; Yin et al., 2016). Conversely, a recent report demonstrated histopathological evidence of neurodegenerative progression by a ketogenic diet in a transgenic mouse model of abnormal mitochondrial DNA repair, despite increases in mitochondrial biogenesis and antioxidant signatures (Lauritzen et al., 2016). Other conflicting reports suggest that exposure to high ketone body concentrations elicits oxidative stress. High ?OHB or AcAc doses induced nitric oxide secretion, lipid peroxidation, reduced expression of SOD, glutathione peroxidase and catalase in calf hepatocytes, while in rat hepatocytes the MAPK pathway induction was attributed to AcAc but not ?OHB (Abdelmegeed et al., 2004; Shi et al., 2014; Shi et al., 2016).

Taken together, most reports link ?OHB to attenuation of oxidative stress, as its administration inhibits ROS/superoxide production, prevents lipid peroxidation and protein oxidation, increases antioxidant protein levels, and improves mitochondrial respiration and ATP production (Abdelmegeed et al., 2004; Haces et al., 2008; Jain et al., 1998; Jain et al., 2002; Kanikarla-Marie and Jain, 2015; Maalouf et al., 2007; Maalouf and Rho, 2008; Marosi et al., 2016; Tieu et al., 2003; Yin et al., 2016; Ziegler et al., 2003). While AcAc has been more directly correlated than ?OHB with the induction of oxidative stress, these effects are not always easily dissected from prospective pro-inflammatory responses (Jain et al., 2002; Kanikarla-Marie and Jain, 2015; Kanikarla-Marie and Jain, 2016). Moreover, it is critical to consider that the apparent antioxidative benefit conferred by pleiotropic ketogenic diets may not be transduced by ketone bodies themselves, and neuroprotection conferred by ketone bodies may not entirely be attributable to oxidative stress. For example during glucose deprivation, in a model of glucose deprivation in cortical neurons, ?OHB stimulated autophagic flux and prevented autophagosome accumulation, which was associated with decreased neuronal death (Camberos-Luna et al., 2016). d-?OHB induces also the canonical antioxidant proteins FOXO3a, SOD, MnSOD, and catalase, prospectively through HDAC inhibition (Nagao et al., 2016; Shimazu et al., 2013).

Non-Alcoholic Fatty Liver Disease (NAFLD) and Ketone Body Metabolism

Obesity-associated NAFLD and nonalcoholic steatohepatitis (NASH) are the most common causes of liver disease in Western countries (Rinella and Sanyal, 2016), and NASH-induced liver failure is one of the most common reasons for liver transplantation. While excess storage of triacylglycerols in hepatocytes >5% of liver weight (NAFL) alone does not cause degenerative liver function, the progression to NAFLD in humans correlates with systemic insulin resistance and increased risk of type 2 diabetes, and may contribute to the pathogenesis of cardiovascular disease and chronic kidney disease (Fabbrini et al., 2009; Targher et al., 2010; Targher and Byrne, 2013). The pathogenic mechanisms of NAFLD and NASH are incompletely understood but include abnormalities of hepatocyte metabolism, hepatocyte autophagy and endoplasmic reticulum stress, hepatic immune cell function, adipose tissue inflammation, and systemic inflammatory mediators (Fabbrini et al., 2009; Masuoka and Chalasani, 2013; Targher et al., 2010; Yang et al., 2010). Perturbations of carbohydrate, lipid, and amino acid metabolism occur in and contribute to obesity, diabetes, and NAFLD in humans and in model organisms [reviewed in (Farese et al., 2012; Lin and Accili, 2011; Newgard, 2012; Samuel and Shulman, 2012; Sun and Lazar, 2013)]. While hepatocyte abnormalities in cytoplasmic lipid metabolism are commonly observed in NAFLD (Fabbrini et al., 2010b), the role of mitochondrial metabolism, which governs oxidative disposal of fats is less clear in NAFLD pathogenesis. Abnormalities of mitochondrial metabolism occur in and contribute to NAFLD/NASH pathogenesis (Hyotylainen et al., 2016; Serviddio et al., 2011; Serviddio et al., 2008; Wei et al., 2008). There is general (Felig et al., 1974; Iozzo et al., 2010; Koliaki et al., 2015; Satapati et al., 2015; Satapati et al., 2012; Sunny et al., 2011) but not uniform (Koliaki and Roden, 2013; Perry et al., 2016; Rector et al., 2010) consensus that, prior to the development of bona fide NASH, hepatic mitochondrial oxidation, and in particular fat oxidation, is augmented in obesity, systemic insulin resistance, and NAFLD. It is likely that as NAFLD progresses, oxidative capacity heterogenity, even among individual mitochondria, emerges, and ultimately oxidative function becomes impaired (Koliaki et al., 2015; Rector et al., 2010; Satapati et al., 2008; Satapati et al., 2012).

Ketogenesis is often used as a proxy for hepatic fat oxidation. Impairments of ketogenesis emerge as NAFLD progresses in animal models, and likely in humans. Through incompletely defined mechanisms, hyperinsulinemia suppresses ketogenesis, possibly contributing to hypoketonemia compared to lean controls (Bergman et al., 2007; Bickerton et al., 2008; Satapati et al., 2012; Soeters et al., 2009; Sunny et al., 2011; Vice et al., 2005). Nonetheless, the ability of circulating ketone body concentrations to predict NAFLD is controversial (M�nnist� et al., 2015; Sanyal et al., 2001). Robust quantitative magnetic resonance spectroscopic methods in animal models revealed increased ketone turnover rate with moderate insulin resistance, but decreased rates were evident with more severe insulin resistance (Satapati et al., 2012; Sunny et al., 2010). In obese humans with fatty liver, ketogenic rate is normal (Bickerton et al., 2008; Sunny et al., 2011), and hence, the rates of ketogenesis are diminished relative to the increased fatty acid load within hepatocytes. Consequently, ?-oxidation-derived acetyl-CoA may be directed to terminal oxidation in the TCA cycle, increasing terminal oxidation, phosphoenolpyruvate-driven gluconeogenesis via anaplerosis/cataplerosis, and oxidative stress. Acetyl-CoA also possibly undergoes export from mitochondria as citrate, a precursor substrate for lipogenesis (Fig. 4) (Satapati et al., 2015; Satapati et al., 2012; Solinas et al., 2015). While ketogenesis becomes less responsive to insulin or fasting with prolonged obesity (Satapati et al., 2012), the underlying mechanisms and downstream consequences of this remain incompletely understood. Recent evidence indicates that mTORC1 suppresses ketogenesis in a manner that may be downstream of insulin signaling (Kucejova et al., 2016), which is concordant with the observations that mTORC1 inhibits PPAR?-mediated Hmgcs2 induction (Sengupta et al., 2010) (also see Regulation of HMGCS2 and SCOT/OXCT1).

Preliminary observations from our group suggest adverse hepatic consequences of ketogenic insufficiency (Cotter et al., 2014). To test the hypothesis that impaired ketogenesis, even in carbohydrate-replete and thus �non-ketogenic� states, contributes to abnormal glucose metabolism and provokes steatohepatitis, we generated a mouse model of marked ketogenic insufficiency by administration of antisense oligonucleotides (ASO) targeted to Hmgcs2. Loss of HMGCS2 in standard low-fat chow-fed adult mice caused mild hyperglycemia and markedly increased production of hundreds of hepatic metabolites, a suite of which strongly suggested lipogenesis activation. High-fat diet feeding of mice with insufficient ketogenesis resulted in extensive hepatocyte injury and inflammation. These findings support the central hypotheses that (i) ketogenesis is not a passive overflow pathway but rather a dynamic node in hepatic and integrated physiological homeostasis, and (ii) prudent ketogenic augmentation to mitigate NAFLD/NASH and disordered hepatic glucose metabolism is worthy of exploration.

How might impaired ketogenesis contribute to hepatic injury and altered glucose homeostasis? The first consideration is whether the culprit is deficiency of ketogenic flux, or ketones themselves. A recent report suggests that ketone bodies may mitigate oxidative stress-induced hepatic injury in response to n-3 polyunsaturated fatty acids (Pawlak et al., 2015). Recall that due to lack of SCOT expression in hepatocytes, ketone bodies are not oxidized, but they can contribute to lipogenesis, and serve a variety of signaling roles independent of their oxidation (also see Non-oxidative metabolic fates of ketone bodies and ?OHB as a signaling mediator). It is also possible that hepatocyte-derived ketone bodies may serve as a signal and/or metabolite for neighboring cell types within the hepatic acinus, including stellate cells and Kupffer cell macrophages. While the limited literature available suggests that macrophages are unable to oxidize ketone bodies, this has only been measured using classical methodologies, and only in peritoneal macrophages (Newsholme et al., 1986; Newsholme et al., 1987), indicating that a re-assessment is appropriate given abundant SCOT expression in bone marrow-derived macrophages (Youm et al., 2015).

Hepatocyte ketogenic flux may also be cytoprotective. While salutary mechanisms may not depend on ketogenesis per se, low carbohydrate ketogenic diets have been associated with amelioration of NAFLD (Browning et al., 2011; Foster et al., 2010; Kani et al., 2014; Schugar and Crawford, 2012). Our observations indicate that hepatocyte ketogenesis may feedback and regulate TCA cycle flux, anaplerotic flux, phosphoenolpyruvate-derived gluconeogenesis (Cotter et al., 2014), and even glycogen turnover. Ketogenic impairment directs acetyl-CoA to increase TCA flux, which in liver has been linked to increased ROS-mediated injury (Satapati et al., 2015; Satapati et al., 2012); forces diversion of carbon into de novo synthesized lipid species that could prove cytotoxic; and prevents NADH re-oxidation to NAD+ (Cotter et al., 2014) (Fig. 4). Taken together, future experiments are required to address mechanisms through which relative ketogenic insufficiency may become maladaptive, contribute to hyperglycemia, provoke steatohepatitis, and whether these mechanisms are operant in human NAFLD/NASH. As epidemiological evidence suggests impaired ketogenesis during the progression of steatohepatitis (Embade et al., 2016; Marinou et al., 2011; M�nnist� et al., 2015; Pramfalk et al., 2015; Safaei et al., 2016) therapies that increase hepatic ketogenesis could prove salutary (Degirolamo et al., 2016; Honda et al., 2016).

Ketone Bodies and Heart Failure (HF)

With a metabolic rate exceeding 400 kcal/kg/day, and a turnover of 6�35 kg ATP/day, the heart is the organ with the highest energy expenditure and oxidative demand (Ashrafian et al., 2007; Wang et al., 2010b). The vast majority of myocardial energy turnover resides within mitochondria, and 70% of this supply originates from FAO. The heart is omnivorous and flexible under normal conditions, but the pathologically remodeling heart (e.g., due to hypertension or myocardial infarction) and the diabetic heart each become metabolically inflexible (Balasse and Fery, 1989; BING, 1954; Fukao et al., 2004; Lopaschuk et al., 2010; Taegtmeyer et al., 1980; Taegtmeyer et al., 2002; Young et al., 2002). Indeed, genetically programmed abnormalities of cardiac fuel metabolism in mouse models provoke cardiomyopathy (Carley et al., 2014; Neubauer, 2007). Under physiological conditions normal hearts oxidize ketone bodies in proportion to their delivery, at the expense of fatty acid and glucose oxidation, and myocardium is the highest ketone body consumer per unit mass (BING, 1954; Crawford et al., 2009; GARLAND et al., 1962; Hasselbaink et al., 2003; Jeffrey et al., 1995; Pelletier et al., 2007; Tardif et al., 2001; Yan et al., 2009). Compared to fatty acid oxidation, ketone bodies are more energetically efficient, yielding more energy available for ATP synthesis per molecule of oxygen invested (P/O ratio) (Kashiwaya et al., 2010; Sato et al., 1995; Veech, 2004). Ketone body oxidation also yields potentially higher energy than FAO, keeping ubiquinone oxidized, which raises redox span in the electron transport chain and makes more energy available to synthetize ATP (Sato et al., 1995; Veech, 2004). Oxidation of ketone bodies may also curtail ROS production, and thus oxidative stress (Veech, 2004).

Preliminary interventional and observational studies indicate a potential salutary role of ketone bodies in the heart. In the experimental ischemia/reperfusion injury context, ketone bodies conferred potential cardioprotective effects (Al-Zaid et al., 2007; Wang et al., 2008), possibly due to the increase mitochondrial abundance in heart or up-regulation of crucial oxidative phosphorylation mediators (Snorek et al., 2012; Zou et al., 2002). Recent studies indicate that ketone body utilization is increased in failing hearts of mice (Aubert et al., 2016) and humans (Bedi et al., 2016), supporting prior observations in humans (BING, 1954; Fukao et al., 2000; Janardhan et al., 2011; Longo et al., 2004; Rudolph and Schinz, 1973; Tildon and Cornblath, 1972). Circulating ketone body concentrations are increased in heart failure patients, in direct proportion to filling pressures, observations whose mechanism and significance remains unknown (Kupari et al., 1995; Lommi et al., 1996; Lommi et al., 1997; Neely et al., 1972), but mice with selective SCOT deficiency in cardiomyocytes exhibit accelerated pathological ventricular remodeling and ROS signatures in response to surgically induced pressure overload injury (Schugar et al., 2014).

Recent intriguing observations in diabetes therapy have revealed a potential link between myocardial ketone metabolism and pathological ventricular remodeling (Fig. 5). Inhibition of the renal proximal tubular sodium/glucose co-transporter 2 (SGLT2i) increases circulating ketone body concentrations in humans (Ferrannini et al., 2016a; Inagaki et al., 2015) and mice (Suzuki et al., 2014) via increased hepatic ketogenesis (Ferrannini et al., 2014; Ferrannini et al., 2016a; Katz and Leiter, 2015; Mudaliar et al., 2015). Strikingly, at least one of these agents decreased HF hospitalization (e.g., as revealed by the EMPA-REG OUTCOME trial), and improved cardiovascular mortality (Fitchett et al., 2016; Sonesson et al., 2016; Wu et al., 2016a; Zinman et al., 2015). While the driver mechanisms behind beneficial HF outcomes to linked SGLT2i remain actively debated, the survival benefit is likely multifactorial, prospectively including ketosis but also salutary effects on weight, blood pressure, glucose and uric acid levels, arterial stiffness, the sympathetic nervous system, osmotic diuresis/reduced plasma volume, and increased hematocrit (Raz and Cahn, 2016; Vallon and Thomson, 2016). Taken together, the notion that therapeutically increasing ketonemia either in HF patients, or those at high risk to develop HF, remains controversial but is under active investigation in pre-clinical and clinical studies (Ferrannini et al., 2016b; Kolwicz et al., 2016; Lopaschuk and Verma, 2016; Mudaliar et al., 2016; Taegtmeyer, 2016).

Ketone Bodies in Cancer Biology

Connections between ketone bodies and cancer are rapidly emerging, but studies in both animal models and humans have yielded diverse conclusions. Because ketone metabolism is dynamic and nutrient state responsive, it is enticing to pursue biological connections to cancer because of the potential for precision-guided nutritional therapies. Cancer cells undergo metabolic reprogramming in order to maintain rapid cell proliferation and growth (DeNicola and Cantley, 2015; Pavlova and Thompson, 2016). The classical Warburg effect in cancer cell metabolism arises from the dominant role of glycolysis and lactic acid fermentation to transfer energy and compensate for lower dependence on oxidative phosphorylation and limited mitochondrial respiration (De Feyter et al., 2016; Grabacka et al., 2016; Kang et al., 2015; Poff et al., 2014; Shukla et al., 2014). Glucose carbon is primarily directed through glycolysis, the pentose phosphate pathway, and lipogenesis, which together provide intermediates necessary for tumor biomass expansion (Grabacka et al., 2016; Shukla et al., 2014; Yoshii et al., 2015). Adaptation of cancer cells to glucose deprivation occurs through the ability to exploit alternative fuel sources, including acetate, glutamine, and aspartate (Jaworski et al., 2016; Sullivan et al., 2015). For example, restricted access to pyruvate reveals the ability of cancer cells to convert glutamine into acetyl-CoA by carboxylation, maintaining both energetic and anabolic needs (Yang et al., 2014). An interesting adaptation of cancer cells is the utilization of acetate as a fuel (Comerford et al., 2014; Jaworski et al., 2016; Mashimo et al., 2014; Wright and Simone, 2016; Yoshii et al., 2015). Acetate is also a substrate for lipogenesis, which is critical for tumor cell proliferation, and gain of this lipogenic conduit is associated with shorter patient survival and greater tumor burden (Comerford et al., 2014; Mashimo et al., 2014; Yoshii et al., 2015).

Non-cancer cells easily shift their energy source from glucose to ketone bodies during glucose deprivation. This plasticity may be more variable among cancer cell types, but in vivo implanted brain tumors oxidized [2,4-13C2]-?OHB to a similar degree as surrounding brain tissue (De Feyter et al., 2016). �Reverse Warburg effect� or �two compartment tumor metabolism� models hypothesize that cancer cells induce ?OHB production in adjacent fibroblasts, furnishing the tumor cell�s energy needs (Bonuccelli et al., 2010; Martinez-Outschoorn et al., 2012). In liver, a shift in hepatocytes from ketogenesis to ketone oxidation in hepatocellular carcinoma (hepatoma) cells is consistent with activation of BDH1 and SCOT activities observed in two hepatoma cell lines (Zhang et al., 1989). Indeed, hepatoma cells express OXCT1 and BDH1 and oxidize ketones, but only when serum starved (Huang et al., 2016). Alternatively, tumor cell ketogenesis has also been proposed. Dynamic shifts in ketogenic gene expression are exhibited during cancerous transformation of colonic epithelium, a cell type that normally expresses HMGCS2, and a recent report suggested that HMGCS2 may be a prognostic marker of poor prognosis in colorectal and squamous cell carcinomas (Camarero et al., 2006; Chen et al., 2016). Whether this association requires or involves ketogenesis, or a moonlighting function of HMGCS2, remains to be determined. Conversely, apparent ?OHB production by melanoma and glioblastoma cells, stimulated by the PPAR? agonist fenofibrate, was associated with growth arrest (Grabacka et al., 2016). Further studies are required to characterize roles of HMGCS2/SCOT expression, ketogenesis, and ketone oxidation in cancer cells.

Beyond the realm of fuel metabolism, ketones have recently been implicated in cancer cell biology via a signaling mechanism. Analysis of BRAF-V600E+ melanoma indicated OCT1-dependent induction of HMGCL in an oncogenic BRAF-dependent manner (Kang et al., 2015). HMGCL augmentation was correlated with higher cellular AcAc concentration, which in turn enhanced BRAFV600E-MEK1 interaction, amplifying MEK-ERK signaling in a feed-forward loop that drives tumor cell proliferation and growth. These observations raise the intriguing question of prospective extrahepatic ketogenesis that then supports a signaling mechanism (also see ?OHB as a signaling mediator and Controversies in extrahepatic ketogenesis). It is also important to consider independent effects of AcAc, d-?OHB, and l-?OHB on cancer metabolism, and when considering HMGCL, leucine catabolism may also be deranged.

The effects of ketogenic diets (also see Therapeutic use of ketogenic diet and exogenous ketone bodies) in cancer animal models are varied (De Feyter et al., 2016; Klement et al., 2016; Meidenbauer et al., 2015; Poff et al., 2014; Seyfried et al., 2011; Shukla et al., 2014). While epidemiological associations among obesity, cancer, and ketogenic diets are debated (Liskiewicz et al., 2016; Wright and Simone, 2016), a meta-analysis using ketogenic diets in animal models and in human studies suggested a salutary impact on survival, with benefits prospectively linked to the magnitude of ketosis, time of diet initiation, and tumor location (Klement et al., 2016; Woolf et al., 2016). Treatment of pancreatic cancer cells with ketone bodies (d-?OHB or AcAc) inhibited growth, proliferation and glycolysis, and a ketogenic diet (81% kcal fat, 18% protein, 1% carbohydrate) reduced in vivo tumor weight, glycemia, and increased muscle and body weight in animals with implanted cancer (Shukla et al., 2014). Similar results were observed using a metastatic glioblastoma cell model in mice that received ketone supplementation in the diet (Poff et al., 2014). Conversely, a ketogenic diet (91% kcal fat, 9% protein) increased circulating ?OHB concentration and diminished glycemia�but had no impact on either tumor volume or survival duration in glioma-bearing rats (De Feyter et al., 2016). A glucose ketone index has been proposed as a clinical indicator that improves metabolic management of ketogenic diet-induced brain cancer therapy in humans and mice (Meidenbauer et al., 2015). Taken together, roles of ketone body metabolism and ketone bodies in cancer biology are tantalizing because they each pose tractable therapeutic options, but fundamental aspects remain to be elucidated, with clear influences emerging from a matrix of variables, including (i) differences between exogenous ketone bodies versus ketogenic diet, (ii) cancer cell type, genomic polymorphisms, grade, and stage; and (iii) timing and duration of exposure to the ketotic state.

Dr Jimenez White Coat
Ketogenesis is created by ketone bodies through the breakdown of fatty acids and ketogenic amino acids. This biochemical process provides energy to various organs, specifically the brain, under circumstances of fasting as a response to an unavailability of blood glucose. Ketone bodies are mainly produced in the mitochondria of liver cells. While other cells are capable of carrying out ketogenesis, they are not as effective at doing so as liver cells. Because ketogenesis occurs in the mitochondria, its processes are regulated independently. Dr. Alex Jimenez D.C., C.C.S.T. Insight

Therapeutic Application of Ketogenic Diet and Exogenous Ketone Bodies

The applications of ketogenic diets and ketone bodies as therapeutic tools have also arisen in non-cancerous contexts including obesity and NAFLD/NASH (Browning et al., 2011; Foster et al., 2010; Schugar and Crawford, 2012); heart failure (Huynh, 2016; Kolwicz et al., 2016; Taegtmeyer, 2016); neurological and neurodegenerative disease (Martin et al., 2016; McNally and Hartman, 2012; Rho, 2015; Rogawski et al., 2016; Yang and Cheng, 2010; Yao et al., 2011); inborn errors of metabolism (Scholl-B�rgi et al, 2015); and exercise performance (Cox et al., 2016). The efficacy of ketogenic diets has been especially appreciated in therapy of epileptic seizure, particularly in drug-resistant patients. Most studies have evaluated ketogenic diets in pediatric patients, and reveal up to a ~50% reduction in seizure frequency after 3 months, with improved effectiveness in select syndromes (Wu et al., 2016b). The experience is more limited in adult epilepsy, but a similar reduction is evident, with better response in symptomatic generalized epilepsy patients (Nei et al., 2014). Underlying anti-convulsant mechanisms remain unclear, although postulated hypotheses include reduced glucose utilization/glycolysis, reprogrammed glutamate transport, indirect impact on ATP-sensitive potassium channel or adenosine A1 receptor, alteration of sodium channel isoform expression, or effects on circulating hormones including leptin (Lambrechts et al., 2016; Lin et al., 2017; Lutas and Yellen, 2013). It remains unclear whether the anti-convulsant effect is primarily attributable to ketone bodies, or due to the cascade metabolic consequences of low carbohydrate diets. Nonetheless, ketone esters (see below) appear to elevate the seizure threshold in animal models of provoked seizures (Ciarlone et al., 2016; D’Agostino et al., 2013; Viggiano et al., 2015).

Atkins-style and ketogenic, low carbohydrate diets are often deemed unpleasant, and can cause constipation, hyperuricemia, hypocalcemia, hypomagnesemia, lead to nephrolithiasis, ketoacidosis, cause hyperglycemia, and raise circulating cholesterol and free fatty acid concentrations (Bisschop et al., 2001; Kossoff and Hartman, 2012; Kwiterovich et al., 2003; Suzuki et al., 2002). For these reasons, long-term adherence poses challenges. Rodent studies commonly use a distinctive macronutrient distribution (94% kcal fat, 1% kcal carbohydrate, 5% kcal protein, Bio-Serv F3666), which provokes a robust ketosis. However, increasing the protein content, even to 10% kcal substantially diminishes the ketosis, and 5% kcal protein restriction confers confounding metabolic and physiological effects. This diet formulation is also choline depleted, another variable that influences susceptibility to liver injury, and even ketogenesis (Garbow et al., 2011; Jornayvaz et al., 2010; Kennedy et al., 2007; Pissios et al., 2013; Schugar et al., 2013). Effects of long-term consumption of ketogenic diets in mice remain incompletely defined, but recent studies in mice revealed normal survival and the absence of liver injury markers in mice on ketogenic diets over their lifespan, although amino acid metabolism, energy expenditure, and insulin signaling were markedly reprogrammed (Douris et al., 2015).

Mechanisms increasing ketosis through mechanisms alternative to ketogenic diets include the use of ingestible ketone body precursors. Administration of exogenous ketone bodies could create a unique physiological state not encountered in normal physiology, because circulating glucose and insulin concentrations are relatively normal, while cells might spare glucose uptake and utilization. Ketone bodies themselves have short half-lives, and ingestion or infusion of sodium ?OHB salt to achieve therapeutic ketosis provokes an untoward sodium load. R/S-1,3-butanediol is a non-toxic dialcohol that is readily oxidized in the liver to yield d/l-?OHB (Desrochers et al., 1992). In distinct experimental contexts, this dose has been administered daily to mice or rats for as long as seven weeks, yielding circulating ?OHB concentrations of up to 5 mM within 2 h of administration, which is stable for at least an additional 3h (D’Agostino et al., 2013). Partial suppression of food intake has been observed in rodents given R/S-1,3-butanediol (Carpenter and Grossman, 1983). In addition, three chemically distinct ketone esters (KEs), (i) monoester of R-1,3-butanediol and d-?OHB (R-3-hydroxybutyl R-?OHB); (ii) glyceryl-tris-?OHB; and (iii) R,S-1,3-butanediol acetoacetate diester, have also been extensively studied (Brunengraber, 1997; Clarke et al., 2012a; Clarke et al., 2012b; Desrochers et al., 1995a; Desrochers et al., 1995b; Kashiwaya et al., 2010). An inherent advantage of the former is that 2 moles of physiological d-?OHB are produced per mole of KE, following esterase hydrolysis in the intestine or liver. Safety, pharmacokinetics, and tolerance have been most extensively studied in humans ingesting R-3-hydroxybutyl R-?OHB, at doses up to 714 mg/kg, yielding circulating d-?OHB concentrations up to 6 mM (Clarke et al., 2012a; Cox et al., 2016; Kemper et al., 2015; Shivva et al., 2016). In rodents, this KE decreases caloric intake and plasma total cholesterol, stimulates brown adipose tissue, and improves insulin resistance (Kashiwaya et al., 2010; Kemper et al., 2015; Veech, 2013). Recent findings indicate that during exercise in trained athletes, R-3-hydroxybutyl R-?OHB ingestion decreased skeletal muscle glycolysis and plasma lactate concentrations, increased intramuscular triacylglycerol oxidation, and preserved muscle glycogen content, even when co-ingested carbohydrate stimulated insulin secretion (Cox et al., 2016). Further development of these intriguing results is required, because the improvement in endurance exercise performance was predominantly driven by a robust response to the KE in 2/8 subjects. Nonetheless, these results do support classical studies that indicate a preference for ketone oxidation over other substrates (GARLAND et al., 1962; Hasselbaink et al., 2003; Stanley et al., 2003; Valente-Silva et al., 2015), including during exercise, and that trained athletes may be more primed to utilize ketones (Johnson et al., 1969a; Johnson and Walton, 1972; Winder et al., 1974; Winder et al., 1975). Finally, the mechanisms that might support improved exercise performance following equal caloric intake (differentially distributed among macronutrients) and equal oxygen consumption rates remain to be determined. Clues may emerge from animal studies, as temporary exposure to R-3-hydroxybutyl R-?OHB in rats was associated with increased treadmill time, improved cognitive function, and an apparent energetic benefit in ex vivo perfused hearts (Murray et al., 2016).

Future Perspective

Once largely stigmatized as an overflow pathway capable of accumulating toxic emissions from fat combustion in carbohydrate restricted states (the �ketotoxic� paradigm), recent observations support the notion that ketone body metabolism serves salutary roles even in carbohydrate-laden states, opening a �ketohormetic� hypothesis. While the facile nutritional and pharmacological approaches to manipulate ketone metabolism make it an attractive therapeutic target, aggressively posed but prudent experiments remain in both the basic and translational research laboratories. Unmet needs have emerged in the domains of defining the role of leveraging ketone metabolism in heart failure, obesity, NAFLD/NASH, type 2 diabetes, and cancer. The scope and impact of ‘non-canonical� signaling roles of ketone bodies, including regulation of PTMs that likely feed back and forward into metabolic and signaling pathways, require deeper exploration. Finally, extrahepatic ketogenesis could open intriguing paracrine and autocrine signaling mechanisms and opportunities to influence co-metabolism within the nervous system and tumors to achieve therapeutic ends.

Acknowledgments

Ncbi.nlm.nih.gov/pmc/articles/PMC5313038/

Footnotes

Ncbi.nlm.nih.gov

In conclusion, ketone bodies are created by the liver in order to be used as an energy source when there is not enough glucose readily available in the human body. Ketogenesis occurs when there are low glucose levels in the blood, particularly after other cellular carbohydrate stores have been exhausted. The purpose of the article above was to discuss the multi-dimensional roles of ketone bodies in fuel metabolism, signaling, and therapeutics. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.

Curated by Dr. Alex Jimenez

Referenced from:�Ncbi.nlm.nih.gov/pmc/articles/PMC5313038/

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Additional Topic Discussion:�Acute Back Pain

Back pain�is one of the most prevalent causes of disability and missed days at work worldwide. Back pain attributes to the second most common reason for doctor office visits, outnumbered only by upper-respiratory infections. Approximately 80 percent of the population will experience back pain at least once throughout their life. The spine is a complex structure made up of bones, joints, ligaments, and muscles, among other soft tissues. Injuries and/or aggravated conditions, such as�herniated discs, can eventually lead to symptoms of back pain. Sports injuries or automobile accident injuries are often the most frequent cause of back pain, however, sometimes the simplest of movements can have painful results. Fortunately, alternative treatment options, such as chiropractic care, can help ease back pain through the use of spinal adjustments and manual manipulations, ultimately improving pain relief. �

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EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

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What Are The Risks Of Nrf2 Overexpression?

What Are The Risks Of Nrf2 Overexpression?

The nuclear erythroid 2-related factor 2 signaling pathway, best known as Nrf2, is a protective mechanism which functions as a “master regulator” of the human body’s antioxidant response. Nrf2 senses the levels of oxidative stress within the cells and triggers protective antioxidant mechanisms. While Nrf2 activation can have many benefits, Nrf2 “overexpression” can have several risks. It appears that a balanced degree of NRF2 is essential towards preventing the overall development of a variety of diseases in addition to the general improvement of these health issues. However, NRF2 can also cause complications. The main cause behind NRF2 “overexpression” is due to a genetic mutation or a continuing chronic exposure to a chemical or oxidative stress, among others. Below, we will discuss the downsides of Nrf2 overexpression and demonstrate its mechanisms of action within the human body.

Cancer

Research studies found that mice that don’t express NRF2 are more inclined to develop cancer in response to physical and chemical stimulation. Similar research studies, however, showed that NRF2 over-activation, or even KEAP1 inactivation, can result in the exacerbation of certain cancers, particularly if those pathways have been interrupted. Overactive�NRF2 can occur through smoking, where continuous NRF2 activation is believed to be the cause of lung cancer in smokers. Nrf2 overexpression might cause cancerous cells not to self-destruct, while intermittent NRF2 activation can prevent cancerous cells from triggering toxin induction. Additionally, because NRF2 overexpression increases the human body’s antioxidant ability to function beyond redox homeostasis, this boosts cell division and generates an unnatural pattern of DNA and histone methylation. This can ultimately�make�chemotherapy and radiotherapy less effective against cancer. Therefore, limiting NRF2 activation with substances like DIM, Luteolin, Zi Cao, or salinomycin could be ideal for patients with cancer although Nrf2 overactivation should not be considered to be the only cause for cancer. Nutrient deficiencies can affect genes, including NRF2. This might be one way as to how deficiencies contribute to tumors.

Liver

The overactivation of Nrf2, can also affect the function of specific organs in the human body. NRF2 overexpression can ultimately block the production of the insulin-like growth factor 1, or IGF-1, from the liver, which is essential for the regeneration of the liver.

Heart

While the acute overexpression of Nrf2 may have its benefits, continuous overexpression of NRF2 may cause long-term harmful effects on the heart, such as cardiomyopathy. NRF2 expression can be increased through high levels of cholesterol, or the activation of HO-1. This is believed to be the reason why chronic elevated levels of cholesterol might cause cardiovascular health issues.

Vitiligo

NRF2 overexpression has also been demonstrated to inhibit the capability to repigment in vitiligo as it might obstruct Tyrosinase, or TYR, action which is essential for repigmentation through melaninogenesis. Research studies have demonstrated that this process may be one of the primary reasons as to why people with vitiligo don’t seem to activate Nrf2 as efficiently as people without vitiligo.

Why NRF2 May Not Function Properly

Hormesis

NRF2 has to be hormetically activated in order to be able to take advantage of its benefits. In other words, Nrf2 shouldn’t trigger every minute or every day,�therefore, it’s a great idea to take breaks from it, by way of instance, 5 days on 5 days off or every other day. NRF2 must also accomplish a specific threshold to trigger its hormetic response, where a small stressor may not be enough to trigger it.

DJ-1 Oxidation

Protein deglycase DJ-1, or just DJ-1, also called the Parkinson’s disease protein, or PARK7, is a master regulator and detector of the redox status in the human body. DJ-1 is essential towards regulating how long NRF2 can perform its function and produce an antioxidant response. In the case that DJ-1 becomes overoxidized, the cells will make the DJ-1 protein less accessible. This process induces NRF2 activation to expire too fast since DJ-1 is paramount for maintaining balanced levels of NRF2 and preventing them from being broken down in the cell. In case the DJ-1 protein is non-existent or overoxidized, NRF2 expression will probably be minimal, even using DIM or alternative NRF2 activators. DJ-1 expression is imperative to restore impaired NRF2 action.

Chronic Illness

If you have a chronic illness, including CIRS, chronic infections/dysbiosis/SIBO, or heavy metal build up, such as mercury and/or that from root canals, these can obstruct the systems of NRF2 and phase two detoxification. Rather than oxidative stress turning NRF2 into an antioxidant, NRF2 will not trigger and oxidative stress can remain in the cell and cause damage, meaning, there is no antioxidant response. This is a significant reason why many people with CIRS have several sensitivities and reach to numerous factors. Some people believe they may be�having a herx response, however, this reaction may only be damaging the cells farther. Treating chronic illness, however, will permit the liver to discharge toxins into the bile, gradually developing the hormetic response of NRF2 activation. If the bile remains toxic and it’s not excreted from the human body, it will reactivate NRF2’s oxidative stress and cause you to feel worse once it’s reabsorbed from the gastrointestinal, or GI, tract. For example, ochratoxin A may block NRF2. Aside from treating the problem, histone deacetylase inhibitors can block the oxidative reaction from a number of the factors which trigger NRF2 activation but it might also prevent NRF2 from triggerring�normally, which might ultimately fail to serve its purpose.

Fish Oil Dysregulation

Cholinergics are substances which boost acetylcholine, or ACh, and choline in the brain through the increase of ACh, particularly when inhibiting the breakdown of ACh. Patients with CIRS often have problems with the dysregulation of acetylcholine levels in the human body, especially in the brain. Fish oil triggers NRF2, activating its protective antioxidant mechanism within the cells. People with chronic illnesses might have problems with cognitive stress and acetylcholine excitotoxicity, from organophosphate accumulation, which might cause fish oil to create�inflammation within the human body. Choline deficiency additionally induces NRF2 activation. Including choline into your diet, (polyphenols, eggs, etc.) can help enhance the effects of cholinergic dysregulation.

What Decreases NRF2?

Decreasing NRF2 overexpression is best for people that have cancer, although it may be beneficial for a variety of other health issues.

Diet, Supplements, and Common Medicines:

  • Apigenin (higher doses)
  • Brucea javanica
  • Chestnuts
  • EGCG (high doses increase NRF2)
  • Fenugreek (Trigonelline)
  • Hiba (Hinokitiol / ?-thujaplicin)
  • High Salt Diet
  • Luteolin (Celery, green pepper, parsley, perilla leaf, and chamomile tea – higher doses may increase NRF2 – 40 mg/kg luteolin three times per week )
  • Metformin (chronic intake)
  • N-Acetyl-L-Cysteine (NAC, by blocking the oxidative response, esp at high doses)
  • Orange Peel (have polymethoxylated flavonoids)
  • Quercetin (higher doses may increase NRF2 – 50 mg/kg/d quercetin)
  • Salinomycin (drug)
  • Retinol (all-trans retinoic acid)
  • Vitamin C when combined with Quercetin
  • Zi Cao (Purple Gromwel has Shikonin/Alkannin)

Pathways and Other:

  • Bach1
  • BET
  • Biofilms
  • Brusatol
  • Camptothecin
  • DNMT
  • DPP-23
  • EZH2
  • Glucocorticoid Receptor signaling (Dexamethasone and Betamethasone as well)
  • GSK-3? (regulatory feedback)
  • HDAC activation?
  • Halofuginone
  • Homocysteine (ALCAR can reverse this homocysteine induce low levels of NRF2)
  • IL-24
  • Keap1
  • MDA-7
  • NF?B
  • Ochratoxin A(aspergillus and pencicllium species)
  • Promyelocytic leukemia protein
  • p38
  • p53
  • p97
  • Retinoic acid receptor alpha
  • Selenite
  • SYVN1 (Hrd1)
  • STAT3 inhibition (such as Cryptotanshinone)
  • Testosterone (and Testosterone propionate, although TP intranasally may increase NRF2)
  • Trecator (Ethionamide)
  • Trx1 (via reduction of Cys151 in Keap1 or of Cys506 in the NLS region of Nrf2)
  • Trolox
  • Vorinostat
  • Zinc Deficiency (makes it worse in the brain)

Nrf2 Mechanism Of Action

Oxidative stress triggers through CUL3 where NRF2 from KEAP1, a negative inhibitor, subsequently enters the nucleus of these cells, stimulating the transcription of the AREs, turning sulfides into disulfides, and turning them into more antioxidant genes, leading to the upregulation of antioxidants, such as GSH, GPX, GST, SOD, etc.. The rest of these can be seen in the list below:
  • Increases AKR
  • Increases ARE
  • Increases ATF4
  • Increases Bcl-xL
  • Increases Bcl-2
  • Increases BDNF
  • Increases BRCA1
  • Increases c-Jun
  • Increases CAT
  • Increases cGMP
  • Increases CKIP-1
  • Increases CYP450
  • Increases Cul3
  • Increases GCL
  • Increases GCLC
  • Increases GCLM
  • Increases GCS
  • Increases GPx
  • Increases GR
  • Increases GSH
  • Increases GST
  • Increases HIF1
  • Increases HO-1
  • Increases HQO1
  • Increases HSP70
  • Increases IL-4
  • Increases IL-5
  • Increases IL-10
  • Increases IL-13
  • Increases K6
  • Increases K16
  • Increases K17
  • Increases mEH
  • Increases Mrp2-5
  • Increases NADPH
  • Increases Notch 1
  • Increases NQO1
  • Increases PPAR-alpha
  • Increases Prx
  • Increases p62
  • Increases Sesn2
  • Increases Slco1b2
  • Increases sMafs
  • Increases SOD
  • Increases Trx
  • Increases Txn(d)
  • Increases UGT1(A1/6)
  • Increases VEGF
  • Reduces ADAMTS(4/5)
  • Reduces alpha-SMA
  • Reduces ALT
  • Reduces AP1
  • Reduces AST
  • Reduces Bach1
  • Reduces COX-2
  • Reduces DNMT
  • Reduces FASN
  • Reduces FGF
  • Reduces HDAC
  • Reduces IFN-?
  • Reduces IgE
  • Reduces IGF-1
  • Reduces IL-1b
  • Reduces IL-2
  • Reduces IL-6
  • Reduces IL-8
  • Reduces IL-25
  • Reduces IL-33
  • Reduces iNOS
  • Reduces LT
  • Reduces Keap1
  • Reduces MCP-1
  • Reduces MIP-2
  • Reduces MMP-1
  • Reduces MMP-2
  • Reduces MMP-3
  • Reduces MMP-9
  • Reduces MMP-13
  • Reduces NfkB
  • Reduces NO
  • Reduces SIRT1
  • Reduces TGF-b1
  • Reduces TNF-alpha
  • Reduces Tyr
  • Reduces VCAM-1
  • Encoded from the NFE2L2 gene, NRF2, or nuclear erythroid 2-related factor 2, is a transcription factor in the basic leucine zipper, or bZIP, superfamily which utilizes a Cap’n’Collar, or CNC structure.
  • It promotes nitric enzymes, biotransformation enzymes, and xenobiotic efflux transporters.
  • It is an essential regulator at the induction of the phase II antioxidant and detoxification enzyme genes, which protect cells from damage caused by oxidative�stress and electrophilic attacks.
  • During homeostatic conditions, Nrf2 is sequestered in the cytosol through bodily attachment of the N-terminal domain of Nrf2, or the Kelch-like ECH-associated protein or Keap1, also referred to as INrf2 or Inhibitor of Nrf2, inhibiting Nrf2 activation.
  • It may also be controlled by mammalian selenoprotein thioredoxin reductase 1, or TrxR1, which functions as a negative regulator.
  • Upon vulnerability to electrophilic stressors, Nrf2 dissociates from Keap1, translocating into the nucleus, where it then heterodimerizes with a range of transcriptional regulatory protein.
  • Frequent interactions comprise with those of transcription authorities Jun and Fos, which can be members of the activator protein family of transcription factors.
  • After dimerization, these complexes then bind to antioxidant/electrophile responsive components ARE/EpRE and activate transcription, as is true with the Jun-Nrf2 complex, or suppress transcription, much like the Fos-Nrf2 complex.
  • The positioning of the ARE, which is triggered or inhibited, will determine which genes are transcriptionally controlled by these variables.
  • When ARE is triggered:
  1. Activation of the�synthesis of antioxidants is capable of detoxifying ROS like catalase, superoxide-dismutase, or SOD, GSH-peroxidases, GSH-reductase, GSH-transferase, NADPH-quinone oxidoreductase, or NQO1, Cytochrome P450 monooxygenase system, thioredoxin, thioredoxin reductase, and HSP70.
  2. Activation of this GSH synthase permits a noticeable growth of the�GSH intracellular degree, which is quite protective.
  3. The augmentation of this synthesis and degrees of phase II enzymes like UDP-glucuronosyltransferase, N-acetyltransferases, and sulfotransferases.
  4. The upregulation of HO-1, which is a really protective receptor with a potential growth of CO that in conjunction with NO allows vasodilation of ischemic cells.
  5. Reduction of iron overload through elevated ferritin and bilirubin as a lipophilic antioxidant. Both the phase II proteins along with the antioxidants are able to fix the chronic oxidative stress and also to revive a normal redox system.
  • GSK3? under the management of AKT and PI3K, phosphorylates Fyn resulting in Fyn nuclear localization, which Fyn phosphorylates Nrf2Y568 leading to nuclear export and degradation of Nrf2.
  • NRF2 also dampens the TH1/TH17 response and enriches the TH2 response.
  • HDAC inhibitors triggered the Nrf2 signaling pathway and up-regulated that the Nrf2 downstream targets HO-1, NQO1, and glutamate-cysteine ligase catalytic subunit, or GCLC, by curbing Keap1 and encouraging dissociation of Keap1 from Nrf2, Nrf2 nuclear translocation, and Nrf2-ARE binding.
  • Nrf2 includes a half-life of about 20 minutes under basal conditions.
  • Diminishing the IKK? pool through Keap1 binding reduces I?B? degradation and might be the elusive mechanism by which Nrf2 activation is proven to inhibit NF?B activation.
  • Keap1 does not always have to be downregulated to get NRF2 to operate, such as chlorophyllin, blueberry, ellagic acid, astaxanthin, and tea polyphenols may boost NRF2 and KEAP1 at 400 percent.
  • Nrf2 regulates negatively through the term of stearoyl CoA desaturase, or SCD, and citrate lyase, or CL.

Genetics

KEAP1

rs1048290

  • C allele – showed a significant risk for and a protective effect against drug resistant epilepsy (DRE)

rs11085735 (I’m AC)

  • associated with rate of decline of lung function in the LHS

MAPT

rs242561

  • T allele – protective allele for Parkinsonian disorders – had stronger NRF2/sMAF binding and was associated with the higher MAPT mRNA levels in 3 different regions in brain, including cerebellar cortex (CRBL), temporal cortex (TCTX), intralobular white matter (WHMT)

NFE2L2 (NRF2)

rs10183914 (I’m CT)

  • T allele – increased levels of Nrf2 protein and delayed age of onset of Parkinson’s by four years

rs16865105 (I’m AC)

  • C allele – had higher risk of Parkinson’s Disease

rs1806649 (I’m CT)

  • C allele – has been identified and may be relevant for breast cancer etiology.
  • associated with increased risk of hospital admissions during periods of high PM10 levels

rs1962142 (I’m GG)

  • T allele – was associated with a low level of cytoplasmic NRF2 expression (P = 0.036) and negative sulfiredoxin expression (P = 0.042)
  • A allele – protected from forearm blood flow (FEV) decline (forced expiratory volume in one second) in relation to cigarette smoking status (p = 0.004)

rs2001350 (I’m TT)

  • T allele – protected from FEV decline (forced expiratory volume in one second) in relation to cigarette smoking status (p = 0.004)

rs2364722 (I’m AA)

  • A allele – protected from FEV decline (forced expiratory volume in one second) in relation to cigarette smoking status (p = 0.004)

rs2364723

  • C allele – associated with significantly reduced FEV in Japanese smokers with lung cancer

rs2706110

  • G allele – showed a significant risk for and a protective effect against drug resistant epilepsy (DRE)
  • AA alleles – showed significantly reduced KEAP1 expression
  • AA alleles – was associated with an increased risk of breast cancer (P = 0.011)

rs2886161 (I’m TT)

  • T allele – associated with Parkinson’s Disease

rs2886162

  • A allele – was associated with low NRF2 expression (P = 0.011; OR, 1.988; CI, 1.162�3.400) and the AA genotype was associated with a worse survival (P = 0.032; HR, 1.687; CI, 1.047�2.748)

rs35652124 (I’m TT)

  • A allele – associated with higher associated with age at onset for Parkinson’s Disease vs G allele
  • C allele – had increase NRF2 protein
  • T allele – had less NRF2 protein and greater risk of heart disease and blood pressure

rs6706649 (I’m CC)

  • C allele – had lower NRF2 protein and increase risk for Parkinson’s Disease

rs6721961 (I’m GG)

  • T allele – had lower NRF2 protein
  • TT alleles – association between cigarette smoking in heavy smokers and a decrease in semen quality
  • TT allele – was associated with increased risk of breast cancer [P = 0.008; OR, 4.656; confidence interval (CI), 1.350�16.063] and the T allele was associated with a low extent of NRF2 protein expression (P = 0.0003; OR, 2.420; CI, 1.491�3.926) and negative SRXN1 expression (P = 0.047; OR, 1.867; CI = 1.002�3.478)
  • T allele – allele was also nominally associated with ALI-related 28-day mortality following systemic inflammatory response syndrome
  • T allele – protected from FEV decline (forced expiratory volume in one second) in relation to cigarette smoking status (p = 0.004)
  • G allele – associated with increased risk of ALI following major trauma in European and African-Americans (odds ratio, OR 6.44; 95% confidence interval
  • AA alleles – associated with infection-induced asthma
  • AA alleles – exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked
  • AA alleles – had a significantly higher risk for developing T2DM (OR 1.77; 95% CI 1.26, 2.49; p = 0.011) relative to those with the CC genotype
  • AA alleles – strong association between wound repair and late toxicities of radiation (associated with a significantly higher risk for developing late effects in African-Americans with a trend in Caucasians)
  • associated with oral estrogen therapy and risk of venous thromboembolism in postmenopausal women

rs6726395 (I’m AG)

  • A allele – protected from FEV1 decline (forced expiratory volume in one second) in relation to cigarette smoking status (p = 0.004)
  • A allele – associated with significantly reduced FEV1 in Japanese smokers with lung cancer
  • GG alleles – had higher NRF2 levels and decreased risk of macular degeneration
  • GG alleles – had higher survival with Cholangiocarcinoma

rs7557529 (I’m CT)

  • C allele – associated with Parkinson’s Disease
Dr Jimenez White Coat
Oxidative stress and other stressors can cause cell damage which may eventually lead to a variety of health issues. Research studies have demonstrated that Nrf2 activation can promote the human body’s protective antioxidant mechanism, however, researchers have discussed that Nrf2 overexpression can have tremendous risks towards overall health and wellness. Various types of cancer can also occur with Nrf2 overactivation. Dr. Alex Jimenez D.C., C.C.S.T. Insight

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below. Key sections:
  • 00:01:14 – Cancer and mortality
  • 00:19:04 – Aging
  • 00:26:30 – Brain and behavior
  • 00:38:06 – Final recap
  • 00:40:27 – Dose
Full timeline:
  • 00:00:34 – Introduction of sulforaphane, a major focus of the video.
  • 00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
  • 00:02:12 – Prostate cancer risk.
  • 00:02:23 – Bladder cancer risk.
  • 00:02:34 – Lung cancer in smokers risk.
  • 00:02:48 – Breast cancer risk.
  • 00:03:13 – Hypothetical: what if you already have cancer? (interventional)
  • 00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
  • 00:04:38 – Sulforaphane and cancer.
  • 00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
  • 00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
  • 00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
  • 00:08:32 – Inhibition of breast cancer stem cells.
  • 00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
  • 00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
  • 00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
  • 00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
  • 00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
  • 00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
  • 00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
  • 00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
  • 00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
  • 00:19:04 – Beginning of aging section.
  • 00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
  • 00:20:34 – Importance of low inflammation for longevity.
  • 00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
  • 00:23:40 – Mid-video recap: cancer, aging sections
  • 00:24:14 – Mouse studies suggest sulforaphane might improve adaptive immune function in old age.
  • 00:25:18 – Sulforaphane improved hair growth in a mouse model of balding. Picture at 00:26:10.
  • 00:26:30 – Beginning of brain and behavior section.
  • 00:27:18 – Effect of broccoli sprout extract on autism.
  • 00:27:48 – Effect of glucoraphanin on schizophrenia.
  • 00:28:17 – Start of depression discussion (plausible mechanism and studies).
  • 00:31:21 – Mouse study using 10 different models of stress-induced depression show sulforaphane similarly effective as fluoxetine (prozac).
  • 00:32:00 – Study shows direct ingestion of glucoraphanin in mice is similarly effective at preventing depression from social defeat stress model.
  • 00:33:01 – Beginning of neurodegeneration section.
  • 00:33:30 – Sulforaphane and Alzheimer’s disease.
  • 00:33:44 – Sulforaphane and Parkinson’s disease.
  • 00:33:51 – Sulforaphane and Hungtington’s disease.
  • 00:34:13 – Sulforaphane increases heat shock proteins.
  • 00:34:43 – Beginning of traumatic brain injury section.
  • 00:35:01 – Sulforaphane injected immediately after TBI improves memory (mouse study).
  • 00:35:55 – Sulforaphane and neuronal plasticity.
  • 00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
  • 00:37:19 – Sulforaphane and duchenne muscular dystrophy.
  • 00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
  • 00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
  • 00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
  • 00:41:01 – Anecdotes on sprouting at home.
  • 00:43:14 – On cooking temperatures and sulforaphane activity.
  • 00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
  • 00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
  • 00:44:56 – Cooking techniques and cruciferous vegetables.
  • 00:46:06 – Isothiocyanates as goitrogens.
According to research studies, Nrf2, is a fundamental transcription factor which activates the cells’ protective antioxidant mechanisms to detoxify the human body. The overexpression of Nrf2, however, can cause health issues. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�. Curated by Dr. Alex Jimenez
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Additional Topic Discussion:�Acute Back Pain

Back pain�is one of the most prevalent causes of disability and missed days at work worldwide. Back pain attributes to the second most common reason for doctor office visits, outnumbered only by upper-respiratory infections. Approximately 80 percent of the population will experience back pain at least once throughout their life. The spine is a complex structure made up of bones, joints, ligaments, and muscles, among other soft tissues. Injuries and/or aggravated conditions, such as�herniated discs, can eventually lead to symptoms of back pain. Sports injuries or automobile accident injuries are often the most frequent cause of back pain, however, sometimes the simplest of movements can have painful results. Fortunately, alternative treatment options, such as chiropractic care, can help ease back pain through the use of spinal adjustments and manual manipulations, ultimately improving pain relief.  
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EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

***
The Role Of Nrf2 Activation

The Role Of Nrf2 Activation

Many current research studies on cancer have allowed health professionals to understand the way the body detoxes. By analyzing upregulated genes in tumorous cells, researchers discovered the nuclear erythroid 2-related factor 2 signaling pathway, best known as Nrf2. NRF2 is an important transcription factor which activates the human body’s protective antioxidant mechanisms in order to regulate oxidation from both external and internal factors to prevent increased levels of oxidative stress.

Principles of Nrf2

NRF2 is essential towards maintaining overall health and wellness because it�serves the primary purpose of regulating how we manage everything we’re exposed to on a daily basis and not become sick. NRF2 activation plays a role in the phase II detoxification system.�Phase II detoxification takes lipophilic, or�fat soluble, free radicals and converts them into hydrophilic, or water soluble,�substances for excretion while inactivating exceptionally reactive metabolites and chemicals as a consequence of phase I.

NRF2 activation reduces overall oxidation and inflammation of the human body through a hormetic effect. To trigger NRF2, an inflammatory reaction due to oxidation must occur in order for the cells to produce an adaptive response and create antioxidants, such as glutathione. To break down the principle of Nrf2, essentially, oxidative stress activates NRF2 which then activates an antioxidant response in the human body. NRF2 functions to balance redox signaling, or the equilibrium of oxidant and antioxidant levels in the cell.

A great illustration of how this process functions can be demonstrated with exercise. Through every workout, the muscle adapts so that it can accommodate another workout session. If NRF2 becomes under- or over-expressed due to chronic infections or increased exposure to toxins, which may be observed in patients who have chronic inflammatory response syndrome, or CIRS, the health issues may worsen�following NRF2 activation. Above all, if DJ-1 becomes over-oxidized, NRF2 activation will end�too quickly.

Effects of NRF2 Activation

NRF2 activation is highly expressed in the lungs, liver, and kidneys. Nuclear erythroid 2-related factor 2, or NRF2, most commonly functions by counteracting increased levels of oxidation in the human body which can lead to oxidative stress. Nrf2 activation can help treat a variety of health issues, however, over-activation of Nrf2 may worsen various problems, which are demonstrated below.

Periodic activation of Nrf2 can help:

  • Aging (ie Longevity)
  • Autoimmunity and Overall Inflammation (ie Arthritis, Autism)
  • Cancer and Chemoprotection (ie EMF Exposure)
  • Depression and Anxiety (ie PTSD)
  • Drug Exposure (Alcohol, NSAIDs )
  • Exercise and Endurance Performance
  • Gut Disease (ie SIBO, Dysbiosis, Ulcerative Colitis)
  • Kidney Disease (ie Acute Kidney Injury, Chronic Kidney Disease, Lupus Nephritis)
  • Liver Disease (ie Alcoholic Liver Disease, Acute Hepatitis, Nonalcoholic Fatty Liver Disease, Nonalcoholic Steatohepatitis, Cirrhosis)
  • Lung Disease (ie Asthma, Fibrosis)
  • Metabolic And Vascular Disease (ie Atherosclerosis, Hypertension, Stroke, Diabetes)
  • Neurodegeneration (ie Alzheimer’s, Parkinson’s, Huntington’s and ALS)
  • Pain (ie Neuropathy)
  • Skin Disorders (ie Psoriasis, UVB/Sun Protection)
  • Toxin Exposure (Arsenic, Asbestos, Cadmium, Fluoride, Glyphosate, Mercury, Sepsis, Smoke)
  • Vision (ie Bright Light, Sensitivity, Cataracts, Corneal Dystrophy)

Hyperactivation of Nrf2 can worsen:

  • Atherosclerosis
  • Cancer (ie Brain, Breast, Head, Neck Pancreatic, Prostate, Liver, Thyroid)
  • Chronic Inflammatory Response Syndrome (CIRS)
  • Heart Transplant (while open NRF2 may be bad, NRF2 can help with repair)
  • Hepatitis C
  • Nephritis (severe cases)
  • Vitiligo

Furthermore, NRF2 can help make specific nutritional supplements, drugs,�and medications work. Many natural�supplements can also help trigger NRF2. Through current research studies, researchers have demonstrated that a large number of compounds which were once believed to be antioxidants were really pro-oxidants. That’s because nearly all of them need NRF2 to function, even supplements like curcumin and fish oil. Cocoa, for example, was shown to generate antioxidant effects in mice which possess the NRF2 gene.

Ways To Activate NRF2

In the case of neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease, stroke or even autoimmune diseases, it’s probably best to have Nrf2 upregulated, but in a hormetic fashion. Mixing NRF2 activators may also have an additive or synergistic effect, as occasionally it can be dose-dependent. The top ways to increase Nrf2 expression are listed below:

  • HIST (Exercise) + CoQ10 + Sun (these synergize very well)
  • Broccoli Sprouts + LLLT on my head and gut
  • Butyrate + Super Coffee + Morning Sun
  • Acupuncture (this is an alternative method, laser acupuncture may also be used)
  • Fasting
  • Cannabidiol (CBD)
  • Lion’s Mane + Melatonin
  • Alpha-lipoic acid + DIM
  • Wormwood
  • PPAR-gamma Activation

The following comprehensive listing containing over 350 other ways to activate Nrf2 through diet, lifestyle and devices, probiotics, supplements, herbs and oils, hormones and neurotransmitters, drugs/medications and chemicals, pathways/transcription factors, as well as other ways, is only a brief guide as to what can trigger Nrf2. For the sake of brevity in this article, we have left out over 500 other foods, nutritional supplements and compounds which can help activate Nrf2. The following are listed below:

Diet:

  • Acai Berries
  • Alcohol (Red wine is better, especially if there is a cork in it, as protocatechuic aldehyde from corks can also activate NRF2. In general, alcohol is not recommended, although acute intake increases NRF2. Chronic intake may decrease NRF2.
  • Algae (kelp)
  • Apples
  • Black Tea
  • Brazil Nuts
  • Broccoli Sprouts (and other isothiocyanates, sulforaphane as well as cruciferous vegetables like bok choy that have D3T)
  • Blueberries (0.6-10 g/day)
  • Carrots (falcarinone)
  • Cayenne Pepper (Capsaicin)
  • Celery (Butylphthalide)
  • Chaga (Betulin)
  • Chamomile Tea
  • Chia
  • Chinese Potato
  • Chokeberries (Aronia)
  • Chocolate (Dark or Cocoa)
  • Cinnamon
  • Coffee (such as chlorogenic acid, Cafestol and Kahweol)
  • Cordyceps
  • Fish (and Shellfish)
  • Flaxseed
  • Garlic
  • Ghee (possibly)
  • Ginger (and Cardamonin)
  • Gojiberries
  • Grapefruit (Naringenin – 50 mg/kg/d naringenin)
  • Grapes
  • Green Tea
  • Guava
  • Heart Of Palm
  • Hijiki/Wakame
  • Honeycomb
  • Kiwi
  • Legumes
  • Lion’s Mane
  • Mahuwa
  • Mangos (Mangiferin)
  • Mangosteen
  • Milk (goat, cow – via regulation of microbiome)
  • Mulberries
  • Olive Oil (pomace – hydroxytyrosol and Oleanolic Acid)
  • Omega 6 Fatty Acids (Lipoxin A4)
  • Osange Oranges (Morin)
  • Oyster Mushrooms
  • Papaya
  • Peanuts
  • Pigeon Peas
  • Pomegranate (Punicalagin, Ellagic Acid)
  • Propolis (Pinocembrin)
  • Purple Sweet Potatoes
  • Rambutan (Geraniin)
  • Onions
  • Reishi
  • Rhodiola Rosea (Salidroside)
  • Rice Bran (cycloartenyl ferulate)
  • Riceberry
  • Rooibos Tea
  • Rosemary
  • Sage
  • Safflower
  • Sesame Oil
  • Soy (and isoflavones, Daidzein, Genistein)
  • Squash
  • Strawberries
  • Tartary Buckwheat
  • Thyme
  • Tomatoes
  • Tonka Beans
  • Turmeric
  • Wasabi
  • Watermelon

Lifestyle and Devices:

  • Acupuncture and Electroacupuncture (via collagen cascade on ECM)
  • Blue light
  • Brain Games (increases NRF2 in the hippocampus)
  • Caloric Restriction
  • Cold (showers, plunges, ice bath, gear, cryotheraphy)
  • EMFs (low frequency, such as PEMF)
  • Exercise (Acute exercise like HIST or HIIT seems to be more beneficial for inducing NRF2, while longer exercise doesn�t induce NRF2, but does increase glutathione levels)
  • High Fat Diet (diet)
  • High Heat (Sauna)
  • Hydrogen Inhalation and Hydrogen Water
  • Hyperbaric Oxygen Therapy
  • Infrared Therapy (such as Joovv)
  • Intravenous Vitamin C
  • Ketogenic Diet
  • Ozone
  • Smoking (not recommended – acutely smoking increase NRF2, chronically smoking decreases NRF2. If you choose to smoke, Holy Basil may help protect against downregulation of NRF2)
  • Sun (UVB and Infrared)

Probiotics:

  • Bacillus subtilis (fmbJ)
  • Clostridium butyricum (MIYAIRI 588)
  • Lactobacillus brevis
  • Lactobacillus casei (SC4 and 114001)
  • Lactobacillus collinoides
  • Lactobacillus gasseri (OLL2809, L13-Ia, and SBT2055)
  • Lactobacillus helveticus (NS8)
  • Lactobacillus paracasei (NTU 101)
  • Lactobacillus plantarum (C88, CAI6, FC225, SC4)
  • Lactobacillus rhamnosus (GG)

Supplements, Herbs, and Oils:

  • Acetyl-L-Carnitine (ALCAR) and Carnitine
  • Allicin
  • Alpha-lipoic acid
  • Amentoflavone
  • Andrographis paniculata
  • Agmatine
  • Apigenin
  • Arginine
  • Artichoke (Cyanropicrin)
  • Ashwaganda
  • Astragalus
  • Bacopa
  • Beefsteak (Isogemaketone)
  • Berberine
  • Beta-caryophyllene
  • Bidens Pilosa
  • Black Cumin Seed Oil (Thymoquinone)
  • Boswellia
  • Butein
  • Butyrate
  • Cannabidiol (CBD)
  • Carotenioids (like Beta-carotene [synergy with Lycopene – 2 � 15 mg/d lycopene], Fucoxanthin, Zeaxanthin, Astaxanthin, and Lutein)
  • Chitrak
  • Chlorella
  • Chlorophyll
  • Chrysanthemum zawadskii
  • Cinnamomea
  • Common Sundew
  • Copper
  • Coptis
  • CoQ10
  • Curcumin
  • Damiana
  • Dan Shen/Red Sage (Miltirone)
  • DIM
  • Dioscin
  • Dong Ling Cao
  • Dong Quai (female ginseng)
  • Ecklonia Cava
  • EGCG
  • Elecampane / Inula
  • Eucommia Bark
  • Ferulic Acid
  • Fisetin
  • Fish Oil (DHA/EPA – 3 � 1 g/d fish oil containing 1098 mg EPA and 549 mg DHA)
  • Galangal
  • Gastrodin (Tian Ma)
  • Gentiana
  • Geranium
  • Ginkgo Biloba (Ginkgolide B)
  • Glasswort
  • Gotu Kola
  • Grape Seed Extract
  • Hairy Agrimony
  • Haritaki (Triphala)
  • Hawthorn
  • Helichrysum
  • Henna (Juglone)
  • Hibiscus
  • Higenamine
  • Holy Basil/Tulsi (Ursolic Acid)
  • Hops
  • Horny Goat Weed (Icariin/Icariside)
  • Indigo Naturalis
  • Iron (not recommended unless essential)
  • I3C
  • Job’s Tears
  • Moringa Oleifera (such as Kaempferol)
  • Inchinkoto (combo of Zhi Zi and Wormwood)
  • Kudzu Root
  • Licorice Root
  • Lindera Root
  • Luteolin (high doses for activation, lower doses inhibit NRF2 in cancer though)
  • Magnolia
  • Manjistha
  • Maximowiczianum (Acerogenin A)
  • Mexican Arnica
  • Milk Thistle
  • MitoQ
  • Mu Xiang
  • Mucuna Pruriens
  • Nicotinamide and NAD+
  • Panax Ginseng
  • Passionflower (such as Chrysin, but chyrisin may also reduce NRF2 via dysregulation of PI3K/Akt signaling)
  • Pau d�arco (Lapacho)
  • Phloretin
  • Piceatannol
  • PQQ
  • Procyanidin
  • Pterostilbene
  • Pueraria
  • Quercetin (high doses only, lower doses inhibit NRF2)
  • Qiang Huo
  • Red Clover
  • Resveratrol (Piceid and other phytoestrogens essentially, Knotweed)
  • Rose Hips
  • Rosewood
  • Rutin
  • Sappanwood
  • Sarsaparilla
  • Saururus chinensis
  • SC-E1 (Gypsum, Jasmine, Licorice, Kudzu, and Balloon Flower)
  • Schisandra
  • Self Heal (prunella)
  • Skullcap (Baicalin and Wogonin)
  • Sheep Sorrel
  • Si Wu Tang
  • Sideritis
  • Spikenard (Aralia)
  • Spirulina
  • St. John’s Wort
  • Sulforaphane
  • Sutherlandia
  • Tao Hong Si Wu
  • Taurine
  • Thunder God Vine (Triptolide)
  • Tocopherols (such as Vitamin E or Linalool)
  • Tribulus R
  • Tu Si Zi
  • TUDCA
  • Vitamin A (although other retinoids inhibit NRF2)
  • Vitamin C (high dose only, low dose does inhibit�NRF2)
  • Vitex/Chaste Tree
  • White Peony (Paeoniflorin from Paeonia lactiflora)
  • Wormwood (Hispidulin and Artemisinin)
  • Xiao Yao Wan (Free and Easy Wanderer)
  • Yerba Santa (Eriodictyol)
  • Yuan Zhi (Tenuigenin)
  • Zi Cao (will reduce NRF2 in cancer)
  • Zinc
  • Ziziphus Jujube

Hormones and Neurotransmitters:

  • Adiponectin
  • Adropin
  • Estrogen (but may decrease NRF2 in breast tissue)
  • Melatonin
  • Progesterone
  • Quinolinic Acid (in protective response to prevent excitotoxicity)
  • Serotonin
  • Thyroid Hormones like T3 (can increase NRF2 in healthy cells, but decrease it in cancer)
  • Vitamin D

Drugs/Medications and Chemicals:

  • Acetaminophen
  • Acetazolamide
  • Amlodipine
  • Auranofin
  • Bardoxolone methyl (BARD)
  • Benznidazole
  • BHA
  • CDDO-imidazolide
  • Ceftriaxone (and beta-lactam antibiotics)
  • Cialis
  • Dexamethasone
  • Diprivan (Propofol)
  • Eriodictyol
  • Exendin-4
  • Ezetimibe
  • Fluoride
  • Fumarate
  • HNE (oxidized)
  • Idazoxan
  • Inorganic arsenic and sodium arsenite
  • JQ1 (may inhibit NRF2 as well, unknown)
  • Letairis
  • Melphalan
  • Methazolamide
  • Methylene Blue
  • Nifedipine
  • NSAIDs
  • Oltipraz
  • PPIs (such as Omeprazole and Lansoprazole)
  • Protandim – great results in vivo, but weak/non-existent at activating NRF2 in humans
  • Probucol
  • Rapamycin
  • Reserpine
  • Ruthenium
  • Sitaxentan
  • Statins (such as Lipitor and Simvastatin)
  • Tamoxifen
  • Tang Luo Ning
  • tBHQ
  • Tecfidera (Dimethyl fumarate)
  • THC (not as strong as CBD)
  • Theophylline
  • Umbelliferone
  • Ursodeoxycholic Acid (UDCA)
  • Verapamil
  • Viagra
  • 4-Acetoxyphenol

Pathways/Transcription Factors:

  • ?7 nAChR activation
  • AMPK
  • Bilirubin
  • CDK20
  • CKIP-1
  • CYP2E1
  • EAATs
  • Gankyrin
  • Gremlin
  • GJA1
  • H-ferritin ferroxidase
  • HDAC inhibitors (such as valproic acid and TSA, but can cause NRF2 instability)
  • Heat Shock Proteins
  • IL-17
  • IL-22
  • Klotho
  • let-7 (knocks down mBach1 RNA)
  • MAPK
  • Michael acceptors (most)
  • miR-141
  • miR-153
  • miR-155 (knocks down mBach1 RNA as well)
  • miR-7 (in brain, helps with cancer and schizophrenia)
  • Notch1
  • Oxidatives stress (such as ROS, RNS, H2O2) and Electrophiles
  • PGC-1?
  • PKC-delta
  • PPAR-gamma (synergistic effects)
  • Sigma-1 receptor inhibition
  • SIRT1 (increases NRF2 in the brain and lungs but may decrease it overall)
  • SIRT2
  • SIRT6 (in the liver and brain)
  • SRXN1
  • TrxR1 inhibition (attenuation or depletion as well)
  • Zinc protoporphyrin
  • 4-HHE

Other:

  • Ankaflavin
  • Asbestos
  • Avicins
  • Bacillus amyloliquefaciens (used in agriculture)
  • Carbon Monoxide
  • Daphnetin
  • Glutathione Depletion (depletion of 80%�90% possibly)
  • Gymnaster koraiensis
  • Hepatitis C
  • Herpes (HSV)
  • Indian ash tree
  • Indigowoad Root
  • Isosalipurposide
  • Isorhamentin
  • Monascin
  • Omaveloxolone (strong, aka RTA-408)
  • PDTC
  • Selenium Deficiency (selenium deficiency can increase NRF2)
  • Siberian Larch
  • Sophoraflavanone G
  • Tadehagi triquetrum
  • Toona sinensis (7-DGD)
  • Trumpet Flower
  • 63171 and 63179 (strong)
Dr Jimenez White Coat
The nuclear erythroid 2-related factor 2 signaling pathway, best known by the acronym Nrf2, is a transcription factor which plays the major role of regulating the protective antioxidant mechanisms of the human body, particularly in order to control oxidative stress. While increased levels of oxidative stress can activate Nrf2, its effects are tremendously enhanced through the presence of specific compounds. Certain foods and supplements help activate Nrf2 in the human body, including the isothiocyanate sulforaphane from broccoli sprouts. Dr. Alex Jimenez D.C., C.C.S.T. Insight

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below.

Key sections:

  • 00:01:14 – Cancer and mortality
  • 00:19:04 – Aging
  • 00:26:30 – Brain and behavior
  • 00:38:06 – Final recap
  • 00:40:27 – Dose

Full timeline:

  • 00:00:34 – Introduction of sulforaphane, a major focus of the video.
  • 00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
  • 00:02:12 – Prostate cancer risk.
  • 00:02:23 – Bladder cancer risk.
  • 00:02:34 – Lung cancer in smokers risk.
  • 00:02:48 – Breast cancer risk.
  • 00:03:13 – Hypothetical: what if you already have cancer? (interventional)
  • 00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
  • 00:04:38 – Sulforaphane and cancer.
  • 00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
  • 00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
  • 00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
  • 00:08:32 – Inhibition of breast cancer stem cells.
  • 00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
  • 00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
  • 00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
  • 00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
  • 00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
  • 00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
  • 00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
  • 00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
  • 00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
  • 00:19:04 – Beginning of aging section.
  • 00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
  • 00:20:34 – Importance of low inflammation for longevity.
  • 00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
  • 00:23:40 – Mid-video recap: cancer, aging sections
  • 00:24:14 – Mouse studies suggest sulforaphane might improve adaptive immune function in old age.
  • 00:25:18 – Sulforaphane improved hair growth in a mouse model of balding. Picture at 00:26:10.
  • 00:26:30 – Beginning of brain and behavior section.
  • 00:27:18 – Effect of broccoli sprout extract on autism.
  • 00:27:48 – Effect of glucoraphanin on schizophrenia.
  • 00:28:17 – Start of depression discussion (plausible mechanism and studies).
  • 00:31:21 – Mouse study using 10 different models of stress-induced depression show sulforaphane similarly effective as fluoxetine (prozac).
  • 00:32:00 – Study shows direct ingestion of glucoraphanin in mice is similarly effective at preventing depression from social defeat stress model.
  • 00:33:01 – Beginning of neurodegeneration section.
  • 00:33:30 – Sulforaphane and Alzheimer’s disease.
  • 00:33:44 – Sulforaphane and Parkinson’s disease.
  • 00:33:51 – Sulforaphane and Hungtington’s disease.
  • 00:34:13 – Sulforaphane increases heat shock proteins.
  • 00:34:43 – Beginning of traumatic brain injury section.
  • 00:35:01 – Sulforaphane injected immediately after TBI improves memory (mouse study).
  • 00:35:55 – Sulforaphane and neuronal plasticity.
  • 00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
  • 00:37:19 – Sulforaphane and duchenne muscular dystrophy.
  • 00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
  • 00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
  • 00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
  • 00:41:01 – Anecdotes on sprouting at home.
  • 00:43:14 – On cooking temperatures and sulforaphane activity.
  • 00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
  • 00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
  • 00:44:56 – Cooking techniques and cruciferous vegetables.
  • 00:46:06 – Isothiocyanates as goitrogens.

According to many current research studies, the nuclear erythroid 2-related factor 2 signaling pathway, best known as Nrf2, is a fundamental transcription factor which activates the cells’ protective antioxidant mechanisms to detoxify the human body from both external and internal factors and prevent increased levels of oxidative stress. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.

Curated by Dr. Alex Jimenez

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Additional Topic Discussion:�Acute Back Pain

Back pain�is one of the most prevalent causes of disability and missed days at work worldwide. Back pain attributes to the second most common reason for doctor office visits, outnumbered only by upper-respiratory infections. Approximately 80 percent of the population will experience back pain at least once throughout their life. The spine is a complex structure made up of bones, joints, ligaments, and muscles, among other soft tissues. Injuries and/or aggravated conditions, such as�herniated discs, can eventually lead to symptoms of back pain. Sports injuries or automobile accident injuries are often the most frequent cause of back pain, however, sometimes the simplest of movements can have painful results. Fortunately, alternative treatment options, such as chiropractic care, can help ease back pain through the use of spinal adjustments and manual manipulations, ultimately improving pain relief. �

blog picture of cartoon paper boy

EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

***

What Are The Benefits Of Nrf2?

What Are The Benefits Of Nrf2?

Oxidative stress is a major contributor in the development of a variety of health issues, including cancer, heart disease, diabetes, accelerated aging and neurodegeneration. Antioxidant rich foods, herbs and supplements can be utilized to protect the human body from high levels of oxidative stress. Recent research studies have demonstrated that the Nrf2 gene pathway can help amplify the effects of antioxidants. The benefits of Nrf2 are described below.

Protects the Body Against Toxins

NRF2 is an intrinsic substance which can protect the cells from harmful, internal and external compounds. NRF2 may help enrich the human body’s reaction to drugs/medications and toxins, improving the production of�proteins that help eliminate compounds from the cell, known as multidrug resistance-associated proteins, or MRPs.�By way of instance, NRF2 is triggered upon cigarette smoke inhalation to allow the lungs to detox.

Additionally, it is essential for the lungs to protect themselves against allergens, viral diseases, bacterial endotoxins, hyperoxia, and various environmental pollutants. The constant trigger of Nrf2 however, can decrease the levels of a substance known as glutathione throughout the human body. NRF2 may also protect the liver from toxicity and it can protect the liver from arsenic hepatotoxicity. Moreover, NRF2 protects the liver and brain from alcohol consumption. By way of instance, Nrf2 can protect�against acetaminophen toxicity.

Fights Inflammation And Oxidative Stress

NRF2 activation can help battle against inflammation by diminishing inflammatory cytokines, such as those present in psoriasis. NRF2 may also decrease inflammation associated with a variety of health issues like arthritis and fibrosis of the liver, kidney, and lungs. NRF2 may also help control allergies by lowering Th1/Th17 cytokines and raising TH2 cytokines. This can be beneficial for ailments like asthma.

NRF2 additionally protects against cellular damage from blue light�and from UVA/UVB� found in sunlight. Nrf2 deficiencies can make it a whole lot easier to get sunburnt. One rationale behind this is because NRF2 is capable of regulating collagen in response to UV radiation. Advanced Glycation End-Products, or AGEs, contribute to the development of many health issues, including diabetes and neurodegenerative diseases. NRF2 can decrease the oxidative stress of AGEs within the body. NRF2 may also protect the human body from higher levels of heat-based stress.

Enhances Mitochondria And Exercise Performance

NRF2 is a mitochondrial booster. NRF2 activation contributes to a rise in ATP energy for mitochondria, in addition to enhanced use of oxygen, or citrate, and fat. With no NRF2, mitochondria would just have the ability to function with sugar, or glucose, rather than fat. NRF2 is also essential for mitochondria to develop through a process known as biogenesis. NRF2 activation is vital in order to�take advantage of� the benefits of exercise.

Because of�Nrf2’s activity, exercise raises mitochondrial function, where this result may be amplified with CoQ10, Cordyceps, and Caloric Restriction. Moderate exercise or acute exercise induces mitochondrial biogenesis and an elevated synthesis of superoxide dismutase, or SOD, and heme-oxygenase-1, or HO-1, through NRF2 activation. Alpha-Lipoic Acid,�or ALA, and Dan Shen can boost NRF2 mediated mitochondrial biogenesis. Furthermore,�NRF2 can also improve exercise tolerance where NRF2 deletion makes exercise harmful.

Protects Against Hypoxia

NRF2 also helps protect the human body from cellular oxygen loss/depletion, a health issue called hypoxia. Individuals with CIRS have reduced levels of oxygen since their NRF2 is obstructed, resulting in reduced levels of both VEGF, HIF1, and HO-1. Ordinarily, in healthy individuals with hypoxia, miR-101, which is required for the creation of stem cells, are overexpressed and enhance amounts of NRF2/HO-1 and VEGF/eNOS, therefore preventing brain damage, but that does not appear to occur in CIRS.

Hypoxia, characterized by low HIF1, in CIRS can also result in a leaky blood brain barrier due to an NRF2 imbalance. Salidroside, located in the Rhodiola, functions on NRF2 activation and assists with hypoxia by increasing levels of VEGF and HIF1 within the human body. NRF2 can also ultimately protect against lactate buildup in the heart. NRF2 activation may also stop hypoxia-induced Altitude Motion Sickness, or AMS.

Slows Down Aging

Several compounds which may be fatal in massive quantities may increase longevity in rather tiny quantities due to xenohormesis through NRF2, PPAR-gamma, and FOXO. A�very small quantity of toxins raises the cell’s ability to become better equipped for the next time it’s challenged with a toxin, however, this is not an endorsement to consume poisonous�chemicals.

A good illustration of this process is with caloric restriction. NRF2 can improve the lifespan of cells by raising their levels of mitochondria and antioxidants as well as lowering the cells’ capability to die. NRF2 declines with aging because NRF2 prevents stem cells from dying and assists them to�regenerate. NRF2 plays a part in enhancing wound healing.

Boosts the Vascular System

Done correctly with the production of sulforaphane, NRF2 activation may protect against heart diseases like high blood pressure, or hypertension, and hardening of the arteries, or atherosclerosis. NRF2 can enhance Acetylcholine’s, or ACh, relaxing activity on the vascular system whilst reducing cholesterol-induced stress. Nrf2 activation may strengthen the heart, however, over-activated Nrf2 can raise the probability of cardiovascular disease.

Statins may prevent or lead to cardiovascular disease. NRF2 also plays a major part in balancing iron and calcium which may shield the human body from having elevated levels of iron. By way of instance, Sirtuin 2, or SIRT2, can regulate iron homeostasis in cells by activation of NRF2 which is believed to be required for healthy levels of iron. NRF2 can also help with Sickle Cell Disease, or SCD. NRF2 dysfunction might be a reason behind endotoxemia like with dysbiosis or lectins induced hypertension. Nrf2 may also protect the human body against amphetamine induced damage to the vascular system.

Fights Neuroinflammation

NRF2 can shield against and assist with inflammation of the brain, commonly referred to as neuroinflammation. Furthermore, NRF2 can help with an Assortment of Central Nervous System, or CNS, disorders, including:

  • Alzheimer’s Disease (AD) – reduces amyloid beta stress on mitochondria
  • Amyotrophic Lateral Sclerosis (ALS)
  • Huntington’s Disease (HD)
  • Multiple Sclerosis (MS)
  • Nerve Regeneration
  • Parkinson’s disease (PD) – protects dopamine
  • Spinal Cord Injury (SCI)
  • Stroke (ischemic and hemorrhagic) – aids hypoxia
  • Traumatic Brain Injury

NRF2 has revealed a decrease of neuroinflammation in teens with Autism Spectrum Disorders�or ASD. Idebenone pairs properly with NRF2 activators contrary to neuroinflammation. NRF2 may also improve the Blood Brain Barrier,�or BBB. By way of instance, NRF2 activation with carnosic acid obtained from rosemary and sage can cross the BBB and cause neurogenesis. NRF2 has also been demonstrated to raise�Brain Derived Neurotrophic Factor, or BDNF.

NRF2 also modulates some nutritional supplements capacity to cause Nerve Growth Factor, or NGF as it� can also aid with brain fog and glutamate-induced issues by modulating N-Methyl-D-Aspartate,�or NMDA receptors. It may also lower the oxidative stress from quinolinic acid, referred to as QUIN. NRF2 activation can protect against seizures and large doses can decrease the brink of a seizure. At regular doses of stimulation, NRF2 can enhance cognitive abilities following a seizure by lowering extracellular glutamate in the brain and by it’s ability to draw cysteine from glutamate and glutathione.

Relieves Depression

In depression, it’s normal to notice inflammation in the brain, especially from the prefrontal cortex and hippocampus, as well as decreased BDNF. In some versions of depression, NRF2 can improve depressive symptoms by lowering inflammation within the brain and increasing BDNF levels. Agmatine’s capability to decrease depression by raising noradrenaline, dopamine, serotonin, and BDNF in the hippocampus depends upon NRF2 activation.

Contains Anti-Cancer Properties

NRF2 is equally a tumor suppressor as it is a tumor promoter if not managed accordingly. NRF2 can protect against cancer caused by free radicals and oxidative stress, however, NRF2 overexpression can be found in cancer cells as well. Intense activation of NRF2 can assist with a variety of cancers. By way of instance, the supplement Protandim can reduce skin cancer by NRF2 activation.

Relieves Pain

Gulf War Illness, or GWI, a notable illness affecting Gulf War Veterans, is a collection of unexplained, chronic symptoms which may include tiredness, headaches, joint pain, indigestion, insomnia, dizziness, respiratory ailments, and memory issues. NRF2 can improve symptoms of GWI by diminishing hippocampal and general inflammation, in addition to decreasing pain. NRF2 can additionally assist with pain from bodily nerve injury and improve nerve damage from diabetic neuropathy.

Improves Diabetes

High glucose levels, best referred to as hyperglycemia, causes oxidative damage to the cells due to the disruption of mitochondrial function. NRF2 activation may shield the human body against hyperglycemia’s harm to the cell, thereby preventing cell death. NRF2 activation can additionally protect, restore, and enhance pancreatic beta-cell function, while reducing insulin resistance.

Protects Vision And Hearing

NRF2 can protect against harm to the eye from diabetic retinopathy. It might also avoid the formation of cataracts and protect photoreceptors contrary to light-induced death. NRF2 additionally shield the ear, or cochlea, from stress and hearing loss.

Might Help Obesity

NRF2 may help with obesity primarily due to its capacity to regulate variables that operate on fat accumulation in the human body. NRF2 activation with sulforaphane can raise inhibit of Fatty Acid Synthesis, or FAS, and Uncoupling Proteins, or UCP, resulting in less fat accumulation and more brown fat, characterized as fat which includes more mitochondria.

Protects The Gut

NRF2 helps protect the gut by safeguarding the intestine microbiome homeostasis. By way of instance, lactobacillus probiotics will trigger NRF2 to guard the gut from oxidative stress. NRF2 can also help prevent Ulcerative Colitis, or UC.

Protects Sex Organs

NRF2 can shield the testicles and keep sperm count from harm in people with diabetes. It can also assist with Erectile Dysfunction, or ED. Some libido boosting supplements like Mucuna, Tribulus, and Ashwaganda�may enhance�sexual function via NRF2 activation. Other factors that boost NRF2, such as sunlight or broccoli sprouts, can also help improve libido.

Regulates Bones And Muscles

Oxidative stress may result in bone density and strength reduction, which is normal in osteoporosis. NRF2 activation could have the ability to improve antioxidants in bones and protect against bone aging. NRF2 can also prevent muscle loss and enhance Duchenne Muscular Dystrophy, or DMD.

Contains Anti-Viral Properties

Last but not least, NRF2 activation can ultimately help defend the human body against several viruses. In patients with the dengue virus, symptoms were not as intense in individuals who had greater levels of NRF2 compared to individuals who had less degrees of NRF2. NRF2 can also help people who have Human Immunodeficiency-1 Virus,�or HIV. NRF2 can protect against the oxidative stress from Adeno-Associated Virus,�or AAV, and H. Pylori. Finally, Lindera Root may suppress Hepatitis C virus with NRF2 activation.

Dr Jimenez White Coat
Nrf2, or NF-E2-related factor 2, is a transcription factor found in humans which regulates the expression of a specific set of antioxidant and detoxifying genes. This signaling pathway is activated due to oxidative stress as it enhances numerous antioxidant and phase II liver detoxification enzymes to restore homeostasis in the human body. Humans are adapted to function throughout a state of homeostasis or balance. When the body is confronted with oxidative stress, Nrf2 activates to regulate oxidation and control the stress it causes. Nrf2 is essential to prevent health issues associated with oxidative stress. Dr. Alex Jimenez D.C., C.C.S.T. Insight

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below.

Key sections:

  • 00:01:14 – Cancer and mortality
  • 00:19:04 – Aging
  • 00:26:30 – Brain and behavior
  • 00:38:06 – Final recap
  • 00:40:27 – Dose

Full timeline:

  • 00:00:34 – Introduction of sulforaphane, a major focus of the video.
  • 00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
  • 00:02:12 – Prostate cancer risk.
  • 00:02:23 – Bladder cancer risk.
  • 00:02:34 – Lung cancer in smokers risk.
  • 00:02:48 – Breast cancer risk.
  • 00:03:13 – Hypothetical: what if you already have cancer? (interventional)
  • 00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
  • 00:04:38 – Sulforaphane and cancer.
  • 00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
  • 00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
  • 00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
  • 00:08:32 – Inhibition of breast cancer stem cells.
  • 00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
  • 00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
  • 00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
  • 00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
  • 00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
  • 00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
  • 00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
  • 00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
  • 00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
  • 00:19:04 – Beginning of aging section.
  • 00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
  • 00:20:34 – Importance of low inflammation for longevity.
  • 00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
  • 00:23:40 – Mid-video recap: cancer, aging sections
  • 00:24:14 – Mouse studies suggest sulforaphane might improve adaptive immune function in old age.
  • 00:25:18 – Sulforaphane improved hair growth in a mouse model of balding. Picture at 00:26:10.
  • 00:26:30 – Beginning of brain and behavior section.
  • 00:27:18 – Effect of broccoli sprout extract on autism.
  • 00:27:48 – Effect of glucoraphanin on schizophrenia.
  • 00:28:17 – Start of depression discussion (plausible mechanism and studies).
  • 00:31:21 – Mouse study using 10 different models of stress-induced depression show sulforaphane similarly effective as fluoxetine (prozac).
  • 00:32:00 – Study shows direct ingestion of glucoraphanin in mice is similarly effective at preventing depression from social defeat stress model.
  • 00:33:01 – Beginning of neurodegeneration section.
  • 00:33:30 – Sulforaphane and Alzheimer’s disease.
  • 00:33:44 – Sulforaphane and Parkinson’s disease.
  • 00:33:51 – Sulforaphane and Hungtington’s disease.
  • 00:34:13 – Sulforaphane increases heat shock proteins.
  • 00:34:43 – Beginning of traumatic brain injury section.
  • 00:35:01 – Sulforaphane injected immediately after TBI improves memory (mouse study).
  • 00:35:55 – Sulforaphane and neuronal plasticity.
  • 00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
  • 00:37:19 – Sulforaphane and duchenne muscular dystrophy.
  • 00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
  • 00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
  • 00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
  • 00:41:01 – Anecdotes on sprouting at home.
  • 00:43:14 – On cooking temperatures and sulforaphane activity.
  • 00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
  • 00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
  • 00:44:56 – Cooking techniques and cruciferous vegetables.
  • 00:46:06 – Isothiocyanates as goitrogens.

When the human body is confronted with harmful internal and external factors like toxins, the cells must rapidly trigger their antioxidant abilities to counteract oxidative stress. Because increased levels of oxidative stress have been determined to cause a variety of health issues, it’s important to use Nrf2 activation to take advantage of its benefits. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.

Curated by Dr. Alex Jimenez

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Additional Topic Discussion:�Acute Back Pain

Back pain�is one of the most prevalent causes of disability and missed days at work worldwide. Back pain attributes to the second most common reason for doctor office visits, outnumbered only by upper-respiratory infections. Approximately 80 percent of the population will experience back pain at least once throughout their life. The spine is a complex structure made up of bones, joints, ligaments, and muscles, among other soft tissues. Because of this, injuries and/or aggravated conditions, such as�herniated discs, can eventually lead to symptoms of back pain. Sports injuries or automobile accident injuries are often the most frequent cause of back pain, however, sometimes the simplest of movements can have painful results. Fortunately, alternative treatment options, such as chiropractic care, can help ease back pain through the use of spinal adjustments and manual manipulations, ultimately improving pain relief. �

blog picture of cartoon paper boy

EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

***

What Is Sulforaphane?

What Is Sulforaphane?

Sulforaphane is a phytochemical, a substance within the isothiocyanate group of organosulfur compounds, found in cruciferous vegetables, such as broccoli, cabbage, cauliflower, and Brussels sprouts. It can also be found in bok choy, kale, collards, mustard greens and watercress. Research studies have shown that sulforaphane can help prevent various types of cancer by activating the production of Nrf2, or nuclear factor erythroid 2-related factor, a transcription factor which regulates�protective antioxidant mechanisms that control the cell’s response to oxidants. The purpose of the following article is to describe the function of sulforaphane.

Abstract

The KEAP1-Nrf2-ARE antioxidant system is a principal means by which cells respond to oxidative and xenobiotic stresses. Sulforaphane (SFN), an electrophilic isothiocyanate derived from cruciferous vegetables, activates the KEAP1-Nrf2-ARE pathway and has become a molecule-of-interest in the treatment of diseases in which chronic oxidative stress plays a major etiological role. We demonstrate here that the mitochondria of cultured, human retinal pigment epithelial (RPE-1) cells treated with SFN undergo hyperfusion that is independent of both Nrf2 and its cytoplasmic inhibitor KEAP1. Mitochondrial fusion has been reported to be cytoprotective by inhibiting pore formation in mitochondria during apoptosis, and consistent with this, we show Nrf2-independent, cytoprotection of SFN-treated cells exposed to the apoptosis-inducer, staurosporine. Mechanistically, SFN mitigates the recruitment and/or retention of the soluble fission factor Drp1 to mitochondria and to peroxisomes but does not affect overall Drp1 abundance. These data demonstrate that the beneficial properties of SFN extend beyond the activation of the KEAP1-Nrf2-ARE system and warrant further interrogation given the current use of this agent in multiple clinical trials.

Keywords: Sulforaphane, Nrf2, Drp1, Mitochondria, Fission, Fusion, Apoptosis

Introduction

Sulforaphane is an Nrf2-Independent Inhibitor of Mitochondrial Fission

Sulforaphane (SFN) is an isothiocyanate compound derived in the diet most commonly from cruciferous vegetables [56]. It is generated in plants as a xenobiotic response to predation via vesicular release of the hydrolytic enzyme myrosinase from damaged cells; this enzyme converts glucosinolates to isothiocyantes [42]. Over the last two decades, SFN has been extensively characterized for its reported anticancer, antioxidant, and antimicrobial properties [57]. Much of this efficacy has been attributed to the capacity of SFN to modulate the KEAP1-Nrf2-antioxidant response element (ARE) signaling pathway, although additional activities of the compound have been identified, including the inhibition of histone deacetylase activity and cell cycle progression [29]. Nrf2 is the master antioxidant transcription factor and under conditions of homeostasis, its stability is suppressed through the action of the cytoplasmic Cullin3KEAP1 ubiquitin ligase complex [20]. Specifically, Nrf2 is recruited to the Cullin3KEAP1 ligase by binding to the dimeric substrate adaptor KEAP1 and is subsequently modified with polyUb chains that target the transcription factor for proteasome-mediated degradation. This constitutive turnover limits the half-life of Nrf2 in unstressed cells to ~15 min [30], [33], [46], [55]. In response to numerous types of stress, most notably oxidative stress, KEAP1, a cysteine-rich protein, acts as a redox sensor, and oxidative modification of critical cysteines, particularly C151, of KEAP1 dissociates Nrf2-KEAP1 from CUL3 thereby preventing Nrf2 degradation [8], [20], [55]. Notably, SFN, and possibly other Nrf2 activators, mimic oxidative stress by modifying C151 of KEAP1 e.g. [21]. Stabilization of Nrf2 allows for its translocation to the nucleus where it induces the expression of a battery of Phase II antioxidant and detoxification genes. Nrf2 binds to the antioxidant response promoter elements (ARE) of its cognate target genes through heterodimerization with small Maf proteins [19]. This system presents a dynamic and sensitive response to indirect antioxidants like SFN, free radicals generated by the mitochondria [16], or other physiologic sources of oxidative stress [41].

Mitochondria are dynamic, subcellular organelles that regulate a host of cellular functions ranging from ATP production and intracellular calcium buffering to redox regulation and apoptosis [13], [49]. Mitochondria also represent the principal source of reactive oxygen species (ROS) within the cell. Proper regulation of mitochondrial function is therefore necessary for optimizing ATP production to meet cellular needs while simultaneously minimizing the potentially harmful effects of excessive free radical production. A critical requirement for fine modulation of mitochondrial function is the capacity for mitochondria to function both independently as biochemical machines and as part of a vast, responsive network.

Mitochondrial network morphology and function are determined by a regulated balance between fission and fusion. Mitochondrial fission is required for daughter cell inheritance of mitochondria during cell division [28] as well as for the selective, autophagic degradation of depolarized or damaged mitochondria, termed mitophagy [1]. Conversely, fusion is required for complementation of mitochondrial genomes and sharing of electron transport chain components between neighboring mitochondria [54]. At the molecular level, mitochondrial fission and fusion are regulated by large, dynamin-like GTPases. Three enzymes primarily regulate fusion: Mitofusins 1 and 2 (Mfn1/2) are two-pass outer membrane proteins that mediate outer membrane fusion via heterotypic interactions between adjacent mitochondria [15], [25], [37], while OPA1 is an inner membrane protein that simultaneously ensures matrix connectivity by regulating the melding of inner membranes [5]. The GTPase activity of all three proteins is required for robust fusion [5], [18], and OPA1 is further regulated by complex proteolysis within the mitochondrial inner membrane by the proteases OMA1 [14], PARL [6], and YME1L [45]. Importantly, intact mitochondrial membrane potential is required for efficient fusion in order to suppress integration of damaged and healthy mitochondria [26].

Mitochondrial fission is primarily catalyzed by a cytosolic protein called Dynamin-related protein 1 (Drp1/DNM1L). Drp1 is recruited from the cytosol to prospective sites of fission on the mitochondrial outer membrane [43]. The major receptors for Drp1 on the outer membrane are mitochondrial fission factor (Mff) [32] and, to a lesser extent, Fission 1 (Fis1) [51]. Additionally, a decoy receptor, MIEF1/MiD51, was discovered that acts to further limit the activity of Drp1 protein at potential fission sites [58]. Once docked at the mitochondrial outer membrane, Drp1 oligomerizes into spiral-like structures around the body of the mitochondrion and then utilizes the energy derived from GTP hydrolysis to mediate the physical scission of the mitochondrial outer and inner membranes [17]. Endoplasmic reticulum-derived tubules act as an initial constrictor of mitochondria prior to Drp1 oligomerization, underscoring the revelation that non-constricted mitochondria are wider than the permissive circumference of a completed Drp1 spiral [12]. Actin dynamics are also important for the ER-mitochondria interactions that precede mitochondrial fission [24]. In addition to its role in mitochondrial fission, Drp1 catalyzes the fission of peroxisomes [40].

Drp1 is very similar to the well-characterized dynamin protein in that both proteins contain an N-terminal GTPase domain, a Middle domain that is critical for self-oligomerization, and a C-terminal GTPase effector domain [31]. Drp1 achieves selectivity for mitochondrial membranes through a combination of interactions with its receptor proteins Mff and Fis1 and also through its affinity for the mitochondria-specific phospholipid cardiolipin via the unique B-insert domain of Drp1 [2]. Drp1 typically exists as a homotetramer in the cytoplasm, and higher order assembly at mitochondrial fission sites is mediated by the Middle domain of Drp1 [3].

Given the implicit link between mitochondrial function and the KEAP1-Nrf2-ARE pathway, we investigated the effects of Nrf2 activation on mitochondrial structure and function. We demonstrate here that SFN induces mitochondrial hyperfusion that, unexpectedly, is independent of both Nrf2 and KEAP1. This effect of SFN is through an inhibition of Drp1 function. We further demonstrate that SFN confers resistance to apoptosis that is Nrf2-independent and mimics that observed in cells depleted of Drp1. These data collectively indicate that in addition to stabilizing and activating Nrf2, SFN modulates mitochondrial dynamics and preserves cellular fitness and survival.

Results

Sulforaphane Induces Nrf2/KEAP1-Independent Hyperfusion of Mitochondria

In the course of studying the effects of Nrf2 activation on mitochondrial network dynamics, we discovered that treatment of immortalized, human retinal pigment epithelial (RPE-1) cells with sulforaphane (SFN), a potent activator of Nrf2 signaling, induced a robust fusion of the mitochondrial network when compared with vehicle-treated control cells (Fig. 1A and B). The morphology of the mitochondria in these cells greatly resembled that of the mitochondria in cells depleted by siRNA of endogenous Drp1, the principal mitochondrial fission factor (Fig. 1A). This result raised the intriguing idea that mitochondrial fission and fusion status responds directly to Nrf2 levels in the cell. However, stimulation of cells with other Nrf2 stabilizers and activators such as the proteasome inhibitor MG132, the pro-oxidant tBHQ, or knockdown of the Nrf2 inhibitor KEAP1 did not induce mitochondrial fusion (Fig. 1A and B). Stabilization of Nrf2 by these manipulations was confirmed by western blotting for endogenous Nrf2 (Fig. 1C). Furthermore, expression of Nrf2 was dispensable for SFN-induced mitochondrial fusion, as knockdown of endogenous Nrf2 with siRNA failed to counter this phenotype (Fig. 1D�F). Because SFN stimulates the KEAP1-Nrf2-ARE pathway by covalently modifying cysteine residues of KEAP1 [21], we knocked down KEAP1 to address whether SFN-induced mitochondrial hyperfusion is stimulated through a KEAP1-dependent, but Nrf2 independent pathway. However, depletion of KEAP1 also failed to abrogate SFN-induced mitochondrial fusion (Fig. 1G�I). In fact, SFN reversed the pro-fission morphology induced by depletion of KEAP1 (Fig. 1G, panel b versus panel d). These results indicate that SFN treatment causes mitochondrial fusion independent of the canonical KEAP1-Nrf2-ARE pathway and led us to interrogate whether SFN directly affects components of the mitochondrial fission or fusion machinery.

Figure 1 SFN induces Nrf2/KEAP1-independent mitochondrial fusion. (A) RPE-1 cells were transfected with the indicated siRNAs and 3 days later treated with DMSO or the Nrf2 activators SFN (50 ?M), MG132 (10 ?M), or tBHQ (100 ?M) for 4 h. Mitochondria (red) are labeled with an anti-Tom20 antibody, and nuclei (blue) are counterstained with DAPI. (B) Graph showing quantification of mitochondrial morphology scoring from (A). >50 cells per condition were evaluated in a blinded fashion. (C) Representative western blots from (A). (D) RPE-1 cells were transfected with 10 nM siRNA and 3 days later treated with SFN for 4 h prior to being fixed and stained as in (A). (E) Graph showing quantification of mitochondrial phenotype scoring from (D). >100 cells per condition were evaluated in a blinded fashion. (F) Representative western blots from (D). (G) Cells were transfected and treated as in (D) with siCON or siKEAP1. (H) Cells from (G) were scored as in (B) and (E) on the basis of mitochondrial morphology. (I) Representative western blots from (G). Data in (B), (E), and (H) were compiled from 3 independent experiments each and statistical significance was determined by two-tailed Student’s t-test. Error bars reflect +/- S.D. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Sulforaphane Impairs the Mitochondrial Association of Drp1

Based on the finding that SFN-treatment induces mitochondrial hyperfusion, we reasoned that this phenotype was either a consequence of excessive fusion activity or an inhibition of fission activity. To discriminate between these two possibilities, we compared the morphology of peroxisomes in the presence and absence of SFN. Peroxisomes are similar to mitochondria in that they are dynamic organelles the shape and length of which are constantly in flux [44]. Peroxisomes contain both Fis1 and Mff in their outer membrane and, as a consequence, are targets for Drp1-mediated fission [22], [23]. However, peroxisomes do not utilize the fusion machinery of the mitochondrial network and consequently, do not undergo fusion [39]. Rather, peroxisomal fission is opposed by the lengthening of existing peroxisomes via de novo addition of membranes and proteins [44]. Because peroxisomes lack Mfn1/2 and OPA1, we reasoned that if SFN activates the fusion machinery rather than inhibiting the fission machinery, peroxisome length would not be affected. In vehicle-treated cells, peroxisomes are maintained as short, round, punctiform organelles (Fig. 2, panels b and d). However, SFN treatment increased peroxisome length by ~2-fold as compared to control cells (Fig. 2, panels f and h). Furthermore, many of the peroxisomes were pinched near the center, indicating a potential scission defect (Fig. 2, panel h, arrowheads). Likewise, peroxisomes in cells transfected with Drp1 siRNA were abnormally long (Fig. 2, panels j and l), confirming that Drp1 is required for peroxisomal fission and suggesting that SFN-treatment causes mitochondrial and peroxisomal phenotypes by disrupting the fission machinery.

Figure 2 SFN induces peroxisomal lengthening. (A) RPE-1 cells were transfected with 10 nM of the indicated siRNA and 3 days later treated with DMSO or 50 ?M SFN for 4 h. Peroxisomes (green) were labeled with an anti-PMP70 antibody, mitochondria with MitoTracker (red), and DNA counterstained with DAPI. Enlarged insets of peroxisomes are shown on the right (panels d, h, and l) to facilitate visualization of the changes in morphology induced by SFN and Drp1 depletion. Arrowheads highlight constriction points. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

We next determined how SFN restricts Drp1 function. Possibilities included reductions in expression levels, recruitment/retention at mitochondria, oligomerization, or enzymatic activity of the GTPase. A deficit in any one of these would result in reduced mitochondrial fission and hyperfusion. We did not detect reproducible changes in Drp1 protein levels after SFN-treatment (Figs. 1C and 3A), and therefore concluded that SFN does not alter Drp1 stability or expression, consistent with Drp1 having a half-life of >10 h [50] and our SFN treatments being of shorter duration. Next, we investigated whether SFN affected the recruitment or retention of Drp1 to mitochondria. Fractionation studies showed that SFN induced a loss of Drp1 from the mitochondrial fraction (Fig. 3A, lanes 7�8 and Fig. 3B). As reported previously [43], only a minor fraction of Drp1 (~3%) is associated with the mitochondrial network at any given time during steady state conditions with most of the enzyme residing in the cytoplasm (Fig. 3A, lanes 5�8). These fractionation data were confirmed using co-localization analysis which showed a ~40% reduction in mitochondria-localized, punctate Drp1 foci after SFN-treatment (Fig. 3C and D). Together, these data indicate that the mitochondrial fusion induced by SFN is, at least partially, due to the attenuated association of Drp1 with the mitochondria. Our data do not distinguish between whether SFN interferes with the mitochondrial recruitment versus the mitochondrial retention of Drp1, or both, as the analysis of endogenous Drp1 was not amenable to visualizing the GTPase by live-cell microscopy.

Figure 3 SFN causes a loss of Drp1 from the mitochondria. (A) Subcellular fractionation of RPE-1 cells following 4 h of DMSO or SFN. Whole-cell lysates (WCL), nuclear (Nuc), cytosolic (Cyto), and crude mitochondrial (Mito) fractions were resolved by SDS-PAGE and processed for western blotting with the indicated antibodies. The migration of molecular weight markers is indicated on the left. (B) Graphs showing densitometric quantification of Drp1 in the indicated fractions from (A). (C) RPE-1 cells were transfected with 10 nM siCON or siDrp1 and 3 days later treated with DMSO or SFN for 4 h. Drp1 (green) was visualized with an anti-Drp1 antibody, mitochondria with MitoTracker (red), and nuclei with DAPI (blue). (D) Automated co-localization analysis of Drp1 and MitoTracker signal from (C). Data in (B) and (D) were compiled from 3 and 5 independent experiments, respectively, and statistical significance was determined by two-tailed Student’s t-test. Error bars reflect +/- S.D and asterisks denote statistical significance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Sulforaphane Confers Protection Against Staurosportine-Induced Apoptosis Independent of Nrf2

Previous work has shown that mitochondrial fission is permissive in the formation of pores in the outer mitochondrial membrane generated by Bax/Bak during apoptosis [11]. Drp1 has been shown to be selectively recruited to mitochondria during apoptosis [11] and, consistent with this, fragmented mitochondria have been observed early in the process [27]. Conversely, inhibiting mitochondrial fission is thought to inhibit apoptosis by blocking the formation of the outer membrane pores that allow for cytochrome c release [53]. Accordingly, stimulating mitochondrial fusion delays the progression of apoptosis induced by compounds including staurosporine (STS) [14]. To determine whether SFN protects RPE-1 cells from STS-mediated apoptosis and if so, whether this requires Nrf2, we established an assay to readily induce poly ADP ribose polymerase (PARP) cleavage, a substrate of activated caspase-3 and definitive marker of apoptosis. Treatment of RPE-1 cells with 1 �M STS for 6 h only caused a very modest cleavage of PARP yet this was prevented by SFN co-treatment (e.g., Fig. 4A, lane 3 versus 4). To increase the robustness of this assay, we further sensitized cells to STS-induced apoptosis by pre-treating them with siRNA targeting the anti-apoptotic factor, Bcl-XL. This pretreatment reduced the expression of Bcl-XL and markedly promoted PARP cleavage as a function of time exposed to STS (Fig. 4B, compare lane 2 to lanes 4�10). Importantly, 2 h of pre-treatment with SFN mitigated PARP cleavage in cells exposed to STS (Fig. 4C, lane 3 versus 4 and lane 5 versus 6). Likewise, cells stably depleted of Nrf2 by CRISPR/Cas9 were comparably protected from STS toxicity by SFN pre-treatment (Fig. 4C, lane 11 versus 12 and lane 13 versus 14 and Fig. 4D). This protection was observed using both PARP cleavage (Fig. 4C and D) and cellular morphology (Fig. 4E) as readouts. The efficacy of Nrf2 depletion by CRISPR/Cas9 was confirmed by western blotting (Fig. 4C, Nrf2 blot). As predicted, depleting cells of Drp1, which also yields a hyperfusion phenotype (Fig. 1A), also blocked PARP cleavage in response to STS as compared to control cells incubated with SFN (Fig. 4F and G). Together, these findings are consistent with SFN conferring protection against apoptosis through its capacity to restrict Drp1 function, independent of the stabilization and activation of Nrf2.

Figure 4 The cytoprotective effects of SFN are independent of Nrf2 expression (A) RPE-1 cells were pre-treated with DMSO or 50 ?M SFN for 2 h prior to treatment with DMSO, 1 ?M staurosporine (STS), or 50 ?M etoposide for 6 h and were processed for anti-PARP western blotting. (B) RPE-1 cells were transfected with 2.5 nM siCON, 1 nM siBcl-XL, or 2.5 nM siBcl-XL and 3 days later were treated with DMSO or 1 ?M STS for 2, 4, or 6 h. Representative western blots are shown and the migration of molecular weight markers is indicated on the left. (C) CRISPR/Cas9-generated wild-type (Nrf2WT) and Nrf2 knockout (Nrf2KO) RPE-1 cells were transfected with 1 nM siBcl-XL and 3 days later were pre-treated with DMSO or 50 ?M SFN for 2 h. Subsequently, the cells were treated with 1 ?M STS for 2, 4, or 6 h. Representative western blots with the indicated antibodies are shown. (D) Quantification of cleaved PARP as a percentage of total PARP (cleaved+uncleaved) from 3 independent experiments. Importantly, the levels of cleaved PARP were comparable whether cells expressed Nrf2 or not, indicating that SFN protection from STS is independent of the transcription factor. (E) 20X phase-contrast images taken immediately prior to harvest of lysates from (C). Scale bar=65 �m. (F) Representative western blots demonstrating that depletion of Drp1 confers near-comparable protection from STS as SFN treatment. RPE-1 cells were transfected with 1 nM siBcl-XL and additionally transfected with either 10 nM siCON or 10 nM siDrp1. 3 days later, siCON cells were pre-treated with SFN as in (A) and (C) and then exposed to STS for 4 h prior to being harvested and processed for western blotting with the indicated antibodies. (G) Same as (D) for the data presented in (F) compiled from 3 independent experiments. Error bars reflect +/- S.E.M.

Discussion

We have discovered that SFN modulates mitochondrial fission/fusion dynamics independent of its effects on the KEAP1-Nrf2-ARE pathway. This is intriguing because of an assumed link between mitochondrial dysfunction and ROS production and the necessity of squelching mitochondria-derived free radicals through the activation of Nrf2. This additional functional impact of SFN is of potential importance given the more than 30 clinical trials currently underway testing SFN for the treatment of a variety of diseases including prostate cancer, obstructive pulmonary disease, and sickle cell disease [7], [10], [47].

Because SFN is an isothiocyanate [56] and it activates Nrf2 signaling by directly acylating critical KEAP1 cysteines to suppress Nrf2 degradation [21], it follows that SFN exerts its pro-fusion effects by modulating the activity of a fission or fusion factor via cysteine modification. Our data strongly support Drp1 being negatively regulated by SFN although whether the GTPase is a direct target of acylation remains to be elucidated. Despite this knowledge gap, the function of Drp1 is clearly being compromised by SFN as both mitochondria and peroxisomes become hyperfused in response to SFN treatment and these organelles share Drp1 for their respective scission events [38]. In addition, SFN decreases the amount of Drp1 that localizes and accumulates at mitochondria (Fig. 3). Because our experiments were done with all endogenous proteins, our detection of Drp1 at mitochondrial fission sites is under steady-state conditions, and consequently, we cannot distinguish between a recruitment versus a retention defect of the enzyme caused by SFN. Further, we cannot eliminate the possibility that SFN acylates a receptor at the mitochondria (Fis1 or Mff) to block Drp1 recruitment yet, we suspect that Drp1 is directly modified. Drp1 has nine cysteines, eight of which reside within the Middle Domain that is required for oligomerization [3], and one of which resides in the GTPase Effector Domain (GED) at the C-terminus of Drp1. Direct acylation of any of these cysteines could cause an activity defect in Drp1 and therefore underlie the effect of SFN on mitochondrial dynamics. Notably, prior work suggests that defects in oligomerization and catalytic activity can abrogate the retention of Drp1 at the mitochondria [52]. Cys644 in the GED domain is a particularly attractive target based on previous work showing that mutation of this cysteine phenocopies mutations that impair Drp1 GTPase activity [4] and that this particular cysteine is modified by thiol-reactive electrophiles [9]. Resolution of this outstanding question will require mass spectrometric validation.In summary, we have identified a novel, cytoprotective function for the clinically-relevant compound SFN. In addition to activating the master anti-oxidant transcription factor Nrf2, SFN promotes mitochondrial and peroxisomal fusion, and this effect is independent of Nrf2. The mechanism underlying this phenomenon involves a reduction in the function of the GTPase Drp1, the primary mediator of mitochondrial and peroxisomal fission. A major consequence of SFN-mediated mitochondrial fusion is that cells become resistant to the toxic effects of the apoptosis inducer staurosporine. This additional cytoprotective action of SFN could be of particular clinical utility in the numerous neurodegenerative diseases for which age is the leading risk factor (e.g., Parkinson’s Disease, Alzheimer’s Disease, Age-related Macular Degeneration) as these maladies have been associated with apoptosis and reduced levels and/or dysregulation of Nrf2 [35], [36], [48]. Together, these data demonstrate that the cytoprotective properties of SFN extend beyond activation of the KEAP1-Nrf2-ARE system and warrant further studies given the current use of this agent in multiple clinical trials.

Materials and Methods

Apoptosis Assays

Cells were seeded and transfected with siRNA as indicated below. The cells were pre-treated with 50 ?M sulforaphane for 2 h to induce mitochondrial fusion and were then treated with 1 ?M staurosporine to induce apoptosis. At the time of harvest, media was collected in individual tubes and subjected to high speed centrifugation to pellet apoptotic cells. This cell pellet was combined with adherent cells and solubilized in 2 times-concentrated Laemmli buffer. Samples were subjected to anti-PARP western blotting.

CRISPR/Cas9 Construct Generation

To create LentiCRISPR/eCas9 1.1, LentiCRISPR v2 (addgene #52961) was first cut with Age1 and BamH1. Next, SpCas9 from eSpCas9 1.1 (addgene #71814) was PCR amplified with Age1 and BamH1 overhangs using the following primers (Forward AGCGCACCGGTTCTAGAGCGCTGCCACCATGGACTATAAGGACCACGAC, Reverse AAGCGCGGATCCCTTTTTCTTTTTTGCCTGGCCGG) and ligated into the cut vector above. sgRNA sequences were determined by using Benchling.com. Parameters were set to target the coding sequence with the highest on-target and lowest off-target scores. The following sequences (targeting sequence underlined, hs sgNFE2L2#1 sense CACCGCGACGGAAAGAGTATGAGC, antisense AAACGCTCATACTCTTTCCGTCGC; hs sgNFE2L2#2 sense CACCGGTTTCTGACTGGATGTGCT, antisense AAACAGCACATCCAGTCAGAAACC; hs sgNFE2L2#3 sense CACCGGAGTAGTTGGCAGATCCAC, antisense AAACGTGGATCTGCCAACTACTCC) were annealed and ligated into BsmB1 cut LentiCRISPR/eCas9 1.1. Lentivirally infected RPE-1 cells were selected with puromycin and maintained as a pooled population. Knockout was confirmed by immunofluorescence and western blotting.

Cell Culture and Transfections

Human retinal pigment epithelial cells transformed with telomerase (RPE-1) (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 1 g/L glucose supplemented with penicillin, streptomycin, 1X non-essential amino acid cocktail (Life Technologies), and 10% Fetal Bovine Serum (Life Technologies). For siRNA-transfections, 30,000�35,000 cells/mL were seeded overnight. Cells received 10 nM siRNA diluted in serum-free DMEM and combined with 0.3% Interferin transfection reagent (PolyPlus). For apoptosis sensitization, cells received 1 nM Bcl-XL siRNA. Cells were harvested 2�3 days post-transfection.

Chemicals, Antibodies, and siRNA Oligos

Antibodies against ?-tubulin (Cell Signaling), ?-tubulin (Sigma), Drp1 (BD Biosciences), KEAP1 (Proteintech), Lamin B1 (Abcam), PARP (Cell Signaling), PMP70 (Abcam), and Tom20 (BD Biosciences) were used at 1:1000 dilutions for western blotting and for immunofluorescence. In-house, anti-Nrf2 rabbit antibody was used at 1:2000 for western blotting [34], [59]. Sulforaphane (Sigma) and staurosporine (Tocris) were used at 50 ?M and 1 ?M respectively. siRNAs against Drp1 (Dharmacon), Nrf2 (Dharmacon), KEAP1 (Cell Signaling), and Bcl-XL (Cell Signaling) were used at 10 nM unless otherwise noted.

Immunofluorescence and in Vivo Labeling

Cells seeded on 18 mm glass coverslips were treated with vehicle or drug, fixed in 3.7% formaldehyde and then permeabilized in 0.2% Triton X-100/PBS on ice for 10 min. Primary antibodies were incubated in 3% bovine serum albumin (BSA) in PBS overnight at 4 �C. Following PBS washes, cells were incubated for 1 h in species-appropriate, Alexa488- or Alexa546-, conjugated secondary antibodies (diluted 1:1000) and 0.1 ?g/mL DAPI (Sigma) in 3% BSA/PBS. Mitochondria were visualized either by anti-Tom20 immunofluorescence or by incubating cells in 200 nM MitoTracker Red CMXRos (Molecular Probes, Inc.) in serum-free DMEM for 30 min at 37 �C prior to fixation.

Microscopy and Image Analysis

Immunofluorescence samples were viewed on an LSM710 Confocal microscope (Carl Zeiss). Micrographs were captured using 63X or 100X oil immersion objectives and images adjusted and enhanced using Adobe Photoshop CS6. Co-localization analysis was performed using Carl Zeiss LSM710 co-localization feature with thresholds manually set while blinded to the identity of the samples. Scale bars throughout, unless otherwise indicated, are 10 �m. Mitochondrial morphology was assessed by blinded scoring. If the mitochondria of a cell were maintained as multiple, round, discriminate puncta, the cell was scored as �fission�. If individual mitochondria were indistinguishable and the whole mitochondrial network appeared continuous, the cell was scored as �fusion�. All other cells, including those with clustering mitochondria, were scored as �intermediate�.

Subcellular Fractionations

RPE-1 cells were grown to confluence. Following a PBS wash, cells were subjected to centrifugation at 600�g for 10 min and resuspended in 600 ?L isolation buffer (210 mM Mannitol, 70 mM Sucrose, 5 mM MOPS, 1 mM EDTA pH 7.4+1 mM PMSF). The suspension was lysed 30 times in a Dounce homogenizer. A fraction of the homogenate was preserved as a �whole cell lysate.� The remainder was subjected to centrifugation at 800�g for 10 min to pellet nuclei. Supernatants were subjected to centrifugation at 1500�g for 10 min to clear remaining nuclei and unlysed cells. This supernatant was subjected to centrifugation at 15,000�g for 15 min to pellet mitochondria. The supernatant was preserved as the �cytosolic fraction�. The pellet was washed gently with PBS and resuspended in isolation buffer. The protein concentration of each fraction was measured by bicinchoninic acid (BCA) assay and equivalent amounts of protein were resolved by SDS-PAGE.

Western Blotting

Cells were washed in PBS and solubilized in 2 times concentrated Laemmli solubilizing buffer (100 mM Tris [pH 6.8], 2% SDS, 0.008% bromophenol blue, 2% 2-mercaptoethanol, 26.3% glycerol, and 0.001% Pyrinin Y). Lysates were boiled for 5 min prior to loading on sodium dodecyl sulfate (SDS) polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and the membranes were blocked for 1 h in 5% Milk/TBST. Primary antibodies were diluted in 5% Milk/TBST and incubated with the blot overnight at 4 �C. Horseradish peroxidase (HRP)-conjugated secondary antibodies were diluted in 5% Milk/TBST. Blots were processed with enhanced chemiluminescence and densitometric quantifications were performed using ImageJ software.

Dr Jimenez White Coat

Sulforaphane is a chemical from the isothiocyanate collection of organosulfur substances obtained from cruciferous vegetables, including broccoli, cabbage, cauliflower, kale, and collards, among others. Sulforaphane is produced when the enzyme myrosinase transforms glucoraphanin, a glucosinolate, into sulforaphane, also known as sulforaphane-glucosinolate. Broccoli sprouts and cauliflower have the highest concentration of glucoraphanin or the precursor to sulforaphane. Research studies have demonstrated that sulforaphane enhances the human body’s antioxidant capabilities to prevent various health issues. Dr. Alex Jimenez D.C., C.C.S.T. Insight

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below.

Key sections:

  • 00:01:14 – Cancer and mortality
  • 00:19:04 – Aging
  • 00:26:30 – Brain and behavior
  • 00:38:06 – Final recap
  • 00:40:27 – Dose

Full timeline:

  • 00:00:34 – Introduction of sulforaphane, a major focus of the video.
  • 00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
  • 00:02:12 – Prostate cancer risk.
  • 00:02:23 – Bladder cancer risk.
  • 00:02:34 – Lung cancer in smokers risk.
  • 00:02:48 – Breast cancer risk.
  • 00:03:13 – Hypothetical: what if you already have cancer? (interventional)
  • 00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
  • 00:04:38 – Sulforaphane and cancer.
  • 00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
  • 00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
  • 00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
  • 00:08:32 – Inhibition of breast cancer stem cells.
  • 00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
  • 00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
  • 00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
  • 00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
  • 00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
  • 00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
  • 00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
  • 00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
  • 00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
  • 00:19:04 – Beginning of aging section.
  • 00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
  • 00:20:34 – Importance of low inflammation for longevity.
  • 00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
  • 00:23:40 – Mid-video recap: cancer, aging sections
  • 00:24:14 – Mouse studies suggest sulforaphane might improve adaptive immune function in old age.
  • 00:25:18 – Sulforaphane improved hair growth in a mouse model of balding. Picture at 00:26:10.
  • 00:26:30 – Beginning of brain and behavior section.
  • 00:27:18 – Effect of broccoli sprout extract on autism.
  • 00:27:48 – Effect of glucoraphanin on schizophrenia.
  • 00:28:17 – Start of depression discussion (plausible mechanism and studies).
  • 00:31:21 – Mouse study using 10 different models of stress-induced depression show sulforaphane similarly effective as fluoxetine (prozac).
  • 00:32:00 – Study shows direct ingestion of glucoraphanin in mice is similarly effective at preventing depression from social defeat stress model.
  • 00:33:01 – Beginning of neurodegeneration section.
  • 00:33:30 – Sulforaphane and Alzheimer’s disease.
  • 00:33:44 – Sulforaphane and Parkinson’s disease.
  • 00:33:51 – Sulforaphane and Hungtington’s disease.
  • 00:34:13 – Sulforaphane increases heat shock proteins.
  • 00:34:43 – Beginning of traumatic brain injury section.
  • 00:35:01 – Sulforaphane injected immediately after TBI improves memory (mouse study).
  • 00:35:55 – Sulforaphane and neuronal plasticity.
  • 00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
  • 00:37:19 – Sulforaphane and duchenne muscular dystrophy.
  • 00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
  • 00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
  • 00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
  • 00:41:01 – Anecdotes on sprouting at home.
  • 00:43:14 – On cooking temperatures and sulforaphane activity.
  • 00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
  • 00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
  • 00:44:56 – Cooking techniques and cruciferous vegetables.
  • 00:46:06 – Isothiocyanates as goitrogens.

Acknowledgements

Sciencedirect.com/science/article/pii/S2213231716302750

How is Sulforaphane Produced?

Heating Decreases Epithiospecifier Protein Activity and Increases Sulforaphane Formation in Broccoli

Abstract

Sulforaphane, an isothiocyanate from broccoli, is one of the most potent food-derived anticarcinogens. This compound is not present in the intact vegetable, rather it is formed from its glucosinolate precursor, glucoraphanin, by the action of myrosinase, a thioglucosidase enzyme, when broccoli tissue is crushed or chewed. However, a number of studies have demonstrated that sulforaphane yield from glucoraphanin is low, and that a non-bioactive nitrile analog, sulforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that in Arabidopsis, nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase. Our objectives were to examine the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture. Heating fresh broccoli florets or broccoli sprouts to 60 �C prior to homogenization simultaneously increased sulforaphane formation and decreased sulforaphane nitrile formation. A significant loss of ESP activity paralleled the decrease in sulforaphane nitrile formation. Heating to 70 �C and above decreased the formation of both products in broccoli florets, but not in broccoli sprouts. The induction of QR in cultured mouse hepatoma Hepa lclc7 cells paralleled increases in sulforaphane formation.

 

Pre-heating broccoli florets and sprouts to 60 �C significantly increased the myrosinase-catalyzed formation of sulforaphane (SF) in vegetable tissue extracts after crushing. This was associated with decreases in sulforaphane nitrile (SF Nitrile) formation and epithiospecifier protein (ESP) activity.

Keywords: Broccoli, Brassica oleracea, Cruciferae, Cancer, Anticarcinogen, Sulforaphane, Sulforaphane nitrile, Epithiospecifier protein, Quinone reductase

In conclusion, sulforaphane is a phytochemical found in broccoli,and other cruciferous vegetables. An uncontrolled amount of oxidants caused by both internal and external factors can cause oxidative stress in the human body which may ultimately lead to a variety of health issues. Sulforaphane can activate the production of Nrf2, a transcription factor that helps regulate�protective antioxidant mechanisms that control the cell’s response to oxidants. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.

Curated by Dr. Alex Jimenez

Referenced from: Sciencedirect.com

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Additional Topic Discussion:�Acute Back Pain

Back pain�is one of the most prevalent causes of disability and missed days at work worldwide. Back pain attributes to the second most common reason for doctor office visits, outnumbered only by upper-respiratory infections. Approximately 80 percent of the population will experience back pain at least once throughout their life. The spine is a complex structure made up of bones, joints, ligaments, and muscles, among other soft tissues. Because of this, injuries and/or aggravated conditions, such as�herniated discs, can eventually lead to symptoms of back pain. Sports injuries or automobile accident injuries are often the most frequent cause of back pain, however, sometimes the simplest of movements can have painful results. Fortunately, alternative treatment options, such as chiropractic care, can help ease back pain through the use of spinal adjustments and manual manipulations, ultimately improving pain relief.

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EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

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The Emerging Role Of Nrf2 In Mitochondrial Function

The Emerging Role Of Nrf2 In Mitochondrial Function

Oxidants are generally produced in a controlled manner in order to regulate essential processes in the human body, including cell division, inflammation, immune function, autophagy, and stress response. However, the uncontrolled production of these oxidants can contribute to oxidative stress, which may affect cellular function, leading to the development of toxicity, chronic disease and cancer. The human body’s protective antioxidant mechanisms are regulated by a series of vital pathways that control the cell’s response to oxidants. The nuclear factor erythroid 2-related factor, otherwise known as Nrf2, is an emerging regulator of cellular resistance to oxidants. The purpose of the article below is to discuss and demonstrate the emerging role of Nrf2 in mitochondrial function.

Abstract

The transcription factor NF-E2 p45-related factor 2 (Nrf2; gene name NFE2L2) allows adaptation and survival under conditions of stress by regulating the gene expression of diverse networks of cytoprotective proteins, including antioxidant, anti-inflammatory, and detoxification enzymes as well as proteins that assist in the repair or removal of damaged macromolecules. Nrf2 has a crucial role in the maintenance of cellular redox homeostasis by regulating the biosynthesis, utilization, and regeneration of glutathione, thioredoxin, and NADPH and by controlling the production of reactive oxygen species by mitochondria and NADPH oxidase. Under homeostatic conditions, Nrf2 affects the mitochondrial membrane potential, fatty acid oxidation, availability of substrates (NADH and FADH2/succinate) for respiration, and ATP synthesis. Under conditions of stress or growth factor stimulation, activation of Nrf2 counteracts the increased reactive oxygen species production in mitochondria via transcriptional upregulation of uncoupling protein 3 and influences mitochondrial biogenesis by maintaining the levels of nuclear respiratory factor 1 and peroxisome proliferator-activated receptor ? coactivator 1?, as well as by promoting purine nucleotide biosynthesis. Pharmacological Nrf2 activators, such as the naturally occurring isothiocyanate sulforaphane, inhibit oxidant-mediated opening of the mitochondrial permeability transition pore and mitochondrial swelling. Curiously, a synthetic 1,4-diphenyl-1,2,3-triazole compound, originally designed as an Nrf2 activator, was found to promote mitophagy, thereby contributing to the overall mitochondrial homeostasis. Thus, Nrf2 is a prominent player in supporting the structural and functional integrity of the mitochondria, and this role is particularly crucial under conditions of stress.

Keywords: Bioenergetics, Cytoprotection, Keap1, Mitochondria, Nrf2, Free radicals

Highlights

  • Nrf2 has a crucial role in maintaining cellular redox homeostasis.
  • Nrf2 affects the mitochondrial membrane potential and ATP synthesis.
  • Nrf2 influences mitochondrial fatty acid oxidation.
  • Nrf2 supports the structural and functional integrity of the mitochondria.
  • Nrf2 activators have beneficial effects when mitochondrial function is compromised.

Introduction

The transcription factor NF-E2 p45-related factor 2 (Nrf2; gene name NFE2L2) regulates the expression of networks of genes encoding proteins with diverse cytoprotective activities. Nrf2 itself is controlled primarily at the level of protein stability. Under basal conditions, Nrf2 is a short-lived protein that is subjected to continuous ubiquitination and proteasomal degradation. There are three known ubiquitin ligase systems that contribute to the degradation of Nrf2. Historically, the first negative regulator of Nrf2 to be discovered was Kelch-like ECH-associated protein 1 (Keap1) [1], a substrate adaptor protein for Cullin 3 (Cul3)/Rbx1 ubiquitin ligase [2], [3], [4]. Keap1 uses a highly efficient cyclic mechanism to target Nrf2 for ubiquitination and proteasomal degradation, during which Keap1 is continuously regenerated, allowing the cycle to proceed (Fig. 1A) [5]. Nrf2 is also subjected to degradation mediated by glycogen synthase kinase (GSK)3/?-TrCP-dependent Cul1-based ubiquitin ligase [6], [7]. Most recently, it was reported that, during conditions of endoplasmic reticulum stress, Nrf2 is ubiquitinated and degraded in a process mediated by the E3 ubiquitin ligase Hrd1 [8].

Figure 1 The cyclic sequential binding and regeneration model for Keap1-mediated degradation of Nrf2. (A) Nrf2 binds sequentially to a free Keap1 dimer: first through its high-affinity ETGE (red sticks) binding domain and then through its low-affinity DLG (black sticks) binding domain. In this conformation of the protein complex, Nrf2 undergoes ubiquitination and is targeted for proteasomal degradation. Free Keap1 is regenerated and able to bind to newly translated Nrf2, and the cycle begins again.(B) Inducers (white diamonds) react with sensor cysteines of Keap1 (blue sticks), leading to a conformational change and impaired substrate adaptor activity. Free Keap1 is not regenerated, and the newly synthesized Nrf2 accumulates and translocates to the nucleus.

In addition to serving as a ubiquitin ligase substrate adaptor protein, Keap1 is also the sensor for a wide array of small-molecule activators of Nrf2 (termed inducers) [9]. Inducers block the cycle of Keap1-mediated degradation of Nrf2 by chemically modifying specific cysteine residues within Keap1 [10], [11] or by directly disrupting the Keap1:Nrf2 binding interface [12], [13]. Consequently, Nrf2 is not degraded, and the transcription factor accumulates and translocates to the nucleus (Fig. 1B), where it forms a heterodimer with a small Maf protein; binds to antioxidant-response elements, the upstream regulatory regions of its target genes; and initiates transcription [14], [15], [16]. The battery of Nrf2 targets comprises proteins with diverse cytoprotective functions, including enzymes of xenobiotic metabolism, proteins with antioxidant and anti-inflammatory functions, and proteasomal subunits, as well as proteins that regulate cellular redox homeostasis and participate in intermediary metabolism.

Nrf2: a Master Regulator of Cellular Redox Homeostasis

The function of Nrf2 as a master regulator of cellular redox homeostasis is widely recognized. The gene expression of both the catalytic and the regulatory subunits of ?-glutamyl cysteine ligase, the enzyme catalyzing the rate-limiting step in the biosynthesis of reduced glutathione (GSH), is directly regulated by Nrf2 [17]. The xCT subunit of system xc-, which imports cystine into cells, is also a direct transcriptional target of Nrf2 [18]. In the cell, cystine undergoes conversion to cysteine, a precursor for the biosynthesis of GSH. In addition to its role in GSH biosynthesis, Nrf2 provides the means for the maintenance of glutathione in its reduced state by the coordinated transcriptional regulation of glutathione reductase 1 [19], [20], which reduces oxidized glutathione to GSH using reducing equivalents from NADPH. The required NADPH is provided by four principal NADPH-generating enzymes, malic enzyme 1 (ME1), isocitrate dehydrogenase 1 (IDH1), glucose-6-phosphate dehydrogenase (G6PD), and 6-phosphogluconate dehydrogenase (PGD), all of which are transcriptionally regulated in part by Nrf2 (Fig. 2) [21], [22], [23], [24]. Curiously, Nrf2 also regulates the inducible gene expression of the cytosolic, microsomal, and mitochondrial forms of aldehyde dehydrogenase [25], which use NAD(P)+ as a cofactor, giving rise to NAD(P)H. Indeed, the levels of NADPH and the NADPH/NADP+ ratio are lower in embryonic fibroblasts isolated from Nrf2-knockout (Nrf2-KO) mice compared to cells from their wild-type (WT) counterparts, and the NADPH levels decrease upon Nrf2 knockdown in cancer cell lines with constitutively active Nrf2 [26]. As expected, the levels of GSH are lower in cells in which Nrf2 has been disrupted; conversely, Nrf2 activation by genetic or pharmacological means leads to GSH upregulation [27], [28], [29]. Importantly, Nrf2 also regulates the gene expression of thioredoxin [30], [31], [32], thioredoxin reductase 1 [28], [29], [32], [33], and sulfiredoxin [34], which are essential for the reduction of oxidized protein thiols.

Figure 2 The role of Nrf2 in the metabolism of rapidly proliferating cells. Nrf2 is a positive regulator of genes encoding enzymes in both the oxidative arm [i.e., glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (PGD)] and the nonoxidative arm [i.e., transaldolase 1 (TALDO1) and transketolase (TKT)] of the pentose phosphate pathway. G6PD and PGD generate NADPH. Nrf2 also regulates the gene expression of the other two NADPH-generating enzymes, malic enzyme 1 (ME1) and isocitrate dehydrogenase 1 (IDH1). The gene expression of phosphoribosyl pyrophosphate amidotransferase (PPAT), which catalyzes the entry into the de novo purine biosynthetic pathway, is also positively regulated by Nrf2, as is the expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme with a critical role in providing one-carbon units for de novo purine biosynthesis. Pyruvate kinase (PK) is negatively regulated by Nrf2 and is expected to favor the buildup of glycolytic intermediates and, together with G6PD, metabolite channeling through the pentose phosphate pathway and the synthesis of nucleic acids, amino acids, and phospholipids. Nrf2 negatively regulates the gene expression of ATP-citrate lyase (CL), which may increase the availability of citrate for mitochondrial utilization or (through isocitrate) for IDH1. Red and blue indicate positive and negative regulation, respectively. The mitochondrion is shown in gray. Metabolite abbreviations: G-6-P, glucose 6-phosphate; F-6-P, fructose 6-phosphate; F-1,6-BP, fructose 1,6-bisphosphate; GA-3-P, glyceraldehyde 3-phosphate; 3-PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; 6-P-Gl, 6-phosphogluconolactone; 6-PG, 6-phosphogluconate; R-5-P, ribulose 5-phosphate; PRPP, 5-phosphoribosyl-?-1-pyrophosphate; THF, tetrahydrofolate; IMP, inosine monophosphate; AMP, adenosine monophosphate; GMP, guanosine monophosphate.

Given the crucial role of Nrf2 as a master regulator of cellular redox homeostasis, it is not surprising that, compared to WT cells, the levels of reactive oxygen species (ROS) are higher in cells in which Nrf2 has been disrupted (Nrf2-KO) [35]. This difference is particularly striking upon challenge with agents causing oxidative stress. Moreover, cells deficient in Nrf2 are much more sensitive to the toxicity of oxidants of various types and cannot be protected by Nrf2 inducers, which, under the same conditions, provide efficient and long-lasting protection to WT cells [29], [36], [37]. In addition to the overall cellular redox homeostasis, Nrf2 is also critical for the maintenance of the mitochondrial redox homeostasis. Thus, compared to WT, the total mitochondrial NADH pool is significantly increased in Keap1-KO and dramatically decreased in Nrf2-KO cells [35].

Using live cell imaging, we recently monitored the rates of ROS production in primary glioneuronal cocultures and brain tissue slices isolated from WT, Nrf2-KO, or Keap1-knockdown (Keap1-KD) mice [38]. As expected, the rate of ROS production was faster in Nrf2-KO cells and tissues compared to their WT counterparts. However, we made the unexpected observation that, compared to WT, Keap1-KD cells also have higher rates of ROS production, although the magnitude of the difference between the WT and the Keap1-KD genotypes was smaller than that between WT and Nrf2-KO. We then analyzed the mRNA levels of NOX2 and NOX4, the catalytic subunits of the two NADPH oxidase (NOX) isoforms that have been implicated in brain pathology, and found that NOX2 is dramatically increased under conditions of Nrf2 deficiency, whereas NOX4 is upregulated when Nrf2 is constitutively activated, although to a smaller extent. Quantitatively, the magnitude of upregulation in cells and tissues from the mutant mice parallels the corresponding increases in ROS production [38]. Interestingly, not only does Nrf2 regulate NADPH oxidase, but the ROS produced by NADPH oxidase can activate Nrf2, as shown in pulmonary epithelial cells and cardiomyocytes [39], [40]. Furthermore, a very recent study has demonstrated that the NADPH oxidase-dependent activation of Nrf2 constitutes an important endogenous mechanism for protection against mitochondrial damage and cell death in the heart during chronic pressure overload [41].

In addition to the catalytic activity of NADPH oxidase, mitochondrial respiration is another major intracellular source of ROS.By use of the mitochondria-specific probe MitoSOX, we have examined the contribution of ROS of mitochondrial origin to the overall ROS production in primary glioneuronal cocultures isolated from WT, Nrf2-KO, or Keap1-KD mice [38]. As expected, Nrf2-KO cells had higher rates of mitochondrial ROS production than WT. In agreement with the findings for the overall ROS production, the rates of mitochondrial ROS production in Keap1-KD were also higher compared to WT cells. Importantly, blocking complex I with rotenone caused a dramatic increase in mitochondrial ROS production in both WT and Keap1-KD cells, but had no effect in Nrf2-KO cells. In contrast to the expected increase in mitochondrial ROS production in WT cells after addition of pyruvate (to enhance the availability of NADH, increase the mitochondrial membrane potential,and normalize respiration), the production of ROS decreased in Nrf2-KO cells. Together, these findings strongly suggest that, in the absence of Nrf2: (i) the activity of complex I is impaired, (ii) the impaired activity of complex I is due to limitation of substrates, and (iii) the impaired activity of complex I is one of the main reasons for the increased mitochondrial ROS production, possibly owing to reverse electron flow from complex II.

Nrf2 Affects Mitochondrial Membrane Potential and Respiration

The mitochondrial membrane potential (??m) is a universal indicator of mitochondrial health and the metabolic state of the cell. In a healthy cell, ??m is maintained by the mitochondrial respiratory chain. Interestingly, a stable isotopic labeling with amino acids in culture-based proteomics study in the estrogen receptor-negative nontumorigenic human breast epithelial MCF10A cell line has shown that the mitochondrial electron transport chain component NDUFA4 is upregulated by pharmacological activation (by sulforaphane) of Nrf2, whereas genetic upregulation of Nrf2 (by Keap1 knockdown) leads to downregulation of the cytochrome c oxidase subunits COX2 and COX4I1 [42]. A study of the liver proteome using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry has found that Nrf2 regulates the expression of ATP synthase subunit ? [43]. In addition, the mitochondrial protein DJ-1, which plays a role in the maintenance of the activity of complex I [44], has been reported to stabilize Nrf2 [45], [46], although the neuroprotective effects of pharmacological or genetic activation of Nrf2 are independent of DJ-1 [47]. However, the consequences of these observations for mitochondrial function have not been investigated.

In agreement with the impaired activity of complex I under conditions of Nrf2 deficiency, the basal ??m is lower in Nrf2-KO mouse embryonic fibroblasts (MEFs) and cultured primary glioneuronal cells in comparison with their WT counterparts (Fig. 3,inset) [35]. In contrast, the basal ??m is higher when Nrf2 is genetically constitutively upregulated (by knockdown or knockout of Keap1). These differences in ??m among the genotypes indicate that respiration is affected by the activity of Nrf2. Indeed, evaluation of the oxygen consumption in the basal state has revealed that, compared to WT, the oxygen consumption is lower in Nrf2-KO and Keap1-KO MEFs, by ~50 and ~35%, respectively.

Figure 3 Proposed mechanism for compromised mitochondrial function under conditions of Nrf2 deficiency. (1) The decreased levels of ME1, IDH1, G6PD, and PGD result in lower NADPH levels. (2) The levels of GSH are also low. (3) The low activity of ME1 may decrease the pool of pyruvate entering the mitochondria. (4) The generation of NADH is slower, leading to impaired activity of complex I and increased mitochondrial ROS production. (5) The reduction of FAD to FADH2 in mitochondrial proteins is also decreased, lowering the electron flow from FADH2 to UbQ and into complex III. (6) The slower formation of UbQH2 may lower the enzyme activity of succinate dehydrogenase. (7) The increased levels of ROS may further inhibit the activity of complex II. (8) The lower efficiency of fatty acid oxidation contributes to the decreased substrate availability for mitochondrial respiration. (9) Glycolysis is enhanced as a compensatory mechanism for the decreased ATP production in oxidative phosphorylation. (10) ATP synthase operates in reverse to maintain ??m. Red and blue indicate upregulation and downregulation, respectively. The boxes signify availability of experimental evidence. The inset shows images of mitochondria of WT and Nrf2-KO cortical astrocytes visualized by the potentiometric fluorescent probe tetramethylrhodamine methyl ester (TMRM; 25 nM). Scale bar, 20 �m.

These differences in ??m and respiration among the genotypes are reflected by the rate of utilization of substrates for mitochondrial respiration. Application of substrates for the tricarboxylic acid (TCA) cycle (malate/pyruvate, which in turn increase the production of the complex I substrate NADH) or methyl succinate, a substrate for complex II, causes a stepwise increase in ??m in both WT and Keap1-KD neurons, but the rate of increase is higher in Keap1-KD cells. More importantly, the shapes of the response to these TCA cycle substrates are different between the two genotypes, whereby the rapid rise in ??m in Keap1-KD cells upon substrate addition is followed by a quick drop rather than a plateau, suggesting an unusually fast substrate consumption. These findings are in close agreement with the much lower (by 50�70%) levels of malate, pyruvate, and succinate that have been observed after a 1-h pulse of [U-13C6]glucose in Keap1-KO compared to WT MEF cells [24]. In Nrf2-KO neurons, only pyruvate is able to increase the ??m, whereas malate and methyl succinate cause mild depolarization. The effect of Nrf2 on mitochondrial substrate production seems to be the main mechanism by which Nrf2 affects mitochondrial function. The mitochondrial NADH redox index (the balance between consumption of NADH by complex I and production of NADPH in the TCA cycle) is significantly lower in Nrf2-KO cells in comparison with their WT counterparts, and furthermore, the rates of regeneration of the pools of NADH and FADH2 after inhibition of complex IV (by use of NaCN) are slower in the mutant cells.

In mitochondria isolated from murine brain and liver, supplementation of substrates for complex I or for complex II increases the rate of oxygen consumption more strongly when Nrf2 is activated and less efficiently when Nrf2 is disrupted [35]. Thus, malate induces a higher rate of oxygen consumption in Keap1-KD compared to WT, but its effect is weaker in Nrf2-KO mitochondria. Similarly, in the presence of rotenone (when complex I is inhibited), succinate activates oxygen consumption to a greater extent in Keap1-KD compared to WT, whereas the response in Nrf2-KO mitochondria is diminished. In addition, Nrf2-KO primary neuronal cultures and mice are more sensitive to the toxicity of the complex II inhibitors 3-nitropropionic acid and malonate, whereas intrastriatal transplantation of Nrf2-overexpressing astrocytes is protective [48], [49]. Similarly, Nrf2-KO mice are more sensitive to, whereas genetic or pharmacological activation of Nrf2 has protective effects against, neurotoxicity caused by the complex I inhibitor 1-methyl-4-phenylpyridinium ion in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine animal model of Parkinson?s disease [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61].

The respiratory control ratio (RCR), the ratio of State 3 (ADP-stimulated) to State 4 respiration (no ADP present), is decreased in the absence of Nrf2, but the RCR is similar between Keap1-KD and WT mitochondria [35]. As the RCR is an indication of the degree of coupling of the mitochondrial respiratory chain activity to oxidative phosphorylation, this finding indicates that the higher rate of respiration in Keap1-KD mitochondria is not due to uncoupling of oxidative phosphorylation. It further suggests that oxidative phosphorylation is more efficient when Nrf2 is activated. The higher rate of respiration in Keap1-KD mitochondria is consistent with the higher levels of mitochondrial ROS production [38] as higher respiration rates may lead to increased electron leak. However, under conditions of oxidative stress, the increased ROS production is counteracted by the Nrf2-dependent transcriptional upregulation of uncoupling protein 3 (UCP3), which increases the proton conductance of the mitochondrial inner membrane and consequently decreases the production of superoxide [62]. Very recently, it was shown that the lipid peroxidation product 4-hydroxy-2-nonenal mediates the Nrf2-dependent upregulation of UCP3 in cardiomyocytes; this might be particularly important for protection under conditions of oxidative stress such as those during ischemia�reperfusion [63].

Nrf2 Affects the Efficiency of Oxidative Phosphorylation and the Synthesis of ATP

In agreement with the effect of Nrf2 on respiration, in brain and liver mitochondria, Nrf2 deficiency results in a decreased efficiency of oxidative phosphorylation (as estimated by the ratio of ADP to oxygen, which is consumed for ATP synthesis), whereas Nrf2 activation (Keap1-KD) has the opposite effect [35]. Compared to WT, the ATP levels are significantly higher in cells with constitutive upregulation of Nrf2 and lower when Nrf2 is knocked down [64] or disrupted [35]. Furthermore, the use of inhibitors of oxidative phosphorylation (oligomycin) or glycolysis (iodoacetic acid) has revealed that Nrf2 changes the way by which cells produce ATP. Thus, in WT neurons, oligomycin causes a complete drop in ATP and iodoacetic acid has no further effect. Remarkably, in Nrf2-KO cells, oligomycin increases the ATP levels, which are then slowly, but completely, depleted by iodoacetic acid, indicating that in the absence of Nrf2, glycolysis, and not oxidative phosphorylation, is the main source of ATP production. Interestingly, despite the increased efficiency of oxidative phosphorylation in Keap1-KD cells, addition of oligomycin results in an ~80% decrease in ATP levels, and iodoacetic acid causes a further ~20% decrease. Thus, either Nrf2 deficiency or its constitutive activation reduces the contribution of oxidative phosphorylation and increases the contribution of glycolysis toward the synthesis of ATP. This effect is particularly pronounced when Nrf2 is absent and is consistent with the dependence of the ??m on the presence of glucose in the medium [35] and the increased levels of glycolytic intermediates (G-6-P, F-6-P, dihydroxyacetone phosphate, pyruvate, and lactate) after knockdown of Nrf2 [24].

The increase in ATP levels after inhibition of the F1F0-ATPase by oligomycin indicates that in the absence of Nrf2, the F1F0-ATPase functions as an ATPase and not an ATP synthase, i.e., it operates in reverse. Such reversal in activity most likely reflects the need to pump protons across the inner mitochondrial membrane in an attempt to maintain the ??m, which is crucial for the functional integrity of this organelle. The reversal of the function of the F1F0-ATPase is also evidenced by the observed mitochondrial depolarization upon oligomycin administration to Nrf2-KO cells, which is in sharp contrast to the hyperpolarization occurring in their WT or Keap1-deficient counterparts [35]. Overall, it seems that under conditions of Nrf2 deficiency ATP is produced primarily in glycolysis, and this ATP is then used in part by the F1F0-ATPase to maintain the ??m.

Nrf2 Enhances Mitochondrial Fatty Acid Oxidation

The effect of Nrf2 deficiency on the ??m is particularly pronounced when cells are incubated in medium without glucose, and the ??m is ~50% lower in Nrf2-KO compared to WT cells [35]. Under conditions of glucose deprivation, mitochondrial fatty acid oxidation (FAO) is a major provider of substrates for respiration and oxidative phosphorylation, suggesting that Nrf2 may affect FAO. Indeed, the efficiency of FAO for both the long-chain (C16:0) saturated fatty acid palmitic acid and the short-chain (C6:0) hexanoic acid is higher in Keap1-KO MEFs and isolated heart and liver mitochondria than in their WT counterparts, whereas it is lower in Nrf2-KO cells and mitochondria [65]. These effects are also highly relevant to humans: indeed, metabolic changes indicative of better integration of FAO with the activity of the TCA cycle have been reported to occur in human intervention studies with diets rich in glucoraphanin, the precursor of the classical Nrf2 activator sulforaphane [66].

During the first step of mitochondrial FAO, the pro-R hydrogen of the ?-carbon leaves as a hydride that reduces the FAD cofactor to FADH2, which in turn transfers electrons to ubiquinone (UbQ) in the respiratory chain, ultimately contributing to ATP production. Whereas stimulation of FAO by palmitoylcarnitine in the absence of glucose causes the expected increase in the ATP levels in WT and Keap1-KO cells, with the ATP rise being faster in Keap1-KO cells, the identical treatment produces no ATP changes in Nrf2-KO MEFs [65]. This experiment demonstrates that, in the absence of Nrf2, FAO is suppressed, and furthermore, it implicates suppression of FAO as one of the reasons for the lower ATP levels under conditions of Nrf2 deficiency [35], [64].

Notably, human 293 T cells in which Nrf2 has been silenced have a lower expression of CPT1 and CPT2[67], two isoforms of carnitine palmitoyltransferase (CPT), the rate-limiting enzyme in mitochondrial FAO. In agreement, the mRNA levels of Cpt1 are lower in livers of Nrf2-KO compared to WT mice [68]. CPT catalyzes the transfer of the acyl group of a long-chain fatty acyl-CoA from coenzyme A to l-carnitine and thus permits the import of acylcarnitine from the cytoplasm into the mitochondria. Although this has not been examined to date, it is possible that in addition to the transcriptional effects on CPT1 expression, Nrf2 may also affect the function of this enzyme by controlling the levels of its main allosteric inhibitor, malonyl-CoA. This is because, by a mechanism that is currently unclear, Nrf2 regulates negatively the expression of stearoyl CoA desaturase (SCD) [69] and citrate lyase (CL) [69], [70]. Curiously, knockout or inhibition of SCD leads to increased phosphorylation and activation of AMP-activated protein kinase (AMPK) [71], [72], [73], and it can be speculated that, in the absence of Nrf2, the SCD levels will increase, in turn lowering AMPK activity. This could be further compounded by the reduced protein levels of AMPK that have been observed in livers of Nrf2-KO mice [68], a finding that is in close agreement with the increased AMPK levels, which have been reported in livers of Keap1-KD mice [74]. One consequence of the decreased AMPK activity is the relief of its inhibitory phosphorylation (at Ser79) of acetyl-CoA carboxylase (ACC) [75], which could be further transcriptionally upregulated in the absence of Nrf2 because it is downregulated by Nrf2 activation [70]. The high ACC activity, in combination with the upregulated CL expression that will increase the production of acetyl-CoA, the substrate for ACC, may ultimately increase the levels of the ACC product, malonyl-CoA. The high levels of malonyl-CoA will inhibit CPT, thereby decreasing the transport of fatty acids into the mitochondria. Finally, Nrf2 positively regulates the expression of CD36 [76], a translocase that imports fatty acids across plasma and mitochondrial membranes. Thus, one mechanism by which Nrf2 may affect the efficiency of mitochondrial FAO is by regulating the import of long-chain fatty acids into the mitochondria.

In addition to direct transcriptional regulation, Nrf2 may also alter the efficiency of mitochondrial FAO by its effects on the cellular redox metabolism. This may be especially relevant when Nrf2 activity is low or absent, conditions that shift the cellular redox status toward the oxidized state. Indeed, several FAO enzymes have been identified as being sensitive to redox changes. One such enzyme is very long-chain acyl-CoA dehydrogenase (VLCAD), which contributes more than 80% to the palmitoyl-CoA dehydrogenation activity in human tissues [77]. Interestingly, Hurd et al. [78] have shown that VLCAD contains cysteine residues that significantly change their redox state upon exposure of isolated rat heart mitochondria to H2O2. Additionally, S-nitrosylation of murine hepatic VLCAD at Cys238 improves the catalytic efficiency of the enzyme [79], and it is likely that oxidation of the same cysteine may have the opposite effect, ultimately lowering the efficiency of mitochondrial FAO. It is therefore possible that, although the expression levels of VLCAD are not significantly different in WT, Nrf2-KO, or Keap1-KO MEFs [65], the enzyme activity of VLCAD could be lower in the absence of Nrf2 owing to the higher levels of ROS.

Based on all of these findings, it can be proposed that (Fig. 3): in the absence of Nrf2, the NADPH levels are lower owing to decreased expression of ME1, IDH1, G6PD, and PGD. The levels of reduced glutathione are also lower owing to decreased expression of enzymes that participate in its biosynthesis and regeneration and the lower levels of NADPH that are required for the conversion of the oxidized to the reduced form of glutathione. The low expression of ME1 will decrease the pool of pyruvate entering the mitochondria, with glycolysis becoming the major source of pyruvate. The generation of NADH is slower, leading to impaired activity of complex I and increased mitochondrial ROS production. The reduction of FAD to FADH2 is also slower, at least in part owing to less efficient fatty acid oxidation, compromising the electron flow from FADH2 to UbQ and into complex III. As UbQH2 is an activator of succinate dehydrogenase [80], slowing down its formation may lower the enzyme activity of succinate dehydrogenase. The increased levels of superoxide and hydrogen peroxide can inhibit complex II activity further [81]. The lower efficiency of fatty acid oxidation contributes to the decreased substrate availability for mitochondrial respiration and ATP production in oxidative phosphorylation. As a compensatory mechanism, glycolysis is enhanced. ATP synthase functions in reverse, as an ATPase, in an attempt to maintain the ??m.

Nrf2 and Mitochondrial Biogenesis

It has been reported that, compared to WT, the livers of Nrf2-KO mice have a lower mitochondrial content (as determined by the ratio of mitochondrial to nuclear DNA); this is further decreased by a 24-h fast in both WT and Nrf2-KO mice; in contrast, although no different from WT under normal feeding conditions, the mitochondrial content in mice with high Nrf2 activity is not affected by fasting [82]. Interestingly, supplementation with the Nrf2 activator (R)-?-lipoic acid [83], [84], [85] promotes mitochondrial biogenesis in 3T3-L1 adipocytes [86]. Two classes of nuclear transcriptional regulators play critical roles in mitochondrial biogenesis. The first class are transcription factors, such as nuclear respiratory factors11 and 2, which control the expression of genes encoding subunits of the five respiratory complexes, mitochondrial translational components, and heme biosynthetic enzymes that are localized to the mitochondrial matrix [88]. Piantadosi et al. [89] have shown that the Nrf2-dependent transcriptional upregulation of nuclear respiratory factor 1 promotes mitochondrial biogenesis and protects against the cytotoxicity of the cardiotoxic anthracycline chemotherapeutic agent doxorubicin. In contrast, Zhang et al. [82] have reported that genetic activation of Nrf2 does not affect the basal mRNA expression of nuclear respiratory factor 1 in the murine liver.

The second class of nuclear transcriptional regulators with critical functions in mitochondrial biogenesis are transcriptional coactivators, such as peroxisome proliferator-activated receptor ? coactivators (PGC)1? and 1?, which interact with transcription factors, the basal transcriptional and RNA-splicing machinery, and histone-modifying enzymes [88], [90], [91]. The expression of the PGC1 family of coactivators is influenced by numerous environmental signals. Treatment of human fibroblasts with the Nrf2 activator sulforaphane causes an increase in mitochondrial mass and induction of PGC1? and PGC1? [92], although the potential dependence on Nrf2 was not examined in this study. However, diabetic mice in which Nrf2 is either activated by Keap1 gene hypomorphic knockdown (db/db:Keap1flox/?:Nrf2+/+) or disrupted (db/db:Keap1flox/?:Nrf2?/?) have lower hepatic PGC1? expression levels than control animals (db/db:Keap1flox/+:Nrf2+/+) [93]. No differences in the mRNA levels for PGC1? are seen in livers of nondiabetic mice that are either WT or Nrf2-KO, whereas these levels are lower in Nrf2-overexpressing (Keap1-KD and liver-specific Keap1-KO) animals [82]. Notably, a 24-h fast increases the levels of PGC1? mRNA in the livers of mice of all genotypes, but the increase is significantly greater in livers of Nrf2-KO compared to WT or Nrf2-overexpressing mice. Compared to WT, Nrf2-KO mice experiencing septic infection or acute lung injury due to infection show attenuated transcriptional upregulation of nuclear respiratory factor 1 and PGC1? [94], [95]. Together, these observations suggest that the role of Nrf2 in maintaining the levels of both nuclear respiratory factor 1 and PGC1? is complex and becomes most prominent under conditions of stress.

In addition to expression of genes encoding mitochondrial proteins, mitochondrial biogenesis requires the synthesis of nucleotides. Genetic activation of Nrf2 enhances purine biosynthesis by upregulating the pentose phosphate pathway and the metabolism of folate and glutamine, particularly in rapidly proliferating cells (Fig. 2) [24]. Analysis of the transcriptome of mutant Drosophila deficient for the mitochondrial serine/threonine protein kinase PTEN-induced putative kinase 1 (PINK1) has shown that mitochondrial dysfunction leads to the transcriptional upregulation of genes affecting nucleotide metabolism [96], suggesting that the enhanced nucleotide biosynthesis represents a mechanism for protection against the neurotoxic consequences of PINK1 deficiency. Nrf2 regulates the expression of phosphoribosyl pyrophosphate amidotransferase (PPAT), which catalyzes the entry into the de novo purine nucleotide biosynthetic pathway, and mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) (Fig. 2). The latter is a bifunctional enzyme with dehydrogenase and cyclohydrolase activities that is critical in providing both glycine and formate as sources of one-carbon units for purine biosynthesis in rapidly growing cells [97]. It is therefore likely that Nrf2 activation might be protective and might reverse mitochondrial dysfunction in PINK1 deficiency. Indeed, pharmacological activation of Nrf2 by sulforaphane, or the triterpenoid RTA-408, restores ??m and protects PINK1-deficient cells against dopamine toxicity [98]. Although the underlying mechanisms seem to be complex, together, these findings indicate that Nrf2 activity may affect mitochondrial biogenesis by influencing the expression levels of critical transcription factors and coactivators, as well as by enhancing nucleotide biosynthesis.

Nrf2 and Mitochondrial Integrity

Although direct evidence is not always available, there are strong indications that Nrf2 is important for mitochondrial integrity, particularly under conditions of oxidative stress. Mitochondria isolated from the brain and liver of rats that had been administered a single dose of the Nrf2 activator sulforaphane are resistant to opening of the mitochondrial permeability transition pore (mPTP) caused by the oxidant tert-butylhydroperoxide [99], [100]. The mPTP, a complex that allows the mitochondrial inner membrane to become permeable to molecules with masses up to 1500 Da, was recently identified to be formed from dimers of the F0F1-ATP synthase [101]. The sulforaphane-mediated resistance to mPTP opening correlates with increased antioxidant defenses, and the levels of mitochondrial GSH, glutathione peroxidase 1, malic enzyme 3, and thioredoxin 2 are all upregulated in mitochondrial fractions isolated from sulforaphane-treated animals [100].

Mitochondrial protein damage and impairment in respiration caused by the electrophilic lipid peroxidation product 4-hydroxy-2-nonenal are attenuated in mitochondria isolated from the cerebral cortex of sulforaphane-treated mice [102]. In rat renal epithelial cells and in kidney, sulforaphane is protective against cisplatin- and gentamicin-induced toxicity and loss of ??m[103], [104]. Protection against a panel of oxidants (superoxide, hydrogen peroxide, peroxynitrite) and electrophiles (4-hydroxy-2-nonenal and acrolein) and an increase in mitochondrial antioxidant defenses have been also observed upon treatment of rat aortic smooth muscle cells with sulforaphane [105]. In a model of contrast-induced acute kidney injury, limb ischemic preconditioning was recently shown to have protective effects, including inhibition of the opening of the mPTP and mitochondrial swelling, by activation of Nrf2 consequent to the inhibition of GSK3? [106].

Mitophagy, the process by which dysfunctional mitochondria are selectively engulfed by autophagosomes and delivered to lysosomes to be degraded and recycled by the cell, is essential for mitochondrial homeostasis [107], [108]. Whereas no causative relation between Nrf2 and mitophagy has been established, there is evidence that the transcription factor may be important in mitochondrial quality control by playing a role in mitophagy. This might be especially prominent under conditions of oxidative stress. Thus, in a model of sepsis, the increases in the levels of the autophagosome marker MAP1 light chain 3-II (LC3-II) and the cargo protein p62 at 24 h postinfection are suppressed in Nrf2-KO compared to WT mice [109]. A small-molecule inducer of mitophagy (called p62-mediated mitophagy inducer, PMI) was recently discovered; this 1,4-diphenyl-1,2,3-triazole compound was originally designed as an Nrf2 activator that disrupts the interaction of the transcription factor with Keap1 [110]. Similar to cells in which Nrf2 is genetically upregulated (Keap1-KD or Keap1-KO), cells exposed to PMI have higher resting ??m. Importantly, the increase in mitochondrial LC3 localization that is observed after PMI treatment of WT cells does not occur in Nrf2-KO cells, suggesting the involvement of Nrf2.

Last, ultrastructural analysis of liver sections has revealed the presence of swollen mitochondria with reduced crista and disrupted membranes in hepatocytes of Nrf2-KO, but not WT, mice that had been fed a high-fat diet for 24 weeks; notably, these livers show clear evidence of oxidative stress and inflammation [68]. It can be concluded that Nrf2 has a critical role in maintaining mitochondrial integrity under conditions of oxidative and inflammatory stress.

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below.

Key sections:

  • 00:01:14 – Cancer and mortality
  • 00:19:04 – Aging
  • 00:26:30 – Brain and behavior
  • 00:38:06 – Final recap
  • 00:40:27 – Dose

Full timeline:

  • 00:00:34 – Introduction of sulforaphane, a major focus of the video.
  • 00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
  • 00:02:12 – Prostate cancer risk.
  • 00:02:23 – Bladder cancer risk.
  • 00:02:34 – Lung cancer in smokers risk.
  • 00:02:48 – Breast cancer risk.
  • 00:03:13 – Hypothetical: what if you already have cancer? (interventional)
  • 00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
  • 00:04:38 – Sulforaphane and cancer.
  • 00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
  • 00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
  • 00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
  • 00:08:32 – Inhibition of breast cancer stem cells.
  • 00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
  • 00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
  • 00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
  • 00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
  • 00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
  • 00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
  • 00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
  • 00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
  • 00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
  • 00:19:04 – Beginning of aging section.
  • 00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
  • 00:20:34 – Importance of low inflammation for longevity.
  • 00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
  • 00:23:40 – Mid-video recap: cancer, aging sections
  • 00:24:14 – Mouse studies suggest sulforaphane might improve adaptive immune function in old age.
  • 00:25:18 – Sulforaphane improved hair growth in a mouse model of balding. Picture at 00:26:10.
  • 00:26:30 – Beginning of brain and behavior section.
  • 00:27:18 – Effect of broccoli sprout extract on autism.
  • 00:27:48 – Effect of glucoraphanin on schizophrenia.
  • 00:28:17 – Start of depression discussion (plausible mechanism and studies).
  • 00:31:21 – Mouse study using 10 different models of stress-induced depression show sulforaphane similarly effective as fluoxetine (prozac).
  • 00:32:00 – Study shows direct ingestion of glucoraphanin in mice is similarly effective at preventing depression from social defeat stress model.
  • 00:33:01 – Beginning of neurodegeneration section.
  • 00:33:30 – Sulforaphane and Alzheimer’s disease.
  • 00:33:44 – Sulforaphane and Parkinson’s disease.
  • 00:33:51 – Sulforaphane and Hungtington’s disease.
  • 00:34:13 – Sulforaphane increases heat shock proteins.
  • 00:34:43 – Beginning of traumatic brain injury section.
  • 00:35:01 – Sulforaphane injected immediately after TBI improves memory (mouse study).
  • 00:35:55 – Sulforaphane and neuronal plasticity.
  • 00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
  • 00:37:19 – Sulforaphane and duchenne muscular dystrophy.
  • 00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
  • 00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
  • 00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
  • 00:41:01 – Anecdotes on sprouting at home.
  • 00:43:14 – On cooking temperatures and sulforaphane activity.
  • 00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
  • 00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
  • 00:44:56 – Cooking techniques and cruciferous vegetables.
  • 00:46:06 – Isothiocyanates as goitrogens.
Dr Jimenez White Coat
Nrf2 is a transcription factor which plays an important role in the cellular antioxidant defense system of the human body. The antioxidant responsive element, or ARE, is a regulatory mechanism of genes. Many research studies have demonstrated that Nrf2, or NF-E2-related factor 2, regulates a wide variety of ARE-driven genes throughout several types of cells. Nrf2 was also found to play an essential role in cellular protection and anti-carcinogenicity, which demonstrates that Nrf2 may be an effective treatment in the management of neurodegenerative diseases and cancers believed to be caused by oxidative stress. Dr. Alex Jimenez D.C., C.C.S.T. Insight

Concluding Remarks

Although many questions still remain open, the available experimental evidence clearly indicates that Nrf2 is an important player in the maintenance of mitochondrial homeostasis and structural integrity. This role becomes particularly critical under conditions of oxidative, electrophilic, and inflammatory stress when the ability to upregulate Nrf2-mediated cytoprotective responses influences the overall health and survival of the cell and the organism. The role of Nrf2 in mitochondrial function represents another layer of the broad cytoprotective mechanisms orchestrated by this transcription factor. As many human pathological conditions have oxidative stress, inflammation, and mitochondrial dysfunction as essential components of their pathogenesis, pharmacological activation of Nrf2 holds promise for disease prevention and treatment. Comprehensive understanding of the precise mechanisms by which Nrf2 affects mitochondrial function is essential for rational design of future clinical trials and may offer new biomarkers for monitoring therapeutic efficacy.

Acknowledgments

Sciencedirect.com/science/article/pii/S0891584915002129

The purpose of the article above was to discuss�as well as demonstrate�the emerging role of Nrf2 in mitochondrial function. Nrf2, or nuclear factor erythroid 2-related factor, is an emerging regulator of cellular resistance to oxidants which can contribute to oxidative stress, affecting cellular function and leading to the development of toxicity, chronic disease, and even cancer. While the production of oxidants in the human body can serve�various purposes,�including cell division, inflammation, immune function, autophagy, and stress response, it’s essential to control their overproduction to prevent health issues. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.

Curated by Dr. Alex Jimenez

Referenced from: Sciencedirect.com

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Additional Topic Discussion:�Acute Back Pain

Back pain�is one of the most prevalent causes of disability and missed days at work worldwide. Back pain attributes to the second most common reason for doctor office visits, outnumbered only by upper-respiratory infections. Approximately 80 percent of the population will experience back pain at least once throughout their life. The spine is a complex structure made up of bones, joints, ligaments, and muscles, among other soft tissues. Because of this, injuries and/or aggravated conditions, such as�herniated discs, can eventually lead to symptoms of back pain. Sports injuries or automobile accident injuries are often the most frequent cause of back pain, however, sometimes the simplest of movements can have painful results. Fortunately, alternative treatment options, such as chiropractic care, can help ease back pain through the use of spinal adjustments and manual manipulations, ultimately improving pain relief. �

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EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

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Nrf2 Signaling Pathway: Pivotal Roles in Inflammation

Nrf2 Signaling Pathway: Pivotal Roles in Inflammation

Nrf2 supports the activation of a group of antioxidant and detoxifying enzymes and genes which protect the human body from the effects of health issues associated with increased levels of oxidative stress, such as Alzheimer’s disease. A variety of natural substances have been demonstrated to activate the Nrf2 pathway, which can help manage the symptoms of neurodegenerative diseases. The purpose of the article below is to discuss the pivotal role of Nrf2 caused by chronic inflammation.

Abstract

Inflammation is the most common feature of many chronic diseases and complications, while playing critical roles in carcinogenesis. Several studies have demonstrated that Nrf2 contributes to the anti-inflammatory process by orchestrating the recruitment of inflammatory cells and regulating gene expression through the antioxidant response element (ARE). The Keap1 (Kelch-like ECH-associated protein)/Nrf2 (NF-E2 p45-related factor 2)/ARE signaling pathway mainly regulates anti-inflammatory gene expression and inhibits the progression of inflammation. Therefore, the identification of new Nrf2-dependent anti-inflammatory phytochemicals has become a key point in drug discovery. In this review, we discuss the members of the Keap1/Nrf2/ARE signal pathway and its downstream genes, the effects of this pathway on animal models of inflammatory diseases, and crosstalk with the NF-?B pathway. In addition we also discuss about the regulation of NLRP3 inflammasome by Nrf2. Besides this, we summarize the current scenario of the development of anti-inflammatory phytochemicals and others that mediate the Nrf2/ARE signaling pathway.

Keywords: Nrf2, Keap1, ARE, Inflammation, Oxidative stress, Phytochemical

Abbreviations

Sciencedirect.com/science/article/pii/S0925443916302861#t0005

Introduction

Inflammation is a complex process that occurs when tissues are infected or injured by harmful stimuli such as pathogens, damage, or irritants. Immune cells, blood vessels, and molecular mediators are involved in this protective response [1]. Inflammation is also a pathological phenomenon associated with a variety of disease states induced mainly by physical, chemical, biological, and psychological factors. The aim of inflammation is to limit and eliminate the causes of cellular damage, clear and/or absorb necrotic cells and tissues, and initiate tissue repair. Two distinct forms of inflammation are distinguished: acute and chronic. Acute inflammation is self-limiting and beneficial to the host, but prolonged chronic inflammation is a common feature of many chronic diseases and complications. Direct infiltration by many mononuclear immune cells such as monocytes, macrophages, lymphocytes, and plasma cells, as well as the production of inflammatory cytokines, lead to chronic inflammation. It is recognized that chronic inflammation plays a critical role in carcinogenesis [2]. In general, both pro- and anti-inflammatory signaling pathways interact in the normal inflammatory process.

In the pathological inflammatory process, mast cells, monocytes, macrophages, lymphocytes, and other immune cells are first activated. Then the cells are recruited to the site of injury, resulting in the generation of reactive oxygen species (ROS) that damage macromolecules including DNA. At the same time, these inflammatory cells also produce large amounts of inflammatory mediators such as cytokines, chemokines, and prostaglandins. These mediators further recruit macrophages to localized sites of inflammation and directly activate multiple signal transduction cascades and transcription factors associated with inflammation. The NF-?B (nuclear factor kappa B), MAPK (mitogen-activated protein kinase), and JAK (janus kinase)-STAT (signal transducers and activators of transcription) signaling pathways are involved in the development of the classical pathway of inflammation [3], [4], [5]. Previous studies have revealed that the transcription factor Nrf2 (NF-E2 p45-related factor 2) regulates the expression of phase II detoxifying enzymes including NADPH, NAD(P)H quinone oxidoreductase 1, glutathione peroxidase, ferritin, heme oxygenase-1 (HO-1), and antioxidant genes that protect cells from various injuries via their anti-inflammatory effects, thus influencing the course of disease [6], [7], [8].

Considering these remarkable findings, the development of targeted therapeutic drugs for inflammatory diseases via signaling pathways has attracted much interest in recent years. In this review, we summarize research on the Keap1 (Kelch-like ECH associated protein)/Nrf2 (NF-E2 p45-related factor 2)/ARE (antioxidant response element) signaling pathway in inflammation.

Structure and Regulation of Nrf2

Keap1-Dependent Nrf2 Regulation

Nrf2 belongs to the Cap �n� Collar (CNC) subfamily and comprises in seven functional domains, Neh (Nrf2-ECH homology) 1 to Neh7 [9], [10]. Neh1 is a CNC-bZIP domain that allows Nrf2 to heterodimerize with small musculoaponeurotic fibrosarcoma (Maf) protein, DNA, and other transcription partners as well as forming a nuclear complex with the ubiquitin-conjugating enzyme UbcM2 [11], [12]. Neh2 contains two important motifs known as DLG and ETGE, which are essential for the interaction between Nrf2 and its negative regulator Keap1 [13], [14].

Keap1 is a substrate adaptor for cullin-based E3 ubiquitin ligase, which inhibits the transcriptional activity of Nrf2 via ubiquitination and proteasomal degradation under normal conditions [15], [16], [17]. The KELCH domains of the Keap1 homodimer bind with the DLG and ETGE motifs of the Nrf2-Neh2 domain in the cytosol, where ETGE acts as a hinge with higher affinity and DLG acts as a latch [18]. Under oxidative stress or upon exposure to Nrf2 activators, Nrf2 dissociates from Keap1 binding due to the thiol modification of Keap1 cysteine residues which ultimately prevents Nrf2 ubiquitination and proteasomal degradation [19]. Then Nrf2 translocates into the nucleus, heterodimerizes with small Maf proteins, and transactivates an ARE battery of genes (Fig. 1A). The carboxy-terminal of Neh3 acts as a transactivation domain by interacting with the transcription co-activator known as CHD6 (chromo-ATPase/helicase DNA binding protein) [20]. Neh4 and Neh5 also act as transactivation domains, but bind to another transcriptional co-activator known as CBP (cAMP-response-element-binding protein-binding protein) [21]. Moreover, Neh4 and Neh5 interact with the nuclear cofactor RAC3/AIB1/SRC-3, leading to enhanced Nrf2-targeted ARE gene expression [22]. Neh5 has a redox-sensitive nuclear-export signal which is crucial for the regulation and cellular localization of Nrf2 [23].

Figure 1 Keap1-dependent and -independent regulation of Nrf2. (A) Under basal conditions, Nrf2 is sequestered with Keap1 by its two motifs (ETGE and DLG) that leads to CUL3-mediated ubiquitination followed by proteasome degradation. Under oxidative stress, Nrf2 dissociates from Keap1, translocates to the nucleus and activates the ARE-gene battery. (B) GSK3 phosphorylates Nrf2 and this facilitates the recognition of Nrf2 by ?-TrCP for CUL1-mediated ubiquitination and subsequent proteasome degradation. (C) p62 is sequestered with Keap1, leading to its autophagic degradation, the liberation of Nrf2, and increased Nrf2 signaling.

Keap1-Independent Nrf2 Regulation

Emerging evidence has revealed a novel mechanism of Nrf2 regulation that is independent of Keap1. The serine-rich Neh6 domain of Nrf2 plays a crucial role in this regulation by binding with its two motifs (DSGIS and DSAPGS) to ?-transducin repeat-containing protein (?-TrCP) [24]. ?-TrCP is a substrate receptor for the Skp1�Cul1�Rbx1/Roc1 ubiquitin ligase complex that targets Nrf2 for ubiquitination and proteasomal degradation. Glycogen synthase kinase-3 is a crucial protein involved in Keap1-independent Nrf2 stabilization and regulation; it phosphorylates Nrf2 in the Neh6 domain to facilitate the recognition of Nrf2 by ?-TrCP and subsequent protein degradation [25] (Fig. 1B).

Other Nrf2 Regulators

Another line of evidence has revealed a non-canonical pathway of p62-dependent Nrf2 activation in which p62 sequesters Keap1 to autophagic degradation that ultimately leads to the stabilization of Nrf2 and the transactivation of Nrf2-dependent genes [26], [27], [28], [29] (Fig. 1C).

Accumulating evidence suggests that several miRNAs play an important role in the regulation the Nrf2 activity [30]. Sangokoya et al. [31] demonstrated that miR-144 directly downregulates Nrf2 activity in the lymphoblast K562 cell line, primary human erythroid progenitor cells, and sickle-cell disease reticulocytes. Another interesting study in human breast epithelial cells demonstrated that miR-28 inhibits Nrf2 through a Keap1-independent mechanism [32]. Similarly, miRNAs such as miR-153, miR-27a, miR-142-5p, and miR144 downregulate Nrf2 expression in the neuronal SH-SY5Y cell line [33]. Singh et al. [34] demonstrated that the ectopic expression of miR-93 decreases the expression of Nrf2-regulated genes in a 17?-estradiol (E2)-induced rat model of mammary carcinogenesis.

A recent discovery from our lab identified an endogenous inhibitor of Nrf2 known as retinoic X receptor alpha (RXR?). RXR? is a nuclear receptor, interacts with the Neh7 domain of Nrf2 (amino-acid residues 209�316) via its DNA-binding domain (DBD), and specifically inhibits Nrf2 activity in the nucleus. Moreover, other nuclear receptors such as peroxisome proliferator-activated receptor-?, ER?, estrogen-related receptor-?, and glucocorticoid receptors have also been reported to be endogenous inhibitors of Nrf2 activity [9], [10].

Anti-Inflammatory Role of Nrf2/HO-1 Axis

HO-1 is the inducible isoform and rate-limiting enzyme that catalyzes the degradation of heme into carbon monoxide (CO) and free iron, and biliverdin to bilirubin. Enzymatic degradation of pro-inflammatory free heme as well as the production of anti-inflammatory compounds such as CO and bilirubin play major roles in maintaining the protective effects of HO-1 (Fig. 2).

Figure 2 Overview of the Nrf2/HO-1 pathway. Under basal conditions, Nrf2 binds to its repressor Keap1 which leads to ubiquitination followed by proteasome degradation. During oxidative stress, free Nrf2 translocates to the nucleus, where it dimerizes with members of the small Maf family and binds to ARE genes such as HO-1. Upregulated HO-1 catalyzes the heme into CO, bilirubin, and free iron. CO acts as an inhibitor of the NF-?B pathway which leads to the decreased expression of pro-inflammatory cytokines, while bilirubin also acts as antioxidant. Furthermore, HO-1 directly inhibits the proinflammatory cytokines as well as activating the anti-inflammatory cytokines, thus leads to balancing of the inflammatory process.

Nrf2 induces the HO-1 gene by increasing mRNA and protein expression and it is one of the classic Nrf2 regulated gene which is widely used in numerous in vitro and in vivo studies. Several studies have demonstrated that HO-1 and its metabolites have significant anti-inflammatory effects mediated by Nrf2. Elevation of HO-1 expression which is mediated by activated Nrf2 leads to the inhibition of NF?B signaling results in the reduced intestinal mucosal injury and tight-junction dysfunction in male Sprague-Dawley rat liver transplantation model [35]. Upregulation of Nrf2-dependent HO-1 expression may protect mouse derived C2C12 myoblasts from H2O2 cytotoxicity [36]. Nrf2-dependent HO-1 has an impact on lipopolysaccharide (LPS)-mediated inflammatory responses in RAW264.7- or mouse peritoneal macrophage-derived foam cell macrophages. Nrf2 activity desensitized foam cell macrophages phenotype and prevent immoderate inflammation of macrophages, those play important role in progression of atherosclerosis [37]. The Nrf2/HO-1 axis affects LPS induced mouse BV2 microglial cells and mouse hippocampal HT22 cells, with impact on neuroinflammation. Upregulation of HO-1 expression via Nrf2 pathway in mouse BV2 microglial cells which defend cell death of mouse hippocampal HT22 cells [38]. Furthermore, cobalt-based hybrid molecules (HYCOs) that combine an Nrf2 inducer with a releaser of carbon monoxide (CO) increases Nrf2/HO-1 expression, liberate CO and exert anti-inflammatory activity in vitro. HYCOs also up-regulate tissue HO-1 and deliver CO in blood after administration in vivo, supporting their potential use against inflammatory conditions [39]. Nrf2/HO-1 upregulation reduces inflammation by increasing the efferocytic activity of murine macrophages treated with taurine chloramines [40]. Altogether, the above-explained experimental models revealed that Nrf2/HO-1 axis plays a major role in anti-inflammatory function, suggesting that Nrf2 is a therapeutic target in inflammation-associated diseases.

In addition, the byproducts of HO-1 such as CO, bilirubin, acts as a powerful antioxidant during oxidative stress and cell damage [41], [42]; it suppresses autoimmune encephalomyelitis and hepatitis [43], [44]; and it protects mice and rats against endotoxic shock by preventing the generation of iNOS and NO [45], [46], [47]. Moreover, Bilirubin reduces endothelial activation and dysfunction [48]. Interestingly, bilirubin reduces the transmigration of endothelial leukocytes via adhesion molecule-1 [49]. These specific references indicating not only HO-1 acts as a potent anti-inflammatory agent but also its metabolites.

Inflammatory Mediators and Enzymes Inhibited by Nrf2

Cytokines and Chemokines

Cytokines are low molecular-weight proteins and polypeptides secreted by a variety of cells; they regulate cell growth, differentiation, and immune function, and are involved in inflammation and wound-healing. Cytokines include interleukins (ILs), interferons, tumor necrosis factor (TNF), colony-stimulating factor, chemokines, and growth factors. Some cytokines are counted as pro-inflammatory mediators whereas others have anti-inflammatory functions. Exposure to oxidative stress results in the overproduction of cytokines which causes oxidative stress in target cells. Several pro-inflammatory cytokines are overproduced when NF-?B is activated by oxidative stress. Furthermore, pro-inflammatory oxidative stress causes further activation of NF-?B and the overproduction of cytokines. Activation of the Nrf2/ARE system plays an important role in disrupting this cycle. Chemokines are a family of small cytokines, the major role of which is to guide the migration of inflammatory cells. They function mainly as chemoattractants for leukocytes, monocytes, neutrophils, and others effector cells.

It has been reported that activation of Nrf2 prevents LPS-induced transcriptional upregulation of pro-inflammatory cytokines, including IL-6 and IL-1? [50]. IL-1? and IL-6 production is also increased in Nrf2?/? mice with dextran sulfate-induced colitis [51], [52]. Nrf2 inhibits the production of downstream IL-17 and other inflammatory factors Th1 and Th17, and suppresses the disease process in an experimental model of multiple sclerosis, autoimmune encephalitis [53]. The Nrf2-dependent anti-oxidant genes HO-1, NQO-1, Gclc, and Gclm block TNF-?, IL-6, monocyte chemo attractant protein-1 (MCP1), macrophage inflammatory protein-2 (MIP2), and inflammatory mediators. But in the case of Nrf2-knockout mice, the anti-inflammatory effect does not occur [54]. Peritoneal neutrophils from Nrf2-knockout mice treated with LPS have significantly higher levels of cytokines (TNF-? and IL-6) and chemokines (MCP1 and MIP2) than wild-type (WT) cells [54]. In vitro, transferring the Nrf2 gene to human and rabbit aortic smooth muscle cells suppresses the secretion of MCP1 [8], [55], and Nrf2-dependent HO-1 expression suppresses TNF-?-stimulated NF-?B and MCP-1 secretion in human umbilical vein endothelial cells [56]. These findings hint that, in response to inflammatory stimuli, upregulation of Nrf2 signaling inhibits the overproduction of pro-inflammatory cytokines and chemokines as well as limiting the activation of NF-?B.

Cell Adhesion Molecules

Cell adhesion molecules (CAMs) are proteins that bind with cells or with the extracellular matrix. Located on the cell surface, they are involved in cell recognition, cell activation, signal transduction, proliferation, and differentiation. Among the CAMs, ICAM-1 and VCAM-1 are important members of the immunoglobulin superfamily. ICAM-1 is present in low concentrations in leukocyte and endothelial cell membranes. Upon cytokine stimulation, the concentration significantly increases. ICAM-1 can be induced by IL-1 and TNF and is expressed by the vascular endothelium, macrophages, and lymphocytes. It is a ligand for integrin, a receptor found on leukocytes. When the ICAM-1-integrin bridge is activated, leukocytes bind to endothelial cells and then migrate into subendothelial tissues [57]. VCAM-1 mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium and contributes to leukocyte recruitment, which ultimately leads to tissue damage due to oxidative stress. Nrf2 inhibits the promotor activity of VCAM-1 [58]. The Nrf2-regulated downstream gene HO-1 can affect the expression of E-selectin and VCAM-1, adhesion molecules associated with endothelial cells [59]. The pulmonary expression of several CAMs such as CD-14, TREM1, SELE, SELP, and VCAM-1 are significantly higher in Nrf2?/? mice than in Nrf2+/+ mice [60]. Nrf2 in human aortic endothelial cells suppress TNF-?-induced VCAM-1 expression and interfere with TNF-?-induced monocytic U937 cell adhesion [8]. Overexpression of Nrf2 also inhibits TNF-?-induced VCAM-1 gene expression in human microvascular endothelial cells [61]. The naturally occurring antioxidant 3-hydroxyanthranilic acid (HA), one of l-tryptophan metabolites formed in vivo along the metabolic route known as the kynurenine pathway during inflammation or infection, is found to induce HO-1 expression and to stimulate Nrf2 in human umbilical vein endothelial cells (HUVECs). Nrf2-dependent HO-1 expression induced by HA inhibits MCP-1 secretion, VCAM-1 expression and NF-kB activation associated with vascular injury and inflammation in atherosclerosis [56]. The anti-proliferative and anti-inflammatory synthetic chalcone derivative 2?,4?,6?-tris (methoxymethoxy) chalcone inhibits ICAM-1, the pro-inflammatory cytokine IL-1?, and TNF-? expression in colonic tissue from mice treated with trinitrobenzene sulfonic acid [62]. Upregulation of Nrf2 inhibits the TNF-?-induced ICAM-1 expression in human retinal pigment epithelial cells treated with lycopene [63]. All these studies suggest that Nrf2 plays a key role in the inflammatory process by regulating the migration and infiltration of inflammatory cells to inflamed tissue.

Matrix Metalloproteinases (MMPs)

MMPs are widely present in the extracellular matrix and are involved in physiological and pathological processes such as cell proliferation, migration, differentiation, wound-healing, angiogenesis, apoptosis, and tumor metastasis. It has been reported that the Nrf2/HO-1 axis inhibits MMP-9 in macrophages and MMP-7 in human intestinal epithelial cells, and this is beneficial in the treatment of inflammatory bowel disease [62], [64]. UV irradiation-induced skin damage is more severe in Nrf2-knockout than in WT mice and the MMP-9 level is significantly higher, indicating that Nrf2 reduces MMP-9 expression. Therefore, Nrf2 is considered to be protective against UV irradiation [65]. Another study also reported that the downregulated transcriptional activation of MMP-9 in tumor cell invasion and inflammation is regulated through inhibition of the NF-kB signaling pathway [66]. In traumatic spinal cord injury, the NF-kB signaling pathway also takes part in regulating the mRNA levels of MMP-9 [67]. Therefore, in inflammation the regulation of MMPs is affected directly by the Nrf2 pathway or indirectly through the Nrf2-influenced NF-?B pathway.

Cyclooxygenase-2 (COX2) and Inducible Nitric Oxide Synthase (INOS)

A series of experiments on Nrf2-knockout mice have demonstrated its crucial role in inflammation and the regulation of pro-inflammatory genes such as COX-2 and iNOS. For the first time, Khor et al. reported increased expression of pro-inflammatory cytokines such as COX-2 and iNOS in the colonic tissues of Nrf2?/? mice compared with WT Nrf2+/+ mice, indicating that Nrf2 suppresses their activity [51]. Another report on pretreatment with sulforaphane, one of the well-known Nrf2 activators present in cruciferous vegetables, demonstrated its anti-inflammatory effect of inhibiting the expression of TNF-?, IL-1?, COX-2, and iNOS at both the mRNA and protein levels in primary peritoneal macrophages from Nrf2+/+ mice compared with those from Nrf2?/? mice [68]. Similarly, the hippocampus of Nrf2-knockout mice with LPS-induced inflammation also shows higher expression of inflammation markers such as iNOS, IL-6, and TNF-? than WT mice [69]. Likewise, Nrf2-knockout mice are hypersensitive to the oxidative stress induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as well as showing increased mRNA and protein levels of inflammation markers such as COX-2, iNOS, IL-6, and TNF-? [70]. Moreover, livers from Nrf2?/? mice challenged with a methionine- and choline-deficient diet have ~ 5-fold higher mRNA expression of Cox2, and iNOS than those from WT mice on the same diet, suggesting an anti-inflammatory role of Nrf2 [71]. Recently, Kim et al. demonstrated that the phytochemical ethyl pyruvate exerts its anti-inflammatory and anti-oxidative effects by decreasing the expression of iNOS through Nrf2 signaling in BV2 cells. They showed that ethyl pyruvate induces the nuclear translocation of Nrf2, which ultimately inhibits the interaction between p65 and p300, leading to decreased expression of iNOS [72]. Furthermore, the carbazole analogue LCY-2-CHO activates Nrf2 and causes its nuclear translocation, leading to the suppression of COX2 and iNOS expression [73] in rat aortic vascular smooth muscle cells.

Paradoxical Role of Nrf2 in the Regulation of NLRP3 iIflammasome�Activity

The NLR family, pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein complex that functions as a pathogen recognition receptor (PRR) and recognizes the wide range of microbial, oxidative stress signals such as pathogen-associated molecular patterns (PAMPs), Damage-associated molecular pattern molecules (DAMPs) and ROS [74]. The activated NLRP3 inflammasome mediates the cleavage of caspase-1 and secretion of pro-inflammatory cytokine interleukin-1? (IL-1?) that ultimately induces the process of cell death known as pyroptosis that protects hosts against a wide range of pathogens [75]. However, aberrant activation of the inflammasome is associated with protein misfolding diseases such as transmissible spongiform encephalopathies, Alzheimer’s disease, Parkinson’s disease and also type 2 diabetes [76], cancer [77], gout, and atherosclerosis [78].

A recent observation from Rong Hu group on association of Nrf2 with negative regulation of inflammasome revealed that, Nrf2 induces the NQO1 expression that leads to the inhibition of NLRP3 inflammasome activation, caspase-1 cleavage and IL-1? generation in macrophages. Furthermore, a well known Nrf2 activator, tert-butylhydroquinone (tBHQ) negatively regulated NLRP3 transcription by activating the ARE by Nrf2-dependent manner [79]. In addition to the above observation, the same group has also been revealed that, dimethyl fumarate (DMF) prevents DSS-induced colitis via activating Nrf2 signaling pathway which is involved in Nrf2 nuclear translocation and inhibition of NLRP3 inflammasome assembly [80].

A series of experiments using natural and synthetic compounds have also revealed the inhibitory effect of Nrf2 on NLRP3 inflammasome activation. For instance, treatment of epigallocatechin-3-gallate (EGCG) in lupus nephritis mice has shown to decreasing renal NLRP3 inflammasome activation which is mediated by Nrf2 signaling pathway [81]. Likewise, citral (3,7-dimethyl-2,6-octadienal), a major active compound in a Chinese herbal medicine Litsea cubeba, inhibits the NLRP3 inflammasome activation via Nrf2 antioxidant signaling pathway in Accelerated and Severe Lupus Nephritis (ASLN) mouse model [82]. Similarly, biochanin protected against LPS/GalN-induced liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation in male BALB/c mice [83]. Furthermore, mangiferin was also shown to up-regulate the expression of Nrf2 and HO-1 in a dose-dependent manner and inhibited LPS/D-GalN-induced hepatic NLRP3, ASC, caspase-1, IL-1? and TNF-? expression [84].

Despite the negative regulation of NLRP3 by Nrf2, it also activates the NLRP3 and AIM2 inflammasome function. Haitao Wen and colleagues discovered that, Nrf2 ?/? mouse macrophages have shown the defective activation of the NLRP3 and AIM2 Inflammasome but not the NLRC4 inflammasome [85]. Interestingly, this observation is depicting the unknown functions of Nrf2 in the context of inflammation associated diseases; hence it is very important to study further to reveal the mechanism in which Nrf2 activates the inflammasome function before considering it as a therapeutic target.

Suppression of Pro-Inflammatory Cytokine Transcription by Nrf2

A very recent investigation based on chromatin immunoprecipitation (ChIP)-seq and ChIP-qPCR results in mouse macrophages revealed that Nrf2 binds to the promoter regions of pro-inflammatory cytokines such as IL-6 and IL-1? and inhibits RNA Pol II recruitment. As a result, RNA Pol II is unable to process the transcriptional activation of IL-6 and IL-1? that ultimately leads to the inhibition of gene expression. For the first time, Masayuki Yamamoto’s group revealed the novel mechanism by which Nrf2 not only transactivates its downstream genes through AREs but also suppresses the transcriptional activation of specific genes with or without an ARE through inhibiting the recruitment of RNA Pol II [50].

Crosstalk Between Nrf2 and NF-?B Pathways

NF-?B is a protein complex responsible for DNA transcription found in almost all types of animal cells and involved in various processes such as inflammation, apoptosis, the immune response, cell growth, and development. p65, a Rel protein of the NF-?B family, has a transactivation domain whereas p50 does not and requires heterodimerization with Rel protein to activate transcription. During oxidative stress, I?B kinase (IKK) is activated and causes the phosphorylation of I?B, resulting in the release and nuclear translocation of NF-?B. NF-?B causes the transcription of pro-inflammatory mediators such as IL-6, TNF-?, iNOS, IL-1, and intracellular adhesion COX-2.

Abnormal regulation of NF-?B has been connected to rheumatoid arthritis, asthma, inflammatory bowel disease, and Helicobacter pylori infection-induced gastritis [86]. It is currently considered that NF-kB activity influences the Keapl/Nrf2/ARE signaling pathway mainly in three aspects: first, Keap1 degrades IKK? through ubiquitination, thus inhibiting the activity of NF-?B [87]. Second, the inflammatory process induces inflammatory mediators like COX2 derived from the cyclopentenone prostaglandin 15d-PGJ2, a strong electrophile that reacts with Keap1 and activates Nrf2, thus initiating gene transcription with simultaneous inhibition of NF-kB activity [58], [88] (Fig. 3 A, B). Third, NF-?B can combine with the competitive Nrf2 transcriptional co-activator CBP [89], [90] (Fig. 3 C, D).

Figure 3 Crosstalk between the Nrf2 and NF-?B pathways. (A) Keap1 directs the IKK to CUL3-mediated ubiquitination and proteasome degradation which ultimately leads to the inhibition of NF-?B phosphorylation and this mechanism also works as competitive binding of Nrf2 and IKK with Keap1. (B) Oxidative stress activates IKK which phosphorylates NF-?B, leading to its translocation into the nucleus and activation of proinflammatory cytokines such as COX-2. The terminal product of COX-2 known as 15d-PGJ2 acts as an inducer of Nrf2 that ultimately leads to the suppression of oxidative stress. (C) Nrf2 binds with its transcriptional cofactor CBP along with small Maf and other transcriptional machinery to initiate ARE-driven gene expression. (D) When NF-?B binds with CBP in a competitive manner, it inhibits the binding of CBP with Nrf2, which leads to the inhibition of Nrf2 transactivation.

It is assumed that the Nrf2 and NF-?B signaling pathways interact to control the transcription or function of downstream target proteins. In justification of this assumption many examples show that direct or indirect activation and inhibition occur between members of the Nrf2 and NF-?B pathways (Fig. 4). In response to LPS, Nrf2 knockdown significantly increases the NF-?B transcriptional activity and NF-?B-dependent gene transcription, showing that Nrf2 impedes NF-?B activity [60], [91]. In addition, increased expression of Nrf2-dependent downstream HO-1 inhibits NF-?B activity. When prostate cancer cells are briefly exposed to ?-tochopheryl succinate, a derivative of vitamin E, HO-1 expression is upregulated. The end-products of HO-1 inhibit the nuclear translocation of NF-?B [92]. These in vivo studies suggest that Nrf2 negatively regulates the NF-kB signaling pathway. LPS stimulates NF-?B DNA binding activity and the level of the p65 subunit of NF-?B is significantly higher in nuclear extracts from the lungs of Nrf2?/? than from WT mice, suggesting a negative role of Nrf2 in NF-?B activation. Moreover, Nrf2?/? mouse embryo fibroblasts treated with LPS and TNF-? show more prominent NF-?B activation caused by IKK activation and I?B-? degradation [60]. And respiratory syncytial virus clearance is significantly decreased while NF-?B DNA-binding activity is increased in Nrf2?/? mice compared with WT mice [93]. Pristane-induced lupus nephritis in Nrf2?/? mice co-treated with sulforaphane have severe renal damage and pathological alterations as well as elevated iNOS expression and NF-?B activation compared to the WT, suggesting that Nrf2 improves lupus nephritis by inhibiting the NF-?B signaling pathway and clearing ROS [94]. NF-?B activity also occurs when cells are treated with an Nrf2 inducer together with LPS and TNF-?. For example, a synthetic chalcone derivative inhibits TNF-?-induced NF-?B activation both directly and indirectly and partly through the induction of HO-1 expression in human intestinal epithelial HT-29 cells [62]. Suppression of NF-?B translocation and DNA-binding activity as well as the suppression of iNOS expression in hepatocytes are found when F344 rats are treated with 3H-1,2-dithiole-3-thione (D3T) [95]. After co-treatment with sulforaphane and LPS, the LPS-induced expression of iNOS, COX-2, and TNF-? in Raw 264.7 macrophages is downregulated, suggested that sulforaphane has anti-inflammatory activity via inhibition of NF-?B DNA binding [96]. Though several experimental studies have been done to explain the link between the Nrf2 and NF-?B pathways, conflicting results remain. Both positive and negative regulations have been reported between Nrf2 and NF-kB [97]. Usually, chemopreventive electrophiles 3H-1,2-dithiole-3-thione, sulforaphane and Triterpenoid CDDO-Me activate Nrf2 by inhibiting NF-kB and its downregulated genes [98], [99], [100]. In contrast, several agents or conditions such as ROS, LPS, flow shear stress, oxidized LDL, and cigarette smoke have been shown to increase both Nrf2 and NF-kB activity [97]. In addition, in vivo studies have revealed that NF-kB activity is decreased in livers isolated from Nrf2?/? mice and NF-?B binding activity is lower in Nrf2?/? than in Nrf2+/+ mice [101]. However, human aortic endothelial cells treated with adenoviral vector Nrf2 inhibit NF-?B downstream genes without affecting the activity of NF-?B [8]. Therefore, crosstalk between the Nrf2 and NF-?B pathways needs further investigation.

Figure 4 Regulatory loop of Nrf2 and NF-?B. The Nrf2 pathway inhibits NF-?B activation by preventing the degradation of I?B-? and increasing HO-1 expression and antioxidant defenses which neutralize ROS and detoxifying chemicals. As a result, ROS-associated NF-?B activation is suppressed. Likewise, NF-?B-mediated transcription reduces Nrf2 activation by reducing�ARE�gene transcription and free CREB binding protein by competing with Nrf2 for CBP. Moreover, NF-?B increases the recruitment of histone deacetylase (HDAC3) to the ARE region and hence Nrf2 transcriptional activation is prevented.
Dr Jimenez White Coat
The activation of the Nrf2 signaling pathway plays a major role in the expression of enzymes and genes involved in the detoxification of reactive oxidants by enhancing the antioxidant capacity of the cells in the human body. While many research studies are available today, the regulatory mechanisms in Nrf2 activation are not fully understood. A possible role of the Nrf2 signaling pathway in the treatment of inflammation has also been found. Dr. Alex Jimenez D.C., C.C.S.T. Insight

Role of Nrf2 in Inflammatory Diseases

In vivo studies have shown that Nrf2 plays an important role in inflammatory diseases affecting different systems; these include gastritis, colitis, arthritis, pneumonia, liver damage, cardiovascular disease, neurodegenerative disease, and brain damage. In these studies, Nrf2?/? animals showed more severe symptoms of inflammation and tissue damage than WT animals. Therefore, it is believed that the Nrf2 signaling pathway has a protective effect in inflammatory diseases. Intra-tracheal installation of porcine pancreatic elastase induces chronic obstructive pulmonary disease, particularly emphysema. Nrf2-deficient mice are highly susceptible to emphysema, and decreased expression of HO-1, PrxI, and the antiprotease gene SLPI occur in alveolar macrophages. Nrf2 is considered to be a key regulator in the macrophage mediated defense system against lung injury [102]. Nrf2-deficient mice with emphysema induced by tobacco smoke exposure for 6 months show increased bronchoalveolar inflammation, upregulated expression of oxidative stress markers in alveoli, and increased alveolar septal cell apoptosis, suggesting that Nrf2 acts against tobacco-induced emphysema through the increased expression of antioxidant genes [102], [103]. With Nrf2 disruption, allergen-mediated airway inflammation and asthma using ovalbumin complex show increased airway inflammation, airway hyper-reactivity, hyperplasia of goblet cells, and high levels of Th2 in bronchoalveolar lavage and splenocytes, whereas the Nrf2-mediated signaling pathway limits airway eosinophilia, mucus hypersecretion, and airway hyper-reactivity as well as inducing many antioxidant genes that prevent the development of asthma [104]. Carrageenan injection into the pleural cavity induces pleurisy, and 15d-PGJ2 accumulation in Nrf2 inflammatory cells is confined to mouse peritoneal macrophages. During the early phase of inflammation, 15d-PGJ2 activates Nrf2 and regulates the inflammatory process via the induction of HO-1 and PrxI. A study also suggested that COX-2 has an anti-inflammatory effect in the early phase by the production of 15d-PGJ2 [105]. Oral administration of 1% dextran sulfate sodium for 1 week induces colitis associated with histological alterations that include shortening of crypts and infiltration of inflammatory cells in colon tissue. To protect intestinal integrity in colitis, Nrf2 could play an important role by regulating pro-inflammatory cytokines and inducing phase II detoxifying enzymes [51]. In an Nrf2-knockout mouse model of LPS-induced pulmonary sepsis, NF-?B activity regulates the influence of inflammatory cytokines such as COX-2, IL-113, IL-6, and TNF? which are essential for initiating and promoting inflammation [60]. Nrf2 reduces inflammatory damage by regulating these inflammatory factors. In these models of acute inflammation, the increased regulation of antioxidant enzymes, pro-inflammatory cytokines, and mediators by the Nrf2 signaling pathway reduces the inflammatory injury in WT animals. Interestingly, this has also been reported in Nrf2-knockout mice in which the symptoms are markedly exacerbated compared with WT mice. Nrf2-related inflammatory diseases are summarized (Table 1).

Research on Nrf2-Dependent Anti-Inflammatory Drugs

In summary, we have discussed experiments showing that the Nrf2 signal pathway plays a regulatory role in many areas of inflammation, so Nrf2-dependent anti-inflammatory agents are important for the treatment of inflammatory diseases.

Plants have been extraordinarily rich sources of compounds that activate Nrf2 transcription factor, leading to the up-regulation of cytoprotective genes. Recently, several studies were conducted to investigate the effects of different anti-inflammatory agents, mostly of plant origin. For example, curcumin is the active ingredient of turmeric and is also found in small amounts in ginger; isothiocyanates, specifically phenylisothiocyanates are from broccoli, celery, and other vegetables; and anthocyanins are from berries and grapes [124]. Studies have shown that all these agents are not only good antioxidants but also have potent anti-inflammatory effects via Nrf2 induction [125], [126]. Therefore, the development of new anti-inflammatory Nrf2 activators from plant extract has attracted much interest in medical research.

In recent years, many animal experiments have been conducted to confirm the actions of these compounds. Artesunate is used mainly for severe malaria, cerebral malaria, and rheumatic autoimmune diseases; it is also effective in septic lung injury. Artesunate activates Nrf2 and HO-1 expression, and the latter reduces the inflow of pro-inflammatory cytokines and leukocytes into tissue to prevent inflammation [127]. Isovitexin, extracted from the hulls of Oryza sativa rice, is thought to have anti-inflammatory and antioxidant properties; it plays a protective role against LPS-induced acute lung injury by activating the Nrf2/HO-1 pathway and inhibiting MAPK and NF-?B [128]. Fimasartan, a newly popular angiotensin II receptor blocker acting on the renin-angiotensin system, reduces blood pressure; using fimasartan to treat mice with surgically-induced unilateral ureteral obstruction reduces oxidative stress, inflammation, and fibrosis via upregulating Nrf2 and the antioxidant pathway and inhibiting RAS and MAPKs [129]. Sappanone is widely distributed in Southeast Asia, where it is used as an anti-influenza, anti-allergic, and neuroprotective medication; it activates Nrf2 and inhibits NF-?B and so may be beneficial in the treatment of Nrf2- and/or NF-?B-related diseases [130]. Bixin extracted from the seeds of Bixin orellana is used for infectious and inflammatory diseases in Mexico and South America; it decreases inflammatory mediators, alveolar capillary leakage, and oxidative damage in an Nrf2-dependent manner to alleviate ventilation-induced lung injury and restore normal lung morphology [131]. Other plant compounds, such as epigallocatechin gallate, sulforaphane, resveratrol, lycopene, and green tea extract have therapeutic effects on inflammatory diseases through the Nrf2 signaling pathway [132], [133], [134]. Recently another phytochemical, eriodictyol, which is present in citrus fruit, has been reported to have anti-inflammatory and antioxidant effects on cisplatin-induced kidney injury and sepsis-induced acute lung injury by regulating Nrf2, inhibiting NF-?B, and inhibiting the expression of cytokines in macrophages [135], [136]. However, numerous phytochemicals show great promise for the prevention and treatment of various human diseases, and some have already entered the clinical trials stage (Table 2).

These plant compounds activate the Nrf2 signaling pathway mainly in the form of electrophilic materials that modify the cysteine residues of Keap1, leading to free nuclear Nrf2 binding with the ARE, resulting in activation of transcription of the corresponding gene.

Sulforaphane and Its Effects on Cancer, Mortality, Aging, Brain and Behavior, Heart Disease & More

Isothiocyanates are some of the most important plant compounds you can get in your diet. In this video I make the most comprehensive case for them that has ever been made. Short attention span? Skip to your favorite topic by clicking one of the time points below. Full timeline below.

Key sections:

  • 00:01:14 – Cancer and mortality
  • 00:19:04 – Aging
  • 00:26:30 – Brain and behavior
  • 00:38:06 – Final recap
  • 00:40:27 – Dose

Full timeline:

  • 00:00:34 – Introduction of sulforaphane, a major focus of the video.
  • 00:01:14 – Cruciferous vegetable consumption and reductions in all-cause mortality.
  • 00:02:12 – Prostate cancer risk.
  • 00:02:23 – Bladder cancer risk.
  • 00:02:34 – Lung cancer in smokers risk.
  • 00:02:48 – Breast cancer risk.
  • 00:03:13 – Hypothetical: what if you already have cancer? (interventional)
  • 00:03:35 – Plausible mechanism driving the cancer and mortality associative data.
  • 00:04:38 – Sulforaphane and cancer.
  • 00:05:32 – Animal evidence showing strong effect of broccoli sprout extract on bladder tumor development in rats.
  • 00:06:06 – Effect of direct supplementation of sulforaphane in prostate cancer patients.
  • 00:07:09 – Bioaccumulation of isothiocyanate metabolites in actual breast tissue.
  • 00:08:32 – Inhibition of breast cancer stem cells.
  • 00:08:53 – History lesson: brassicas were established as having health properties even in ancient Rome.
  • 00:09:16 – Sulforaphane’s ability to enhance carcinogen excretion (benzene, acrolein).
  • 00:09:51 – NRF2 as a genetic switch via antioxidant response elements.
  • 00:10:10 – How NRF2 activation enhances carcinogen excretion via glutathione-S-conjugates.
  • 00:10:34 – Brussels sprouts increase glutathione-S-transferase and reduce DNA damage.
  • 00:11:20 – Broccoli sprout drink increases benzene excretion by 61%.
  • 00:13:31 – Broccoli sprout homogenate increases antioxidant enzymes in the upper airway.
  • 00:15:45 – Cruciferous vegetable consumption and heart disease mortality.
  • 00:16:55 – Broccoli sprout powder improves blood lipids and overall heart disease risk in type 2 diabetics.
  • 00:19:04 – Beginning of aging section.
  • 00:19:21 – Sulforaphane-enriched diet enhances lifespan of beetles from 15 to 30% (in certain conditions).
  • 00:20:34 – Importance of low inflammation for longevity.
  • 00:22:05 – Cruciferous vegetables and broccoli sprout powder seem to reduce a wide variety of inflammatory markers in humans.
  • 00:23:40 – Mid-video recap: cancer, aging sections
  • 00:24:14 – Mouse studies suggest sulforaphane might improve adaptive immune function in old age.
  • 00:25:18 – Sulforaphane improved hair growth in a mouse model of balding. Picture at 00:26:10.
  • 00:26:30 – Beginning of brain and behavior section.
  • 00:27:18 – Effect of broccoli sprout extract on autism.
  • 00:27:48 – Effect of glucoraphanin on schizophrenia.
  • 00:28:17 – Start of depression discussion (plausible mechanism and studies).
  • 00:31:21 – Mouse study using 10 different models of stress-induced depression show sulforaphane similarly effective as fluoxetine (prozac).
  • 00:32:00 – Study shows direct ingestion of glucoraphanin in mice is similarly effective at preventing depression from social defeat stress model.
  • 00:33:01 – Beginning of neurodegeneration section.
  • 00:33:30 – Sulforaphane and Alzheimer’s disease.
  • 00:33:44 – Sulforaphane and Parkinson’s disease.
  • 00:33:51 – Sulforaphane and Hungtington’s disease.
  • 00:34:13 – Sulforaphane increases heat shock proteins.
  • 00:34:43 – Beginning of traumatic brain injury section.
  • 00:35:01 – Sulforaphane injected immediately after TBI improves memory (mouse study).
  • 00:35:55 – Sulforaphane and neuronal plasticity.
  • 00:36:32 – Sulforaphane improves learning in model of type II diabetes in mice.
  • 00:37:19 – Sulforaphane and duchenne muscular dystrophy.
  • 00:37:44 – Myostatin inhibition in muscle satellite cells (in vitro).
  • 00:38:06 – Late-video recap: mortality and cancer, DNA damage, oxidative stress and inflammation, benzene excretion, cardiovascular disease, type II diabetes, effects on the brain (depression, autism, schizophrenia, neurodegeneration), NRF2 pathway.
  • 00:40:27 – Thoughts on figuring out a dose of broccoli sprouts or sulforaphane.
  • 00:41:01 – Anecdotes on sprouting at home.
  • 00:43:14 – On cooking temperatures and sulforaphane activity.
  • 00:43:45 – Gut bacteria conversion of sulforaphane from glucoraphanin.
  • 00:44:24 – Supplements work better when combined with active myrosinase from vegetables.
  • 00:44:56 – Cooking techniques and cruciferous vegetables.
  • 00:46:06 – Isothiocyanates as goitrogens.

Conclusions

Currently, much research has focused on the role of the Nrf2/Keap1/ARE signaling pathway in inflammation. Among the enzymes upregulated by Nrf2, HO-1 is one of the representative stress response enzymes. HO-1 has prominent anti-inflammatory and antioxidant properties. In general, the Nrf2 signaling pathway also negatively regulates cytokines, chemokine releasing factors, MMPs, and other inflammatory mediators COX-2 and iNOS production, which directly or indirectly affect the relevant NF-kB and MAPK pathways and other networks that control inflammation. It is suggested that the Nrf2 and NF-?B signaling pathways interact to regulate the transcription or function of downstream target proteins. Suppression or inactivation of NF-?B-mediated transcriptional activity through Nrf2 most probably occurs in the early phase of inflammation, as NF-?B regulates the de novo synthesis of an array of pro-inflammatory mediators. However, there are still some limitations in the research such as whether there are connections between Nrf2 and other signaling pathways such as JAK/STAT, the significance of the current Nrf2 activators derived from natural plant sources in inflammation, and how to improve the biological activity and enhance the targeting of these compounds. These require further experimental validation.

In addition, the Nrf2 signaling pathway can regulate > 600 genes [163], of which > 200 encode cytoprotective proteins [164] that are also associated with inflammation, cancer, neurodegenerative diseases, and other major diseases [165]. Growing evidences suggesting that, Nrf2 signaling pathway is deregulated in many cancers, resulting in aberrant expression Nrf2 dependent gene battery. Moreover, inflammation plays a major role in oxidative stress related diseases especially in cancer. Application of several Nrf2 activators to counteract the inflammation may result in aberrant expression of Nrf2 downstream genes which induces oncogenesis and resistance to chemo and/or radio therapy. Therefore, highly specific activators of Nrf2 may be developed to minimize its pleiotropic effects. Several activators of Nrf2 have shown a significant improvement of the anti-inflammatory functions in oxidative stress related diseases. The best example of Nrf2 activator approved by FDA and widely used for the treatment of inflammatory disease such as Multiple sclerosis (MS) is dimethyl fumarate. Tecfidera� (registered name of dimethyl fumarate by Biogen) used effectively to treat relapsing forms of multiple sclerosis in large number of patients [152]. However, the efficacy of using Nrf2 activators to treat inflammatory diseases requires further validation to avoid the deleterious effects of Nrf2. Therefore, development of therapies for the anti-inflammation activity mediated by Nrf2 could have significant clinical impact. Ongoing studies of the Nrf2 signaling pathway around the world are devoted to developing highly-targeted therapeutic agents to control the symptoms of inflammation, and to prevent and treat cancer as well as neurodegenerative and other major diseases.

Acknowledgments

Sciencedirect.com/science/article/pii/S0925443916302861#t0005

In conclusion, Nrf2 senses the levels of oxidative stress in the human body and ultimately helps promote the regulation of antioxidant and detoxifying enzymes and genes. Because chronic inflammation caused by increased levels of oxidative stress has been associated with neurodegenerative diseases, Nrf2 can play an essential role in the treatment of health issues like Alzheimer’s disease, among others. The scope of our information is limited to chiropractic and spinal health issues. To discuss the subject matter, please feel free to ask Dr. Jimenez or contact us at�915-850-0900�.

Curated by Dr. Alex Jimenez

Referenced from: Sciencedirect.com

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Additional Topic Discussion: Relieving Knee Pain without Surgery

Knee pain is a well-known symptom which can occur due to a variety of knee injuries and/or conditions, including�sports injuries. The knee is one of the most complex joints in the human body as it is made-up of the intersection of four bones, four ligaments, various tendons, two menisci, and cartilage. According to the American Academy of Family Physicians, the most common causes of knee pain include patellar subluxation, patellar tendinitis or jumper’s knee, and Osgood-Schlatter disease. Although knee pain is most likely to occur in people over 60 years old, knee pain can also occur in children and adolescents. Knee pain can be treated at home following the RICE methods, however, severe knee injuries may require immediate medical attention, including chiropractic care. �

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EXTRA EXTRA | IMPORTANT TOPIC: Recommended El Paso, TX Chiropractor

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