The Counterintuitive Catabolic & Pro-Diabetic Effects of Leucine Supplementation in Rodents on Corticosteroids

Not the mice from this study, but still a nice example of the effects of dexamethasone on skeletal muscle (right; Quin. 2013)

"Leucine-laced water + stress = insulin resistance" - This simple equation is the net result of a recent study by Nelo Eidy Zanchi and his colleagues from the Laboratory of Applied Nutrition and Metabolism at the School of Physical Education and Sports of the University of Sao Paulo in Brazil. Inspired by previous research which clearly indicated that leucine does not only have pro-anabolic, but also insulin sensitizing effects, Zanchi et al. speculated that the provision of adequate amounts of leucine would blunt the catabolic and pro-diabetic effects of 7 days of intraperitoneally injections of  dexamethasone, an artificial corticosteroid that's used to treat all sorts of inflammatory diseases.

Remember SuppVersity Rule of Smart Supplementation No. 2? Right. Specificity!

In order to test their hypothesis that leucine supplementation either in low doses in the drinking water or as higher dosed oral gavage would ameliorate the negative side effects of DEXA treatment, the scientists randomized groups of 10 male Wistar rats to six groups receiving either low dose or high dose leucine supplements with and without dexmethasone.

"During  the duration of the experiment, which lasted  seven  days,  DEXA (a synthetic glucocorticoid analogue that does not bind to plasma binding proteins) was given daily (at 9:00 a.m.) through intraperitoneal injection (5 mg/kg/day); control groups received  an equivalent volume of saline (0.9% NaCl). As DEXA was reported to decrease food intake, all groups were  fed the same amount of food (in terms of caloric intake) equal to the DEX group. Thus, differences among groups did not originate from different food intakes. We measured the caloric content of our standard chow (16.32 kJ/g) as well as leucine (25 kJ/g) in a calorimetric bomb (FTT Oxygen Bomb Calorimeter) in  order to avoid differences in the caloric ingestion between experimental groups and observed that the total caloric consumption was not statistically different among groups." (Zanchi. 2013)

The leucine was administered either in dosages of 0.068g/kg body weight per day (low dose) or 1.35 g/kg per day (high-dose) twice daily at 8:00  a.m. and  2:00  p.m. through gavage over seven days. And while the scientists had selected the high dose (LH) "to induce a maximal increase in muscle protein synthesis and insulin plasmatic levels", the dosage in the LL (=low leucine) group was too low to increase either muscle protein synthesis or plasma insulin levels. The third, non-supplemented control group received an NaCl (sodium) placebo, the volume of which was identical to the supplement to make sure that any possible volume-induced effects of oral gavage that could for example be induced by gastric expansion would not skew the study results.

"But leucine has been shown to be anabolic! So it must help."

Aside from the usual basal fasting glucose, insulin, tryacilglycerol (TAG) and HOMA-IR values, the scientists did also assess the motor performance of the animals by the means of two standardized strength and ambulation tests (Kennel. 1996; Anderson. 2004; Viera. 2008).

Figure 1: Effect of 7 days of low (LL) and high (LH) dose leucine supplementation with and with out dexamethasone on total body mass, soleus (slow twitch) and EDL (fast twitch) muscle mass in male Wistar rats (left; values expressed relative to non-supplemented control) and corresponding changes in mean ambulation and grip strength (right; Zanchi. 2013)

As you can see in figure 1 the supplemental leucine failed to reduce the negative side effects of dexamethasone. As far as the total body weight and the fast-twitch muscle mass (EDL) are concerned, you could even argue that the high dose treatment (DEX-LH) did even amplify the catabolic effects of the synthetic corticosteroid:

"Thus, leucine supplementation at both low and high doses did not counteract body weight loss in both food restricted (control groups) and DEXA-treated animals. Soleus muscle mass did not differ among groups. Leucine supplementation at  high doses  attenuated food  restriction-induced EDL muscle loss (CON-LH group) when compared with the CON-NS group  (p < 0.05). All DEXA-treated animals presented reduced EDL muscle mass when compared with the CON-NS group (p < 0.05), and leucine supplementation at both low and high doses of amino acid did not attenuate it." (Zanchi. 2013)

Now, you may well argue that the mere fact that the muscle weight was "statistically significant" reduced, this does not mean that these reductions would be physiologically significant and that the minimal differences between the DEX groups would not matter, anyway. If you just go by the data on the left side of figure 1, this is certainly right, if you do yet also consider the significant reductions in muscle function (figure 1, right) and the fact that all that happened within no more than 7 days, the overall result should actually remind you of the "Three Simple Rules of Smart Supplementation" - and here specifically the 2nd one: Specificity!

Figure 2: Time course of the dexamethasone-induced detoriations in fed serum glucose levels and ameliorative effect of low and high dose leucine supplementation (left) and effects of the treatment on fasting insulin levels and HOMA-IR (index of insulin resistance) at the end of the study (Zanchi. 2013)

In fact, the data in figure 2 only confirms the notion that things that you cannot define "good and bad", "black and white" and "beneficial or detrimental" without a context and the outcome you are expecting. If you are trying to keep the postprandial blood sugar in check, for example he addition of an effective (high dose) of leucine to the diet would appear to be a good idea. If, on the other hand, you are more concerned about insulin resistance, you would be better advised to use minimal amounts of leucine or simply refrain from supplementation altogether.

Figure 3: If the ingestion of bolus amounts of leucine is not helpful, lacing the water of the rodents DEXA treated rodents with leucine turned them into full-blown diabetics (Zanchi. 2013)

As these results clearly demonstrate the provision of additional leucine is not useful to counter the negative side-effects of synthetic corticosteroids. On the contrary, the negative effects on insulin resistance are apparently even augmented and the muscle function is further compromised by the purpotedly anabolic high dose leucine supplement.

And while the overall effects of the bolus administration may still be negligible, the scientists ingenious idea that the provision of similar amounts of leucine in the drinking water in a second follow-up experiment turned out to be "capable of inducing a massive diabetic state" (Zanchi. 2013; see figure 3 for the ensuing surge in fasting blood glucose levels) while decreasing the mass of the fast-twich EDL muscles even further.

Bottom line: Overall these results only confirm the simple, but often neglected truth that inductive reasoning is a futile undertaking in the realms of exercise and nutrition sciences: What is good for an athlete is rarely optimal for an obese person, the same diet that helps the obese lose weight, will make the athlete feel miserable, and lacing the drinking water of rodents on corticosteroids with the exact same amount of leucine that has had highly beneficial effects on the insulin sensitivity of diabetic rodents in previous studies (Guo. 2010) will not only fail to ameliorate the glucocorticoid-induced detoriations in blood glucose, it will even exasperate them.

So, does that mean you should not take your whey protein or BCAAs any longer? No, if you did that you would make the exact same mistake as someone who laces his water with leucine in order to avoid the catabolic effects of the synthetic corticosteroid he is taking for medical reasons. On the other hand, the results of the study at hand should make you re-evaluate the necessity and even benefits of guzzling BCAAs all-day long, at least if the reason for doing so is that you believe that you are so stressed that you would otherwise fall into a catabolic black hole.

That said, there may even be implications for the average pre-diabetic inhabitant of the Western hemisphere who is eating his hamburger and French fries on the parking lot of the local fast food restaurant, because he cannot make room to prepare and consume a real meal somewhere in his busy and stressful schedule. I mean, despite the fact that the aforementioned specificity principle does not allow for anything but a still to be verified hypothesis, it does at least appear not to far-fetched that this chronic endogenous stress, despite being very different from the "stress" that's induced by the administration of a synthetic corticosteroid that does not bind to serum proteins, could have similar negative modulatory effects on the purported benefits of chronic leucine supplementation ... but as I've said before, this would be something to investigate in another study. So unless you are actually taking dexamethasone for medical reasons, you are probably not at risk of developing diabetes due to a high amount of leucine in your diet.

In the unfortunate case that you are actually on synthetic corticosteroids, a previous study by the same group of scientists, in the same rodent model does suggests that three workouts with three sets of squats (10 reps each) per week may offer the protection against corticosteroid induced muscle loss decreased skeletal muscle GLUT-4 expression and insulin resistance, leucine does not have to offer.... well, at least as long as you abstain from leucine supplementation, because the latter had the exact same detrimental effects in the 2011 study where it was administered to one of the experimental groups in conjunction with resistance training as it had in these more recent experiments in the absence of any type of workout (Nicastro. 2011). 

References:

• Anderson,  K.D.; Abdul, M.; Steward, O. Quantitative assessment of deficits and recovery of
  forelimb motor function after cervical spinal cord injury in mice.  Exp. Neurol.  2004,  190,
  184–191.
• Kennel,  P.F.; Fonteneau, P.; Martin, E.;  Schmidt,  J.M.; Azzouz, M.; Borg, J.; Guenet,  J.L.;
  Schmalbruch, H.; Warter, J.M.; Poindron, P. Electromyographical and motor performance studies
  in the pmn mouse model of neurodegenerative disease. Neurobiol. Dis. 1996, 3, 137–147.
• Nicastro H, Zanchi NE, da Luz CR, de Moraes WM, Ramona P, de Siqueira Filho MA, Chaves DF, Medeiros A, Brum PC, Dardevet D, Lancha AH Jr. Effects of leucine supplementation and resistance exercise on dexamethasone-induced muscle atrophy and insulin resistance in rats. Nutrition. 2013 Apr;28(4):465-71. Epub 2011 Nov 12.
• Qin J, Du R, Yang YQ, Zhang HQ, Li Q, Liu L, Guan H, Hou J, An XR. Dexamethasone-induced skeletal muscle atrophy was associated with upregulation of myostatin promoter activity. Res Vet Sci. 2013 Aug 29.
• Vieira, N.M.; Bueno,  C.R., Jr.; Brandalise, V.; Moraes,  L.V.; Zucconi, E.; Secco, M.; Suzuki, M.F.; Camargo, M.M.; Bartolini, P.; Brum, P.C.; Vainzof, M.; Zatz, M. SJL dystrophic mice express a significant amount of human muscle proteins following systemic delivery of human adipose-derived stromal cells without immunosuppression.  Stem Cells  2008,  26, 2391–2398.  
• Zanchi NE, Guimarães-Ferreira L, de Siqueira-Filho MA, Felitti V, Nicastro H, Bueno C, Jr, Lira FS, Naimo MA, Campos-Ferraz P, Nunes MT, Seelaender M, de Oliveira Carvalho CR, Blachier F, Lancha AH, Jr. Dose and Latency Effects of Leucine Supplementation in Modulating Glucose Homeostasis: Opposite Effects in Healthy and Glucocorticoid-Induced Insulin-Resistance States. Nutrients. 2013; 4(12):1851-1867.

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. 2013; Busquets. 2013).

"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. 2013) 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).

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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. 2013;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. 2013;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.
• Salama AF, Kasem SM, Tousson E, Elsisy MK. Protective role of L-carnitine and vitamin E on the testis of atherosclerotic rats. Toxicol Ind Health. 2013 Feb 13.
• Viganò A, Mora S, Brambilla P, Schneider L, Merlo M, Monti LD, Manzoni P. Impaired growth hormone secretion correlates with visceral adiposity in highly active antiretroviral treated HIV-infected adolescents. AIDS. 2003 Jul 4;17(10):1435-41.
• 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.