Effects of β-hydroxybutyrate treatment on glycogen repletion and its related signaling cascades in epitrochlearis muscle during 120 min of postexercise recovery

Abstract

We investigated the effects of β-hydroxybutyrate (β-HB), the most abundant type of ketone body in mammals, on postexercise glycogen recovery in skeletal muscle by using an in vitro experimental model. Male ICR mice swam for 60 min and then their epitrochlearis muscles were removed and incubated with either physiological levels of glucose (8 mmol/L) and insulin (60 μU/mL) or glucose and insulin plus 1, 2, or 4 mmol/L of sodium β-HB. Four millimoles per liter β-HB had a significant positive effect on glycogen repletion in epitrochlearis muscle at 120 min after exercise (p < 0.01), while 2 mmol/L of β-HB showed a tendency to increase the glycogen level (p < 0.09), and 1 mmol/L of β-HB had no significant effect. We further investigated the effect of 4 mmol/L β-HB treatment on the signaling cascade related to glycogen repletion in the epitrochlearis muscles throughout a 120-min recovery period. After incubating the muscles in 4 mmol/L of β-HB for 15 min postexercise, the Akt substrate of 160 kDa Thr642 (p < 0.05) and Akt Thr308 (p < 0.05) phosphorylations were significantly increased compared with the control treatment. At the same time point, 5′-AMP–activated protein kinase and acetyl-coenzyme A carboxylase phosphorylations were significantly lower (p < 0.05) in the epitrochlearis muscle incubated with 4 mmol/L of β-HB than in the control muscle. Our results demonstrate that postexercise 4 mmol/L β-HB administration enhanced glycogen repletion in epitrochlearis muscle. Four millimoles per liter β-HB treatment was associated with alternation of the phosphorylated status of several proteins involved in glucose uptake and metabolic/energy homeostasis at the early stage of postexercise.

Effects of Nutrient Intake Timing on Post-Exercise Glycogen Accumulation and Its Related Signaling Pathways in Mouse Skeletal Muscle

“…However, higher phosphorylation levels in AMPK signaling pathways did not result in higher glycogen repletion in the plantaris muscle. This result is consistent with our previous study showing that greater glycogen repletion induced by post-exercise ketone body administration was accompanied with lower phosphorylation levels in AMPK and ACC [31]. Other studies also reported that post-exercise enhancement of glucose transport with insulin treatment at 3 h after the exercise occurred without increased AMPK activation at the same time point [23,27].…”

“…Protein isolation from plantaris muscles was performed as described previously [31] in a radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, and 1 mM ethylenediaminetetraacetic acid (EDTA)) supplemented with a protease inhibitor mixture (cOmplete Mini, EDTA-free; Roche Applied Science, Mannheim, Germany), and a phosphatase inhibitor mixture (PhosSTOP; Roche Applied Science). After centrifugation at 600 g for 20 min at 4 °C, the supernatants were collected, and their protein concentrations were determined by a bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Western blotting was performed as described previously [31,32]. The protein samples (5–10 μg) and a pre-stained molecular weight marker (Bio-Rad, Hercules, CA, USA) were run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for 60 min at 150 V. The proteins were then transferred from the gels to polyvinylidene difluoride (PVDF) membranes using Transblot Turbo (Bio-Rad).…”

Ketones for Post-exercise Recovery: Potential Applications and Mechanisms

“…This was evident as the muscle glycogen was 50% higher in the ketone group compared to the control group, explaining the upstream drivers that are increased insulin secretion and uptake of glucose. This is consistent with the in vitro work by Takahashi et al (2019) that showed improved glycogen repletion after exercise by incubation mice epitrochlearis muscles with physiological concentrations of glucose, insulin and 4 mM BHB. The question remains, does ketone increase insulin sensitivity and responsiveness per se or simply magnifying the effects of exercise has on both of these parameters as explained by Zorzano et al (1986).…”

Modulation of Cellular Biochemistry, Epigenetics and Metabolomics by Ketone Bodies. Implications of the Ketogenic Diet in the Physiology of the Organism and Pathological States

“…Insulin injection may revert KB levels [19]. The in vitro studies performed on skeletal muscle isolated form mice subjected to physical exercise (swimming) for 60 min at 35 • C, have shown that 4 mM BHB significantly improves glycogen repletion in epitrochlearis muscle, the major determinant of exercise performance [22].…”