Intermittent Thoughts On Intermittent Fasting - AMPK III/III: Natural Rythmicity for Maximum Fat & Minimal Muscle Loss

Image 1: The quest for fat loss, muscle size, health and longevity reminds me of the famous egg-laying wool-milk-sow.
(img austria-lexicon)

At the end of last weeks installment, I provided you with an extensive, yet obviously "incomplete" list of AMPK-"promoters", among which the  organosulfur compound alpha lipoic acid (ALA) turned out to be of chief interest in the subsequent enjoyable and inspiring "intellectual intercourse" I had with Banga, Dr. J, Bomb Jack and of course Lerner in the comments section of the post. Now, the fundamental question that arose from this discussion was how supplementation with ALA (or agents with similar effects on 5' adenosine monophosphate-activated protein kinase expression) could be used as a tool to promote fat, not weight loss while preserving or even building lean muscle mass and optimizing health and longevity - basically it's the quest for the egg-laying wool-milk-sow, an imaginary animal and a commonly used German metaphor for a jack-of-all-trades device. And I can assure you that an ultimate solution to this problem is just as hard to find as this mythical animal an image of which you can see on the right.

When AMPK is the good guy, mTOR must not necessary be the bad guy

We have learned in the past couple of installments that its modulatory effect on the AMPK/mTOR seesaw, which, even in your average citizen of the affluent Western hemisphere, is oftentimes imbalanced towards the "anabolic" mTOR side, these days. Against that background I have pointed towards the myriad of beneficial metabolic effects of AMPK activation, which could in fact ameliorate, if not reverse, many of the ailments that have befallen our fat-anabolic (remember without mTOR, fat cells cannot differentiate, cf. Bell. 2000, where blockage of the mTOR pathway with rapamycin inhibited adipocyte differentiation) society.

Illustration 1: Lifestyle factors like nutrition, nutrient timing, sleep etc. determine the fundamental balance between AMPK and mTOR, but supps can skew / tweak the balance

I hope that highlighting the merits of AMPK in the lat two installments of the series did not give the impression that mTOR its anabolic counterpart on the seesaw (cf. illustration 1) was "useless" or even dangerous, as the sheer amount of recently published studies on the implication of mTOR in the etiology of cancer could make you believe.

Image 2: A breast cancer cell (img from The Guardian); if you want it (not yourselves!) to live longer, don't feed it with leucine, Ladies!

The mTOR-cancer connection: In the course of these first 10 months of 2011 more than 16,000 studies have been published that focus or at leas mention the involvement of an (over-)stimulation of the mTOR pathway in cancer development. It is yet plain short-sighted stupidity to believe that measures like dietary leucine restriction (and subsequent down-regulation of the mTOR pathway) would protect you, let alone cure cancer. It is certainly correct that mTOR promotes proliferation in cancer cells, but it does so in about every other cell in your body. Moreover, studies on the effect of leucine deprivation on mTOR signalling in breast cancer patients showed that not only was "leucine restriction is not sufficient to inhibit mTOR signaling in most breast cancer cell lines", but was "associated with activation of survival molecule Akt, making leucine deprivation an undesirable approach for breast cancer therapy" (Singh. 2011). And with regard to the use of "true" mTOR inhibitors such as rapamycin, David Sabanti remarks in a recent review:

As induction of apoptosis rather than cytostasis is increasingly considered a prerequisite for an effective anticancer agent, it will be crucial to understand when rapamycin has such effects and where it does not, and to learn how to trigger apoptosis with additional therapies.

Now, the use of novel pharmacological mTOR-inhibitors aside, any dietary approach (such as leucine deprivation) would obviously prevent apoptosis, because it would act via mTORC1 inhibition to increase Akt-expression and (cancer-)cell survival (Sun. 2005; O'Reilly. 2005). Would you risk your "metabolic currency", i.e. your muscle, and sacrifice quality of life for the futile hope that you could thusly keep cancer "at bay"? I hope not, because in that case you still have not grasped the fundamental idea that in life its not about black and white, but about black and white and a balance between the two.

This takes us back to the egg-laying wool-milk sow or, without the metaphorical ornamentation, the issue of losing fat weight, while gaining muscle weight. From all you have learned before, it appears quite obvious that - intermittendly fasted or not - gaining muscle and losing fat literally at the same time is virtually impossible. On the other hand, you could obviously first milk and fleece your egg-laying wool-milk sow and then collect the eggs, it has been laying before you grabbed, milked and fleeced it. I assume you will notice that, again, this is a cycle: milk, fleece, collect eggs,... milk, fleece, collect eggs, ... fast, train, feed... fast, train, feed. As you see, nature is not very inventive in her fundamental concepts. She will always rely on her tried and proven cyclicality. Now, if we cannot escape that, we can at least try to tweak it depending on our goals. While for Mr. Supp in illustration 1 this would be only a step to the left to help Mr. AMPK to get the better of Mr. mTOR) or a step to the right to help Mr. mTOR gain control over the AMPK/mTOR seesaw. For us, in the real world, the most obvious thing we could resort to in order to achieve similar results are supplements.

When to take what to optimize fat loss and minimize muscle loss in the AMPK phase

In the comments related to the last installment, Bomb Jack suggested that taking ALA during a fast to jack the naturally high AMPK level up even more, would theoretically make sense, but he mentions that he had seen "studies about [ALA] causing lean tissue loss on the long run". One of these studies was conducted by Yi Wang et al. in 2010 on 24 month old male C57BL/6 mice (Wang. 2010). Half of the mice received 0.75% alpha-lipoic acid in their drinking water for one month, the rest of the mice served as a unsupplemented control.

Image 3: 22 month old C57BL/6 mouse, in view of the fact that mice live ~2.5 years this is already a granny; in other words, the 24 month old mice in the Wang study were really old.

How much is 0.75% in the drinking water of mice in human equivalents? To calculate that you need to know that the mice in the study weighed ~30g, that the average mouse drinks ~5.8ml per day (Bachmanov. 2002) and that a 0.75% solution means that 100ml contain 0.75g of the given solute. Now you calculate the absolute dose per day, which would be 5.8ml/day * 0.75g /100ml = 0.435mg/day divide that by 30 to get the dose per gram of body weight and multiply it by 1000 to get the dose per kg => 14.5mg/kg. To get the human equivalent dose (HED) you then use the formula I explained before and your calculator will tell you that the HED of 14.6mg/kg in a mouse would be roughly 1.2mg/kg in a human being (~95mg for the average adult).

Although the human equivalent (~95mg/day; cf. red box above) of the dose the mice received was way less than most commercially sold supplements contain and by far less than the 800mg - 1,200mg of alpha lipoic acid that has been used with positive results in many trials that involved diabetic patients, the results the Wang et al. observed were pretty pronounced. In the ALA group...

• food consumption decreased: -18% - 4.50 ± 0.30 g/d vs. 5.50 ± 0.30 g/d in control mice
• body weight decreased: -15.8% - 5.27 ± 0.62 g total body weight loss
• energy expenditure increased:  +25% - 7.64 ± 0.10 kcal/kg^0.75/h vs. 5.90 ± 0.10 kcal/kg^0.75/h
• glucose utilization increased: +10% judged by the respiratory quotient
• insulin sensitivity increased: -47% area under the curve in glucose tolerance test
• mitochondrial biogenesis increased: +138% relative abundance of mtDNA content
• PGC-1α* in skeletal muscle increased: + 80.0% 
• GLUT-4 in skeletal muscle increased: + 105.0%
  
  * PGC-1α ramps up thermogenesis, stimulates mitochondrial biogenesis, promotes the remodeling of muscle tissue, controls lipid and glucose metabolism (Liang. 2006)

And all that was accompanied by significant increases in AMPK and decreases in mTOR and P70S6K phosphorylation. In view of the results of the Wilson study, which showed that a much smaller (~40%) increase in AMPK activity went hand in hand with decrease in protein synthesis we can safely assume that the latter is (unfortunately) responsible for both the desirable increases in energy expenditure, glucose metabolism and mitochondrial biogenesis, as well as the not so desirable loss of appetite (you do not need to eat, when you run on stored substrate) and body mass (cf. figure 2).

Figure 1: Relative changes in phosphorylation status of AMPK, mTOR, p70S6K and 4E-BP1 in old mice supplemented with 0.75% ALA in their drinking water for 1 month compared to unsupplemented control (data calculated based on Wang. 2010)

On the other hand, the decrease of the amount of phosphorylated Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), of which Anthony et al. have shown in 2002 that its phosphorylation (which usually occurs consequent to the activation of the mTOR pathway) is not necessary for the increase in protein synthesis that occurs upon leucine administration (Anthony. 2002), was not statistically significant. This could be important, as dephosphorylation of 4E-BP1 and not decreases in p70S6K appear to be hallmark features of cancer cachexia, i.e. profound muscle loss in cancer patients (Tisdale. 2008).

Figure 2: Absolute (left) and relative (right) changes in body composition in old mice supplemented with 0.75% ALA in their drinking water for 1 month compared to unsupplemented control (data calculated based on Wang. 2010)

And indeed, if you, as a diligent student of the SuppVersity who knows about the subtle differences between absolute and relative data, do not focus on the absolute (figure 3, left) but rather on the relative (firgure 3, right) changes in body composition, you will have to concede that the mouse grandpas underwent a transformation you would usually expect to see in the course of the contest preparation for a bodybuilding show. While the mice "on ALA" did lose ~20% of their body weight, they also cut down from 18% to 9% body fat and (-60% fat mass) and increased the relative amount of lean mass from 74% to 87%. For a 300lbs bodybuilder this would be like cutting 32lbs of fat while losing "only" 16lbs of muscle, to end up 48lbs lighter and with a body fat percentage of 9% at 252lbs - not yet really stage ready, but certainly not bad, given the fact that he would not even have to diet to achieve that result (remember: his appetite would have decreased and his energy expenditure increased).

Rythmicity is the key when it comes to seesawing and supplementation 

The mouse vs. human issue aside, the Wang study has another caveat in terms of drawing conclusions regarding the use of ALA and other AMPK promoters during an intermittent fast - the mice did not fast ;-) Reason would dictate, though, that ingesting ALA with drinking water 24/7 on a dietary regimen with constant food availability would actually be a disadvantage compared to taking ALA specifically at the onset of the fast (i.e. when your last meal would be digested) to keep AMPK, which would already been raising at that point (and thus mTOR declining) maximized in the course of the fasting period. On the other hand, taking ALA or any other AMPK promoter after your workout and before your meal would appear to be plain out stupid, as intermittent fasting does only make sense if you really reap the maximal anabolic benefit from the short feeding window. Against that background, popping alpha lipoic acid post workout and thus potentially increasing AMPK appears (remember that we do not have any studies explicitly investigating the effects of taking ALA post-workout vs. at other times of the day, so that these are just yet to be validated hypotheses!) to be counter-indicated, as it would potentially blunt the mTOR and p70S6K response to exercise and food-intake.

Image 2: R- and S- isomer of alpha lipoic acid (Shay. 2010)

"Alpha lipoic acid, yeah! But can I buy the cheap racemic mixture or is it worth paying the extra bucks for the R-ALA?" Actually, it was Daniel Spasic who posted a similar question on my Facebook pinwall and thus made me look into the purported advantages of R-ALA, again. 

I mean we all "know" that R-ALA is the preferable form, but do you remember where you know that from? 

Well, me neither and so I dug back into the host of studies BASF was doing back in the 1990s, until their business department finally realized that a natural anti-diabetes drug is not only non-patentable, but could also compromise the sales of patentable pharmacological drugs like Chlorpropamide (patented in the mid-1980s; cf. DrugPatentWatch) which happens to be made of Propylamine, which in turn - you guessed it - is produced by BASF and sold to BigPharma and the Agrobusiness who use the same ingredient to produce of Prochloraz, and other well-known fungicide... but I am getting taken away, here.

Image 2: The scientific
evidence pro R-ALA
is quite conclusive;
the S-isomer, however,
could potentially negate
its benefits and is
useless, at best!

The main point is that there is clear-cut evidence that the R-isomer has not only a longer half-life and a greater bioavailability (Herrmann. 1996), but is also the "true" anti-oxidant: In a 1997 study by Streeper et al., for example, S-lipoic acid had no effect on insulin-mediated glucose uptake in obese Zucker rats, while R-lipoic acid increased the latter by +64%. In the same study chronic intake of R-lipoic acid reduced plasma insulin and free fatty acid levels. S-lipoic acid, on the other hand, increased insulin levels and had no effect on free fatty acids - probably a direct consequence of the reduced expression of glucose-transporter protein (GLUT4) the scientists observed in the S-ALA supplemented rats. This may now sound like the racemic mixture, which is in fact the ~50/50 R-ALA/S-ALA mixture you can buy for a few bucks at every GNC would be toxic - this certainly ain't the case. On the other hand, would you buy an anticoagulant with an with vitamin K in it, i.e. a product with two active ingredients with partly antagonistic effect? I don't think so...

And for those of you who like to think in images: Taking ALA right at the beginning of your short feeding window would be as if Mr. Supps from illustration 1 took a step to the left, just when Mr. mTOR turn on the right of the seesaw was about to gain momentum - Mr. Supps with his alpha lipoic acid would be a real spoilsport, then, wouldn't he? If, on the other hand, Mr. Supps knows how to play the game, he will follow the AMPK/mTOR seesaw's natural rythm, grab some alpha lipoic acid and step to the left, when, at the beginning of the postprandial phase (~2h after the last meal), it actually is Mrs. AMPK turn, wait until she has had her share and then jump to the right in order to hand Mr. mTOR a leucine-rich protein shake. By doing this, i.e. taking ALA at the onset of the fast, ~2h after the last meal and a leucine rich (post-workout-)protein shake to ramp up protein synthesis right at the beginning of the feeding phase, the natural rythm would not only be preserved, it would also be amplified. And what would translate into a more energetic seesawing in our metaphorical world or Mr. Supps, Mrs. AMPK and Mr. mTOR, should translate to improved fat loss during the fast and a pronounced muscle anabolic response to the daily "refeeds", as you may well call your meals if you compress the feeding window to <6h. If the mice in the Wang study had gotten this rythm right, they would probably have made it to the Mouse-Bodyduilding Masters ;-)

The "hungry" side of neuronal AMPK activation

Before I end, this pretty epic (in terms of the details we have covered) yet not very productive (in terms of how much ground we have made) installment of the Intermittent Thoughts, I want to briefly mention a very important and actually completely logical, since natural difference between the effects of increased AMPK phosphorylation in skeletal muscle and increased AMPK phosphorylation in the brain. While the former triggers all the fat-loss friendly adaptations you read about in this, as well as in previous Intermittent Thoughts, the later will have you forage through your fridge in no time - regardless of whether it's feeding time or not ;-)

Illustration 2: The differential role of hypothalamic, liver and skeletal muscle AMPK expression (Long. 2006)

You will certainly remember from previous installments that, in our eukarocyte ancestors, AMPK was the major cellular energy sensing mechanism and has been preserved by humans (and all other mammals) even in these days of nutritional abundance. AMPKs activation is thus a signal for your body that the respective cell is starving (to be precise that the intracellular ratio of ADP+AMP to ATP is rising) - if your brain cells are not already starving, it should therefore be obvious that, contrary to AMPK signalling from the muscle tissue, which usually means "Hey, if you want me to do some work for you, you better provide me with some energy! I see those lazy adipocytes over there have more than enough stored energy...", AMPK signaling from the brain indicates an acute emergency - after all, the biological equivalent of "women and children first" is "brain first! Who cares about the rest?" It should thus not surprise you that

• an increase in cerebral AMPK phosphorylation results in increased food intake (Andersson. 2004), while dephosphorylation decreases food intake (Kim. 2004); 
• genetically modified mice with no AMPK activity in the AgRP neurons were leaner and had an increased energy expenditure compared to wild-type mice (Claret. 2007)
• ghrelin, the hunger hormone, triggers AMPK phosphorylation in the brain and thus increases food intake (Kola. 2008)

With all that being said, you are probably happy to hear that alpha lipoic acid decreases or at least counters potential increases in AMPK activity in the hypothalamus (Kim. 2004).

Update (10/29/2011) - The two AMPK-isoforms: As Mounier et al. report in a very recent paper the two isoforms of AMPK, i.e. AMPKα1 and AMPKα2 have very distinct effects on the mTOR induced increase in muscle protein synthesis, and thusly, muscle size (Mounier. 2011). As the scientists point out, AMPKα1 plays "a predominant role in the control of muscle cell size" (meaning it prevents exuberant hypertrophy), while AMPKα2 mediates "muscle metabolic adaptation" (increased glucose uptake, mitochondrial biogenesis, etc.), of which we have learned that they are so vital for our metabolic health.

Close your eyes and lean out

On that note, I will close today's lesson with an some interesting information from a study Jonathan P. has brought back onto my radar, recently. It's a study by Dworak et al. on ATP changes during sleep (Dworak. 2010), which underlines the importance of sleep, specifically when you want to lose weight, because at the onset of sleep, the reduction in neuronal activity goes hand in hand with a surge in brain ATP levels, which (as you have learned today) will reduce cerebral AMPK phosphorylation and its negative metabolic consequences. Prolonged waking, on the other hand, has been shown to increase AMPK activity in the brain (Wigren. 2009). So, what are you waiting for? That's all for today... lights out ;-)!