Leucine, Citrulline or a Non-Essential Amino Acid Mix - Which Amino Acid(s) are Most Effective in Preventing Muscle Loss During an 18h (Intermittent) Fast?

Image 1: If Chris, "the Techician", Aceto's usually well-informed sources are right and the former Mr Olympia Jay Cutler is currently trying to lose muscle (I heard him say that on Heavy Muscle Radio), Cutler would be ill advised if he ingested ~20g of non-essential amino acids during and / or in-between extended fasts and hours of arduous low-intensity cardio sessions (img  MuscleTech)

Those of you who followed the "Amino Acids for Super Humans" series I did earlier this year on Carl Lanore's Super Human Radio may remember the arginine < > citrulline < > ornitine cycle and how I tried to explain that, from a physiological perspective, arginine's role in ammonia detox is probably as, if not more important than its role in the production of nitric oxide. What most of you will probably have overheard, or, in the respective shownotes, over-read, was my reference to a 2006 study from the University of Paris, which was - at least to my knowledge - the first study to show that citrulline (much like leucine) increases protein synthesis and thusly reduces the loss of muscle protein in old malnourished rats (Osowska. 2006). As it is often the case with isolated study results like that, these observations have not gotten much attention within the research community, so that it is not very surprising that the latest information on citrulline's putative role in whole body protein homeostasis come from the same laboratory at the Sorbonne, as the previously cited ones.

Citrulline vs. Leucine, and non-essential aminos as a control!?

What is particularly interesting about these results, the scientists from the Département Biologie Expérimentale, Métabolique et Clinique at the Pharmaceutical Faculty of the venerable Université Paris Descartes published in the (btw. highly recommendable) Journal Amino Acids, is that they allow for a direct comparison of the magnitude and the mechanism the ingestion of citrulline, leucine or a mix of other non-essential amino acids has on the fractional protein synthesis in skeletal muscle tissue (Tibialis anterior) in a fasted state (18h food deprivation).

Figure 1: Fractional protein synthesis (in %/h) in tibialis anterior muscle of fasted rats 50 minutes after administration of leucine, l-citrulline or isonitrogenous (to leucine) non-essential amino acids (data adapted from Plenier. 2011)

To my own surprise the winner of the battle of the "protein anabolic amino acids" is neither the usual (leucine), nor the unusual suspect (citrulline), but rather the non-essential amino acid combo which consisted of 1.35g/kg of alanine, glycine, proline, histidine, asparagine and serine.

Alanine, glycine, proline, histidine, asparagine, serine - Non-essential high potentials?

Let's briefly put this surprising result into (a human) perspective: If we assume that you are on an extended intermittent fast, traveling or had - for whatever other reason - no access to food for 18h, then the ingestion of 0.22g/kg of a non-essential amino acid mixture (if you weigh 80kg that would be 17.5g), would induce a 9.37% greater increase in muscle protein synthesis than the same amount of leucine and a 16.67% greater increase than 23g of l-citrulline.

Figure 1: Phosphorylation of Akt, s6K, 4EBP1 (left) and AMPK (right) 60min after administration of leucine, l-citrulline or isonitrogenous (to leucine) non-essential amino acids (data adapted from Plenier. 2011)

If we combine the previous calculations with the data from the Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component, it becomes even more obvious that this study provides further evidence against the current over-emphasis of l-leucine which is so prevalaent especially among the bodybuilding-oriented physical culturists. As I have pointed out in previous posts, here at the SuppVersity, pushing the "protein-anabolic gas-pedal" through the floor (=ingesting huge amounts of leucine on its own) makes no sense if your car has long run out of fuel (=there are no amino acids to synthesize).

Against that background it is actually not very surprising that the protein synthesis in the fasted leucine group was reduced, although the phosphorylation of  p70S6K was identical and the one of 4EBP1 even greater (both indicate that the protein synthetic machinery was set into gear) than in the fed control. What is surprising, though, is the fact that the actual protein synthetic response in the leucine group fell 10% short of the one that was observed in the tibialis muscle of the rodents which receive an isonutrogenous amount of non-essential amino acids. After all, previous studies have suggested that the induction of measurable increases in protein synthesis was an exclusive property only branched chain (BCAA) or essential (EAA) amino acid mixtures would posses. Methodological differences in the design of respective studies aside, Servane Lé Plenier and his colleagues suggest the following two possible explanations for the surprising effects the alanine, glycine, proline, histidin, asparagine and serine combo exhibited on skeletal muscle protein synthesis in the fasted state:

[firstly,] in the fasted state, NEAA homeostasis is maintained by catabolism of essential amino acids (EAA) - alanine, for example, is produced in muscle from LEU and pyruvate - and limited EAA availability affects MPS since it is well known that a deficiency in one amino acids may be a limiting step for protein synthesis. Hence, in the fasted state, NEAA administration could spare EAA utilization and thereby preserve MPS.

[secondly,] one or more amino acids in the NEAA mixture could display specific anabolic properties. For example, alanine has been shown to stimulate liver protein synthesis in starved rats (Perez-Sala. 1987), but to the best of our knowledge this effect has not been shown in muscle. Similarly, proline and glycine may possess pharmacological properties that could indirectly modulate protein synthesis.

Personally, I don't believe that any of the non-essential amino acids (NE-AA) in the NE-AA formula actually had an individual effect on protein synthesis beyond its ability to spare essential amino acids and its availability as a substrate for inter-organ amino acid transfer (especially for alanine and asparagine, which are transaminated in the liver, this could be an important factor). So that the practical implications of this study should be clear: if you want to minimize muscle loss during a(n) (intermittent) fast, you better have some non-essential amino acids with your leucine!

One question answered, 999 new ones raised

Image 2: If you have read all Intermittent Thoughts articles which dealt with the AMPK/mTOR Metabolic Seesaw and the respective follow-ups, you will probably already have noticed that the ingestion of non-essential amino acids had the least impact on the fasting-induced increase in AMPK-phosphorylation of all three treatments. And I guess I don't have to tell you that this is good news for all intermittent fasters out there - spare the muscle, improve your health and burn the fat, what more can you as for?

Unfortunately, this study leaves us with way more questions than answers. I personally, for example would venture the guess that the ingestion of a complete EAA product would result in an even more profound amelioration of the fasting induced reduction in fractional protein synthesis. That being said, the latter could also compromise another advantage of the non-essential amino acids, I have not even mentioned, yet: their almost non-existent effect on intra-muscular AMPK-expression (cf. figure 2, right). If you read all Intermittent Thoughts articles which dealt with the AMPK/mTOR Metabolic Seesaw and the respective follow-ups, you will be familiar with notion that the fasting-induced phosphorylation of intra-muscular AMPK is responsible for the majority of the health, as well as the closely related fat-burning effects of (intermittent) fasting. Now, if the ingestion of a ~20g bolus of alanine, glycine, proline, histidine, asparagine and serine could increase your skeletal muscle protein synthesis back to almost normal levels (NE-AA -12.5% vs. leucine-only -20%), while keeping the AMPK-alpha levels maxed out (cf. figure 2, right), it would at least warrant an experiment before we totally discard the possibility that, under certain circumstances, such as the fasting window of an intermittent fast, the oftentimes disregarded "non-essential amino acids" could perhaps be more than just a band-aid when you have run out of essential ones.

Whether there will be a place for citrulline in particular is questionable, though. With the least effect on protein synthesis and the greatest impact on AMPK, it would de facto be a "band-aid" solution, for everyone who fasts, deliberately. In other contexts, however, l-citrulline supplementation could well have its merits. In cancer patients it could for example be used to ameliorate muscle loss without triggering the pro-carcinogenic (Garcia-Maceira. 2009), but I guess this would be the topic of another study and another blogpost, here at the SuppVersity ;-)

Green, Oolong, Pu'Erh or Black, All Teas May Help Keep Fat in Check. 750ml of Tea Per Day Could Ward Off Some of Your Holiday Weight Gain.

Image 1: Though color does matter to some extend, the most important thing appears to be that you drink your tea, whether it is green, black, white or well... pu-erh ;-)

Christmas time is approaching and my personal experience tells me that for many of my fellow human beings Santa has more than just iPads, iPhones and iPods in his gunnysack. The two, three or more pounds of body weight, Santa, or rather the festivities in his honor (unfortunately most people have forgotten that we are actually celebrating the birth of Jesus Christ) leave behind on their hips are yet mostly unwanted, so that the results of a recent study which was conducted by scientists from the National Hsinchu University of Education and the National Taiwan University may come pretty handy (Huang. 2011), especially for those of you who plan on having some "healthy" low sugar, but high fructose junk-food over the holidays.

Tea in lieu of hot wine punch could ward off your holiday weight gain 

In their 12-week study, Hsiu-Chen Huang and Jen-Kun Lin fed a group of initially healthy five-weeks-old male wistar rats (body weight: 150-200g) either a standard rat chow (Purina, Ralston Purina, St. Louis, MO) or a chow that consisted of 60% fructose and 40% of the chow.  Now, the latter group was again divided into a fructose control group and 4 experimental groups, where 4% of the standard chow were replaced by ground green, oolong, black or pu-erh tea leaves.

Figure 1: Catechin content of the leaves of green, oolong, black and pu-erh tea.

The scientists' reasoning was that the addition of the catechin-rich tea leaves (cf. figure 1) to the chow would ameliorate the detrimental effects of the high-fructose diets on blood lipids and other measures of metabolic health. Now, the whole rodent vs. human data aside, you are probably not willing to eat tea leaves, are you?

If we do yet assume that the main effect is induced by the "active ingredients" of the leaves and take the catechin content as a measure of how much of that we would have to ingest, eating the whole leaves is probably not even necessary. With their daily food intake of ~27.5g the rodents got roughly 46mg of catechins (calculated for the green tea).

That in turn would equal a dose of 153mg/kg (this calculation takes the weight gain into account) for the rats and 25mg/kg for a human being (cf. my article on human equivalent doses). So, if you weighed 80kg you would have to get 2g of catechins, which is the equivalent of 7.5 mini-cups of 100ml tea each brewed from 1g of tea (the last step in this calculation is based on USDA data from 2007).

Figure 2: Body weight of rats fed either the 40% fructose diet or the same diet enriched with 4% of tea leaves from green, black, oolong or pu-erh tea (data adapted from Huang. 2011)

But let's forget the math and the speculations about whether or not the results are going to translate to human beings for a moment and just have a look at the ameliorative effect the different types of tea had on the weight gain of the high fructose fed rats in the course of the study period. In that, it was not the catechin-laden green tea which had the most profound anti-obesity effect, but the highly oxidized black and the oxidized and fermented pu-erh teas.

Gaining the least weight does not necessarily mean having the "best" blood lipids

Interestingly, a different picture emerges, when we focus exclusively on the blood lipids, where the green tea leaves produce a lipid profile of which current medical "wisdom" tells us that it is the "most beneficial" one ;-)

Figure 3: Relative changes in serum lipid levels due to fructose feeding and fructose + tea leave feeding (data calculated based on Huang. 2011)

As a studious student of the SuppVersity, you will probably already be thinking about possible underlying mechanisms of these anti-obesity and anti-hyperlipidemic effects of the different types of tea leaves. Well, part of those are probably mediated by an old acquaintance: 5' AMP-activated protein kinase or AMPK.

Figure 4: Relative changes in AMPK phosphorylation and fatty acid synthethase in rats in response to feeding with 4% of black, green, oolon or pu-erh tea; my quantification on the left, original blots on the right  (data calculated based on Huang. 2011)

In view of the fact that Huang and Lin were either to lazy or did not consider it necessary to quantify their immunoblots (figure 4, right), I used a standard image processor to come up with the data in figure 4. Since this is obviously not very reliable, I also included the original blots so that you can convince yourselves that both the increase in AMPK phosphorylation, as well as the amelioration of the fructose induced increases in fatty acid synthethase activity correlate with the effects the different types of teas had on lipid metabolism of the rats.

Green, Black, Olong, Pu-Erh? Which tea to chose?

If we take a final look at the "whole picture", it appears that the exotic pu-erh tea would be the tea of choice, when you totally discard taste, price and whatever other factors may influence your choice. After all, it is was exactly as effective in ameliorating the weight gain as the black tea, induced an impressive +244% increase in AMPK phosphorylation and had what I, contrary to the medical establishment, would consider the "optimal" effect on the lipid profile of the rodents (a profound decreases in triglycerides, even below the level of rats fed the normal chow, and a +11% increase in HDL over the control diet despite high fructose feeding). The "classics", black and green tea, on the other hand, share a close and still notable second place, while the fancy oolong tea appears to be the least effective in this quartet of natural health promoters, the potency of which, and this is something I cannot emphasize enough, can hardly be explained based on their catechin content alone (cf. figure 1) and thusly renders the use of respective abstracts for the same purpose more than questionable.

Dietary Fiber - Friend or Foe? Addition of Hydroxpropyl-Methylcellulose, a Non-Fermentable Viscous Fiber, to Standard(!) Rodent Chow Reduces Fat Gain by -22%

Image 1: Just as about everything, these days, you can buy the semisynthetic non-fermentable viscous fiber Hydroxpropyl-Methylcellulose pound-wise from China - this is probably also where the producers of the junk food you hopefully are not eating get their E464 from ;-)

If the health and fitness community on the Internet was a battlefield (personally, I sometimes think it is ;-) one of the ongoing skirmishes would certainly be fought over the question whether the deliberate ingestion of great amounts of dietary fiber was a good or rather a bad thing. I must admit that I have not really made up my mind on the benefits and caveats of increasing or decreasing your fiber intake, partly because the available science appears to be quite inconclusive. This, obviously does not hinder the "ANTI faction" in the fitness and nutrition world to add "fiber" as the 1001 item on their never-ending list of ultimate dietary evils. My gut feeling does yet tells me this has more to do with the fact that mainstream dietary recommendations list dietary fiber as a "healthy food" to eat, than with a thorough research of the available literature, which has, as of lately, been extended by a particularly interesting study from scientists from the Department of Food Science and Nutrition at the University of Minnesota and the Pennington Biomedical Research Center in Baton Rouge, Louisiana (Islam. 2011).

When fiber ain't fiber, natural is not naturally better

Being aware of the partly contradictory results previous studies on the metabolic effect (as measured in the lab and not by the way your poo-poo looks or how often you have to go to the toilette, like the "experts" like to do it ;-), Ajmila Islam and her colleagues fed a group of 6 week-old male Wistar rats a standardized rodent chow (AIN-93G, composition see figure 1 in previous blogpost) with either hydroxypropyl-
methylcellulose (HPMC) or standard cellulose as a source of dietary fiber, which comprised 5% of the animals' otherwise totally identical diets. This obviously sounds nonsensical if you follow the usual black or white approach to nutrition (which btw. is propagated by the mainstream and the ANTIs, as well), after all fiber is fiber and should be good or bad!? Well, it turns out that there are more than subtle differences in

1. the viscosity of the fiber / fiber food mixture, and
2. the fermentability of different types of dietary fiber

Now, cellulose the main component in plant cell walls and "the fiber" most mainstream dietitians (and their ANTI opponents) have in mind, when they are talking about the beneficial / detrimental effects of "dietary fiber", is neither viscous nor readily fermentable. Hydroxypropyl-methylcellulose (HPMC) is also non-fermentable, but contrary to its naturally occurring cousin it has a high viscosity.

Hydroxypropyl-methylcellulose, short HPMC, is a semisynthetic, inert, viscoelastic polymer that is used as an ophthalmic lubricant, as well as an excipient and controlled-delivery component in oral medicaments, and has, under the disguise of the "E-number" E464, already found its way into a lot of commercially produced foodstuff, where it is used as an emulsifier, thickening and suspending agent, and as an alternative to animal gelatin.

While its artificial origin will obviously make the ANTI faction cry out loud, again, I suggest you first read about the effect HPMC had on the animals, before you totally discard it as being "not natural", "non paleo", "the work of Monsatan" or whatever...(btw. it is at least kosher ;-)

Figure 1: Changes in body composition (weights in g) after 6 weeks on standard diet with either non-viscous cellulose or viscous hydroxypropyl-methylcellulose as the primary source (5%) of dietary fiber (data adapted from Islam. 2011).

If you look at the data in figure 1, you will have to assert that the body composition of the lab animals did benefit from 6 weeks on the 5% HPMC diet. With a -29% reduction in the increases in purportedly "dangerous visceral fat", a -22% reduction in total adipose tissue gain and non-significant changes in lean mass accrual HMPC easily outperform the "Allis" and "Orlistats" of the pharmaceutical industry. Moreover, while the latter simply reduce the amount of dietary fat that is actually digested (an idiotic approach to weight loss, if you asked me), the non-fermentable viscous fiber in the Islam study worked its fat burning magic right via increases in AMPK, COX, citrate synthase, PGC-alpha, PPAR-delta and UCP3 expression, or, put simply: The 5% HPMC diet ramped up mitochondrial fatty acid oxidation.

Figure 2: Changes (relative to cellulose group) in gene expression in liver and soleus muscle of HMPC fed rats (data adapted from Islam. 2011).

Nevertheless, the slight, yet statistically significant smaller increases in bone density in the HPMC group (+6.4g vs. +7.4g) do suggest that in addition to these inert metabolic effect, the mere excretion of parts of the diets (with respect to bone density probably minerals like calcium and phosphorus), of which the animals in both groups consumed about identical amounts, could also have contributed to the otherwise beneficial effects of HPMC feeding, which, as the profound decrease (-41%) in liver PEPCK expression suggests, also included reductions in the hepatic rate of gluconeogenesis.

Figure 3: Changes (relative to cellulose group) in glucose metablism and adipokine expression of HMPC fed rats (data adapted from Islam. 2011).

The overall beneficial effects on glucose and fatty acid metabolism, by the way, are also reflected in the changes in blood glucose and adipokine concentrations I plotted in figure 3. The lower insulin and leptin levels in the presence of reduced body fat stores indicate increased insulin and leptin sensitivity and could be partly mediated by the marked increase in adiponectin expression, as the latter, as Islam et al. point out, has been found to have an "insulin sensitizing effect in both muscle and liver and a thermogenic effect (enhanced lipid metabolism) in skeletal muscle".

"Fiber is good, then! Right?"

Contrary to cellulose and fermentable viscous fiber, such as guar gum, of which a 2010 study by Isken et al. (Isken. 2010) found that it had, fed at 10% of the diet of mice, no effect on body fat levels in the short term (15 weeks) and even increased adiposity in the long-term (from 27 weeks to 43 weeks), short term feeding with 5% hydroxypropyl-methylcellulose (HPMC) exhibited unexpectedly profound beneficial effects on the metabolism of these otherwise healthy and normally fed (this is important, because we usually see fiber supplementation in the context of "high fat" diets) rodents.

Whether it would be advisable to deliberately look for the number E464 on the foods you consume is yet still highly questionable. For one, every food with an "E -number" on its ingredient list should disappear from your grocery list, anyways. I do not care which number it is, but if food has "E"'s in it chances that this is highly processed garbage are 99% and in that case the supposedly insignificant amount of HPMC will not turn junk into health food. And secondly, and certainly more importantly, we are just beginning to understand how the viscosity of the foods we eat and their susceptibility to fermentation interact and which impact(s) these characteristics have on our digestion and metabolism. After all, it could well be possible that, just as in the Isken study, this beneficial short term effects eventually fire back and the formerly lean HMPC rats suddenly start gaining weight (and body fat) like crazy... you see, as usual things are more complicated than the innocent (yet actually invalid) question "Is fiber good or bad?" might suggest.

Intermittent Thoughts On Intermittent Fasting - Exercise (3/3): How Training Solves the AMPK/mTOR Antagonism.

Image 1: Just like Two-Face, a character from the Batman comic books, AMPK turns out to have two faces,... ah I mean isoforms the differential expression of which explain why exercise, contrary to starving yourself, maintains or even builds muscle mass while reducing your love handles (img batman.wikia.com).

In the last installment of the Intermittent Thoughts on Intermittent Fasting series, we have revisited the idea of different training modalities, i.e. endurance and strength training, for the promotion of AMPK-related reductions in body fat and mTOR-dependent increases in muscle mass. We have also busted the long-standing myth of the "anabolic window of opportunity", which, upon closer examination, turned out to have the size of a barn door (>24h) that is unlocked with the key of exercise and nutrition sciences. Related findings showed that even in the absence of additional nutritional stimuli a single intense strength training session led to a profound and (>24h sustained) increase in mTOR phosphorylation in 24 untrained, young, healthy, male subjects (Vissing. 2011). In conjunction with the results of Burd et al. (Burd. 2011), who found that the beneficial effects of strength training on the subsequent response to protein feeding depend on exercise intensity and volume and last for >24h, these results further underline the synergistic effects the fasting, training, feeding cycle of classical intermittent fasting regimens had and still has on the health and physiqueof its practitioners.

Unfortunately, both the concept of "fat loss", as well as that of "muscle gain" are still largely associated with notion of what is commonly referred to as "energy balance". If you read my recent blogpost on the  "High(er) Reps for Fat Loss"-Myth, you will be aware of the fallacy behind the idea of "going to the gym to burn fat". And while more and more trainees (also thanks to the educational work of BodyRX Radio ;-) are getting the idea that you have already lost the fight against your love handles, when you go to the gym solely "to burn calories", the notion that you go to the gym to either "pump up" or "totally exhaust", "damage" and "break down" muscle tissue is similarly illusive. Contrary to what the more is more mentality of the western society may suggest, simple linear causality is nothing you will ever see as the underlying "reason" for the success of a given exercise regimen.

Gain muscle or lose fat? AMPK vs. mTOR and the unique effect of exercise
 

Image 2: "Immunocytochemistry/ Immunofluorescence - AMPK alpha 1 + AMPK alpha 2 (phospho S485 + S491) antibody (ab39400)" ... and if you do not understand this lingo, what you see here is nothing else but one of the unspecific markers for both isoforms of AMPK that is used in most of the studies (img abcam)

Regardless of whether you intend to lose fat, to build muscle or strength, the previous installments should have made it pretty clear that you will always be dealing with two-way processes, or I should say cycles. Now, interestingly enough, exercise, contrary to dieting or overeating, appears to have the unique quality of driving both at the same time - fat loss and protein synthesis, AMPK and mTOR. This works, and this is going to be the main message of this concise piece of the Intermittent Thoughts series, because the exercise induced muscular(!) AMPK-response differs from the one your brain and many other organs will exhibit, when you starve yourself during a diet. Actually we have been knowing for quite some time that the predominant isoform of AMPK that is expressed during exercise is AMPK-alpha2. Back in 2000, already, Wojtaszewski et al. found that "high" (in this case >70% of the individual VO2max) intensity exercise for 60min selectively increased AMPK-alpha2 activity almost threefold (Wojtaszewski 2011). Similar to the results of previously discussed studies, the increased AMPK levels returned to baseline within 3h after exercise-cessation.

Unfortunately, only few of the subsequent studies, which investigated the effects of different exercise regimen, used iso-form specific tests to determine which of the two AMPK isoforms was expressed consequent to the respective training protocols. According to the ground-laying work of Stapleton et al. (Stapleton. 1996) and supported by a study by Stephens et al., it is yet likely that the relative exercise-induced expression of AMPK-a1 in human muscle tissue is negligable.

Figure 2: AMPK-a2 expression (arbitrary units measured in the absence of AMP) and fat oxidation in g/min in 7 healthy individuals during 30 minutes cycling at 62.8% of VO2Max (data adapted from Stephens. 2002).

Moreover, the results of Stephens et al. underline that the exercise-induced increase in AMPK-alpha2 does not only increases fatty acid oxidation, but that both exhibit an excellent correlation with exercise induced glucose depletion (Stephens. 2002).

Figure 2: Glycogen content (mmol/kg) and phosphorylation of AMPK (arbitrary units) in human vastus lateralis muscle before (0 min) and at the cessation of 120 min of one-legged knee-extensor exercise, while consuming either a glucose containing drink or a placebo drink.  (data adapted from Thorbjorn. 2006)

It is thus not surprising that Thorbjorn et al. were able to show that the ingestion of 0.7 g of glucose/kg of body weight/hour did not only blunt the exercise induced AMPK-a2 response but also reduces its beneficial effects on fat oxidation by -47% (cf. figure 2)!

The results of older studies sometimes begin to shine in the light of novel findings 

Now, you probably knew all that before - after all we have been talking about this effect, its beneficial effects on fatty acid oxidation and glucose uptake, as well as its supposedly negative impact on protein synthesis in previous installments of this series. And in fact, these results begin to shine only, in the light of the results of a a recently published study by Mounier et al., who were able to show that only the increased expression of the alpha1 isoform of AMPK, but not AMPK-alpha2 does impair mTOR signalling. Against that background, the systemic antagonism of AMPK-alpha1 (expressed in liver, brain, and other organs) and mTORc1 mediated protein synthesis stands in stark contrast to the metabolically highly beneficial synergism of concomittant exercise-induced AMPK-alpha2 and mTORc1 expression.

To make a long story short: Exercise is unique in its ability to help you shed fat and build muscle "at the same time", because it activates a specific isoform of the "starvation sensor" AMPK, which does not block the concomitant increase in protein synthesis subsequent to the (likewise) exercise-induced increase in mTOR phosphorylation. On that note, my schedule forces me to end this abbreviated version of the Intermittent Thoughts, yet not without the promise that I am finally going to tie all the knots together in the next installments of this series.

Intermittent Thoughts On Intermittent Fasting - Exercise (2/3): Opening the "Anabolic Barn Door" With the Key of Exercise and Nutrition Science!

Image 1: The "anabolic window" turns out to be more of a barn door, which is unlocked by the key of exercise and nutrition science (Random House Books)

Looking back, the main take-aways from the last installment were the dependence of exercise performance on adequate and not so much constant energy supply, as discussed in the context of the Ramadan fasting soccer players, the increased AMPK response to fasted training on a hypercaloric diet, which would suggest that things like "fasted cardio" in the morning could well have it's place in an intermittent fasting regimen even when you are bulking (in order to ward off fat gains), and, last but not least, the differential AMPK- and p70S6K protein synthetic response of cyclists and powerlifters to unaccustomed training stimuli. Accordingly, a versatile training routine that is timed in a way that allows you to train fasted or semi-fasted training, i.e. having your first easily digestible high protein meal / supplement ~30min-1h before you hit the gym, will certainly help with lean gains and muscle-sparing fat loss.

How to train if someone "just wants to look good naked"?

While the observations of the Coffey study (Coffey. 2005) did underline the importance of versatility, or, I should say constant "novelty", or at least modification of the training stimuli, they did not really provide any clues on how someone, who "just wants to look good naked" (and I assume this applies to the majority of non-athletes, today) should train to transform his formerly at best non-obese physique to the cover-model'ish look everybody is aspiring these days.

Figure 1: Study design of the Vissing study with its 10-week preconditioning phase for the strength and endurance training groups (generated based on information from Vissing. 2011)

In regard to this question, a similar, yet more recent study on non-athletes comes to mind. In the course of the latter, K. Vissing and his colleagues from Aarhus, Denmark, and Geelong, Australia, took a closer look at the response of the "AMPK/mTOR seesaw" to either endurance or strength training (Vissing. 2011) after a comparatively brief per-conditioning period of 10 weeks (cf. illustration 1) - a scenario of which we can expect more reliable results than from its "highly trained recreational athletes" counterpart from the Coffey study, where the participants have been focusing on training for their respective sport (cycling or powerlifting) for years. Accordingly, Vissing et al. expected to see that...

[...] mTORC1 signaling would be selectively activated by SE [strength training], whereas AMPK signaling would be activated by both types of exercise but to a relatively higher degree after EE [endurance exercise] compared with SE [...]

Thus, their research hypothesis was in accordance with the publicly accepted idea that only strength training builds muscle (obviously the role of mTOR-activation in this process is widely unknown in the general public), while endurance exercise would be the better form to train if one wanted to lose fat - as a diligent reader of the SuppVersity, you will obviously be aware that the reduction in adipose tissue you will hopefully observe, when you are dieting, is primarily a result of the depletion of muscular (and hepatic) ATP stores, which brings the AMPK energy emergency police on the scene which will concomitantly tell your muscles to suck up all extra (i.e. more than your brain needs) glycogen from your blood stream and kick your adipocytes' asses, so that they release some of their fatty energy reserves as metabolic firewood for your mitochondria.

I hope you remember "The 'hungry' side of neuronal AMPK activation", i.e. the differential effects of AMPK phosphorylation in reaction to energy shortage in muscle or liver tissue vs. its effects in the brain. If not, I suggest you (re-)read the respective passage in "AMPK III/III: Natural Rythmicity for Maximum Fat & Minimal Muscle Loss", as a thorough understanding of this difference if of utmost importance if you want to be able to compare and interpret the data from various studies correctly.

The Coffey study (discussed in the last installment) did however show that this assumption, i.e. both endurance, as well as strength training will always increase AMPK, does not hold true, when we are talking about highly trained athletes - neither in the cyclists nor in the powerlifters from the Coffey study did engaging in their respective discipline produce statistically significant increases in AMPK phosphorylation.

Figure 2: AMPK phosphorylation (0, 2.5, 5 and 22h post) and approximate area under the respective curces (small graph) during post-exercise recovery from single-bout exercise, conducted with an exercise mode to which the exercise subjects were accustomed through 10 weeks of prior training (data calculated based on Vissing. 2011)

Conversely, in the Vissing study, AMPK phosphorilation did transiently increase in both the strength and endurance trained groups immediately post (at 0h) exercise (cf. figure 2). However, with the subsequent drop of the phosphorylated AMPK (pAMPK) below the values of the control groups, the estimated area under the curve (AUC; I simply used weighed averages for the calculation), i.e. the absolute AMPK phosphorylation over the whole 22h post-exercise window, for which the scientists have data (cf. figure 2, right), was -12% and -17% lower in the strength training group than in the control and endurance group, respectively.  

Without the AMPK elevation of an intermittent fast (or calorie reduction), it is thus unlikely that strength training alone is going to trigger significant AMPK responses.

Interestingly, the scientists state that the protein expression "of any of the reported signaling proteins" was "not altered" by the 10 weeks of pre-training, which would indicate that, contrary to years of competitive endurance exercise (cf. cyclists in illustration 1 in previous installment), 10 weeks with three weekly sessions of combined steady-state and interval exercises on stationary bikes do not blunt AMPK phosphorylation in response to 120 min of bicycle exercise at 60% of the individual VO2 max.

The induction of mTOR phosphorylation is and will remain the real strength of strength training

Likewise, the protein synthetic response (as evidenced by mTOR and p70S6K expression) did not change in response to a 10-week pre-conditioning phase comprising 30 leg workouts (3 exercises; 3-5 sets; 10 reps in the first 15 sessions, 4-6 reps in the last 15 sessions). Interestingly, and contrary to the often heard assertion that mTOR phosphorylation would be a strength training exclusive, figure 3 shows that there is still a minor, yet over the course of the post-exercise period, non-negligible increase in mTOR phosphorylation in the endurance trained subjects, whose 45min cycling session effectively blunted the mTOR dephosphorylisation the control group, who, just like all of the previously (before the preconditioning) 22 untrained healthy male subjects (79.1 kg; 182 cm; 23.3 years), fasted for the first 5h "post exercise" (their exercise consisted of sitting on the couch, doing nothing ;-).

Figure 3: mTOR phosphorylation (0, 2.5, 5 and 22h post) and approximate area under the respective curces (small graph) during post-exercise recovery from single-bout exercise, conducted with an exercise mode to which the exercise subjects were accustomed through 10 weeks of prior training (data calculated based on Vissing. 2011)

Even without looking at the data in figure 3 it should be obvious that the meager increase in mTOR phosphorylation in the endurance group cannot compete with what we see in the strength trained subjects, whose p-mTOR ( = phosphorylated mTOR) levels skyrocket in the post exercise phase, peaking at +218% (control: 56%; endurance: 130%) not immediately or maybe 1h post exercise but 5h after. Thus, the purported "anabolic window" of 1-2h after a workout turns out to be a barn door, in the real world - a barn door which is wide open right in the middle of your intermittent fasting feeding window!

Strength training = opening the "anabolic barn door"

Yet, while we do now know how to unlock the barn door, we still do not know if there ain't a way to push it open even further / faster, and how to keep it wide open for as long as possible. In this context, a study by Burd et al. from Steward Phillips group at the Department of Kinesiology of  McMaster University in Hamilton, Ontario (Burd. 2011) could provide further clues into the "optimal" way(s) to push the "anabolic barn door" open, as wide as possible.

After all that has been said about the over-expression of mTOR in our current society in the previous installments, it should be said that the problem does not lie with mTOR itself, as it is not the latter which inhibits AMPK, but the energy abundance that triggers the mTOR response in our western obesity scenario. This chronic nutritionally induced suppression of AMPK is something we need to distinguish from both the training-induced increase in mTOR phosphorylation and the temporary and strategically used dietary stimuli that are so characteristic of intermittent fasting.

Figure 4: If we disregard the nutritional component, the training induced "anabolic barn door" does not only coincide with the feeding window, it would also keep you nicely "anabolic" in the course of the fasting period.

In figure 4, I have extrapolated the missing two hours to complete a 24 hour intermittent fasting period, in the course of which you would do your training session early in the morning, head towards the gym at 8:00am, change your clothes, warm up, training for about an hour and break the fast at 10:00am. Thereafter, you would have a pretty long feeding window of about 6 hours, to then begin another fast... in that, your meal pattern would differ profoundly from the one of the study subjects, because the latter had to fast for the first 5 hours post exercise, so that the mTOR response was not augmented and the study results distorted by meal ingestion (afterwards they were allowed to eat whatever they wanted until 22:00pm and had to report back for the 8:30am blood draw (mTOR still +89% elevated) on the next morning. Due to these differences it is difficult to predict how your overall (i.e. exercise + food induced) mTOR response would look like on the above regimen.

Will the "anabolic barn door" stay open in the course of the fast and thusly prevent muscle breakdown?

This is where the data from the Burd study comes into play (Burd. 2011). In their study, Bird et al. had measured the fractional protein synthesis rate in response to feeding (15g of whey protein) and feeding and exercise (unilateral leg raises) at different intensities, i.e. 90% 1RM to failure, 30% 1RM with matched work-load and 30% 1RM to failure. What they found was that

regardless of condition, rates of mixed muscle protein and sarcoplasmic protein synthesis were similarly stimulated at FED and EX-FED (Burd. 2011)

- an observation, the scientist attribute to the fact that the sarcoplasmic constituents of the muscle may be more susceptible to hydration flux, so that the results may not adequately represent the "actual" protein synthetic response.Thusly, the researchers rely in their interpretation of the data mainly on the myofibrillar protein synthesis rate (cf. figure 5).

Figure 5: Changes (% per hour) in absolute myofibrillar protein synthesis (adapted from Burd. 2011)

As you would expect and actually can see in figure 5, the latter did respond to the additional exercise stimulus. Pumping away at 30% of your 1RM max without going to failure, is yet not enough to augment the statistically hardly significant increase in fractional protein synthesis that was triggered by protein ingestion, alone. It takes some effort, or, in other words, heavy weights and training to failure to trigger elevations in AKT phosphorylation (90% 1RM to failure) or mTOR phosphorylation (30% 1RM to failure) to get that done (note: neither of the two, i.e. protein kinase B = AKT or mTOR was significantly elevated by feeding, alone).

[...] protein ingestion stimulated rates of myofibrillar protein synthesis above fasting rates by 0.016 ± 0.002%/h and the response was enhanced 24 h after resistance exercise, but only in the 90FAIL and 30FAIL conditions, by 0.038 ± 0.012 and 0.041 ± 0.010, respectively. Phosphorylation of protein kinase B on Ser473 was greater than FED at EX-FED only in 90FAIL, whereas phosphorylation of mammalian target of rapamycin on Ser2448 was significantly increased at EX-FED above FED only in the 30FAIL condition.(Burd. 2011)

Moreover, and this may be of even greater importance in the context of exercising on an intermittent fast, muscle protein synthesis stayed elevated way beyond what is usually considered the <4h "anabolic window".

Our results suggest that resistance exercise performed until failure confers a sensitizing effect on human skeletal muscle for at least 24 h that is specific to the myofibrillar protein fraction. (Burd. 2011)

While this is obviously important for everyone who wants to accrue as much muscle muss as possible, any elevations in protein synthesis will also help a dieter to keep is hardly earned muscle, because in essence our muscles are continuously build up and broken down  - proteolysis, i.e. the breakdown of muscle tissue, and protein synthesis are going hand in hand and it is the ratio of one to the other, which decides whether we are in an "anabolic" (synthesis > breakdown) or catabolic (breakdown > synthesis) state. Consequently, any elevation in protein synthesis will ameliorate muscle loss - no matter how proteolytic a dieter may become during the fasting phase.

It takes >24h for the barn door to close itself - use this time to get rid of fat, not muscle

Fine, we unlocked the "anabolic barn door", it stays open for "at least 24h"... blah blah... wtf! how does all that translate from the metaphorical into the real world of intermittent fasting? Well, the answer is pretty simple, as hundreds of trainees have been practicing exactly that with extreme success over the past couple of months:

1. fast until min. 1h before your training
2. spike your protein synthesis with a protein shake (~20g of whey), EAAs (~10g) or BCAAs (~8g)
3. train semi-fasted and heavy
4. feast within a 5-8h window
5. repeat the same litany again

Now, the sheer size of the barn door, ahm... sorry, the long-lasting anabolic and thusly anti-catabolic effect of intense strength training should allow you to either skip or replace "3. train semi-fasted and heavy" with "3. passive or active recovery" (in that case you also do not want to ingest the protein shake / EAA / BCAA) or even some "3. semi-fasted cardio" (see notes in red box) if you feel that your conditioning or weight loss will benefit from that, every other day without running the risk of either gaining too much fat weight.

Image 2: Your "anabolic barn" is huge enough to accommodate one or two steady state, low intensity or high intensity "cardio" sessions per week.

If you want to incorporate "cardio" training into your routine, the pre-conditioning protocol from the Vissing study could actually be a very good, since diversified, regimen. In that, you would cycle between doing "standard" steady state conditioning work, longer medium-intensity interval training and short, but intense HIIT sessions. The result would be a very complete "cardio" protocol, of which the Vissing study showed that it will help you ramp up your AMPK levels pretty profoundly, even if you are only sitting on one of those cycle ergometers pedaling away jovially at 60% of your VO2 max. And in case you are now concerned about possibly shutting the barn door - look at figure 3 again, the mTOR response to this kind of exercise may not be earth-shattering, but a plus of 25% @5h post exercise is better than what you would get if you just lay around lazily, as the control group in the Vessing study did.

With these insights into why that of which you already knew that it works actually works, I conclude this week's installment of the Intermittent Thoughts and hope that I did not bore you so much that you do not come back next Sunday for another installment of this series ;-)

Lose(!) 33% Body Fat in 10 Days!? The Heavy Metal Obesity Link: Study Shows "Preventive Role" for Inorganic Cobalt in Obesity-Related Diseases.

Image 1: Cobalt - certainly not what you would expect to see at a health food store or pharmacy; with Kawakami et al.'s study this may change in the future (img Alchemist-hp)

"We are living in a toxic world!" - you have probably heard or read this sentence more than once and while I cannot deny that the environmental load of, among others, heavy metals appears to be increasing, I can however tell you that, according to a recent study from scientists from the Tukushima Bunri University in Japan, exposure to some of those heavy metals produces quite unexpected results in a rodent model of the metabolic syndrome and in lean controls. Instead of making them gain weight even more rapidly, the "toxic" (maybe we will have to reconsider that, just as we did in the case of chromium) heavy metal cobalt did not only reduce the weight of the white adipose tissue of the rodents, it increased leptin, adiponectin, and HDL-cholesterol, as well, and thusly "may have a preventive role in obesity-related diseases" (Kawakami. 2011)

This is certainly the 1001st time I am writing this, but I cannot emphasize often enough that the "high fat diet" researchers use in their studies has (in most cases) nothing to do with the Atkins or even a low-carb diet. Its main characteristic is that it is hypercaloric and high in fat and carbs. Please keep that in mind whenever you read about another study on the detrimental health effects of "high fat diets".

For 24days Kawakami et al. fed a group of seven-weeks-old male mice either a standard diet with 357.6kcal/100g or a hypercaloric (cf. red box above) high fat diet (HFD), where the latter induced obesity and dislepidemia within 2 weeks. After this initial phase, i.e. when the HFD mice were already obese and metabolically deranged, the scientists injected the animals with Sodium Arsenite (NaAsO2: 1.0 mg/kg bw), Mercuric Chloride (HgCl2: 1.0 mg/kg bw), Manganese Chloride (MnCl2: 5.0 mg/kg bw), Cobalt Chloride (CoCl2: 0, 1.3, 5.0, 7.5 mg/kg bw) or saline (control).

Figure 1: Modulatory effects of 10 days of heavy metal injection in mice on a high fat diet; values expressed as changes relative to animals on a normal diet (data calculated based on Kawakami. 2011)

Now, if you look at the data in figure 1, you will notice that the administration of Mercuric Chloride may have been most "effective" in ameliorating the HFD-induced increase in white adipose tissue (WAT) mass (HFD +70%; HgCl2 -14% vs. normal fed control), but those "fat burning" effects went hand in hand with profound elevations of the liver enzymes AST, ALT (in this case we can safely assume that these were not coming from the muscle tissue of the animals) and the blood urea nitrogen (BUN) levels, which indicate deteriorations of the kidney metabolism. Manganese and cobalt, on the other hand, had negligible or even beneficial effects (compared to HFD alone) on liver and kidney health and ameliorated the weight gain to +10% and +17%, respectively.

Figure 2: Adiponectin and leptin serum levels and mRNA expression in mice after 10 days on a high fat diet with simulatenous injection of mercury or cobalt; data expressed relative to normal fed control (calculated based on Kawakami. 2011)

What is particularly interesting about cobalt, though, is that it did not simply starve out the adipose tissue by poisoning it (like that was probably the case for mercury), but triggered exactly those metabolic adaptations scientists have been trying to provoke with drugs for years now: elevations in adiponectin and leptin (cf. figure 2), the two adipokines, researchers currently believe to be essential for successful weight loss / maintenance.

Figure 3: pAMPK/AMPK ratio after injection of different dosages of Cobalt chloride (calculated based on Kawakami. 2011)

In a follow up experiment, the scientists, also found that cobalt dose-dependently increases AMPK phosphorylation (for more on AMPK, I would like to refer you to the Intermittent Thoughts series) in white adipose tissue (WAT), muscle and liver of the animals (cf. figure 3). Of the three tested dosages, administration of 5mg/kg CoCl2 per day resulted in the most beneficial AMPK response, while with the maximal dose of 7.5mg/kg the negative / toxic effects appear to prevail (another of these bell-shaped dose-response curves, I guess).

Figure 4: Glucose tolerance test in mice on high fat diet with or without cobalt injections compared to mice on standard diet (control); values in mg/dl (data adapted from Kawakami. 2011)

Now, you are probably asking yourselves: "So what's the catch?". A brief look at figure 4 tells you that is ain't glucose intolerance, as the cobalt treated animals had the exact same response to the glucose tolerance test, as the mice on the normal diet - in other words: cobalt completely reversed the HFD induced glucose intolerance, and it did so not only without negative effects on blood lipids, but in the presence of a profound elevation of HDL levels and a reduction in LDL levels (cf. figure 5).

Figure 5: Relative (to normal fed control) changes in blood lipid in mice on a high fat diet with or without heavy metal injections (calculated based on Kawakami. 2011)

And as if that was not enough, the cobalt injections also eradicated the iincreases in free fatty acids and ameliorated the increase in triglycerides.

From the lab to the bedside?

Last but not least, and I hardly dare showing you this graph, because I would expect that some of you will already be googling a source of injectable cobalt (which would be plain out stupid, before any reliable safety data and confirmation of these results in controlled human trials are available), cobalt had almost identical effects when it was injected to the mice on the normal diet.

Figure 6: Relative changes in body composition and liver and kidney parameters due to heavy metal injection in non-obese mice on a standard diet (calculated based on Kawakami. 2011)

As figure 6 goes to show the mice lost 33% of their white adipose tissue and liver, as well as kidney function did not take a beating (HDL stayed the same, LDL decreased by -1%). Whether we will see a obesity or even just a weight-loss drug based on cobalt in the near future, does yet still seem questionable. In view of the fact that the number of "bad things" (cobalt is in fact an essential nutrient as it is the active center of vitamin B12 = cobal-amin) that have unexpectedly positive health effects is increasing day by day, we do yet obviously have to ask ourselves, whether there may be some major flaws in our current understanding of how our body works and how it deals and is effected by "toxins", oxidants and co.

Intermittent Thoughts On Intermittent Fasting - AMPK III/III: Natural Rythmicity for Maximum Fat & Minimal Muscle Loss

Image 1: The quest for fat loss, muscle size, health and longevity reminds me of the famous egg-laying wool-milk-sow.
(img austria-lexicon)

At the end of last weeks installment, I provided you with an extensive, yet obviously "incomplete" list of AMPK-"promoters", among which the  organosulfur compound alpha lipoic acid (ALA) turned out to be of chief interest in the subsequent enjoyable and inspiring "intellectual intercourse" I had with Banga, Dr. J, Bomb Jack and of course Lerner in the comments section of the post. Now, the fundamental question that arose from this discussion was how supplementation with ALA (or agents with similar effects on 5' adenosine monophosphate-activated protein kinase expression) could be used as a tool to promote fat, not weight loss while preserving or even building lean muscle mass and optimizing health and longevity - basically it's the quest for the egg-laying wool-milk-sow, an imaginary animal and a commonly used German metaphor for a jack-of-all-trades device. And I can assure you that an ultimate solution to this problem is just as hard to find as this mythical animal an image of which you can see on the right.

When AMPK is the good guy, mTOR must not necessary be the bad guy

We have learned in the past couple of installments that its modulatory effect on the AMPK/mTOR seesaw, which, even in your average citizen of the affluent Western hemisphere, is oftentimes imbalanced towards the "anabolic" mTOR side, these days. Against that background I have pointed towards the myriad of beneficial metabolic effects of AMPK activation, which could in fact ameliorate, if not reverse, many of the ailments that have befallen our fat-anabolic (remember without mTOR, fat cells cannot differentiate, cf. Bell. 2000, where blockage of the mTOR pathway with rapamycin inhibited adipocyte differentiation) society.

Illustration 1: Lifestyle factors like nutrition, nutrient timing, sleep etc. determine the fundamental balance between AMPK and mTOR, but supps can skew / tweak the balance

I hope that highlighting the merits of AMPK in the lat two installments of the series did not give the impression that mTOR its anabolic counterpart on the seesaw (cf. illustration 1) was "useless" or even dangerous, as the sheer amount of recently published studies on the implication of mTOR in the etiology of cancer could make you believe.

Image 2: A breast cancer cell (img from The Guardian); if you want it (not yourselves!) to live longer, don't feed it with leucine, Ladies!

The mTOR-cancer connection: In the course of these first 10 months of 2011 more than 16,000 studies have been published that focus or at leas mention the involvement of an (over-)stimulation of the mTOR pathway in cancer development. It is yet plain short-sighted stupidity to believe that measures like dietary leucine restriction (and subsequent down-regulation of the mTOR pathway) would protect you, let alone cure cancer. It is certainly correct that mTOR promotes proliferation in cancer cells, but it does so in about every other cell in your body. Moreover, studies on the effect of leucine deprivation on mTOR signalling in breast cancer patients showed that not only was "leucine restriction is not sufficient to inhibit mTOR signaling in most breast cancer cell lines", but was "associated with activation of survival molecule Akt, making leucine deprivation an undesirable approach for breast cancer therapy" (Singh. 2011). And with regard to the use of "true" mTOR inhibitors such as rapamycin, David Sabanti remarks in a recent review:

As induction of apoptosis rather than cytostasis is increasingly considered a prerequisite for an effective anticancer agent, it will be crucial to understand when rapamycin has such effects and where it does not, and to learn how to trigger apoptosis with additional therapies.

Now, the use of novel pharmacological mTOR-inhibitors aside, any dietary approach (such as leucine deprivation) would obviously prevent apoptosis, because it would act via mTORC1 inhibition to increase Akt-expression and (cancer-)cell survival (Sun. 2005; O'Reilly. 2005). Would you risk your "metabolic currency", i.e. your muscle, and sacrifice quality of life for the futile hope that you could thusly keep cancer "at bay"? I hope not, because in that case you still have not grasped the fundamental idea that in life its not about black and white, but about black and white and a balance between the two.

This takes us back to the egg-laying wool-milk sow or, without the metaphorical ornamentation, the issue of losing fat weight, while gaining muscle weight. From all you have learned before, it appears quite obvious that - intermittendly fasted or not - gaining muscle and losing fat literally at the same time is virtually impossible. On the other hand, you could obviously first milk and fleece your egg-laying wool-milk sow and then collect the eggs, it has been laying before you grabbed, milked and fleeced it. I assume you will notice that, again, this is a cycle: milk, fleece, collect eggs,... milk, fleece, collect eggs, ... fast, train, feed... fast, train, feed. As you see, nature is not very inventive in her fundamental concepts. She will always rely on her tried and proven cyclicality. Now, if we cannot escape that, we can at least try to tweak it depending on our goals. While for Mr. Supp in illustration 1 this would be only a step to the left to help Mr. AMPK to get the better of Mr. mTOR) or a step to the right to help Mr. mTOR gain control over the AMPK/mTOR seesaw. For us, in the real world, the most obvious thing we could resort to in order to achieve similar results are supplements.

When to take what to optimize fat loss and minimize muscle loss in the AMPK phase

In the comments related to the last installment, Bomb Jack suggested that taking ALA during a fast to jack the naturally high AMPK level up even more, would theoretically make sense, but he mentions that he had seen "studies about [ALA] causing lean tissue loss on the long run". One of these studies was conducted by Yi Wang et al. in 2010 on 24 month old male C57BL/6 mice (Wang. 2010). Half of the mice received 0.75% alpha-lipoic acid in their drinking water for one month, the rest of the mice served as a unsupplemented control.

Image 3: 22 month old C57BL/6 mouse, in view of the fact that mice live ~2.5 years this is already a granny; in other words, the 24 month old mice in the Wang study were really old.

How much is 0.75% in the drinking water of mice in human equivalents? To calculate that you need to know that the mice in the study weighed ~30g, that the average mouse drinks ~5.8ml per day (Bachmanov. 2002) and that a 0.75% solution means that 100ml contain 0.75g of the given solute. Now you calculate the absolute dose per day, which would be 5.8ml/day * 0.75g /100ml = 0.435mg/day divide that by 30 to get the dose per gram of body weight and multiply it by 1000 to get the dose per kg => 14.5mg/kg. To get the human equivalent dose (HED) you then use the formula I explained before and your calculator will tell you that the HED of 14.6mg/kg in a mouse would be roughly 1.2mg/kg in a human being (~95mg for the average adult).

Although the human equivalent (~95mg/day; cf. red box above) of the dose the mice received was way less than most commercially sold supplements contain and by far less than the 800mg - 1,200mg of alpha lipoic acid that has been used with positive results in many trials that involved diabetic patients, the results the Wang et al. observed were pretty pronounced. In the ALA group...

• food consumption decreased: -18% - 4.50 ± 0.30 g/d vs. 5.50 ± 0.30 g/d in control mice
• body weight decreased: -15.8% - 5.27 ± 0.62 g total body weight loss
• energy expenditure increased:  +25% - 7.64 ± 0.10 kcal/kg^0.75/h vs. 5.90 ± 0.10 kcal/kg^0.75/h
• glucose utilization increased: +10% judged by the respiratory quotient
• insulin sensitivity increased: -47% area under the curve in glucose tolerance test
• mitochondrial biogenesis increased: +138% relative abundance of mtDNA content
• PGC-1α* in skeletal muscle increased: + 80.0% 
• GLUT-4 in skeletal muscle increased: + 105.0%
  
  * PGC-1α ramps up thermogenesis, stimulates mitochondrial biogenesis, promotes the remodeling of muscle tissue, controls lipid and glucose metabolism (Liang. 2006)

And all that was accompanied by significant increases in AMPK and decreases in mTOR and P70S6K phosphorylation. In view of the results of the Wilson study, which showed that a much smaller (~40%) increase in AMPK activity went hand in hand with decrease in protein synthesis we can safely assume that the latter is (unfortunately) responsible for both the desirable increases in energy expenditure, glucose metabolism and mitochondrial biogenesis, as well as the not so desirable loss of appetite (you do not need to eat, when you run on stored substrate) and body mass (cf. figure 2).

Figure 1: Relative changes in phosphorylation status of AMPK, mTOR, p70S6K and 4E-BP1 in old mice supplemented with 0.75% ALA in their drinking water for 1 month compared to unsupplemented control (data calculated based on Wang. 2010)

On the other hand, the decrease of the amount of phosphorylated Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), of which Anthony et al. have shown in 2002 that its phosphorylation (which usually occurs consequent to the activation of the mTOR pathway) is not necessary for the increase in protein synthesis that occurs upon leucine administration (Anthony. 2002), was not statistically significant. This could be important, as dephosphorylation of 4E-BP1 and not decreases in p70S6K appear to be hallmark features of cancer cachexia, i.e. profound muscle loss in cancer patients (Tisdale. 2008).

Figure 2: Absolute (left) and relative (right) changes in body composition in old mice supplemented with 0.75% ALA in their drinking water for 1 month compared to unsupplemented control (data calculated based on Wang. 2010)

And indeed, if you, as a diligent student of the SuppVersity who knows about the subtle differences between absolute and relative data, do not focus on the absolute (figure 3, left) but rather on the relative (firgure 3, right) changes in body composition, you will have to concede that the mouse grandpas underwent a transformation you would usually expect to see in the course of the contest preparation for a bodybuilding show. While the mice "on ALA" did lose ~20% of their body weight, they also cut down from 18% to 9% body fat and (-60% fat mass) and increased the relative amount of lean mass from 74% to 87%. For a 300lbs bodybuilder this would be like cutting 32lbs of fat while losing "only" 16lbs of muscle, to end up 48lbs lighter and with a body fat percentage of 9% at 252lbs - not yet really stage ready, but certainly not bad, given the fact that he would not even have to diet to achieve that result (remember: his appetite would have decreased and his energy expenditure increased).

Rythmicity is the key when it comes to seesawing and supplementation 

The mouse vs. human issue aside, the Wang study has another caveat in terms of drawing conclusions regarding the use of ALA and other AMPK promoters during an intermittent fast - the mice did not fast ;-) Reason would dictate, though, that ingesting ALA with drinking water 24/7 on a dietary regimen with constant food availability would actually be a disadvantage compared to taking ALA specifically at the onset of the fast (i.e. when your last meal would be digested) to keep AMPK, which would already been raising at that point (and thus mTOR declining) maximized in the course of the fasting period. On the other hand, taking ALA or any other AMPK promoter after your workout and before your meal would appear to be plain out stupid, as intermittent fasting does only make sense if you really reap the maximal anabolic benefit from the short feeding window. Against that background, popping alpha lipoic acid post workout and thus potentially increasing AMPK appears (remember that we do not have any studies explicitly investigating the effects of taking ALA post-workout vs. at other times of the day, so that these are just yet to be validated hypotheses!) to be counter-indicated, as it would potentially blunt the mTOR and p70S6K response to exercise and food-intake.

Image 2: R- and S- isomer of alpha lipoic acid (Shay. 2010)

"Alpha lipoic acid, yeah! But can I buy the cheap racemic mixture or is it worth paying the extra bucks for the R-ALA?" Actually, it was Daniel Spasic who posted a similar question on my Facebook pinwall and thus made me look into the purported advantages of R-ALA, again. 

I mean we all "know" that R-ALA is the preferable form, but do you remember where you know that from? 

Well, me neither and so I dug back into the host of studies BASF was doing back in the 1990s, until their business department finally realized that a natural anti-diabetes drug is not only non-patentable, but could also compromise the sales of patentable pharmacological drugs like Chlorpropamide (patented in the mid-1980s; cf. DrugPatentWatch) which happens to be made of Propylamine, which in turn - you guessed it - is produced by BASF and sold to BigPharma and the Agrobusiness who use the same ingredient to produce of Prochloraz, and other well-known fungicide... but I am getting taken away, here.

Image 2: The scientific
evidence pro R-ALA
is quite conclusive;
the S-isomer, however,
could potentially negate
its benefits and is
useless, at best!

The main point is that there is clear-cut evidence that the R-isomer has not only a longer half-life and a greater bioavailability (Herrmann. 1996), but is also the "true" anti-oxidant: In a 1997 study by Streeper et al., for example, S-lipoic acid had no effect on insulin-mediated glucose uptake in obese Zucker rats, while R-lipoic acid increased the latter by +64%. In the same study chronic intake of R-lipoic acid reduced plasma insulin and free fatty acid levels. S-lipoic acid, on the other hand, increased insulin levels and had no effect on free fatty acids - probably a direct consequence of the reduced expression of glucose-transporter protein (GLUT4) the scientists observed in the S-ALA supplemented rats. This may now sound like the racemic mixture, which is in fact the ~50/50 R-ALA/S-ALA mixture you can buy for a few bucks at every GNC would be toxic - this certainly ain't the case. On the other hand, would you buy an anticoagulant with an with vitamin K in it, i.e. a product with two active ingredients with partly antagonistic effect? I don't think so...

And for those of you who like to think in images: Taking ALA right at the beginning of your short feeding window would be as if Mr. Supps from illustration 1 took a step to the left, just when Mr. mTOR turn on the right of the seesaw was about to gain momentum - Mr. Supps with his alpha lipoic acid would be a real spoilsport, then, wouldn't he? If, on the other hand, Mr. Supps knows how to play the game, he will follow the AMPK/mTOR seesaw's natural rythm, grab some alpha lipoic acid and step to the left, when, at the beginning of the postprandial phase (~2h after the last meal), it actually is Mrs. AMPK turn, wait until she has had her share and then jump to the right in order to hand Mr. mTOR a leucine-rich protein shake. By doing this, i.e. taking ALA at the onset of the fast, ~2h after the last meal and a leucine rich (post-workout-)protein shake to ramp up protein synthesis right at the beginning of the feeding phase, the natural rythm would not only be preserved, it would also be amplified. And what would translate into a more energetic seesawing in our metaphorical world or Mr. Supps, Mrs. AMPK and Mr. mTOR, should translate to improved fat loss during the fast and a pronounced muscle anabolic response to the daily "refeeds", as you may well call your meals if you compress the feeding window to <6h. If the mice in the Wang study had gotten this rythm right, they would probably have made it to the Mouse-Bodyduilding Masters ;-)

The "hungry" side of neuronal AMPK activation

Before I end, this pretty epic (in terms of the details we have covered) yet not very productive (in terms of how much ground we have made) installment of the Intermittent Thoughts, I want to briefly mention a very important and actually completely logical, since natural difference between the effects of increased AMPK phosphorylation in skeletal muscle and increased AMPK phosphorylation in the brain. While the former triggers all the fat-loss friendly adaptations you read about in this, as well as in previous Intermittent Thoughts, the later will have you forage through your fridge in no time - regardless of whether it's feeding time or not ;-)

Illustration 2: The differential role of hypothalamic, liver and skeletal muscle AMPK expression (Long. 2006)

You will certainly remember from previous installments that, in our eukarocyte ancestors, AMPK was the major cellular energy sensing mechanism and has been preserved by humans (and all other mammals) even in these days of nutritional abundance. AMPKs activation is thus a signal for your body that the respective cell is starving (to be precise that the intracellular ratio of ADP+AMP to ATP is rising) - if your brain cells are not already starving, it should therefore be obvious that, contrary to AMPK signalling from the muscle tissue, which usually means "Hey, if you want me to do some work for you, you better provide me with some energy! I see those lazy adipocytes over there have more than enough stored energy...", AMPK signaling from the brain indicates an acute emergency - after all, the biological equivalent of "women and children first" is "brain first! Who cares about the rest?" It should thus not surprise you that

• an increase in cerebral AMPK phosphorylation results in increased food intake (Andersson. 2004), while dephosphorylation decreases food intake (Kim. 2004); 
• genetically modified mice with no AMPK activity in the AgRP neurons were leaner and had an increased energy expenditure compared to wild-type mice (Claret. 2007)
• ghrelin, the hunger hormone, triggers AMPK phosphorylation in the brain and thus increases food intake (Kola. 2008)

With all that being said, you are probably happy to hear that alpha lipoic acid decreases or at least counters potential increases in AMPK activity in the hypothalamus (Kim. 2004).

Update (10/29/2011) - The two AMPK-isoforms: As Mounier et al. report in a very recent paper the two isoforms of AMPK, i.e. AMPKα1 and AMPKα2 have very distinct effects on the mTOR induced increase in muscle protein synthesis, and thusly, muscle size (Mounier. 2011). As the scientists point out, AMPKα1 plays "a predominant role in the control of muscle cell size" (meaning it prevents exuberant hypertrophy), while AMPKα2 mediates "muscle metabolic adaptation" (increased glucose uptake, mitochondrial biogenesis, etc.), of which we have learned that they are so vital for our metabolic health.

Close your eyes and lean out

On that note, I will close today's lesson with an some interesting information from a study Jonathan P. has brought back onto my radar, recently. It's a study by Dworak et al. on ATP changes during sleep (Dworak. 2010), which underlines the importance of sleep, specifically when you want to lose weight, because at the onset of sleep, the reduction in neuronal activity goes hand in hand with a surge in brain ATP levels, which (as you have learned today) will reduce cerebral AMPK phosphorylation and its negative metabolic consequences. Prolonged waking, on the other hand, has been shown to increase AMPK activity in the brain (Wigren. 2009). So, what are you waiting for? That's all for today... lights out ;-)!