Is There a Silky Way to a Leaner, Healthier You? BPI Sports Introduces the First Silk Protein Supplement to the Market

Image 1: I wonder whether the silk worms, themselves, aren't a "highly anabolic" source of dietary protein as well... for the time being, BPI (and I assume a handful of soon to follow copy cats) rely on their produce, though.

If you have been following the protein supplement market over the last couple of years, you will certainly have noticed that, flavoring and packaging aside, most products are as alike as two peas in a pod... a pros pos, peas. About 2 years ago, LG Sciences tried to establish a pea-protein concentrate as a "fat-burning" (that was what LG said ;-) alternative to Whey and failed equally miserably as their competition whose initially omnipresent advertisements for their "revolutionary" beef protein product have disappeared from most of the pertinent websites as of late. Now, January 2012, it appears as if BPI Sports, who have hitherto not exactly been known for their "innovative" products, are about to launch another assault against the ever-increasing predominance of milk proteins: BLOX is the name of the breakthrough BPI obviously thinks I have been waiting for... and guess what, they are right!

A silky ergogenic from the domesticated silk moth?!

In fact, my interest in the silk proteins, which are the major (as far as the hitherto available information is concerned, even the "only") ingredient of BPI's new product, dates back to 2010, when Shin et al. published the results of a rodent study, which found that supplementation with 500mg/kg (HED ~40mg/kg) silk amino acids for 42 days increased the maximal (loaded) swimming time in mice by more than +100% while concomittantly increasing the gain in total lean body mass by ~15% over the unsupplemented, yet exercised control (Shin. 2010).

Figure 1: Blood glucose, liver glycogen, muscle glycogen, plasma protein, corticosteroid and testosterone levels in mice after 42days of 30min daily swimming exercise; data expressed relative to sedentary control (data calculated based on Shin. 2010)

In view of the fact that this regimen also reduced the exercise induced depletion of liver and muscle glycogen, and kept the cortisol levels in check, and boosted the total testosterone levels to levels which make me doubt whether those increases were not due to some sort of methodological error (cf. figure 1), I was actually quite surprised that it took so long for someone to find an affordable source (probably in China) for the, in the case of the Shin study, hydrolyzed proteins that are produced by Bombyx mori, the worm of the domesticated silk moth.

Figure 2: Amino acid composition of the silk protein hydrosolate from Worldway Co., Ltd. (Jeoneui, Korea) that was used in the exercise study (data adapted from Shin. 2010)

Now, the interesting thing about these proteins (remember: a protein is a complex that consists of several amino acids) is that despite their nondescript amino acid composition (cf. figure 2) their specific structure within the proteins gives them an unexpected biological activity, of which its anti-diabetic and anti-hyperlipidemic effects have received the greatest interest among researchers (I mean, who would be interested in getting jacked and horny, anyways *rofl*). The underlying mechanisms are yet still not fully understood.

So, how does that stuff work? I mean, it does work, right?

A recently published study by scientists from the Korea University in Seoul does yet shed some light at least onto the not immediately ergogenic effects of silk protein hydrosolates. In an in-vitro experiment with 3T3-L1 fibroblasts (stem cells that would usually form connective tissue), Hyun-Sun Lee, Hyun Jung Lee, Hyung Joo Suh were able to show that the fundamental physiological processes that are responsible for the improvements in glucose and lipid metabolism reported in previous studies (Gao. 2000; Hyun. 2004; Lee. 2007) are in fact attributable to two of our old acquaintances: Increases in GLUT-4, the glucose transporter on the cell surface of eg. muscle tissue, and leptin, the adipokine that tells your body that there is more than enough fat to burn (Lee. 2011).

Figure 3: Glucose uptake in fibroblasts incubated with different amounts of silk protein with and without insulin and insulin and piogliazone controls; data expressed relative to untreated control (data adapted from Lee. 2011)

As the data in figure 3 goes to show, the effect size of the transporter specific (the scientists found no statistically significant increase in GLUT-1 expression) increase in glucose uptake is comparatively small and depends, contrary to the diabetes drug pioglitazone, the silk protein hydrosolate increased GLUT-4 expression and subsequent glucose uptake only in the presence of albeit low amounts insulin.

Figure 4: Relative mRNA expression of selected transcription factors in adipogenesis pathway after incubation of fibroblasts with either 1mg/mL silk protein hydrosolate or 50nM pioglitazone (data adapted from Lee. 2011)

What is interesting, however, is that the silk protein hydrosolate did not only produce a significantly more pronounced increase in leptin expression (175.9% ± 11.1%) that the thiazolidinedione class anti-diabetes drug, pioglitazone, it also left the fatty acid synthase (FAS) activity, which is partly responsible for the obesogenic side-effects of many of pioglitazone untouched.

BPI is right, I have been waiting for this, but I am still waiting for more studies, as well ;-)

Image 2: Even if it turns out to be another supplement non-starter, the BLOX box looks nice, doesn't  it?

These latest results are yet unsuitable to explain the way more exciting findings from the initially discussed study by Shin et al. who invoke the activity of PGC-alpha, the transcription factor that is responsible for the release of irisin, and subsequent increases in the capacity to oxidize fatty acids as a possible, yet hitherto not established mechanism behind the perseverance of muscle and liver glycogen. The non-existent increase in PPAR-gamma activity in the Lee study would however go against this hypothesis, if... well, if we even knew if the composition of the silk proteins the two groups used were similar, let alone identical and, of course, whether they survive the passage through the human digestive tract and exhibit similarly beneficial effects, when they hit your bloodstream... after all, I may assume you are neither a rat, nor a mouse, right?

So, even if the currently available research does suggest that "silk proteins" hold some promise, these products are fundamentally different from your usual amino acid supplements, because their biological activity is a function of both their amino acid composition, as well as the structural arrangement of those aminos within individual proteins and peptides. The source, the way the product is processed and God knows what else will thusly decide whether BPI's BLOX will become a valid addition (I am sure it won't replace whey proteins) to the established supplemental protein sources, or whether it will end up in one of those niches of the supplement stores right next to pea and other exotic protein sources.

Intermittent Thoughts: Dihydrotestosterone (DHT) - Bigger, Stronger, Faster or just Balder, Fatter and Unhealthier?

Image 1: The ancient Greek ideal of the male body has probably more to do with DHT than the freaky physiques of today's IFBB Pro bodybuilders.

I guess after the revelations about the importance of estrogen in the process of skeletal muscle hypertrophy in the last installment of the Intermittent Thoughts you will probably be eager to hear what its male counterpart dihydrotestosterone (DHT), is able to do... I mean, with DHT being the male hormone par excellence it is only reasonable to assume that its effects on skeletal muscle mass and strength, two characteristic features of the male persuasion, must be significant, right? Before we are going to address this vitally important questions, let's briefly take a look at what the dihydrotestosterone actually is.

DHT the hormone to which testosterone is just another prohormone

Similar to estrogen, DHT (exact name 17β-hydroxy-5α-androstan-3-one) is a testosterone metabolite. The process by which your body (male and female, btw.) generates this powerful androgen, the receptor-affinity of which is about 3x-10x higher than that of testosterone (depending on which source you cite and which assay the researchers used; for more detailed data on receptor binding, check out my previous blogpost "Beyond Vida's Book") is called 5-alpha reductase (5-ar). In the course of the "reduction" process one of hitherto three identified mammalian isoforms of the 5-alpha reductase enzyme (3-oxo-steroid-4-ene dehydrogenase). Of these three isoforms, which catalyze the reduction process, type III (predominantly) and type I (to a lesser extend) are expressed in human skeletal muscle (cf. Yarrow. 2011)...

Illustration 1: (1) Testosterone (either preformed or locally formed from DHEA) arrives at the target tissue, (2) is reduced to DHT by one out of three locally expressed reductase enzymes and (3) either acts intracrine, i.e. right inside the cell, where it was formed or is released into circulation.

I do not want to lose myself in too many details at this point, but a rough grasp of the local reduction of testosterone and the subsequent intracrine (meaning right where the hormone is created, cf. illustration 1) effects of DHT is of paramount importance to understand some of the initially counter-intuitive effects of DHT, you are going to read about in the following paragraphs.

"DHT makes you strong bro!" - correct!

At least for those of you who have been on some of the bulletin boards, where people discuss the effects of various androgenic compounds, the first statements that pop into your mind, when you hear the three letters D, H and T, could be "brutal strength gains", "hit new personal records on each lift" or "doubled my bench within 2 weeks". And although I suppose that statements such as the latter lack any empirical basis, the broscientitific evidence that DHT and DHT-like designer steroids exert profound effects on muscle strength cannot be denied.

In this context, the results of a 2010 study from the Biomedical and Clinical Sciences Research Institute at the School of Medicine, Health Policy and Practice of the University of East Anglia in Norwich, UK, is of particular significance (Hamdi. 2010). Using isolated extensor digitorum longus (EDL, a mainly fast twitch muscle in adult mice) and extensor digitorum longus (EDL, a mainly fast twitch muscle in adult mice) muscles from male and female mice, M.M. Hamdi and G. Mutungi established that the strength promoting effects of DHT are mediated mainly via the ERK, i.e. the extracellular signal-regulating kinase (also known as MAPK), pathway and thusly in a non-androgen receptor mediated way.

Figure 1: Maximal isometric force production in slow an fast twitch fibers after incubation with 630pg/ml DHT; data expressed relative to initial isometric force production P0 (data calculated based on Hamdi. 2010)

As the data in figure 1 goes to show, incubation of isolated rat myofibers with 630pg/ml androstanolone (17β-hydroxy-5α-androstan-3-one, DHT) increased the isometric force (P0 = 100%) of the fast twitch muscle fibers in the EDL from both male and female mice by ~30%. If we take a look at the SuppVersity's Motto  "Where Bro- and Pro-Science Meet in the Spirit of True Wisdom", this is thusly one of the (as of late rare) occasions, where bro- and pro-science actually "meet", not "clash", in the "Spirit of True Wisdom".

DHT works via the MAPK pathway and not via the androgen receptor

Without the "pro"-aspect of science we would yet not know that it this is neither a androgen receptor mediated action (as the use of a DHT-inhibitor did not block the effects) nor a downstream effect of IGF-1 (the inhibition of which by co-incubation with an IGR-R inhibitor left the effects similarly unchanged), but a direct effect of the DHT induced increase in ERK-1/2 phosphorylation and the subsequent accumulation of myosin light chain in the DHT treated rodent muscle:

Our hypothesis is that DHT activates the epidermal growth factor receptor (EGFR), either directly or indirectly, and this leads to an increase in the phosphorylation of ERK1/2. The activated ERK1/2 then phosphorylates MLCK which in turn phosphorylates the 20 kDa RMLCs and this increases force production in fast twitch fibres but decreases it in slow twitch fibres. (Hamdi. 2010)

In that, it is not really important that you understand all the intermediate steps which eventually lead to the increase in force production. What is important though is the hypothesis that the changes, you are seeing in figure 2 are not mediated via the androgen receptor, which were equally distributed in both the slow- and fast-twitch fibers in the study at hand - this is particularly noteworthy, because after all DHT is the androgen per se.

Figure 2: Changes (a.u.) in phosphorylated ERK-1/2 and myosin light chain content of slow twitch and fast twitch muscle fiber treated with either DHT or testosterone propionate; * p < 0.05 (data calculated based on Hamdi. 2010)

The experiments also revealed that, despite the increase in p-ERK-1/2 in the slow-twitch muscle fibers, testosterone treatment did not induce similar changes in myosin light chain content like DHT. In view of the fact that the scientists have used female DGL and soleus muscle fibers for this experiment to minimize the local reduction of DHT to testosterone and isolate the effects of DHT, it should also be stated that the last-mentioned effects on slow-twitch fiber ERK-1/2 phosphorylation may well be a downstream effect of the aromatization of testosterone to estrogen (cf. "Estrogen: Friend or Foe of Skeletal Muscle Hypertrophy").

Against that background it is actually quite astonishing that a series of rodent studies which were conducted by scientists from Japan (Aizawa. 2010; 2011) found statistically significant increases in intra-muscular DHT in response to an endurance type of exercise. If you add to that the results of a 2008 human study by Hawkins et al. (Hawkins. 2008), which found a similar increase in systemic DHT (and SHBG) levels in 102 sedentary men (ages 40-75 yr) who were randomly assigned to a 12-month aerobic exercise intervention, while DHT levels did not budge in a 2008 study by Vingren et al. (Vingren. 2008), which used a resistance training protocol, this raises the question whether our current understanding of the strength promoting intracrine effects of DHT is not only part of a larger picture, which would be characterized by distinct intra-, auto-, para- and endocrine effects of DHT on skeletal muscle and other exercise related physiological functions.

The litmus test: Does DHT "build muscle"?

The absence of increased levels of DHT in response to strength training as well as the fact that the increase in myosin light chain is at best "facilitative" to building bigger already suggest that, with respect to its "muscle-building effect", your most potent androgen is somewhat of a non-starter... let me give you a three of the rare examples, where scientists even dared to administer DHT to their study participants, to substantiate (not prove) this hypothesis:

• in 1992, Marin et al. found that 3 months of transdermal DHT administration to middle-aged obese men increased muscle strength and diameter of type II muscle fibers, albeit to a lesser extent than
  testosterone administration (Marin. 1992);
   
• in a 3 month trial using transdermal DHT Ly et al  found a reduction in body fat mass and improved isokinetic knee flexion strength of the dominant leg, but no improvements in lean body mass, knee extension strength, or shoulder flexion/extension strength in hypogonadal elderly men (Ly. 2001);
   
• in 2010, Idan et al. conducted a trial on the effects of DHT administration on prostate growth in 114 healthy men over 50 and found neither beneficial nor negative effects on prostate growth (please understand that I will not address the prostate issue in detail, as it is not directly related to the topic at hand and would require a whole installment of its own) and a very modest increase in lean mass (2.4%) in response to 70mg DHT gel for 2 years (!)

Now, if you take a look at these examples and compare that to what you know about the muscle-building effects of testosterone, it should be obvious why most "chemical athletes" (i.e. steroid users) take 5-ar inhibitors like finasteride when they are "on" high doses of testosterone. Since the latter will reduce the circulating levels of DHT by "only" 50% this practice allows them to keep any unwanted DHT-related androgenic side effects (which are going to occur when you reach supra-physiological DHT levels) at bay, while still having enough 5-alpha reductase activity to benefit from the highly appreciated effects on muscle strength.

Note: Contrary to finasteride, which is highly selective for the type II isoform of the 5-ar enzyme, dustasteride, which has been found to reduce circulating DHT levels by >90% is a pan-5-ar inhibitor. It is thusly no wonder that 0.5mg/day of dustasteride prevented the increase in lean mass in female-to-male transsexuals who were treated with 1,000mg testosterone-undeconate for 54 weeks (Meriggiola. 2008).

Although testosterone and not DHT appears to be the major hormonal driving force of actual increases in muscular size (not strength!), the results of the Meriggiola study, where the total (>90%) blockade of all three of the 5-ar isoforms by dustasteride (see red box, above) inhibited the muscle-building effects of 1,000mg testosterone-undeconate clearly suggest that the reduction of at least small amounts of testosterone to dihydrotestosterone is a necessary prerequisite for the testosterone-induced increases in lean muscle mass. Whether a critical threshold as for circulating DHT levels exists, or whether it was the dustasteride induced blockade of the local reduction of testosterone to DHT by 5-ar type III right in the skeletal muscle that was responsible for this effect will yet have to be established in future studies.

High serum DHT = lower chance of alopecia! High local 5-ar = hair loss, though.

Image 2: Is your hair line receding? Could be DHT, but local not systemic! In young men high DHT levels correlate with full hair, in older men the local increase in 5-ar or the and the reduction in SHBG can elevate DHT beyond a healthy threshold.

Now muscle is obviously not the only thing you want... and when it comes to DHT, hair, respectively the loss of the latter, obviously is the first thing that comes to mind. Notwithstanding that it is an established fact that bathing your hair follicles in excess amounts of dihydrotestosterone will eventually kill them, you may be surprised to hear that a 1992 paper by Knussmann et al. (Knussmann. 1992) showed that contrary to common believe the correlation between allopecia and serum DHT levels in the 110 healthy young men in their study is a negative one (r = -.25, p < 0.01). Yet although the same is true for total testosterone (r = -.25, p < 0.01), the correlation between the ratio of free / total testosterone (T_free/T_total) is positive and statistical significant (r = .02; p < 0.05)!

Now, how can that be? Is it testosterone that is "shaving your head from within?" - well, in a way it is, but most probably due to its local conversion to DHT (I hope by now you understand, why I stressed this factor in the introduction). Contrary to bound testosterone, which cannot be reduced by the 5-ar reductase enzymes in your scalp, the free testosterone can and will thusly - as a prohormone - do its bit to the thinning of your hairline:

[...] DHT in the hair follicle is thought to lead to hypoplasia of the scalp follicle, and a higher formation of testosterone metabolites was observed in the scalp of bald men as compared to hair obtained from nonbalding men. Yet we found a relationship, not between the disposition to balding and the ratio DHT/T, but between the diposition to balding and T_free/T_total. An elevated rate of dissociation from the binding globulin fits in well with the findings of Cipriani et al. (1983) that men with androgenic alopecia exhibit a significant reduction in sex hormone binding globulin (the same is true for bald-headed women). (Knussmann. 1992)

The overall increase in both aromatization and 5-a reduction with age, as well as the tissue specific expression of those enzymes thusly explains why your men begin losing their hair, as they get older although their total androgen levels begin to decline. A similar pattern, i.e. decreased SHBG levels and consequently increases in local 5-a reduction are implicated in female androgenic alopecia, as well (De Villez. 1986).

Note: If you want to judge your serum DHT levels by your body hair, the most prudent way to do so would be look at your legs. While the correlation (r = .16) Knussmann et al. found for DHT, alone, was not statistically significant, it was still the best indicator for "high" DHT levels.

Now, if we assume you have full hair and your legs have some resemblance to those of a bear (an unrealiable indicative of "high" DHT levels), does that predispose you to an increase in visceral body fat, as some sources on the Internet would have it? I mean, designer steroids that are structurally related to DHT are not particularly known for their obesogenic effect. They rather seem help their (ab-)users to lean out pretty rapidly, so the last question I will address in this installment of the Intermittent Thoughts will be ...

If testosterone helps you to lean out, will DHT make you fat?

To answer that I want to go back to the study, I presented in Friday's SuppVersity post on how eccentric training is able to recruit mesenchymal stem cells for muscular repair / hypertrophy. From either this post or the discussion of the underlying mechanisms by which testosterone works its muscle building, fat burning magic (cf. "Understanding the Big T"), you should remember that those pluripotent stem cells are unfortunately capable of becoming fat cells, as well. Luckily, dihydrotestosterone, the "big brother" of the "big T" shares testosterones anti-differential effect on pre-adipocytes (Singh. 2003).
Unfortunately, though, DHT does not prevent their proliferation (i.e. the generation of new pre-adipocytes; cf. Gupta. 2008). Instead, gene assays suggest that it stimulates all aspects of adipocyte metabolism, i.e. the beneficial ones like glycolosis (helps blood sugar management) and lipolysis (helps getting the fat out of the adipose tissue) and not (generally) beneficial ones as the production of fatty acids and triacylglyceroles, cell proliferation and differentiation (Bolduc. 2004).

Whether there is an overall negative effect of "normal" DHT levels on visceral fat, as it is sometimes suggested (esp. in the "lay press" = Internet ;-) appears however questionable. After all, Vandenput et al. (Vandenput. 2007) have shown that not DHT, but rather androstane-3 α,17-β-diol-17-glucuronide (17G), one of its metabolites correlates with visceral adiposity in healthy young men (r = 0.16; p < 0.05).

Figure 3: Correlation of the bioactive androgens (total and free testosterone and DHT) with DXA-measurements of body fat in different compartments; data obtained from n = 1068 young men (data adapted from Vandenput. 2007)

Serum DHT levels, on the other hand, showed the strongest negative correlation with total body fat, total body fat (% total mass), arm fat, leg fat and trunk fat of all three measured androgens (cf. figure 3) and was a close second to total testosterone as far as its negative, i.e. diminishing, effects on central fat distribution is concerned (r = -0.07; p <0.05).

Note: In view of the fact that, as of late, leptin has become a focus of attention even for the average person trying to lose weight, it might be of interest that there were statistically significant negative correlations (r = -0.23 and r = -0.25; young vs. elderly) in both study groups.

Interestingly, things look somewhat different for the 1001 elderly study participants. The pattern that emerges here should remind you of the previously discussed allopecia issue. While there are still negative correlations for the total and relative amount of body fat in all compartments for serum DHT, there is a statistically yet not significant positive correlation between free testosterone and the central fat distribution in the elderly (mean age 75y) subjects that was not present in their young (mean age 19) counterparts. Moreover, the overall correlation between 17G and central obesity and the 17G/DHT ratio and central obesity raises from 0.08 and 0.20 (p < 0.05) in young men to 0.14 and 0.34 (p < 0.05) in elderly men.

Lean, mean, strong... are these "all things male"?

If we discard the important role of DHT in the brain, which would explain the "mean" (not necessarily defined as mean in aggressive, but rather as "alpha-male mean") in "lean, mean, strong" and expand "strong" to the established bone-building effects of DHT, which apparently surpass those of testosterone (eg. Capur. 1989), being as muscular as Mr. Olympia obviously is not one of the "things male". As, contrary to some of its synthetic cousins, the current research suggests that the original father of all androgens may be an indispensable bystander, when its precursor testosterone is blowing up your muscles, its immediate effects do yet appear to be restricted to strength and body composition.

Collectively, this as well as the previous installments on testosterone (Part 1, Part 2, Part 3) and estrogen should have made it quite clear that even the ostensibly straight forward role of the sex steroids in the concert of skeletal muscle hypertophy is way more complex than the commonly accepted notion that "you just inject your weekly test and become Mr. O" would suggest. It is in fact so complex that I will devote the next installment of the Thoughts to revamp the main ideas and to try to connect the dots between mTOR, myostatin, IGF, inflammation, testosterone, estrogen, DHT and co...

If a High Fat Diet was a Pill, the Lay Press Would Celebrate it as "Exercise in a Pill": Two days on High Fat Diet Increase the Activity of Irisin Releasing Transcription Factor PGC1-α

Image 1: If a high fat diet was a patentable pill, I guess BigPharma would already be selling it.

It's crazy, whenever I go to the groceries, there are new products popping up with colorful slogans like "fat free", "diet", "particularly low in fat", "fat reduced", "only 0.1%" etc. What is yet all the more disturbing is that these products are "placed" right next to a more recent "invention" of the industry - products with a "reduced sugar content"... I could now go on a rampage on how the Kellog's and co. just replace table sugar with other sugars in order to be able to put these stickers on their products, but I actually want to make a different point today. If you stand still for a moment and watch the people pass by, there are two things you will notice.

1. The healthy and lean (and not anorexic) will pass by without even noticing the great "0.00001% fat, reduced sugar whatever" with the Weight Watchers sticker on it.
2. The hungry-looking slightly overweight to morbidly obese people will pick up the aforementioned "0.00001% fat, reduced sugar whatever" with the Weight Watchers sticker on it, look at the price-tag, sigh and put three packets of that stuff into their cart.

Now, if we, for the time being, disregard that these products are full of chemicals and hidden sugars and just focus on the notion that eating a product that is both low in carbs and low in fats will help you lose weight we have identified what, at least in my humble opinion, is the #1 reason that many self-perceived "low-carbers" stop losing weight after they dropped the initial few pounds of water: A low carb, low fat diet that - irrespective of their protein intake - is telling their bodies that they are in the midst of a famine, where you better hold on to your fat and burn the wasteful muscle tissue so that you can survive for another few weeks...

Please understand: Low carb = high fat! And high fat burns, not stores fat.

I guess, I better stop preaching now and get to the nitty gritty of the latest study on the effects of a high fat vs. low fat diet on short-term (2-days) metabolic adaptations in lean (BMI 19-25kg/m²) and obese (BMI 30-40kg/m²) human subjects.

Figure 1: Relative energy content of the low and high fat diets the lean and obese study participants were fed (data adapted from Bergouignon. 2012)

As you can see from the data in figure 1, the scientists designed the study in a way that facilitates a direct comparison of high fat (50%) vs. low fat (20%). This is different from high carb vs. low carb, because the high fat diet (35% carbs vs. 65% in the low fat diet) is by no means so "low" in carbs as the average low-carber would have it. That the metabolic adaptations were still pretty profound - after only two days - is something, I personally would not have expected.

Figure 2: Total 24h kcal expenditure (left axis) and protein, carbohydrate and fat oxidation (right axes) in lean and obese subjects on the low and high fat diets (data adapted from Bergouignon. 2012)

Both, lean and obese subjects showed almost identical responses with regard to their 24h kcal expenditure and substrate utilization (cf. figure 1). Most prominent among these was the profound increases in 24h fat oxidation (+88% in the lean and +109% in the obese subjects) in the high fat groups that was yet accompanied by a -26% (lean) and -37% (obese) reduction in carbohydrate oxidation. The overall caloric expenditure was thusly "the same" - unless you still believe that 50kcal per day would make a difference ;-)

Figure 3: Free fatty acid and triglyceride levels (24h AUC in mmol/dl and mg/dl, right axis) and free fatty acid to triglyceride ratio (left axis; data calculated based on Bergouignon. 2012)

If we now take a look at the blood lipids (cf. figure 2), i.e. the free fatty acid (FFA) and triglyceride levels, on the different diets, we see a "phenomenon" low-carbers know all to well. After only a few days on a low-carb diet, triglyceride levels start to plumed. The amount of free fatty acids that are released into the circulation from the liver and the adipose tissue, on the other hand, rises. This time with a significantly greater FFA response in the obese individuals (p=0.04), which does support the hypothesis that a significant amount of the FFAs came from the well-stocked adipose tissue of the obese study participants.

Figure 3: Total mRNA expression in vastus lateralis muscle of study participants expressed relative to the subjects in the lean low fat group; please note the discontinuity of the axis between 200% and 600%, which became necessary to fit the data into the graph (data calculated based on  Bergouignon. 2012)

The most intriguing changes this two-day high fat "challenge" induced took yet place on a transcriptional level in the skeletal muscle tissue of the subjects. However, of the changes in total mRNA expression, only the increase in CD36, a protein which regulates the fatty acid uptake of skeletal muscle, and pyruvate dehydrogenase kinase isozyme 4 (PDK4), which reduced the oxidation of glucose, reached statistical significance.

Is a high fat diet exercise in a pill?  ...

If we discard the statistical shenanigan for a moment, you may yet notice the "non-significant" +33% increase in Sirtuin 1 (Sirt 1) expression in both high fat diet groups. The intelligent and well-read SuppVersity student you are, you will also remember that this protein, which is downregulated in states of insulin resistance, has been identified as one of the key players in the anti-cancer effects of compounds such as resveratrol and the longevity effects that have been ascribed to low-calorie diets. And as if that was not enough, Sirt 1 also plays a key role in the beneficial adaptations that come with exercise, where it is part of a triumvirate of AMPK + SIRT1 + PGC1-alpha,  which is induces the production of irisin the purported "exercise hormone" that is supposed to "brown your adipose tissue" (meaning to convert the sluggish white into the metabolically more active brown fat) has gotten quite some attention in the lay press as of late (eg. LA Times).

Figure 4: Skeletal muscle mRNA expression (in au) of acetylated (=inactive) PGC1-alpha and phosphorylated (=active) AMPK in lean and obese subjects subjects on low and high fat diets (data adapted from Bergouignon. 2012)

So, if we fell for the, in my book, stupid idea that a single cellular messenger, in this case irisin, would work like "exercise in a pill" (think about it - how often have you heard that in the last years?) and acknowledge that an increase in PGC1-alpha activity in skeletal muscle will increase the release of this myokine (i.e. a messenger protein that is released from the muscle; compare "adipokine" like leptin, which is a messenger protein that is released from adipose tissue), the logical conclusion would be that a high fat diet works like "exercise in a pill". Why? Because the data in figure 4 shows that the active, i.e. de-acetylated amount of PGC1-alpha (which is obviously the exact opposite of what the scientists measured) in the muscle of the subjects who consumed the high fat diet increased (p < 0.05) - ah,... and did I mention that this is human data, while the irisin craze is based solely on rodent studies?

... it sure looks like that!

In combination with the increase in phosphorylated AMPK (p-AMPK, click here to learn more about the "mTOR/AMPK Seesaw"), which was likewise statistically significant, the short-term adaptation to high fat feeding in lean and obese human beings shares two of the main characteristics of the "healthy endurance training" the same Dr's who warn their patients about the dangers of a high fat diet usually prescribe to their patients. Now tell me, is the voice in your stomach still telling you that low-carb may be good, but that eating a high fat diet is not an option? Well, in that case I guess you are beyond help... go buy some low-fat sugar reduced cornflakes ;-)

Beyond Satellite Cells: Eccentric Training Bullies Stem Cells, Destined to Become Bone, Cartilage, Adipose or Nerve Tissue into Contributing to Skeletal Muscle Growth

Image 1: Apart from their epistemic value, these stained tissue samples from the Valero study are actually pretty aesthetic.The arrows, by the way, mark NG2 monocytes, which "coerce" the non-myogenic stem cells (perycites) to get going ;-)

I guess, the term "satellite cells" has been mentioned so often in the course of the past couple of weeks that it actually would not be necessary to tell you (once again) that these myogenic precursor cells are a necessary prerequisite for the repair and long-term growth of your pecs, legs, back, biceps, triceps, delts and the rest of the skeletal muscle tissue in your body. Those of you, who have read all the latest installments of the Intermittent Thougths will also be familiar with the notion that both the local expression of MGF-1 and inflammatory cytokines, as well as systemic hormones, such as testosterone and estrogen play an important role not only in the recruitement and migration of satellite cells into the muscle, but also in their regeneration and maintenance (cf. "Are You Serming Away Your Growth Potential"). A group of scientists from the University of Illinois must however have figured that this is not yet complicated enough and began digging even deeper into the (re-)generation of (new) muscle tissue... and let me tell you, what they found is intriguing.

Eccentric training is "numero uno" for satellite cell recruitment

I guess you will be aware that it is a relatively well-established fact that of all real-world physical activities eccentric, or lengthening, contractions of skeletal muscle appears to constitute the most potent inducer of both "productive" exercise induced muscle damage, as well as subsequent increases in satellite cell recruitment.

Figure 1: Changes in intramuscular architecture in young and old subjects in response to 6 sets of eccentric leg extensions (data calculated based on Dreyer. 2006)

Back in 2006 Dreyer et al. published the results of a study which compared the satellite cell response to an eccentric exercise regimen (1x12 + 5x16 eccentric-only reps on leg-extension machine) in young and old subjects (Dreyer. 2006). As the data in figure 1 goes to show, this unquestionably exhaustive bout of exercise produced a quite remarkable (>150%) increase in satellite cell volume per muscle fiber and a likewise highly significant (>100%) increase in the number of satellite cells compared to the total number of cells in the sublaminar compartment. It is also evident from the data in figure 1 that the preparatory accumulation of satellite cells 24h after the eccentric exercise-bout was profoundly blunted in the older subjects.

Figure 2: Satellite cell count per myonucleus before and 8d after 100reps of eccentric leg extensions in untrained young subjects (data adapted from Mikkelson. 2009)

Note: As you can see in figure 2, age is yet not the only factor that can compromise the adaptive response to eccentric lengthening contraction of skeletal muscle. Mikkelson et al. who had the (young) participants of their study perform 100 eccentric reps on a similar leg-extension machine (Mikkelson. 2009), for example, found that an indomethacin (NSAID; COX-inhibitor) infusion for 7.5 h during the exercise day did not only blunt the increase in pax-7+/myonuclei, or in "layman's terms", satellite cells 8 days after the exercise session, the combination of exercise and NSAID actually led to a slight and statistically obviously non-significant reduction in satellite cells per myonucleus. This does support the findings, I discussed in one of the previous installments of the Intermittent Thoughts, which dealt with the importance of "inflammation" as a vital constituent of both the repair and hypertrophy response to exercise induced muscle-damage (cf. "IGF-1, IL-15 & Co").

The age-related decline in satellite cell activity unquestionably raises a question Lerner addressed in one of his insightful comments on my post on the important role of estrogen for the maintenance of the satellite cell pool, which is whether or not the latter would be limited... I mean, when the whole craze about stem-cell therapy began, the general accepted notion appeared to be that the average adult human being has a very limited / if any of those "super cells".

If you have been following recent publications, you will yet be aware that as of late researchers (interestingly also from the University, I work at ;-) have made quite some progress in "producing" and "reprogramming" stem cells from all sorts of human and animal tissue. Assuming that you have also read everything about how testosterone works its "muscle building and fat burning magic" (cf. "Understanding the Big T"), you should also be aware that the latter, i.e. the "fat burning" effect is at least partly mediated by the reprogramming of stem cells which are actually supposed to become fat cells into "satellite cells". All that being said, it should actually not surprise you that the main finding of the initially mentioned study is that eccentric contractions of skeletal muscle have a very similar effect on "non-myogenic" stem cells, which "happen to be in the vicinity of the exercised muscle fibers".

A study on You, Wolverine and the Ultimate Hard Gainer

Unfortunately, the design of the study by M. Carmen Valero and her colleagues from the Department of Kinesiology and Community Health at the University of Illinois is... well, let's say not exactly straight-forward, or easy to understand (Valero. 2012). Basically, the scientists took 3 types of muscle fibers from rodents,

Figure 3: Localization of stem cell antigen-1 (Sca-1) (arrows, TRITC-red) positive mononuclear cells and a-7 integrin (FITC-green) in the different muscle tissue before (SED) or 24 hr post-exercise (Ex) at 20x magnification (adapted from Valero. 2012)

• wild type, as a normal control (that would be your muscle ;-)
   
• alpha-7 integrin transgenic (a7TG), which is resistant to injury, but still responsive to strain (I guess that is either the "Wolverine" or "Unbreakable" type of muscle tissue ;-), and
   
• alpha-7 (-/-), which is the "ultimate hard gainer muscle" that does not respond with alpha-7 integrin expression to overload

Now, even if you don't understand a word of what I am talking about here, I guess that you will be able to see the significant color-differences in the immuno-stained (=marked by antibodies) tissue samples in figure 3. If you just focus on the arrows, the red and green areas, that should suffice to grasp the idea that the both the stretch induced alpha-7 integrin response, as well as the number (arrows) and area (red staining) of stem cells in are maximal in the Wolverines, "normal" in guys and gals like you and minimal to non-existant in the "ultimate hard gainer".

The first take-home message of this study is thusly that the strain that is induced by eccentric training activates "dormant" stem cells via alpha-7 integrin. Why this is the case, becomes clear when you look at the structure of those heterodimers, which transverse the cell membranes of regular muscles and adhere the extracellular matrix to the cytoskeletal network. If you strain the muscle, this will obviously affect the integrin system, which thusly functions as a "sensor" for mechanical signals.

"No satellite cell available? Well I guess we just take this one, then..."

Upon closer analysis of the "satellite cells" that actually responded to the alpha-7 integrin signals, the scientists did yet realize that the resource from which the majority of the new muscle cells were recruited were actually mesenchymal stem cells, which are usually destined to become osteoblasts (bone), chrondocytes (cartilage) and adipocytes (fat cells):

In this study, we provide the first demonstration that muscle resident mesenchymal-like stem cells (mMSCs), predominantly pericytes, are increased in muscle in an a-7 integrin dependent
manner following an acute bout of eccentric exercise. mMSCs maximally appeared in a7BX2 transgenic muscle resistant to injury following eccentric exercise and were rarely present in
muscle lacking the a-7 integrin, suggesting that factors other than injury or inflammation are primary regulators of mMSC accumulation in skeletal muscle.

In view of the fact that a preliminary experiment in which the scientists transplanted exogenous mMSCs into the muscle of living mice, led to a very localized, but distinctly measurable increase in new fibers opens a whole new venue for research into the artificial enhancement of skeletal muscle hypertrophy... for you as an avid (and probably "natural") trainee, however, the main take home message is that exercise, in this case, the strain (not the damage!) that is induced by eccentric lengthening contractions of your muscles still appears to be the major determinant of all aspects of skeletal muscle hypertrophy. Don't forget that, when you are about to invest the next few hundred bucks into whatever "myogenic" supplement the guy at GNC is trying to persuade you into buying... without hard and consistent work at the gym those powders and caps are pretty useless.

Adelfo Cerame - Road to The Wheelchair Nationals '12: Bring Your Cucumbers to Vegas! Or, How Intermittent Fasting Can Make a Road-Trip Bodybuilding Compatible

Image 1: When a bodybuilder heads to Vegas, its either for the Arnold or because he knows the difference between passion and obsession.

I don't know what to say, but somehow I think I am kind of jealous. My friend Adelfo is heading to Vegas and I am sitting here in the rainy cold, reading about how he is preparing not for the contest, but for three wild days in Las Vegas! I mean if it were for the Arnold's... but no, it is just for fun! And you know what? That is exactly what it takes to be successful - making bodybuilding your passion, not your obsession. How that works? Well, I guess I will hand over to Adelfo give you his rundown of the fundamentals, like choosing hard liquor over beer and making sure to be in a drunken stupor not before the bar, but before your feeding window closes ;-)

Viva Las Vegas! There is no life beyond, but a life with bodybuilding!

Las Vegas! That's the place to be... well, it will be for me over the next three days - belated birthday celebrations ;-) And yes, I remember that I have a show coming up in just a few weeks. Do you, by the way remember, that I told you these days were coming right at the beginning of my prep? You know that I am pretty meticulous, when it comes to planning things ahead and this belated birthday present is part of the plan, so to say. So, yes: I am nine weeks out, and going to be out f@cking around in Las Vegas for 3 days. Why? Simply because I can get away with it. Remember what the rapper Suga Free once said? "If you stay ready, then you ain't gotta get ready!"... exactly that, "staying ready", is what I have been doing since October 1st and this is why I am now able to enjoy life without the slightest touch of OCD.

Recipe of the week: My Leafless Crunchy Salad

I don’t know about you all, but I hate lettuce! It’s not filling at all! And it takes a whole bag just to fill you up! I prefer crunchy cucumbers, squash, zucchinis and fruit in my salad!

Ingredients:

• sliced cucumbers, zucchinis and squash
• a couple sticks of sliced celery
  (slice into small pieces)
• 2 cups of sliced strawberries
• 1-2 sliced kiwis
• ½ cup of blueberries
• ½ cup of cherry tomatoes
• 2-3 tbs. of sliced almonds
• 1 packet of truvia (stevia)
• red wine vinegar
• sea salt & pepper 

Macros: 30-40g of carbs (all from the fruits)/ 5g fat

Tip: Sprinkle sea salt, pepper, truvia, and red wine vinegar to liking. I don’t count macros in veggies, so load up on as much cucumbers, zucchini and celery as you want.

I am - and I hope this does not sound too narcissistic - at a point where I am really confident with my work ethic and the current level of my physique. So confident, in fact, that I can actually enjoy the upcoming days in Vegas without feeling guilty. And let's be honest, even if I made an effort to really f*** things up - how much permanent damage can you possibly do within 3 days?

A modified routine can keep you on track even when you are "behind the enemy lines"

Image 2: A recipe from Martin Berkhan’s LeanGains website - Vanilla Vodka, lime or lemon wedges, cinnamon & brown sugar… light in calories & carbs, and straight to the point after a couple shots!
. 

Will I try to mess things up? I guess, you should know me too well by now to buy that off me, aren't you? Obviously I will still be sticking to my routine: My fast and feeding protocols, my commitment to eat whole nutrient dense foods, and I will still train. To make that work, I will have to apply a few tweaks to my usual routine. First and foremost, I will have to reschedule the feeding and fasting windows, so that they allow me to stay up late and consume the one or other alcoholic beverage... and yes, practically, this means that I will be drinking alcohol only during my feeding hour. With regard to the type of beverages I will be selecting, it should be obvious that beer, with its tons of carbs and useless calories is a complete no-go.

I'll stick to the real man's choice: hard liquor ;-) 

 For a lightweight like me this has another advantage. My tolerance for alcohol is so low nowadays that all I would need is 5-6 shots… maybe 10, and I’m feeling a good mellow buzz for the night.

Traveling is no excuse to let yourself go!

Bodybuilder or traveling salesman, being on the road is not excuse to let yourself go. It may take a little planning and probably ain't as convenient as going to McDonald's but even a bodybuilder can maintain his regular nutritional regimen while traveling or being on vacationing. For me this means that

• Boiled chicken breast
• Cucumbers
• Fruits
• Whey protein
• Creatine monohydrate
• Quest bars (I’ll buy some at my gym after I train this Thursday morning before I hit the road)

are part of the luggage I am going to bring with me to Vegas. Five items... plus the simple rule to make sure to order animal meats and veggies, when I go out to dinner - that is all it takes!

Traveling-Tip: Whenever you are out on a short vacation like mine, make sure one of the days is a re-feed day ;-) I, for my part, am already looking forward to the all-you-can-eat buffets I am going to hit on Sunday...

Diet to live, don't live to diet!

I just realized that the thrill of anticipation has carried me away, somewhat, but I guess you got my main message, anyway. Whatever diet you are on, if it is not flexible enough to incorporate a short vacation weeks before you step on stage, it probably ain't worth sticking to, anyway. I mean, if you start living to diet, things begin to get out of hand... it's all about balance and setting the correct priorities at the right times.

Right now I am, by the way, at 1600 kcal per day, I decided not to take it down to 1400 kcal yet. I will re-evaluate this decision on the 1st of February, but as of now I've been enjoying the perks of increasing my calories, adding starchy carbs back into my PWO meals and having re-feed days every Sunday. And though I was initially afraid of water- or even fat gains, up to now it seems as if there have been nothing but benefits from the increase in calories, the re-inclusion of starchy carbs in my post-workout meals and the frequent re-feeds: My muscles feel fuller, my vascularity has improved, and to me it looks as if I was still getting leaner!? What do you think?

Image 3: December 24, 2011, diet was strictly protein + fat @1400kcal for the two weeks prior (left); January 13, 2012 back at 1600kcal and with 100g of carbs added back into my post-workout meals (right).

I’m still kind of baffled how my vascularity has improved. I guess it’s the organic russet potatoes, I’ve been eating after my workouts along with the SlinShot "nutrient repartitioner" by PurusLabs. I guess, you could also argue, though that the fuller muscles just shine a bit more, now that I am at an overall lower body fat level. But with all the nutritional, and training methods that I’ve been playing around with for the past couple of months it is difficult, ... no, it is impossible to pinpoint the one factor that is responsible for the success and that, as surprising as it may seem, actually is "the one" factor that will eventually make you succeed: Finding the right mix of dietary and training techniques that works for you, person A at timepoint T1. For me that was no-to-low carb at T1, but now that I have achieved a body fat level, where it becomes increasingly harder for my body to run on fat for fuel exclusively, an adequate intake of carbohydrates post-workout that will suffice to replenish my glycogen stores appeared to be exactly what was necessary to avoid stagnation or regress.

Three days in Vegas and still 8 weeks to go!

Image 4: The re-addition of starchy carbs into my Slinshot supplemented post-workout meals made a hell of difference in terms of vascularity.

After my Vegas weekend, I will be hitting my 8-week mark. On Monday, to be precise. And from there on, it is no more planned distractions, no more holidays and no more birthdays to celebrate! It’s just going to be eight solid weeks of proper dieting and intense training. More than enough time to make all the necessary adjustments to get ridiculously shredded without burning myself out. For now, however, I am going to enjoy the little mini vacation... my well-deserved break before I will be bound to the gym and the kitchen for eight full weeks, in the course of which I will try really hard to push to my personal limits with both my training and my nutritional regimen... so I guess we will see if, in those weeks to come, I will still be able to say that "this has been one of the easiest preps by far"... but for now, I will pack my clothes and food for the trip and hope that I am going to see you here, next Thursday, eager to hear, or I should say, read how the road-trip effected  my physique :-)

6mg Melatonin 30min Before HIT Will Increase Fatty Acid Oxidation, Boost Your Antioxidant Capacity, Reduce MDA Levels and Modulate Your Immune Response

Image 1: Other than you may have expected, this is exactly not what happened when the soccer players took 6mg of melatonin before their workouts.

The blogpost on melatonin's anti-Alzheimer's + anti-obesity effects from last week caused quite a stir, both in the comment area, here at the SuppVersity, as well as on facebook. Even friends in the gym came up with questions. Therefore, I suppose that you won't mind, if I re-address the topic today. This time, however, with data from a human study, of which I bet that it will catch your interest... after all, the study, which has been published ahead of print two days before Christmas could hold the key to winning the FIFA World Cup 2014 ;-)

Melatonin makes you sleepy? I don't think so!

I guess, even after last week's news, most of you will still think of melatonin as the "sleep hormone". At least those of you who follow my recommendation to make sure that they get their daily dose of SuppVersity news should yet already be familiar with the notion that melatonin may also be a very effective ergogenic aid / adaptogen to be taken not at night, but right before an event! Contrary to the Ococha study, which, due to its awkward "loading protocol" and the ultra-endurance setting, had little relevance for the average trainee, the recently published study by M.D. Maldanado and his (or her) colleagues (Maldando. 2011) from the Department of Medical Biochemistry and Molecular Biology at the University of Seville Medical School and the Andalusian Centre of Sports Medicine in Spain is approaching an area of physical activity that is much more dear to my heart: Football! Or as you, my American friends would say, "soccer" ;-)

Note: The scientists make a very useful comment with regards to the timing of exogenous melatonin, I guess you will be interested in: "Melatonin itself has a very short half-life in the blood (range of 20–40 min, depending on condition), so that elevated levels cannot persist after many hours. According with concentration-time curve of our laboratory and other researchers, 30-45 min is [sic!] the necessary time so that oral melatonin is absorbed in the gastrointestinal tract (GIT) and can be detected in blood before its metabolism and elimination. On the other hand, considering that melatonin is not toxic and has no undesirable effects, the 6 mg administered assured us their absorption by the mucous and is within the ranges recom- mended for use in humans (3–20 mg)." I hope that will spare me at least a few of the question some of you are probably already harboring ;-)

To study the effects of pre-workout melatonin supplementation on markers of oxidative damage, the Spanish scientists recruited sixteen 18 to 20 year old professional soccer players, and randomized them to receive either placebo or 6mg of melatonin 30 minutes prior to an intense (HR >135bpm; speed 25km/h) 60-min training session on stationary bikes.

Figure 1: Plasma total antioxidant activity (TAS) and lipid oxidation (MDA) before, during and right after 60min of high intensity (heart rate >135bpm) exercise on stationary bike (data adapted from Maldando. 2011)

As you can see in figure 1 the 6mg of melatonin the subjects received immediately before the arduous steady state high intensity "cardio" exercise, did not just ameliorate the -22% (60min mark) decrease in total antioxidant capacity (TAS) in the soccer players, it did in fact raise the TAS levels to +15% over baseline, while the subjects were pedaling on their bikes. Consequently, the increase in lipid oxidation (MDA) levels was profoundly reduced, yet not completely blunted (let alone reversed).

Figure 2: Plasma triglyceride before, during and after 60min HIT training (data adapted from Maldando. 2011)

For those of you who, besides being athletic, also like to look athletic, it may also be of interest that Maldando et al. ascribe the statistically signifant decrease in plasma triglyceride levels in the melatonin group to what they call an increased "catchment and lipids consumption by cells" - and for the native speakers out there - this is the Spanish way of saying that melatonin increases fatty acid oxidation during high intensity steady state exercise and could thusly be highly beneficial for anyone who wants to shed some additional pounds (in addition to what you diet will do for you) on the treadmill, bike or similar "cardio equipment" - and before you ask, I guess this will work for HIIT, as well ;-)

Melatonin increases immune response to 60-min HIT exercise

Its antioxidant and fat-burning activity aside, the 6mg of melatonin eight of the sixteen soccer players consumed before they hopped onto their stationary bikes exerted a statistically significant modulatory effect on plasma IgA levels (ca. +40% after 60min), about which Maldano et al. state that it

[...] indicates the start of a humoral immune response, for which it takes between 5 and 7 days [...so that melatonin] could act to promote the adaptation between plasma and mucosal IgA during the exercise.

Overall, I am becoming more and more convinced that my statement from last week that "melatonin is probably one of the most underrated supplements you can still buy without a script" is actually pretty accurate and that melatonin, as Maldono et al. put it, "could be a plausible therapeutic option for professional athletes that should make major exercises throughout their working lives" ;-)

Biotin Ameliorates Skeletal Muscle Insulin Resistance in Model of Type II Diabetes. Increase in GLUT-4 Expression Yet not Accompanied by Translocation to Cell-Membrane

Image 1: Otsuka Long-Evans Tokushima fatty rats (OLETF, right) have the genetical disposition to become type II diabetics.

Are your nails brittle? Is your hair falling out? No? Then, are you obese or insulin resistant? Yes? ... I guess, by know you are asking yourselves what your hair and nails have to do with your pre-diabetic beer-belly. Well, according to the recently published restults from a study by Yuka Saki and his Japanese colleagues, biotin, the water-soluble nutrient that has been discovered by Bateman in 1916 and is also known as vitamin B7, vitamin H or coenzyme R could well be the missing link. After all, the well-known, but often misunderstood micronutrient plays a central role in both fatty acid, as well as glucose metabolism and is by no means just a "hair & nails" vitamin.

Biotin could help even if "it's in your genes" ;-)

The Japanese researchers used the infamous Otsuka Long-Evans Tokushima fatty rats whose name already implies that their have the unfortunate propensity to gain tons of body fat and develop type II diabetes, even if they are not fed the "high fat" diet (of which at least those of you who are not the first time here at the SuppVersity should know by now that it is high in fat and carbs) to check, whether supplementation could ameliorate the inevitable development of skeletal muscle insulin resistance in these poor critters.

Figure 1: Body weight, fasting glucose and insulin levels at the beginning and end of the 8-week study period (data adapted from Sasaki. 2012)

If you look at the results of the 8 week treatment period in the course of which the 34-week old, already overweight (compared to the non-diabetic LETO group) OLETF rats received either plain water or water with 3.3mg/L biotin (i.e. ~400µg/kg biotin for a rat and 65µg/kg for a human being), the treatment did in fact have the desired effect on glucose and insulin levels (cf. figure 1). The increase in insulin sensitivity, on the other hand, "allowed" the biotin-treated OLETF rats to gain even more weight than their profoundly diabetic peers. This, by the way, is something you will also see in humans, who - just as their omniscient Dr. told them - take their diabetic drugs and / or insulin, but refuse to make the necessary dietary and lifestyle changes.

Figure 2: Glucose uptake, total and membrane GLUT-4 expression in hindlimb muscle of 42-week old OLETF rats with or without biotin treatment (data calculated based on Sasaki. 2012 and expressed relative to non-diabetic LETO control)

What is also interesting is that despite a significant increase in total GLUT-4 (glucose transporter) expression in the skeletal muscle of the OLETF rats, a concomitant increase in receptor translocation to the cell-membrane, of which you have probably read in previous blogposts that it is responsible for the exercise-induced increase in glucose uptake, did not take place (cf. figure 2). Consequently, the glucose uptake in the hindlimb muscle did increase in response to biotin supplementation, but failed to reach the same (100% in figure 2) level as in the non-diabetic control group.

Biotin does not via AMPK... good or bad news?

These observations lead the scientists to conclude that the mechanism that is responsible for the nevertheless beneficial effects of supplemental biotin can not be a direct consequence of AMPK activation, which is at the heart of both exercise, as well as drug (esp. Metformin) induced ameliorations in insulin sensitivity. This is something, I actually find pretty interesting, because a) the elucidation of the exact mechanism by which biotin is able to increase insulin sensitivity and skeletal muscle glucose uptake could lead to novel insights into skeletal muscle glucose metabolism which could also be of relevance for "physical culturists", and b) if biotin, despite being essential for the activation of Acetyl-CoA carboxylase (ACC), a major downstream target of AMPK, does not work by "simply" stimulating the AMPK pathway, it is actually likely that the effects of biotin supplementation and exercise could add up.

Study Identifies Caffeic Acid Induced AMPK-α2 Activity Behind the Fat Burning, Insulin-Sensitizing & Life-Extending Effects of Coffee. Plus: Why Creatine & Coffee Don't Mix!

Image 1: I've got news for you: Real coffee does not come in brown sterophome cups ;-)

Ever since Starbucks came out with the "McDonald's version" of what used to be the drink of the popes and kings, coffee has gotten sort of a bad reputation. It is supposed to "burn out your adrenal glands" (I bet most people who support this hypothesis don't even know where the adrenal glands are situated), derive you of  vital nutrients and water, reduce insulin sensitivity, give you palpitations and high blood pressure, ... I guess, you know the whole litany. If we take a closer look at the contemporary scientific consensus, many of these arguments against your 2-3 cups of coffee per day lack any scientifically verified basis. Others apply only if you (ab-)use the uber-potent high-caffeine, high sugar, low polyphenol Starbucks brew as "rocket fuel" on your mission not to mars, but to your first (or next?) heart attack.

Live longer, live leaner and live diabetes-free with...coffee!

I guess, some of you will probably remember my pre-Christmas blogpost on the "Anti-Diabesity Effects of Coffee", in which I elaborated on the results of a recently published paper by Matsuda et al. who observed significant reductions in weight gain in rodents receiving either diluted coffee or pure caffeine in addition to a fattening high fat (+high carb) diet. Contrary to the body weight gain, which was ameliorated about equally effective by both treatments, the "whole coffee" treatment had a much more pronounced effect on the particularly unhealthy visceral fat in the epididymal area (cf. previous news, figure 2).

Figure 1: Molecular structure of chlorogenic and caffeic acid, two of the major phenolic compounds in coffee beans (adapted from Tsuda. 2012)

Although these results clearly indicate that there is more to coffee than the world's most popular recreational drug, caffeine, they cannot answer the question what exactly this "more" would be. The most likely candidates obviously are the two major plant phenols in coffee beans, caffeic acid and its ester, cholorogenic acid (cf. figure 1). Both, caffeic, as well as cholorogenic acid have already been studied for their antihyperglycemic (=blood glucose lowering) effects in animal models (e.g. Rodriguez de Sotillo. 2002; Bassoli 2008), but the molecular mechanism by which they perform their blood sugar reducing magic had not been fully elucidated until Satoshi Tsuda and his colleagues from the Laboratory of Sports and Exercise Medicine at the Graduate School of Human and Environmental Studies of the University of Kyoto in Japan conducted an experiment which identified the AMPK-pathway about which you have already learned so much, here at the SuppVersity, as one, if not the underlying cause of the beneficial health effects of coffee.

In view of the fact that cacao, just like coffee contains both chlorogenic and caffeic acid (Duke. 2000), it is almost certain that the health benefits which have been ascribed to the consumption of phenol rich dark chocolate within the last couple of years can be traced back to increases in skeletal muscle AMPK-phosphorylation, as well.

To prove their hypothesis that caffeic acid and / or chlorogenic acid act directly on the AMPK-pathway in skeletal muscle,  the scientists incubated isolated rat epitrochlearis muscles with different amounts of the coffee phenols and measured the phosphorylation of AMPKα Thr¹⁷² and ACC Ser⁷⁹

Figure 2: Relative AMPK-phosphorylation in isolated rat epitrochlearis muscles in response to incubation with 0.01, 0.1 and 1mM of chologenic and caffeic acid (left); relative increase in AMPK-phosophorylation after incubation with 1mM of caffeic acid for 5, 15, 30 and 60 min (right; data adapted from Tsuda. 2012)

As you can see, caffeic acid, but not chlorogenic acid lead to dose- and time-dependent increase in skeletal muscle AMPK-phosphorylation (cf. figure 2). A similar response was observedfor its downstream target (data not shown in figure 2), Acetyl-CoA carboxylase (ACC), an enzyme that is directly involved in the regulation of mitochondrial fatty acid oxidation.

Figure 3: Relative increase in AMPK isoform phosophorylation (left) and increase in glucose transport measured with 3O-methyl-glucose as a marker (right) in skeletal muscle of rats after incubation with 1mM of caffeic acid for 30 min ( data adapted from Tsuda. 2012)

And while it does not come as a surprise that the increase in AMPK went hand in hand with the previously observed increase in glucose uptake the underlying mechanisms of which the scientists tried to uncover (cf. figure 3), it is of particular importance for physical culturists and anybody else who is interested to burn fat, while maintaining / building muscle that this effect was mediated by the alpha 2, not the alpha 1 isoform of AMPK. As I hope those of you who have been following my dissertations on AMPK in the Intermittent Thought (Part 1, Part 2, Part 3) will be aware of, the former, i.e. AMPK-alpha-2 is also expressed in response to exercise and does not reduce muscle protein synthesis by compromising the mTOR response (note: due to the isoform-specificity of caffeic could be called a true exercise mimetic).

Figure 4: Relative amounts of ATP and phosphocreatine (PCr) and phosphorylated AKT in skeletal muscle after incubation with 1mM of caffeic acid for 30 min (data adapted from Tsuda. 2012)

That you do not have to be afraid of losing muscle, if you ramp up your skeletal muscle AMPK expression by caffeic acid, is also supported by the absence of any detrimental effects on skeletal muscle protein kinase B (AKT) expression and the adenosin-triphosphate (ATP) levels in the skeletal muscle samples (cf. figure 4). What is intriguing, though, is the statistically significant decrease in the amount of phosphocreatine, a phosphorylated creatine molecule your muscle (and brain) tissue uses as a rapidly mobilizable energy reserve.

Caffeic acid won't decrease protein synthesis, but could reduce the effectiveness of creatine

And while Tsuda et al. mention the detrimental effect caffeic acid exerts on intra-muscular phosphocreatine stores in the discussion of the results, they are not able fully explain this observation which reminds me of the old "myth" that the caffeine in coffee would compromise the beneficial effects of creatine supplementation... I guess we have just found why some studies did in fact support this hypothesis. If the caffeic acid induced increase in AMPK-phosphorylation goes hand in hand with a reduction in the amount of stored creatine phosphate (PCr), this could mean that you would need more creatine to achieve and maintain "maximal" levels of this high-performance energy reserve.

As you can see, it is always the same, with every question we answer a new one arises. What did Socrates say? Yeah: "I know that I know nothing!" I suppose this is a good concluding word for today's blogpost. Come back tomorrow if you want to know what else you do not know ;-)