NMN Plus – Dosage Recommendations


We include a very small dose of Niacin in the formula for NMN PLUS.

NIACIN  is well known to cause an uncomfortable “Niacin Flush in some people.  

We used a dosage that is about 1/2 of what  research shows affects less than 1% of users.  Read more about this, and what you can do to minimize niacin flush.

In addition to dosage, avoid taking NMN PLUS on an empty stomach to further minimize the chance you might experience a flush.

After the first trial or 2, you can increase to 2 capsules at a time if you desire.

Tolerance to Niacin Flush builds up rapidly. The few people that do experience it find it diminishes and no longer occurs after 4-7 days of continued use with a particular dosage, and can be increased further.

For those that want ONLY NMN, we offer  NMN PURE, which contains 125 mg of NMN per capsule and nothing else, but that product will not be available to ship until Dec 8, 2017.

Time of Day

Humans have a natural Circadian rhythm with a peak NAD+ levels around Noon, and a second, smaller peak in the middle of the night.

It is not yet known if supplementing with 2 doses to emulate the 2 natural peaks is beneficial, or, just one peak in the middle of the day. Experts such as Dr Brenner, Dr Sinclair, and others are on both sides of this issue.

If you chose to take NMN Plus twice a day, we recommend:

  • 1 Capsule between 8-10am
  • 1 Capsule before bedtime

If you chose to take NMN Plus once per day, we recommend

  • 1 Capsule between 8-10 am, until you are sure you don’t experience Niacin Flush
  • after acclimated for a few days

  • 2 Capsules between 8-10 am


In addition to NMN, which is the  IMMEDIATE PRECURSOR used by our bodies to produce NAD+, we include the other 3  major precursors:

  • NAM (Nicotinamide)
  • Tryptophan
  • NA (Niacin)

Obviously, it would be easier to produce only the single ingredient NMN PURE, but we are so convinced the proven NAD+ boosting ability of the secondary precursors make this product more effective, we have gone many months of research, development, and testing, in addition to the expense, to offer this Complete NAD+ Booster.

to read more about NMN  and other NAD+ precursors  Click here

to read more about NAD+  Click here

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Best Anti-aging supplements other than NR and NMN

22 Best Anti-Aging Supplements

Geroprotectors are substances that support healthy aging, slow aging, or extend healthy life. Sometimes people refer to them as “aging suppressants,” “anti-aging drugs,” “gerosuppressants,” “longevity therapeutics,” “senolytics,” or “senotherapeutics.” They include various foods, nutraceuticals (supplements), and pharmaceuticals (drugs). Unfortunately none comes close to realizing the age-old aspiration of ending aging altogether (yet), but some may make a practical difference for many people.

I’ve used several geroprotectors for years. And I’m exploring ways to incorporate others into my diet, if they’re applicable to my personal situation and meet a few general criteria:

First, I look for geroprotectors supported by multiple studies on humans – not just anecdotal evidence, one study, or studies on non-human animals. Although I’ve nothing against the health benefits of placebo, I prefer knowing that something more than only placebo is at work.

Second, I look for geroprotectors with the highest ratios of efficacy to expense. Given innumerable options and a limited budget, I want to do more than just empty my wallet.

Third, I look for geroprotectors that are legal and generally safe. If it’ll put me in a hospital or a prison, it’s not worth it.

Based on those criteria, I’ve compiled a list of top tier natural geroprotectors. These are, to the best of my knowledge, the most well-researched and effective geroprotectors available in the United States without a prescription. I’ve excluded from this list any geroprotectors that are primarily nootropic geroprotectors (such as ginkgo and melatonin), which you can find in my list of top tier nootropics. This information is for educational purposes only. It is not medical advice. Please consult a physician before and during use of these and other geroprotectors.

1) Berberine


Berberine is a compound of extracts from herbs such as barberry. Supplementation may provide a strong decrease to blood glucose, and a notable decrease to total cholesterol, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Berberine may also provide a subtle increase to HDL-C; and a subtle decrease to insulin, LDL-C, and triglycerides. Evidence for these effects may not be as reliable. See the Berberine article  for more studies and details.

2) Blueberry


Blueberry is the fruit of a perennial flowering plant native to North America. Supplementation may provide a notable decrease to DNA damage, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

See the Blueberry article at Examine.com for more studies and details.

3) Boswellia Serrata (Frankincense)


Boswellia Serrata is a plant native to India and Pakistan. Supplementation may provide notable support for long-term joint function, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

See the Boswellia Serrata article at Examine.com for more studies and details.

4) Cocoa


Cocoa comes from the seeds of evergreen trees native to tropical regions of Central and South America. Supplementation may provide a notable increase to blood flow, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Cocoa may also provide a subtle increase to insulin sensitivity, and photoprotection; and a subtle decrease to general oxidation, platelet aggregation, and LDL-C. Evidence for these effects may not be as reliable.

5) Coenzyme Q10

Coenzyme Q10

Coenzyme Q10 is a molecule found in the mitochondria of humans and other organisms. Supplementation may provide a notable decrease to lipid peroxidation, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Coenzyme Q10 may also provide a subtle increase to blood flow, endothelial function, and exercise capacity; and a subtle decrease to blood pressure, exercise-induced oxidation, and general oxidation. Evidence for these effects may not be as reliable. See the Coenzyme Q10 article at Examine.com for more studies and details.

6) Creatine


Creatine is a nitrogenous organic acid that occurs naturally in vertebrates. Supplementation may provide a strong increase to power output and a notable increase to hydration, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Creatine may also provide a subtle increase to anaerobic running capacity, lean mass, bone mineral density, muscular endurance, testosterone, VO2 max, and glycogen resynthesis; and a subtle decrease to blood glucose, lipid peroxidation, and muscle damage. Evidence for these effects may not be as reliable. See the Creatine article at Examine.com for more studies and details.

7) Curcumin


Curcumin is the bioactive in Turmeric, which is a perennial plant native to Southern Asia. Supplementation may provide a notable increase to antioxidant enzyme profile and a notable decrease to inflammation and pain, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Curcumin may also provide a subtle increase to HDL-C, and functionality in the elderly or injured; a subtle decrease to blood pressure, general oxidation, lipid peroxidation, and triglycerides; and subtle support for long-term joint function. Evidence for these effects may not be as reliable. See the Curcumin article for more studies and details.

8) DHEA (Dehydroepiandrosterone)


DHEA is a natural hormone in humans and other animals. Supplementation may provide a notable increase to estrogen or testosterone (depending on the need of the body), according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

See the Dehydroepiandrosterone article at Examine.com for more studies and details.

9) Fish Oil


Fish Oil, as the name suggests, is an oil that accumulates in the tissues of some fish species. Supplementation may provide a strong decrease to triglycerides, thereby supporting a healthy cardiovascular system, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Fish Oil may also provide a subtle increase HDL-C, endothelial function, and photoprotection; and a subtle decrease to blood pressure, inflammation, natural killer cell activity, platelet aggregation, and LDL-C. Evidence for these effects may not be as reliable. See the Fish Oil article at Examine.com for more studies and details.

10) Garlic


Garlic is a bulbous plant native to Central Asia. Supplementation may provide a notable increase to HDL-C and a notable decrease to LDL-C, total cholesterol, and blood pressure, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Garlic may also provide a subtle decrease to triglycerides and a strong decrease to rate of sickness. Evidence for these effects may not be as reliable. See the Garlic article at Examine.com for more studies and details.

11) Horse Chestnut (Aesculus Hippocastanum)

Horse Chestnut

Horse Chestnut is a deciduous flowering tree native to South East Europe. Supplementation may provide notable support to long-term circulatory function, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Horse Chestnut may also provide a subtle decrease to pain. Evidence for this effect may not be as reliable. See the Horse Chestnut article at Examine.com for more studies and details.

12) Magnesium


Magnesium is an essential dietary mineral found in food like nuts, cereals, and vegetables. Supplementation may provide a notable decrease to blood pressure (only in cases of high blood pressure), according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Magnesium may also provide a subtle increase to insulin sensitivity, aerobic exercise, and muscle oxygenation; and a subtle decrease to blood glucose, and insulin. Evidence for these effects may not be as reliable. See the Magnesium article at Examine.com for more studies and details. also check out my article on Magnesium Glycinate supplementation. Magnesium is an ingredient in Thrivous Serenity.

13) Nitrate


Nitrate is a molecule produced in the body in small amounts and available in vegetables like beetroot. Supplementation may provide a notable decrease to blood pressure, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Nitrate may also provide a notable increase to anaerobic running capacity; and a notable decrease to oxygenation cost of exercise. Evidence for these effects may not be as reliable.

14) Olive Leaf

Olive Leaf

Olive Leaf comes from an evergreen tree native to the Mediterranean, Africa, and Asia. Supplementation may provide a notable decrease to blood pressure and oxidation of LDL, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Olive Leaf may also provide a subtle increase to HDL-C; and a subtle decrease to LDL-C, total cholesterol, cell adhesion factors, and DNA damage. Evidence for these effects may not be as reliable. See the Olive Leaf Extract article at Examine.com for more studies and details.

15) Pycnogenol (Pine Bark)

Maritime Pine

Pycnogenol is an extract from bark of the maritime pine, native to the Mediterranean. Supplementation may provide a notable increase to blood flow, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Pycnogenol may also provide a subtle decrease to leg swelling; and subtle support for long-term joint function. Evidence for these effects may not be as reliable. See the Pycnogenol article at Examine.com for more studies and details.

16) Salacia Reticulata

Salacia Reticulata

[“Kothala Himbutu” by Satheesan.vn under CC BY-SA 3.0 / cropped]

Salacia Reticulata is a plant native to the forests of Sri Lanka. Supplementation may provide a notable decrease to blood glucose and insulin, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

See the Salacia Reticulata article at Examine.com for more studies and details.

17) SAMe (S-Adenosyl Methionine)


SAMe is a naturally-occurring compound found in most tissues and fluids of the human body. Supplementation may provide notable support for long-term joint function, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with SAMe may also provide a subtle increase to functionality in elderly or injured; and a notable decrease to pain. Evidence for these effects may not be as reliable. See the S-Adenosyl Methionine article at Examine.com for more studies and details.

18) Spirulina


[“Spirulina” by Lara Torvi under CC BY 2.0 / cropped]

Spirulina is a blue-green algae. Supplementation may provide a notable decrease to lipid peroxidation and triglycerides, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Spirulina may also provide a strong decrease to allergies, nasal congestion, and liver fat; a notable increase to power output; a notable decrease to blood pressure and general oxidation; a subtle increase to HDL-C and muscular endurance; and a subtle decrease to LDL-C and total cholesterol. Evidence for these effects may not be as reliable. See the Spirulina article at Examine.com for more studies and details.

19) TUDCA (Tauroursodeoxycholic Acid)


TUDCA is a bile acid found naturally in trace amounts in humans and in large amounts in other animals like bears. Supplementation may provide a notable decrease to liver enzymes, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with TUDCA may also provide a notable increase to insulin sensitivity. Evidence for this effect may not be as reliable. See the Tauroursodeoxycholic Acid article at Examine.com for more studies and details.

20) Vitamin B3 (Niacin)


Vitamin B3, also known as Niacin, is an essential dietary vitamin found in foods like liver, chicken, beef, fish, peanuts, cereals, and legumes. Supplementation may provide a strong increase to HDL-C and a notable decrease to LDL-C and triglycerides, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Vitamin B3 may also provide a subtle increase to blood glucose and insulin; and a subtle decrease to insulin sensitivity and vLDL-C. Evidence for some of these effects may not be as reliable. See the Vitamin B3 article at Examine.com for more studies and details.

21) Vitamin D

Vitamin D3

Vitamin D is an essential dietary vitamin naturally synthesized in the skin from sun exposure. Supplementation may provide a notable decrease to risk of falls, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Vitamin D may also provide a notable increase to functionality in elderly or injured; and a subtle decrease to blood pressure, bone fracture risk, and fat mass. Evidence for some of these effects may not be as reliable. See the Vitamin D article at Examine.com for more studies and details.

22) Vitamin K

Vitamin K1

Vitamin K is an essential dietary vitamin found in foods like leafy green vegetables and some fruits. Supplementation may provide a notable increase to bone mineral density, according to multiple peer-reviewed, double-blind, placebo-controlled studies in humans:

Supplementation with Vitamin K may also provide a notable decrease to bone fracture risk. Evidence for this effect may not be as reliable. See the Vitamin K article at Examine.com for more studies and details.

Ben Greenfield on NAD+

This is the transcript from Ben’s podcast you can listen to here:

Ben: Hey folks, it’s Ben Greenfield, and I wanna tell you about an article I recently read in Scientific American.  This article was called “Beyond Resveratrol: The Anti-Aging Nad Fad”, and NAD referring to NAD.  And what this article includes, among many other things, is, for example, quote, “Recent research suggests it may be possible to reverse mitochondrial decay with dietary supplements that increase cellular levels of a molecule called NAD.”  And there’s another part of the article that says, quote, “The mitochondria in muscles of elderly mice were restored to a youthful state after just a week of injections with NMN, a molecule that naturally occurs in cells and boosts levels of NAD.”  Well, since that article was published a few months ago, I have received an onslaught of questions about this mysterious molecule called NAD.

And it just so happens that a friend of mine, named Thomas Ingoglia, he knows one of the best NAD scientists on the face of the planet.  He’s in contact with one of the best NAD clinicians on the planet, both with decades of experience, second to none when it comes to NAD, and I actually consider Thomas himself to be one of the most knowledgeable and frequent users of NAD who I’ve ever met.  He’s one of the few guys that’s been playing around with it in combination with things like cryotherapy, and blueberry extract, and hyperbaric oxygen, and all these other biohacks that have allowed him to turn completely around from being bedridden sick and losing half his family in a car crash, to being in the best health of his life, including crushing his first Spartan race with me last year, prior to which he actually took high doses of NAD.

And the problem is a lot of NAD clinical researchers seem to mostly be underground at the moment.  The FDA doesn’t look kindly at NAD supplement companies and integrative doctors who use NAD.  They’re skeptical of naturopathy, and the first impulse is to turn these type of compounds into patentable drugs because that’s a language that the FDA speaks, and NAD can be dangerous.  I’ve spoken with Thomas and he knows a guy personally who has poisoned himself while using NAD incorrectly and hospitalized himself with the same substances we’re gonna be talking about in this podcast episode.

So, yeah.  You need to proceed with caution and with the type of formal clinical information that Thomas has opened my eyes to, and Thomas is actually on the call with me today, but I don’t just have Thomas here with me today.  Along with Thomas, first of all, we have Dr. Ross Grant, PhD., and Dr. Grant is one of the most prolific authors in the field of NAD, and he specializes in the effect of NAD on the brain.  He’s been researching it since 1994, back when nobody was doing NAD research.  He’s the clinical associate professor at the University of Sydney Medical School, and the CEO of the Australasian Research Institute, and a biochemical pharmacologist himself.  So he’s a smart cookie, and he specifically researches NAD and its role in oxidative stress, and the human cellular response to oxidative stress and how NAD affects that.  And in addition to Dr. Grant, we’re also, as if that weren’t enough, joined by Dr. Philip Milgram, who is an M.D., and Philip Milgram is based out of the NAD Treatment Center in San Diego, California, and he specifically helps people in recovery from addiction using NAD protocols.

So, between Dr. Grant, Dr. Milgram, and Thomas, we have quite a few folks who specialize in NAD.  So if you’re curious about this stuff, you are in the right place.  Now before I jump in, and I’m gonna jump in starting, with Thomas, and Thomas telling us his story and how he first kinda discovered NAD, I wanna tell you that all the show notes for everything that we’re gonna talk about, including a link to that article that first kinda sparked my own interest in this, you can find at bengreenfieldfitness.com/nad.  That’s bengreenfieldfitness.com/nad.  So, with that being said, let’s just go through here real quick so everybody can know everybody else’s voices.

Dr. Grant, welcome to the show and thank you for coming on at 4 A.M., Australian time.


Ben:  Alright.  Well, fantastic.  Well, Thomas, like I mentioned, you’re the guy who kinda first became my outlet into the wide world of NAD.  So tell me a little bit about how you first discovered NAD, and how you got to the point where you were kind of dug deep into a health hole, so to speak when it came to needing some serious healing.

Thomas:  Well, I’d like to start by saying that this all really began when I lost half my family in a car crash a few weeks after I lost them, old friends to drugs and alcohol, I had a friend who knocked on my door the day that he killed himself, and he had abused drugs, and he had severe psychological problems because of it.  But I had lost my dad, and my brother, and my nephew instantly in a head-on collision, and it was very hard for me because I was chronically ill for about seven years.  I was suffering from what doctors called Fibromyalgia and Chronic Fatigue Syndrome, and I took opiate drugs at that time.

My illness was brutal.  I spent a lot of time in bed.  I had trouble standing for periods of, let’s say, over 45 minutes.  I was very sick.  I would get headaches, there were so many different symptoms that I had, and I was very fatigued all the time.  And going to the funerals, because we had a funeral in Costa Rica, and I had to go see my mom in Hawaii where the accident took place, the car crash, it was just, it was very hard on me, and I was very angry.  And I made a point that at the funeral, to myself, that I would do anything and everything to cure myself.

Ben:  So, do you know what exactly it was that was causing you to be in a position where like you couldn’t stand up for 45 minutes, or for more than 45 minutes?  Or what exactly the infection was?

Thomas:  Well, it’s crazy because when people ask me, I’m forced to tell them that, you know, I don’t know.  I mean, the Mayo Clinic couldn’t figure out what was going on.  I do know that there were some tests for Lyme that showed positive results.  I do believe I had a lot of the symptoms for antibiotic adverse reaction, and I put some of the claim on that.  And then also, there’s a concept called opiate-induced hyperalgesia which is this idea that longer term use of opiates will cause pain throughout your body.  So, you know, I think it was probably sort of a mixture of those things, and we can go down a rabbit hole on this great deal and I’d rather, maybe save that for another time, but needless to say that it was a nightmare, and I was looking for something that would be beneficial for me, no matter what I had.

Ben:  So you were more or less addicted to opiates and extremely sick?

Thomas:  Yeah, and tired.

Ben:  Okay, and so what did you do from there?

Thomas:  Well, I was up late at night in one of my support groups, and someone would mention NAD, and I was like, “Okay.  I’ll give it a shot.”  And I called Springfield Wellness Clinic, which was the longest running NAD clinic in the country for 15 years.  They may have treated over a thousand patients, and they told me, “You know what?  We have a place in San Diego where it’s being done.”  And so, I was like, “Great!  I live in San Diego.”  So, I started the treatment and it’s intravenous NAD+, and that’s very important that you get the right molecule.  It’s not NADH, which is completely different.

Ben:  Okay.  So, when you say intravenous, you’re actually getting this stuff injected?

Thomas:  Yeah, it’s into your vein.  It’s a lengthy treatment.  I ended up going for 12 days.  It might be like seven hours a day, and then I was a noisy patient, I was not easy to be around.  They asked me to give up opiates and I was scared and, you know, just kind of grumpy.  But after that, about the day seven, the day nine, things began to change.  I remember Anne Rogers saying, “Your eyes are shining.  You’re changing.  I can see it,” and I was a little bit surprised that she could see that I was changing physically, and that happens to quite a lot of chronically ill patients.  Their face and cheeks just start to get color, it doesn’t happen with everyone, and then a day later, they get more color and gloss in their eyes.  Their eyes no longer look, like dull.  So…

Ben:  I mean, I’ve done IV injections before, like Myer’s cocktails where you get high dose glutathione, and vitamin C, and a host of different vitamins, but with this NAD, is it just NAD that you’re injecting when you do something like this intravenously?  Or are you including other things like vitamin complex, or things along those lines?

Thomas:  You know, the NAD doctors that are involved with the Mestayer model, where Mestayer is the doctor in Springfield.  They worked to customize the protocol, her patient, and so, other things that you might see in Myer’s cocktails might be added at some point, just so you know that the supplements aren’t mixed in the same bag.

Ben:  Okay.  Gotcha.

Philip:  Let me jump in real quick.  There are definite protocols that Dr. Mestayer from Louisiana has worked on, and we’re also working on on improving and yes, it is similar to getting Myer’s cocktail.  It’s an intravenous slow drip at the rate that the person can tolerate it, and then we add other minerals, and we individualize the treatment according to testing and what the patient’s personal needs are to enhance the NAD experience.  The NAD is the most active ingredient but we use these other compounds, because there’s an art to this, it’s not just starting an IV and running NAD in.

Ben:  Okay.  Gotcha.  Now, I do wanna definitely ask you about some of the protocols that you do there Dr. Milgram, but back to you Thomas, in terms of starting into this, you’re literally saying within days of beginning NAD injections that your health completely turned around?

Thomas:  Yeah, I mean, I remember being in the ocean, swimming in the ocean about a week afterwards, and I just remember looking around and everything I sensed was just richer and full of more color, and my pain went down by 50% in the first 10 days of treatment, and then I started to go down further over the next six months and a year, and it said it might be beneficial to keep coming in every so often to get a booster, and I was diligent in doing that.  And I felt healthier, and less pain, and more energy, you know, every time I came in.  So…

Ben:  Interesting.

Thomas:  At that point, I went back to old doctors and just sort of talk to them, and some of these doctors were like, they said, you have no idea what you just stumbled upon.  We just came to a conference, we were just at a conference, I was just talking about this, and you should pursue this and then they gave the whole “you-should-go-to-medical-school” thing, but it was really motivational, and they encouraged me go to medical conferences which I did, and I got the same responses that these integrative medical conferences that I went.  And I also remember meeting with a scientist who was working in the NAD field for coffee, and he asked me about my story.  I remember telling him, I was like, “You know there’s one thing that I gotta tell you about, and that’s the music sounds beautiful.  Like, I can really have an emotional response to music.  I can really hear all the nuances in it.”  And he responded, “Well, it’s because your nerves are revived.”

Ben:  Wow.

Thomas:  I dissected mice on NAD and we noticed a difference in their nerve connections to their ears, and I don’t know, I was sort of, at that point, that was incredible.  But it was six months after I did the NAD that I saw the article by David Sinclair.  Well, I saw it in Time magazine, the journal article he did and so, at that point I was like, “What did I stumble upon?”  They’re using a precursor, and we’re using, you know…

Philip:  The actual NAD.

Thomas:  The actual NAD.  Yeah.

Ben:  Okay.  Gotcha.  So there is a difference, I know, and I wanna delve into that here in a moment, but Dr. Milgram, in terms of you and your story, are you the person who Thomas actually hooked up with, in terms of his first experience with NAD?

Philip:  Yes.  He hooked up with this lady that I worked with together with Dr. Mestayer in Louisiana, and her name was Anne Rogers, she was looking to bring it to San Diego, and she found me as an addiction medicine doctor, and also somebody interested integrative medicine and nutrition, and individualized medical care with metabolic nutrition, and just serendipity, she found me.  I’ve been doing addiction medicine for 25 years.  I’ve been in treatment myself for opiates, in recovery since March 23rd, 1988.  Then I studied intervention with Vernon Johnson, the guy that invented intervention back in 1991, and I’ve dedicated my life to this because this really changes people’s lives.  I’ve been involved in Tom’s therapy, I was trained by Dr. Mestayer in Louisiana, and who’s been doing it since 2001, and then I’ve been involved with administering NAD to Tom, yes.

Ben:  Okay.  Gotcha.  And you yourself, if I’m not mistaken, didn’t you used to have some kind of like addiction issues that you overcame with NAD as well?

Philip:  I didn’t overcome them with NAD.  I’ve been medical director of several detox places but originally, I was just exposed to the old, white knuckle type of detox where they prescribe high levels of drugs to counteract withdrawal symptoms like valium, benzodiazepines, and other heavy duty drugs, and I’ve been doing that for 25 years.  When we came to NAD, it’s a game changer.  There’s virtually no withdrawal symptoms, the hyperalgesia is decreased, and…

Ben:  What’s hyperalgesia?

Philip:  What Tom referred to that opiates, in a way makes it a cycle that makes you use more opiates.  It actually increases your sensation of pain, then you require more opiates.

Ben:  Okay.  Gotcha.

Philip:  It’s a vicious cycle.

Ben:  Gotcha.  How is NAD actually breaking addiction?  Like how does this work?

Philip:  Well, I can’t give you the answer in 25 words or less, but it actually works at the epigenetic level to decrease withdrawal symptoms.  Okay?  It also works at the mu-opioid receptor level to decrease this exaggerated response of pain through the sodium and calcium-gated channels.  And it also decreases anxiety by having an effect on the bundled up chromatin that comes from lack of NAD.

Ben:  Okay.  So someone who’s like anxious, who’s having withdrawal symptoms, and who has like a chemical addiction to opiates.  It’s working on all three of those different platforms to decrease the addiction to opiates, and let me know if I’m correct about that, but also decrease addiction to other substances as well?

Philip:  Yes.  And in fact, it has the greatest effect on alcohol addiction.  It’s amazing.  It actually acts at the metabolism and genetic expression of alcohol predisposition.  Again, I can go as deep into this as you’d like.

Ben:  Well, what I’m curious, I guess the first thing and I’m sure a lot of our listeners are wondering this too, and perhaps this is a question for you, Dr. Milgram, or for Dr. Grant, but what exactly is NAD?  Like, what is it and what does it do?

Philip:  Well, it’s like we’re on a phone call talking about relativity, and we have Einstein on the phone with us.  Why don’t we have Dr. Grant, who’s one of the world’s experts on this, how he answers this question.

Ross:  Yeah.  Look, I’m happy to make a few comments and I’ll try and make them fairly brief, but NAD probably, is what could be considered one of the master regulators of cell metabolism and often when people talk about different molecules having this amazing impact, they’re often sort of talking at a level which is on molecules functioning at a fairly high level in a cell.  But here, we actually do have a molecule which is fundamental to the way the cell functions, and its levels as it goes up and down has an influence on multiple different areas.  So NAD itself, I mean apart from the clinical benefit as Dr. Milgram has talked about, and obviously Tom has experience, but at its fundamental level it’s actually involved in a number of different things.

It’s a co-factor, which we’ve known for many decades now.  So, you know, alcohol dehydrogenase as you need NAD, in fact it’s the NAD that runs out when people are trying to metabolize alcohol, and so they’re ending up with high levels of alcohol as a result, but it’s needed as a co-factor.  Another one is to lactate dehydrogenase, et cetera, which is also involved with that buildup of lactate, when you’re using your muscles.  It’s an electron transporter.  Now what that means is that you need to, when you’re turning the food you eat into the energy that the body needs to the muscles, et cetera, you need to be able to transfer the electrons from that food that you took down, what we call a respiratory chain in the mitochondrion, and that’s how we generate the energy, ATP.

Ben:  Okay.

Ross:  NAD is critical for that.  You know, it’s also needed for DNA repair, so, our DNA is getting damaged all the time, and I guess we’ll talk about aging a little bit later on, but it’s a really important molecule.  Well for now call a substrate, so it’s actually used up by the enzymes, or one of the key group of enzymes, there are a number of them, but basically it repairs particularly, and it’s actually used by those enzymes in order to help prepare the DNA.

Ben:  So, your body makes NAD on its own?

Ross:  It makes NAD on its own, and it makes it from a few different precursors.  So I know that was mentioned before, about nicotinamide mononucleotide or NMN, but there’s a number of precursors that it can actually make it from.  It can make it actually, originally also, from the molecule tryptophan, which is an amino acid people would have linked up in their minds to things like serotonin, which is one of these neurotransmitters, but happy to get into that a little bit later on, the epigenetic signaling its involved with as we know, the switching on and switching off genes by changing acetylation patterns, and these are sorta linked to people might have heard in a sort of aging medicine, or aging sort of like chemistry with the sirtuins, and NAD is a precursor to that, so sirtuins might be important.

And I guess a lot of the enthusiasm about resveratrol is the fact that it was theoretically driving sirtuin activity.  What’s interesting is that sirtuins are like a factory, like any enzyme, they won’t function unless they have, if you like, the raw material for that factory to work on, and NAD is that raw material.

Ben:  Okay.  Gotcha.  Now, I took physiology in college, like physiology and biochemistry, and what we were basically told was that NAD, or this nicotinamide adenine dinucleotide was basically, all it does is it carries electrons, right.  Like it’s involved in redox reactions in the cell, and you basically have it, this NAD+ gets an electron from other molecules, it gets reduced, and that makes this stuff called NADH, which then can donate electrons, and it’s just essentially one of the ways that the human body kinda keeps working as a giant battery.  We never learned about any of this stuff regarding like anti-aging, or addiction, or it acting on these sirtuin pathways you’re talking about, and so, I guess that’s my first question, especially after hearing you and reading this Scientific American article about how this could be the new anti-aging drug of the future.  How exactly is it working when it comes to NAD?  I know that you just mentioned this sirtuin pathway, and I know that’s intimately involved with aging, so can you go into, not only what this sirtuin pathway is, but what’s the proposed mechanism between, or as to how NAD would actually help someone when it comes to anti-aging?

Ross:  Yeah.  I mean, it’s a very good question and all of the answers aren’t known in here.  What we do know is that when it comes to aging, so if you think of what’s actually happening when somebody is aging, they’re actually getting an accumulation of damage within the cell, and that accumulation of damage particularly around the DNA, is affecting the way those cells function and therefore the way the organ functions.  And a couple of the key things that characterize aging is that part from the cumulative damage is also this decrease in energy, and just a decrease in what we might call the viability of the cell.

So very simply, where does NAD set in?  NAD, we know, can improve energy efficiency because one of the key things that’s needed in order for the mitochondrion to work well, in order for the energy to be produced, is that we need to have an efficient supply of these NAD molecules, as you said, to be able to transfer those electrons so that we end up having a mitochondrion being able to convert literally the energy we take in, mix it with the oxygen that we also breathe, and finally produce ATP at the end.  If you don’t have NAD, the mitochondrion doesn’t work.  Therefore, you don’t generate the energy.

Now, at the epigenetic level, then we have again NAD as a master regulator at that point.  If NAD levels drop, then the epigenetic switches, and people would be aware that now we know we have methylation patterns, and acetylation patterns, and what these are is just molecules that set on and off, both the DNA as well as the chromatin that sits around with the proteins, to allow the genes to either be switched on or switched off.  If you’ve got lots of NAD around, it is able to switch on, so sirtuins act, they deacetylate things, and they’re able to switch on pathways that are linked to improving cell viability, and in other words improving the health of the cell.  Now, remember this is at a global level, so it’s not just happening in one organ, it’s actually happening across the body, including the brain, as well as the muscles, as well as other tissue.  And so this increases things like a radio oxidant production, and basically keep cells working in what we would consider to be a younger state of metabolism.

Ben:  Okay.  Gotcha.  So essentially all we’re doing is we’re allowing our mitochondria to function more efficiently when we have adequate NAD, and we’re also fighting a lot of the oxidation that could cause aging, but by giving our body extra NAD via something like the intravenous injections that Thomas and Dr. Milgram were talking about, we’re not just able to, for example, fight off some of the bad things that can happen when we’re addicted to a substance, but we can literally activate these anti-aging pathways.

Ross:  Correct, and I guess the other important thing is that it looks like we were able to increase the repair of DNA that might get damaged in the course of normal metabolism.

Philip:  Ben, can I chime in here?

Ben:  Yeah.  For sure.

Philip:  I’ve got a list, you know, I want you to get out of thinking that it’s just NAD to NAD+ in the Krebs cycle. That’s what we learned, making ATP.  That’s what we learned back in Biology 101, but this is much greater than this.  It’s an epigenetic co-factor in blocking over-reactive genes that are not being expressed properly.

Ben:  So what do you mean by that?

Philip:  In histone acetylation, methylation, phosphorylation, and DNA methylation, and micro RNA, it has an effect to allow the histones to get tightened up in the chromatin, and of course, very greatly simplifying this, and the NAD actually helps unbundle this.  Though it actually works to remodel your chromatin by its effect on these sirts and the PARPS, it actually affects DNA repair.  It affects the CD38 gene for increasing immune function.  It causes the mitochondrial biogenesis.  It increases the production, and effectiveness of each individual mitochondria.  It actually offers a neuroprotective device qualities protecting the nerves from demyelination and other things.  It being explored at Harvard for use in ALS, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease.  It also again, it’s very important that the sensation of pain by the gated calcium and sodium channels, the voltage-gated chain, the NAD helps that function properly so you don’t have this hyperalgesia.  It may act itself as a neurotransmitter, and it has an effect on autophagy.

Ben:  Really?  So it can also act as an actual neurotransmitter?

Philip:  That’s still being studied.  That’s sort of nebulous, probably beyond the scope of this discussion.

Ben:  Okay, but basically it could potentially be involved in like cell-to-cell communication?

Philip:  Yes.

Ben:  Okay.  Interesting.

Ross:  Yeah.  And I can jump in there and yeah, it certainly support that, it looks like NAD probably does serve as a neurotransmitter by systemically as well as in the central nervous system.

Ben:  Interesting.  Okay.  So, this Scientific American article it, obviously, based on some of the cool things about NAD that you guys have just talked about, it goes into how there’s a lot of different NAD sources being created right now.  Like there’s this stuff called Niagen.  There’s something called Basis by I think a pharmaceutical company Elysium.  They talked in this article about things that can assist with your body’s own production of NAD, like resveratrol, that we’d find in red wine or in supplement form, or something called pterostilbene.  Can you help us cut through all this clutter, Dr. Grant, as far as what the best way is to actually get NAD into our bodies, and what exactly the status is as far as the development of pharmaceuticals or the development of supplements?

Ross:  Yeah, sure.  Look, to get it into the body, and this is one of the reasons why clinics like Dr. Milgram’s and Dr. Mestayer will use IV NAD, if you want to get NAD into the body efficiently, what we call a 100% bioavailability, you know, when it’s all getting in, then the best way is to take an IV.  The unfortunate thing is, and even though there’s a lot of work that needs to be done still in this area, it looks like if you take NAD orally, and I’ve seen supplemental tablets for NAD out there, but taking it orally, unfortunately, you’re pretty much not going to absorb it across the gastrointestinal tracks.  So from the stomach and the gut, you’re not gonna absorb NAD very efficiently.  So, to what to get NAD up efficiently, it’s intravenous.

Now, there are a few other ways in which the body can actually make NAD, so that what we call precursors, and you mentioned one of them, niagen.  Niagen’s a fairly new one, and that was identified actually originally from milk in around about 2007, but that is called nicotinamide riboside.  And nicotinamide, yeah, nicotinamide riboside actually gets converted into NMN, which is one of the other precursors that are also available, the one you talked about in one of the other article.  So you can get those two, and those two will be absorbed across the gap.  So you can get effectively NAD increased from taking those orally.

The other two ways of getting them is by the classic vitamin B3’s that you’ll get from over-the-counter at the drugstore, and that would be nicotinic acid which is the acid form of vitamin B3, and this is the one that the…

Ben:  You said B like boy?

Ross:  Yeah, B like boy.

Ben:  Okay.

Ross:  So, vitamin B3.  So the nicotinic acid form is the one that’s been used for actually quite a few decades now to reduce cholesterol.  It’s the one unfortunately that has a side effect that gives you flushing.  We can talk about why that happens.  But then the other molecule that you’ll also get, again across the counter and in many supplements, is the amide form called nicotinamide.  Now, you’ll notice that NAD is nicotinamide adenine dinucleotide.  So that is the nicotinamide version.  Now nicotinamide can also be, what we call, recycled.  It’s in what we call the salvage pathway.  It can be recycled through to NAD.  The negative thing with having too much nicotinamide, as opposed to the other types of NAD precursor that I’ve mentioned, is that nicotinamide is a byproduct.  Now, you’ve heard we’ve talked about CD38, we’ve talked about sirtuins, and talked about the PARPS.  So CD38, the immune modulators, the Sirts, the epigenetic modulators, and PARPS, the DNA repair enzymes.  Now all of those three, when they use NAD, they will actually generate nicotinamide as a byproduct.

Ben:  Okay.

Ross:  And the important thing about that is that that nicotinamide, as the levels start to go up in the cell, the nicotinamide starts to inhibit those enzymes, the CD38s, the sirtuins, the PARPs.  And so too much nicotinamide, unfortunately, can stop the very reactions that you want to have acting.

Ben:  Interesting.  Okay.

Ross:  So, we would probably suggest that getting it from some of the other sources, the nicotinamide riboside, NMN, or nicotinic acid would be better.

Ben:  Okay.  And the NMN, is that something that one can, for example, purchase in like a supplement form, or this nicotinamide riboside?

Ross:  Both NMN and nicotinamide riboside can be purchased.

Ben:  Okay.  And the absorption of those in the gut is sufficient to actually increase NAD levels inside the human body?

Ross:  Yes, they do.  And there has been some studies in animals particularly, in fact we’re in the middle of preparing to conduct what we call a head-to-head on these so that we can see which one is actually the better one.  And most of them there is evidence that both of them will significantly increase NAD.  We’re probably favoring the riboside at the moment as opposed to the NMN, but both of them seem to be able to do it.

Ben:  Okay.  So…

Philip:  And again, Ben, it’s dose related.  You know, think of the breakdown of nicotinamide adenine dinucleotide into nicotinamide and adenine, okay?  That reaction of breaking the NAD down to nicotinamide is where the Sirt and the CD38, and the PARPs are generated and activated.  So if you have too much of the nicotinamide, it can actually force that to not happen.  So I would say like people that are taking too much nicotinamide is probably not a good idea.

Ben:  And how much would be too much? ‘Cause if you look on like Amazon, right, like if you go to Amazon, you do a search for like nicotinamide supplements, you’ll find like NOW Foods, and Life Extension, and all these other companies selling like 500mg capsules of nicotinamide, like that’s about the dose you’ll see in a lot of these.  When you say, “If you take too much, you’re gonna shut down those positive pathways that you’re trying to activate,” how much would be too much if someone were just gonna like try to get a nicotinamide supplement from one of these companies?

Philip:  Well, usually they come as 250 or 300mg, and they suggest two a day, and I think that’s the proper dose.  But people will think, “Oh, wow.  If a little bit is good, a lot is better,” and they’re taking more than that, I think they may actually be doing themselves disfavor.

Ben:  Okay.  So once you’re exceeding, like close to about 600mg, you’re saying that that could be Bad News Bears?

Philip:  Correct.  Just like too much vitamin D.

Ben:  Okay.  So, nicotinamide, we can buy nicotinamide, also known as NMN supplements on a website, like Amazon for example, from a good company, and we could take anywhere from 300 to 600mg of that to activate some of these anti-aging, or antioxidant pathways, or new mitochondrial-building pathways.  Now this nicotinamide riboside, which is not the NMN form, is that also something that you can find in supplemental form?

Ross:  Yes, you can find nicotinamide riboside as a supplement, and nicotinamide riboside, like NMN, will be converted through to NAD.  In fact, nicotinamide riboside is converted first to NMN, and then through to NAD, and for reasons that we still don’t understand, it seems to be [0:41:35] ______ way of getting there, and you can take, unlike the nicotinamide, which is actually having to be recycled through the pathway, and it recycles through NMN as well, but nicotinamide riboside, you probably can take up to, I would think, easily 500 to even possibly 1,000mg a day without too much concern ’cause you’re not gonna be generating nicotinamide directly.  If you’re supplementing with nicotinamide on its own, so that’s the vitamin B3 that’s often out there, if you supplement that on its own, you’re already increasing the body’s nicotinamide, and that’s doing the inhibition.  But taking the other two, the NR, the nicotinamide riboside, or the NMN, neither of those will produce nicotinamide until it goes through NAD.

Ben:  Okay.  Gotcha.  Now this article for example, the one in Scientific American talked a lot about resveratrol, which you find in wine, and the fact that resveratrol can kinda like rev up the sirtuin pathways.  And when we rev up the sirtuin pathways, we can do things like form new mitochondria, or keep mitochondria running smoothly.  Now, when it comes to resveratrol, is that something that you would take in conjunction with something like NAD injections, or one of these nicotinamide supplements, or is that something that one would use on its own?  What are your thoughts on the use of something like a resveratrol supplement?

Ross:  Yeah, I’m happy to make a comment.  I think that there is some positives with certainly the antioxidant effects that come from the stilbenes, which resveratrol is one, and…

Ben:  What’d you call, the stil, the pterostilbene?

Ross:  Yeah.  The pterostilbenes.  They’re a class of molecules that are what we call in the phytonutrient class.  So, there’s mention of them coming from things like the skin of red grapes, and from blueberries, and in fact, you even get them from things like peanuts, et cetera.

Ben:  Is that why Thomas that you told me that you were using like a blueberry extract in conjunction with your NAD injections?

Thomas:  That was just something that I had originally gotten from a brain conference that I went to, but as soon as I found that out, yeah, I’m very pretty religious on taking my blueberry extract.

Ben:  Okay.  And that’s because this pterostilbene that Dr. Grant was talking about, you’re gonna find that in addition to resveratrol in things like blueberries, and grapes, and dark purple, and blue type of fruits, or berries?

Ross:  Mhmm.

Thomas:  Yeah.

Ben:  Okay.  Gotcha.

Philip:  And we’ve found that NAD really is the most important thing to increase your Sirt 1 through 7 activity, but resveratrol specifically, you need that for Sirt 2.

Ben:  Okay.  Gotcha.  So there’s different sirtuin pathways that are activated by different forms of supplements and you would, for example, take resveratrol in conjunction with NAD to get the best of both worlds.

Philip:  Correct.

Ross:  Yeah.  Though theoretically, there seems to be a fairly good benefit.  I mean all the sirtuins need NAD, some of them get switched on a little more efficiently than others in different parts of the cell.

Ben:  Now, in terms of the best delivery mechanism, it sounds like intravenously that you can really get very, very high levels of NAD compared to some of these supplements that we’re talking about, but how much of a difference is it?  I mean, are we talking about like a night and day difference between intravenous injections versus someone using a supplement, like a resveratrol mixed with an NAD supplement?  Are we talking about a slight difference?  I mean, in terms of comparison between intravenous delivery versus oral delivery, how do these different delivery mechanisms vary?

Thomas:  In my personal experience, I just think that intravenous NAD is, you know, I haven’t seen anything that’s like it in comparison.

Philip:  Let me respond on a clinical level.  You know, we suffer from lack of NAD because we’re using it up and not replenished in proper levels, and it’s the cause of underlying aging, and other disease processes.  So I think that you have to sort of flood the body with NAD to start off with, but then you can supplement, and that’s why we use intravenous.  Then you can supplement NAD with a pure source of NAD that we give intranasally that I think is a very good method of continuing to substitute to boost your NAD levels.

But, you know, the NAD business has been around actually for many, many years.  But because of human greed, it’s sort of been kept underground.  People wanted to make money on it, so they called it different things.  They called it Coenzyme 1, they called it amino acid therapy when Dr. Hitt was doing it down in Mexico.  But then it was analyzed by Dr. Mestayer and his friends in Louisiana, and found that the active ingredient was actually pure NAD.  And they now have the pure source of, you wanted NAD that doesn’t have a contamination in it, you want it so that it has a high level, it’s not very stable at room temperature for a prolonged period of time, so it has to be gotten fresh, and be made up fresh and used quickly, and that’s why we have these protocols, and then we also use it with other nutrients to individualize therapy for people’s individual needs.

Ben:  Okay.  Gotcha.  So, in terms of other things, are you talking about things aside from like blueberry extract and resveratrol that play well with something like NAD when it comes to the anti-aging or the addiction mitigating effect?

Philip:  Correct.  Not only that we need to add things to enhance the NAD experience, and we know what those are, certain minerals and, but also, it seems like a lot of the things that we’ve been doing for detox for years actually go and work against these…

Ben:  Like what?

Philip:  Like benzodiazepines.  We do not give benzodiazepines when we do NAD therapy.

Ben:  You mean like valium, and stuff like that?

Philip:  It’s like anti-NAD.

Ben:  Would that be, for example, like a valium?

Philip:  Valium, Xanax.

Ben:  Okay.  So basically, a lot of these will actually have an effect that inhibits mitochondrial function?

Philip:  Correct.

Ben:  Okay.  Gotcha.  And what are some of the   things  that  combine  well  with  NAD?    You mentioned minerals as one.

Philip:  Well, magnesium, calcium, potassium, but we also handling the side effects, there’s drugs that we use and drugs that we don’t use that we’ve found to not inhibit NAD, but decrease some of the withdrawal symptoms, and enhance the NAD thing. I mean, we’ve developed this over the years.  Dr. Mestayer has been working on it, and we’ve develop these and increased these even more.

Ben:  It’s really interesting because I did a podcast with a guy named Dr. David Minkoff a few weeks ago where we talked about cancer and mitochondria, and anti-aging, and it seems that a lot of this kinda overlaps in terms of, you know, we spoke of hyperbaric oxygen therapy to enhance oxygen availability for enhancing mitochondrial function.  I mean, I know that’s something that Thomas, you told me that you had experimented a little bit with.  I’ve talked about cold therapy, and cold thermogenesis for enhancing mitochondrial function and the amount of nitric oxide that you have in your body.

You know, you guys are now bringing up other ways to enhance mitochondrial function such as mixing NAD with resveratrol, with minerals.  It seems to me that when we’re talking about like anti-aging, that there are all these different things that seem to kind of be going after the same effect, which is, more or less, improving mitochondrial health, while also improving the ability of DNA to repair, in the case of NAD.  But I know that, and I have a question for Dr. Grant actually about this, I know that the effect of this stuff can go beyond anti-aging and, for example, could kinda reach into the realm of fitness, which I think a lot of our listeners might be interested in.  As far as the research on NAD and anything such as VO2 Max, or lactate tolerance, or time to exhaustion, or anything like that, Dr. Grant, have there been studies on NAD, and how it affects the actual fitness or exercise performance?

Ross:  There’s been very few and I suspect that that’s going to rapidly change.  We’re involved in a study a little while ago now where we were looking at the influence of a particular supplement that was being provided to athletes, and seeing what the effect was, that will be getting a 15% improvement in what they called ‘Time to Fatigue’, as you’ve mentioned, so they could exercise about 15% minutes longer, and it was the exercise physiologists who brought the problem to our lab, and asked us to have a look, and see what we thought could be the issue.

So we checked around various pathways and it looked like NAD levels were actually increased with this supplement.  Now we thought that that was probably functioning as a preservation of NAD.  So it makes sense because of NAD’s role in being able to efficiently or make the mitochondrion where you’re producing the energy, where you’re making that happen efficiently.  And, as I mentioned, with lactate dehydrogenase being able to convert that more efficiently through the pyruvate.  So increasing NAD, it makes sense that you would have the potential for increase in fitness.

Ben:  Okay.  Gotcha.  So, what they’re saying is it may improve time to exhaustion by somehow working on the ability to turn over lactic acid more quickly, but it’s not actually something that’s been fleshed out in actual human research?

Ross:  In great detail.

Ben:  Okay.  Gotcha.

Thomas:  But then also, by acting through specifically the Sirt1, we found some of the things that you and your experience have shown to increase, improve the human condition.  Things like fasting, mechanically stretching muscles, exercise, low glucose diets, proper circadian rhythm sleep cycles, actually act to let the Sirt1 and the NAD work properly.  And that’s why they work, it works through the NAD and Sirt pathway for these things that you can do to be a healthy person, that’s why they work.  A lot of the better diets, the ketogenic diet, Atkin’s diet, Paleo diets, that’s how they work, they also work to increase Sirt1.

Ben:  Right.

Ross:  And that’s true, and the good thing with an activation of things like Sirt1 is it looks like we’ll end up getting an increase generation of mitochondrions.  You actually increase the number of mitochondrion as well as the efficiency of energy productions.  So, it [0:53:01] ______.

Philip:  And especially in the brain.

Ross:  Mhmm.  In the brain and in the muscles as well.  But, yes, certainly both.

Ben:  We’re talking now about, like, you know, some different lifestyle strategies that could, for example, enhance these Sirt1 pathways, you know, in a similar way as NAD, such as some of the things you were talking about, like ketosis, we mentioned calorie restriction, we mentioned cryotherapy, hyperbaric oxygen chambers.  What are some of the best ways to actually raise NAD levels naturally with other food strategies, or other lifestyle strategies, or other biohacks, for example?

Ross:  Yeah.  Look, I’m happy to jump in there with some of the lifestyle elements, which I think are particularly important.  If you want to turn over your NAD fast, in other words, if you want to drop your NAD, which is not what we’re gonna do and I’m just using that, then you’re gonna have any condition which has an increase in inflammation and what we call oxidative stress, or free radical damage.  Both of those, not only does it signal through to increase the CD38 activity, but it also increases PARP activity, which is the DNA repair.  So inflammation, free radical damage, both of those are gonna decrease your NAD, which is what you don’t want.

So, very simply, any of the lifestyle behaviors that are going to improve that is going to increase your NAD naturally.  And this includes taking in less calories, we know that using whole foods and some of the work in the brain that we’ve done which is interesting, showing that the caroteniods particularly, so these are the molecules that are coming from the red, yellow, and green leafy sort of veggies, these are particularly high in caroteniods and these can actually help preserve, particularly in the brain, NAD levels.  Exercising, eating whole grains, I mean these are gonna be able to provide things like your nicotinic acid and vitamin B3, et cetera, on their own.  But all of these, essentially doing those things that you’d be [0:54:52] ______ , most people would recognize were healthy for them is going to help to increase their NAD and maintain high NAD levels, and therefore high sirtuin activity, et cetera.

Ben:  Okay.  Gotcha.  What about light therapy?  You hear a lot about like infrared light and your infrared intranasal light, things along those lines, how about light?  Does it play a role here?

Ross:  Look, I don’t know, nobody’s done any work on that directly, as far as I’m aware, not specifically with NAD, but there is work that’s been done on light therapy of different wavelengths, even through the blue wavelengths decreasing inflammation within certain parts of the body, particularly in people with pain.  So, where there’s benefit that’s there, and I guess there’s still a lot of work still going on there, that photomodulation, that if it’s going to decrease pain, which is associated with cytokine signaling and increase in inflammatory cytokines drive or increase your pain, then there is going to be benefits with NAD.  And that probably works both ways, so the higher NAD is able to shut down some of those pathways.

Ben:  Okay.  Gotcha.  What about you, Thomas?  I know you’ve experimented with some things when it comes to enhancing your response to NAD therapy and supplementation.  What are some ways that you’re using food or lifestyle strategies to increase your own levels of NAD?

Thomas:  Personally, I had a very substantial decrease in pain and a very strong uptake in energy in that 12 days I initially did NAD therapy, but I wasn’t a 100% back to recovery and so, I was still desperately trying to make some changes and I think, sort of accidentally, I was really cutting down on my caloric intake, I was eating more vegetables, and my carbohydrate intake had come down, and I think that that may have contributed to sort of the positive impact that sort of accentuated the high levels of NAD that I had.  And when that happened to me in over the course of, let’s say, a year, I had lost about 40 pounds in fat ’cause I did a body comp.  And then it was a lot, I think it was actually a year and a half, and then at that point, I met up with you at the Spartan race in Las Vegas and it was my first Spartan, and I ended up placing at the top one and a half percent.

Ben:  Prior to that race, you actually took some NAD.  I remember we were in the condo, and you were taking something, but you didn’t do an IV, right?  You just basically were using like one of these nicotinamide type of supplements we were talking about?

Thomas:  No, actually I did IV NAD a few days before I arrived, and I wanted to see what kind of impact that had.  I don’t, I’m not sure, I mean this is just anecdotal, I definitely, I mean it’s obvious, I already said that, yeah, I mean, I had no energy before, and now I’m doing all these things.  But as far as me personally, having this immediate effect, I don’t know where but that’s something that needs to be looked at a little bit more.

Ben:  Okay.  Gotcha.  Now, I wanna get into anti-aging here real quick.  Dr. Grant, I know that you mentioned that NAD levels will drop when we age, which is perhaps why NAD is being proposed as an anti-aging supplement, but how much?  Like is this a significant drop?  Has this been studied in terms of the actual decrease?

Ross:  Yeah, absolutely.  I mean, we did some early studies back in 2011, we were just looking at animals, and across the age range you can see, while you know, accumulation of damage within the body, and things like PARPs go up the enzyme that’s actually trying to repair the DNA go up, NAD drops.  And then we said, “Alright, what we saw in animals, we thought we’d better have look at that in humans”.  So we looked at it in, with the pelvic, non-sun exposed skin, all the eight way from, sort of eight day old circumcisions up to 79 year old hip replacement, and you could see again the damage sort of accumulating with time, and particularly after the age of 60.  But then you could see the NAD drop fairly consistently with inflammation and oxidative damage, and in 2014 we published some work showing the same sort of decline within the brain, and this can be anywhere up to 40%.  So there is a significant drop that can happen…

Ben:  Forty percent is how much it could decrease as you age.

Ross:  Yeah.

Ben:  Is that just due to everything from oxidation to DNA damage, to living in an industrialized area where you’ve got lots of inflammation, free radicals, and things like that?

Ross:  Yeah, it’s predominantly what causes that inflammation and oxidative damage.  So as were saying lock-in formation of cell will drive oxidative damage inflammation which then damage the DNA, so you’ve got an increase in [0:59:51] ______ that drive the CD38.  Now, CD38 is definitely involved in the immune function as Dr. Milgram is saying earlier, but CD38 probably has some other function.  We have some other what we called NAD like a hydrolases that function in ways that were not really quite sure in a cell but these seem to go up to certain CD38 when we have inflammation going on and that seems to be also a primary driver of decreasing NAD as we get older.

So, there is a significant drop in NAD and it depends where you’ll look for I mean, whether or not it’s enough in the plasma or whether it’s in the tissue itself.  So it can vary depending on the different tissues, but certainly in the plasma they can give quite some significant shift.  And also in the fluid that drains the brain called the cerebral spinal fluid which is a good indicator of what actually happening clinically within the tissues.

Ben:  Okay.

Philip:  I remember reading some studies that they tested people under 45 and over 45 for their NAD levels and they found that 300% increase in the average between the pre- 45 and post-45.  Also, another study shows that it seems to exponentially increase after age 60 the loss of NAD.

Ross:  The loss of NAD that’s certainly what we found after the age of 60.  So, there seems to be an acceleration of damage and therefore an acceleration of the rapid loss of NAD as a result.

Ben:  Can you actually test your own NAD levels, like is there a blood test for that?

Ross:  Yeah look, we have actually offered that to one or two of our clinics here in Australia or any because we know that they can actually get sample to us in a particular way which preserve NAD.  The trouble with doing it and sending it through a normal pathology lab is a) they won’t do the test, and b) the samples itself once it collected needs to be preserved extremely quickly otherwise NAD will change very rapidly, and so you’ll get abnormal results.  So it is possible to do it but it’s a bit tricky to do it sort of in a regular basis in a normal lab at this stage.

Philip:  We are gonna be dealing clinical trials in our facility and in conjunction with Dr. Mathai in Louisiana to do this the right way, and actually test people with the special testing that Dr. Grant is talking about.  We had to purchase this special centrifuge and we have to handle the samples in a certain way, and we are gonna be deforming clinical studies on this.

Ben:  Well, vitamin B3 is also known as nicotinic acid or niacin or nicotinamide, couldn’t you just measure your vitamin B3 levels?

Ross:  Vitamin B3 is an important precursor to NAD and as we mentioned nicotinamide, if you take nicotinic acid, yes you’ll be able to generate NAD through that pathway.  If you take nicotinamide, nicotinamide is that sort of complicated situation where it’s both can be made back into NAD but it’s actually a byproduct of NAD.  So nicotinamide is going up doesn’t necessarily mean that NAD is going up.  Nicotinamide going up could actually mean that you’re turning over NAD quite a lot.  So, difficulty there.

Ben:  Okay, gotcha.  And I’ve got a few other questions: the first is I know that a lot of people listening in and Thomas kinda briefly mentioned that he thought that some of his symptoms may have been indicative of something like Lyme disease, and I’ve heard a few snippets here and they’re about the use of NAD for the use of something like Lyme which I know is a frustrating issue for a lot of people who have chronic fatigue is this Lyme infection.  What’s the link between NAD and the treatment of Lyme disease?

Ross:  Well, I’m not sure if Dr. Milgram wants to make comment there.  I can sort of begin that one off.  Lyme disease is originally as a Borrelia infection which is a spirochetal bacteria.  And people think to end up with chronic fatigue, reduced energy, and there are some central nervous system dysfunction that can happen with people at a chronic stage, and one of the things that NAD will do as we’ve talked about in essentially from the beginning of our discussion is that it has a significant improvement in being able to increase the efficiency of energy production.  So it’s probably working on at that level, I don’t know of anybody who’s done any specific studies to look at mechanism and I guess that’s where we probably still lacking a bit of information but improving energy production, improving the way the immune system probably functions which is the primary driver often behind and this is sub-clinical so it’s hard to test a little bit.  It seems almost certain that you’ve got disregulated immune function.  Again, maybe through CD38, some of these [1:04:50] ______ maybe out of half ways but we probably able to modulate more efficiently the immune function as well as increasing mitochondrial function.

Ben:  Okay, got you.

Philip:  It’s a controversial subject, Ben, about treating Lyme disease, and I know doctors, well intentioned, wonderful doctors that have lost their medical license by treating Lyme disease.  But I can say that for sure the NAD in many cases can help decrease some of the symptoms of Lyme disease.  We’re not treating the cause of Lyme disease which is antibiotics treating a bacteria but some of the symptoms can be lessened with NAD.

Ben:  Now Dr. Milgram and Dr. Grant, as we’re coming up towards the end here, is there anything else that you want to throw in as far as things that you find exciting in the realm of NAD research or the use of NAD as an anti-aging drug or for the use in other conditions?

Philip:  Well, even the MTHFR gene has improved with NAD.  The…

Ben:  When you say the MTHFR gene has improved with NAD, what do you mean?

Philip:  Well, again, this unspooling of the DNA, it may make the expression of the MTHFR gene change through a snip called C677T.

Ben:  And for people who are listening, can you explain this MTHFR gene?

Philip:  I’d rather like to talk about, more about some other things that I’m really excited about NAD.

Ben:  Okay.

Philip:  I have been doing addiction treatment for 25 years, and I’ve treated hundreds of patients, the good old-fashioned way of white knuckling it, and seeing them go through terrible withdrawal symptoms, through cravings, and the NAD virtually stops that.  It’s just absolutely amazing.  They have like a brain, people say they feel like they’ve never used before, or drank.  Their cravings are decreased.  It’s just amazing, and I’m really excited about, not only the anti-aging things that they’ve talked about, and there’s people like Dr. Watson, and if you have a chance to hear Dr. Watson talk from UCLA, he is extremely excited about the effects of NAD for an anti-aging.  And also, these different disease processes that we’ve before had no way to treat them.  Things like fibromyalgia, or chronic fatigue syndrome, Alzheimer’s disease, Parkinson’s disease, ALS.  They’re finding that NAD can make a difference in the therapy of these patients.  I think, in my opinion, and this is just an opinion, NAD will turn out to be one of the greatest advances in medical science since Fleming invented penicillin.

Ben:  Wow.  That’s quite the claim.  And in terms of people using NAD, you’re thinking that it’s gonna be just like these type of injections that you’re doing, that are gonna get the most efficacy?  Or are you just talking about NAD in general use as a supplement?

Philip:  I think you need to flood the body with NAD to start off with, and then we’re looking that this place that found out where, what is actually was in Dr. Hitt’s amino acid therapy from Mexico, it was pure NAD.  They are manufacturing a pure source of NAD that we are now using as supplementation.  And then you can also use the nicotinamide riboside, and other dietary things to enhance this.  And also it’s important that you do all of the things that, you’re expert in Ben, with the fasting, the mechanical stretch, the exercise, the low glucose, proper circadian rhythms, and sleep, all these things will enhance the effectiveness of your own NAD to work.

Ben:  Yeah, and I think that’s the key here whenever we talk about like a new anti-aging drug, or we talk about like this Scientific American article is they’ll talk about the use of things like resveratrol, or NAD, or nicotinamide, or many of these other compounds, and they’ll say that in terms of like mice and mitochondria, that the effects of NAD simulate the effect of calorie restriction, for example.  And the way I like to think about things is that you first do the lifestyle strategies, right.  Like consume more dark berries, blueberries, or grapes, or resveratrol containing compounds, engage in calorie restriction, expose your body to good amounts of oxygen, pay attention to things like air, and light, and water, and electricity, and all those other things that can affect mitochondrial variables.

I’m not saying you cover up a bad lifestyle with something like NAD injections, or the use of some of these NAD supplements that we’ve talked about, but it sounds like, especially as you age, and especially based off of what Dr. Grant said as you get past 60, if you want to engage in some type of anti-aging protocol, it sounds like this might be a smart one to throw into the mix based on actual research that’s been done on what this can do to everything from DNA to cellular damage.

Thomas:  Correct.

Ross:  Yeah.  Absolutely.

Philip:  And also, when Dr. Grant is the world’s expert on oxidative stress and NAD, and he brought up a point as we were talking about preparing for this conference.  He talked about, you know, if you have a lot of rust around, you have a lot of oxidation going around, the NAD’s not gonna be as effective.  So you have to have a healthy lifestyle which will enhance your NAD expression.

Ben:  Yeah.  Yeah.  Exactly.  As with a lot of these things we talked about here, you can’t cover up a bad lifestyle with a pill.  Well guys, first of all, Dr. Grant, I wanna thank you so much for coming on the show, as I know it’s extremely early over in Australia.

Ross:  You’re most welcome.

Ben:  And Dr. Milgram also.  Thank you for coming on and devoting your time.  And Thomas, thank you for your introduction to Dr. Milgram and Dr. Grant, and for kinda opening my eyes to NAD.

I have a couple more quick questions.  As far as any further resources Thomas, for folks, you had mentioned to me a book written by a guy named Dr. Nady Braidy, called “NAD+ Metabolism In Neurodegeneration and Ageing,” which I know is available on Amazon.  Is that what you would consider to be the best resource for people to delve more into NAD, or are there other places you would point people to?

Thomas:  I think that’s a great resource.  It’s heavy reading.  That was written by Dr. Grant’s student, and now colleague of Dr. Grant, and I think that would that’s a great place to start if you wanna go heavy into the science.  Dr. Milgram also has a blog at his website, nadtreatmentcenter.com, and the blog gets updated with new science as it unfolds.

Ben:  Okay.

Thomas:  So that’s nadtreatmentcenter.com.

Ben:  Cool.  I’ll link to that, and I’ll also link to everything else that we talked about including this Scientific American article, some of the resveratrol supplements that we discussed, the book by Nady Braidy, et cetera if you just go to bengreenfieldfitness.com/nad.  And if you go to bengreenfieldfitness.com/nad, that is also where you can leave any questions, any comments, any feedback that you have about today’s episode, or anything else that you wanna pipe in on in terms of your own personal experience with NAD, or other anti-aging protocols that we discussed in today’s show.  So gentlemen, thank you all for joining on the show today.

Philip:  Thank you.

Ross:  You’re welcome.

Thomas:  My privilege.

Ben:  Alright, folks.  So, this Ben Greenfield, along with Dr. Grant, Dr. Milgram, and Thomas Ingoglia signing out from bengreenfieldfitness.com.  Once again, you could check out the show notes at bengreenfieldfitness.com/nad.  Thanks for listening and have a healthy week.

The Ketone body β-Hydroxybutyrate a key signaling metabolite in disease and aging

However, in its oxidized form (NAD+), NAD is also a cofactor for sirtuin enzymes and poly-ADP-ribose polymerase (PARP), both of which consume NAD in the course of removing acyl groups from and adding poly-ADP to proteins, respectively. Sirtuins and PARP thereby regulate cellular functions ranging from gene expression and DNA damage repair to fatty acid metabolism (133). During times of fasting, or relative scarcity of cellular energy, more NAD is in the oxidized state, and sirtuins and PARP can be more active.

NAD+, a simple energy carrier, thereby acts as a fulcrum around which many cellular processes can be regulated in response to changes in the external environment. Such signaling metabolites include acetyl-CoA (coenzyme A), another carrier of high-energy bonds that is also substrate for a widely prevalent protein posttranslational modification (lysine acetylation), and S-adenosylmethionine, which similarly is substrate for a common posttranslational modification of histones and other proteins (methylation) (43).

Independent manipulation of these signaling molecules can recapitulate, or abrogate, some of the broader biological effects of environmental changes such as fasting or dietary restriction. For example, long-term dietary restriction can prevent the onset of common age-related hearing loss in C57BL/6 mice. However, dietary restriction in mice that carry a genetic knockout of the NAD-dependent sirtuin gene SIRT3 has no such beneficial effect (121).

Inhibition of the TOR (target of rapamycin) signaling complex by rapamycin (46), activation of AMPK by metformin (81), or provision of NAD+ precursors (159) recapitulates some of the beneficial effects of dietary restriction on diseases of aging and longevity. Understanding the specific molecular actions of these signaling pathways and signaling metabolites that link changes in the environment to broad regulation of cellular functions will permit researchers to more precisely capture the therapeutic potential of metabolic or dietary changes to treat disease. It might also help explain the heterogeneous responses of individuals to such environmental changes, depending on their genetic or epigenetic capacity to generate and respond to these signaling metabolites.

Here, we review the signaling activities of the endogenous metabolite β-hydroxybutyrate (BHB). BHB is the most abundant ketone body in mammals. Ketone bodies are small molecules synthesized primarily in the liver from fats that circulate through the bloodstream during fasting, prolonged exercise, and when carbohydrates are restricted.

They are taken up by tissues in need of energy, converted first to acetyl-CoA and then to ATP. Emerging evidence, however, shows that BHB not only is a passive carrier of energy but also has a variety of signaling functions both at the cell surface and intracellularly that can affect, for example, gene expression, lipid metabolism, neuronal function, and metabolic rate. Some of these effects are direct actions of BHB itself. Some are indirect effects governed by downstream metabolites into which BHB is converted, such as acetyl-CoA. We focus this review on BHB itself, referring to ketogenic diets only when necessary for translational context.

Although ketogenic diets have been widely used both for research into the effects of ketone bodies and as therapeutics for conditions ranging from epilepsy to obesity, a ketogenic diet is a complex physiological state with many possible active components of which BHB is only one. Still, the signaling effects of BHB we summarize are likely relevant to the molecular mechanisms of interventions such as fasting, dietary restriction, and ketogenic diets. Altogether, these observations present a picture of a powerful molecule that offers both opportunities and cautions in its therapeutic application to common human diseases.


Ketone bodies are small, lipid-derived molecules that provide energy to tissues when glucose is scarce, such as during fasting or prolonged exercise. Over 80% of the human body’s stored energy resides in the fatty acids contained in adipose tissue (7). During fasting, after muscle and liver stores of glycogen are depleted, fatty acids are mobilized from adipocytes and transported to the liver for conversion to ketone bodies. Ketone bodies are then distributed via blood circulation to metabolically active tissues, such as muscle or brain, where they are metabolized into acetyl-CoA and eventually ATP (7).

In humans, serum levels of BHB are usually in the low micromolar range but begin to rise to a few hundred micromolar after 12–16 h of fasting, reaching 1–2 mM after 2 days of fasting (13, 108) and 6–8 mM with prolonged starvation (12). Similarly, serum levels of BHB can reach 1–2 mM after 90 min of intense exercise (64). Consistent levels above 2 mM are also reached with a ketogenic diet that is almost devoid of carbohydrates (60). The term ketone bodies usually includes three molecules that are generated during ketogenesis: BHB, acetoacetate, and acetone. Most of the dynamic range in ketone body levels is in the form of BHB.

When ketogenesis is activated, such as during fasting, blood levels of BHB rise much faster than either acetoacetate or acetone (74).

Regulation of Ketone Body Metabolism 

The biochemistry of ketone body production and utilization is well understood and has been recently summarized both in the literature and in textbooks (e.g., 7, 66, 92) (Figure 1). Two points are particularly relevant to understanding the signaling activities of BHB. First, the same enzyme, β-hydroxybutyrate dehydrogenase (BDH1; EC, interconverts BHB and acetoacetate in both the final step of ketogenesis and the first step of BHB utilization.

BDH1 imparts chirality to BHB, as described below. Second, regulation of BHB synthesis is controlled via two principal mechanisms: substrate availability in the form of fatty acids and expression and activity of the enzyme HMG-CoA synthase (HMGCS2; EC Ketogenesis occurs mostly in the liver (7), although expression of HMGCS2 may be sufficient to produce ketogenesis in other tissues (127, 158). Insulin and glucagon regulate ketogenesis primarily by modulating the availability of fatty acid substrates at the levels of mobilization from adipose tissue and importation into hepatic mitochondria (71).

HMGCS2 gene expression is regulated by insulin/glucagon via acetylation and deacetylation of the FOXA2 transcription factor (143, 144, 135). FOXA2 deacetylation is controlled in part by the NAD-responsive enzyme SIRT1 (135). HMGCS2 gene expression is also regulated indirectly by the target of rapamycin complex mTORC1; mTORC1 inhibition is required for the activation of peroxisome proliferator-activated receptor alpha (PPARα) and fibroblast growth factor 21 (FGF21), both of which are required to induce ketogenesis (3, 4, 49, 116).

The activity of HMGCS2 is regulated posttranslationally by succinylation and acetylation, regulated by the mitochondrial desuccinylase SIRT5 (104) and deacetylase SIRT3 (117), respectively. Altogether, this network of regulation centered on HMGCS2, involving substrate availability, transcriptional control, and posttranslational modification, lends tight temporal and spatial precision to BHB synthesis.

BHB Transport 

BHB transport is relatively less well understood than its synthesis and utilization. As a small, polar molecule, BHB is readily soluble in water and blood (7). Several monocarboxylic acid transporters, including MCT1 and MCT2, carry BHB across the blood-brain barrier (99), and their expression can regulate brain BHB uptake (5). The monocarboxylate transporter SLC16A6 may be the key transporter for exporting BHB from the liver (52), but the putative transporters that facilitate the uptake of BHB into target tissues or its intracellular movement remain to be identified.

BHB Chirality 

BHB is a chiral molecule at the 3′ hydroxyl group, an important feature in consideration of its signaling activities and possible therapeutic applications. There are two enantiomers, R/D and S/L. R-BHB is the normal product of human and mouse metabolism. This chiral specificity is introduced by BDH1, which catalyzes the final step in BHB synthesis by reducing the nonchiral 3′ carbonyl group of acetoacetate to the chiral 3′ hydroxyl group of BHB.

BDH1 is also required for the utilization of BHB, by catalyzing the same reaction in reverse. As a result of the chiral specificity of BDH, only R-BHB is produced by normal metabolism and only R-BHB can be readily catabolized into acetyl-CoA and ATP. Fasting, exercise, caloric restriction, ketogenic diet, and any other state that results in endogenous production of BHB will produce only R-BHB.

S-BHB itself is not a normal product of human metabolism. However, S-BHB-CoA is a transient intermediate in the final round of β-oxidation of fatty acids. Under normal circumstances, it should not persist long enough to leave the mitochondrion or circulate in the blood. Experiments involving infusions of labeled R-BHB, S-BHB, or mixtures thereof into rats or pigs found that S-BHB is converted mostly to R-BHB (74); the molecular pathway for this is not known, but it may occur through conversion of S-BHB to acetyl-CoA and then production of R-BHB from acetyl-CoA.

At least some of the S-BHB is eventually converted to CO2, presumably also after being metabolized to acetyl-CoA. S-BHB is metabolized much more slowly than R-BHB is (138), so that infusion of the same amount of S-BHB may result in higher and more sustained blood levels of S-BHB compared with a similar infusion of R-BHB (19). We discuss the chiral specificity of BHB signaling activities below.


Several direct signaling actions of BHB have been described, including binding to cell-surface receptors, competitive inhibition of enzymes, as a substrate for protein posttranslational modification, and modulation of ion channel activity (Figure 2).

Schematic of direct and indirect signaling functions of the ketone body BHB. Indirect signaling functions require catabolism to other molecules, whereas direct signaling functions are actions of BHB itself. The major downstream effects of signaling functions are noted. Abbreviations: BHB, β-hydroxybutyrate; CoA, coenzyme A; FFAR3, free fatty acid receptor 3; GABA, γ-amino-butyric acid; HDAC, histone deacetylase; HCAR2, hydroxycarboxylic acid receptor 2; NAD, nicotinamide adenine dinucleotide; VGLUT, vesicular glutamate transporter.

BHB Inhibits Class I Histone Deacetylases 

BHB inhibits class I histone deacetylases (HDACs) (41), a family of proteins that have important roles in regulating gene expression by deacetylating lysine residues on histone and nonhistone proteins (reviewed in References 85, 90, and 150). Class I HDACs (e.g., HDAC1, HDAC2, HDAC3, and HDAC8) are small, mostly nuclear proteins that consist primarily of a deacetylase domain, and are usually found in large regulatory multiprotein complexes. Histone hyperacetylation is generally associated with activation of gene expression; so as a broad generality, class I HDAC activity suppresses gene expression. Many nonhistone proteins, including NF-κB, TP53, MYC, and MYOD1, among others (34), are also subject to HDAC-mediated deacetylation and regulation.

BHB inhibits HDAC1, HDAC3, and HDAC4 (classes I and IIa) in vitro with an IC50 of 2– 5 mM. BHB treatment of certain cultured cells induces dose-dependent histone hyperacetylation, particularly at histone H3 lysines 9 and 14 (118). Fasting, which increases plasma BHB levels, is associated with increases in histone acetylation in a number of mouse tissues by Western blot analysis (118), as well as in the liver by quantitative mass spectrometry (147). BHB can also increase histone H3 acetylation in vitro in macrophages (153) and neurons (120).

Treating mice with BHB via an osmotic pump increases histone hyperacetylation, particularly in the kidney, and causes specific changes in gene expression, including induction of forkhead box O3 (Foxo3a), the mammalian ortholog of the stress-responsive transcriptional factor DAF16 that regulates life span in C. elegans (59). Induction of Foxo3a appears to be a direct effect of HDAC inhibition, as HDAC1 and HDAC2 are found on its promoter, knockdown of both relieves HDAC-mediated Foxo3a repression, and BHB causes hyperacetylation of histones at the Foxo3a promoter that results in increased Foxo3a expression (118).

BHB regulates Bdnf (brain derived neurotrophic factor) expression in the mouse brain by a similar mechanism after exercise. Exercise increases BHB levels and Bdnf expression in the hippocampus, and Bdnf expression is increased by treating hippocampal slices with BHB or by infusing BHB intraventricularly. Treating primary neurons with BHB increases histone H3 acetylation, reduces Bdnf promoter occupancy by HDAC2 and HDAC3, and increases Bdnf expression (120).

Inhibition of class I HDACs appears to be a direct effect of BHB. Enzymatic inhibition in vitro is observed using immunopurified FLAG-tagged HDAC proteins with a synthetic peptide substrate, which should provide no opportunity for BHB metabolism or secondary effects (118). Competitive inhibition of the catalytic site is the likely mechanism.

Crystal structures of the human class I HDAC8 bound to several hydroxamic acid inhibitors (122, 130), as well as modeling of other inhibitors, show that a carbonic or hydroxamic acid group of an inhibitor is commonly bound to the catalytic zinc at the bottom of the hydrophobic active site channel of the HDAC (137). BHB is structurally similar to the canonical HDAC inhibitor butyrate.

Butyrate demonstrates the kinetics of a competitive inhibitor (115), suggesting that its carboxylic acid group might chelate the catalytic zinc in a manner similar to that of other acidic moieties on HDAC inhibitors. The structures of butyrate, BHB, and acetoacetate differ only in the oxidation state of the 3′ carbon. Increasing oxidation may be a barrier to binding within the hydrophobic channel of the HDAC active site, and as expected, the IC50 of the three compounds for HDAC1 increases with the oxidation state (115, 118).

β-Hydroxybutyrylation of Proteins 

In addition to inhibiting enzymes involved in the regulation of protein posttranslational modifications such as HDACs, BHB can itself modify proteins at the posttranslational level. Lysine β-hydroxybutyrylation [K(BHB)] was detected via mass spectrometry as a histone modification in yeast, fly, mouse, and human cells (147). Western blot analysis detected that histone K(BHB) increases in human cells in proportion to treatment with exogenous BHB, and increases in mouse liver in proportion to increases in plasma BHB levels with either fasting of normal mice or induction of diabetic ketoacidosis with streptozotocin. At least 40 BHB-modified histone lysine sites were detected in human cells or mouse liver by mass spectrometry, including sites critical for transcriptional regulation such as H3K9. Fasting increased the relative abundance of K(BHB) at these sites by up to 40-fold (147).

Chromatin immunoprecipitation (ChIP) with specific antibodies also revealed that K(BHB) of H3K9 is preferentially associated with the promoters of actively transcribed genes (147). A comparison of ChIP data and RNAseq expression data from fasted mouse liver found a strong correlation between genes with the greatest increase in expression and genes with the greatest increase in K(BHB) at the promoter.

H3K9 K(BHB) is correlated with other activation markers, such as acetylated H3K9 and trimethylated H3K4, but has an association with gene activation independent of these other two markers, suggesting an independent function (147). What that function is remains to be determined. The scope of β-hydroxybutyrylation as a posttranslational modification is not clear: It is not known whether nonhistone proteins are modified, or whether K(BHB) exists in organs other than the liver.

It is also not known whether any enzymes catalyze the addition or removal of K(BHB), providing opportunities for specific targeting and regulation. The wide diversity of lysine acylations removed by the various mammalian sirtuins (1) hints that hydroxybutyrylation might be removed by a specific sirtuin. In all, histone K(BHB) may be the nexus of an important new network of gene regulatory activity associated with fasting and other conditions linked to increase BHB production.

Cell Surface Receptors: HCAR2 and FFAR3 

BHB binds to at least two cell surface G-protein-coupled receptors (GPCRs), HCAR2 (hydroxycarboxylic acid receptor 2) and FFAR3 (free fatty acid receptor 3). These are among several GPCRs with fatty acid ligands that have important roles in metabolism and metabolic disease (9, 70). HCAR2 (also known as HCA2, PUMA-G, and Gpr109) is a Gi/o-coupled GPCR that was first identified as a nicotinic acid receptor (129). It was later shown to bind and be activated by BHB, with an EC50 of 0.7 mM (124).

HCAR2 activation reduces lipolysis in adipocytes (95, 124), which might perhaps represent a feedback mechanism to regulate the availability of the fatty acid precursors of ketone body metabolism. However, elevated levels of free fatty acids in plasma from dysregulated adipocytes are also thought to contribute to insulin resistance through a variety of mechanisms, including proinflammatory cytokines, oxidative stress, and endoplasmic reticulum stress (10).

Pharmacological agonists of HCAR2 reduce both plasma free fatty acids and plasma glucose levels in humans with type 2 diabetes (21). Infusion of BHB has long been observed to reduce the concentrations of nonesterified fatty acids in plasma, even when insulin levels (which also regulate fatty acid release from adipocytes) are held constant (108). In retrospect, this reduction of free fatty acids in plasma may be due to HCAR2 activation.

HCAR2 is also expressed in a variety of other cell types, including immune cells, microglia, and colonic epithelial cells, in which its activation induces anti-inflammatory effects (38). In the central nervous system, this is associated with neuroprotection mediated in part by the activation of IP3dependent intracellular calcium release, alterations in prostaglandin production, and downstream inhibition of NF-κB activation (reviewed in Reference 96).

More specifically, HCAR activates a neuroprotective subset of macrophages via the production of PGD2 by COX1 (103). The neuroprotective effect of ketogenic diet in a mouse stroke model is abrogated in Hcar2 knockout mice, and it appears that activation of HCAR2 on brain-infiltrating macrophages/monocytes is crucial to this neuroprotection (103). Finally, HCAR2 activation in neurons can potentiate glutaminergic signaling, which in one particular region of the brain helps regulate blood pressure and heart rate (106).

Activation of HCAR2 in the gut epithelium by short-chain fatty acids produced by bacterial fermentation of dietary fiber activates the NLRP3 inflammasome and maintains gut membrane integrity (79). The mechanism for inflammasome activation appears to be potassium efflux stimulated by intracellular calcium release. Inflammasome activation is beneficial in mouse models of colitis, and appears to explain the benefit of a high-fiber diet in these conditions, but stands in interesting contrast to the role of BHB in suppressing NLRP3 inflammasome activation described below.

BHB is also a ligand for FFAR3 (also known as GPR41). FFAR3 is another Gi/o-proteincoupled receptor that is highly expressed in sympathetic ganglions (62) throughout the body of mice (94). Ffar3 knockout mice have reduced basal oxygen consumption and body temperature but are then insensitive to the usual further sympathetic depression seen during prolonged fasting (62).

Antagonism of FFAR3 by BHB suppresses sympathetic tone and heart rate and may be responsible for the sympathetic depression commonly seen during fasting (62). However, an electrophysiological study in rats later reported that BHB in fact acts as an agonist of FFAR3 (145), which regulates voltage-dependent calcium channels. In fact, other endogenous short-chain fatty acids, including acetate, butyrate, and propionate, have been reported as agonists of FFAR3 (11). Further work is needed to confirm the activity of BHB on FFAR3, but a substantial literature is emerging on other biological functions of FFAR3 relevant to human health.

One such function is glucose homeostasis. Deletion of Ffar3 (together with Ffar2) in mouse pancreatic β cells improves insulin secretion and glycemic control on a high-fat diet (125). Genetic gainand loss-of-function neurotransmitter models of Ffar3 alone similarly show that lower FFAR3 signaling increases insulin secretion from
pancreatic β islet cells (132). An alternative route by which FFAR3 affects glucose metabolism
is via activation by gut-derived propionate in the periportal afferent neural system. This feeds

into a brain-gut signaling circuit that induces intestinal gluconeogenesis, with beneficial effects on glucose and energy homeostasis (17).

FFAR3 also regulates inflammation. Activation of FFAR3 by gut-derived propionate helps rescue allergic airway inflammation in mice by reducing the capacity of lung-resident dendritic cells to promote TH2 -mediated inflammation (128). Similarly, activation of FFAR2 and FFAR3 by acetate suppresses the expression of inflammatory cytokines in human monocytes, which suggests FFAR2/3 agonists may help ameliorate inflammatory bowel diseases (2). As these examples illustrate, whether activation or inhibition of FFAR3 is beneficial to health may be highly dependent on the specific tissue and disease contexts.

Membrane Channel and Transporter Regulation 

Two threads of indirect evidence imply a role for BHB in the modulation of potassium flux across the plasma membrane. Inhibition of potassium efflux is the suggested mechanism by which BHB inhibits activation of the NLRP3 inflammasome (153). An extensive series of careful in vitro experiments excluded other known direct and indirect signaling functions of BHB, including HDACs, HCAR2, and metabolism to acetyl-CoA. The event most upstream of inflammasome activation that is affected by BHB treatment is prevention of the decline in intracellular potassium in response to inflammasome-activating signals (153). This effect is the reverse of HCAR2 activation stimulating potassium efflux.

Application of acetoacetate or R-BHB to brain slices reduces the rate of firing from γ-aminobutyric acid (GABA)ergic neurons, and pharmacological inhibition of ATP-sensitive K (KATP) channels or knockout of the Kir6.2 subunit abrogates the effect (78). Although this might suggest that BHB could increase K channel opening, several lines of evidence indicate that the change in KATP activity is more likely an indirect effect of the absence of glucose on local intracellular ATP concentrations.

The nonmetabolized enantiomer S-BHB did not have a similar effect on neuron firing (78); a later study showed that the change in KATP channel opening is due to reduced glycolysis in the presence of BHB (76) and that providing sufficient BHB and oxygen to maintain normal cellular ATP production can prevent the change in KATP activity (77).

BHB appears to have a direct regulatory effect on the neuronal vesicular glutamate transporter VGLUT2 (56). Cl− is an allosteric activator of glutamate uptake in an in vitro system consisting of proteoliposomes containing purified VGLUT2. Acetoacetate, BHB, and pyruvate inhibit this Cl−-dependent glutamate uptake, with kinetics indicating competition for the Cl− binding site. All these effects require the anions to be exposed to the extravesicular side of the membrane transporter.

Acetoacetate is >10-fold more potent than BHB at inhibiting VGLUT2, but the IC50 of 3.75 mM for BHB still suggests that biologically relevant effects on glutamate uptake might occur at physiological low-millimolar BHB concentrations. Cl−-dependent activation of other VGLUT transporters were similarly inhibited by acetoacetate, but the vesicular GABA transporter VGAT was not.

Consistent with these biochemical findings, acetoacetate inhibits glutamate release from neurons in a manner suggesting a reduced vesicular quantity of glutamate (56). In other contexts, however, BHB enhances glutaminergic transmission by increasing neurotransmitter release (120). Nevertheless, VGLUT inhibition may be a mechanism by which BHB can reduce excitatory glutamate neurotransmission without affecting inhibitory GABA neurotransmission.

Enantiomeric Specificity of Direct Signaling Activities 

As noted above, BHB is a chiral molecule, and R-BHB is the enantiomer generated and readily consumed in normal mammalian metabolism. Signaling functions or other effects that depend on the rapid catabolism of BHB therefore are relevant only to the R-enantiomer. Signaling functions that are direct actions of BHB, however, might be recapitulated in part or in full by S-BHB depending on the stereoselectivity of the proteins involved. Indeed, several of the direct signaling functions described here have been reported to be nonstereoselective in the literature.

S-BHB can bind the GPCR HCAR2, albeit with somewhat lower affinity than R-BHB. An in vitro assay with modified Chinese hamster ovary cells expressing human HCAR2, using calcium flux as the readout, found that both R-BHB and S-BHB showed robust, similar receptor activation at concentrations of 15 mM. A quantitative binding assay using radiolabeled GTP to measure nucleotide exchange found that the EC50 was 0.7 mM for R-BHB and 1.6 mM for S-BHB with human HCAR2, and 0.3 mM and 0.7 mM, respectively, with mouse HCAR2. Acetoacetate was inactive (124). The chirality of BHB used to test activation of FFAR was not reported (62).

S-BHB can also block inflammasome activation (153). The use of caspase-1 activation in lipopolysaccharide-treated bone marrow–derived macrophage cells as an in vitro assay of NLRP3 inflammasome activation showed that S-BHB effectively blocks inflammasome activation, albeit perhaps with lower efficacy than R-BHB.

The stereoselectivity of HDAC inhibition by BHB remains to be explored, but if, as described above, the relevant biochemical action is chelation of a catalytic zinc at the base of a hydrophobic channel, then the chirality of the trailing 3′ hydroxyl group may not be a critical determinant of activity. There is at least one suggestion that both R-BHB and S-BHB can increase histone acetylation in vitro (154).

The chirality of histone K(BHB) may be important to its biological function. The method used to detect K(BHB) could not distinguish modification with R-BHB from modification with S-BHB (147), but it is likely that both K(S-BHB) and K(R-BHB) are present. First, K(BHB) was detected in yeast and flies, and although generation of S-BHB-CoA as an intermediate of lipid β-oxidation is a conserved pathway in these metazoans, mitochondrial ketogenesis of R-BHB is not. Nor is Saccharomyces cerevisiae known to use polymers of R-BHB as an energy store, as is common in prokaryotes (18).

Second, although fasting increases synthesis of R-BHB in mouse liver, it also increases flux of S-BHB-CoA as an intermediary of fatty acid oxidation. A number of factors probably influence whether K(R-BHB) or K(S-BHB) predominates. By analogy with other protein acylations, K(BHB) is likely formed from CoA-activated BHB. β-Oxidation produces SBHB-CoA from an acyl-CoA precursor.

Free R-BHB (as well as free S-BHB) must undergo enzymatic activation by a CoA synthase, which has long been observed to occur (108), although the precise CoA synthase involved and cellular compartment in which the activation occurs are unknown. The balance of K(R-BHB) versus K(S-BHB) in the liver might therefore depend on the rate and site of activation of R-BHB and on the efficiency of extramitochondrial transport of both activated forms.

The balance in extrahepatic tissue might also depend on the relative utilization of fatty acids versus BHB for energy. Neurons, for example, which do not utilize fatty acids (7), would likely favor K(R-BHB) under ketogenic conditions. Whether the chirality of K(BHB) matters depends on its biological function, which is not yet understood. If the function is simply to occupy the lysine site and prevent alternative modifications, the chirality may be irrelevant. If the function is to serve as a binding site for other proteins, the 3′ hydroxyl group of BHB should remain both chiral and accessible to influence the binding of any putative K(BHB) recognition proteins.


In addition to its direct signaling activities, BHB might exert additional signaling effects in the course of its catabolism to acetyl-CoA and ATP. These reflect the signaling activities of other intermediate metabolites, including acetyl-CoA, succinyl-CoA, and NAD. Finally, catabolism of BHB can alter the steady state of downstream metabolic pathways, such as those that regulate neurotransmitter synthesis. When considering the possible medical applications of BHB precursors, note that R-BHB should be much more potent at eliciting these indirect signaling actions than S-BHB is, as S-BHB is catabolized much more slowly and through a different route.

Production of Acetyl-CoA, Substrate for Protein Acetylation 

Catabolism of BHB into acetyl-CoA should raise intracellular acetyl-CoA levels, favoring both enzymatic and nonenzymatic protein acetylation. This effect complements HDAC inhibition by BHB but may have broader effects in multiple cellular compartments. Protein acetylation in mitochondria appears to be particularly sensitive to acetyl-CoA flux, as a variety of states associated with increased lipid utilization—including dietary restriction, fasting, and high-fat diets—increase mitochondrial protein acetylation. This effect on mitochondrial acetylation occurs despite the fact that neither acetyltransferases nor the HDACs that are inhibited by BHB enter mitochondria (48). Acetylation—and deacetylation by SIRT3—is widespread in mitochondria (105) and regulates the function of several mitochondrial enzymes, including HMGCS2 (117) and the long-chain acylCoA dehydrogenase (51).

Increased acetyl-CoA pools also affect nuclear protein acetylation. Mitochondrial acetylcarnitine is a source of acetyl-CoA for histone acetylation (80). Export of acetyl-CoA from the mitochondria is accomplished via a citrate shuttle mediated by citrate synthase inside mitochondria and ATP citrate lyase inside the cytoplasm (140).

ATP citrate lyase is a key enzyme in fatty acid biosynthesis, but its role in facilitating acetyl-CoA export from mitochondria is also required for the increase in histone acetylation that occurs with growth factor stimulation (140). An alternative pathway for acetyl-CoA export from mitochondria is via the enzymes carnitine acetyltransferase and carnitine/acylcarnitine translocase (89). Indeed, a muscle-specific knockout of carnitine acetyltransferase in mice compromises glucose tolerance and decreases metabolic flexibility (89), demonstrating the importance of intracellular acetyl-CoA transport to overall metabolic health.

Consumption of Succinyl-CoA, Substrate for Protein Succinylation 

Utilization of BHB in peripheral tissues uses succinyl-CoA to donate CoA to acetoacetate. This consumption of succinyl CoA may affect the balance of lysine succinylation, which, like acetylation, is widespread in mitochondria (104) and present across diverse organisms (139). A substantial fraction of these succinylation sites are regulated by the mitochondrial desuccinylase, the sirtuin SIRT5 (104). HMGCS2 has long been known to be succinylated, a modification that reduces its activity (102); HMGCS2 enzymatic activity in the liver is suppressed by succinylation and restored by desuccinylation mediated by SIRT5 (104). The mechanism of lysine succinylation is not clearly understood; succinyltransferase is not known to exist in mammalian cells, and because both liver succinyl-CoA abundance and succinylation of HMGCS2 are reduced in rats after treatment with glucagon (101, 102), it is possible that succinylation is primarily a nonenzymatic process dependent on local concentrations of succinyl-CoA. Although HMGCS2 itself is not expressed in the peripheral tissues that utilize BHB, enzymes in many other key mitochondrial pathways, including fatty acid oxidation, branched-chain amino acid catabolism, and the tricarboxylic acid (TCA) cycle, are heavily succinylated and regulated by SIRT5 (104). By analogy with acetylation (and the effect of succinylation on HMGCS2), these pathways may be activated by a reduction in succinylation. Consumption of succinyl-CoA during BHB utilization and consequent reduction in mitochondrial protein succinylation may therefore regulate many of these crucial mitochondrial pathways in peripheral tissues, perhaps favoring the switch to lipid-dependent energy usage.

Cytoplasmic and Mitochondrial NAD:NADH Equilibrium 

Cellular NAD balance is emerging as a crucial factor in metabolic disease and aging (123). NAD utilization during BHB metabolism differs from that during glucose metabolism in two important respects. Fewer NAD+ molecules are consumed per acetyl-CoA produced when BHB is used than when glucose is used, and the cellular compartment in which NAD+ is consumed is different. Metabolism of one molecule of glucose to two molecules of acetyl-CoA involves conversion of four molecules of NAD+ into NADH. Two of these molecules are converted in the cytosol during glycolysis; the other two are converted in the mitochondrion by pyruvate decarboxylase. The cytosolic NADH are shuttled into mitochondria, potentially depleting the cytoplasmic NAD+ pool with high glucose utilization.

By contrast, metabolism of one BHB molecule to the same two molecules of acetyl-CoA involves conversion of only two molecules of NAD+ into NADH, both in the mitochondrion by BDH1, thereby preserving the cytoplasmic NAD+ pool (7). The cytoplasmic and mitochondrial NAD pools are relatively distinct, so the preservation of cytoplasmic NAD+ by BHB may have important cellular effects. NAD+ is a cofactor for sirtuin deacylases (such as nuclear/cytoplasmic SIRT1) as well as poly-ADP-ribose polymerase (PARP) (133).

Consumption of NAD+ by PARP or overproduction of NADH may promote age-related diseases by decreasing the activity of sirtuins (53). Conversely, repletion of NAD+ by exogenous feeding with nicotinamide mononucleotide improves glucose tolerance in both high-fat-diet-fed and aged mice (152). The relative sparing of NAD+ by utilizing BHB vis a` vis glucose may therefore have important consequences for metabolic diseases and diabetes.

In the mitochondrion, BHB utilization is both determined by and changes the NAD:NADH equilibrium, with implications for signaling. The mitochondrial BHB:AcAc equilibrium is so closely linked to the NAD:NADH equilibrium that early metabolic studies of the liver used the former as a proxy for the latter (65). This tight relationship suggests that any influence that pushes the mitochondrial NAD:NADH equilibrium toward NADH (such as consumption of NAD by sirtuins or reduced activity of NADH dehydrogenase/complex I) will reduce BHB consumption. This reduction could increase the local concentration of BHB available for direct signaling functions within and around the mitochondrion, but also potentially reduce the flux of BHB transiting the cytoplasm and nucleus toward mitochondria.

However, BHB utilization also affects the mitochondrial redox state. Increased utilization of BHB is associated with pushing the NAD:NADH equilibrium toward NADH, as well as increasing the oxidation state of coenzyme Q (114). Owing to the increased heat of combustion of BHB compared with that of pyruvate, BHB also increases the efficiency of ATP production from the mitochondrial proton gradient and reduces the production of free radicals (131). Changes in free radical production might alter the activity of signaling networks that sense and respond to mitochondrial free radicals, such as p66Shc (33).

Neurotransmitter Synthesis 

The potential mechanisms of action of ketogenic diets in treating epilepsy remain complex and controversial and have been the subject of several thorough reviews (47, 83, 154). One mechanism consistently proposed, however, involves how the downstream effects of BHB catabolism on the abundance or flux of other intermediate metabolites might alter the biosynthesis of the inhibitory neurotransmitter GABA.

The biosynthesis of GABA in inhibitory GABAergic neurons begins with the synthesis of glutamine in astrocytes. Glutamine is exported from astrocytes to neurons, where it undergoes conversion to glutamate and then decarboxylation to GABA. An alternative fate for glutamate in neurons is donation of its amino moiety to oxaloacetate, producing aspartate and α-ketoglutarate. Studies of isotopically labeled BHB show that it is used as a substrate for the synthesis of glutamine and other amino acids (156). Data from clinical studies of children on ketogenic diet for epilepsy show that cerebrospinal fluid GABA levels are higher on ketogenic diet, and the highest levels correlate with best seizure control (16).

BHB may affect GABA production via increased synthesis and/or pushing the fate of glutamate toward GABA and away from aspartate. Studies in synaptosomes show that BHB increases the content of glutamate and decreases that of aspartate (25). Studies of ketogenic diet in rodents similarly show that less glutamate is converted to aspartate (156). In cultured astrocytes, the presence of acetoacetate reduces the conversion of labeled glutamate to aspartate (155). Infusion of labeled BHB into rats fed a normal diet rapidly increases the levels of all components of this pathway (glutamine, glutamate, GABA, and aspartate) (112).

Even when ketotic states are not associated with an increase in overall GABA levels, the proportion of glutamate shunted to GABA production is increased (84). The reason for this shunt may be the effect of BHB catabolism on TCA cycle intermediates. The relatively greater efficiency of BHB at generating acetyl-CoA compared with that of glucose, described above, increases the flux of acetyl-CoA through the TCA cycle. This increases the proportion of oxaloacetate required to condense with acetyl-CoA to permit its entry into the TCA cycle, reducing the availability of free oxaloacetate to participate in glutamate deamination to aspartate. By indirectly tying up oxaloacetate, BHB pushes the fate of glutamate toward GABA (154). Altogether, one effect of BHB catabolism, alone or as part of a ketogenic diet, appears to increase the capacity of GABAergic neurons to rapidly generate GABA from glutamate (154).

Although BHB is structurally reminiscent of GABA itself, evidence of a direct effect of BHB on activating GABA receptors is lacking. Neither BHB nor acetoacetate alters GABA currents in cultured rodent cortical neurons (22) or in rat hippocampal neurons (126). BHB did enhance the function of GABAA receptors expressed in Xenopus oocytes, but only modestly and at concentrations of 10 mM or higher (149), leaving the physiological significance of this effect unclear.

However, GABOB (γ-amino-β-hydroxybutyric acid) is an endogenous agonist of GABA receptors that differs structurally from BHB only in the presence of the γ-amino moiety. It is biochemically plausible that BHB might be a direct substrate for GABOB synthesis, but no such aminotransferase is known to exist. Nor has any pathway for conversion of GABA to GABOB yet been identified in mammals. Of interest in consideration of BHB precursors as therapeutics, the enantiomer of GABOB that would be derived from S-BHB has the more potent antiepileptic affect and is a stronger GABAB receptor agonist (148).


The integration of the various direct and indirect signaling functions of BHB appears to broadly help the organism adapt to a fasting state. The transition to a fasting state is already under way when ketogenesis is activated in the liver. BHB production might further promote that transition in extrahepatic tissues while also fine-tuning the control of lipid and glucose metabolism.

The combinatorial effects of BHB on gene expression, described below, might be expected to generally facilitate the activation of new transcriptional programs. The enrichment of histone K(BHB) at genes activated by fasting suggests that this might be particularly important for activating fasting-related gene networks, although this could also reflect a nonspecific association with activated transcription.

Inhibition of class I HDACs by BHB could play a major role in metabolic reprogramming, according to studies of HDAC knockout mice and of HDAC inhibitors. HDAC3 regulates expression of gluconeogenic genes (86), and HDAC3 knockout mice have reduced fasting glucose and insulin levels (8, 26, 63). In fact, chronic treatment with the HDAC inhibitor butyrate essentially keeps mice metabolically normal on a high-fat diet, with lower glucose and insulin levels, better glucose tolerance, reduced weight gain, and improved respiratory efficiency (32). Butyrate also provides some of these benefits even to mice already obese from being fed a high-fat diet (32).

Similarly, inhibition of class I HDACs, but not class II HDACs, increases mitochondrial biogenesis, improves insulin sensitivity, and increases metabolic rate and oxidative metabolism in a mouse diabetes model (31). The mechanism for these metabolic benefits of class I HDAC inhibition may be upregulation of PGC1α (Ppargc1a) in in a variety of tissues by relief of HDAC3-mediated transcriptional repression (31, 32). Transcription of Fgf21 is similarly upregulated via inhibition of HDAC3 by butyrate, activating ketogenesis in obese mice (72). Several single nucleotide polymorphisms in HDAC3 have been associated with an elevated risk of type 2 diabetes in a Chinese population (157).

Activation of HCAR2 reduces lipolysis in adipocytes; because the availability of fatty acids in the liver is a critical determinant of ketogenesis (and is strongly regulated by insulin), this may provide a self-feedback mechanism to limit the production of BHB. Why this mechanism is insufficient to prevent dysregulated BHB production in insulin-deficient states such as diabetic ketoacidosis, and whether it could be potentiated to treat such states, such as through HCAR2 agonists, is unclear.

HCAR2 was originally identified as a niacin receptor, spurring efforts to develop more specific agonists to capture the therapeutic benefits of niacin on cardiovascular risk or glycemic control that were thought to be due to HCAR2’s effects on the levels of free fatty acid in blood. An HCAR2 agonist, GSK256073, transiently reduces levels of free fatty acids in blood but has only a modest effect on glycemic control in type 2 diabetes mellitus (20). Two other HCAR2 agonists similarly lowered free fatty acids but without otherwise altering the lipid profile in humans, while niacin was found to produce beneficial changes to the lipid profile in Hcar2 knockout mice (69).

Thus, the model that niacin (and by extension BHB) exerts beneficial effects on cardiovascular risk through activation of HCAR2 in adipose tissue is probably too simplistic. It may even be the case that other cell types such as macrophages might mediate the therapeutic effects of HCAR2 agonists (30).

Interpreting the metabolic effects of BHB mediated by FFAR3 depends on whether BHB acts as an agonist or antagonist, and in which contexts. Strong evidence suggests that BHB antagonizes FFAR3 to reduce sympathetic activity, resulting in reduced heart rate, body temperature, and metabolic rate. If BHB also antagonizes FFAR3 in other contexts, it could improve insulin secretion from pancreatic β islet cells and impair intestinal gluconeogenesis. Altogether, these findings suggest that BHB would improve glycemic control, though a decrease in metabolic rate could be obesogenic.

The important role of the NLRP3 inflammasome in regulating obesity-associated inflammation and metabolic dysfunction has been extensively reviewed (15, 45). Briefly, the NLRP3 inflammasome appears to mediate an inflammatory response to nutrient excess and mitochondrial dysfunction. Mice deficient in NLRP3 are grossly normal when fed chow but are protected from obesity and insulin resistance when fed a high-fat diet. One proposed mechanism is through a reduction in inflammasome-induced IL-1β, which otherwise inhibits insulin signaling in adipocytes and hepatocytes while inducing pancreatic β-cell dysfunction.

While NLRP3 may have an important homeostatic role in the response to day-to-day nutrient fluctuations, its chronic activation by nutrient excess may contribute to the development of metabolic disease—and inhibition of the NLRP3 inflammasome by BHB might ameliorate these maladaptations.


Regulation of gene expression is the most common theme that emerges from the direct and indirect signaling functions of BHB. Histones are at the nexus of this theme, with their posttranslational modifications, including acetylation, succinylation, and β-hydroxybutyrylation. Histone acetylation is a well-understood mechanism for both broad and specific regulation of gene expression, and BHB can alter histone acetylation by directly inhibiting HDACs and by indirectly promoting acetyltransferase activity via acetyl-CoA flux.

The biology of histone β-hydroxybutyrylation largely remains to be elucidated but is an area that should receive strong interest for its potential relevance to gene expression reprogramming in response to metabolic stimuli. Lysine succinylation is another histone posttranslational modification (146) and is regulated by the sirtuin SIRT7 in the context of the DNA damage response (73). Finally, alterations in cytoplasmic and nuclear NAD+ levels can affect the activity of sirtuins, which deacylate a number of histone tail residues (54).

The net integration of all these effects might be expected to facilitate gene transactivation, particularly of quiescent genes that are activated in response to stimuli. Two well-characterized examples of how HDAC inhibition (and acetyltransferase activation) can activate gene expression include reactivation of HIV latency and activation of lineage-specific genes during muscle differentiation. The HIV provirus sits in a transcriptionally inactive state in resting T cells, providing a reservoir of potential virus production that is impossible for current antiretroviral therapy to eradicate (44).

The class I HDACs HDAC1, HDAC2, and HDAC3 help maintain this transcriptionally silenced state, as components of several transcriptional repressor complexes, by deacetylating histone tails. Ordinarily, a key step in the reactivation of HIV transcription is the recruitment of complexes containing the acetyltransferase p300 to acetylate histone tails. Inhibition of HDACs with vorinostat or panobinostat also promotes histone acetylation, resulting in the loss of latency and reactivation of HIV transcription (44). In vivo, such HDAC inhibitors broadly reactivate transcription from a diverse pool of latent proviruses (6).

The differentiation of a satellite cell (i.e., muscle stem cell) into the muscle lineage involves the transcriptional activation of a sequence of differentiation-promoting transcription factors (see Reference 119 for a review). One of these factors, MYOD1, further activates a wide range of muscle-specific genes. HDACs, including HDAC1 and HDAC2, reside at the promoters of many of these MYOD1 targets, maintaining histones in a deacetylated state.

Activation of the genes requires dissociation of the HDAC from the promoter, and often recruitment of a p300 complex to promote histone acetylation. HDAC inhibitors potentiate this process, much as in HIV latency, and both increase acetylation at MYOD1 target promoters and increase myogenic differentiation. One contrast with HIV latency is that many gene promoters in satellites cells are maintained in a poised or bivalent state, possessing both activating and repressing histone modifications (119).

Thus, whereas HIV latency is an example of HDAC inhibition waking a gene from deep silencing, muscle differentiation is an example of potentiating or lowering the threshold of activation for a gene already poised to do so. A similar example of activating poised genes may be relevant to cognition and dementia (see below).

How histone K(BHB) participates in epigenetic regulation largely remains to be determined. K(BHB) was particularly enriched after fasting at genes that were upregulated by fasting (147). Other stimuli or contexts expected to change gene expression were not examined, nor were cells or animals treated with BHB in the context of such stimuli.

Thus, we do not yet know whether K(BHB) is specifically associated with a gene network that is responsive to fasting, or whether it is generally associated with newly transactivated promoters. We also have yet to understand the biological meaning of K(BHB)—whether it is an active signal that stimulates transcription through regulatory or binding interactions with the protein machinery of chromatin remodeling and transactivation, or whether it is a bystander modification emplaced at fasting-activated promoters owing to the confluence of increased BHB-CoA levels and newly accessible histone tails.

An additional layer of regulation can occur on nonhistone proteins involved in gene expression, as these posttranslational modifications are not restricted to histones. Acetylation and succinylation occur throughout the proteome and can affect protein function. It is possible that β-hydroxybutyrylation will be found to as well. Acetylation of nonhistone proteins is a critical step in both HIV latency and muscle differentiation, where acetylation activates the transcription factors Tat (44) and MYOD1 (119), respectively. The multilayered effects of BHB on gene expression can be illustrated with HMGCS2 itself, the rate-limiting enzyme in BHB synthesis. As described above, the transcription factor FOXA2 helps control Hmgcs2 transcription.

FOXA2 is itself acetylated by EP300 in a reaction using acetyl-CoA as the acetyl group donor (increased by BHB). FOXA2 is deacetylated by a class I HDAC (inhibited by BHB) and/or by the sirtuin SIRT1, which requires NAD+ as a cofactor (increased by BHB). Initiation of Hmgcs2 transcription by activated FOXA2 might involve displacing or deactivating HDACs (inhibited by BHB) to permit emplacement of activating histone marks such as H3K9 acetylation by acetyltransferases (via acetyl-CoA increased by BHB). As this example illustrates, the overall impact of BHB might generally favor initiation or upregulation of transcription, but the effects on any specific loci could vary depending on the posttranslational regulation of the proteins involved at that site.


The ketogenic diet has been clinically used for decades to treat epilepsy and currently has a wide range of therapeutic applications, mostly in childhood epilepsies (142). Despite this extensive clinical history, the mechanism of action of the ketogenic diet remains controversial (107). In fact, whether BHB itself is necessary or even active in the therapeutic effect of ketogenic diets is controversial, and the evidence varies between animal models (107). The various possible mechanisms of the antiepileptic effect of ketogenic diets have been reviewed (83, 107).

Several of the signaling activities described above may be relevant, particularly modulation of potassium channels, FFAR3 activation, and promotion of GABA synthesis. Epigenetic modifications may also contribute to neuronal hyperexcitability and the long-term effects of epilepsy on the brain through persistent changes in gene expression (111). REST (RE1-silencing transcription factor) is a transcriptional repressor that recruits HDACs, among other chromatin-modifying enzymes, to help silence target genes.

REST expression is increased in neurons after seizures and promotes aberrant neurogenesis. However, whether REST activity is helpful or harmful for seizure control differs in different seizure models (111). The contribution of epigenetic modifiers, including those that are modulated by BHB, to epilepsies and their long-term effects requires much further study.

Ironically, given the decades of study on the role of ketogenic diets in epilepsy, more molecular detail is known about the potential mechanisms of BHB in ameliorating dementia. Two major threads link BHB signaling with dementia: epigenetic modifications and neuronal hyperexcitability. There is a growing literature on the importance of epigenetic regulation in learning and memory, specifically in mouse models of dementia. Age-related impairments in learning and memory in wild-type mice are associated with alterations in histone acetylation (98), and treatment with HDAC inhibitors improves memory performance in both young and aged mice (42, 98).

HDAC inhibitors also improve cognition in the CK-p25 dementia mouse model (28). HDAC2 appears to be the crucial mediator of these effects, as overexpression of HDAC2, but not HDAC1, impairs learning and memory in wild-type mice (42). Conversely, Hdac2 knockout mice show improved memory formation, which is not further improved by HDAC inhibitors (42). HDAC2 expression is increased in the brains of two mouse dementia models as well as the brains of humans with Alzheimer’s disease (39). One model of how HDAC inhibitors regulate cognition is via epigenetic priming, reminiscent of the poised transcriptional state of genes involved in muscle differentiation (40). The broader role of epigenetics in cognition and neurodegenerative disease has been reviewed (67, 68, 100).

The worlds of epilepsy and dementia have been linked through the finding that mouse models of Alzheimer’s disease show neuronal hyperexcitability and epileptiform spikes from dysfunctional inhibitory interneurons (97, 134). Epilepsy, an extreme manifestation of this hyperexcitability, is associated with more rapid cognitive decline in patients with Alzheimer’s disease (136). Promising treatments that reduce epileptiform spikes, including at least one commonly used antiepileptic drug, improve cognition in these models (113, 134).

The various signaling activities by which BHB acts in epilepsy may thus be relevant to ameliorating cognitive decline in Alzheimer’s disease. In small studies, provision of BHB precursor molecules improves cognition in an Alzheimer’s mouse model (58) and in a patient with Alzheimer’s disease (93). Further exploration of the links between BHB signaling, epilepsy, and dementia may prove fruitful in generating new translational therapies.

Inhibition of the NLRP3 inflammasome could also prevent cognitive decline and dementia. β-amyloid protein, which aggregates into the amyloid plaques characteristic of Alzheimer’s disease, activates the NLRP3 inflammasome in microglia, the resident macrophage population in the brain, releasing inflammatory cytokines including IL-1β (reviewed in References 29 and 35).

This activation is evident in the brain of humans with both mild cognitive impairment and Alzheimer’s disease, and Alzheimer’s mouse models that carry deficiencies in NLRP3 inflammasome components are protected from β-amyloid deposition and cognitive decline (50). Microglia, as critical mediators of brain inflammation, may be the site of integration of various BHB-related signals, including HCAR2 activation.


The hypothesis that BHB may play a broad role in regulating longevity and the effects of aging comes in part from the observation that many of the interventions that most consistently extend longevity across a wide range of organisms, such as dietary restriction and fasting, intrinsically involve ketogenesis and the production of BHB in mammals (92).

The effects of such regimens on invertebrate, rodent, and human health have been reviewed and can include extended longevity, cognitive protections, reductions in cancer, and immune rejuvenation (75, 82). More specific interventions that promote ketogenesis, such as transgenic overexpression of FGF21, also extend life span in rodents (161). BHB itself extends longevity in C. elegans (24), and whether it would do so in rodents remains to be investigated.

Several of the signaling functions of BHB described above broadly regulate longevity and diseases of aging pathways, most prominently HDAC inhibition and inflammasome inhibition. The data from invertebrate organisms showing that reduction in class I HDAC activity extends life span, and generally acts through similar pathways as dietary restriction, have been reviewed (92). Briefly, deletion of Rpd3, the yeast and fly homolog of mammalian class I HDACs, extends replicative life span by 40–50% in S. cerevisiae (61).

Rpd3 deletion enhances ribosomal DNA silencing (61), the same mechanism by which overexpression of the sirtuin Sir2 enhances replicative longevity in S. cerevisiae (57). Drosophilids heterozygous for a null or hypomorphic Rpd3 allele show a 30–40% extension of life span, with no further increase with caloric restriction (110). Both caloric restriction and reduced Rpd3 activity increase expression of Sir2 (110). Conversely, mutations in Sir2 block life span extension by either caloric restriction or Rpd3 mutations (109). In both organisms, then, modest reductions in HDAC activity (stronger reductions are lethal) extend life span via the same mechanisms as in dietary restriction and Sir2 expression.

Other possible longevity mechanisms downstream of Rpd3 in invertebrates include autophagy, which is regulated by histone acetylation of specific genes (151), and enhanced proteostasis through increased chaperone expression (162).

No life span data yet exist for reduced HDAC function in rodents. However, Hdac2 knockout mice display impaired IGF-1 signaling and are 25% smaller than normal (163), a potential longevity phenotype (87). Hdac2 knockout is also protective in models of tumorigenesis (163). Conditional knockouts in mouse embryonic fibroblasts and embryonic stem cells demonstrated roles for HDAC1 and HDAC2 in hematopoiesis (141) and stem cell differentiation (23). By analogy to the modest reductions in class I HDACs that enhance longevity in invertebrates, it may be of interest to determine whether heterozygous HDAC1/2 knockout mice, or mice treated with low-dose pharmacological HDAC inhibitors, have enhanced longevity.

An inducible compound heterozygote knockout of HDAC1 and HDAC2 does suppress one translatable age-related phenotype, cardiac hypertrophy, as do HDAC inhibitors (88). HDAC inhibitors ameliorate cardiac dysfunction in mouse diabetes models (14) and prevent maladaptive cardiac remodeling (160). The mechanism for the effect on cardiac hypertrophy appears to be inhibition of HDACs that suppress the activity of a mechanistic mTOR complex (88). This is one of several examples of intersections between BHB, its signaling effects, and mTOR/rapamycin, a canonical longevity-regulating pathway (55). As described above, mTOR is also a checkpoint in the activation of ketogenesis; inhibition of mTORC1 is required to activate the transcription factors and hormones that control ketogenesis (4, 116).

Inhibition of NLRP3 inflammasome activation might also have broad effects on aging and longevity, as reviewed in References 27 and 37. The NLRP3 inflammasome in particular has a wide range of activating stimuli, many of which accumulate with age such as urate, amyloid, cholesterol crystals, and excess glucose. The age-related phenotypes that may be ameliorated by its inhibition are similarly diverse: insulin resistance, bone loss, cognitive decline, and frailty.

In two such examples, BHB inhibited NLRP3 inflammasome activation in urate crystal–activated macrophages, and ketogenic diet ameliorated flares of gout arthritis in rats (36). Whether genetic or pharmacological inhibition of the NLRP3 inflammasome would extend mammalian life span remains unknown, but the potential certainly exists for translational application to human diseases of aging.


The ketone body BHB expresses a variety of molecular signaling functions, in addition to its role as a glucose-sparing energy carrier, that may influence a broad range of human diseases. There is sufficient evidence for several significant human diseases, including type 2 diabetes mellitus and Alzheimer’s dementia, in model organisms to justify human studies of BHB or a BHB-mimetic intervention. The diversity of age-associated diseases and pathways affected by BHB signaling suggests that therapies derived from BHB may hold promise for broadly enhancing health span and resilience in humans (91).

The translation of these effects into therapies that improve human health span requires the pursuit of two converging strategies: deeper mechanistic understanding of the downstream effects of BHB signals and improved systems for the targeted delivery of BHB for both experimental and therapeutic goals. Deeper mechanistic understanding would solidify some of the transitive connections described above. For example, BHB inhibits HDACs, and HDAC inhibition protects against cognitive decline in rodents; but does BHB protect against cognitive decline? Via HDAC inhibition? Which gene promoters are targeted? Establishing such links would permit rational design of human studies to test specific effects of BHB, with plausible biomarkers and intermediate outcomes. Improved delivery systems would facilitate both animal and human studies.

BHB-mimetic drugs, or ketomimetics, would recapitulate the desired activity of BHB. The key obstacles to exogenous delivery of BHB are its nature as an organic acid and the rapid catabolism of R-BHB. The quantity of exogenous BHB required to sustain blood levels over a long period would likely be harmful because of either excessive salt load or acidosis. Alternatively, approaches to ketomimetics include (a) the use of agents that activate endogenous ketogenesis in an otherwise normal dietary context, (b) the delivery of BHB prodrugs or precursors that avoid the acid/salt problem, and (c) the use of agents that phenocopy specific downstream signaling events.

The last approach, such as using HCAR2 agonists or HDAC inhibitors, is tempting, but a perhaps crucial advantage of adapting BHB itself is utilizing the existing endogenous transporters and metabolite gradients to bring BHB to its sites of action. Esters of BHB are a promising approach to delivering BHB as a prodrug, but the expense of synthesis is challenging. Confirming whether such synthetic compounds need be enantiomerically pure, or indeed whether S-BHB has better pharmacokinetics for the desired signaling function, might help reduce cost.

The ketone body BHB, a fasting fuel and fasting signal, is emerging as a poster child of the endogenous metabolite that transmits signals from the environment to affect cellular function and human health. Researchers have made important strides in understanding the signaling functions of BHB, many of which have crucial implications for the management of human diseases. A deeper knowledge of the endogenous actions of BHB, and improved tools for delivering BHB or replicating its effects, offers promise for the improvement of human health span and longevity.

Does Basis still use Nicotinamide Riboside (NR) from Chromadex?

In 2012, Research published by Dr Sinclair  of Harvard stunned researchers by showing that short term supplementation with Nicotinamide Mono-Nucleotide (NMN) reversed many aspects of aging, making the cells of old mice resemble those of much younger mice, and greatly improving their health.

Since then Chromadex has been marketing a related product named Nicotinamide Riboside (NR or Niagen) that seems to work on the same cellular pathways as NMN.

1000s of articles have teased us with the possibility that taking a supplement called Niagen could halt or maybe even actually reverse some of the signs of aging. Of course there is a lot of skepticism with such claims.

Chromadex has been producing the only commercially available form of Niagen and supplies it to 20 or more different companies that put their own brand name on the bottle and sell to customers.

Elysium Health is one of these sellers, although they have fairly deep pockets and extensive connections among top researchers in the field.

To overcome the skepticism, Elysium Health decided they would enlist several Nobel Prize wining scientists as advisors and consultants.

This does add a lot of credibility to the company, even though the 7 nobel prize winning scientists Elysium pays had little or no input in creating this product, but are mostly used for marketing purposes.

Elysium Health makes a change to the formula for Basis

Basis contracted to buy Nicotinamide Riboside Chloride (often referred to as NR, or the brand name Niagen), and combine it with another off the shelf product from Chromadex, Pterostilbene.

But in June 2016, Elysium got into a contract dispute with Chromadex and later sued Chromadex for abusing the patent process.

Their goal seems to be to invalidate Chromadex patent(s) so they could produce their own version, which appears to be what they are doing now.

Elysium has changed the product information on their website to list “Nicotinamide Riboside”, without the Chloride.  A key patent Chromadex has licensed the rights to involves adding the Chloride to make it more stable.

This new ingredient used by Basis likely has the same effects in the human body, but we don’t really know for sure.

It seems Elysium Health plans to go forward with this revised formula for now, and continue to use previous test results obtained with their prior  formula that used Chromadex Nicotinamide Riboside Chloride.  It will be interesting to see how that strategy holds up going forward.

Elysium Health has some very slick Marketing people

So now they have changed their product to use a slightly different ingredient, but want to smooth it over and not cause customers to worry about it.

Their answer is to portray the change as something they did on purpose – to MAKE THE PRODUCT BETTER.

When a customer questioned the change, this is how they answered on their Facebook page:

Hi Anne–

Thank you for reaching out to us with your questions. I’d be happy to provide additional information for you here!

From the start, Elysium has always been committed to bringing superior, high-quality supplements to market. As part of that effort, we have established a new supply chain, located in the United States, that utilizes a proprietary process to produce Nicotinamide Riboside — the first of its kind for the production of Basis, and this does fully meet the GMP standard as outlined by the FDA. We believe our vertically integrated supply chain benefits our customers as it enables us to better manage manufacturing, packaging, shipment and eventually the expansion of our new product line.
While the specific Basis formulation and the amount of each ingredient have not changed, this new production process has allowed us to take an exceptional product and make it even purer. This reflects our ongoing commitment to being a trusted source for our customers by continually exceeding the highest standards in the industry.

In regard to your question about GMO’s, this does not apply to Basis as we don’t have food products in our ingredients eligible for genetic modification. Basis is produced by nature identical synthesis, meaning that the active molecules are constructed to be nature identical. This process is preferable to attempting to distill down the ingredients from food as the final product is purer than what the bi-product would be via distillation.
If you have other questions or if there is anything we can do to help, my team can be reached directly here or by email at care@elysiumhealth.com or phone at 888-220-6436.

Pretty slick, I thought. No, they didn’t substitute something they just threw together to get around the patent and supply problem – they made something more pure and trustworthy.

I don’t know who is going to with the legal battles between Chromadex and Elysium Health, but I see Elysium as way ahead in the marketing department even if I don’t really trust their honesty.

(I have added the above update on the Elysium Health Basis onto an earlier review of the product below)

Basis is the same Nicotinamide Riboside that many other brands sell, with the addition of Pterostilbene, a form of resveratrol.

It is produced by Elysium Health, a new company founded by MIT biologist Lenny Guarente, one of the preeminent researchers in the anti-aging field.

They have also enlisted 6 Nobel Laurete scientists to serve as advisors to the company, which lends a great deal of credibility.

However, these scientists have had no significant role in researching,creating or testing Basis or either of the ingredients used.



hpn-single-bottleNicotinamide Riboside is a recently discovered version of Vitamin B that recent research has shown to raise NAD+ levels in humans.

The only manufacturer of Nicotinamide Riboside is Chromadex, as they have bought up all the patents on production methods for Nicotinamide Riboside.

Niagen is the brand name used by Chromadex.

The two names are synonymous. Basis uses Niagen supplied by Chromadex.



Pterostilbene is also manufactured by Chromadex.

Pterostilbene is described by Dr Guarente as “a close relative of resveratrol, but is potentially more potent and effective”.

So, Basis MIGHT be better than taking Nicotinamide Riboside plus resveratrol.

But there has been no testing at all to show any synergy from combining Nicotinamide Riboside with resveratrol or Pterostilbene.

The original research by Dr Sinclair with mice used NMN, but it is not commercially available yet.   The recent excitement and dozens or research studies about slowing or halting the aging process is centered around Nicotinamide Riboside.


Since all Nicotinamide Riboside is supplied by Chromadex, we recommend the less expensive options below.

Note: If you do want to try NR with Pterostilbene, we have a listing for 1 bottle of Pterostilbene and 1 bottle of Nicotinamide Riboside.


[product_category category=”basis” columns=”3″ per_page=”6″ orderby=”menu_id” order=”asc”]


screen-shot-2016-12-01-at-2-21-53-pmOne of the main selling points for Basis is the company cofounder, Dr Guarente, and the Nobel laureate scientists shown here that are on the advisory board.

They certainly lend a lot of credibility. It is very doubtful they would lend their name to some scammy fly by night product, which makes me believe in the potential for Nicotinamide Riboside.


Why Logo White red You’ll find very little mention of Niagen in the sales and marketing literature about Basis.

Elysium would like you to think that Basis is some exclusive formula created by their founders.

In fact, they purchase Niagen from Chromadex like several other brands.

The research and testing that has generated so much excitement has been done with Nicotinamide Riboside from Chromadex.

There has been no research published to show that Pterostibene makes Niagen work any better.

[box]Conclusion: ALL Niagen comes from Chromadex, there is no proof that Basis is any more effective than other brands of Niagen.[/box]


dollar sign moneybagBasis is available only thru their website for $60 a bottle, which would last for one month if taking 2 pills per day (250mg of Niagen).

Recent research indicates that the optimum dosage for maximum increase in NAD+ levels is at least 250mg per day, or more.

In fact, the best evidence on recommended dosage will hopefully soon be available from a recently completed study sponsored by Elysium Health themselves.

This study of 120 elderly patients tested blood NAD+ levels of 250mg and 500mg of Elysium Health Basis vs placebo.

Once this study is published we’ll have a lot better idea if one bottle per month is sufficient


If you know that Basis is Niagen + Pterostilbene, and start searching for “Niagen”, you quickly realize you can get the same thing for 50% less elsewhere.

Of course other brands don’t have the impressive scientific pedigree that Elysium’s founders have, which some people don’t mind paying the extra $ for.

That pedigree might also lead you to trust Basis more.

Screenshot 2016-01-25 15.21.18

[box]Conclusion: Since ALL Niagen comes from Chromadex, there is no difference in the quality among brands[/box]

What does Basis do?

The field of Anti-Aging supplements is littered with scams and hoax products that are supposed to miraculously stop the aging process.

It is for that exact reason that Elysium Health DOES NOT market Basis specifically as an anti-aging pill like some of their competitors.

Rather, they focus on some specific areas that their pill may help with such as:

— DNA repair
— Energy production
— Cellular detoxification
— Protein function

Basis – Conclusion

  • Niagen plus Pterostilbene (similar to Resveratrol)
  • Pre-eminent antiaging researcher as cofounder of company
  • 6 Nobel laureate scientists on advisory board
  • By far the most expensive Niagen on the market
  • One bottle a month may not be enough for optimum results


[box]Conclusion: We don’t believe Basis by Elysium Health to be a scam. They go out of their way to avoid making exaggerated claims.

If you want to spend $60-120 a month, Basis is probably a good choice.

If not, you might want to spend your money on some other brand where you will get twice as much Niagen per dollar.


Mitochondrial dysfunction and the inflammatory response

The .pdf version of this research can be found at https://www.researchgate.net/

1. Introduction
Inflammation, a basic biological response against invading pathogens, contributes to repairing damage and preventing further tissue or cell injury (Medzhitov, 2008). However, when the inflammatory response cannot be appropriately attenuated, multi-organ failure or chronic inflammation can develop (Medzhitov, 2008).

In this sense, increasing levels of pro-inflammatory cytokines have been associated with disease evolution and unfavorable outcomes in several pathological scenarios, such as chronic heart failure, peritoneal dialysis, pancreatitis, and cancer (Berres et al., 2011; Gravante et al., 2009; Hiller et al., 2011; Nevado et al., 2006; Yndestad et al., 2012).

While each of these disorders has a specific pathogenesis and disease evolution, they all involve inflammatory processes. In addition, the inflammatory process also has a role in mediating the aging process via changes in redox status and oxidative stress-induced inflammatory responses (Gilca et al., 2007); this process has been called inflammaging (Salminen et al., 2012). In fact, aging and low-grade inflammation involves many of the same molecular pathways, including oxidative stress and DNA damage pathways.

The molecular inflammation hypothesis of aging postulates that chronic low-grade inflammation in aging causes cumulative damage to biological macromolecules through the formation of oxygen radicals (Chung et al., 2009).

This process would eventually render cells more susceptible to damage and more sensitive to catabolism, processes associated with an exacerbated response to age-related diseases (Chung et al., 2009; Jian et al., 2011; Loeser, 2009). Hence, the risk of degenerative diseases and poor outcomes of acute processes rise exponentially with age.

This is also supported by studies showing that healthy centenarians exhibit low levels of inflammatory biomarkers (Franceschi, 2007).

Altered mitochondrial function is linked to several acute and chronic inflammatory diseases (Naik and Dixit, 2011). Additionally, mitochondria have long been proposed to play a key role in aging (Green et al., 2011).

As a consequence of their central role in ATP formation via the mitochondrial respiratory chain (MRC), mitochondria are the major source of reactive oxygen species (ROS) and are thus highly involved in oxidative stress processes (Brookes et al., 2004). However, mitochondria are also targets of these molecules (Brookes et al., 2004).

Under physiological conditions, about 1–3% of molecular oxygen is incompletely reduced during redox reactions in the MRC, and this in turn leads to production of the ROS superoxide anion (O2−) as a by-product of these reactions. In this scenario, complex interactions in antioxidant defense systems repress oxidative stress within mitochondria (Kienhöfer et al., 2009).

However, under pathological conditions, an excess of O2− ions are produced, which causes the activation of redox-sensitive transcription factors, such as the key regulator of tissue inflammation, nuclear factor-κB (NF-κB), and a subsequent increase in the expression of cytokines, chemokines, eicosanoids, inducible nitric oxide synthase (iNOS), and adhesion molecules (Chung et al., 2009).

In addition to alteration of signaling pathways regulated by ROS, the excess of ROS also drives oxidation of membrane lipids, proteins, and mitochondrial DNA (mtDNA) (Henze and Martin, 2003).

In particular, mtDNA seems to be especially sensitive to oxidative damage, and therefore susceptible to mutations, because of its close proximity to the site of ROS production and the lack of protective histones. In fact, aged individuals, whose cells have accumulated a high level of oxidative damage, exhibit an increased rate of mutagenesis in their mtDNA (Kujoth et al., 2005).

Moreover, superoxide anions can combine with nitric oxide (NO) to produce peroxynitrite (ONOO-), a powerful oxidant capable of affecting mitochondrial integrity (Brown, 2003; Escames et al., 2011).

Mitochondrial dysfunction drives mitochondrial genome mutagenesis, affecting genes encoding respiratory chain complexes and compromising the efficiency of oxidative phosphorylation (OXPHOS), which may lead to further mtDNA mutations and even mutations in the nuclear genome, thereby causing increased cell damage (Escames et al., 2011).

In this review, we aim to highlight the significance of mitochondrial dysfunction and to discuss the contribution of mitochondria to the development of inflammatory human diseases and the aging process.

2. Mitochondrial dysfunction may modulate inflammatory processes

It is well known that mitochondria are not only the primary source of ATP, but also participate in many other cell signaling events, such as regulation of calcium (Ca2 +) homeostasis, orchestration of apoptosis, and production of ROS, as mentioned above (Brookes et al., 2004).

In this sense, ROS are the major host defense mechanism against infection and harmful agents (Chen et al., 2012), and mitochondria are the principal mediators of inflammation (Tschopp, 2011).

During viral infection the pattern recognition receptors RIG-I and MDA5 attach to viral RNA allowing its interaction with a mitochondrial polypeptide adaptor, MAVS, which finally drives the production of type I interferon (Saitoh and Akira, 2010).

Interestingly, pathogens have recently been shown to have developed their own reciprocal defense systems; for example, Leishmania infection has been shown to cause marked upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation, potentially preventing ROS-mediated inactivation to suppress macrophage defense mechanisms and facilitate parasite survival (Basu Ball et al., 2011).

However, under dysregulated processes, when mitochondria are compromised by damage or mutations, excess O2− ions are produced, and cellular stress cannot be effectively resolved. Thus, mitochondria are the principal arbitrators of the pro-inflammatory status; they act through modulating innate immunity via redox-sensitive inflammatory pathways or direct activation of the inflammasome, a group of protein complexes whose activation results in immediate activation of caspase-1, thereby allowing for cleavage and subsequent activation of the inactive precursors IL-1β and IL-18 (Strowig et al., 2012).

Additionally, both of these pathways, i.e., the redox-sensitive inflammatory pathway and the inflammasome pathway, may work together to activate inflammatory cytokines, leading to an overstimulation of the inflammatory response (Escames et al., 2011).

Taken together, these recent studies have suggested the unexpected supplementary role of mitochondria as drivers of the inflammatory process.

2.1. Activation of redox-sensitive inflammatory pathways by mitochondrial impairment
Cellular systems that protect against oxidants involve antioxidative defense enzymes (superoxide dismutase [SOD], glutathione peroxidase [GPx], and catalase) (Kienhöfer et al., 2009), oxidant scavengers (vitamin E, vitamin C, carotenoids, uric acid, and polyphenols) and mechanisms to repair oxidant-induced damage to lipids, proteins, or DNA (Karger et al., 2012; Ugarte et al., 2010).

Despite these protective mechanisms, uncontrolled generation of ROS can overwhelm the capacity of cellular antioxidant protection causing mitochondrial dysfunction. In this sense, in vivo studies in transgenic mice showed that the overexpression of catalase targeted to mitochondria reduces age-associated pathologies and the mice have an extension in lifespan (Schriner et al., 2005).

Additionally, several studies have demonstrated that mitochondrial dysfunction may generate low-grade inflammatory and matrix degradation responses in several cell types via mitochondrial Ca2 + exchange, ROS generation, and NF-κB activation (Amma et al., 2005; Cillero-Pastor et al., 2008; Ichimura et al., 2003).

Thus, the flow of mitochondrial Ca2 + plays a crucial role in the evolution of cell signals and in the regulation of mitochondrial activity. Moreover, excessive mitochondrial Ca2+ accumulation has been shown to be critical for disease development (Dada and Sznajder, 2011; Tanaka et al., 2004).

For example, increased mitochondrial Ca2+ levels in cardiomyocytes contribute to cardiac inflammation and dysfunction after burn injury or sepsis (Maass et al., 2005).

Additionally, when cells are treated with inhibitors of mitochondrial Ca2 + exchange, ROS levels decrease significantly, leading to subsequent reductions in the levels of pro-inflammatory mediators. The rise in mitochondrial Ca2 +, a consequence of importing cytosolic Ca2 +, augments the mitochondrial production of ROS by different mechanisms, including 1) Ca2+ stimulation of the tricarboxylic acid cycle, which would enhance electron flow into the respiratory chain; 2) Ca2 + stimulation of NO synthase, elevating NO levels, which would in turn inhibit respiration at complex IV and enhance ROS generation; and 3) Ca2+ binding to cardiolipin (Brookes et al., 2004).

Thus, ROS production by mitochondrial electron transport could represent an intermediate step in pro-inflammatory gene expression/NF-κB signaling.
Mitochondrial dysfunction has also been shown to increase the responsiveness of several types of cells to cytokine-induced inflammatory responses, resulting in a significant amplification of the inflammatory response through ROS generation and NF-κB activation (Vaamonde-García et al., 2012; Nakahira et al., 2011).

When mitochondrial dysfunction was induced in normal synovial fibroblasts by an inhibitor of mitochondrial ATP synthase, oligomycin, an otherwise less-efficient concentration of IL-1β was as effective as a 10-times greater concentration of IL-1β would be in the absence of pretreatment with the mitochondrial inhibitor (Valcárcel-Ares et al., 2010).

In lung epithelial cells, pre-existing mitochondrial dysfunction induced by antisense oligonucleotides to complex III increased mitochondrial ROS generation, resulting in marked potentiation of ragweed pollen extract-induced accumulation of inflammatory cells in the airways (Aguilera-Aguirre et al., 2009).

In other cell types, mitochondrial impairment also increased the generation of ROS, resulting in potentiation of cytotoxicity or inflammatory cell accumulation (Bulua et al., 2011; Schulze-Osthoff et al., 1993).

Moreover, several studies have reported enhanced sensitivity to NF-κB activation in the context of mitochondrial dysfunction, suggesting that NF-κB binding sites may be essential for inflammatory gene expression (Ungvari et al., 2007; Vaamonde-García et al., 2012).

As expected, this inflammatory response is modulated by antioxidants. Additionally, these in vitro studies are supported by in vivo results. Thus, the effects of in vivo NF-kB inhibition (with pyrrolidinedithiocarbamate, PDTC or resveratrol) in aged rats significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness in vessels (Ungvari et al., 2007).

2.2. Mitochondrial modulation of the NLRP3 inflammasome.
Recent studies have shown mitochondria as important players in NLRP3 inflammasome activation (Green et al., 2011; Kepp et al., 2011; Nakahira et al., 2011; Shimada et al., 2012; Zhou et al., 2011).

Cell damage can initiate the accumulation of damage-associated molecular patterns (DAMPs), molecules recognized by the innate immune system and able to develop an inflammatory response (Krysko et al., 2011b) through the framework of the inflammasome and subsequent caspase-1 activation and pro-inflammatory cytokines release. Mitochondria have been recently identified as key sources of DAMPs (mito-DAMPs) playing a role in DAMP-modulated inflammation in different disorders, such as systemic inflammatory response syndrome (SIRS), rheumatoid arthritis (RA), cirrhosis, cancer, and heart diseases (Green et al., 2011; Zhang et al., 2010; Zitvogel et al., 2012), as well as the aging process (Salminen et al., 2012).

DAMPs activate the same receptors that detect pathogen-associated molecular patterns (PAMPs) (Krysko et al., 2011), such as Toll-like membrane receptors (TLRs) and cytoplasmic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs).

One of the best-studied NLRs, because of its solid association with several inflammatory diseases, is NLRP3. NLRP3, a cytosolic receptor, is one of 22 described human NLR family members; it functions to monitor the cytosol for stressful situations and has a crucial role in regulating immune responses (Kepp et al., 2011; Schroder and Tschopp, 2010; Shimada et al., 2012; Tschopp, 2011).

Once activated, the NLRP3 receptor induces NLRP3 oligomerization and recruitment of the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1, generating a multiprotein platform called the NLRP3 inflammasome. Then, NLRP3 activates caspase-1 and in turn drives the maturation of pro-inflammatory cytokines, such as IL-1β and IL-18. The NLRP3 inflammasome also conducts caspase-1-mediated cleavage of other substrates such as cytoskeletal proteins, glycolytic enzymes and caspase-7 (Tschopp, 2011; Zitvogel et al., 2012).

Mitochondria through ROS generation act as integrators across different stimuli activating the NLRP3 inflammasome (Kepp et al., 2011).

Increased levels of ROS are essential for NLRP3 activation; therefore, scavenging ROS with different chemicals suppresses inflammasome activation (Tschopp, 2011). Initially, Tschopp’s group, performing experiments in the human acute monocyte leukemia cell line, THP-1, suggested that NLRP3 inflammasome-activating ROS were generated by an NADPH oxidase stimulated upon particle phagocytosis, since in their model, ROS production was inhibited by NADPH oxidase inhibitors (Dostert et al., 2008).

However, later experiments performed by the same group (Zhou et al., 2011) and others showed that this result was ambiguous since immune cells from chronic granulomatous disease (CGD) patients, which have defective NADPH activity and thereby lack NADPH-dependent ROS generation, achieved normal levels of active caspase-1 and, in turn, active IL-1β.

Thus, these studies provided evidence of the existence of an alternative source of ROS (Meissner et al., 2010; van Bruggen et al., 2010; van de Veerdonk et al., 2010).

Since mitochondria are the main source of ROS, it is not surprising that mitochondria could also be the principal initiator of NLRP3 inflammasome activation. For example, the subcellular localization of NLRP3 suggests an important link between mitochondria and NLRP3. Under unstimulated conditions, most NLRP3 and ASC proteins are associated with the endoplasmic reticulum (ER).

Once the inflammasome is activated, NLRP3 redistributes to the perinuclear space and colocalizes with mitochondria (Zhou et al., 2011). Additionally, inhibition of voltage-dependent anion channels (VDACs), which are regulated by Bcl-2 family members through a decrease in mitochondrial Ca2 + levels and the resulting ROS production, significantly decreases NLRP3 inflammasome activation (Zhou et al., 2011).

Intriguingly, VDAC is also required for pro-apoptotic activity by Bax.
Other danger signals, specifically mtDNA and ATP, have been implicated in NLRP3 inflammasome activation; these signals can be released following different types of cell death (discussed below).

Indeed, it has been reported that mtDNA in the plasma of patients with femur trauma reaches values several thousand times higher than that of healthy volunteers, with functionally important immune repercussions (Zhang et al., 2010).

Additionally, mtDNA has been detected in RA synovial fluid and induces in vivo and in vitro inflammatory responses (Collins et al., 2004). Also, recent results have identified degraded mtDNA as a new DAMP subtype and a possible trigger of neurodegeneration (Mathew et al., 2012).

With regard to ATP, studies have shown that shedding of ATP from necrotic hepatocytes activates the NLRP3 inflammasome to generate an inflammatory microenvironment, alerting circulating neutrophils to adhere within liver sinusoids (McDonald et al., 2010).

Also, it has been shown that the release of extracellular ATP during apoptosis could be required for the successful immunogenic tumor cell death elicited by cancer treatment (Hahn et al., 2012).

Moreover, ATP can decrease intracellular K+ concentrations, a necessary event for NLRP3inflammasome activation, and mitochondria could also have a crucial role in modulating this process since they possess several K+ channels and can modulate intracellular K+ levels (Heinen et al., 2007).

Finally, NFPs is also considered a crucial mito-DAMP in several pathologies, including SIRS; this molecule can attract and stimulate neutrophils through high-affinity formyl peptide receptors (Zhang et al., 2010).

Interestingly, DAMPs and mito-DAMPs can activate antigenpresenting cells (APCs), as well as other nonimmune cell types, such as mesenchymal stem cells and astrocytes (Mathew et al., 2012; Pistoia and Raffaghello, 2011). In one study, degraded mtDNA strongly induced pro-inflammatory IL-1β, IL-6, MCP-1, and TNFα in mouse primary astrocytes, demonstrating the role of degraded mtDNA in inflammasome activation (Mathew et al., 2012).

The NLRP3 inflammasome and NF-κB pathways could work together to activate inflammatory cytokines, leading to overstimulation of the inflammatory response (Escames et al., 2011).

Once constituted, inflammasomes activate inflammatory caspases, mainly caspase-1, and subsequently induce IL-1β and IL-18 secretion. Then, pro-inflammatory cytokines can also activate NF-κB, generating a vicious cycle that increases the level and duration of the inflammatory response. The activation of inflammasomes may represent a possible explanation for this synergistic effect between mitochondrial impairment and cytokines.

Thus, studies investigating the role of mitochondria in inflammasome activation are revealing novel concepts in the development of inflammatory response, and these mechanisms may be of help in explaining the usual association of mitochondria with several inflammatory disorders and the aging process.

2.2.1. Role of mitochondria in the inflammatory response modulated by cell death.
Depending on the type of cell death, the dying cells could release and present at their surface specific signals that could modulate the type of immune response (Zitvogel et al., 2010).

Mitochondria have a key role in cell death (Kepp et al., 2011). Cell death can result from a variety of processes, including the apoptotic pathway and the necrotic pathway, 2 well-described extremes.

Apoptosis involves a series of orchestrated events requiring protein synthesis and ATP consumption. The dying cell forms vesicles that fragment into several apoptotic bodies; these are phagocytosed by macrophages or adjacent cells, thus avoiding the inflammatory reaction. Interestingly, contradictory recent data show that this paradigm is not always true, and depending on the inflammatory scenery, apoptosis could elicit a powerful inflammatory response (Shimada et al., 2012).

In particular, this study provides a link between apoptosis and inflammasome activation via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome. In this sense, Nakahira et al. also find that cyclosporine A, which modulates intrinsic apoptosis through inhibition of mitochondrial permeability transition, prevents the release of IL-1β by apoptotic stimuli (Nakahira et al., 2011).

Moreover, in vitro studies showed that silencing of antiapoptotic protein Bcl-2 increases IL-1β secretion while Bcl-2 overexpression resulted in the contrary (Shimada et al., 2012). In contrast, necrosis results from a rapid loss of cell homeostasis as intracellular ATP stores become depleted.

Cells increase their volume, leading to loss of cell membrane integrity and leakage of cellular components, thus inducing inflammation. In this study by Shimada above, necrotic stimuli did not secrete IL-1β (Shimada et al., 2012).

Mitochondrial DAMPs can also trigger autophagy. Autophagy is usually considered a type of cell death, although this process is thought to be an attempt to adapt and survive during periods of stress, such as nutrient deprivation, hypoxia, etc. (Edinger and Thompson, 2004).

Autophagy can control inflammation through macrophage-mediated clearing of apoptotic corpses or by inhibiting NLRP3 inflammasome activation by removing permeabilized or ROS-producing mitochondria (mitophagy). Thus, the inhibition of mitophagy results in spontaneous inflammasome activation (Goldman et al., 2010; Levine and Kroemer, 2008; Nakahira et al., 2011). Besides, mitochondria also play an important role in autophagia as they supply membranes for the biogenesis of autophagosomes (Hailey. 2010). Decreases in the expression of several autophagy-related genes, as well as the presence of mutations in these genes, have been found both in different inflammatory diseases, including neurodegenerative diseases, cardiovascular diseases, and Crohn’s disease (Gegg and Schapira, 2011; Travassos et al., 2010), and in the aging process (Salminen et al., 2012). For example, alterations in PINK1 and Parkin genes, which cooperate to identify and label damaged mitochondria for selective degradation via autophagy or mutations in ATG16 and IRGM, respectively, have been described. Additionally, the in vivo relevance of autophagic regulation of caspase-1 mediated inflammatory responses has been shown in two animal models of sepsis (the endotoxic shock model and the cecal ligation and puncture model of polymicrobial sepsis) (Nakahira et al., 2011). Furthermore, autophagy also declines during aging, generating the inflammatory condition via activation of inflammasomes, which in turn can actually accelerate the aging process (Salminen et al., 2012).

Mitochondria are triggers for the apoptotic pathway; several soluble pro-apoptotic proteins, including cytochrome c, are released from the mitochondrial intermembranous space after mitochondrial outer membrane permeabilization (MOMP). This leads to the formation of the apoptosome and the subsequent activation of the caspase cascade, which will trigger the execution phase of apoptosis. On the other hand, the mitochondrial permeability transition (MPT), which depends on the mitochondrial matrix protein cyclophilin D (Nakagawa et al., 2005), results in the instantaneous dissipation of the mitochondrial transmembrane potential and cessation of OXPHOS, thus rapidly increasing ROS, depleting ATP levels, and triggering necrotic cell death (Green et al., 2011; Hahn et al., 2012; Sasi et al., 2009).

As mentioned before, mtDNA can directly bind NLRP3; however, the current results do not rule out that mtDNA may bind to other members of the NLRP3 complex. ATP released from the dying cell constitutes a “find me” signal that acts as a chemo-attractant for monocytes and macrophages. Apoptosis releases much higher levels of cellular ATP related to necrosis (Hahn et al., 2012). It is therefore possible that ATP binding would be necessary for mtDNA binding (Shimada et al., 2012). DAMPs are often modified by processes of proteolysis and oxidation, which are related to cell death mechanisms. In this sense, HMGB1, a member of the high mobility group box family of DNA, can be released by necrotic cells, triggering a powerful immune response. During apoptosis, the generation of ROS by mitochondrial oxidized HMGB1 in turn enabled its immunostimulatory capacity (Hahn et al., 2012; Krysko et al., 2011).

Overall, depending on the specific circumstances and environment, cells can suffer from different lethal stress pathways promoting specific inflammatory responses, i.e., sterile inflammation or pathogen-induced inflammation. A better understanding of these processes is necessary for the development of new successful therapies to mitigate inflammatory diseases.

3. Pro-inflammatory mediators impair mitochondrial activity
A great number of inflammatory mediators, including the cytokines TNFα and IL-1β and the reactive nitrogen intermediate NO, may induce mitochondrial damage (López-Armada et al., 2006a; Maneiro et al., 2005; Stadler et al., 1992; Zell et al., 1997). TNFα and IL-1β decrease the activity of MRC complex I, ATP production, and mitochondrial membrane potential (Δψm). These mediators also induce the accumulation of significant amounts of ROS (Guidarelli et al., 2007; Kim et al., 2010). Interestingly, complex I together with complex III have been suggested to be major sources of ROS (Guzy et al., 2005), and the activities of complexes II and IV were also decreased in other cell types (Biniecka et al., 2011; Maneiro et al., 2003). Moreover, in adipocytes, the changes induced by TNFα cause pronounced morphological changes in the mitochondria, presumably due to variations in the levels of several mitofusion proteins (Chen et al., 2010). Another important study by Rowlands et al. showed that the severity of inflammation in mouselung microvessels is modulated by the inhibitory effect of TNFR1 ectodomain shedding by mitochondrial Ca2+ (Rowlands et al., 2011). Thus, mitochondrial Ca2+ and ROS are primary mediators of TNFαmediated inflammatory responses (Dada and Sznajder, 2011).

NO is another particularly important mediator in the pathophysiology of inflammatory processes. A variety of NO donors have been shown to suppress mitochondrial energy production in different cell types. Moreover, most of the catabolic effects of NO are potentially related to the ability of NO to combine with the superoxide anion to generate peroxynitrite, which, as mentioned earlier, is a powerful oxidant capable of inhibiting important enzymes and affecting mitochondrial integrity (Fermor et al., 2010; Johnson et al., 2000; Maneiro et al., 2005). In particular, NO induced a significant, reversible decrease in the activity of complex IV as well as a reduction in Δψm (Tomita et al., 2001). NO can also irreversibly inhibit respiration, most likely through ATP synthase and the strong oxidant peroxynitrite, which inactivates all respiratory complexes. Other studies demonstrated that, in intact U937 cells, peroxynitrite enhances the formation of superoxide ions in the mitochondria through a Ca2+-dependent mechanism that involves the inhibition of complex III, which then dismutates to H2O2 (Guidarelli et al., 2007).

Mitochondria play an important role during apoptosis. As mentioned above, severe mitochondrial damage can even lead to cell death by necrosis or by activation of the apoptotic signaling pathway and related caspases (Cillero-Pastor et al., 2011; Irrinki et al., 2011; López-Armada et al., 2006b; Maneiro et al., 2005). Indeed, the classic signs of cell death are preceded by mitochondrial alterations, which include changes in MRC activities, loss of Δψm, decreases in energy production, and/or induction of the mitochondrial permeability transition (MPT), and all of these alterations are modulated by both cytokines and NO. Thus, such pro-inflammatory mediators can modulate cell survival by exerting their effects on mitochondria. In addition, mito-DAMPs can be generated as consequence of these cell death processes.

To summarize, mitochondrial complexes could suffer permanent alterations caused by mutations in DNA or inflammatory mediators, such as reactive nitrogen species (RNS). Other stimuli, including cytokines and NO, induce functional and reversible modification of mitochondrial complexes at physiological concentrations. The combination of both effects (permanent and functional alteration of mitochondrial complexes) may cause a more substantial modification of mitochondrial activity, establishing continual, sublethal mitochondrial damage and thereby generating a vicious cycle of increasing inflammation that is difficult to break. Therefore, preserving mitochondrial function could reduce excessive oxidative stress and may represent a novel therapeutic advantage for patients with inflammatory diseases and chronic low-grade inflammation in aging.

4. Physiological function of ROS in the regulation of cell signaling
Although ROS has been traditionally linked to disease states, its crucial roles in normal physiological processes (vascular tone, oxygen sensing, and skeletal muscle physiology) and protective mechanisms, such as the above-mentioned host defense system and control of inflammation and immune responses, are becoming obvious. In this sense, there is a tight balance between appropriate redox states and oxidative stress. The final balance results from the magnitude and class of ROS, as well as the persistence of ROS production. Thus, high ROS concentrations may drive unspecific damage to nucleic acids, proteins, carbohydrates, and lipids or overtake the signaling pathways regulated by ROS.

In contrast, low to moderate levels of ROS lead to physiological regulation of cell signaling pathways (protein phosphorylation, ion channels, and transcription factors) and subsequently control cellular processes, such as differentiation, apoptosis, and migration.

A brief description of the most relevant physiological roles of ROS is as follows. ROS participate in immune system, at both innate and acquired levels. These molecules produced by phagocytes represent one of the early actions of defense against pathogen invasion. Notably, some pathogens have been shown to downregulate mitochondrial ROS generation and thereby prevent macrophage defense mechanisms, favoring pathogen persistence (Basu Ball et al., 2011). An example of the importance of ROS in immune system signaling is reflected in chronic granulomatous disease (CGD), which is caused by a lack of ROS-generating phagocytes. In this manner, CGD patients inefficiently kill invader bacteria by ROS, suffering recurrent infections (Mauch et al., 2007). Interestingly, CGD patients and mice defective for ROS production also show evidence of noninfectious inflammatory states, supporting the role of ROS in controlling inflammation and immune responses (van de Veerdonk et al., 2010). With respect to acquired immunity, ROS participates in signal transduction cascades within T lymphocytes (Alfadda and Sallam, 2012). Furthermore, as detailed earlier, recent evidence highlights a role for ROS in immune responses through NRPL3 activation (Nakahira et al., 2011; Shimada et al., 2012; Zhou et al., 2011).

Other illustrations of the crucial role of ROS in normal physiological processes are the key regulation of vascular system, the role of ROS in oxygen sensing through stabilization of hypoxia-inducible factor-1 (HIF-1) and the subsequent stimulation of angiogenesis and bioenergetic responses to rescue redox homeostasis, the role of ROS in skeletal muscle physiology regulation through glucose uptake during contraction, and the role of ROS in regulating gene stability, transcription, and signal transduction (Alfadda and Sallam, 2012).
Finally, increasing evidence that antioxidants may be harmful highlights the physiological role of ROS. Specifically, Ristow et al. reported that antioxidant supplementation may prevent increased insulin sensitivity by reducing the expression of ROS-sensitive transcriptional regulators of insulin sensitivity associated with exercise training in humans (Ristow et al., 2009). Also, the nuclear factor-erythroid 2-related factor 2 (Nrf2), a redox stress-sensitive transcription factor that induces several antioxidant and detoxification genes, is kept inactive in the absence of redox stress by its binding to Kelch-like ECH-protein 1 (Keap1). Thus, disruption of Nrf2 signaling impaired angiogenesis and microvascular rarefaction in aging (Valcarcel-Ares et al., 2012). In this sense, it is worth bearing in mind that antioxidant strategies will likely not be successful because their activity is too unspecific, too insufficient, and too delayed; however, it is also plausible that the ineffectiveness of antioxidant strategies may arise from their inhibition of essential cell functions that require ROS.

However, excessive levels of ROS cause not only oxidative damage to macromolecules, but can also affect the physiological signaling pathways regulated by ROS. Among other consequences, increased ROS production will lead to augmented activation of the transcription factors HIF-1, NF-κB, and activator protein-1 (AP-1) as well as the NLRP3 inflammasome. This will increase the release of pro-inflammatory cytokines that in turn will enhance ROS production, and hence, feed-back in a self-stimulatory manner, thereby amplifying the inflammatory response (Dröge, 2002).

5. Mitochondrial dysfunction and inflammatory responses in chronic human pathologies
Extensive research over the past decade has revealed that continued mitochondrial dysfunction can lead to chronic inflammation, which in turn may mediate most chronic diseases, including rheumatoid, cardiovascular, neurological, or metabolic diseases.

5.1. Rheumatoid diseases
Osteoarthritis (OA) and RA are the most common rheumatoid pathologies. In both of these conditions, the mitochondrial characteristics, including MRC activity, ATP synthesis, and Δψm, are adversely affected in several cell types. In particular, ex vivo studies have revealed that OA chondrocytes, as compared with normal chondrocytes, showed significant decreases in the activities of MRC complexes II and III and Δψm (Johnson et al., 2000; Liu et al., 2010; Maneiro et al., 2003). In RA and

systemic juvenile idiopathic arthritis, studies have demonstrated that there is a deficiency in a subunit of the MRC complex IV in the synovium (Biniecka et al., 2011; Ishikawa et al., 2009). Furthermore, human RA synoviocytes (Da Sylva et al., 2005; Ospelt and Gay, 2005) and normal synoviocytes under arthritis-like conditions, such as hypoxia (Biniecka et al., 2011), which causes a significant increase in Δψm in synovial cells, have been shown to exhibit significantly higher mutation rates than normal-aged or OA synoviocytes. These data support the conclusion that mitochondrial mutagenesis is correlated with the local inflammatory environment in arthritis (Harty et al., 2011). Other cells, such as T lymphocytes, show mitochondrial impairment in patients with RA and systemic lupus erythematosus (Gergely et al., 2002; Moodley et al., 2008).

Oxidative stress can contribute to the activated phenotype of synoviocytes in inflammatory arthritis and OA chondrocytes (Davies et al., 2008; Filippin et al., 2008; Henrotin et al., 2005). Interestingly, studies have shown that inhibition of complex III or V induces ROS production and NF-κB activation in human articular chondrocytes (CilleroPastor et al., 2008; Milner et al., 2007) and synovial cells (Valcárcel-Ares et al., 2010) in vitro. Additionally, inhibition of complex III or V in chondrocytes/synoviocytes induces low-grade inflammatory and matrix degradation processes through the synthesis of pro-inflammatory stimuli, including prostaglandin E2 (PGE2), the chemokines IL-8 and monocyte chemotactic protein-1, and several matrix metalloproteinases (MMPs) (Cillero-Pastor et al., 2010; Cillero-Pastor et al., 2008; VaamondeGarcía et al., 2012).

Specifically, it has been shown that the inhibition of MRC activity induces cyclooxygenase-2 (COX-2) expression and PGE2 production through redistribution of mitochondrial Ca2+, generation of ROS, and activation of the transcription factor NF-κB (Biniecka et al., 2010; Cillero-Pastor et al., 2008). Mitochondrial dysfunction can also induce PGE2 liberation in human OA chondrocytes through 4-hydroxy-2nonenal (4-HNE) (Vaillancourt et al., 2007), a lipid peroxidation byproduct that is associated with a higher frequency of mtDNA mutations, which are often increased in articular tissues of patients with inflammatory arthritis (Biniecka et al., 2010; Vaillancourt et al., 2007).

In addition to the above processes, the decline in mitochondrial function can also increase the inflammatory responsiveness of both normal human chondrocytes and synoviocytes to cytokines, promoting mechanisms that may contribute to joint destruction and pain (Valcárcel-Ares et al., 2010; Vaamonde-García et al., 2012). Interestingly, the hypoxic state of arthritic joints in vivo, which correlates with increased disease activity and mtDNA mutagenesis rates, exacerbates the inflammatory response in synoviocytes by increasing COX-2 expression and the release of MMPs in response to IL-1β (Demasi et al., 2004). In fact, inflammasome activation may explain these synergistic phenomena. NF-κB signaling and the NLRP3 inflammasome pathway could work together to activate inflammatory cytokines, thereby leading to overstimulation of the inflammatory response (Escames et al., 2011). As additional support for the critical role of the inflammasome in the pathogenesis of OA, the NLRP3 inflammasome could also mediate the pathological effects of hydroxyapatite crystals in vitro and in vivo (Jin et al., 2011).

Other DAMPs/mito-DAMPs are also highly secreted in tissues of rheumatoid patients. In fact, oxidized mtDNA, which has immunostimulatory properties and is capable of inducing arthritis in mice, can be detected in the synovial fluid of RA patients, but not in that of control subjects (Collins et al., 2004). Some studies have also considered cytochrome c a mitoDAMP that can induce arthritis (Pullerits et al., 2005); however, recent data suggest that this implication is not mediated by a direct role in NLRP3 activation (Shimada et al., 2012). Moreover, as mentioned earlier, autophagy prevents the accumulation of dysfunctional mitochondria. In this regard, OA cartilage has been shown to be deficient in autophagic processes, and the pharmacological activation of autophagy may be an effective therapeutic approach for OA (Caramés et al., 2012). Together, these findings support the critical role of mitochondrial dysfunction in rheumatoid disorders.

5.2. Cardiovascular diseases
Abnormalities in mitochondrial functions are increasingly recognized in association with cardiomyopathies. In fact, the myocardium is a highly energy-demanding tissue, with mitochondria supplying greater than 90% of ATP. Consequently, mitochondria are essential for cardiomyocyte function and viability (Dutta et al., 2012). Since ROS are constantly generated during mitochondrial respiration, it is not surprising that a dysregulation in the production of free radicals can cause oxidative damage in the heart. This oxidative damage may cause mtDNA mutations that result in mitochondrial dysfunction. Moreover, increased mitochondrial Ca2 + levels modulate myocardial inflammation and dysfunction in states of injury, such as sepsis and burn trauma (Maass et al., 2005). Another important study showed that escape of mtDNA from autophagy-mediated degradation leads to TLR9-mediated inflammatory responses in cardiomyocytes and may induce myocarditis and dilated cardiomyopathy (Oka et al., 2012).

With advanced age, mitochondrial ROS production significantly increases both in the heart (Judge et al., 2005) and vasculature (Ungvari et al., 2007), making the vascular endothelium and smooth muscle susceptible to ROS oxidation. In fact, mitochondria-derived ROS likely contribute to the development of chronic low-grade vascular inflammation in aging by activating redox signaling pathways (Dai et al., 2012). This low-grade vascular inflammation actives the redox-sensitive transcription factor NF-kB, stimulating endothelial activation by production and secretion of cytokines, growth factors, and proteases in the vascular wall, thereby promoting atherosclerosis. This damage is augmented by excessive ROS generation in the heart and the vasculature, as well as defective oxidant scavenging. These mechanisms disturb endothelial homeostasis (i.e., induce endothelial dysfunction), a characteristic feature of patients with coronary atherosclerosis (Munzel et al., 2010).

Traditionally, it has been hypothesized that cardiovascular diseases are precipitated by atherosclerosis due to arterial blockage from fatty deposits. However, in recent years, diagnostic and therapeutic strategies have been based not only on control of cholesterol levels, healthy diet, and smoking, but also taking into account other factors, such as inflammatory markers and mitochondria (Garg, 2011; Marchant et al., 2012). In particular, the roles of inflammasomes in the development of cardiovascular diseases are currently being studied in depth (Garg, 2011). Finally, pathways that improve mitochondrial function and attenuate mitochondrial oxidative stress (i.e., antioxidant therapies) show efficacy in various animal models and promising results for the treatment of cardiovascular diseases.

5.3. Neurological diseases
Increasing evidence has demonstrated the role of mitochondrial dysfunction in neurological and neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), dementia, multiple sclerosis, ataxia, and encephalomyopathies (Monje et al., 2003; Morán et al., 2012; Witte et al., 2010). A common feature in neurodegenerative diseases is the presence of neuroinflammation and oxidative stress. In both AD and PD, oxidative stress activates inflammatory signaling pathways through several mechanisms, including exacerbating the production of ROS, inducing mitochondrial dysfunction, activating microglia and astrocytes to release pro-inflammatory cytokines, and others (Schapira, 2010; von Bernhardi and Eugenín, 2012; Whitton, 2007). In fact, animal models of PD, induced by the mitochondrial inhibitor rotenone, exhibit increased production of IL-1β in the hypothalamus (Yi et al., 2007).

Furthermore, pro-inflammatory cytokines released in activated glia have been shown to stimulate increased ROS production in the brain, contributing to other mechanisms that amplify ROS production and lead to deterioration of mitochondrial function. In addition to exacerbating disease progression, this process may also suppress neurogenesis. Severe mitochondrial damage may even lead to cell death by necrosis or by activation of apoptotic signaling and activation of caspases (Voloboueva and Giffard, 2011).

Because neurodegenerative diseases are accompanied by chronic inflammation, the induction of mitophagy may have beneficial effects by removing permeabilized or ROS-producing mitochondria (Green et al., 2011). Moreover, as mentioned earlier, alterations in several genes involved in autophagy, such as PINK1 or Parkin, have been described in PD. Furthermore, downregulation of mitochondrial ROS may also decrease astrocyte activation (Voloboueva and Giffard, 2011). Finally, it has been recently described in mouse primary astrocytes how degraded mtDNA could be a new identified subtype of DAMP inducing inflammasome activation and ensuing neurodegeneration (Mathew et al., 2012).

5.4. Metabolic diseases
Insulin resistance, pancreatic islet β-cell failure, and elevated plasma free fatty acid (FFA) levels are features commonly associated with the development of several metabolic diseases, including obesity, type 2 diabetes mellitus, and metabolic syndrome. Accumulating evidence indicates that mitochondrial dysfunction is a central contributor or mediator of these processes (Coletta and Mandarino, 2011; Ma et al., 2012; Martins et al., 2012; Vial et al., 2010).

Diverse sources of inflammation can contribute to the insulin resistance observed in obesity and type 2 diabetes. Elevated plasma FFA levels play an important role in the development of skeletal muscle insulin resistance through different mechanisms, including oxidative stress, inflammation, and mitochondrial dysfunction (Coletta and Mandarino, 2011; Martins et al., 2012). The inflammation induced by FFAs may induce impairment in mitochondrial function in adipocytes (Ji et al., 2011; Youssef-Elabd et al., 2012). Furthermore, FFAs can activate these inflammatory signaling pathways directly through interactions with members of the TLR family and inflammasome activation (Reynolds et al., 2012; Wen et al., 2011; Youssef-Elabd et al., 2012).
Since mitochondrial dysfunction drives NLRP3 inflammasome activation, it is not surprising that this pathway can underlie cellular metabolic disorders. Specifically, a large body of evidence demonstrates a pathological role for the association of NLRP3 with a nonmitochondrial protein, thioredoxin-interacting protein (TXNIP), in type 2 diabetes and obesity. For example, TXNIP deficiency impairs activation of the NLRP3 inflammasome, improving glucose tolerance and insulin sensitivity in β-cells and adipocytes and decreasing fat deposits in the liver (Vandanmagsar et al., 2011; Zhou et al., 2010). Interestingly, high levels of oxidized DNA have been related with diabetes (Simone et al., 2008). It is possible that the elevated levels of damaged DNA detected in diabetes could be related with the activation of NLRP3 (Shimada et al., 2012). Consequently, mitochondria may represent novel pharmacological targets for therapeutic interventions in metabolic disorders.

5.5. Cancer and immunogenic cell death
Mitochondrial processes play an important role in tumor initiation and progression. Otto Warburg made the first association between mitochondrial dysfunction and cancer in the 1930s. He observed increased rates of aerobic glycolysis in a variety of tumor cell types, and he hypothesized that the cell’s choice of the glycolytic route to produce ATP instead of the more productive oxidative phosphorylation route may be due to the impaired respiratory capacity of these cells (Warburg, 1956). Years after this discovery, a number of cancer-related mitochondrial defects have been identified. In this sense, altered expression and activity of respiratory chain subunits as well as mtDNA mutations have been associated with human tumors. For example, MRC Complex I dysfunction has been associated with the pathogenesis of Hürtle cell tumors of the thyroid (Máximo et al., 2005). In the same way, low activity of Complex III has been found in breast cancer (Putignani et al., 2008) and Hürtle cell tumors (Bonora et al., 2006; Stankov et al.,

The aggressiveness of renal cell tumors is related to lower Complex II, III, and IV activities (Simonnet et al., 2002). As we know, dysfunction in MRC complexes is associated with mtDNA mutations. In fact, mutations in mtDNA have been reported in a variety of cancers, including ovarian, thyroid, salivary, kidney, liver, lung, colon, gastric, brain, bladder, head and neck, and breast cancers, as well as leukaemia (Modica-Napolitano and Singh, 2004).

In the context of mitochondrial dysfunction, we found that ROS is a key player in tumor formation and progression. It is now recognized that ROS plays an important role as a signaling molecule, mediating changes in cell proliferation, differentiation, migration, and invasiveness, as well as large-scale changes in gene transcription (Weinberg and Chandel, 2009). It is well known that mitochondrial dysfunction increases ROS levels, and tumor cells generally exhibit higher levels of ROS than normal cells (Weinberg and Chandel, 2009). One signaling pathway that may be particularly important to ROS-mediated tumorigenesis involves the activation and stabilization of HIF. HIF is a key transcription factor in cancer progression that regulates the enhancement of both glucose metabolism and angiogenesis. HIF activation occurs under hypoxic conditions, but ROS also appears to stabilize HIF under conditions of normal oxygen concentrations, leading to aberrant activation of HIF and promoting tumorigenesis (Chandel et al., 1998).

In addition, it has been demonstrated that tumors lacking the tumor-suppressor succinate dehydrogenase (Complex I) and fumarate hydratase exhibit impaired HIF degradation (King et al., 2006), once again linking mitochondrial impairment with oncongenesis.
As was discussed earlier, mitochondrial ROS production results in activation of the NLRP3 inflammasome with the consequent release of pro-inflammatory cytokines (Zitvogel et al., 2012). In vivo experiments suggest that inflammasome products, such as IL-1β, can directly drive oncogenesis or suppress immunosurvellaince mechanisms, thereby facilitating tumor development (Zitvogel et al., 2012). In the same way, many human cancers are etiologically linked to chronic inflammatory processes, such as those that occur with gastric cancer, hepatic cancer, and colorectal cancer (Coussens and Werb, 2002). However, the role of the inflammasome in carcinogenesis is controversial. Yet, in sharp contrast, the inflammasome or its products, depending on the specific tumor and its grade, can reduce tumorogenesis by causing cell death and promoting antitumor immune responses (Zitvogel et al., 2012).

In this context, recently developed therapies have been able to induce cell death by triggering ROS production. These include a specific group of mitochondrial-targeted anticancer drugs (mitocans) (Hahn et al., 2012). An example of these mitocans is the vitamin E succinate. This pro-oxidant selectively targets cancer cell mitochondria and is a potent inducer of ROS, leading to apoptosis in cancer cells but not in related normal cells. This is because cancer cells are under much more oxidative stress than normal cells and thus are more vulnerable to further damage by ROS-generating agents. Interestingly, these types of therapies have immune-enhancing properties, meaning that they are able to modulate antitumor immune responses, stimulating the immune system to cause immunogenic tumor cell death by upregulating the release of DAMP molecules. Of note, the activation or inhibition of the inflammasome as cancer therapy will likely depend on the specific cancer and its grade (Hahn et al., 2012).

6. Mitochondrial dysfunction and inflammatory responses in acute human pathologies
Generally, the acute inflammatory response to infection and tissue damage should be able to reduce injury to the organism, preventing further damage to the cells or tissues. However, as mentioned above, when the acute inflammatory responses cannot be effectively resolved, progressive damage and multi-organ failure can occur. Sepsis, a representative acute inflammatory disease, is unfortunately the main cause of death in critical care units. Mitochondrial dysfunction and impaired oxygen consumption play a role in sepsis, and the severity of sepsis has

been shown to correlate with mitochondrial damage in both humans and experimental models (Garrabou et al., 2012; Kung et al., 2012). Thus, changes to the ultrastructure of the mitochondria and significant inhibition of mitochondrial complex activities have been described in several cell types isolated from septic patients. Also, during overwhelming sepsis, increased Ca2+ influx into the mitochondria leads to mitochondrial dysfunction, resulting in the release of cytochrome c and cell death (Dada and Sznajder, 2011).

In this context, as mentioned before, mitochondrial damage may release several mito-DAMPs, including mtDNA. Consistent with this, septic plasma samples have shown significantly increased amounts of mtDNA and inflammatory cytokines, with corresponding NF-κB activation, and a correlation between adverse outcomes in sepsis and levels of extracellular mtDNA has been demonstrated (Garrabou et al., 2012). Since mtDNA can activate the NLRP3 inflammasome, its participation in the pathogenesis of sepsis is not surprising.

Interestingly, NLRP3 polymorphisms may be used as relevant risk estimates for the development of sepsis, which highlights the importance of NLRP3 in the pathogenesis of sepsis (Zhang et al., 2011). As expected, mitochondrial antioxidants have been shown to alleviate oxidative and nitrosative stress in several in vitro and in vivo model of sepsis (Apostolova et al., 2011; Zang et al., 2012). Taken together, these findings have led to a better understanding of the pathophysiological
processes of acute diseases and will be fundamental in improving the outcome of sepsis-related disorders.

7. Mitochondrial protection attenuates inflammation
Excessive mitochondrial oxidative stress plays a central role in triggering the deleterious cascade of events associated with inflammatory diseases and chronic low-grade inflammation in aging. In this regard, several approaches to preserve mitochondria are under investigation, including the use of antioxidant compounds, development of specific mitophagy-inducing therapies, and the pharmacological manipulation of mitochondrial sirtuins (SIRTs).

Strategies that control ROS production could limit either the redox-sensitive inflammatory pathway or the direct activation of the inflammasome. Thus, several meta-analyses have supported that dietary antioxidant intake is associated with a lower incidence of inflammatory diseases (Alissa and Ferns, 2012; Demetriou et al., 2012; Lahiri et al., 2012; Patelarou et al., 2011). In particular, resveratrol, a natural antioxidant found in grape skin and red wine, improves mitochondrial function by preventing oxidative stress and subsequent inflammation (Catalgol et al., 2012). In vitro, this molecule attenuates mitochondrial oxidative stress in several cell types, i.e., by restoring mitochondrial

complex III activity, which is considered to be the major source of mitochondrial oxidative stress (Xu et al., 2012). Moreover, resveratrol pretreatment efficiently prevents inhibition of mitochondrial membrane depolarization and ATP depletion, preserving mtDNA content and inhibiting COX-2 activity through a decrease in NF-κB activation in different cells (Dave et al., 2008; Lin et al., 2011; Ungvari et al., 2009; Xu et al., 2012). In animal models, resveratrol protects from developing age-related and acute diseases by improving mitochondrial function (Elmali et al., 2007; Jian et al., 2012; Lin et al., 2012; Ungvari et al., 2010).
As described earlier, an imbalance between mitophagy and mitochondrial biogenesis could be involved in inflammatory pathology. Autophagy/mitophagy decreases ROS production and mtDNA release after MPT opening, in turn affecting the NF-κB pathway and inflammasome activation. Thus, pharmacological induction of autophagy could mitigate inflammatory reactions. For example, activation of autophagy by rapamycin reduces the severity of experimental OA (Caramés et al., 2012). Interestingly, resveratrol also improves mitochondrial function through induction of mitochondrial biogenesis (Csiszar et al., 2009; Ungvari et al., 2009).

Recent studies revealed that SIRTs or class III histone deacetylases, have an essential role in mitochondrial protection against oxidative stress (Pereira et al., 2012). Three of the 7 SIRTs described in humans, SIRT3, SIRT4, and SIRT5, are localized inside mitochondria, which lends support to the potential role of SIRTs in mitochondrial biology (Hirschey et al., 2010). SIRTs can indirectly regulate the expression of several inflammatory genes through deacetylation of several signaling proteins, including the transcription factors FOXO (Salminen et al., 2008) and NF-κB (Natoli, 2009) and the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) (Sugden et al., 2010). Deacetylation of FOXO by SIRT1 or SIRT3 upregulates the expression of SOD2 and catalase, thereby decreasing cellular ROS levels. Also, deacetylation of PGC-1α by both SIRT1 and SIRT3 stimulates mitochondrial biogenesis (Pereira et al., 2012). As mentioned above, several studies have demonstrated that resveratrol can protect mitochondrial functions through activation of SIRTs (Shinmura et al., 2011).

While the role of mitochondria in the development of the inflammatory response is now beginning to be understood, there is still a lot to be discovered. Understanding the mechanisms that induce inflammation through mitochondrial impairment may help to identify relevant therapeutic targets for the treatment of multiple degenerative and acute disorders as well as the aging process. For these reasons, it is plausible that, in few years, specific mitochondrial-targeting pharmacological treatments of inflammatory responses may become a reality. Indeed, some pharmaceutical companies have already begun designing new molecules that are the equivalent of super-antioxidants and have demonstrated exciting results in several preclinical studies in vitro and in vivo (McManus et al., 2011; Mukhopadhyay et al., 2012).

8. Limitations and future research directions
The use of effective strategies to increase our knowledge about the role of mitochondrial dysfunction in the inflammatory components of multiple degenerative and acute diseases, as well as in the aging process, is necessary for the development of new successful therapies aimed at improving human health. Thereby, an appropriate way to avoid contradictory results is the use of unmanipulated cells from healthy and diseased donors. Most in vitro studies are conducted in isolated cells kept in standard media with high glucose concentrations and under aerobic conditions, quite different from effects in tissues in a live organism, which could lead cells to obtain their energy predominantly from anaerobic glycolysis. Thus, the hypoxic nature of rheumatoid joints in vivo, which is correlated with increased mtDNA mutagenesis rate and disease activity, increases the inflammatory response in synoviocytes by increasing COX-2 expression and MMPs release in response to IL-1β (Demasi et al., 2004). For this reason, it is likely that the effects of mitochondrial impairment observed in cultures

maintained in normal atmosphere will even be more crucial in damaged tissue, where mitochondrial activity is probably lower due to genetic predisposition, somatic mutations in the mtDNA of the MRC as a consequence of the elevated levels of pro-inflammatory mediators (notably ROS), or the direct effects of pro-inflammatory mediators on the MRC. Future in vitro studies must take this into account. In addition, an important tool in these studies could be the transmitochondrial cybrid system, which allows the study of the effects of mitochondrial dysfunction and inflammatory responses with a common nuclear background, thereby avoiding the large number of inflammatory properties regulated by nuclear-encoded genes (Kaipparettu et al., 2010).

The majority of studies to date were conducted using in vitro or ex vivo strategies. Thus, it is necessary to translate the results to in vivo designs to support a clear role for mitochondrial dysfunction in the development of the inflammatory response. Animal models are also useful tools in medical science research. In this sense, different studies have reported that transgenic expression of mitochondrial-targeted catalase in mouse models of chronic diseases, such as cancer or Alzheimer’s disease, reduced primary tumor invasiveness and decreased metastasis incidence and burden (Goh et al., 2011) and several typical events of the disease (Mao et al., 2012).

Finally, in transgenic mouse lines, the overexpression of catalase targeted to mitochondria reduced age-associated pathologies and led to an extension in lifespan (Schriner et al., 2005). Overall, these in vivo studies support that mitochondria-targeted molecules may be an effective therapeutic approach to treat or prevent inflammation linked to numerous degenerative and acute diseases. In addition, other interesting models to be kept in mind are represented by mice with increased mtDNA mutations that exhibit pathology associated to human aging (Trifunovic, 2006) Analysis of the inflammasome has not been performed in these animals, but could be a useful model in which to study the role of mitochondria in NLRP3 inflammasome activation.

However, despite our increasing knowledge on the role of mitochondria in the development of the inflammatory response, there is still much to learn. Several key questions remain to be addressed. For example, which mechanisms determine whether mitochondria induce the inflammasome or apoptosome? Along these lines, recent research has suggested that oxidized mtDNA released during apoptosis results in activation of the NLRP3 inflammasome, providing a link between apoptosis and inflammasome activation (Shimada et al., 2012).

Interestingly, VDAC, whose activity is modulated by Bcl-2 family members, is decisive for inflammasome activation, and also in apoptosis induction by Bax (Yamagata et al., 2009; Zhou et al., 2011). However, what is the exact mechanism through which mtDNA activates the NLRP3 inflammasome? It also remains to be determined the importance of inflammasome in non-immune cell types.

Furthermore, other factors, such as tissue specificity, the effects of environmental factors, and age, may determine the final outcome. Clearly, more work is necessary to shed light on how mitochondrial dysfunction modulates the inflammatory response associated with distinct degenerative and acute diseases as well as the aging process.

9. Conclusions

Both acute and chronic inflammatory diseases, as well as the aging process, have been linked to accumulation of ROS and RNS, which could potentially be a main source of mitochondrial genomic instability leading to respiratory chain dysfunction. Indeed, mitochondrial impairment could modulate innate immunity through both redox-sensitive inflammatory pathways and direct activation of the inflammasome. Mitochondria could integrate these 2 pathways, leading to an overstimulation of the inflammatory response (Fig. 1). In conclusion, there is substantial evidence supporting the hypothesis that a decline in mitochondrial function is essential to the development of the inflammatory phenotype observed in multiple human degenerative or acute diseases as well as in advanced age.

All of these findings support mitochondria as new pharmacological targets. The preservation of mitochondrial function could reduce oxidative stress and may represent a novel therapeutic advantage for patients with degenerative or acute diseases as well as functional decline in aging. Despite these advances in our knowledge of inflammatory alterations, it is necessary to keep in mind that the best treatment is prevention; this is true for both inflammatory-related diseases and in healthy aging. Although we are becoming increasingly aware of the importance of a healthy diet, there is still a long way to go. Adequate nutrition could in fact be the best way to prevent aging declines and inflammatory degenerative or acute diseases. Thus, as Hippocrates said 25 centuries ago, “Let food be the medicine and medicine be the food”

Inflammation Is a key participant in all aging and disease

Inflammation influence on aging process itself, not just age related disease


We know NAD+ levels go down dramatically as we age.   Many recent and ongoing research studies are   investigating protocols for boosting NAD+ levels in elderly individuals to combat both disease and infirmities related to aging.  Supplementing with Nicotinamide Riboside and/or NMN are 2 that are attracting a great deal of attention, especially after Dr David Sinclair demonstrated that raising NAD+ levels in elderly mice could “turn back the clock” leaving them with muscular function of young mice.


  • NAD+ is key for mitochondria to perform all functions within the body
  • NAD+ levels go down as we age
  • Lower NAD+ levels impair all functions in the body
  • Inflammation lower NAD+ levels


What you can do about it:


  • HIIT
  • Weight Training
  • Cardio

Diet and Nutrition

  • Weight loss – especially visceral fat
  • foods  – dr axe, perricone, weill (trufood restaurants)
  • fasting

Herbs & Supplements

NAD+ boosters

  • Niagen
  • NMN
  • Grape Seed Extract

Anti-inflammatories, Anti-oxidants

  • Curcumin
  • omega 3 oils
  • CoQ10
  • PQQ
  • Boswellia
  • Green Tea
  • Pomegranate
  • Lycopene

AMPK activators

  • Berberine
  • Metformin


What is Inflammation?

from watson at antiagingfirewalls.com

Inflammation is a necessary part of our bodies defensive response to injury and disease, as it strives to eliminate the cause of irritation and is an essential initial stage of healing injured tissues.  We could not survive without it.   Unfortunately, if the cause of the irritation or illness is not eliminated, inflammation can often get out of control.  Such Chronic Inflammation is now recognized to be  both a cause and effect of nearly ALL age related conditions such as cancer, arthritis, metabolic syndrome, heart disease, osteoporosis, Alzheimers, IBS, asthma, COPD, depression, fatigue and more (456).


Anything that can help fight chronic inflammation is of potential importance in preventing and even treating these diseases. For example, Curcumin  a powerful anti-inflammatory that  matches the effectiveness of many prescription drugs in treating some chronic conditions, but without the side effects (71011121314).   You can read more about some of the amazing benefits of Curcumin here.

Chronic inflammation, sometimes referred to as constitutive inflammation, other times as inflammaging, can persist over an extended period of weeks to months and even years.  It is often associated with the presence of macrophages and lymphocytes, fibrosis, vascular proliferation, and tissue destruction.  Moreover, chronic inflammation plays critical roles in many disease processes including cancers, dementias, diabetes, pulmonary diseases, cardiovascular diseases, atherosclorsis, sarcopenia, and anaemia.  Chronic inflammation occurs in the case of incurable autoimmune diseases such as arthritis, lupus, scleroderma, asthma and chronic obstructive pulmonary disease (COPD).

The biological mechanisms of chronic inflammation can be very complex,  Nuclear factor- B (NF-κB) is activated by more than 200 different stimuli has for good reason been thought of as the master activator of inflammation.  It is a central topic in this blog entry.  For example, during inflammation immune system macrophage cells could be activated by Toll-like receptors (TLRs), through the recognition of a pathogen endotoxin such as lipopolysaccharide (LPS). This event initiates a signaling pathway that releases NF-κB into the cell nucleus, activating genes associated with the transcription of proteins related to the inflammatory process, such as iNOS, responsible for NO synthesis, COXs, which synthetize prostaglandins, and cytokines like IL-6. The generation of ROS is also triggered by the TLR signaling pathway.

Among the highly technical topics related to chronic inflammation and its consequences are Activating protein-1 (AP-1), AGEs, RAGE receptor, PAMPs, DAMPs, RNS, leukotrienes, LOX, prostaglandins, COX1, COX2, Resolvins, Protectins, Maresins, the NLRP3 inflammasome, lipoxins, Ca++ induced inflammation, pyroptosis, cellular senescence-induced inflammation, roles of inflammation in aging, potassium efflux out of a cell, mitochondrial ROS, translocation of NLRP3 to the mitochondria, cytosolic release of mitochondrial DNA, cardiolipin release, release of lysosomal cathepsin D into the cytosol, extracellular LPS “priming” of NLRP3, amyloid-beta “triggering” of NLRP3  via TLR4,  ATP, and pore-forming toxins.  We expect to touch on most of these in this blog series on inflammation.

Inflammation and aging

Since chronic inflammation plays central roles in numerous deleterious health processes and in aging, it is often referred to as “inflammaging” and is the subject of much ongoing research.  From the 2014 publication Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases:  “Human aging is characterized by a chronic, low-grade inflammation, and this phenomenon has been termed as “inflammaging.” Inflammaging is a highly significant risk factor for both morbidity and mortality in the elderly people, as most if not all age-related diseases share an inflammatory pathogenesis.  Nevertheless, the precise etiology of inflammaging and its potential causal role in contributing to adverse health outcomes remain largely unknown.  The identification of pathways that control age-related inflammation across multiple systems is therefore important in order to understand whether treatments that modulate inflammaging may be beneficial in old people.”

The 2016 publication Inflammaging and Anti-Inflammaging: The Role of Cytokines in Extreme Longevity points out the central linkages between inflammation and aging.  “Longevity and aging are two sides of the same coin, as they both derive from the interaction between genetic and environmental factors.  Aging is a complex, dynamic biological process characterized by continuous remodeling. One of the most recent theories on aging focuses on immune response, and takes into consideration the activation of subclinical, chronic low-grade inflammation which occurs with aging, named “inflammaging.”  Long-lived people, especially centenarians, seem to cope with chronic subclinical inflammation through an anti-inflammatory response, called therefore “anti-inflammaging.”  In the present review, we have focused our attention on the contrast between inflammaging and anti-inflammaging systems, by evaluating the role of cytokines and their impact on extreme longevity.  Cytokines are the expression of a network involving genes, polymorphisms and environment, and are involved both in inflammation and anti-inflammation.  We have described the role of IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IL-22, IL-23, TNF-α, IFN-γ as pro-inflammatory cytokines, of IL-1Ra, IL-4, IL-10, TGF-β1 as anti-inflammatory cytokines, and of lipoxin A4 and heat shock proteins as mediators of cytokines.  We believe that if inflammaging is a key to understand aging, anti-inflammaging may be one of the secrets of longevity.”  This is an opinion I (Vince) hold. In a later blog entry in this series, I will share some of the approaches to anti-inflammaging I have been personally and professionally pursing.




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Chronic Inflammation accelerate aging and disease

 The following is from Oprah.com site from 2005!!!

Dr. Perricone’s years of research have shown that the inflammation-aging connection is the single greatest cause of aging and age-related diseases such as heart disease, diabetes, Alzheimer’s disease, arthritis, certain forms of cancer, diminished mental and physical energy, the loss of muscle mass and wrinkled, sagging skin.

This chronic inflammation goes on day after day, year in and year out, leading to disease states as well as the disease of aging. In fact, aging is a chronic, uniformly progressive, inflammatory disease that is always fatal.

Our food choices are critical when it comes to causing and controlling inflammation. This is good to know because it actually means we are in control of the situation!

This is the key to health, longevity, mental clarity, well-being and beautiful youthful skin. Foods that are pro-inflammatory, such as all forms of sugar, processed foods, pasta, breads, pastry, baked goods, and snack foods such as rice and corn cakes, chips, pretzels, etc., cause a highly destructive pro-inflammatory response in our bodies. If we choose sugary or starchy foods, we trigger this pro-inflammatory release of sugar into our bloodstream, which causes our body to store fat rather than burn it for energy.

The result? Acceleration of the aging process of all organ systems in our body, including the skin, causing an increased risk of degenerative disease and inflexible, wrinkled, sagging skin. In addition, by eating that muffin or couple of cookies, the resulting insulin response triggers our appetite—causing us to crave more and more of these types of carbohydrates, resulting in a vicious cycle of overeating.

That is the bad news! Now for the good news:

Fortunately we can control inflammation in our bodies. It starts with the very foods we eat. All we have to do is avoid foods that provoke a “glycemic” response in the body, i.e. cause a rapid rise in blood sugar.

Read more: http://www.oprah.com/health/dr-perricones-inflammation-aging-connection-concept#ixzz4cDz9vQmG

below from DrAxe.com

Inflammation has been found to be associated with just about every health condition.  Researchers are furiously investigating chronic inflammation’s effects on health and possible preventive medical applications.

It’s “an emerging field,” says UCLA’s Dr. David Heber. “It’s a new concept for medicine.” (1)

Why is it a new concept? Because modern medicine focuses on treating symptoms, not addressing the root cause of an issue.Arthritis is inflammation of the joints. Heart disease is inflammation of the arteries. Instead of taking a medication to reduce joint pain or lower cholesterol, we would be better served by reducing inflammation in the body.

Dr. Tanya Edwards, director of the Center for Integrative Medicine, writes that inflammation is now recognized as the “underlying basis of a significant number of diseases.”

Although inflammation has long been known to play a role in allergic diseases like asthma, arthritis and Crohn’s disease, Edwards says that Alzheimer’s diseasecancer, cardiovascular disease, diabetes, high blood pressurehigh cholesterol levels and Parkinson’s disease may all be related to chronic inflammation in the body.


What Inflammation is, and Why You Should Care

Just to make sure that we’re all on the same page, I want to briefly explain what inflammation is.

I’m not going to get into much detail, because inflammation is extremelycomplicated.

It involves dozens of cell types and hundreds of different signalling molecules, all of which communicate in immensely complex ways.

Older Male Doctor, Smaller

Put simply, inflammation is the response of the immune system to foreign invaders, toxins or cell injury.

The purpose of inflammation is to affect the function of immune cells, blood vessels and signalling molecules, to initiate an attack against foreign invaders or toxins, and begin repair of damaged structures.

We’re all familiar with acute (short-term) inflammation.

For example, if you get bitten by a bug, or hit your big toe on the doorstep, then you will become inflamed.

The area will become red, hot and painful. This is inflammation at play.

Inflammation is generally considered to be a good thing. Without it, pathogens like bacteria and viruses could easily take over our bodies and kill us.

However, there is another type of inflammation that may be harmful, because it is inappropriately deployed against the body’s cells (7).

This is a type of inflammation that is active all the time, and may be present in your entire body. If is often called chronic inflammation, low-grade inflammation, or systemic inflammation (8).

For example, your blood vessels (like your coronary arteries) may be inflamed, as well as structures in your brain (910).

It is now believed that chronic, systemic inflammation is one of the leading drivers of some of the world’s most serious diseases (11).

This includes obesity, heart disease, type 2 diabetes, metabolic syndrome, Alzheimer’s diseasedepression and numerous others (1213141516).

However, it is not known exactly what causes the inflammation in the first place.

Bottom Line: Inflammation is the response of the immune system to foreign invaders, toxins and cell injury. Chronic inflammation, involving the entire body, is believed to drive many killer diseases.


Why Care About Omega-6 and Omega-3 Fatty Acids?

Omega-6 and Omega-3 fatty acids are called polyunsaturated because they have many double bonds (poly = many).

Our bodies don’t have the enzymes to produce them and therefore we must get them from the diet.

If we don’t get any from the diet, then we develop a deficiency and become sick. That is why they are termed the “essential” fatty acids.

However, these fatty acids are different than most other fats. They are not simply used for energy or stored, they are biologically active and have important roles in processes like blood clotting and inflammation.

The thing is… Omega-6s and Omega-3s don’t have the same effects. Omega-6s are pro-inflammatory, while Omega-3s have an anti-inflammatory effect (1).

Of course, inflammation is essential for our survival. It helps protect our bodies from infection and injury, but it can also cause severe damage and contribute to disease when the inflammatory response is inappropriate or excessive.

In fact, excess inflammation may be one of the leading drivers of the most serious diseases we are dealing with today, including heart disease, metabolic syndrome, diabetes, arthritis, Alzheimer’s, many types of cancer, etc.

Put simply, a diet that is high in Omega-6 but low in Omega-3 increases inflammation, while a diet that includes balanced amounts of each reduces inflammation (2).

The problem today, is that people who eat a typical Western diet are eating way too many Omega-6s relative to Omega-3s.

Bottom Line: An Omega-6:Omega-3 ratio that is too high can contribute to excess inflammation in the body, potentially raising the risk of all sorts of diseases.





We may not realize that our intestinal flora can be a driver of inflammation. Gut-associated inflammation has been linked to insulin resistance, some forms of cancer, and even mental health concerns.

The trillions of bacteria that live within our gut have an intimate connection to our immune system, helping to strike a balance between tolerance and regulation. One type of bacteria that can cause inflammation is gram-negative bacteria.


Some gram-negative bacteria exist naturally in a balance with gram-positive bacteria in our gut. But excessive or harmful gram-negative bacteria may appear due to an infection or in response to poor lifestyle choices, such as a high-fat, low-fibre diet.

Gram-negative bacteria have molecules in their cell walls called lipopolysaccharides (LPS), which are a little like a coat of gnarly armour. If the barrier function of the gut is diminished (which can result from a high-fat, high-sugar diet, stress, or other causes), these LPS can enter the bloodstream, where they incite an inflammatory response.


While we see evidence that altered intestinal flora can lead to increased inflammatory markers, the ability of probiotics to reduce chronic inflammation is still being researched. In some studies, selected strains or blends of probiotic bacteria have outcompeted gram-negative bacteria. In addition, some probiotics have been shown to physically reinforce the gut barrier to prevent LPS passage. Through research to date, we see that certain probiotic strains within the Lactobacillus group are strong enough to act this way.

Some probiotic bacteria have also shown promise in reducing the production of messengers called pro-inflammatory cytokines. Probiotic bacteria produce substances known as short chain fatty acids, which can lower inflammatory markers in addition to strengthening the gut barrier.

Chronic inflammation can be debilitating, but it is not a life sentence. Inflammation is best addressed through an integrative approach to healthy living: eat more plants, move more, manage stress, and don’t forget to use beneficial bacteria to your immune advantage.





You know what inflammation looks like: You get a cut or bruise, and the area around it soon turns red, gets warm, and swells up. This is called the acute inflammatory response, and it’s your immune system’s defensive reaction to infection or injury. A complex array of immune cells congregate at the site and release a variety of chemicals to deal with the infectious organisms or debris from the injury and to allow tissue repair to begin; normally the inflammation gradually subsides. This immune response is essential to life.

But there’s another way inflammation works—it can be chronic and cause a low-grade systemic reaction. Because it increases with aging, it has been dubbed “inflammaging.” Chronic systemic inflammation has been the focus of a great deal of scientific attention during the past two decades (especially the past few years) and is now viewed as a sort of “unified field” explanation for many, if not most, age-related chronic diseases.

Accordingly, factors (genetic, lifestyle, and environmental) that promote chronic inflammation or disrupt the body’s protective mechanisms against it may increase the risk of premature aging and the disorders that go with it. On the other hand, healthy aging and longevity may be related to reduced levels of inflammation and/or strong protective mechanisms that guard against its adverse effects.

This was suggested by the results of a study in the Canadian Medical Association Journal last year, which included 3,000 British civil servants. It found a strong link between higher levels of chronic inflammation (as measured by blood levels of an inflammatory marker) and a decreased likelihood of “successful aging,” defined as optimal physical and cognitive health and the absence of chronic diseases. In fact, elevated levels of inflammation appeared to reduce the odds of successful aging by half over the next decade and to markedly increase the odds of cardiovascular disease and death.


Many complex roles

It can be both a cause andan effect of some disorders—setting up a vicious cycle that helps explain their chronic nature.

For example, chronic inflammation plays reciprocal roles with obesity and insulin resistance. It contributes to the development of insulin resistance, which in turn may help promote obesity. Conversely, obesity worsens insulin resistance and increases chronic inflammation, partly because body fat (especially the type surrounding internal organs) releases pro-inflammatory compounds. In effect, inflammation, obesity, and insulin resistance reinforce one another, often resulting in type 2 diabetes. What’s more, many lifestyle factors that promote inflammation, such as being sedentary and having an unhealthy diet, also promote obesity and insulin resistance.


Hard to pin down

Chronic inflammation is a varied phenomenon that affects nearly every aspect of human physiology and disease development. Many different kinds of specialized cells and chemicals are involved in producing and regulating these inflammatory processes.

Since it is so complex, there is no way to measure chronic inflammation directly. Instead, researchers measure a variety of inflammatory chemical markers in the blood or tissue, notably interleukin-6, tumor necrosis factor (TNF), C-reactive protein, prostaglandins, and leukotrienes. Elevated levels of these factors are good indicators of disease activity for some conditions (such as inflammatory bowel disease). But it’s not clear whether measuring them adequately gauges inflammation and the resulting risks for some other disorders (such as cancer).

Time for CRP Testing?

C-reactive protein, or CRP, is produced by the liver in response to inflammation. Of all markers for inflammation, it has gotten the most attention because research has shown that elevated blood levels are strongly associated with an increased risk of cardiovascular disease, even in people otherwise at low risk.

This was seen in the well-known JUPITER study a few years ago, which focused on people with desirable cholesterol levels but elevated CRP. It found that they greatly reduced their risk of heart attacks and strokes when they took a statin drug. Besides lowering LDL (“bad”) cholesterol, statins have anti-inflammatory effects, as seen in reductions in CRP.

Subsequently the FDA approved rosuvastatin—the statin used in JUPITER—for people who have desirable levels of LDL but high CRP and at least one other coronary risk factor. And according to revised cholesterol guidelines released last year, in cases where there’s uncertainty about statin treatment, CRP level is one of several factors that doctors should consider in making the decision.

Most doctors do not routinely measure CRP, however. It’s not clear what cutoff should be used to define high CRP, nor is it certain that bringing down elevated CRP will, by itself, be beneficial. Still, if you’re at intermediate coronary risk, and you and your doctor are on the fence about starting drug therapy, you should consider CRP testing. A high result could tip the balance toward a statin.


The link to heart disease

For many years atherosclerosis was seen as a kind of plumbing problem—that is, merely a matter of plaque building up in the walls of coronary arteries and clogging them. But blood vessels are nothing like pipes—they are active tissue involved in complex processes. In simplest terms, cells lining the vessels absorb cholesterol (and other substances) from the blood, leading to the build-up of plaque. The body perceives this plaque as an injury and sends inflammatory cells into the vessel walls, where they set off a cascade of events that can ultimately cause plaque to rupture and a clot to form over it. If the clot breaks off or otherwise obstructs blood flow to the heart or brain, this can result in a heart attack or stroke.

It now appears that inflammation plays key roles in all stages of the development of cardiovascular disease. Bacterial or viral infection may also trigger the inflammatory process in blood vessels. Meanwhile, coronary risk factors such as obesity, high blood pressure, undesirable cholesterol levels, and smoking cause or worsen arterial inflammation. Having an inflammatory disorder, such as rheumatoid arthritis, diabetes, or inflammatory bowel disease, also increases coronary risk.

Some medications that help prevent heart attacks and strokes, notably statins, do so at least in part by reducing inflammation. The story is more complicated regarding aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). At the low doses used to protect the heart, aspirin has only a small effect on inflammation; its heart benefit comes primarily from its ability to reduce the risk of blood clots. No other NSAIDs are good for the heart. In fact, some NSAIDs, notably celecoxib (Celebrex), increase the risk of heart attacks slightly.



The cancer connection

As early as the mid-19th century, scientists found links between chronic inflammation (or its markers) and cancer. It’s now estimated that more than 20 percent of cancer cases are associated with inflammation.

Inflammation is involved with cancer development on many levels. Notably, it contributes to tumor initiation by inducing oxidative stress, DNA damage, and chromosomal instability. It promotes tumor cell proliferation and resistance to apoptosis (programmed cell death after a certain number of cell divisions, a good thing when it comes to cancer cells). Simply put, increased inflammation makes it easier for normal cells to transform into malignant cells.

The evidence is strongest concerning gastrointestinal cancers, including certain kinds of colon, liver, esophageal, and stomach cancer. It’s theorized that these organs are at high risk because they are exposed directly to pro-inflammatory dietary and environmental factors. Inflammation can also alter colonic microflora in ways that increase cancer risk.

On the positive side again, evidence is accumulating that aspirin, partly because of its anti-inflammatory effect, can reduce the risk of certain types of colon cancer and possibly certain other cancers.


What does all this mean for you?

What can you do to reduce chronic inflammation and the risks it entails? There is no magic food, pill, or treatment. But many of the same steps that help prevent cardiovascular disease may do so in part by helping to tamp down inflammation.

  • Eat a heart-healthy diet. Lab research has shown that many healthful foods, especially fatty fish, fruits, and vegetables (as well as chocolatewine, and tea) have anti-inflammatory effects. Other studies have shown that the Mediterranean diet tends to reduce inflammation (as measured by CRP). On the other hand, saturated fats, trans fats, sugar, and other refined carbohydrates have pro-inflammatory effects in the body.
  • Aerobic exercise, done regularly and moderately, reduces chronic inflammation via a variety of complex mechanisms. In contrast, being sedentary or training very intensely both increase inflammation.
  • If you are very overweight, and especially if the extra pounds are in your abdomen, lose weight via a healthy diet and exercise. That will reduce inflammation and the risk of chronic diseases.
  • Don’t smoke—it’s a powerful cause of inflammation. Avoid secondhand smoke.
  • If you have had a heart attack or are at elevated risk for one, talk to your doctor about low-dose aspirin. If you have no history of cardiovascular disease, however, the risks of aspirin therapy (bleeding in the stomach or brain) may outweigh its small benefit. Similarly, if you are at high risk for colon cancer because of polyps or family history, discuss aspirin therapy with your doctor.
  • If you’re prescribed a statin, here’s an added reason to take it: It serves double duty—against cholesterol and inflammation.
  • Don’t drink more than moderate amounts of alcohol.
  • Get adequate sleep and try to find ways to deal with stress, anxiety, and depression. Social isolation can also increase chronic inflammation, as was seen in a study in the Journal of Health and Social Behavior last year, so increasing social activities may help.

On the horizon: 


Can NMN really reverse Aging? (backup)

As we age, our levels of the Co-enzyme Nicotinamide Adenine Dinucleotide NAD+ drop significantly in multiple organs in mice and humans  (5,8,10).

NAD+ decrease is described as a trigger in age-associated decline(23), and perhaps even the key factor in why we age (5).

In 2013, research published by Dr David Sinclair demonstranted that short term supplementation with Nicotinamide MonoNucleotide (NMN) reversed many aspects of aging, making the cells of old mice resemble those of much younger mice, and greatly improving their health (8).


The quotes below are directly from that research:

NMN was able to mitigate most age-associated physiological declines in mice”

“treatment of old mice with NMN reversed all of these biochemical aspects of aging”

Since that landmark 2013 study, dozens of others have been published investigating the efficacy of supplementation with NMN in treatment and prevention of a wide range of disease including cancer, cardiovascular disease, diabetes, Alzheimers, Parkinsons, and more (5,6,7,9,10,11,13,14,15,16).

According to Dr Sinclair:

enhancing NAD+ biosynthesis by using NAD+ intermediates, such as NMN and NR, is expected to ameliorate age-associated physiological decline


NR benefits chartNAD+ is a key co-enzyme that the mitochondria in every cell of our bodies depend on to fuel all basic functions. (3,4)

NAD+ play a key role in communicating between our cells nucleus and the Mitochondria that power all activity in our cells (5,6,7)


NAD+ levels decreaseAs we age, our bodies produce less NAD+ and the communication between the Mitochondria and cell nucleus is impaired. (5,8,10).

Over time,  decreasing NAD+ impairs the cell’s ability to make energy, which leads to aging and disease (8, 5) and perhaps even the key factor in why we age (5).


NAD+ can be synthesized in humans from several different molecules (precursors), thru 2 distinct pathways:
De Novo Pathway

  • Tryptophan
  • Nicotinic Acid (NA)

Salvage Pathway

  • NAM – Nicotinamide
  • NR – Nicotinamide Riboside
  • NMN – Nicotinamide MonoNucleotide

The NAD+ supply is constantly being consumed and replenished through the Salvage Pathway, with approximately 3g of NAM metabolized to NMN and then to NAD 2-4 times per day (14).

  • The salvage pathway sustains 85% or more of our NAD+ (14)
  • Nampt is the rate-limiting step in the salvage process (97).
  • As we age, Nampt enzyme activity is lower, resulting in less NAM recycling, less NAD+, more disease and aging (97,101).


NAM, NA, NMN, NR, and Tryptophan ALL elevate levels of NAD+ significantly in the liver, which has many benefits for metabolic health.

This chart from the Trammell thesis shows the impact on liver NAD+ for mice given NR, NAM, and NA by oral gavage 0.25, 1, 2, 4, 6, 8 and 12 hours before testing.

Charts showing NMN impact on NAD+ levels in the liver are below.

* Note:  These charts are somewhat deceptive. It shows NAM (green bar) elevated NAD+ nearly as much as NR (black bar)

However if they used equal mg of each supplement, which is how people actually purchase and use them, it would show NA about equal with NR and NAM far effective than NR at elevating NAD+ in the liver.

Mice in these experiments didn’t receive equal WEIGHTS of each precursor. Instead researchers chose to use quantity of molecules, which makes NR look “better” by comparison.

In this case, “185 mg kg−1 of NR or the mole equivalent doses of Nam and NA”(16).

Molecular weight for NR is 255 grams, NAM is 122 grams, and NA 123 grams.  So this chart used a ratio of  255 grams of NR to 122 and 123 grams of NAM and NA.


  • “NMN makes its way through the liver, into muscle, and is metabolized to NAD+ in 30 minutes” (R)


  • Is much slower, taking 8 hours to reach peak NAD+ in humans (R)


  • Has very similar NAD+ profile to NR, taking 8 hours to reach peak NAD+ in humans (R)
  • Has been shown to increase NAD+ level in liver (47%), but was weaker in kidney (2%), heart (20%), blood (43%) or lungs (17%) (R)

NA (Niacin)

  • Elevates NAD+ to peak levels in liver in 15 minutes (R)
  • raised NAD+ in liver (47%), and impressively raised kidney (88%), heart (62%), blood (43%) and lungs (11%) (R)


  • In the liver  tryptophan is the preferable substrate for NAD+ production (R)
  • Administration of tryptophan, NA, or NAM to rats showed that tryptophan resulted in the highest hepatic NAD+ concentrations(R)


Restoring NAD+ to youthful levels in ALL CELLS throughout the body is the goal.

However, many tissues cannot utilize NAD+ directly from the blood as NAD+ cannot readily pass through the cellular membrane.

Muscle tissue, for example, depends on cells internal recycling of NAD+ through the salvage pathway which is controlled by Nampt.

To restore depleted NAD+ levels in such cells, a precursor must:

  • Be available in the bloodstream
  • Once inside a cell, be able to bypass the Nampt bottleneck

NA and Tryptophan
NA and Tryptophan act through the De Novo pathway, which supplies a small percentage of our NAD+, primarily in the liver

NAM is abundant in the blood and easily carried into such cells throughout the body, but  depends on Nampt, which is the rate limiting enzyme in the salvage pathway.

When taken orally as a supplement, most NR does not make it through the digestive system intact, but is broken down to NAM (97,98,99).

For more info on how NR is converted to NAM in the body.

NR can bypass the Nampt bottleneck, but is not normally available in the bloodstream

After oral NMN supplementation, levels of NMN in the bloodstream are quickly elevated and remain high longer than NAM, NA, or NR (18,22,97,98,99)

Oral NMN supplements:

  • Make their way intact thru the digestive system (22)
  • Quickly elevates levels of NMN in the bloodstream for use throughout the body (22)
  • Quickly elevates levels of  NMN in tissues throughout the body (22)
  • Quickly raises levels of NAD+ in blood, liver and tissues  through the body (22,23)
  • Remain elevated longer than NAM, NA, or NR (18)

Only NMN is readily available in the bloodstream to all tissues, and bypasses the Nampt bottleneck in the Salvage pathway


The chart at right shows levels of a double labeled NAD+ (C13-d-nad+) in liver and soleus muscle at 10 and 30 minutes after oral administration of double labeled NMN.

This clearly shows that NMN makes it way through the liver intact, through the bloodstream, into muscle, and is metabolized to NAD+ in 30 minutes (22) .

This quote below is directly from that study.

Orally administered NMN is quickly absorbed, efficiently transported into blood circulation, and immediately converted to NAD+in major metabolic tissues (22).


In this 2016 study, mice were given a single dose of  NMN in water.

NMN  levels in blood showed it is quickly absorbed from the gut into blood circulation within 2–3 min and then cleared from blood circulation into tissues within 15 min





In this 2017 study, NMN supplementation for 4 days significantly elevated NAD+ and SIRT1, which protected the mice from Kidney damage.

NAD+ and SIRT1 levels were HIGHER in OLD Mice than in YOUNG Mice that did not receive NMN.



In a long-term experiment documented in the 2016 study (22) , mice were given 2 different doses of NMN over 12 months.

Testing revealed that NMN  prevents some aspects of  physiological decline in mice, noting these changes:

  • Decreased body weight and fat
  • Increased lean muscle mass
  • Increased energy and mobility
  • Improved visual acuity
  • Improved bone density
  • Is well-tolerated with no obvious bad side effects
  • Increased oxygen consumption and respiratory capacity
  • Improved insulin sensitivity and blood plasma lipid profile

Here are some quotes from  the  study:

NMN suppressed age-associated body weight gain, enhanced energy metabolism, promoted physical activity, improved insulin sensitivity and plasma lipid profile, and ameliorated eye function and other pathophysiologies

NMN-administered mice switched their main energy source from glucose to fatty acids

These results strongly suggest that NMN has significant preventive effects against age-associated impairment in energy metabolism

NMN effectively mitigates age-associated physiological decline in mice


Researchers found that NMN administration suppressed body weight gain by 4% and 9% in the 100 and 300 mg/kg/day groups.

Analyses of  blood chemistry panels and urine did not detect any sign of toxicity from NMN.

Although health span was clearly improved, there was no difference in maximum lifespan observed.

These results suggest that NMN administration can significantly suppress body weight gain without side effects


Oxygen consumption significantly increased in both 100 and 300 mg/kg/day groups during both light and dark periods (Figure 3A).

Energy expenditure also showed significant increases  (Figure 3B).

Respiratory quotient significantly decreased in both groups during both light and dark periods (Figure 3C),

This suggests that NMN-administered mice switched their main energy source from glucose to fatty acids.

The mice that had been receiving NMN for 12 months exhibited energy levels, food and water consumption equivalent to the mice in the control group that were 6 months younger.

NMN administration has significant preventive effects against age associated physical impairment


The first clinical trial of NMN in humans is currently underway by an international collaborative team between Keio University School of Medicine in Tokyo and Washington University School of Medicine (33).

Participants are 50 healthy women between 55 and 70 years of age with slightly high blood glucose,BMI and triglyceride levels.

Using a dose of 2 capsules of 125mg NMN per day over a period of 8 weeks, researchers are testing for:

  • change in insulin sensitivity
  • change in beta-cell function
  • works to control blood sugar
  • blood vessels dilate
  • effects of NMN on blood lipids
  • effects of NMN on body fat
  • markers of cardiovascular and metabolic health

According to the study:

“Data from studies conducted in rodents have shown that NMN supplementation has beneficial effects on cardiovascular and metabolic health, but this has not yet been studied in people”

Testing of metabolic parameter will continue for 2 years after supplementation has ended, so final results will not be published for some time yet, but preliminary results are expected to be announced in early 2018.


NMN is found in many food sources such as edamame, broccoli, cucumber,cabbage, avocado, tomato, beef and shrimp.

As such, it is likely free from serious side effects in humans, and has been available for purchase commercially for over 2 years.


In the long term (12 month) 2016 mouse study (22), both 100 and 300mg/kg per day improved oxygen consumption, energy expenditure, and physical activity more.

According to the FDA guidelines, an equivalent  would be about 560 mg for a 150lb human.

It should be noted that NMN administration did not generate any obvious toxicity, serious side effects, or increased mortality rate throughout the 12-month-long intervention period, suggesting the long-term safety of NMN.

The current Human study uses a dosage of 2 capsules of 125 mg, which seems to be the most commonly used dosage.


NAD+ levels decrease throughout the body as we age, contributing to disease and aging.

Restoring NAD+ levels can ameliorate many age released health issues.

All the NAD+ precursors are effective at raising NAD+ levels in the liver.

Raising NAD+ in the liver has many benefits, but is not effective in tissues and organs that cannot access NAD+ directly from the bloodstream and so depend on internal cellular NAD+ recycling.

For these tissues, utilizing each cells internal Salvage Pathway is necessary to restore NAD+ levels.

NR is not stable in the body and not normally found in the bloodstream, so is not readily available as NR to many tissues. Once metabolized to NAD+ it cannot enter cells. If metabolized to NAM it cannot bypass the Nampt bottleneck.

NMN is the only precursor that is stable and available to cells through the bloodstream, and can bypass the Nampt bottleneck to quickly restore NAD+ throughout the body.


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Head to Head Comparison of Short-Term Treatment with the NAD(+) Precursor Nicotinamide Mononucleotide (NMN) and 6 Weeks of Exercise in Obese Female Mice (Uddin, 2016)

NAD(+) levels were increased significantly both in muscle and liver by NMN
NMN-supplementation can induce similar reversal of the glucose intolerance
NMN intervention is likely to be increased catabolism of fats
NMN-supplementation does mimic exercise

DNA Damage

A conserved NAD+ binding pocket that regulates protein-protein interactions during aging (Sinclair, 2017)

This study showed supplementation with NMN was able to repair the DNA in cells damaged by radiation.

the cells of old mice were indistinguishable from young mice after just one week of treatment.”

Diabetes & Metabolic disease

Nicotinamide Mononucleotide, a Key NAD+ Intermediate, Treats the Pathophysiology of Diet- and Age-Induced Diabetes in Mice (Yoshino, 2011)

NMN was immediately utilized and converted to NAD+ within 15 min, resulting in significant increases in NAD+ levels over 60 min

administering NMN, a key NAD+ intermediate, can be an effective intervention to treat the pathophysiology of diet- and age-induced T2D

Surprisingly, just one dose of NMN normalized impaired glucose tolerance

Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging (Gomes, Sinclair,2013)

raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse

treatment of old mice with NMN reversed all of these biochemical aspects of aging

restore the mitochondrial homeostasis and key biochemical markers of muscle health in a 22-month-old mouse to levels similar to a 6-month-old mouse

CardioVascular Disease

Nicotinamide mononucleotide, an intermediate of NAD+ synthesis, protects the heart from ischemia and repercussion (Yamamoto, 2014)

NMN significantly increased the level of NAD+ in the heart

NMN protected the heart from I/R injury

Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice (de Picciotto, 2016)

NMN reduces vascular oxidative stress
NMN treatment normalizes aortic stiffness in old mice
NMN represents a novel strategy for combating arterial aging

Short-term administration of Nicotinamide Mononucleotide preserves cardiac mitochondrial homeostasis and prevents heart failure (Zhang, 2017)

NMN can reduce myocardial inflammation

NMN thus can cut off the initial inflammatory signal, leading to reduced myocardial inflammation

Nicotinamide mononucleotide requires SIRT3 to improve cardiac function and bioenergetics in a Friedreich’s ataxia cardiomyopathy model

Remarkably, NMN administered to FXN-KO mice restores cardiac function to near-normal levels.

restoration of cardiac function and energy metabolism upon NMN supplementation
remarkable decrease in whole-body EE and cardiac energy wasting

Neurological Injury

Nicotinamide mononucleotide attenuates brain injury after intracerebral hemorrhage by activating Nrf2/HO-1 signaling pathway (Wei, 2017)

NMN treats brain injury in ICH by suppressing neuroinflammation/oxidative stress

NMN treatment protects against cICH-induced acute brain injury
NMN treatment reduces brain cell death and oxidative stress
These results further support the neuroprotection of NMN/NAD+


Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer’s disease-relevant murine model (Long, 2015)

We now demonstrate that mitochondrial respiratory function was restored

Nicotinamide mononucleotide protects against β-amyloid oligomer-induced cognitive impairment and neuronal death (Wang, 2016)

NMN could restore cognition in AD model rats.
The beneficial effect of NMN is produced by ameliorating neuron survival, improving energy metabolism and reducing ROS accumulation.
These results suggest that NMN may become a promising therapeutic drug for AD

Nicotinamide mononucleotide inhibits JNK activation to reverse Alzheimer disease(Yao, 2017)

NMN Treatment Rescues Cognitive impairments
NMN Treatment Improves Inflammatory Responses

Kidney Disease
Nicotinamide Mononucleotide, an NAD+ Precursor, Rescues Age-Associated Susceptibility to AKI in a Sirtuin 1-Dependent Manner (Guan, 2017)

Supplementation with NMN restored kidney SIRT1 and NAD+ content in 20-month-old mice and protected both young and old mice from acute kidney injury.


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Nicotinamide Riboside Optimum Dosage

dosageNicotinamide Riboside (NR) is a form of vitamin B3 closely related to Niacin that is showing great promise for it’s ability to raise  NAD+ levels in older humans, back to the levels normally found in youth to prevent and repair damage to various organs in the body.

NAD+   is a key co-enzyme that enables the mitochondria to power and repair damage in every cell of our bodies.


There have been numerous studies of NR and NMN in mice that showed no negative side effects in Human Equivalent Dosages (HED) of 2.1 to 17 grams per day

The FDA recently granted GRAS (Generally Recognized as Safe) status on the basis of this clinical study, which showed “no observed adverse effect level was 300 mg/kg/day.”

screen-shot-2016-10-17-at-2-25-43-pmUsing the chart here from the  FDA guidelines for calculating this to HED of 2880 mg for a 130lb person.

With the FDA required 10x safety factor, that would equate to a dose of 288 mg per day for a 130lb human.

That is likely the limit on what sellers will recommend, but many people have been taking 500-1,000mg a day with no noticeable side effects.

[box]The 10x safety factor required by the FDA results in a safe dosage of 288 mg a day, although many people take much more and few if any side effects are reported at 1,000 mg a day or less[/box]



The first published research to date that measures the NR supplementation increase in NAD+ levels in humans by Dr Charles Brenner is also documented in the Phd dissertation by Samuel AJ Trammel at the University of Iowa.

Experiment #1
In the first experiment, one Human subject was given a single dose 1,000 mg of NR each morning for 7 days. Blood levels of NAD+ and metabolites were 9 times the first day and every 24 hours thereafter.


From the results shown in chart above, we see NAD+ levels did not rise until 4 hours after ingesting, peaked at around 8 hours,  and remained elevated up to 24 hours.

Experiment #2
The second experiment involving human subjects included 12 individuals that were given 100,300, or 1,000 mg of NR with a washout period of 7 days between doses. Blood levels of NAD+ were recorded at 1, 2, 4, 8, and 24 hours.



100 mg per day
This chart shows 100mg per day (purple) elevates NAD+ levels around 4 hours, dropping significantly by 8 hours and continuing to decline throughout the 24 hours.

300 mg per day
The numbers in this line (red) are slightly elevated at 8 hours, then continue rising to 24 hours.

It appears that a dosage of 300mg achieved the same NAD+ increase as 1,000 mg at the 24 hour mark.

1,000 mg per day
This line (black) looks very similar to the first test with one subject given 1,000 mg daily.

Increased NAD+ noted at 4 hours, with maximum increase reached around 8 hour. It appears NAD+ levels remain at that maximum through 24 hours.

We can see that at all dosages the NAD+ levels were elevated somewhat within 4 hours.

It does appear an upper limit was reached after which, additional NR did not raise NAD+ any further.

Dr Brenner points to the increased NAAD levels that coincide with the peak of NAD+ and suggest NAAD acts as an “overflow pool”, that may later be converted to NAD+ if needed.

Do other Metabolites of NAD+ matter?
The author notes that supplementation with Nicotinamide Riboside elevates the level of many NAD+ metabolites at different rates:

“Because every NAD+ metabolite can be converted to one or more other metabolites, snapshots of the levels of NAD+ , nicotinamide (Nam) or any other NAD+ metabolite without assessment of the NAD+ metabolome on a common scale has the potential to be misleading.”

NAAD is much higher in the 1000mg subjects. However, the first study implies there is a limit to the possible increase of NAD+. Despite repeated usage over seven days, NAD+ tops out.

The second study shows that at 24 hours, NAD+ is elevated by approximately the same amount in the 300mg and 1,000mg test subjects.

[box]Conclusion: The maximum effect appears to be achieved at some dosage around 300mg per day.

Note: Subjects in this study were healthy and between 30-55 years of age. Older, sicker subjects might benefit from higher dosages. The Elysium Basis testing with older individuals (below) will hopefully shed more light on this.[/box]


niagen_basis_elysiumResearch to prove Benefits and Safety for Elysium Health Basis brand of Nicotinamide Riboside

This recently complete, but not yet published study tracked 120 elderly subjects (60-80yrs age) over 8 weeks monitored blood and heart parameters to ensure safety.

They also measured NAD+ levels and several physical performance tests.

Completed in July 2016 but not yet published, it was sponsored by Elysium Health, manufacturer of Basis Nicotinamide Riboside.

A single capsule of BASIS is 125 mg of Chromadex NIAGEN brand of Nicotinamide Riboside, along with 50 mg of Chromadex Pterostilbene.

Participants received either placebo, 1, or 2 capsules of BASIS

Elysium Health did issue a press release that states that 125 mg of NIAGEN resulted in a 40% increase in blood NAD+ levels that was maintained throughout the 8 weeks of the study.

The 250 mg dosage resulted in an increase that was “significantly higher” than the 125 mg dose, and reached 90% at one of the 4 checkpoints (4 weeks).

Since the increase from the 250 mg dosages reached a plateau at 4 weeks, and dropped afterwards, implies that a higher dosage probably would not be any more effective.

This rather speculative interpretation agrees with the results in Study #1 that the most effective dosage is higher than 125 mg, but has peaked out at 250mg a day

[box]Conclusion: Most people will likely get the maximum NAD+ increase at 250mg per day



NAD+ is synthesized in humans by several different molecules (precursors), thru 2 different pathways:
De Novo Pathway

  • Tryptophan
  • Nicotinic Acid (NA)

Salvage Pathway

  • NAM – Nicotinamide
  • NR – Nicotinamide Riboside
  • NMN – Nicotinamide MonoNucleotide

The NAD+ supply is not stagnant – it is constantly being consumed and replenished, with the entire NAD+ pool being turned over 2-4 times per day (14).

This recycling is through the salvage pathway, where the enzyme Nampt catalyzes NAM to NMN, which is then metabolized to NAD+.

Nampt is the rate-limiting step in the salvage process (97).

Many studies have confirmed the importance of Nampt in maintaining sufficient NAD+ levels, such as the quote below from a 2016 study that used mice lacking Nampt in muscle fiber:

“NAD content of muscle was decreased by ~85% confirmed the prevailing view that the salvage route of NAD synthesis from NAM sustains the vast majority of the NAD” (97)

These mice exhibited normal muscle strength and endurance while young, but deteriorated rapidly as they aged which confirmed Nampt is critical to maintaining NAD+ levels.

As we age, Nampt enzyme activity is lower, resulting in less NAM recycling, less NAD+, more disease and aging (97,101).


NR had been known for decades, but was not thought to be that important until 2004 when Dr. Charles Brenner discovered the enzyme NRK1 can phosphorylate NR directly to NMN, bypassing the Nampt “bottleneck” (100).

This newly discovered “shortcut” in the NAD+ salvage pathway found that NR can be metabolized directly to NMN to boost NAD+ levels more effectively than NAM.


When taken orally as a supplement, most NR does not make it through the digestive system intact, but is broken down to NAM (97,98,99).

Even when taken at very high dosages, NR has not been detected in the bloodstream in any research (97,98,99).

“This evidence indicates that NR is converted to NAM before absorption occurs and that this reaction is the rate-limiting step ” (98)

“NR has been shown be converted to Nam before being absorbed or reaching tissues” (99)

“we were surprised to find that NR exerts only a subtle influence on the steady state concentration of NAD in muscles. Our tracer studies suggest that this is largely attributable to breakdown of orally delivered NR into NAM prior to reaching the muscle. ” (97)

Note:NAM does elevate NAD+, but is on the “wrong” side of the Nampt bottleneck, which limits it’s effectiveness


The following five charts are all from the thesis published by Samuel Alan Trammell in 2016 under supervision by Dr Brenner:

Nicotinamide riboside is uniquely and orally bioavailable in mice and humans

This chart above shows the impact on NAD+ metabolites over time for a 52 year old human after ingesting 1000mg of NR daily for 7 days.

NAD+ levels begin to rise at 4.1 hours, and peak at 8.1 hours.

NAM levels double at .6 hours and have a second peak at 7.7 hours, long before NAD+ levels are elevated.

This chart at right shows metabolites found in urine of the subject from the same experiment as above.

The red box shows NAM  is elevated more than 10x baseline at the same time point that NAD+ is elevated, which implies that NR has elevated NAM to such an extent that excess NAM is excreted in urine.

This chart a left shows impact of NR, NA, and NAM supplementation on blood plasma NAD+ (b), and NAM  (d) levels in 12 human subjects.

The red line at 2 hours shows NR supplementation increases NAM perhaps 3x (d), but has not yet elevated NAD+(b).

The 2 hour mark also is the point at which NAM supplementation begins to increase NAD+ levels (b).

The blue line at 8 hours is when both NR (b) and NAM (d) supplementation reach peak NAD+ increase.

Lastly, the green bar and black bar in chart b show that NAM elevates NAD+ slightly less than NR.

NR elevated NAD+ slightly more than NAM, but is much slower acting


The chart above shows the result on NAD+ metabolism of 15 mice fed NR by oral gavage at a dose of 185 mg/kg of bodyweight.

The NR was synthesized with heavy atoms of deuterium at the ribosyl C2 and 13C on the Nam side, to allow tracking.

The measurement at 2 hours shows 54% of the NAD+ has the single heavy molecule (white bar, M+1). This 54% was likely broken down to NAM first, losing the second labelled heavy atom.

At the same time point, 5% of the NAD+ had both labels (Grey bar, M+2).

This 5% of NR made it through the digestive tract intact and was metabolized through the shortcut from NR -> NMN -> NAD+, vs 54% that had been through NR -> NAM -> NMN -> NAD+.

The chart above shows the impact of the same double labeled NR on mouse liver, but this time after IP (Intraperitoneal) Injection.

Note the dramatic difference in the ratio of labelled M+2 over M+1. IP results in much higher levels of intact NR (M+2) being metabolized to NAD+, whereas Oral NR shows far more M+1 labelled NR to NAD+.

This different behavior in IP vs oral NR supplementation also implies oral NR is partially metabolized to NAM before conversion to NAD+.

The above chart shows the resultant increase in select NAD+ metabolites of mice fed NR (unlabeled) at 185 mg/kg of bodyweight.

As noted by the authors, NR and NAR are the only NAD+ precursors tested that did NOT result in elevated levels of the precursor in the liver.

Here is one last quote in discussion section from the Trammell thesis:

“NR has not been detected in the blood cell fraction nor in plasma …NR varied and displayed no response to NR administration … but was detected after IP of double labeled NR in liver (Figure 5.7) and muscle (Figure 5.9), revealing NR does circulate”

They are saying that NR is found in small quantities in the liver, but is not detectable in bloodstream.  Oral supplementation with NR did not show any increase in NR in the body.  However, Injection (IP) of NR does result in a detectable increase of NR in muscle and Liver. So NR does circulate in the bloodstream when injected, but has not yet been detected upon oral supplementation.

The timing and amplitude of the increases in metabolites noted above imply that:

  • Oral NR does not result in a detectable increase of NR in the body
  • It’s likely a majority of the increase in NAD+ is due to NR->NAM->NAD+.

Note: NAM does elevate NAD+, but is on the “wrong” side of the Nampt bottleneck, which limits it’s effectiveness


Further testing with larger sample sizes and more data points is underway that will give a much better estimate on the most effective dosage. For now, some conclusions on dosage we see are:

  • A single dose of NR does increase NAD+ levels
  • NAD+ levels remain elevated 24 hours after a single dose.
  • There is an upper limit on the increase of NAD+ levels with NR supplementation
  • Maximum NAD+ elevation is maintained at a dosage higher than 125 mg per day – likely close to 250mg per day
  • It appears that a single daily dose may be just as effective as 2  smaller dosages.

Most NR ends up as NAM after digestion, so is much slower and less effective than NMN.

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Supplementation to correct NAD+ deficiency repairs vision damage in Mice


Nicotinamide adenine dinucleotide (NAD+) is a coenzyme found in the cells of all living creatures (3,4) and is critical for communication between the cell nucleus and the mitochondria that power the cells (5,6,7)


Everyone experiences lower NAD+ levels throughout the body as we age, effecting the communication within every cell of our bodies.

Scientists have known for some time that this loss of NAD+ leads to many different age related diseases as the cells lose ability to perform basic tasks and repair damage due to oxidative stress. (8)


  • Neural cognitive dysfunction (1,2,3)
  • Decreased Energy and muscle strength (23,24)
  • Higher Blood Sugar Levels and Increased Insulin resistance (20,21,22)
  • Chronic Inflammation resulting in hypertension and heart disease( 12,13,14)
  • Fatty Liver Disease (NAFLD and AFLD)(15,16,17)
  • Increased belly fat (18,19)

[box]Conclusion: Declining NAD+ levels are implicated in many age related disease and chronic conditions[/box]


In addition to the chronic age related diseases found to be related to declining NAD+ levels, the study below finds impaired NAD+ biosynthesis in many diverse retinal diseases among young and older mice.

NAMPT-Mediated NAD+ Biosynthesis Is Essential for Vision In Mice

This study published in cell magazine published sep 27, 2016 found that

  • Limiting the natural NAD+ synthesis in the photoreceptors in Mice results in loss of vision
  • Supplementation to increase NAD+ reverses the damage and restores vision
  • Mouse models of retinal dysfunction exhibit early retinal NAD+ deficiency
  • NAD+ deficiency causes retinal metabolic dysfunction

Vision depends on the 2 classes of photoreceptors the rods and cones. Many different diseases such as Retinitis Pigments (RP), Age-Related Macular Degeneration (AMD), Rod and Cone Dystrophies, and Leber Congenital Amaurosis (LCA) all attack the photoreceptors thru diverse pathways that lead to photoreceptor death and blindness.

Photoreceptors are known to have high energy requirements, but limited reserves so are dependent on constant synthesis of NAD+ to meet energy needs. (Ames et al., 1992, Okawa et al., 2008)

The authors theorized that NAD+ biosynthesis plays a key role in healthy vision. They noted that the leading cause of blindness in children, LCA, is caused by a mutation in the enzyme NMNAT1 which results in impaired synthesis of NAD+.(Falk et al., 2012, Koenekoop et al., 2012, Sasaki et al., 2015)

In mammals, NAM is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT) as the first step in biosynthesis of NAD+.

In this study, researchers created knockout mice that lack NAMPt in rod photoreceptors which disrupts the normal NAD+ biosynthesis, resulting in a 26-43% reduction in retinal NAD+ levels.

Within 6 weeks, these mice exhibited significant photoreceptor death and vision loss. The results very closely matched the degeneration seen in patients with Retinitis Pigments and other degenerative vision disease.

According to the authors:

NAD+ deficiency caused metabolic dysfunction and consequent photoreceptor death…these findings demonstrate that NAD+ biosynthesis is essential for vision


To confirm the cause of vision loss, researchers supplemented knockout mice with daily injections of NMN, bypassing the need for NAMPT in the first step of NAD+ synthesis. Those mice receiving NMN experienced significant recovery of retinal function.
(Figures 3A–3C).

These data clearly demonstrate that NAMPT-mediated NAD+ biosynthesis is necessary for the survival and function of both rod and cone photoreceptors, as promoting NAD+ biosynthesis in the retina with NMN supplementation can compensate for Nampt deletion, thereby reducing photoreceptor death and improving vision.



Researchers were able to determine that NAD+ deficiency is common in many vision problems.

Mice subjected to light exposure retinal damage had significant reduction in NAD+ levels (Figure 3H)

Similar reductions in NAD+ levels were found in mice with streptozotocin (STZ)-induced diabetic retinal dysfunction compared to non-hyperglycemic controls (Figure 3I).

Lastly, they compared 18-month-old vs 6 month old mice. As with the light exposed and diabetic induced mice, the older mice exhibited diminished vision along with decreased retinal NAD+ levels (Figure 3J).

These findings support the idea that NAD+ deficiency may be a shared feature of retinal dysfunction.


screen-shot-2016-10-04-at-10-12-49-amAfter demonstrating that light induced retinal dysfunction was linked to decreased NAD+ levels, researchers were able to show that supplementation to increase NAD+ could protect against retinal damage.

Mice that were given injections of NMN for 6 days prior, and 3 days after light exposure exhibited improved retinal function vs those that did not receive NMN injections (Figures 4A–4C).

[box]Conclusion: Results from this study suggest that supplementation to increase NAD+ deficiencies can help repair macular damage and may be an effective treatment for many common degenerative vision problems.[/box]


This study was very specific for the impact of NAD+ on Retinal disease in Mice, but is also further evidence that the absence of sufficient NAD+ has dire consequences, and that replacement of NAD+ can repair damage.

Researchers are experimenting with various techniques for raising NAD+ levels in mice and humans such as:

The 2013 study by Dr David Sinclair that demonstrated increased levels of NAD+  reverses age related degeneration in mice also used injections of NMN (25).

You can read more about Nicotinamide Mono-Nucleotide (NMN) here.

Other studies with mice and human subjects use supplementation with Nicotinamide Riboside (NR) to raise NAD+ levels.

NR is a precursor the body can use to manufacture NAD+. It has been shown to be safe and effective at raising NAD+ levels in humans in dosages of around 250mg a day.

Another approach to boosting NAD+ levels is preventing the drop in NAD+ levels in the first place.

Recent studies have demonstrated that the enzyme CD38 becomes elevated as we age, possibly in response to increasing inflammation levels, and corresponds with declining NAD+ levels.

Flavonoids such as Quercetin are proving effective at lowering CD38 levels which results in higher circulating levels of NAD+ in the bloodstream.

Dr Sinclair recently published this article on CD38 and concluded that:

    • Combating the rise of CD38 is a promising approach to protect NAD+ levels.
    • The efficacy of NAD+ precursors may be enhanced by co-supplementation with CD38 inhibitors
Conclusion: Inhibiting CD38 to prevent NAD+ destruction AND supplementing with NAD+ precursors so the body can create more NAD+ is a promising new avenue in the anti-aging battle