Intermittent Thoughts On Intermittent Fasting - AMPK II/III: Leucine, HMB and a Glimpse on Other AMPK Modulators

Image 1: You pick a health, diet or diabetes supplement and I find the study that shows that in one way or another its effect is related to AMPK ;-)

I ended yesterday's installment of the Intermittent Thoughts on Intermittent Fasting Series on a pretty bold statement about the benefits of preworkout BCAA supplementation that would, at first sight, contradict common sense, or rather what common sense would dictate based on all you have read about the beneficial effects of BCAA supplementation on mTOR-related muscle protein synthesis (MPS) and the complementarity of mTOR and AMPK as regulators of anabolic (e.g. MPS, adipogensis ,etc.) and non-anabolic "scraping, rebuilding, recycling and repairing" processes. Since, after all, Bomb Jack, who posted a comment on last weeks installment of this series, is right: It would be logical that supplementation with BCAAs (he mentions HMB specifically) during the fast should result in dephosphorylation (~deactivation) of AMPK and thus negate its desirable effect on (metabolic) health.

And in fact, in the Wilson study I wrote about on Saturday the postprandial increase in AMPK phosphorylation, was blunted by the provision of carbohydrates, leucine or a combination of both (cf. yesterday's news) and you would assume that HMB supplementation would do the same, but the latter is - at least for chronic supplementation with low amounts (320mg/kg in rats ~ 52mg/kg in humans) of HMB - not the case (Pimentel. 2011), as the data I plotted in figure 1 clearly shows:

Figure 1: Effect one month of saline (control) or 80mg/day HMB on mTOR and AMPK phosphorylation and GLUT-4 expression in extensor digitorum longus (EDL) muscle of rats (Pimentel. 2011).

In the Pimentel study, there was, if anything, a non-significant increase in the AMPK and its purported downstream effect on GLUT-4 mediated glucose uptake  - both of which common sense would have told us to be compromised by HMB supplementation. While the lack of information on the "timing" or, more specifically, the interval between the last feeding and the intragastric administration (gavage) of 320 mg/kg body weight of HMB is a drawback in view of the significance of these results in an intermittent fasting context, rats usually eat at night and thus the administration of the 80mg of HMB (the rats weighed only 250g) "daily at the same time (during the light period)" will probably have coincided with a "fasting" period.

How can we explain that mTOR expression increased, while AMPK remained constant?

Are the different result a consequence of the metabolic magic of HMB? Well, before we analyze that in detail, there is another significant difference, we have to account for - in fact, a much more obvious one, which the amount of amino acids the rats were given in the Wilson and the Pimentel study, respectively (cf. figure 2).

Figure 2: Dosage, not type of supplement would be the most probable explanation for the different effects of leucine and HMB supplementation on AMPK phosphorylation in the Wilson vs. the Pimentol study.

I hope you did not already forget that, the main function of AMPK is to prevent that your cells run out of fuel or, to be precise, to avoid the ratio of "used" energy ADP and AMP (adenosine di- and monophospate) to ATP (adenosine triphospate) to continue to rise beyond a tolerable level. I further assume that you will be familiar with the fact that branched-chain amino acids bypass oxidation in the liver and thus become readily available energy sources for skeletal muscle (Renny. 2011). Now, if you put one and one together the answer seems pretty obvious: If the dosage of amino acids is sufficient (remember that those 270mg leucine are 4x more leucine than the the rats in the Wilson study got for "breakfast") to restore ATP levels to "appropriate" levels, the decrease in the ADP/ATP ratio will allow part of the AMP-activated protein kinase to be dephosphorylated.

According to our current understanding, BCAAs in general and leucine in particular trigger the ATP related decrease in AMPK and the complementary increase in mTOR by two distinct pathways, of which Tokunaga et al. write (Tokunaga. 2004)

[...]leucine stimulates p70α phosphorylation via mTOR pathway, in part, by serving both as a mitochondrial fuel through oxidative carboxylation and an allosteric activation of glutamate dehydrogenase. This hypothesis may support an idea in which leucine modulates mTOR function, in part by regulating mitochondrial function and AMPK.

In plain English: Leucine increases ATP when it is "burned" as fuel and it docks directly to the the non-active site of glutamate dihydrogenase enzyme and thusly increases the conversion of glutamate to alpha-ketoglutarate which in turn can be fed into the citric cycle to ultimately produce ATP.

Is it all about (cellular) energy ...

Figure 3: AMPK phosphorylation in Escherichia coli at different ADP/ATP ratios (data adapted from Xiao. 2011)

In April 2011 Xiao et al. published a study in Nature with some interesting quantitative data on the ADP/ATP ratio, on the one hand, the phosphorylation status of AMPK, on the other (Xiao. 2011). As my plot of the data in figure 3 shows, with increasing ATP levels (at constant ADP levels of 30µM) the phosphorylation of AMP-activated protein kinase in Escherichia coli BL21 cells declines by roughly -20% from 44% at a 30/0 ADP/ATP ratio to 22% at a 30/800 ADP/ATP ratio.

Yet, although these results would confirm the hypothesis that the main reason for the discrepancy is dose, or rather, energy related, and each and every nutrient that could potentially raise ATP levels, would eventually decrease AMPK, this still does not explain the increase in mTOR Pimentel et al. observed, despite (statistically non-significant) increases in AMPK.

... or is there a place for the "magic" of HMB?

As you probably know, beta-hydroxy-beta-methylbutyrat (HMB) is an oxidation product of leucine and / or its keto-acid alpha-ketoisocaproate (KIC) (Koevering. 1992). In 1998 Lembert et al. found that even KIC is not a direct substrate for ATP production, instead "KIC must transaminate with glutamate or glutamine to yield alpha-ketoglutarate and leucine" (Lembert. 1998). We may thus assume that similarly HMB cannot be used (directly) to restore cellular ATP pools. Moreover, HMB is thought to be the second (non-energetic) pathway by which leucine acts on protein synthesis / breakdown. According to a 2011 review of the literature by Zanchi et al. (Zanchi. 2011)

Nissen et al. (1996) suggested that HMB or some other metabolite (since there is no specific inhibitor to BCAT) is the main component responsible for the anti-catabolic effects of HMB because when adopting inhibitors of BCAA transamination, the only BCAA capable of anti-proteolytic effects is leucine, which undergoes a process capable of generating HMB (Slater and Jenkins 2000). Such effects were not observed when other BCAAs were tested (isoleucine and valine), suggesting that HMB or some metabolite may be the key element in promoting the [anticatabolic] effects.

When usually 5% of the dietary leucine is metabolized into HMB (Wilson. 2008), and these 5% are responsible for the non-ATP dependent effects on phosphorylation of mTOR, p70^S6k, and 4E-BP1 of leucine (Eley. 2007), it is no wonder that chronic intake of 80mg of HMB did stimulate mTOR in the absence of increased ATP levels (which would obviously have led to a decrease in AMPK expression that was not present in the Pimentol study), while 270mg leucine, yielding only 13.5mg HMB, did not stimulate mTOR, but was (ab-)used as a substrate to increase cellular ATP levels, thusly reduced AMPK levels and increased protein anabolism - different pathways, similar results: an increase in net protein synthesis.

Figure 4: Simplified illustration of the two distinct pathways by which leucine can work its muscle protein synthetic (MPS) magic and a hint on the compensatory (/) / amplifying (+) effects of exercise.

There is however, a third major pathway to the metabolic effects that are brought about by common intermittent fasting programs and this third player makes things even more complicated (cf. figure 4) - it's exercise! You probably remember from yesterday's installment that

1. during exercise in the fasted state temporarily AMPK increases and the energetically costly muscle protein synthesis (MPS) is reduced, while
2. after exercise (regardless of whether it was performed fasted or not, cf. "Glycogen-Free Growth") muscle protein synthesis increases due to an exercise-induced stimulation of the mTOR protein synthetic cascade

Before we dig deeper into this modulatory effects of different modes of exercise in the next installment of the Intermittent Thoughts on Intermittent Fasting, however, I want to conclude today's thoughts with a preliminary list of supplements / medications that have been shown to modulate the phosphorylation state of 5' AMP-activated protein kinase.

Image 2: If you insist on trying HMB, don't be stupid and buy a capped products, the prices for bulk HMB powder have lately been crushed - a major European carrier, for example, sells 250g at <13€ atm; HMB is thus cheaper than BCAAs, which cost 16Euros in the small 250g pack - did you hear me say that even 13€ is too much, no - you must be mistaken ;-)

"Should you prefer HMB over leucine as a dietary supplement to promote lean mass gains and prevent muscle loss during the fast?" I assume this is a question many of you will now be pondering about. My answer to this question would be "NO!" Firstly, if you are no construction worker or pursue a similar physically demanding profession, the fear of losing muscle (which is different from "feeling flat", my bodybuilding friends ;-) during a ~16h fast is hilarious, which means that BCAA, Leucine or HMB supplementation, while you sitting fasted at your desk in the office is simply unwarranted. Secondly, when you are exercising the increased energy demand will negate / compensate the negative effect the increase in ATP has on AMPK activity. And thus, thirdly, a large bolus of leucine (or a complete BCAA or EAA product) taken pre-workout will not only ward off proteolysis (as HMB would do) it will also provide the necessary energy to train harder and thus help to increase the exercise induced stimulus on protein synthesis.

All that and the absence of conclusive scientific evidence that would demonstrate the superiority of HMB supplementation over the provision of adequately dosed BCAA or EAA mixtures (it stands to reason that you cannot compare 3g of HMB to 3g of BCAA) are arguments against the use of β-Hydroxy β-methylbutyric acid. If you wanted to try it, anyway (and have no problem swallowing a powder that tastes like poison), the prices for bulk-powders have gone through the floor, lately ;-)

How to modulate AMPK "artificially" -  supplements, medications, hormones and more

In view of the fact, that the discussion of the effects of leucine (BCAAs and HMB) alone took much longer than I had expected and this whole episode took a different turn than I would have expected, the following list is more a preliminary overview than a comprehensive explanation of the effects of various supplements, medications, hormones and hormone-like substances on the AMPK. The latter will follow, as promised, but for today, you will have to content yourselves with what I would like to call a sneak peak on the AMPK-mTOR modulation handbook of which I hope that it will be one of the outcomes of all the past and future work that is going into this series ;-)

AMPK promoters:

• Adiponectin (Yamauchi. 2002; Handy. 2010; Iwabu. 2010)
• Alpha lipoic acid (Lee. 2005; Wang. 2010; Packer. 2011)
• Artemisia sacrorum Ledeb (Yuan. 2010)
• Aspirin (Sung. 2011)
• Astragalus (Zhou. 2009)
• Berberine (Ma. 2011)
• Brain-derived neurotrophic factor (Matthews. 2009)
• Caffeine (Jensen. 2007; Spirydopulos. 2008; Egava. 2009) 
• Capsaicin (Kim. 2010)
• Catechins / green tea (Huang. 2009; Murase. 2009; Reiter. 2010)
• Chromium (Wang. 2009)
• Ciliary neurotrophic factor (CNTF) (Watt. 2006)
• Cinnamon (Huang. 2011)
• Conjugated Linolic Acid (CLA) (HSU. 2011)
• Coptidis Rhizoma (CR), the dried rhizomes of Asian herbs (Egawa. 2011) 
• Cordiceps sinensis (Wong. 2010)
• Creatine (Ceddia. 2004)
• Curcumin (Kim. 2010)
• DHA (Jing. 2011)
• Ecklonia Cava (Kim. 2010)
• Garlic (Lee. 2011)
• Ginseng (Juang. 2010)
• Glucosamine (Kong. 2009)
• Grape seed extract (Meeprom. 2011)
• Hibiscus sabdariffa extract (Yang. 2010)
• Interleukin-6 (Kelly. 2009)
• Kidney bean extract (Lee. 2009)
• Malva verticallata seeds (Jeong. 2011)
• Metformin (Zhou. 2001; Freyer. 2002; Ohira. 2009)
• Mulberry extract (Tsuduki. 2009)
• Nicotine (Cheng. 2007)
• Prostaglandin E2 (Zhu. 2011) 
• Pu-erh tea (Way. 2009) 
• Quercitin (Jung. 2010)
• Raishi Mushroom (Lane. 2011)
• Resveratrol (Fullerton. 2010; Lin. 2010; Breen. 2008)
• Tiozolidinediones (diabetes meds. like rosiglitazone) (Freyer. 2002; LeBrasseur. 2005; Yang. 2011) 
• Vanadium (Hwang. 2011)

I still have two things to add to this list, firstly, this list is the result of a VERY cursory and 100% random search and is not even intended to be complete (at this time ;-). The intention (at least for in this installment) is to show you that an overwhelmingly large percentage of purported health supplements, diabetes and obesity treatments work via the AMPK pathway. And, secondly, I decided to limit the references to 1-3 per compound, even if in cases such as Metformin, ALA & Co the number of relevant studies is probably >500. Therefore you better consider the given references as evidence that I did not make up any associations between compound X and AMPK phosphorylation - and, if you want to know more before the release of the next installment, I suggest you go to PubMed and enter the respective keywords and do some digging on your own (your SuppVersity homework of the day - so to say ;-)

I hope you do not mind that I did not manage to tackle the effects of sleep and exercise in this installment, as I had originally intended. It is, after all, the central characteristic of this series that I sit down in front of the computer and start thinking at point "A", then I dig, here, get distracted there and follow up on "A1" to "A743", so that the output is by no means as structured and straight forward as my lectures and seminars or my SuppVersity blogposts on isolated topics... so, I can only hope that you enjoyed the turn this installment took (at best, because you learned something new) and in the unfortunate case that you did not enjoy what you have just read, you can at least look forward to the next episode of the Intermittent Thoughts on Intermittent Fasting Series ;-)