Different Intermittent Fasting Protocols explained

Yes, you’ve heard of it, but have you experimented with an Intermittent Fasting Protocol to see how many pounds of body fat you can melt away? Here’s several to choose from, guided by doctors who walk their talk. And if none are for you, there’s always soups and smoothies.  Watch the videos!

Choose your Intermittent Fasting protocol and get lean!

[photo credit: PrecisionNutrition.com]


“Hi, My Name is John. And I Haven’t Eaten in 24 Hours.”

Actually, my name is Joe, not John, and I just ate bagels and lox an hour ago, which is now a lump lying in my stomach because I’m not used to eating that much bread stuff. That said, it’s John, not Joe, who’s the subject of this article, the first part anyway.

“Hi, my name is John…” is how Dr. John Berardi of Precision Nutrition fame began his detailed and informative guide, Experiments With Intermittent Fasting, which chronicles his experimentation with an eating regimen that shortens your feeding window, can melt away body fat and help you live healthier, if not longer.

I’m going to summarize John Berardi approach with, and benefits thereby derived by his experiment with Intermittent Fasting (IF). Then I’ll review the various IF methods you can try. Lastly, should no Intermittent Fasting protocol in any form be your thing, there’s always soups and smoothies – I’ll tell you why they can do lots to improve your overall nutrition and get you leaner.

In this article, you’ll discover:

  • How a guy already lean at 10% body fat used IF to drop it to 4%;
  • The three IF protocols most favored by Dr. Berardi;
  • Tips, strategies and outlines of an Intermittent Fasting protocol you can start right away;
  • A list of various intermittent fasts, time-restricted feeding and periodic fasting you can try to get lean and healthy fast;
  • Videos of Dr. Michael Mosley explaining his 5:2 Intermittent Fasting protocol; and
  • A video of Dr. Michael Greger showing the studies that indicate why soups and smoothies might be just good enough if you just don’t want to experience hunger.

Let’s dive in…


Your 10-Minute Intermittent Fasting Primer

Curious but only have 10 minutes to get to the gist of what the heck this Intermittent Fasting thing is all about?

Well, thankfully, Dr. Berardi’s got you covered. What follows in this section is his three favorite IF protocols and his key takeaways.  Perhaps one of this three favorite Intermittent Fasting protocols will become yours.

Dr. Berardi’s Approach

There are many different kinds of IF protocols, so Dr. Berardi tested six different methods over a six month period. He carefully took extensive notes on everything from scale weight, body-fat percentage, and blood/hormonal markers, to lifestyle markers like energy levels and cognitive thoughts.

What Happened To Him?

Before I tell what happened to him, know that Dr. Berardi was anything but fat and out of shape before he began his IF experiment. At the start of it, he was 10% body fat at 190 pounds. And yet, he got leaner — over the course of six months, he dropped twenty pounds of weight, from 190 pounds to 170 pounds, and reduced his body fat from 10% to 4% while maintaining most of his lean muscle mass.

Moreover, he found three IF strategies he could follow indefinitely with little effort; in fact, more easily and time-consuming than the “traditional” dieting about which he is a renowned expert.

Dr. Berardi’s Three Favorite IF Protocols

1. The Trial Fast

This is a fast whereby you simply go 24 hours without food.

The idea is to intentionally experience hunger, to get familiar with it, to learn that hunger won’t kill you.

Do this fast by choosing any 24-hour period during which you don’t eat. Dr. Berardi suggests this approach to make this experiment beneficial to you:

10 PM Saturday:

  • Eat your last meal of the day

    Choose your Intermittent Fasting protocol and get lean like Dr. Berardi

    Dr. John Berardi

  • Drink 500 mL (2 cups) of water

10 AM Sunday:

  • Drink 1 L (4 cups) of water + 1 serving greens powder
  • Drink 250 mL (1 cup) of green tea
  • Take 5 grams BCAA (branched chain amino acids) powder (or take 5 capsules)

3 PM Sunday:

  • Drink 1 L (4 cups) of water + 1 serving greens powder
  • Drink 250 mL (1 cup) green tea
  • Take 5 grams BCAA (branched chain amino acids) powder (or take 5 capsules)

10 PM Sunday:

  • Eat a small snack before bed
  • Drink 500 mL (2 cups) of water


  • Eat normally

Tips and Strategies for the Trial Fast:

  • The tea, greens, and BCAAs aren’t essential to fasting, but they’ll probably make it easier.
  • Drinking water helps to mitigate feelings of hunger. I’ve found that mixing a teaspoon of organic apple cider vinegar with eight ounces of pure water makes drinking lots of water more palatable.
  • Be aware of your body cues. Feeling stressed out or “upset” during your fast? Relax. Take a few deep breaths, and pay close attention — this is what hunger can feel like. You don’t have to react to it by running to the fridge. The more familiar you get with the hunger sensation, the more manageable it will become.
  • Have healthy food (lean meats, veggies, etc.) in the house and ready to go when you “break” the fast on Sunday night with a small meal. Perhaps a tablespoon of almond butter and some celery is a good way to start eating again. Be mindful of eating slowly.

2. The Periodic Fast

As the name suggests, you fast periodically – eat sometimes, fast other times. The reason to do this is to practice hunger management and experience more of the potential health and fat loss benefits of intermittent fasting.

So while you should still eat well (high protein, lots of veggies, a balance of fats, and a moderate intake of minimally processed carbohydrates), you’d periodically take a full day to fast (just like the Trial Fast).

Do this once a week. More than that might be problematic for all but the committed, as Dr. Berardi explains here.

The Periodic Fast is flexible. Choose whichever 24 hours you want:

  • From breakfast to breakfast — Eat breakfast on Monday, and don’t eat again until breakfast on Tuesday.
  • Dinner to dinner — Eat dinner on Wednesday, and don’t eat again until dinner on Thursday.

To do it, follow the rules above from the “Trial Fast”.

Tips and Strategies:

Perhaps a good time to do the Periodic Fast is when traveling, a time when quality food may be scarce. Or if travel stresses you, don’t add to it by doing IF, but instead pick the least stressful day in your week or month.

3. The Daily Fast

The Daily Fast is an 8-hour feeding period followed by a 16-hour fast. It’s very effective at getting lean, lean, lean and is the one I regularly practice.

This Intermittent Fasting protocol is best for people who are already fit, have plenty of experience eating healthily and want to be leaner than the average bear.

Know this:

  • The Daily Fast will typically be harder to adhere to for men over 15% body fat and women over 22% body fat, which frankly is most of us.
  • Men generally respond best to the 16-hour fast, 8-hour eating split .
  • Women seem to need a longer eating window and shorter fast, so might try a 14-hour fast with an 10-hour eating window, or a more relaxed approach.
  • This is not for pregnant women, people who have or have had eating disorders, and people simply looking to be healthy and fit with no particular desire to be unusually lean.

The Daily Fast is outlined in more detail in here, but here’s the essence of this 8-hour feeding/16-hour fasting period:

  • High protein & vegetable intake: During the 8-hour eating window, eat a lot of protein (lean meat, poultry, fish) and vegetables (think green growing things). Err on the side of eating too much of these foods.
  • Fasted training: Do intense resistance training 3 times per week, right before you eat your first meal. In other words, you’ll be training on an empty stomach. (Check out this selection of exercise routines.)
  • Carb cycling: On training days, add carbs (quinoa, rice, whole grain bread, fruit, etc.) to your base diet of protein and veggies.
  • Nutrient timing: On training days, eat as much of your food as soon after training as possible. Your biggest meal should come right after your workout.

Most people who follow this protocol fast from 9 PM until 1PM the next day and exercise around noon while consuming 10 grams of BCAAs (branched chain amino acids) during training, says Dr. Berardi.

(In my case, I fast from 8 PM till 1 PM the next day, usually exercise later in the afternoon and after I’ve eaten my first meal at 1 PM, and I’m light on the BCAAs. Might this be why my six pack remains a steadfast fantasy?)

After exercising, eat two to three large meals before 9 PM, with your biggest meal coming right after exercise, as mentioned.

Of course, many people can’t exit work or other commitments in the middle of the day to work out. As mentioned, this typically includes me. If this is your situation, you won’t have the benefit of exercising in a fasted state, but as long as you follow the rest of the protocol (and in this case exercising later in the day), you’ll get lean.

Sample Single-Day Schedule:

8:00 AM – Wake up, drink 500 mL (2 cups) water

9:00 AM – Drink 1 L (4 cups) water with 1 serving greens+, 250 mL (1 cup) green tea

11:00 AM – 250 mL (1 cup) green tea

12:00 PM – Workout session with 10 g BCAA during session

1:30 PM – Eat first meal, largest of the day

4:30 PM – Eat second meal, moderate sized meal

8:30 PM – Eat third meal, moderate sized meal

Tips and Strategies:

  • Don’t fool yourself into thinking you can just skip breakfast and get shredded; what makes it work is combination of all the principles at play, including the food selection, fasted training and nutrient timing. This is an advanced strategy, not a magic bullet.
  • Even if you think you can do the Daily Fast, consider choosing the Trial or Periodic Fast first.
  • If you find eating this way is too strict, try a) extending the eating window from 8 hours to 9 or even 10 hours, or b) turning your hardest training day into an “eat what you want” day to relax things a little. Or try two “eat what you want” days. These aren’t rules, just guidelines; better to follow a more relaxed plan than abandon a stricter one.
Dr. Berardi’s “Big” Takeaways

He encourages anyone experimenting with IF to remember these four things:

  1. Trial fasting is a great way to practice managing hunger. This is an essential skill for anyone who wants to get in shape and stay healthy and fit.
  2. More regular fasting isn’t objectively better for losing body fat. While Dr. Berardi’s experiments worked quite well, the intermittent fasting approach (bigger meals, less frequently) didn’t produce better fat loss than a more conventional diet approach (smaller meals, more frequently) might have.
  3. More regular fasting did make it easier to maintain a lower body fat percentage. Intermittent fasting isn’t easy, but Dr, Berardi found this approach made it easier to maintain a low body weight and a very low body fat percentage versus. more conventional diets.
  4. Intermittent fasting can work but it’s not for everyone, nor does it need to be. In the end, IF is just one among may ways for improving health, performance, and body composition. To wit, check out my articles on diet and nutrition.

In case you’re wondering if IF really works to improve lean body composition, know that the title picture for this article is the “before and after” pics of Dr. Berardi’s clients, which I’ll copy again so you don’t have to bother scrolling up:

Choose an Intermittent Fasting protocol and get these results for yourself

Impressive, I say, so let’s stay with this IF thing for just a few beats more.


A Quick Review of Intermittent Fasting Protocols

Yes, grasshopper, there are more than the three Intermittent Fasting protocols profiled above that you may choose from.

Ayesha Muttucumaru from Get The Gloss wrote a good review of several fasting plans that reduce your feeding window; meaning, the time period in which you feed yourself.

In her article, From 5:2 to 16:8Which Fasting Plans Do What?, Ms. Muttucumaru segments various fasts into three categories:

  • Intermittent Fasts,
  • Time-restricted Feeding, and
  • Periodic Fasting

She points out something I’ve written quite a bit about in this site regarding the health benefits derived from fasting and other caloric-restriction protocols.

Fasting increases fat loss, specifically of visceral fat – the dangerous type that clings to our middles and that has been linked to increased risk of type 2 diabetes, heart disease, certain cancers and Alzheimer’s. Fasting has also been associated with improved blood pressure and insulin sensitivity, better gut health, lower cholesterol and increased cognitive function as well as supporting an important repair process in the body called autophagy, which only happens when in a fasted state. Think of autophagy as a ‘time-out’ or the ‘repair mode’ that allows the body the chance to rid itself of a buildup of cellular debris, too much of which can increase the risk of age-related diseases such as arthritis and type 2 diabetes.

Another point Ayesha Muttucumaru makes is that the benefits derived from fasting are related to its duration. For example, to benefit by cellular autophagy, you need to fast for a minimum of 12 to 14 hours; whereas it only takes five hours to improve your gut microbes and insulin sensitivity.

Without further adieu, check out these various fasting protocols to see which might resonate with you.

1. Intermittent Fasting

Fasting only on certain days of the week.

The plans:

The 5:2 diet: aka The Fast Diet, the best known of the fasting methods made famous by Dr Michael Mosley. (See his videos below.) It’s five days of regular eating and two ‘fast’ days of 800 calories,  and has been associated with improved DNA repair and brain function, plus an increase in fat loss as demonstrated in a 2011 Manchester University study. Dr Mosley lost 20lb in 12 weeks and saw his blood sugar and cholesterol levels returned to normal after being pre-diabetic and suffering from high cholesterol.

The 1:1 diet: aka Alternate Day Fasting. Published as The Every Other Day Diet by Dr Krista Varady and Bill Gottlieb, it involves eating 500 calories every other day. You’re able to eat what you like during the fast, provided calorie intake is limited on the fast days.

The 6:1 diet: Made famous by Coldplay’s Chris Martin, this diet involves completely fasting for one day and eating normally for the rest of the week. Sounds simple enough.  Drink lots of water on the fast day.

2. Time-restricted feeding

This is about reducing your “feeding widow” per 24 hour period. You can reduce your meal frequency from three per day to two, or just one — or eat within a “window” of time, often eight hours.  As mentioned above, my feeding widow is from 1 PM to 8 PM.

The plans:

The 2 Meal Day: Created by personal trainer and online health coach Max Lowry, this plan involves skipping one meal (either breakfast or dinner) and extending your night fast (that is, while you’re sleeping) to around 16 hours. Read more about the 2 Meal Day here.

The Warrior Diet: Published by Ori Hofmekler in 2001, this approach involves one meal per day in the evening, supposedly emulating warriors of olden times who only ate what they killed and did so at night (I guess). Its emphasis is placed on whole foods and whole grains.

How to Lose Weight Well: Published by Dr. Xand van Tulleken and Georgina Davies, this evening meal focused plan can be adapted to best suit your lifestyle and objectives. To help achieve faster weight loss and the benefits of intermittent fasting, it recommends one 800-calorie meal a day or if that’s too infrequent, two healthy meals a day of 1,200 calories or three meals of 1,500 calories.

Metabolic Balance: Founded by Dr. Wolf Funfack, this diet plan claims to aid weight loss and improve sleep, digestion and energy levels by advocating a five-hour fast in between meals over a three-month time period.

16:8: aka The 8-Hour Diet. Published by Men’s Health Editor David Zinczenko and Peter Moore, this plan comprises of 16 hours fasting and an eight-hour “feeding window”.

3. Periodic fasting

Fasting for a few days or weeks at a time.

The plans:

Fast Mimicking diet: Created by Professor of gerontology and biological sciences at USC, Valter Longo, it comprises of meal boxes designed to be used for 5 days every month. Using natural, gluten-free and plant-based ingredients, meals are low-protein, low-carb and high in good fat, with 770 to 1,100 calories aimed for per day.

The Bodhimaya Method: Founded by brothers Daniel and Cornelius O’Shaugnessy, the method combines the format of the 16:8 plan (outlined above) with a fast day food plan portion ratio of 1:7:2 (carbs to veg to protein). Find out more about The Bodhimaya Method here.

Buchinger Wilhelmi: A selection of medically supervised fasts starting from a minimum of four days (10, 14 and 21 day programs are also available). Fast days feature small amounts of food (of around 250 calories) and a carefully regulated exercise plan to prevent muscle loss.

8-weeks: aka The Blood Sugar Diet. Created by Dr Michael Mosley to prevent and reverse type 2 diabetes, this plan involves eating three small meals totaling 800 calories per day for eight weeks.

As cited above, Dr. Mosely is also the creator of the the 5:2 Diet described by his book, The Fast Diet. Watch this self-described former “skinnyfat” BBC science journalist, executive producer and medical doctor describe his motivation behind his work and how it transformed his life:


(Go here to learn about High Intensity Interval Training, which Dr. Mosely suggests be incorporated with his 5:2 diet.)

If intrigued by the video above, watch this to-the-point video by Kevin Partner, which he calls The Fast Guide to the Fast Diet – for people too lazy to read the book:



Soups and Smoothies

Perhaps you’ve just scrolled past every Intermittent Fasting protocol to get here, because there’s just no way in hell (cause it would be hell) to fast for longer than, say, from breakfast to lunch.

Certainly, you don’t have to fast to whittle away some body fat. What you do have to do is get into a caloric deficit, which basically means to consume fewer calories than you do now.

One way to cope with fewer calories than you’re accustomed to is to make them satiate you more than they otherwise would by employing some tricks. That’s where soups and smoothies enter the picture.

I’ll get right to the bottom line here:

  • Smoothies offer an opportunity to quickly create a very nutritious meal from ingredients you might not ordinarily eat (like kale and spinach), and be as satiating as the meal it replaces — if they’re drunk slowly.
  • Soup will satiate you more that the very same ingredients eaten whole on a plate, as opposed as blended and heated as soup.

Want to know why?

Watch nutrition expert Dr. Michael Greger take you through the experiments on this:


I wholeheartedly recommend that you consume more smoothies and soups.

Just make sure that your smoothies are not dominated by sweet stuff like sugary fruit — dates, bananas, papaya.  Rather, for the fruit ingredients use mainly berries with just a bit of the sugary fruits.  Then add green veggies like kale, watercress, broccoli, beets and spinach.  For protein use whey if the smoothies is to be consumed after a workout, given that whey is quickly absorbed into the bloodstream, or pea, hemp or sprout-derived protein powders.  Finally, use water, almond milk or keifer, or combinations of all three.

For soups, search the Interwebs for good soup blender recipes; meaning, get guidance for what combination of veggies ingredients, stock and herbs you can throw in a blender, heat up and slowly eat.  In my case, I often mix whatever vegetables are handy, along with some vegetable stock I get at the grocer and season to taste.  Fast, nutritious and satiating.


Your Takeaway

Yes, lots to digest here.

Suggest you do this:

  • Grab a friend and together look for an Intermittent Fasting protocol that does not seem overwhelming, one you feel you could do without feeling that you’ve sentenced yourself to torture, and then give it a whirl. You have a better chance getting through it with a friend, because misery loves company.
  • Start small and step up big if IF seems right for you.
  • If you’d rather poke yourself in the eye than feel hunger pangs, try adding soups and smoothies to your diet. Consume them mindfully, slowly.  And let yourself eat less for the next meal, rather than eating the usual amounts of food.

OK, then… get to it!

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.

Alternate-Day Fasting Increases Fullness After Meals

Alternate-day fasting is a version of intermittent fasting, which is currently one of the world’s most popular weight loss trends.

Recently, a group of scientists examined the effects of 8-week alternate-day fasting on appetite ratings, appetite hormones and body weight.

Here is a detailed summary of their results.

Empty Orange Plate


Alternate-day fasting (ADF) is a weight loss technique that has recently become popular (1).

One popular version of ADF restricts calories by 75% every second day, while allowing unrestricted eating in between.

Human trials have shown that this method may lead to 4–8% weight loss in just 2–3 months. Here is a summary of study results from over the years:

  • 2005: 16 non-obese men and women fasted every other day for 22 days and lost 2.5% of their initial body weight (2).
  • 2007: 9 overweight asthma patients consumed 20% of their calorie needs every second day for 8 weeks and lost 8% of their initial body weight (3).
  • 2009: 16 obese people consumed 25% of their calorie needs every second day for 8 weeks and lost 12.4 lbs (5.6 kg) (4).
  • 2013: 32 obese people fasted every other day for 8 weeks and lost 4.8% of their body weight on a high-fat diet (45% fat), but 4.2% on a low-fat diet (25% fat) (5).
  • 2013: 32 obese people fasted every other day for 12 weeks and lost 6.6 lbs (3 kg). When fasting and doing endurance exercise, they lost 13.2 lbs (6 kg) (6).
  • 2013: 15 overweight or obese women fasted every other day for 6 weeks and lost 7% of their initial body weight (7).

Additionally, giving in to hunger and cravings is the main reason why people fail to adhere to a diet (8).

This seems to be less common during ADF, though the reason is not clear (9).

Article Reviewed

This trial examined how ADF affects appetite ratings, appetite hormones and weight.

Changes in hunger and fullness in relation to gut peptides before and after 8 weeks of alternate day fasting.

Study Design

This trial examined the effects of 8-week ADF on subjective ratings of appetite, appetite hormones and body weight.

It should be noted that results from this study have been published before (10).

A total of 59 middle-aged, obese adults completed the trial, or 80% of those who initially started. 84% of the participants were women.

The study was divided into 3 periods:

  • Control period 1: For 2 weeks, the participants followed their habitual diets. Initial measurements took place during this period.
  • Alternate-day fasting: For 8 weeks, the participants fasted every second day.
  • Control period 2: Immediately after the ADF, participants followed their habitual diet for 2–3 days before final appetite measurements were taken.

Bottom Line: This trial examined how ADF affects appetite and body weight. The study included 59 people, and was divided into three periods.

Alternate-Day Fasting

For 8 weeks, the participants fasted every second day, with 75% calorie restriction. Conversely, on “feed days” they were allowed to eat whatever they wanted.

To improve compliance, all participants were provided with the food they were allowed to eat on fast days.

This food contained 60% of calories from carbs, 24% from fat and 16% from protein. Participants were allowed to consume unlimited amounts of calorie-free drinks.

Every week, the researchers measured body weight. Fat mass, lean mass and intra-abdominal fat were also measured using DXA.

Bottom Line: The participants consumed 25% of their calorie needs every other day for 8 weeks.

Appetite Assessment

Subjective ratings of appetite were assessed after a 12-hour fast, before and after a standardized meal.

The standardized meal consisted of a liquid meal, providing 440 calories. It was designed to have a similar macronutrient profile as a typical breakfast, or 60% of calories from carbs, 24% from fat and 16% from protein.

Fasting blood samples were taken before the meal. Blood was also drawn immediately after the meal, and at 30, 90 and 120 minutes afterwards.

The blood was analyzed for total ghrelin, peptide YY (PYY) and several other hormones. Immediately before each of the blood draws, the participants rated their appetite using a visual analog scale (VAS).

Bottom Line: Appetite hormones and subjective ratings of appetite were assessed before and after the 8 weeks of ADF.

Finding 1: Body Weight Decreased

During eight weeks of ADF, the participants lost 8.6 lbs (3.9 kg).

This weight loss was largely due to loss of fat — 4.9 lbs (2.2 kg) — but also lean mass (water and muscle) — 3.1 lbs (1.4 kg).

The chart below shows the changes in fat mass and lean mass.

Fat and Lean Mass Change

Bottom Line: Fasting every other day for 8 weeks led to 8.6 lbs (3.9 kg) weight loss.

Finding 2: Fullness After Meals Increased

Subjective ratings of fullness after the test meal were significantly higher after 8 weeks of ADF, as shown below.

Start and End Fullness

This increase in fullness was associated with higher levels of peptide YY, which is a hormone that reduces appetite after meals (1112).

Similar to fullness, PYY levels after meals increased after 8 weeks of ADF. The reason why ADF increased PYY levels and fullness is unclear.

Bottom Line: Post-meal fullness was greater after 8 weeks of ADF, compared to before.

Finding 3: Ratings of Hunger Remained Unchanged

Eight weeks of ADF did not change hunger ratings.

This is consistent with previous studies suggesting that hunger remains unchanged after 3–12 weeks of ADF (23913).

Conversely, studies on hunger after continuous calorie restriction indicate that compensatory increases in hunger are common (141516).

Taken together, these findings indicate that intermittent calorie restriction is easier to stick to than continuous dieting or fasting.

However, levels of the hunger hormone ghrelin were significantly higher after the test meal at the end of the trial, compared to its levels before the trial.

The reason for these findings is unknown.

Bottom Line: Despite the loss of weight, subjective ratings of hunger didn’t change. This indicates that ADF is easier to adhere to than dieting.


This study had a few limitations.

First, meals were not standardized the day before the appetite measurements took place. Even though the tests were preceded by a 12-hour fast, the previous evening’s meal might have affected the findings.

Second, one of the authors, Krista Varady, wrote the book The Every Other Day Diet, creating a conflict of interest. None of the other authors reported competing interests.

Summary and Real-Life Application

ADF is an effective weight loss strategy, causing significant weight loss after 8 weeks. Yet despite the weight loss, hunger ratings after a standardized meal remained unchanged.

Additionally, when the ADF was over and the participants went back on their regular diets, subjective ratings of fullness were significantly higher after the standardized meal, compared to a similar meal before the fast.

These findings suggest that ADF is relatively easy to stick to. However, further studies need to examine its long-term effects.

Everything in Moderation: Helpful or Harmful Advice?

Most dietary guidelines recommend dietary diversity.

In other words, they say that people should eat lots of different kinds of food in moderation. “Everything in moderation” is a popular phrase.

However, it is unclear whether following this recommendation has any effects on people’s health.

A group of scientists tried to answer this question by examining the association of dietary diversity and abdominal obesity and type 2 diabetes.


Dietary diversity has never been clearly defined.

Most previous studies simply define it as the number of different foods you eat in a certain time frame.

However, this leaves out some important factors, such as how the foods are spaced out or how different the foods actually are.

A few previous studies have investigated the effects of a diverse diet, but most focused only on diversity within selected food groups.

For example, one observational study links eating many different types of fruits and vegetables to a reduced risk of type 2 diabetes (1).

Another observational study showed that eating a variety of vegetables may help prevent weight gain. However, eating many varieties of unhealthy foods may increase the risk of weight gain (2).

These studies suggest that dietary diversity can affect health in different ways, depending on what foods are eaten.

Article Reviewed

A team of researchers from the US examined how a varied diet affects the risk of obesity and type 2 diabetes.

Everything in Moderation — Dietary Diversity and Quality, Central Obesity and Risk of Diabetes.

Basic Study Design

This study was a secondary analysis of the Multi-Ethnic Study of Atherosclerosis, a prospective observational study in people of Caucasian, Hispanic, African and Chinese descent.

The purpose of this secondary analysis was to examine the association of dietary quality with abdominal obesity and type 2 diabetes.

A total of 2505 men and women, 45 to 84 years old, participated. None of them had type 2 diabetes. At the beginning of the study, their diet was estimated using a food frequency questionnaire.

To estimate dietary diversity, the researchers assessed:

  1. Count: How many different foods were eaten more than once per week.
  2. Evenness: How the diversity was spread out over the week.
  3. Dissimilarity: How different the foods were.

After 5–7 years, the study staff measured body weight and waist circumference. Fasting blood sugar was measured 10–11 years later.

Bottom Line: This was a prospective observational study examining how the risk of weight gain and diabetes is linked with dietary diversity or quality.

Finding 1: Dietary Diversity Is Not Associated With Dietary Quality

The major finding of the study is that a diverse diet is not necessarily a healthy diet.

The count and evenness measures of dietary diversity were only weakly linked with dietary quality.

Conversely, greater food dissimilarity was linked with lower dietary quality — a lower intake of healthy foods and a higher intake of unhealthy foods.

Bottom Line: A diverse diet is not necessarily a healthy diet. Eating many different types of food doesn’t guarantee that those foods are high in quality.

Finding 2: Dietary Diversity May Promote Weight Gain

Greater food dissimilarity was linked with higher gain in waist circumference, which is a measure of belly fat.

This is probably because a higher food dissimilarity was associated with a higher intake of unhealthy food.

Supporting this, one observational study showed that greater dietary variation was associated with a higher intake of unhealthy foods (3).

Additionally, observational studies suggest that high dietary diversity may lead to increased calorie intake and fat gain (234).

Bottom Line: Eating many different kinds of food does not necessarily prevent belly fat. In fact, increasing diversity may even promote higher calorie intake and weight gain.

Finding 3: A Varied Diet Was Not Linked With Type 2 Diabetes

During the 10-year follow-up period, 24% of the participants were diagnosed with type 2 diabetes.

However, this was not linked to dietary diversity. Likewise, no significant associations were found when foods were categorized as either healthy or unhealthy.

In fact, a greater variety in fruit and vegetable intake did not seem to be associated with a reduced risk of type 2 diabetes.

This is inconsistent with one observational study suggesting that eating a greater variety of fruits and vegetables may help prevent type 2 diabetes (1).

On the other hand, the present study found high dietary quality to be significantly associated with less risk of type 2 diabetes.

This means that diets should focus on high-quality food, rather than on diversity.

Bottom Line: A diverse diet did not protect against type 2 diabetes. However, a high-quality diet was linked with less risk of type 2 diabetes.


The use of food frequency questionnaires to assess dietary variation is one of the main limitations of the study.

Food frequency questionnaires can only include a few types of food. For example, the fruit and vegetable category included only 23 different foods.

For this reason, dietary diversity may have been biased or underestimated.

Additionally, this was an observational study, not a controlled trial. Although the study was designed well, it can not prove causation.

For example, it can show that people who eat a diverse diet have a higher risk of weight gain, but it can not prove that the diverse diet itself caused the weight gain.

Bottom Line: The study was designed well. However, the use of food frequency questionnaires to assess dietary intake limited the researchers’ ability to fully evaluate dietary diversity.

Summary and Real-Life Application

In short, this study does not support the idea that eating whatever you want, even in moderation, promotes a healthier diet.

However, this doesn’t mean dietary diversity is a bad thing.

For example, in developing countries with limited food supplies, greater dietary diversity would increase nutrient intake (5).

It just means that, for many people, diet should focus on eating quality, whole foods — but not trying to eat “everything in moderation.”

7 Days of Overeating and Inactivity Causes Insulin Resistance

Obesity is one of the most common consequences of the Western lifestyle.

Unsurprisingly, obesity is not only a matter of weight. It is also associated with a variety of health problems.

The most common health problems associated with obesity are collectively known as metabolic syndrome.

Insulin resistance and type 2 diabetes are among the main components of this syndrome, but how they are linked with obesity is not clearly understood.

Article Reviewed

A team of scientists at Temple University, Philadelphia, set out to investigate how overeating and obesity may be linked with insulin resistance.

Boden et al. Excessive Caloric Intake Acutely Causes Oxidative Stress, GLUT4 carbonylation, and insulin resistance in healthy men. Science Translational Medicine 2015.

Materials and Methods

Six healthy, middle-aged men volunteered to participate in the study.

Half of them were normal weight, while the rest were slightly overweight.

None of them had a family history of diabetes or other endocrine disorders, and they were not taking any drugs.

They spent one week at a hospital, where they were overfed and confined to their rooms to minimize physical activity.

Their diet consisted of approximately 6000 kcal per day, a whopping 200–250% increase in their normal calorie intake.

It was a typical US diet, standardized to 50% carbohydrate, 35% fat and 15% protein.

Various blood analyses were done every day, while other measurements, such as fat biopsies, were only performed at the beginning and end of the study. Body weight and composition were also measured.


By the end of the study, the participants had gained an average 3.5 kg, all in the form of fat mass.

On day two, insulin levels rose quickly with signs of increased insulin resistance, gradually becoming worse as the study progressed.

This chart shows how fasting insulin changed during the study (increased by 150%):

In addition, oxidative stress increased significantly during the study.

The source of this oxidative stress was found in fat tissue, as evident from fat biopsies. Apparently, fat tissue is particularly stressed during periods of overeating.

Most importantly, the study strongly suggests that oxidative stress was the cause of insulin resistance in the study participants.

In fact, oxidative stress appears to inactivate GLUT-4, an insulin-regulated glucose transporter mainly found in fat tissue and muscles.

Insulin resistance did not seem to be triggered by elevated levels of free fatty acids (FAA) or increased inflammation. Both remained constant during the study period.

Main Conclusions

This study showed that overfeeding causes insulin resistance.

It indicates that insulin resistance is caused by oxidative stress associated with increased storage of fat in fat tissue.

The oxidative stress seems to disrupt blood sugar regulation by damaging GLUT4, a protein responsible for transporting glucose from the bloodstream into cells.


Apart from the small number of participants, the study doesn‘t have any obvious limitations.

However, for comparison, it would have been useful to have a control group receiving a weight-maintenance diet.

As the researchers point out, inactivity is likely responsible for a part of the adverse changes seen in the study.

What Do Other Studies Say?

A slower and less pronounced increase in insulin resistance has been found in other studies feeding participants less amounts of excess calories.

One 8-week study in 29 healthy men investigated the effect of increasing calorie intake by 40%. It found an 18% increase in insulin resistance (1).

Another study in 40 healthy men and women showed that eating an excess of 1040 kcal/day for a month decreased insulin sensitivity by 8% (2).

Previous research also indicates that insulin resistance is linked with oxidative stress and that adipose tissue is heavily stressed during overeating (34567).

For this reason, several human trials have tried to decrease insulin resistance by using antioxidants (compounds that act against oxidative stress). However, their results have been inconsistent (89101112).

The results of the current study are supported by studies of cells in test tubes, showing that oxidative stress causes insulin resistance on the cellular level (131415).

Studies in humans and animals also indicate that a high-fat diet increases oxidative stress and insulin resistance (7).


In short, this study clearly shows that drastic overeating and inactivity are seriously unhealthy. They increase oxidative stress and cause insulin resistance in as little as two days.

Oxidative stress impairs blood sugar regulation by inactivating a protein called GLUT4, responsible for transporting glucose from the bloodstream into cells.

Simply put, overeating and lack of exercise may explain what causes insulin resistance – a leading driver of metabolic syndrome and type 2 diabetes, which are currently massive health problems worldwide.

The Paleo Diet May Help Fight Metabolic Syndrome

In the past decade, the Paleolithic diet (or paleo diet) has gained considerable popularity due to its claimed health benefits.

However, it differs from official dietary recommendations in many ways. For example, it excludes all grains, dairy products and industrially processed food.

Several studies have investigated the health effects of the paleo diet. This meta-analysis examined its effects on metabolic syndrome.


Metabolic syndrome is a group of adverse conditions associated with abdominal obesity.

These include the following risk factors for type 2 diabetes and heart disease:

  • Abdominal obesity.
  • Elevated blood sugar.
  • Elevated blood pressure.
  • High blood triglycerides.
  • Low HDL-cholesterol.

An underlying cause of metabolic syndrome is thought to be the modern processed diet, causing low-grade inflammation and insulin resistance (12).

For this reason, it is very likely that diets based on unprocessed, whole foods, like the paleo diet, may improve health among people with metabolic syndrome.

Study Reviewed

Scientists from Bahrain and the Netherlands did a meta-analysis of randomized controlled trials that compared the paleo diet with other dietary patterns.

Paleolithic nutrition for metabolic syndrome: systematic review and meta-analysis.

Basic Study Design

The researchers conducted a meta-analysis of randomized controlled trials.

They combined results from many conceptually similar studies and performed new statistical analyses.

This meta-analysis included four randomized controlled trials (IIIIIIIV) with a total of 159 participants.

The researchers then compared these findings to control diets based on nutrition guidelines from around the world.

The main research question of this review was: Does adhering to a Paleolithic diet improve metabolic risk factors?

Finding 1: The Paleo Diet Improved Body Composition

The paleo diet resulted in greater improvements in body composition than the diets based on official health recommendations.

Below is an overview of how the paleo diet differed from the control diets in the short-term. The numbers represent the mean difference.

  • Body weight: -2.69 kg (-5.9 lbs).
  • Waist circumference: -2.38 cm (-0.94 in).

Supporting this, a few other trials have shown that going on the paleo diet may reduce body weight and improve body composition (45).

Bottom Line: The Paleolithic diet resulted in greater reductions in body weight and waist circumference than diets based on official health recommendations.

Finding 2: The Paleolithic Diet Reduced Heart Disease Risk

Compared to the control diet, the Paleolithic diet also improved several other risk factors for heart disease.

  • Triglycerides: -0.40 mmol/L.
  • Systolic blood pressure: -3.64 mm Hg.
  • Diastolic blood pressure: -2.48 mm Hg.

HDL-cholesterol and C-reactive protein levels were also reduced, but the differences between the diets were not significant.

A few other human trials on the paleo diet have provided similar results (345).

Bottom Line: The Paleolithic diet resulted in greater improvements in heart disease risk factors than diets based on official nutritional recommendations.

Finding 3: Effects on Blood Sugar Control

The paleo diet resulted in significant reductions in both fasting blood sugar and insulin.

However, changes in blood sugar and insulin were not significantly different between diets.

One uncontrolled trial also showed significant improvements to blood sugar control on a paleo diet (3).

Bottom Line: The Paleolithic diet resulted in improvements in blood sugar control. However, the difference between diets was not significant.

How Does the Paleolithic Diet Improve Metabolic Syndrome?

There are several possible reasons why the paleo diet may have an advantage over conventional diets.

  • Paleolithic nutrition contains almost no carbs that are high on the glycemic index. Eating lots of high-glycemic foods may increase low-grade inflammation and the risk of insulin resistance (6).
  • Paleolithic diets contain no processed foods. They are exclusively based on whole foods, including fruits and vegetables.
  • Paleolithic diets contain no refined vegetable oils. Many vegetable oils are high in omega-6 fats, and may cause unfavorable ratios between omega-3 and omega-6. This may cause chronic, low-grade inflammation (78).

Bottom Line: Paleolithic diets do not contain any processed foods or foods that are high on the glycemic index. This may help prevent low-grade inflammation and reduce the risk of insulin resistance.


This meta-analysis appears to be well designed and not have any serious limitations. The control diets were also relatively similar.

However, the included studies had a few potential limitations:

  • Most of the studies had few participants and were short in duration.
  • The paleo diet prescribed in the studies may not accurately represent what “paleo” eaters consume in the real world.
  • In three of the studies, the dietary intervention was in the form of advice and recommendations. Compliance may have been an issue in some of these studies (91011).
  • None of the included trials were fully blinded. This might have affected their results.
  • Although the present meta-analysis tried to standardize the time-point data, the duration still ranged from 2 weeks to 6 months (1011).
  • Although the test and control diets in all 4 studies were reasonably similar, there were some differences. Even slight variations in macronutrient composition may have strong effects on health outcomes.
  • One of the included trials had a baseline imbalance. Participants in the Paleolithic group had significantly worse outcome values at the start of the study compared to the control group (10).
  • Only one study reported adverse effects, and none of them assessed quality of life.

Summary and Real-Life Application

In short, the study shows that a paleo diet may have moderate benefits for metabolic syndrome.

However, the authors of the article believe that more evidence is needed before Paleolithic nutrition can be recommended in official guidelines.

Additionally, it is debatable whether total avoidance of dairy and grains is necessary, or even advantageous, for optimal health.

That being said, it is clear that avoiding processed foods and adhering to a diet based on whole foods is sound nutritional advice.

Inflammation, NAD+, and the mitochondria theory of Anti-Aging

Are We Coming Closer to Eating the Holy Grail of Anti-Aging?

In the late 1940s, Denham Harman PhD, an accomplished chemist, became so fascinated with the idea of finding a cure to ageing that he decided to go back to school and study medicine.

In 1953, while still an intern at Berkeley in California, he proposed a radical new theory called the ‘the free radical theory of ageing’. [i] In this theory, he declared that ageing is caused by reactive oxygen species accumulating within cells.

Denham then noticed that it wasn’t simply the accumulation of reactive oxygen species that affected lifespan, but the damage these reactive oxygen species were inflicting on mitochondria.

So, he modified his theory and gave it a new name: ‘the mitochondrial theory of ageing’. Harman changed the course of anti-ageing research forever.

What are reactive oxygen species?

Mitochondria are energy ‘factories’ within cells. They produce ‘packages’ of energy in the form of the molecule ‘ATP’.

In the process of energy production, single electrons escape from the ‘factory line’ and react with oxygen molecules to form free radicals such as peroxide and superoxide.

These free radicals react with everything and can wreak havoc, this is why they are called ‘reactive oxygen species’. Excessive levels of these reactive oxygen species cause damage to the mitochondria themselves.

They can be ‘mopped up’ by anti-oxidants and mitochondria usually contain anti-oxidants that do this but these may decline in number, over time and with age.

As a result, high levels of reactive oxygen species start to accumulate and cause mitochondrial damage.

Dysfunctional mitochondria can no longer produce enough energy and ‘starving’ cells may degenerate and die.

Brain cells are extremely dependent on their mitochondria for energy and most diseases of old age seem to affect the nervous system, giving credibility to Denham’s theory.

In 1999, another anti-ageing expert, Professor Vladimir Skulachev of Moscow State University, proposed a similar theory on ageing which he calls ‘Phenoptosis’.[ii]

In this theory, mitochondrial death from reactive oxygen species leads first to cell death, then organ death and that then kills the whole organism.

The bottom line of these theories is if we want to live longer, we need to look after our mitochondria.

Anti-oxidant advice

The advice universally given out over the past few decades where we have been told to eat plenty of fruit, chocolate, tea and various other things because they all contain anti-oxidants, is based on these theories.

If we eat anti-oxidants, we can prevent our mitochondria from being damaged by excessive reactive oxygen species. The problem is that eating more anti-oxidants doesn’t seem to have much of an effect on longevity, as some studies have shown.[iii]

One reason may be that the anti-oxidants we eat may not actually reach their target and enter into the mitochondria that need them.

Inflammation turns mitochondria into toxic factories that wreak havoc.

Macrophages are a type of white blood cell  whose normal role is to digest cellular debris and foreign substances. These biological dustbins maraud within and between cells throughout the body, destroying pathogens as they roam.  Normally, this is a good thing.  But they are able to reek havoc  with mitochondria, the cells power plants, if they get out of control.

Mitochondria stop producing energy


This articles shows how macrophages signal mitochondria to switch from producing ATP (the cells energy source)  by normal oxidative phosphorylation  to glycolysis.  The end product of the glycolysis is ROS that are pro-inflammatory and can be toxic to surrounding cells.

“Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production.”

(Research article that also describes this process – http://www.medicalnewstoday.com/articles/313090.php)



to produce  Reactive Oxygen Species (ROS).  ROS such as Nitric oxide, superoxide and peroxynitrite induce inflammation and at higher levels damage cells, and are implicated in chronic disease (r) .  Anti-Oxidants are often taken in hopes of combatting these ROS and lowering inflammation, but are often like putting a finger in a leaking dike.


This article about newly elevated role of inflammation in neurological disease like MS.

“Increasing clinical, imaging and biopsy evidence show that inflammation also plays a major role3,4, Indeed, a biopsy study in patients with MS concluded that: “Inflammation alone may be sufficient to cause significant clinical deficits without demyelination5. However, the mechanism by which inflammation causes loss of function remains unresolved, although roles for cytokines6, reactive oxygen species and nitric oxide7,8,9 have been proposed. Some of these factors can impair mitochondrial function10, and increasing evidence points to an energy deficit as a major feature of the brain and spinal cord in multiple sclerosis (MS) e.g. refs 1112131415 implicating an energy deficit in the pathophysiology of the functional deficit.

Our findings reveal profound changes in mitochondrial function that parallel the equally profound changes in neurological function. Loss of mitochondrial function at the start of disease expression was accompanied by the increased expression of a key astrocytic glycolytic enzyme.

These data place mitochondrial dysfunction at the centre of the pathophysiology of demyelinating disease of the CNS.”


ROS impair mitochondrial function

“The mitochondrial deficits described above were accompanied by an increase in astrocytic PFK2 expression, consistent with a shift from oxidative to glycolytic ATP production in these cells…threshold was breached where ATP supply was no longer sufficient to match metabolic demand…”

“The cause of the mitochondrial dysfunction remains unclear, although roles for hypoxia, superoxide and nitric oxide are implicated by the current and other studies…….Nitric oxide, superoxide and peroxynitrite have also been implicated as responsible for the mitochondrial dysfunction apparent as early as three days after immunisation in excised tissue23, and we have confirmed the mitochondrial dysfunction at early days in vivo (data not shown), a time when inflammatory cells are very few in number, if present.”

“It is difficult from our findings to determine the sequence of events between the onset of mitochondrial depolarisation and the arrival of any inflammatory cells within the spinal cord. However, it is noticeable that the mitochondrial dysfunction in EAE started before the arrival of many inflammatory cells”

In summary, the current findings focus attention on mitochondrial dysfunction and energy insufficiency as a major cause of neurological deficits in neuroinflammatory disease



Excess Blood Glucose Fuels Inflammatory Fires

(This section from Lifeextension.com)

When glucose is properly utilized, our cells produce energy efficiently. As cellular sensitivity to insulindiminishes, excess glucose accumulates in our bloodstream. Like spilled gasoline, excess blood glucose creates a highly combustible environment from which oxidative and inflammatory fires chronically erupt.

Excess glucose not used for energy production converts to triglycerides that are either stored as unwanted body fat or accumulate in the blood where they contribute to the formation of atherosclerotic plaque.

As an aging human, you face a daily onslaught of excess glucose that poses a grave risk to your health and longevity. Surplus glucose relentlessly reacts with your body’s proteins, causing damaging glycation reactions while fueling the fires of chronic inflammation and inciting the production of destructive free radicals (Basta 2004; Uribarri 2005; Toma 2009).

Avert Glycation and Inflammation by Controlling Glucose Levels with Green Coffee Extract 

Unroasted coffee beans, once purified and standardized, produce high levels of chlorogenic acid and other beneficial polyphenols that can suppress excess blood glucose levels. Human clinical trials support the role of chlorogenic acid-rich green coffee bean extract in promoting healthy blood sugar control and reducing disease risk.

Scientists have discovered that chlorogenic acid found abundantly in green coffee bean extractinhibits the enzyme glucose-6-phosphatase that triggers new glucose formation and glucose release by the liver (Henry-Vitrac 2010; Andrade-Cetto 2010). Glucose-6-phosphatase is involved in dangerous postprandial (after-meal) spikes in blood sugar.

In another significant mechanism, chlorogenic acid increases the signal protein for insulin receptors in liver cells (Rodriguez de Sotillo 2006). That has the effect of increasing insulin sensitivity, which in turn drives down blood sugar levels.

In a clinical trial, 56 healthy volunteers were challenged with an oral glucose tolerance test before and after a supplemental dose of green coffee extract. The oral glucose tolerance test is a standardized way of measuring a person’s after-meal blood sugar response. In subjects not taking green coffee bean extract, the oral glucose tolerance test showed the expected rise of blood sugar to an average of 144 mg/dL after a 30 minute period. But in subjects who had taken 200 mg of the green coffee bean extract,that sugar spike was significantly reduced, to just 124 mg/dL—a 14% decrease (Nagendran 2011). When a higher dose (400 mg) of green coffee bean extract was supplemented, there was an even greater average reduction in blood sugar—up to nearly 28% at one hour.

Ensuring that fasting glucose levels stay between 70 and 85 mg/dL, and that two hour post-meal glucose levels remain under 125 mg/dL can help combat chronic inflammation.

Conventional Medicine Typically Overlooks Chronic Inflammation

Chronic inflammation or para-inflammation is generally not treated on its own by mainstream physicians. Interventions in conventional medicine are usually only undertaken when the inflammation occurs in association with another medical condition (such as arthritis).

Currently, conventional preventive medical approaches to inflammation are limited to the use of CRP to predict cardiovascular disease in high-risk subjects, and the prophylactic use of drugs like aspirin to inhibit the inflammatory cascade linked to thrombosis (uncontrolled blood clotting).

Indeed, the potentially asymptomatic nature of low grade inflammation is such that elevations of pro-inflammatory cytokines may progress undetected for some time, only being discovered after they have had time to cause enough cellular damage to produce disease symptoms.

As future studies solidify the association between inflammatory mediators and different diseases, early detection of cytokine aberrations and anti-inflammatory therapy to reduce disease risk may gain more mainstream acceptance


[i] Harman D. “Aging: a theory based on free radical and radiation chemistry”. Journal of Gerontology1956, 11 (3): 298–300.

[ii] Skulachev VP, Anisimov VN, Antonenko YN, Bakeeva LE, Chernyak BV, Erichev VP, Filenko OF, Kalinina NI, Kapelko VI, Kolosova NG, Kopnin BP, Korshunova GA, Lichinitser MR, Obukhova LA, Pasyukova EG, Pisarenko OI, Roginsky VA, Ruuge EK, Senin II, Severina II, Skulachev MV, Spivak IM, Tashlitsky VN, Tkachuk VA, Vyssokikh MY, Yaguzhinsky LS, Zorov DB. An attempt to prevent senescence: a mitochondrial approach. Biochim Biophys Acta. 2009 May;1787(5):437-61.

[iii] Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. JAMA 2007, 297:842–857.

[iv] Bough KJ, Wetherington J, Hassel B, Pare JF, Gawryluk JW, Greene JG, et al. Mitochondrial biogenesis in the anticonvulsant mechanism of the ketogenic diet. Ann Neurol 2006;60:223–35.

[v] Jarrett SG, Milder JB, Liang LP, Patel M. The ketogenic diet increases mito- chondrial glutathione levels. J Neurochem 2008;106:1044–51.

[vi] Sullivan PG, Rippy NA, Dorenbos K, Concepcion RC, Agarwal AK, Rho JM. The ketogenic diet increases mitochondrial uncoupling protein levels and activi- ty. Ann Neurol 2004;55:576–80.

[vii] Kashiwaya Y, Takeshima T, Mori N, Nakashima K, Clarke K, Veech RL: D-beta-hydroxybutyrate protects neurons in models of Alzheimer’s and Parkinson’s disease. Proc Natl Acad Sci USA 2000, 97:5440-5444.

[viii] Massieu L, Haces ML, Montiel T, Hernandez-Fonseca K. Acetoacetate protects hippocampal neurons against glutamate-mediated neuronal damage during glycolysis inhibition. Neuroscience 2003;120:365–78.

[ix] Henderson ST, Vogel JL, Barr LJ, Garvin F, Jones JJ, Costantini LC. Study of the ketogenic agent AC-1202 in mild to moderate alzheimer’s disease: A randomized, double-blind, placebo-controlled, multicenter trial. Nutr Metab (Lond) 2009; 6: 31.

[x] T.B.VanItallie,C.Nonas,A.DiRocco,K.Boyar,K.Hyams,and S. B. Heymsfield, “Treatment of Parkinson disease with diet- induced hyperketonemia: a feasibility study,” Neurology, vol. 64, no. 4, pp. 728–730, 2005.

[xi] Scheibye-Knudsen M, Mitchell SJ, Fang EF, Iyama T, Ward T, Wang J, Dunn CA, Singh N, Veith S, Hasan-Olive MM, Mangerich A, Wilson MA, Mattson MP, Bergersen LH, Cogger VC, Warren A, Le Couteur DG, Moaddel R, Wilson DM 3rd, Croteau DL, de  Cabo R, Bohr VA. A high-fat diet and NAD(+) activate Sirt1 to rescue premature aging in cockayne syndrome. Cell Metab. 2014 Nov 4;20(5):840-55.

[xii] Pérez-Guisado J, Muñoz-Serrano A. A pilot study of the Spanish Ketogenic Mediterranean Diet: an effective therapy for the metabolic syndrome. J Med Food.  2011 Jul-Aug;14(7-8):681-7.

[xiii] DiNicolantonio JJ, Lucan SC, O’Keefe JH. The Evidence for Saturated Fat and for Sugar Related to Coronary Heart Disease. Prog Cardiovasc Dis. 2015 Nov 13. pii: S0033-0620(15)30025-6.

Intermittent Fasting has many health benefits

Why has intermittent fasting (IF) become so popular in the past few years?

One of the main reasons is simplicity. There are a million and one diets that involve specific foods or nutrients, but IF skirts all those details. Let’s see what the main types of IF are, and what the evidence shows about weight loss and other health effects.

IF variants

Also known as time-restricted feeding,[1] IF alternates periods of normal food intake with extended periods (usually 16–48 h) of low-to-no food intake. This approach lends itself to different variants, including those:

  • Alternate-Day Fasting (also known as Alternate-Day Modified Fasting). This diet can take different forms: you can eat over 12 hours then fast for 36 hours; you can eat over 24 hours then fast for 24 hours; or you can eat normally over 24 hours then eat very little (about 500 kcal) over the next 24 hours.
  • Eat-Stop-Eat. You fast or severely restrict calories for 24 hours, either at regular intervals (two days per week in the 5:2 Diet) or just from time to time.
  • Random Meal Skipping. You skip meals at random throughout the week.
  • Feeding Window. You can only eat during a set period of time every day (from 10 a.m. to 6 p.m, for instance).

Should you decide to try intermittent fasting, pick a variant you think you can stick with for at least a few weeks.

Effects on Weight

Yes, IF can lead to weight loss,[2] but all variants may not be equally efficacious. For instance, simply skipping breakfast led to weight loss in one study[3] but not in another.[4] In both studies, the control groups were provided with a standard breakfast, such as oatmeal, but neither group was restricted in what they could eat the rest of the day.

It’s also possible that people with more weight to lose may benefit more from an IF approach, but one thing is sure: if you compensate for the meals you skipped by eating more later in the day, or the next day, or the next, you won’t lose weight. The weight-loss equation is simple: you need to ingest less calories than you burn. IF is just one way to make this happen, but it is a way that some people find easier than the more common “eat smaller meals” approach.

To lose weight, you need to burn more than you eat — or, conversely, to eat less than you burn. Some people find this goal easier to reach through intermittent fasting than through the traditional “smaller meals” approach.

Effects on Health

The body of evidence on IF is still relatively small, but a growing number of studies have reported improvements in various health markers aside from weight, notably lipids.[1] Further, those studies suggest that IF may provide unique metabolic benefits over the “eat smaller meals” approach.

The most intriguing of those benefits, but also the most debated, is a longer life. Fasting can kick-start some regenerative processes in the body,[5] and extended lifespans from caloric restriction have been reported in many animal models[6] (but not all[7]). Keep in mind, however, that those animals were either fed low-calorie diets or rotated through periods of fasting for most of their lives. It is unknown if IF can extend the lifespan of human beings, and if it can, which variant is best and how many weeks, months, or years are required to make a difference.

Assessing the potential metabolic benefits of IF is a long-term endeavor. As a systematic review of the literature noted in 2015,[8] preliminary evidence looks promising, but solid research is still sparse, so that “further research in humans is needed before the use of fasting as a health intervention can be recommended”.

Extreme Weight Loss Causes Extreme Metabolic Slowdown

Long-term weight loss is hard to maintain.

Studies show that weight loss reduces the number of calories you burn at rest, resulting in slower weight loss and faster weight regain (1).

A new study checked in on former contestants of The Biggest Loser TV show, and the findings were recently published in Obesity.


The Biggest Loser is a popular reality TV show. It uses an “eat less, move more” approach to help obese participants lose large amounts of weight.

Participants are encouraged to eat a very-low-calorie diet. They also follow a strict exercise regimen, exercising for several hours a day on most days.

While The Biggest Loser may be an effective way to lose weight, there are some downsides to rapid weight loss using extreme calorie restriction and exercise.

One of them is adaptive thermogenesis, often referred to as metabolic adaptation or starvation mode (2).

Metabolic adaptation is the brain’s natural, physiological response to reduced calorie intake for long periods of time. In order to maintain energy balance and prevent starvation, the body responds by reducing calorie expenditure.

This is known as metabolic slowdown. And although it’s a well-known phenomenon accepted by scientists, how it works is still unclear.

Article Reviewed

This observational study investigated changes in resting metabolic rate and body composition 6 years after participants lost a large amount of weight.

Persistent metabolic adaptation 6 years after The Biggest Loser competition.

Study Design

This study involved participants from The Biggest Loser competitors in Season 8.

It was a follow-up study to a previous study, which examined whether The Biggest Loser weight loss program helped preserve lean mass and maintain resting metabolic rate (3).

Out of the 16 participants in the original study, 14 participated in the follow-up that took place 6 years after the weight loss competition ended. This time, the researchers looked into changes in resting metabolic rate and body composition.

For two weeks before the follow-up measurements, body weight was monitored daily.

The participants then stayed at a clinical center for three days. During that time, the researchers measured several factors, including body weight and composition, resting metabolic rate and total energy expenditure.

Blood samples were also collected after an overnight fast, to measure blood sugar, insulin, blood lipids, leptin and several other hormones.

These measurements were then compared to data collected at the beginning and the end of the 30-week competition in 2009.

Bottom Line: This follow-up study examined the changes in resting metabolic rate and body composition 6 years after extreme weight loss.

Finding 1: Contestants Regained a Significant Amount of the Weight They’d Lost

Of the 14 participants who took part in the follow-up study, 13 regained weight within 6 years.

What’s more, four contestants are heavier today than before they took part in The Biggest Loser. Only one contestant weighs less now than 6 years ago.

These findings are shown in the figure below.

However, despite this weight regain, the researchers concluded that compared to other dieting methods the contestants were quite successful at long-term weight loss.

Bottom Line: Most of the contestants regained weight in the years following their extreme weight loss. Only one participant currently weighs less.

Finding 2: Metabolic Adaptation Persists Over Time

Resting metabolic rate (RMR) remained low, as it was 6 years earlier when the competition ended. This is despite substantial weight regain.

That means that to maintain their weight, participants must consume fewer calories.

The average calories burned at rest were around 500 calories lower per day than expected based on current weight and age.

Bottom Line: The contestants had unusually slow metabolisms 6 years after the competition, making long-term weight maintenance difficult.


The study had several limitations. First, it did not include a control group of obese people who did not lose weight.

Second, the subjects in this study were obese, which makes the results hard to translate to the general public.

Third, the results are hard to compare to more typical weight loss programs due to the extreme nature of The Biggest Loser.

Lastly, the study was very small, which limits statistical power.

Bottom Line: This study was small, didn’t include a control group and may not apply to non-obese people or those following a less extreme program.

Summary and Real-Life Application

In short, this study found that contestants of The Biggest Loser had slow metabolisms for their size 6 years after the show ended.

This made it difficult for them to maintain weight loss, and almost all of the participants had put a significant amount of weight back on.

Unfortunately, there is no known way to completely prevent metabolic slowdown.

However, doing some form of resistance training, like weight lifting, can at least partly reduce muscle loss and metabolic slowdown during weight loss. Eating a high-protein diet may also help (456).

Alternate day fasting is effective weight loss plan

Following a standard weight loss diet is difficult for most people who too often depend on the fad weight loss pills like Garcinia Cambogia extract.

Recently different methods such as intermittent fasting are gaining popularity.

A randomized controlled trial compared the safety and effectiveness of alternate-day fasting to a traditional, calorie-reduced diet. Here is a detailed summary of its findings.


When people diet, they eat less than they normally would.

Typically, an effective weight loss diet involves a 20–30% calorie deficit, relative to the amount of calories needed to maintain weight. It generally leads to a moderate 5–10% weight loss over a 6-month period (12).

However, sticking to a calorie-reduced diet for a long period is extremely difficult for most people (3).

For this reason, alternative strategies are growing in popularity. One such strategy is intermittent fasting, which involves eating little or nothing for specified periods and normally the rest of the time.

One common intermittent fasting approach is alternate-day fasting (ADF), which involves eating little or nothing every other day.

Like most other weight loss methods, ADF reduces the risk of heart disease and diabetes. It may also cause beneficial changes in appetite hormones (456).

Studies in overweight or obese adults indicate that ADF may cause 3–8% weight loss over a period of 2–12 weeks (78).

Yet, it’s still unclear whether ADF is an effective weight loss strategy. Until now, no studies have compared ADF to a traditional weight loss diet (9).

Article Reviewed

screen-shot-2016-10-26-at-10-41-04-pmThis was a randomized controlled trial comparing the effectiveness of alternate-day fasting to a standard weight loss diet.

A randomized pilot study comparing zero-calorie alternate-day fasting to daily caloric restriction in adults with obesity.

Study Design

This was a small, 2-month randomized controlled trial examining the safety and effectiveness of alternate-day fasting, compared to a traditional weight loss diet.

A total of 26 obese adults participated in the study. They were randomly assigned to one of two groups:

  • Alternate-day fasting (ADF): Participants fasted every other day. On non-fasting days, they could eat as much as they wanted. On fasting days, they were only allowed to consume water, calorie-free beverages and stocks or broths.
  • Traditional weight loss diet (TWD): Participants followed a calorie-restricted diet (a deficit of 400 calories per day) for two months.

In both groups, all food was provided by the study kitchen, and food intake was closely monitored. Additionally, the participants’ macronutrient intakes were standardized with 55% of calories from carbs, 15% from protein and 30% from fat.

At the start and end of the study, the researchers measured the following:

  • Body weight.
  • Body composition: Measured using dual-energy X-ray absorptiometry.
  • Blood lipids: Total cholesterol, triglycerides and HDL were measured in fasting blood samples.
  • Blood sugar control: Evaluated with a glucose tolerance test.
  • Resting metabolic rate: Assessed in the morning using standard indirect calorimetry.
  • Appetite hormones: Leptin and ghrelin were measured in fasting blood samples.
  • Brain-derived neurotropic factor (BDNF).

When the study was over, the participants received standard weight maintenance advice. The above measurements were repeated after a 6-month unsupervised follow-up.

Summary: This was a randomized controlled trial comparing the safety and effectiveness of alternate-day fasting to a traditional weight loss diet.

Finding 1: Alternate-Day Fasting and Standard Dieting Caused Similar Weight Loss

Alternate-day fasting (ADF) and the traditional weight loss diet (TWD) caused similar weight loss.

Specifically, those who fasted every other day lost 18.1 pounds (8.2 kg), on average, whereas those who dieted every day lost 15.7 pounds (7.1 kg), as shown in the chart below.

Weight Loss ADF TWD

Although the weight loss was slightly higher among those who fasted every other day, the difference was not statistically significant. However, the relative weight loss (percentage of body weight) was nearly significant.

Further studies with a greater number of participants and more statistical power are needed to determine whether this difference is real or just a chance occurrence.

Summary: Alternate-day fasting led to weight loss similar to that of a standard weight loss diet with a moderate calorie deficit.

Finding 2: Alternate-Day Fasting Led to a Greater Calorie Deficit

Participants who fasted every other day achieved a greater calorie deficit.

They consumed 376 fewer calories per day, on average, compared to those who were on the traditional weight loss diet.

The chart below shows the differences in calorie deficit between groups.

Calorie Deficit ADF TWD

This is a large reduction in calories that should lead to considerable weight loss over two months.

However, this extra calorie deficit didn’t seem to significantly affect weight loss, as shown in the previous section.

Possible explanations include the underreporting of food intake in the ADF group or a reduction in the number of calories burned.

Summary: Alternate-day fasting seemed to cause a greater calorie deficit, on average, compared to a traditional weight loss diet.

Finding 3: Alternative-Fasting Had Favorable Effects on Body Composition

After the intervention part of the study had ended, the researchers followed the participants for an additional six months.

During these six months, there were no significant changes in weight regain between groups.

However, when the researchers compared values from the start of the intervention, changes in percent fat mass (FM) and lean mass (LM) were significantly more favorable among those who fasted every other day.

These findings are presented in the chart below.

Change FM Trunk FM LM

These findings should be interpreted with caution since there were some between-group differences in body weight at the start of the study.

Summary: Alternate-day fasting appeared to beneficially affect body composition, compared to a traditional weight loss diet.

Finding 4: Resting Metabolic Rate Decreased in Both Groups

Both alternate-day fasting and traditional dieting caused a drop in the number of calories burned at rest (resting metabolic rate).

This effect is known as metabolic adaptation or starvation mode — the body’s response to a calorie deficit.

When the decrease in resting metabolic rate (RMR) was adjusted for fat mass and lean mass, the difference between groups became marginally significant. The findings are presented in the chart below.

Change In Resting Metabolic Rate

Summary: Both alternate-day fasting and a calorie-reduced diet caused a decrease in resting metabolic rate.

Finding 5: Alternate-Day Fasting Caused an Increase in BDNF

Previous studies suggest that fasting may improve mental performance, possibly due to changes in brain-derived neurotrophic factor (BDNF).

BDNF may also be involved in the regulation of energy balance (10111213).

In the current study, there were no differences in BDNF levels between groups.

However, at the end of the follow-up period, the researchers discovered that levels of BDNF had increased significantly among those in the ADF group, compared to the TWD group, as shown in the chart below.


These findings suggest that ADF may lead to long-term changes in the formation of BDNF, which might promote weight loss maintenance. This needs to be studied further.

Summary: Alternate-day fasting led to an increase in brain-derived neurotropic factor. The health relevance of this is unclear.

Finding 6: Effects of Alternate-Day Fasting on Appetite Hormones

Previous studies indicate that alternate-day fasting increases fullness after meals, as well as levels of the satiety hormone peptide YY (14).

In the present study, the researchers measured leptin (a satiety hormone) and ghrelin (the hunger hormone) at the start and end of the study. The findings are presented in the chart below.

Change Leptin Ghrelin ADF TWD

There were no significant differences in hormone changes between groups.

ADF also led to improvements in blood lipids. Once again, there were no significant between-group differences.

Summary: Alternate-day fasting and a traditional weight loss diet similarly affected the appetite hormones ghrelin and leptin.


The main limitation of the study was its small size. The low statistical power may explain the lack of significant differences in some of the outcomes.

Second, physical activity levels weren’t monitored. This might have affected the results.

Third, the researchers didn’t know how many of the participants continued following the ADF or TWD during the follow-up period.

Finally, food intake was strictly controlled, and the findings may not be generalized to a free-living population.

Summary and Real-Life Application

In short, this study suggests that alternate-day fasting is safe and at least as effective as a moderate, calorie-reduced diet.

It did not raise the risk of weight regain during the first six months after the weight loss program finished.

Although weight loss wasn’t significantly different between groups, there were some signs that alternate-day fasting may be more beneficial than continuous dieting. These findings need to be confirmed by larger studies.