Tuesday, November 22

The gut microbiota regulates the metabolic response to fasting

Metabolic adaptation to fasting is an essential mechanism developed by mammals in order to survive. The transition from the fed to the fasted state is tightly regulated. This metabolic shift includes reducing glucose oxidation and storage, and increasing the supply of free fatty acids (FFA) and ketone bodies (KB) to peripheral tissues. Glucose is spared for obligate glucose-consuming cells (such as some neurons, erythrocytes, kidney cells) by FFA's effects on membrane glucose transporters in peripheral tissues, upregulation of lipolytic enzymes and downregulation of glycolytic enzymes. Overall, if extended, fasting will produce a state of peripheral insulin resistance, which implies that skeletal muscle will become desensitized to insulin's effect, thereby reducing glucose transport into cells. However, this scenario differs from that observed during pathological insulin resistance, in which skeletal muscle, liver and adipose tissue are insulin resistant, so there is no control of glucose and FFA levels, leading to glucotoxicity and lipotoxicity.

Healthy humans should be able to fast without problems (metabolic flexibility). If everything is working as supposed to, there should be no problem when switching to a predominantely lipolytic/ketogenic metabolism. How hard the transition to a fasting state is may be a marker of the functioning of intermediate metabolism. 

Crawford et al. (1) tested a basic hypothesis, based on previous findings:

  • Germ-free (GF) mice are leaner than conventionally raised (CONV-R) mice, even when GF mice consume more food (2).
  • Transplantation of the gut microbiota from obese mice to GF recipients causes a greater increase in adiposity than does a microbiota from lean mice (3).
Overall, there seems to be an alteration in the composition and the microbiome of gut bacteria from obese mice, which increases energy harvest from available food. This is an aberration from its normal function, which is to provide an adequate amount of energy from otherwise indigestable nutrients. If true, then we might expect that this mechanism is beneficial to the host in periods of nutrient deprivation. If not, there would be no obvious evolutionary explanation for this symbiotic relationship. Accordingly,  after withdrawal of nutrients, GF mice die more rapidly that CONV-R mice, even when the rate of body weight loss is the same (4). Because adaptation to fasting involves a shift towards ketogenesis, the gut microbiota might regulate this process.

The gut microbiota regulates ketone body metabolism during fasting

Compared to CONV-D*, GF mice showed 37% lower levels of serum betahydroxybutyrate (BHB) in the fasted state, with no differences in the fed state. Moreover, levels of insulin, glucose, FFA and triglycerides where unchanged. CONV-D mice had increased hepatic triglyceride stores compared to GF mice, difference which was enhanced dramatically with fasting. As expression of Pnpla2 was increased similarly in both CONV-D and GF mice, fasting-induced fatty acid mobilization was not impaired by the absence of gut microbiota. PPARa expression was higher in CONV-D than in GF mice, and the fasting-induced ketogenesis was impaired in CONV-D PPARa-/- mice. 

The liver produces BHB for utilization in peripheral tissues. It lacks the key enzyme 3-oxoacid CoA transferase, so is incapable of oxidizing ketone bodies. When fasted, GF mice had 50% less concentration of liver BHB compared to CONV-D mice. Expression of Fgf21 and Hmgcs2, both targets of PPARa which stimulate ketogenesis, was lower in fasted GF mice compared to CONV-D mice. 

These results suggest that the gut microbiota stimulates ketogenesis during fasting by a PPARa-dependent mechanism. Additionally, it promotes hepatic triacylglycerol synthesis and storage. 

There are two possible ways of generating acetyl CoA in the liver during a fast: from acetate produced in the gut and from oxidation of fatty acids from adipose tissue. In GF mice, the only source is the latter. Cecal acetate levels were very low in fed GF mice and 20-fold greater in CONV-D mice. During fasting, these levels were reduced in CONV-D, but remained significantly higher compared to GF mice, which showed no reduction. 

One unexpected result was the change in the microbiota composition induced by fasting. It was found that there was a significant increase in the proportion of Bacteroidetes and a significant reduction in the proportion of Firmicutes. Previous studies have found a correlation between a reduction in Bacteroidetes and an increase in Firmicutes with obesity (the high Firmicutes/low Bacteroidetes hypothesis) (5, 6). 

The gut microbiota influences myocardial metabolism

Analysis of the myocardial transcriptome of CONV-D and GF mice revealed an enrichment in the ketone body metabolic pathway in both groups, compared to their PPARa -/- counterparts. Expression of genes involved in fatty acid and ketone body metabolism were increased with fasting in both groups, but Oxct1 gene expression was higher in CONV-D mice. Conversely, an increased Glut1 expression was only observed in GF mice. 

The rate of glucose oxidation was significantly increased in isolated working hearts of GF mice, without alteration of fatty acid oxidation. In the absence of BHB, glucose utilization was also significantly greater. Glycogen levels were reduced in the myocardium of fasted GF mice compared to CONV-D mice, and there were no significant differences in heart rate, cardiac hydraulic work, mitochondrial morphology or number, or mitochondrial respiration. 

The shift towards a ketogenic metabolism in the myocardium is one hallmark of adaptation to fasting, as BHB is more energy efficient than glucose. So in periods of deprivation of nutrients, the myocardium maintains its normal functioning by using BHB instead of relying in glucose. This adaptation is impaired in GF mice.

The gut microbiota affects myocardial mass

Heart weight was reduced in fasted GF mice compared to CONV-D mice. Training elicits an hypertrophic response in the heart. However, this response was blunted in the absence of gut microbiota, as the hearts of trained GF mice remained smaller than the hearts of trained CONV-D mice. This correlated with alterations in a subset of pathways, which included ketone body metabolism. Administration of a ketogenic diet rescued heart weight in GF mice and shifted the myocardial transcriptome toward ketone body metabolism.

These results suggest that the gut microbiota is an important component for cardiovascular health, and that ketone bodies represent an essential substrate for the heart

Summing up

This is one of the most interesting studies that I have read lately. It provides a new template for the relationship between metabolism and gut microbiota, and shows the importance of gut bacteria for the normal response to fasting. I have summarized the findings of the study in the following figure (my interpretation):

The gut microbiota regulates ketogenesis during fasting. Fasting induces an increase in the proportion of Bacteroidetes and a reduction in the proportion of Firmicutes. These changes promote the production of acetate, which serves as substrate for hepatic acetyl CoA synthesis. The gut microbiota also stimulates hepatic triglyceride stores, providing another source of energy during fasting. The increase in acetyl CoA levels stimulates ketogenesis by a PPARa-dependent mechanism, increasing serum BHB levels. The elevated concentration of BHB levels supplied to the heart promotes the shift towards a ketone body-based metabolism, and inhibits glucose oxidation. Myocardial ketone body metabolism maintains myocardial mass and the normal hypertrophic response to exercise.

* CONV-D (conventionalized) mice were transplanted with distal gut microbiota from CARB-fed CONV-R lean mice. 

ResearchBlogging.orgCrawford PA, Crowley JR, Sambandam N, Muegge BD, Costello EK, Hamady M, Knight R, & Gordon JI (2009). Regulation of myocardial ketone body metabolism by the gut microbiota during nutrient deprivation. Proceedings of the National Academy of Sciences of the United States of America, 106 (27), 11276-81 PMID: 19549860


  1. Hi Lucas,

    So GF mice absorb less monosaccharide and have more FIAF--does that explain their leanness? Is monosaccharide the only harvest difference?

    A common idea is that obese microbiota, firmicutes, harvest more nutrients: Isn't it a bit counter intuitive then that fasting changes microbiota composition to that of a lean animal, which is normally less "efficient"?

    Don't obese animals typically have the same "problems" as the GF mice, in response to fasting or ketogenic diet; ie, less ketone use?

  2. Hi john,

    GF mice are very interesting, but very complex as well.

    Normally, one of the functions of the microbiota is to absorb indigestable energy. This means a wide range of exogenous and endogenous oligo/polysaccharides. Another way of increasing energy absorption is by specific transporters. So the difference between normal CONV mice and GF mice is the fact that gut flora can digest plant oligo, poly and monosaccharides, so the calories provided by SCFA are lost. FIAF might be a factor, however, this protein is supressed only in the intestine. There is contrary evidence towards the implication of fiaf as the most important factor (http://www.ncbi.nlm.nih.gov/pubmed/20441670). The most accepted protein regulating microbe-host interaction is Gpr41: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2569967/pdf/zpq16767.pdf

    Re: Firmicutes, there is contrary evidence for the high Firmicutes/low Bacteroidetes hypothesis: the cause seems to be more production and absorption of SCFA. Metabolomic analysis has shown an upregulation of enzymes and metabolic pathways supporting this hypothesis, and there is more than just Firmicutes (for example, increases in methanogens). This might happen during fasting, although the relative proportions of bacteria are different, the final effect is the same: more SCFA. There also might be an upregulation of Gpr41 during fasting.

    In the end, dysbiosis might cause a metabolic profile of gut bacteria which resembles fasting, but during the fed state, contributing to overall adiposity. Because the high Firmicutes/low Bacteroidetes hypothesis considers only the proportion of phyla in the gut, the exact species characterizing an obese microbiota are not well known (ie. you can see an increase in the proportion of Firmicutes, but differences between classes increased).

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  4. Could you, please, elaborate more on the sentence "The gut microbiota also stimulates hepatic triglyceride stores, providing another source of energy during fasting". I am interested to know more about physiological effect of the ketogenic diets because I have to be on a such diet for the migraines management. Overall I fell excellent, my other health issues disappeared. However, very recently blogger Eveline-Carbsane posted here
    the abstract from some article that says that

    " Hepatic steatosis, inflammation, and ER stress in mice maintained long term on a very low-carbohydrate ketogenic diet (sorry, abstract only at the moment).

    Longitudinal measurement of body composition, serum metabolites, and intrahepatic fat content, using in vivo magnetic resonance spectroscopy, reveals that mice fed the ketogenic diet over 12 wk remain lean, euglycemic, and hypoinsulinemic but accumulate hepatic lipid in a temporal pattern very distinct from animals fed the Western diet. Ketogenic diet-fed mice ultimately develop systemic glucose intolerance, hepatic endoplasmic reticulum stress, steatosis, cellular injury, and macrophage accumulation, but surprisingly insulin-induced hepatic Akt phosphorylation and whole-body insulin responsiveness are not impaired. Moreover, whereas hepatic Pparg mRNA abundance is augmented by both high-fat diets, each diet confers splice variant specificity. The distinctive nutrient milieu created by long-term administration of this low-carbohydrate, low-protein ketogenic diet in mice evokes unique signatures of nonalcoholic fatty liver disease and whole-body glucose homeostasis."
    What is your opinion?

  5. Hi Galina,

    There are certain factors to take into account for trying to interpret the study:

    - The diet used was http://www.bio-serv.com/pdf/F3666.pdf. Check the Fatty acid profile and the main ingredient (hats off to Chris Masterjohn on the PUFA content in lard used in most studies).

    From the study:

    - "Whereas consumption of KD is relatively reduced by mass, caloric density is higher in KD than in WD or chow. Therefore, caloric intake was higher in KD-fed mice". Calories are important.

    - "It should be noted that the KD studied here would not be studied in or prescribed for humans. Moreover, the minimum protein requirement for normal growth, reproduction, and lactation in mice is ∼14% (29). With regard to the development of NAFLD, low protein content, particularly the amino acid methionine, and prospectively low choline are recognized as confounding variables of the KD."

    My overall recommendation based on this study would be: DON'T EAT A 90-95% FAT DIET.

  6. Thank you for your answer. Sure, calories are important . Probably, Atkins-style fat fast on 1000 calories of mostly fat a day shouldn't cause a fat liver decease. I don't plan to try it - it is just an illustration .