8x Increase in "Mitochondria Building" Protein PGC1-Alpha W/ Medium Intensity Exercise in Glycogen Depleted Elite(!) Cyclists: Training Revolution or Recipe for Disaster?

With only 2-7x increases in PGC1-alpha expression HIIT seems to lag behind compared to this "eat low, train low, gain high" strategy, but not every protein essay that glitters in the petri dish will turn into Olympic Gold in the real world ;-)

As a diligent student of the SuppVersity you should by now have at least a preliminary understanding of how the adaptive machine you call your "body" adapts to the various nutritional and physical challenges most people subsume under the all-encompassing and pretty nondescript terms "diet" and "exercise". Against that background it should not really come as a surprise that researchers from the The Swedish School of Sport and Health Science are soon going to publish the data of an experiment that shows that even (you could probably also say, in particular, although respective evidence is still missing) highly trained athletes can benefit from exercising in a glyocogen depleted state - at least if the yardstick you use to measure the "benefits" is an increase in mitochondrial biogenesis (Psilander. 2012).

Train high, eat low (carb), train low and...?

To elicit the differential effects of 6x10 min bouts of cycling at 60% of the individual VO2max (4min of active rest in between) with normal vs. depleted skeletal muscle glycogen stores, Psilander et al. had their 10 highly trained male national elite level competitive road cyclists and mountain bikers (27.8±1.6 years, 74.7±2.0 kg, 183±2 cm, and 4.9±0.1 l/min VO2Max) perform an 8x4min interval training at 88% of their individual VO2Max ~16.5h before they had to report back at the laboratory on the actual testing day (the intervals were seperated by 4min of active rest, i.e. cycling at 100W+).

Figure 1: Graphical outline of the experimental protocol and its effect on the glyocogen stores of the from the vastus lateralis muscle (based on Psilander.. 2012)

The protocol (see figure 1) was repeated twice, with adequate time in-between and in random order, with the subjects consuming water only and low carbohydrate meals

• low carb meals (LC) were eggs and bacon (0.02 g CHO, 0.6 g protein and 0.8 g fat/kg bw) for dinner and breakfast, providing a total of of <0.04 g CHO, 1.2 g protein and 1.6 g fat/kg bw

before the glycogen depleted trial (LC) and high carbohydrate beverages (maltodextrin-dextrose powder Carbo 134 w/ 1.0g CHO/kg bw) + high carbohydrate meals

• high carb meals (HC) were pasta with meat sauce and lemonade for dinner (1.83 g CHO, 0.53 g protein and 0.14 g fat/kg body weight bw) and oatmeal and orange juice (1.54 g CHO, 0.31 g protein and 0.12 g fat/kg bw) for breakfast and additional bananas with beverage 3,5,7 and 8 for a total of 12.6 g CHO, 0.9 g protein and 0.3 g fat/kg bw

before the glycogen repleted trial (HC).

... get impressive increases in PGC1-alpha, but no AMPK response at all!

As the data in figures 1 & 2 goes to show you the nutritional intervention was not without effect the factual glycogen levels (figure 1, right) and the glucose, insulin and fatty acid levels before and after the workout (figure 2) - and, as you would expect it, the corresponding changes in gene and protein expression in the muscle samples the researchers collected approximately 15 min before the depletion (S1) and test exercise (S2), as well as 3 h after the test exercise:

Figure 2: Free fatty acid levels before depletion (S1) and before (S2) and after (S3) exercise trial, as well as PGC1-alpha and p-AMPK expression (data calculated based on Psilander. 2012)

Now what you probably won't have anticipated, though is the absence of the expected p-AMPK response to exercise in the low glycogen (LC) trial.

"The mRNA content of the master regulator of mitochondrial biogenesis (PGC-1a) was not changed 14 h after depletion exercise (pre-test exercise) but was significantly increased 3h after the test exercise in both conditions (Fig.2). The increase was, however, much more pronounced in LG than in NG (8.1-fold vs. 2.5-fold, P<0.01). The mRNA content of two other regulators of mitochondrial biogenesis (PRC and Tfam) also increased significantly but with no difference between conditions (time-dependent effect, Py0.01; [not shown in my graph]). The mRNA content of genes for oxidative metabolism enzymes (PDK4 and COX I) only increased after LG with a significant difference between the two conditions. The mRNA content of CS, Sirt1, NRF1 and PPAR[-delta] did not change under any conditions." (Psilander. 2012)

Almost 8x elevated levels of PGC-alpha but no change in the "fat burning, GLUT-4 pomoter" AMP-activated protein kinase? How can that be? The answer to this question is actually pretty simple: If the phosphorlyation of AMPK changes in response to changes in the ATP to ADP ratio (the name is misleading, here as scientists have initially believed that the main determinant was the ATP to AMP ratio, which is yet not the case), it should be obvious that it won't change, if the ATP levels are already so low that at most the ADP to AMP, but not the already rock bottom ATP do AMP ratio will be changing.

What happens if your body senses that it cannot fuel his energetic demands with glucose?

In the presence of borderline hypoglycemic glucose levels (the normal range starts at 4.4 mmol/L; after the depleted test the subject were at 4.3 mmol/L!) your body would be ill advised to increase glucose uptake. So if this is not an option the only way to make up for the lack of energy are fatty acids. Unfortunately the amount of fatty acids your skeletal muscle can oxidize is strictly rate-limited by your mitochondrial capacity ... now, I am asking you what's the "natural", the logical and in the case of the 10 cyclists in the study at hand also the factual reaction that will get you out of this mess? Right! To build more powerful mitochondria and thus widen the "bottle neck"! And what's going to do just that? Yeah! The ~8x increase in PGC-1alpha expression you see in figure 2. 

Practical implications: From protein essays to results?  

 Now that we have gotten the mechanisms straight, there is but one question we have to answer - what does that mean for you? When and for whom does it make sense to train with depleted gycogen stores? And in an even broader context - what does that tell us about low-carbing and (intermittent) fasting?

1. 

   Before you even consider making this a staple of your regimen, I would encourage you to read the whole SuppVersity Athlete's Triad Series

   Even (or especially?) for trained athletes competing in largely aerobic sports, training in a state of depleted glycogen store can serve as a viable tool to elicit even higher (2-7x; cf. Gibala. 2009, Nordsorg. 2010. Psilander. 2010) increases in increases (8x!) PGC1-alpha and (allegedly) mitochondrial biogenesis as you would see them in response to high intensity interval training at much lower intensities (but correspondingly longer durations). 
2. Training in a fasted state does not per se guarantee / put you at risk of being glycogen depleted, neither does intermittent fasting and or "training on empty". As long as you replete your glycogen stores after your workouts you won't see similarly pronounced increases in PGC1-alpha in response to "regular" aerobic training at a low intensity. You will, on the other hand, still see increases in AMPK and, what's even more important, you will be able to perform at much higher intensities! A fact that is particularly important for the strength trainees out there.
3. While it may make sense on occasion, and merely based on it's beneficial effects on purported  mitochondrial biogenesis (I don't have to remind you that we don't have any information on whether the increase in PGC1-alpha did even translate into an increase in mitochondrial biogenesis in the absence of adequate glycogen / ATP levels!), I want to reemphasize the scientists very hint that "[l]ongitudinal studies examining protein levels and performance are required" before it can be recommended to include this practice as a staple into your routine!

4. 

   Life is to complex for black-and-white thinking, and so are AMPK, mTOR & co! Learn more in the Intermittent Thoughts.

   Long-term exercise in a glycogen-depleted state without adequate carbohydrate intake and thus glycogen repletion is not for nothing one of the causative factors of the athlete's triad (see Part I & II of the SuppVersity Athlete's Triad Series). I would therefore be very surprised if the long-term outcomes of low-carbing + (intermittent) fasting w/out regular glycogen repletion would be anything but negative, regardless of its beneficial effects on PGC1-alpha. After all, the study at hand clearly shows that you will also be missing out on the benefificl effects of increased p-AMPK expression of which you know based on what you have read in the Intermittent Thoughts on Intermittent Fasting Series that it is one of the, if not the central argument in favor of intermittent fasting.

The practical take home message of this study is therefore that exercise + diet induced targeted glycogen depletion before a workout (not via an overnight fast, only; that would leave your muscle glycogen stores largely intact, while your body is burning fat and tapping into your hepatic glycogen reserves) can become one among a whole host of tools in your workout-toolbox. You can use it sporadically, but you should not need another study to be able to predict that the downsides of chronic use are going to outweigh (purported - again, we are measuring markers only, here!) short term benefits.

On a last note: I guess you know that the SuppVersity is the place where you will hear about respective longitudinal data first, right? To make sure you don't miss that I suggest you go to www.facebook.com/SuppVersity like the page or register for updates at twitter.com/ProfDrAndro!

References:

• Gibala MJ, McGee SL, Garnham AP, Howlett KF, Snow RJ, Hargreaves M. Brief intense interval exercise activates AMPK and p38 MAPK signaling and increases the expression of PGC-1alpha in human skeletal muscle. J Appl Physiol. 2009 Mar;106(3):929-34.
• Nordsborg NB, Lundby C, Leick L, Pilegaard H. Relative workload determines exercise-induced increases in PGC-1alpha mRNA. Med Sci Sports Exerc. 2010 Aug;42(8):1477-84.
• Psilander N, Wang L, Westergren J, Tonkonogi M, Sahlin K. Mitochondrial gene expression in elite cyclists: effects of high-intensity interval exercise. Eur J Appl Physiol. 2010 Oct;110(3):597-606. Epub 2010 Jun 23.
• Psilander N, Frank P,  Flockhart M, Sahlin K. Exercise with low glycogen increases PGC-1agene expression in human skeletal muscle. Eur J Appl Physiol. 02 Oct 2012 [ahead of print]

If a High Fat Diet was a Pill, the Lay Press Would Celebrate it as "Exercise in a Pill": Two days on High Fat Diet Increase the Activity of Irisin Releasing Transcription Factor PGC1-α

Image 1: If a high fat diet was a patentable pill, I guess BigPharma would already be selling it.

It's crazy, whenever I go to the groceries, there are new products popping up with colorful slogans like "fat free", "diet", "particularly low in fat", "fat reduced", "only 0.1%" etc. What is yet all the more disturbing is that these products are "placed" right next to a more recent "invention" of the industry - products with a "reduced sugar content"... I could now go on a rampage on how the Kellog's and co. just replace table sugar with other sugars in order to be able to put these stickers on their products, but I actually want to make a different point today. If you stand still for a moment and watch the people pass by, there are two things you will notice.

1. The healthy and lean (and not anorexic) will pass by without even noticing the great "0.00001% fat, reduced sugar whatever" with the Weight Watchers sticker on it.
2. The hungry-looking slightly overweight to morbidly obese people will pick up the aforementioned "0.00001% fat, reduced sugar whatever" with the Weight Watchers sticker on it, look at the price-tag, sigh and put three packets of that stuff into their cart.

Now, if we, for the time being, disregard that these products are full of chemicals and hidden sugars and just focus on the notion that eating a product that is both low in carbs and low in fats will help you lose weight we have identified what, at least in my humble opinion, is the #1 reason that many self-perceived "low-carbers" stop losing weight after they dropped the initial few pounds of water: A low carb, low fat diet that - irrespective of their protein intake - is telling their bodies that they are in the midst of a famine, where you better hold on to your fat and burn the wasteful muscle tissue so that you can survive for another few weeks...

Please understand: Low carb = high fat! And high fat burns, not stores fat.

I guess, I better stop preaching now and get to the nitty gritty of the latest study on the effects of a high fat vs. low fat diet on short-term (2-days) metabolic adaptations in lean (BMI 19-25kg/m²) and obese (BMI 30-40kg/m²) human subjects.

Figure 1: Relative energy content of the low and high fat diets the lean and obese study participants were fed (data adapted from Bergouignon. 2012)

As you can see from the data in figure 1, the scientists designed the study in a way that facilitates a direct comparison of high fat (50%) vs. low fat (20%). This is different from high carb vs. low carb, because the high fat diet (35% carbs vs. 65% in the low fat diet) is by no means so "low" in carbs as the average low-carber would have it. That the metabolic adaptations were still pretty profound - after only two days - is something, I personally would not have expected.

Figure 2: Total 24h kcal expenditure (left axis) and protein, carbohydrate and fat oxidation (right axes) in lean and obese subjects on the low and high fat diets (data adapted from Bergouignon. 2012)

Both, lean and obese subjects showed almost identical responses with regard to their 24h kcal expenditure and substrate utilization (cf. figure 1). Most prominent among these was the profound increases in 24h fat oxidation (+88% in the lean and +109% in the obese subjects) in the high fat groups that was yet accompanied by a -26% (lean) and -37% (obese) reduction in carbohydrate oxidation. The overall caloric expenditure was thusly "the same" - unless you still believe that 50kcal per day would make a difference ;-)

Figure 3: Free fatty acid and triglyceride levels (24h AUC in mmol/dl and mg/dl, right axis) and free fatty acid to triglyceride ratio (left axis; data calculated based on Bergouignon. 2012)

If we now take a look at the blood lipids (cf. figure 2), i.e. the free fatty acid (FFA) and triglyceride levels, on the different diets, we see a "phenomenon" low-carbers know all to well. After only a few days on a low-carb diet, triglyceride levels start to plumed. The amount of free fatty acids that are released into the circulation from the liver and the adipose tissue, on the other hand, rises. This time with a significantly greater FFA response in the obese individuals (p=0.04), which does support the hypothesis that a significant amount of the FFAs came from the well-stocked adipose tissue of the obese study participants.

Figure 3: Total mRNA expression in vastus lateralis muscle of study participants expressed relative to the subjects in the lean low fat group; please note the discontinuity of the axis between 200% and 600%, which became necessary to fit the data into the graph (data calculated based on  Bergouignon. 2012)

The most intriguing changes this two-day high fat "challenge" induced took yet place on a transcriptional level in the skeletal muscle tissue of the subjects. However, of the changes in total mRNA expression, only the increase in CD36, a protein which regulates the fatty acid uptake of skeletal muscle, and pyruvate dehydrogenase kinase isozyme 4 (PDK4), which reduced the oxidation of glucose, reached statistical significance.

Is a high fat diet exercise in a pill?  ...

If we discard the statistical shenanigan for a moment, you may yet notice the "non-significant" +33% increase in Sirtuin 1 (Sirt 1) expression in both high fat diet groups. The intelligent and well-read SuppVersity student you are, you will also remember that this protein, which is downregulated in states of insulin resistance, has been identified as one of the key players in the anti-cancer effects of compounds such as resveratrol and the longevity effects that have been ascribed to low-calorie diets. And as if that was not enough, Sirt 1 also plays a key role in the beneficial adaptations that come with exercise, where it is part of a triumvirate of AMPK + SIRT1 + PGC1-alpha,  which is induces the production of irisin the purported "exercise hormone" that is supposed to "brown your adipose tissue" (meaning to convert the sluggish white into the metabolically more active brown fat) has gotten quite some attention in the lay press as of late (eg. LA Times).

Figure 4: Skeletal muscle mRNA expression (in au) of acetylated (=inactive) PGC1-alpha and phosphorylated (=active) AMPK in lean and obese subjects subjects on low and high fat diets (data adapted from Bergouignon. 2012)

So, if we fell for the, in my book, stupid idea that a single cellular messenger, in this case irisin, would work like "exercise in a pill" (think about it - how often have you heard that in the last years?) and acknowledge that an increase in PGC1-alpha activity in skeletal muscle will increase the release of this myokine (i.e. a messenger protein that is released from the muscle; compare "adipokine" like leptin, which is a messenger protein that is released from adipose tissue), the logical conclusion would be that a high fat diet works like "exercise in a pill". Why? Because the data in figure 4 shows that the active, i.e. de-acetylated amount of PGC1-alpha (which is obviously the exact opposite of what the scientists measured) in the muscle of the subjects who consumed the high fat diet increased (p < 0.05) - ah,... and did I mention that this is human data, while the irisin craze is based solely on rodent studies?

... it sure looks like that!

In combination with the increase in phosphorylated AMPK (p-AMPK, click here to learn more about the "mTOR/AMPK Seesaw"), which was likewise statistically significant, the short-term adaptation to high fat feeding in lean and obese human beings shares two of the main characteristics of the "healthy endurance training" the same Dr's who warn their patients about the dangers of a high fat diet usually prescribe to their patients. Now tell me, is the voice in your stomach still telling you that low-carb may be good, but that eating a high fat diet is not an option? Well, in that case I guess you are beyond help... go buy some low-fat sugar reduced cornflakes ;-)