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. 2012)

"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. 2012)

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. 2012)

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. 2012)

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. 2012)

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. 2012)

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. 2012; 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,
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• 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. 2012 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. 2012 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. 2012; 4(12):1851-1867.

Eating by The Clock: Is Eating 3x a Day Making You Fat? Scheduled Feeding Results In Adipogenesis in Growing Rats - Less Lean, More Fat Mass.

Image 1: Simplified illustration of the
purported function of ghrelin in the
human body (healthhabits.ca)

Eating whenever you feel "hungry" (I suppose most of us don't even know what hunger is, anyway) seems to be a bad idea in a society where the abundance of readily available (low quality) foods turns out to be at the heart of many of the health problems we are facing these days. Thus, it seems counter-intuitive that our standardized 3xday feeding schedules, which, if nothing else, do prevent us from eating nutritional bullsh** 24/7, could in fact be making us fat. This, i.e. the finding that eating at fixed times 3x a day induces weight, or more specifically, fat gain in rats on an other wise (calorically) unrestricted diet, is yet the surprising result of an investigation (Verbaeys. 2011) the results of which have been published in the March issue of the American Journal of Physiology.

For 14 days the scientists fed growing Wistar rats on either a 3x a day feeding schedule (three meals at fixed time points: at dark onset and at 3 and 6 h after dark onset) or ad libitum (rats had access to food whenever they were hungry). The hypothesis was that the scheduled feeding procedure would influence the rats' ghrelin levels and would thus affect food consumption and utilization. Yet, despite an anticipatory increase in acetylated ghrelin levels before 'feeding time', the rats on the imposed feeding-schedule did consume the exact same amount of calories as their ad libitum feed peers (note: when food was available the rats were allowed to eat to satiety). Notwithstanding, they did display "a slower growth rate compared to ad libitum fed controls", which would suggest that eating on a 3x a day schedule ain't the optimal feeding schedule to "grow" (muscle if you are an athlete, overall if you are still an adolescent or think of your children).

Figure 1: Acetylated ghrelin levels in ad libitum (control) and scheduled fed rats (data adapted from Verbaeys. 2011)

Overall growth aside, the periods of intermittent fasting had detrimental effects on the body composition (cf. figure 2; data measured reliably by DEXA scans) of the growing rats, as well:

[...] scheduled-fed rats exhibiting a feeding pattern with intermittent fasting periods had a higher fat/lean ratio compared to ad libitum fed controls.

In this context, it is particularly interesting that these effects are not attribuable to increased ghrelin levels, exclusively, as an additional ghrelin-treated control group did show a concomitant increase in both fat and lean mass (they were just bulkier), "but the fat/lean ratio was not significantly increased compared to controls".

Figure 2: Body Composition Changes in ad libitum (control), scheduled-fed rats and rats that were injected with endogenous ghrelin (data adapted from Verbaeys. 2011)

Taken together, this data leads the scientists to conclude that

[these] results suggest that scheduled feeding, associated with intermittent fasting periods, even without a nutrient/calorie restriction on a daily basis, results in adipogenesis.

In the above statement, the occurrence of the words "intermittent fasting" and "adipogenesis" within the same sentence, certainly is something Martin Berkhan from LeanGains.com won't like. In Berkhan's defense it has yet to be said that an "intermittent fast", as Berkhan proposes it in his leangains program, is somewhat different from the food-deprived periods between two feedings the rats in this study were exposed to. In a dieting scenario elevated corticosteroid levels, as they were observed in the scheduled fed group (cf. figure 2), are inevitable, anyhow (note: low-carb high protein diets are notorious for raising cortisol). And what most people forget: cortisol breaks down fat, as well. So after all, its not about eradicating corticosteroids all together, but rather about managing them.

Figure 3: Glucose, triacylglycerol and corticosterone levels in rats after 14 days of ad libitum (control), scheduled feeding or ghrelin injection. (data adapted from Verbaeys. 2011)

What this study does show us, is that elevated cortisol levels can become an issue, especially around meal times. If you mimic the rats in this study and starve yourselves up to the next "scheduled feeding", you will exhibit the same anticipatory excitement the rodents did (note: you probably will do so if you worry too much about calories and your next meal making you fat, as well). Your corticosterone levels rise (or keep elevated), your body stops building and starts eating away muscle and you increase the risk of storing what you are about to eat as fat, instead of muscle. Keep that in mind when you lay out your diet. Whether you are going to bulk or going to cut - listening to your body (assuming that you still know what hunger is) may hold the key to success.

My assumption that higher cortisol levels and not elevated ghrelin levels (cf. figure 1) could be the main culprit, here, are corroborated by the results of a recent investigation into the role of baseline leptin and ghrelin levels on body weight and fat Mass changes after an energy-restricted diet intervention in obese women, which revealed that "[o]bese women with higher leptin and lower ghrelin levels at baseline seem to be more resistant to FM loss"(Labyen. 2011). It is also supported by a Finnish study (Cederberg. 2011) reporting that

An increase in UAG [unacetylated ghrelin] level during the exercise intervention [in 552 healthy young men (mean age 19.3 and range 19-28 years)] was associated with reduced weight, fat mass, fat %, and waist circumference, but not with fat-free mass. [...] Associations of changes in UAG level with waist circumference were significantly stronger than with fat % after the adjustment for confounding variables.

Bottom line: Listen to the tummy rumbles, if you want to gain, ignore them and do some Yoga (or use other stress management techniques) if you want to lose weight, but by all means: Don't stress yourself, if you want to get/stay lean!

The Hair-to-Belly Cortisol Connection: Hair Analysis As Reliable Tool for Longterm Cortisol Analyses

Figure 1: 3D model of molecular
structure of cortisol (Wikipedia)

Although cortisol is probably one of the most important hormones, and a lack of cortisol will provoke symptoms which can range from simple fatigue to death, chronically elevated levels of this essential corticosteroid are associated with increases in visceral obesity and its entailing metabolic pathologies, which are commonly described as "the metabolic syndrome". The results of a very recent study (Manenschijn: 2011), that was published in the medical journal Steroids, may thus turn out to be a valuable tool in the a:nalysis of the underlying causes of pathological obesity and its consequences.

Manenschijn et al. collected hair samples of 195 healthy individuals, 9 hypercortisolemic and one hypocortisolemic patient and measured the cortisol levels in the hais and saliva of their subjects. They then correlated the data with waist and hip circumferences, as well as blood pressure values of 46 of the healthy subjects and found:

[...] a positive correlation between hair cortisol and both waist circumference (r = 0.392, p = 0.007) and waist-to-hip ratio (WHR) (r = 0.425, p = 0.003). No correlations were found between hair cortisol levels and BMI, blood pressure or age. There was no decline in cortisol levels in six consecutive hair segments. Hair cortisol levels were elevated in patients with known hypercortisolism (p < 0.0001).

This is an interesting result. On the one hand, it does show that hair analysis may serve as a valuable tool in the assessment of longterm cortisol levels (as long as confounding factors, as washout effects due to certain shampoos and other hair care products are taken into consideration), on the other hand, however, it raises the question whether the well accepted association between stress, high cortisol and elevated blood pressure is practically relevant, if longterm cortisol levels remain within certain "healthy" limits.