Fasting and AMPk Signaling – Lean vs. Obese

Fasting and AMPk Signaling - Lean vs. Obese - 4

Introduction

AMPk is a cellular energy sensor, activated mostly at low energy states. Its activation often depends on ratios such as AMP/ATP and NAD+/NADH. The main purpose of AMPk activation is to restore cellular energy.

“Thus, any change in cellular energy status activates AMPK, leading to concomitant inhibition of energy-consuming processes and stimulation of ATP-generating pathways to restore energy balance” [1]

Stressors that can reduce cellular energy include nutrient deprivation (i.e. caloric restriction), hypoxia (low oxygen conditions), exercise, ischemia, cold exposure, heat shock, fasting, metformin [3] and several others.

High levels of AMP/ATP (low energy status) lead to the activation of AMPk, which triggers processes such as:

– increased oxidation of fatty acids,
– increased glucose uptake inside the muscles,
– increased mitochondrial biogenesis [2],
– ketogenesis,
– reduced cholesterol synthesis,
– etc.

In my current view, higher activity of AMPk is a good thing. However, I assume that most people lack the exposure to such stressors – that I mentioned above. Chances are, then, that AMPk activity inside their cells is reduced or inexistent. Here, I want to briefly discuss the findings of a study relevant to the topic. [1]

Fasting in Lean vs. Obese Subjects

In this study researchers recruited 12 lean and 14 non-diabetic obese subjects and fasted them for 48 hours. Researchers wanted to determine how their bodies respond to fasting by measuring numerous metabolic, energetic, physiologic, and genetic markers. [1]

One possible shortcoming of the study is that the fasting protocol may have been too short for metabolic adaptations to occur – say, compared to the ones that occur after 5 or 7 days of water only fasting.

A 48-hour fast may have only depleted subject’s glycogen stores and modestly kickstarted fat burning processes (higher fatty acid oxidation and ketogenesis). This is a much different metabolic picture compared to someone who is already a couple of days into a fast.

However, we need to understand that they had the subjects under observation for the entire 48 hours. It may be inconvenient for subjects to remain fasted and under strict supervision for longer periods, especially if there is no incentive to be motivated by. Leaving this aside, let’s see what they found..

Subjects

n = 26 (small sample):

– 12 lean subjects: 2 males, 10 females, average BMI = 23.3
– 14 obese subjects: 2 males, 12 females, average BMI = 35.2
– all subjects: healthy, non-smoking, normal blood glucose, no diabetes running in the family
– supervised throughout the entire duration of the experimen

Methods and Measures

Blood samples: at breakfast (t = 90 min), at 24 hours and at 48 hours of fasting
Muscle biopsies: vastus lateralis, at breakfast (t = 135 min) and at 48 hours of fasting
Substrate oxidation: by indirect calorimetry (from exhaled oxygen and carbon dioxide) at t = 45 min, at 24 hours and at 48 hours of fasting

Results

Fasting and AMPk Signaling - Lean vs. Obese - 1

As they point out, adaptation to fasting is characterized by higher lipolysis, higher ketogenesis, and higher lipid oxidation, with a concomitant decrease in glucose uptake and oxidation by peripheral tissues:

“In lean individuals, we observed several of these well-known effects on whole body substrate metabolism together with decreased plasma levels of glucose, insulin, and leptin and a concomitant increase in GH. Not surprisingly, our obese subjects exhibited elevated levels of glucose, insulin, TG, and leptin and lower circulating adiponectin at baseline than lean individuals and a marked whole body metabolic inflexibility characterized by impaired fasting induced switch from glucose toward FA oxidation. In addition, the decrease in plasma leptin and insulin levels in response to fasting was also blunted, in line with previous reports.” [1]

This study confirms what others have found in the past, that growth hormone activity increases with fasting. It is interesting though that GH levels in lean subjects increased 10x from baseline to 48 hours of fasting (0.9 mU/l => 9 mU/l) compared to 4x in obese subjects (0.7 mU/l to 3.1 mU/l).

“The fasting-induced increase in plasma GH levels is believed to play an important role in the regulation of whole body substrate metabolism, notably by inhibiting glucose uptake and enhancing lipid oxidation in skeletal muscle. Interestingly, we found that the change in plasma GH levels was significantly different between groups, with a much larger increase in lean than in obese individuals.” [1]

The higher glucose, leptin, insulin, and TAG levels in obese subjects reflect their metabolic inflexibility. However, I assume that if obese subjects (under optimal conditions) would have kept on with the fast, they may have partially or fully recovered their metabolic flexibility.

Reduction in body weight occurred in a similar way in both groups.
Thyroid markers such as T3 and TSH also decreased in both groups.
Total cholesterol, LDL, cortisol and CRP (this is interesting) increased in both groups.

As pointed out, obese subjects had an attenuated response to fasting compared to lean subjects. This response was characterized by:

– lower reduction in glucose oxidation
– lower increase in fat oxidation

In my translation: they were still burning more sugar and less fat compared to lean subjects.

In terms of energy expenditure (REE), obese subjects burned more than lean subjects. However, correcting for lean body mass made this effect disappear.

Fasting and AMPk Signaling - Lean vs. Obese - 2

Worth mentioning is that “human beings, like all living organisms, have to constantly adjust their metabolism in response to changes in environmental nutrient availability.” [1]

Resting energy expenditure (REE) is thus just a snapshot. I think it may not be wise to follow a fixated daily caloric intake strategy, as the body is always adjusting both REE and TEE.

Metabolic Gene Expression Response to Fasting (mRNA)

Determined in skeletal muscle, from vastus lateralis biopsy:

“At baseline, transcript levels of HK1, PKM2, PPARA, CD36, ACACA, ATP2A1, ACADM, ACOX3, and PDK4 were significantly higher in obese than in lean individuals, whereas LPL mRNA expression was found to be significantly lower (Table 4).” [1]

Then:

“Prolonged fasting induces significant upregulation of INSR, PDK4, PFKFB3, and UCP3 and downregulation of HK2 and PPARGC1A mRNA expression in lean subjects (Table 4), in line with previous studies.” [1]

Fasting and AMPk (surprising)

Off-topic: One should be cautious not to make inappropriate extrapolations from non-human studies. Research on cells and model organisms are really helpful in discovering new mechanisms, in observing and in exploring response to different strategies, but human data is much needed when proposing/introducing/implementing new therapies.

Fasting and AMPk Signaling - Lean vs. Obese - 3

One of their hypotheses was that fasting activates AMPk, which leads to adaptations to food deprivation.

“Indeed, although most of its established functions came from in vitro and/or rodent studies, it is largely acknowledged that AMPK activation promotes both glucose uptake and lipid oxidation in skeletal muscle through direct phosphorylation of key regulatory enzymes or transcription factors.” [1]

They did not observe different basal (post-meal) AMPk activity in lean vs. obese.

“This is in line with most of the previous reports, although one study has reported reduced AMPK activity in skeletal muscle from healthy obese and type 2 diabetes individuals.” [1]

However, they observed that AMPk activity in skeletal muscle of lean subjects is decreased with fasting, unlike in obese individuals. In another study it was reported that AMPk activity in healthy subjects is not significantly affected by fasting (72 hours):

“On top of the cellular energy state, AMPK constantly monitors intracellular glycogen stores. Interestingly, a paradoxical increase in skeletal muscle glycogen content was reported during prolonged fasting.” [1, 4]

They make the following assumptions:

“Taken together, the physiological rationale for a reduced AMPK activity during prolonged fasting still remains unclear. One of the hypotheses builds on modulation of the so-called Randle cycle: a reduced AMPK activity would therefore prevent glucose uptake by reducing AMPK-mediated GLUT4 translocation to the plasma membrane, leading to subsequent inhibition of glucose oxidation and the concomitant shift toward mitochondrial FA.” [1]

Authors also note about the significant decrease in expression of important mitochondrial respiratory chain subunits in the obese subjects. They think this is due to fewer mitochondria in the muscles of obese subjects, rendering them more unable to adapt to higher fatty acid availability in fasting as well as impaired ability to shift from glucose to fatty acid oxidation.

Conclusion

Obese subjects (vs. lean) are characterized by whole body metabolic inflexibility to prolonged fasting, altered AMPk signaling, and fewer muscle mitochondria. Repeating from above:

I think that 48 hours is still too short of a fasting window to be put under the umbrella of prolonged fasting.

A metabolic + physiologic + energetic + transcriptional snapshot of someone who is 5-7 days fasted may be much different from that of someone who barely depleted their glycogen stores. It follows that AMPk activity in prolonged fasting (more than 48 hours) could be increased. But it still remains unclear if that is what happens, which is why, as always, more studies are needed.


References:

  1. Wijngaarden, M. A., van der Zon, G. C., van Dijk, K. W., Pijl, H., & Guigas, B. (2013). Effects of prolonged fasting on AMPK signaling, gene expression, and mitochondrial respiratory chain content in skeletal muscle from lean and obese individuals. American Journal of Physiology-Endocrinology and Metabolism, 304(9), E1012-E1021.
  1. Rafaeloff-Phail, R., Ding, L., Conner, L., Yeh, W. K., McClure, D., Guo, H., … & Brooks, H. (2004). Biochemical regulation of mammalian AMP-activated protein kinase activity by NAD and NADH. Journal of Biological Chemistry, 279(51), 52934-52939.
  1. Cantó, C., Gerhart-Hines, Z., Feige, J. N., Lagouge, M., Noriega, L., Milne, J. C., … & Auwerx, J. (2009). AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature, 458(7241), 1056-1060.
  1. Vendelbo, M. H., Clasen, B. F., Treebak, J. T., Møller, L., Krusenstjerna-Hafstrøm, T., Madsen, M., … & Goodyear, L. J. (2012). Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle. American Journal of Physiology-Endocrinology and Metabolism, 302(2), E190-E200.

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