DHEA Revives Liver of Aged Rats and Improves Antioxidant Reserves and Akt Signaling in Young and Old Rats.

Image 1:  I don't think celebrities realize it, but there is more to anti-aging than an unlined face. New studies show that DHEA could after all help with all sorts of age related diseases (img. antiagingpossible.com)

It's been a while since DHEA was in the news. While I have posted a handfull of mostly beneficial findings related to dehydroepitestosterone (DHEA), the hype that sourrounded its purported anti-aging effect in the late 1990s has completely abated. In view of DHEA's implication  (or rather the lack of the latter) in age-related autoimmune disease, sexual disfunction, osteoporisis, deteroiations of lipid metabolism, type 2 diabetes and cardiovascular and liver disease (Basci. 2007- ignificance of  dehydroepiandrosterone  and  dehydroepiandrosterone  sulfate  in  different  diseases), it is questionable how people beyond the age of 40, when DHEA production declines by 2% per year(!) could not benefit from a carefully planned and monitored DHEA treatment. A group of scientists from Brazil obviously thought the same and decided to take a fresh look at what happens on a molecular level, when 3 (young rats) and 24 months (old rats) old male Wistar-rats are given 10mg/kg deyhdroepitestosterone [human equivalent: 1.62mg/kg; 80kg human: 130mg/day] subcutaneously per day for 5 weeks (Jacob 2011).

Figure 1: Relative changes in total, reduced and oxidized glutathione in young and old rats after 5 weeks on 10mg/kg DHEA (data adapted from Jacob 2011)

As you can see in figure 1, the treatment induced profound increases in total and reduced glutathione and age-dependendly increased (young) or decreased the absolute level of oxidized glutathione (GSSG). Despite the absolute increase in GSSG, usually a marker of oxidative stress, the more important GSH / GSSG ratio, i.e. the ratio of reduced to oxidized glutathion, a more comprehensive marker of the balance of pro- vs. anti-oxidant metabolic processes, improved even more in the twelve young rats, (+6% GSH/GSSG) than in their older companions (+1% GSH/GSSG).

Figure 2: p-Akt levels in young and old rats with and without DHEA supplementation (data adapted from Jacob 2011)

As a faithful student of the SuppVersity, the serine/threonine kinase Akt should not be a stranger to you, after all, mTOR and p706SK (the "muscle builders", you've read about in the context of BCAAs, leucine and exercise-induced protein synthesis), are among its intracellular substrates. In agreement with previous studies, chronic administration of DHEA increased p-Akt-expression in the study at hand (cf. figure 2). The scientists speculate "that the Akt activation in this organ [the liver] is a protective answer" and could, after all, be the underlying reason for the preservation / restauration of hepatic function in the old rats.

Image 2: Oral DHEA supplements are sold for a few bucks over-the-counter (at least in the USA). Yet,
esp. for people under the age of 35, it probably does not make sense to buy and use those. At least, for
as long as it takes for the results of the study at hand to be confirmed in humans. And the allegedly benign 7-Keto DHEA could wreak havoc on your natural corticosteroid metabolism.

So how much DHEA should I take? Given the fact that the name of this blog is SuppVersity, I should have apprehended DeDeRa's question on which form and how much DHEA I would suggest you take. My answer is quite simple: NONE! Why? Well, this is a rodent study done with injectable DHEA. Not only would it be imprudent to extrapolate any dosing suggestions for oral DHEA supplements in humans, without clinical tests, we cannot even be sure that the effects would be identical, even if we hit the right dosage. Thus, while I am convinced that DHEA is probably more benign than many other OTC "supplements", especially people under the age of 30-35 should think twice or better thrice before popping any DHEA supplement - this includes 7-keto, the cortisol-suppressant effects of which can wreak havoc on your natural corticosteroid balance, make you feel tired and sluggish and deprive your body of an important anti-inflammatory pathway.

Most importantly, however, the study does away with the longstanding prejudice that DHEA (at the given dosage) "represent[s] a toxic potential to [the] liver". The isolated finding that endogenous DHEA lead to increased oxidation in the liver (it still does, but the overall pro- vs. anti-oxidant balance still improves!), as well as insufficient funding by the pharmaceutical industry, who obviously is not interested in naturally occuring and thus non-patentable treatment methods, had been one of the primary reasons many scientists decided not to dig deeper into the ameliorative, preventive and restaurative effects of DHEA in the context of age-related diseases. Personally, I hope that the few new studies that have been published, lately, will encourage other researchers to have another look at a hormone with profound yet complex and complicated effects on numerable aspects of the mammalian metabolism.

Essential Amino Acids Stimulate Muscle Glucose Uptake by Exponentiating Insulin's Effect on GLUT4 Expression

Image 1: High EAA smoothies are a good, high sugar one a very bad idea

Scientists from the Exercise Physiology and Metabolism Laboratory at the Department of Kinesiology and Health of the University of University of Texas at Austin, the Abbott Laboratories in Columbus, Ohio, and the Taipei Sports University in Taipaie, Taiwan, report in one of their latest papers that the administration of an amino acid enriched perfusate, a solution that is administered by the means of a canula, into the hindlimb (from the right iliac artery and vein to the tip of the femoral artery of the rat) of 9-week old Sprague Dawley rats stimulates glucose uptake in the presence of insulin, but not by increasing insulin and/or p-Akt (Bernard. 2012).

EAAs stimulate insulin induced GLUT-4 translocation

This is interesting, as we have hitherto assumed that many of the beneficial effects of additional protein or amino acids on glucose clearance were related to BCAA or EAA induced increases in insulin. The data, Jeffrey R. Bernard and his colleagues produced, do now suggest that this process, despite its dependence on the presence of insulin, is not the result of increases in insulin, phosphatidylinositol 3-kinases (PI 3K) or p-Akt levels (cf. figure 1), but rather a consequence of an increasd expression of AS160 a relatively unknown substrate of p-Akt that mediates the effects of the former on GLUT-4 (glucose receptor) translocation to the cell membrane and subsequent glucose uptake into the muscle (and unfortunately fat cells):

Figure 1: Simplified schematic illustration of the insulin > PI3K > p-Akt > AS160 induced increase in GLUT-4 glucose transporter expression and the effects of increased serum amino acids

Practically speaking this would mean that any increase in serum EAA levels should be able to increase the insulin-mediated glucose uptake, without increasing insulin even further. This observation would, at least in part, explain the beneficial effects individual EAAs or EAA rich protein sources have been shown to have on insulin stimulated glucose uptake (Frid. 2005; Kalogeropoulou. 2008, Morifuji. 2009).

EAA solutions, perfused rat hindlimbs and the real world: I guess it is about time to put some things into perspective here. While the locally mediated increase in glucose uptake, as it occurs in a study like this, is not mediated by increases in circulating insulin (would be hard to imagine, I mean, when the pancreas in not "connected" to the "test system" ;-), the real world effect of EAA rich protein sources, above all whey protein isolates and hydrosolates on pancreatic insulin production is very pronounced.

Figure 2: Insulin levels in 16 healthy male subjects after 0-180min after the ingestion of 45g whey protein isolate (white triangle) or hydrosolate (black squares) after an overnight fast (Power. 2009)

Power, Hallihan and Jakeman, for example, observed a peak increase of +100% and +150% in plasma insulin in sixteen healthy men after the ingestion of 45g of whey isolate or whey hydrosolate after an overnight fast (Power. 2009). Now, before you get scared, think about it this way: Through its systemic effect on pancreatic insulin release, whey - you may remember originally a constituent of milk (click here to read all about milk, colostrum & co.) - does thusly facilitate the uptake of the milk sugar via two distinct mechanism: Firstly it ramps up pancreatic insulin production, which in and out of itself would have increased GLUT4 translocation and subsequent glucose uptake. And secondly, its amino acid content will exponentiate this effect via AS160, or, put simply by increasing the insulins ability to stimulate GLUT4 translocation at the tissue level.

That other EAA rich protein sources will have similar beneficial effects on glucose clearance and that those are obviously not restricted to milk sugar, but would apply for every other source of glucose in your diets should be as obvious as the unfortunate fact that  these effect are not muscle specific :-(

Faster glucose clearance, no increase in insulin, no decrease in AMPK-alpha2

In their study, the scientists had used a solution that was particularly high isoleucine and contained 5.28 mg cysteine, 3.36 mg methionine, 6.68 mg valine, 944.8 mg isoleucine and 6.68  128 mg leucine per 50 ml solution. Whether another mixture would have elicited similar, if not superior results is questionable, what can be said, however, is that this mix is already superior to isoleucine, alone (Bernard. 2011).

Figure 3: Glucose uptake, AS160 and GLUT4 expression expressed relative to baseline (no insulin, no amino acids) in the presence and absence of insulin, and an essential amino acid mixture (AA); data calculated based on Bernard. 2012

As you can see in figure 3 these changes, in particular the increase in GLUT4 receptor density, were pretty profound (+800%!), but occurred only, if 200 μU/ml insulin were added to the perfusion solution (according to Bernard, this results in high, but still physiological insulin concentrations).

The expression of AMPK-alpha2, as you remember from the Intermittent Thoughts the "good" isoform of AMPK that is expressed in response to exercise and won't hinder your gains, on the other hand, did not differ between treatments. This would have been a surprise, anyways, but I guess the scientists wanted to check, whether amino acids could induce GLUT4 translocation via AMPK activation and thus by the same mechanism as your workouts do. That the latter is not the case, makes them even more interesting to promote the already improved ability of skeletal muscle glucose uptake after a workout.

Bottom line: Take advantage of the GLUT4 amplifying effects of amino acids...

... not just after your workouts, but with every meal, by making sure that it contains an ample amount of high quality protein (~10g+ EAA content, e.g. ), or, if nothing that falls into this category is available, by adding additional BCAAs, better EAAs in capsule(=more convenient) or powder (=cheaper and in some cases even palatable ;-) to your ready-to-eat-low-protein-whatever.

And in case you are still doubting the real-world significance of this in most people's eyes probably maddish practice, let me briefly quote the results of a study by Loenecke et al. who investigated the relationship between the amount of quality protein, carbohydrate, and dietary fat consumed and the amount of times the ~10 g essential amino acid (EAA) threshold was reached at a meal, with percent central abdominal fat in 27 young and healthy men and women (Loenecke. 2012; my emphases):

Quality protein consumed in a 24-hour period was inversely related with percent CAF (r = -.420, p = 0.041). No associations were found with carbohydrate (r = -.198, p = 0.354) or dietary fat (r = -.196, p = 0.359) with percent CAF. The amount of times reaching the EAA threshold for a meal throughout the day was also inversely related with percent CAF (r = -.547, p = 0.006).

I would say this means you either buy a new fridge for all the eggs, meats, fish, crustaceans, dairy & co. or head over to the supplement vendor of your choice and get yourself a bag of whey, casein or milk protein. And if your digestive tract doesn't like those (try using isolates, first, those should be 100% lactose free), pick egg, pea or beef protein isolates - mostly they don't taste as well, but their essential amino acid content is high and at least pea is also "kosher" for vegans ;-)

References:

1. Bernard JR, Liao YH, Hara D, Ding Z, Chen CY, Nelson JL, Ivy JL. An amino acid mixture improves glucose tolerance and insulin signaling in Sprague-Dawley rats. Am J Physiol Endocrinol Metab. 2011 Apr;300(4):E752-60. Epub 2011 Feb 8.
2. Bernard JR, Liao YH, Doerner PG 3rd, Ding Z, Hsieh M, Wang W, Nelson JL, Ivy JL. An amino acid mixture is essential to optimize insulin-stimulated glucose uptake and GLUT4 translocation in perfused rodent hindlimb muscle. J Appl Physiol. 2012 May 17.
3. Frid AH, Nilsson M, Holst JJ, Björck IM. Effect of whey on blood glucose and insulin responses to composite breakfast and lunch meals in type 2 diabetic subjects. Am J Clin Nutr. 2005 Jul;82(1):69-75
4. Kalogeropoulou D, Lafave L, Schweim K, Gannon MC, Nuttall FQ. Leucine, when ingested with glucose, synergistically stimulates insulin secretion and lowers blood glucose. Metabolism. 2008 Dec;57(12):1747-52.
5. Loenneke JP, Wilson JM, Manninen AH, Wray ME, Barnes JT, Pujol TJ. Quality protein intake is inversely related with abdominal fat. Nutr Metab (Lond). 2012 Jan 27;9(1):5.
6. Morifuji M, Koga J, Kawanaka K, Higuchi M. Branched-chain amino acid-containing dipeptides, identified from whey protein hydrolysates, stimulate glucose uptake rate in L6 myotubes and isolated skeletal muscles. J Nutr Sci Vitaminol (Tokyo). 2009 Feb;55(1):81-6.
7. Power O, Hallihan A, Jakeman P. Human insulinotropic response to oral ingestion of native and hydrolysed whey protein. Amino Acids. 2009 Jul;37(2):333-9.

Speed Up Your Regeneration and Propel Your Gains by Taking a HOT Bath Bath 2-Days Before Arduous Workouts

Image 1: Are women tougher than men, because bathe more often? If you define toughness by your muscles resistance to eccentric exercise damage, the answer could be "YES!"

If you listened to Brooks, Carl and me on Super Human Radio, yesterday (download the podcast), you may remember me stating that 48h appears to be a good rule of thumb, as far as the rest periods between workouts for individual body parts are concerned (this assumes that you are young, healthy, reasonably conditioned and lift heavy). A recently published paper by Chad D. Touchberry  does now suggest that there may be another 48h window before your workout (Touchberry. 2012). One you would use a priori to improve your recovery a posteriori - preconditioning in a hot bath for 20 min at 41°C, 48h before a hard workout or competition!

Eccentric treadmill running = maximum muscle damage

At least in a rodent model, those 20 min of heat exposure in 41°C warm water lead to statistically highly significant decreases in exercise induced muscle damage, improved and accelerated the recovery process and, contrary to what could be assumed based on previous research on the expression of heat proteins (Frier. 2007), did not hamper, but promote muscle gains in response to an exercise protocol consisting that consisted of running at 18m/min down a -16% grade for 5 min. This protocol has been used as a model for injurious exercise repeatedly in the past and constitutes one of the standard tests in rodent, but also in human studies (e.g. Pumpa. 2011).

Figure 1: Creatine kinase (CK) activity and immune cell infiltration after eccentric exercise with (EE+HS) and without (EE) preconditioning via hot bath 48h before (data calculated based on Touchberry. 2012)

As the data in figure 1 goes to show, the hot bath (EE+HS) had significant ameliorative effects on both the muscle damage (indicated by CK and the black sections in the H&E-stained soleus muscle cross-sections in figure 1, right), of which the researchers state that, despite the fact that "the mechanism by which heat shock protects skeletal muscle from damage is currently unknown", the protection of skeletal muscle against damage in mice overexpresssing HSP70 (McArdle. 2004a) as well as the differential HSP72 elevation in the HS group 2h and 48 h following exercise collectively

[...] suggest that HSP72 or another heat sensitive protein (i.e.,alphaB-crystallin) may play a role in mediating cytoprotection of skeletal muscle cells.

Moreover, Touchberry et al. explain the existing discrepancies between their own results and previous results by Mc.Ardle et al. (Mc. Ardle. 2004b), who did not find reduced muscle damage after pre-treatment with hsp-inducing concentric exercise 10h prior to the (in my humble opinion questionable) in-vitro application of eccentric strain to skeletal muscle tissue, with the "greater time for HSP accumulation prior to the exercise stressor" in their (48h) vs. the Mc.Ardle study (10h), which is obviously yet another indicator that rest is one of the most under-appreciated determiners of workout efficiency (cf. my words on SHR ;-)

Regeneration is one thing, but are muscle gains another?

Now, I am well aware that one of the main reasons regeneration isn't sexy, is that it does not trigger the phosphorylation of Akt, m-TOR and all the rest of the sciency terms with which laymen are bombarded by the supplement industry these days.

Figure 2: Total protein, new myosin heavy chain (MHCNEO) content and p-Akt expression in soleus muscle 2h and 48h after the eccentric exercise bout (data calculated based on Touchberry. 2012)

The study at hand does even show that heat pre-treatment will actually reduce, not promote the phosphorylation of AKT 48h after the exercise bout (cf. figure 2). If you do yet take into account that the total protein concentration and MHCneo (novel myosin-heavy-chain motor proteins) content in the soleus muscle of the rodents was increased profoundly, I guess you will have to agree that it is unlikely that less damage, a faster regeneration, and as a consequence less need for protein to be recruited via p-AKT only to repair the damage is going to propel, not diminish your gains!

Practical implications & open questions

Once again, the obvious message of this study is: Not he who trains the most, but he who regenerates and rebuilds the best, gains the most! And adequate rest aside, preconditioning in a hot (not a "cold thermogenic" bath ;-) can help dampen the exercise induced damage and accelerate your recovery.

Note: In February 2012, Bayley et al. published a paper that shows that the application of passive heat in form of a 42°C hot water bath for 40min immediately prior to a bout of HIT leg extensor exercises reduced the time to fatigue in seven healthy men by a whopping -36%  (Bayley. 2012). Impatience, or rather the unwillingness to grant your body the time it needs to recover is thus detrimental even if the stressor is "just" a hot bath!

Whether the same would be true if you train today and do the hot water immersion immediately, 2h, 10h or 12h post and thus 48h, 46h, 38h or 36h before your next workout is yet about as questionable, as whether or not similar effects could be elicited by switching back and forth between light and heavy days every 48h.  Both may appear likely, but aside form the fact that the optimal timing or workout intensity will still have to be elucidated, are still in the state of an interesting research hypothesis, not more, but also not less.

Update - Suggested reading: Since there have been questions pertaining to the usefulness of hydrotherapy post-workout, i.e. as a means of "classic" re- and not "precovery", I thought I rather refer you directly to my buddy Sean's E-book on the issue. Here is a snippet from the book

Image 2: Don't miss Sean's free e-book on classic hydrotherapy

Quick Hit Summary Water therapy is a common modality to enhance muscle recovery post workout. Sitting in chest high thermoneutral water for 20-30 minutes may accelerate waste removal while increasing blood flow to working muscles. Cold, hot and contrast water temps are also commonly used to assist recovery. The goal of cold water therapy is to reduce inflammation whereas hot water purportedly increases muscle blood flow. Contrast water therapy involves alternating between hot and cold water baths to induce a vaso-pumping effect. Current evidence does not support the theory behind these latter 2 therapies simply because the heat (from the water) is incapable of penetrating more than a couple centimeters into the skin. Thus, there is no stimulus to increase muscle blood flow

You can get this e-book alongside two other books for free if you register for Sean's newsletter, which is, take my word on it (!), not a weekly advertisement piece!
 
References:

1. Bailey SJ, Wilkerson DP, Fulford J, Jones AM. Influence of passive lower-body heating on muscle metabolic perturbation and high-intensity exercise tolerance in humans. Eur J Appl Physiol. 2012 Feb 10.
2. Briese E. Normal body temperature of rats: the setpoint controversy. Neurosci Biobehav Rev. 1998 May;22(3):427-36. Review. 
3. Frier BC, Locke M. Heat stress inhibits skeletal muscle hypertrophy. Cell Stress Chaperones. 2007 Summer;12(2):132-41. 
4. McArdle A, Dillmann WH, Mestril R, Faulkner JA, Jackson MJ. Overexpression of HSP70 in mouse skeletal muscle protects against muscle damage and age-related muscle dysfunction. FASEB J. 2004a Feb;18(2):355-7.
5. McArdle F, Spiers S, Aldemir H, Vasilaki A, Beaver A, Iwanejko L, McArdle A, Jackson MJ. Preconditioning of skeletal muscle against contraction-induced damage: the role of adaptations to oxidants in mice. J Physiol. 2004b Nov 15;561(Pt 1):233-44. Epub 2004 Aug 26.
6. Pumpa KL, Fallon KE, Bensoussan A, Papalia S. The effects of Lyprinol(®) on delayed onset muscle soreness and muscle damage in well trained athletes: a double-blind randomised controlled trial. Complement Ther Med. 2011 Dec;19(6):311-8.
7. Touchberry CD, Gupte AA, Bomhoff GL, Graham ZA, Geiger PC, Gallagher PM. Acute heat stress prior to downhill running may enhance skeletal muscle remodeling. Cell Stress Chaperones. 2012 May 17. [Epub ahead of print]

Carnitine as Repartitioning Agent? IGF-1, p-AKT & mTOR Up, Catabolic Proteins Down + 7% Improvement in Lean- to Total Mass Ratio W/ HED of 1-1.5 of Carnitine/Day

It won't spare you the sweat, but carnitine could make it even more worthwhile by ramping up the anabolic and shutting down the catabolic signals.

Until 2006 l-carnitine has been known as a fat-burner, an in-effective fat-burner and an expensive and pretty useless supplement (depending on whom you were asking). Then, in July 2006, Kraemer et al. published a paper (a human study, above all!) in the journal Medicine & Science in Sports and Exercise a consequential paper so to say; a paper in which the authors report that l-carnitine l-tartrate supplementation at a dosage of 2.933g/day (this amount of LCLT contains 2g of pure carnitine) led to a statistically significant increase in androgen receptors in the vastus lateralis after a heavy resistance training protocol in previously strength trained male subjects (Kraemer. 2006).

Still, the evidence has always been inconclusive to say the least

Despite the fact that the concomitantly elevated post-workout luteinizing hormone levels (+19%) Kreamer et al. observed would tell you that the testosterone that would have been necessary to activate those receptors was already on its way, I have never considered this study as convincing evidence of the anabolic prowess of l-carnitine. Plus, let's be honest, differences in whatever serum markers in response to an acute bout of resistance training have failed us way too often, not to look at studies like these with appropriate skepticism.

Do you remember the Ratames study from 2005? The one that showed that high volume training lowers the no. of androgen receptors on the trained muscles? This certainly makes l-carnitine sound like the perfect addition to high volume routines, right? (learn more)

That the same principle of "calm down and don't get too excited over the results of a single trial" does all the more apply to rodent studies should be self-evident and still, science is all about taking each and every experimental result into account to form a theory that can explain all of them, or, alternatively, is able to bust short-comings in previous studies that don't comply with the predictions of the respective theory.

Now, the soon-to-be-published paper by Janine Keller and her colleagues from the University of Giessen (Germany) certainly qualifies as part of the evidence we simply cannot ignore, when we are looking for evidence in support of the theory that l-carnitine could be an overlooked muscle builder or repartitioning agent.

After all, their observation of decreased levels of the proteolytic (=catabolic) MuRF1 protein, as well as the ubiquitin-protein conjugates, which are increased in catabolic states such as starvation and atrophy denervation (cf. Wing. 1995) , alone, would signify that l-carnitine could make a valuable addition to everybody's supplementation regimen.

Lower catabolism + increased anabolism = ???

There is more, however, the addition of 1250 mg L-carnitine/kg to a basally "low carnitine" vegetarian diet also led to significant increases in systemic IGF-1 concentrations in plasma and a local increase in the activity of the PI3K/Akt/FoXO-1 signalling pathway (see figure 1)

Figure 1: IGF-1 mRNA and serum levels, as well as the muscle specific expression and phosphorylation (ph) Akt, mTOR & co after four weeks on the low or high carnitine diets (Keller. 2013)

These results do yet not stand in isolation as the ones by Kraemer et al. still do. Other recent studies by the same research group in Giessen, as well as colleagues from the University of Barcelona have already confirmed the anti-catabolic effects of l-carnitine in piglets and a cancer cachexia model in rodents, respectively (Keller. 2012; Busquets. 2012).

"And you are telling me that works in humans, as well? "

What's the best form of carnitine to take to elicit these effects: I knew you would ask this, so I react to two facebook questions by adding this red box willingly admitting that I just cannot tell you what the best form of carnitine is. There simply is no study that would compare e.g. acetyl-l-carnitine (ALCAR) and l-carnitine l-tartrate (LCLT) in a scenario that would be relevant to the above question. What I can tell you though, is that it appears as if you were better off with LCLT than with ALCAR, if your goal is to top off your intra-muscular carnitine levels. That being said, even normal creatine can do that - you will just have to take more of it. If you are looking for more information you can check out the part of the Amino Acids for Super Humans Series that's dealing with "the carnitines", here.

In this context it does yet also have to be mentioned that the effects of l-carnitine are at least in part species specific. How we know that? Well, in contrast to the said study by Basquets et al. the provision of an carnitine to piglets (Keller. 2012) did not only reduce the MuRF-1 expression, but also the level of its likewise catabolic E3 ligase cousin atrogin-1.

"It has been shown that myofibrillar proteins, like myosin light chain proteins are the main targets of MuRF1for ubiquitination. Thus, carnitine might suppress particularly the degradation of myofibrillar proteins, which under physiological conditions comprise around 60% of total muscle proteins. In contrast to MuRF1, atrogin-1 tags primarily proteins for degradation which are important for controlling protein synthesis and myoblast differentiation, like myogenic factor MyoD, myogenin and the eukaryotic initiation factor of protein synthesis eIF3-f." (Keller. 2013)

With pigs usually being a superior model of the human physiology, this would suggest that the anti-catabolic effects l-carnitine could have on humans are probably more, not less pronounced than those that were observed in previous rodent studies.

Whether the same goes for the IGF-1 response cannot be said, but just like the anticatabolic effects, the pro-anabolic increase in IGF-1 has been observed in previous trials, including a human trial by Di Marzio et al. who observed a significant increase in IGF-1 in HIV patients in response to the provision of 3g/day of acetyl-l-carnitine (Di Marzio. 1999). In the absence of the existing evidence from animal studies, these results would yet have little significance for healthy human beings, whose growth hormone and IGF-1 levels are not rock bottom to begin with (Viganò. 2003).

---

Bottom line: Irrespective of the absence of human data on the IGF-1 boosting effects from non-HIV patients - or even better in training scenarios - it would warrant future studies if an adequate amount of carnitine in the diet can exert beneficial effects in non-obese human beings. For the "sedentary", or let's rather say non-exercised rodents in the study at hand, the latter was a mere fat loss effect - despite the elevations in p-AKT, m-TOR, IGF-1 and the overall more "anabolic" state the rodents were in their lean body mass was not increased compared to their peers on the low carnitine diet.

"Just another set!" ... "I don't know man, we've already pumped away 100,000kg today... do you really believe that's productive, I mean, yeah, we are cuttin', but still" ...learn what this dialog is all about and whether and if / when "another set" is / isn't a good idea (read more)

The lean-to-total mass ratio of the rodents, on the other hand was ~7% higher in the rodents in the high carnitine group. If we do however take into consideration that most of you will not be vegetarians and thus not similarly carnitine deprived as the rodents in the control group on the <1mg/kg carnitine diets, it is highly questionable if the addition of the human equivalent of the 1.25g/kg chow, i.e. 15mg/kg body weight (HED) would actually yield any measurable benefit to non-vegetarians - irrespective of whether they train or not. After all, even the average omnivore human being consumes 100-300mg of carnitine per day (Broquist. 1994), so that the difference between your basal carnitine intake and the supplemental equivalent dose of 1050-1500mg/day is more than 100x lower than the exorbitant difference between the low (if not deficient) carnitine diet in Keller's rodent study at hand (remember: the basal diet had less than 1mg/kg chow; the supplemented diet hat 1250mg/kg diet!).

So what's the verdict then? I guess, I will leave the final words to Burke et al. who reviewed the usefulness of carnitine as an ergogenic aid in one of the first installments of the "A-Z Supplement Review" in the British Journal of Sports Medicine and wrote "future work with l-carnitine may also find some useful outcomes" (Burke. 2009) - needless, to say that the SuppVersity is going to be the place, where you will read about it first ;-)


References:

• Broquist HP. Carnitine. In Shils ME, Olson JA, Shike M (eds): "Modern Nutrition in Health and Disease." Malvern, PA: Lea & Febiger, 1994. 459– 465.
• Burke LM, Castell LM, Stear SJ, Rogers PJ, Blomstrand E, Gurr S, Mitchell N, Stephens FB, Greenhaff PL. BJSM reviews: A-Z of nutritional supplements: dietary supplements, sports nutrition foods and ergogenic aids for health and performance Part 4. Br J Sports Med. 2009 Dec;43(14):1088-90.
• Busquets S, Serpe R, Toledo M, Betancourt A, Marmonti E, Orpí M, Pin F, Capdevila E, Madeddu C, López-Soriano FJ, Mantovani G, Macciò A, Argilés JM:  l-Carnitine: An adequate supplement for a multi-targeted anti-wasting therapy in cancer.  Clin Nutr. 2012;31:889–895.
• Di Marzio L, Moretti S, D'Alò S, Zazzeroni F, Marcellini S, Smacchia C, Alesse E, Cifone MG, De Simone C. Acetyl-L-carnitine administration increases insulin-like growth factor 1 levels in asymptomatic HIV-1-infected subjects: correlation with its suppressive effect on lymphocyte apoptosis and ceramide generation. Clin Immunol. 1999 Jul;92(1):103-10.
• Glass DJ:  Signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Nat Cell Biol. 2003; 5:87–90 .
• Kraemer WJ, Spiering BA, Volek JS, Ratamess NA, Sharman MJ, Rubin MR, French DN, Silvestre R, Hatfield DL, Van Heest JL, Vingren JL, Judelson DA, Deschenes MR, Maresh CM. Androgenic responses to resistance exercise: effects of feeding and L-carnitine. Med Sci Sports Exerc. 2006 Jul;38(7):1288-96.
• Keller J, Ringseis R, Koc A, Lukas I, Kluge H, Eder K:  Supplementation with l-carnitine downregulates genes of the ubiquitin proteasome system in the skeletal muscle and liver of piglets. Animal. 2012;6:70–78.  
• Keller J, Couturie A, Haferkamp M, Most E, Eder K. Supplementation of carnitine leads to an activation of the IGF-1/PI3K/Akt signalling pathway and down regulates the E3 ligase MuRF1 in skeletal muscle of rats. Nutrition & Metabolism. 2013; 10:28. 
• Lösel D, Rehfeldt C. Effects of l-carnitine supplementation to suckling piglets on carcass and meat quality at market age. Animal. 2013 Mar 11:1-8.
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• Wing SS, Haas AL, Goldberg AL. Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation. Biochem J. 1995 May 1;307 ( Pt 3):639-45.

The Glucose Repartioning Effects of Isoleucine: Falsely Underappreciated BCAA and Its Dipeptides Maximize GLUT-4 Expression and Ramp Up Muscular Glucose Uptake

Still waiting for your abs to appear? Maybe you have the wrong "scientifically proven", "superior", or "modern", but unnatural ratio of BCAAs in your peri-workout nutrition - just kiddin' that alone won't be the reason, but still...

In the past week I have posted several facebook news items which were, in one way or another, related to he negative effects of isolating (micro-)nutrients and/or consuming them in what one could call "unnatural" ratios. Now one of those natural ratios most of you will be familiar with is the 2:1:1 ratio of leucine to valine and isoleucine, the three branched-chain amino acids. I have long been eye-balling with more than some skepticism how supplement companies have been trying to monetize on the unwarranted hype around leucine by twisting the ratio from 2:1:1 to 3:1:1 and even 10:1:1 and stating that this would be a "more anabolic" or "scientifically supported" modification. With the impending publication of a paper by a group of Brazilian scientists, I am yet pretty sure that you are soon going to see very different "optimal" ratios being propagated (Morato. 2013).

Leucine was yesterday, isoleucine is the future - at least for lean mass gains

You already know from previous articles that EAAs increase GLUT-4 expression - could it be that this effect was stimulated by isoleucine, alone? As a branch-chained amino acid it is after all one of the EAAs. Or is it rather a synergistic effect of various amino acids?

The data the researchers are presenting in their soon-to-be-published article in the Jornal of Food Chemistry, clearly indicates that not leucine, not glutamine, not cysteine, not arginine, not citrulline and none of the other usually often hailed amino acids, but the hitherto more or less ignored branched chain amino acid l-isoleucine is the driving force of the increase in skeletal muscle GLUT-4 translocation and subsequent glucose uptake in response to BCAAs and BCAA rich protein sources such as whey protein.

To study the individual effects of various components of whey on glucose uptake, the scientists administered a mixture of glucose + whey protein hydrosolate or one of the following amino acids / dipeptides to 49 rats who had been exercised the day before and were kept in a fasted state for 15:30hr afterwards (this protocol was meant to deplete muscle and liver glycogen): L-isoleucine (ILE), L-leucine (LEU), L-leucine plus L-isoleucine (LEU+ILE), L-isoleucyl-L-leucine and (ILE-LEU), L-leucyl-L-isoleucine (LEU-ILE).

Important recent update on isoleucine: I highly suggest you read my more recent article on isoleucine, as well. It discusses the beneficial effects on blood glucose in man, but adds that an amino acid mix containing high amounts of isoleucine blunts glycogen resynthesis after the workout.

After receiving these solutions, the animals (N=7 per group) were sacrificed and the effects on glucose transporter 4 (GLUT-4), p-Akt and AMPK in skeletal muscle, as well as the insulin and glucose levels in the blood and the glycogen content of  liver, skeletal muscle and heart were evaluated.

Figure 1: Skeletal muscle protein expression, insulin and glucose levels and glycogen content of muscle, liver and heart 30min after the ingestion of the glucose + whey / AA / peptide solutions (Morato. 2013)

A brief glance at the data in figure 1 reveals that the isoleucine + glucose solution did produce exactly those non-insulin dependent  increases in GLUT-4 activity, glucose uptake and disappearance from the blood stream we'd expect from a glucose repartitioning agent (check out the low insulin and glucose levels and high GLUT-4 expression in for ILE in figure 1). 

Where did the glucose go, dude?

Training glycogen deplete can be regarded as an intensity technique that can increase markers of mitochondrial biogenesis by +700% (read more), not repleting your glycogen levels for days must be regarded as madness, though.

What may surprise you, though, is the fact that the glucose that disappeared from the blood stream did not reappear in form of glycogen in either of the tested organs. That's yet not due to oxidation, let alone it's deposition in the adipose tissue of the animals, but simply due to the fact that the study protocol, in which the rodents had only 30min to live after the administration of the test solutions. According to previous research, the complete repletion of severely reduced glycogen levels can take up to 24h (Jentjens. 2003) and that's in the presence of sufficient amounts of glucose. The 30% glucose solution in which the 0.55g/kg of whey, amino acids (AA) or dipeptides were dissolved in the study at hand, however, would not have been sufficient to replete the glycogen levels to a significant degree, even if the poor critters had lived for another 24h.

Now you could certainly argue that the scientists would not have had to measure the glycogen content, in the first place, if they already knew it would not change. In a way, this is correct, but since we know that the rate of glucose uptake is inversely related to the glycogen levels in the muscle, measuring the actual glycogen content was necessary simply to make sure that inter-group differences in terms of the amount of glycogen that was still left in muscle, liver and heart tissue of the animals would not interfere with the study outcomes.

Amino acids, lactate dehydrogenase (LDH), creatine kinase and more

No wonder the cup on the left is the only one that smiles. The black coffee it contains may be among the least known, but most consumed GLUT4 promoters worldwide. That's at least what a  paper by Guarino et al. from 2012 suggests. And while this partly explains the reduced diabetes risk in habitual coffee drinkers, the lower incidence of CVD and cancer are brought about by the natural synergy of various nutrients in coffee (read more)

In addition to the previously discussed parameter, Morato et al. also analyzed a whole host of additional parameters, mostly with non-significant or physiologically irrelevant results. One thing that's worth mentioning though are the higher serum levels of the (main) glyconeogenic amino acid l-alanine in the l-isoleucine group.

The scientist ascribe the latter to the competition of isoleucine and alanine for hepatic transport via the neutral amino acid transporter. The decreased uptake of alanine by liver, in turn, could have reduced hepatic (=by the liver) gluconeogenesis and thus contributed to a further reduction (or absence of an increase) in serum glucose levels. This would also explain the lower ALT levels in the isoleucine group. After all, this enzyme that's often misattributed as an indicator of liver damage is actually nothing but a marker of the transamination of alanine (hence "ALT" as in ALanine amino-Transferase), a process that is a necessary step in hepatic gluconeogenesis from alanine to pyruvate, which will subsequently be converted to glucose and released into the blood.

Bottom line: If we recap the results the main take home messages of this study are as follows: (1) Never mess with nature's wisdom *lol*; (2) the hitherto mostly overlooked #3 of the BCAAs could be a very important contributer to the nutrient and thus body recompositioning effects of branch-chained amino acids and BCAA rich protein; (3) while l-isoleucine may be king, when it comes to the non-insulin dependent increase in GLUT-4 activity and the subsequential lowering of blood glucose, the whey(-exclusive?) dipeptide l-leucyl-isoleucine with its profound effects on both Akt and insulin, is probably the more anabolic GLUT-4 stimulant.

Having 10g of essential amino acids (EAAs) or 2030g of "high quality = high EAA" protein with each and every of your meals is one of the easiest an most reliable strategies to become and stay lean (read more). With isoleucine obviously being one of those EAAs and 25g of whey and casein having ~1.75g respectively 1.13g of this carbohydrate repartitioning amino acid in it, this could well explain part of the benefits (note: 125g of chicken breast will also yield 1.5g of isoleucine and the same amounts of beef and pork also have >1g; the same goes for 3-4 eggs or 500g of peas and 2.5kg pumpkin or 4kg of eggplant ;-)

Based on these insights most of you probably won't really have to change anything about their protocol... well, unless you have fallen for the unwarranted advertisement claims of the supplement business and switched from a cheap whey protein to an overexpensive BCAA or EAA product with tons of leucine and almost nothing else in it.

Whether or not there would be real world benefits of isolated l-isoleucine supplementation for people with compromised insulin sensitivity or even full-blown diabetes will have to be elucidated in future studies. In view of the fact that BCAAs have yet already been proposed as viable "treatment" (I'd rather prefer the term "management", though) strategies for type II diabetes and related diseases (e.g. Manders. 2012; Takeshita. 2012) and considering the fact that leucine alone did not have any beneficial effects on glucose management on pro-diabetic diets (Nairizi. 2009), the addition of some isoleucine to products meant to increase glucose uptake from skeletal muscle does certainly look worth investigating. Whether the outcomes will be significantly superior to those of plain whey protein hydrolysates (Sousa. 2012), on the other hand, is something I wouldn't be too certain about.

References:

• Jentjens R, Jeukendrup A. Determinants of post-exercise glycogen synthesis during short-term recovery. Sports Medicine. 2003; 33(2):117-144.
• Manders RJ, Little JP, Forbes SC, Candow DG. Insulinotropic and muscle protein synthetic effects of branched-chain amino acids: potential therapy for type 2 diabetes and sarcopenia. Nutrients. 2012 Nov 8;4(11):1664-78.
• Morato PN, Lollo PCB, Moura CS, Batista TM, Carneiro EM, Amaya-Farfan J. A dipeptide and an amino acid present in whey protein hydrolysate increase translocation of GLUT-4 to the plasma membrane in Wistar rats, Food Chemistry. 2013 [epub ahead of print].
• Nairizi A, She P, Vary TC, Lynch CJ. Leucine supplementation of drinking water does not alter susceptibility to diet-induced obesity in mice. J Nutr. 2009 Apr;139(4):715-9. 
• Sousa GT, Lira FS, Rosa JC, de Oliveira EP, Oyama LM, Santos RV, Pimentel GD. Dietary whey protein lessens several risk factors for metabolic diseases: a review. Lipids Health Dis. 2012 Jul 10;11:67.
• Takeshita Y, Takamura T, Kita Y, Ando H, Ueda T, Kato K, Misu H, Sunagozaka H, Sakai Y, Yamashita T, Mizukoshi E, Honda M, Kaneko S. Beneficial effect of branched-chain amino acid supplementation on glycemic control in chronic hepatitis C patients with insulin resistance: implications for type 2 diabetes. Metabolism. 2012 Oct;61(10):1388-94.