A 2-Hydroxybutyrate- mediated feedback loop regulates muscular fatigue
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Curated by eLife
eLife assessment
The work by Johnson and co-workers has identified an important role of 2-Hydroxybutyrate in skeletal muscle oxidative capacity in the early stages of exercise. Mechanistically, they show convincing data to support a role of 2-Hydroxybutyrate in the regulation of BCAA metabolism via SIRT4, ADP-Ribosylation, and CEBP. However, whether this is the sole mechanism and if these translate to longer exercise training regimes requires future experiments.
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Abstract
The metabolite 2-hydroxybutyrate (2HB) is produced by skeletal muscle acutely during exercise and persists for several hours in the blood post-exertion. We show here that 2HB directly inhibits branched- chain aminotransferase enzymes, and that this inhibition in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBPβ mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity both in vitro and in vivo , with the latter mimicking the effects of exercise training on whole body metabolism. Thus, we show here that 2-HB production by skeletal muscle represents a novel mechanism for the modification of metabolism by exercise.
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eLife assessment
The work by Johnson and co-workers has identified an important role of 2-Hydroxybutyrate in skeletal muscle oxidative capacity in the early stages of exercise. Mechanistically, they show convincing data to support a role of 2-Hydroxybutyrate in the regulation of BCAA metabolism via SIRT4, ADP-Ribosylation, and CEBP. However, whether this is the sole mechanism and if these translate to longer exercise training regimes requires future experiments.
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Reviewer #1 (Public Review):
The authors aimed to investigate if 2-hydroxybutyrate (2HB), a metabolite induced by exercise, influences physiological changes, particularly metabolic alterations post-exercise training. They treated young mice and cultured myoblasts with 2HB, conducted exercise tests, metabolomic profiling, gene expression analysis, and knockdown experiments to understand 2HB's mechanisms. Their findings indicate that 2HB enhances exercise tolerance, boosts branch chain amino acid (BCAA) enzyme gene expression in skeletal muscles, and increases oxidative capacity. They also highlight the role of SIRT4 in these effects. This study establishes 2HB, once considered a waste product, as a regulator of exercise-induced metabolic processes. The study's strength lies in its consistent results across in vitro, in vivo, and ex vivo …
Reviewer #1 (Public Review):
The authors aimed to investigate if 2-hydroxybutyrate (2HB), a metabolite induced by exercise, influences physiological changes, particularly metabolic alterations post-exercise training. They treated young mice and cultured myoblasts with 2HB, conducted exercise tests, metabolomic profiling, gene expression analysis, and knockdown experiments to understand 2HB's mechanisms. Their findings indicate that 2HB enhances exercise tolerance, boosts branch chain amino acid (BCAA) enzyme gene expression in skeletal muscles, and increases oxidative capacity. They also highlight the role of SIRT4 in these effects. This study establishes 2HB, once considered a waste product, as a regulator of exercise-induced metabolic processes. The study's strength lies in its consistent results across in vitro, in vivo, and ex vivo analyses. The authors propose a mechanism in which 2HB inhibits BCAA breakdown, raises NAD+/NADH ratio, activates SIRT4, increases ADP ribosylation, and controls gene expression.
However, some questions remain unclear based on these findings:
This study focused on the effects of short-term exercise (1 or 5 bouts of treadmill running) and short-term 2HB treatment (1 or 4 days of treatment). Adaptations to exercise training typically occur progressively over an extended period. It's important to investigate the effects of long-term 2HB treatment and whether extended combined 2HB treatment and exercise training have independent, synergistic, or antagonistic effects.
Exercise training leads to significant mitochondrial changes, including increased mitochondrial biogenesis in skeletal muscle. It would be valuable to compare the impact of 2HB treatment on mitochondrial content and oxidative capacity in treated mice to that in exercised mice.
The authors demonstrate that 2-ketobutyrate (2KB) can serve as an oxidative fuel, suggesting a role for the intact BCAA catabolic pathway. However, it's puzzling that the knockout of BCKDHA, a subunit crucial for the second step of BCAA catabolism, did not result in changes in oxidative capacity in cultured myoblasts.
Nevertheless, this innovative model of metabolic signaling during exercise will serve as a valuable reference for informing future.
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Reviewer #2 (Public Review):
Summary:
The manuscript entitled "A 2-HB-mediated feedback loop regulates muscular fatigue" by the Johnson group reports interesting findings with implications for the health benefits of exercise. The authors use a combination of metabolic/biochemical in vivo and in vitro assays to delineate a metabolic route triggered by 2-HB (a relatively stable metabolite induced by exercise in humans and mice) that controls branched-chain amino transferase enzymes and mitochondrial oxidative capacity. Mechanistically, the author shows that 2-HB is a direct inhibitor of BCAT enzymes that in turn control levels of SIRT4 activity and ADp-ribosylation in the nucleus targeting C/EBP transcription factor, affecting BCAA oxidation genes (see Fig 4i in the paper). Overall, these are interesting and novel observations and …Reviewer #2 (Public Review):
Summary:
The manuscript entitled "A 2-HB-mediated feedback loop regulates muscular fatigue" by the Johnson group reports interesting findings with implications for the health benefits of exercise. The authors use a combination of metabolic/biochemical in vivo and in vitro assays to delineate a metabolic route triggered by 2-HB (a relatively stable metabolite induced by exercise in humans and mice) that controls branched-chain amino transferase enzymes and mitochondrial oxidative capacity. Mechanistically, the author shows that 2-HB is a direct inhibitor of BCAT enzymes that in turn control levels of SIRT4 activity and ADp-ribosylation in the nucleus targeting C/EBP transcription factor, affecting BCAA oxidation genes (see Fig 4i in the paper). Overall, these are interesting and novel observations and findings with relevance to human exercise, with the potential implication of using these metabolites to mimic exercise benefits, or conditions or muscular fatigue that occurs in different human chronic diseases including rheumatic diseases or long COVID.Weaknesses:
There are several experiments/comments that will strengthen the manuscript-1- A final model in Figure 6 integrating the exercise/mechanistic findings, expanding on Fig 4i) will clarify the findings.
2- In some of the graphs, statistics are missing (e.g Fig 6G).
3- The conclusions on SIRT4 dependency should be carefully written, as it is likely that this is only one potential mechanism, further validation with mouse models would be necessary.
4- One of the needed experiments to support the oxidative capacity effects that could be done in cultured cells, is the use of radiosotope metabolites including BCCAs to determine the ability to produce CO2. Alternatively or in combination metabolite flux using isotopes would be useful to strengthen the current results.
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