Isolated murine skeletal muscles utilize pyruvate over glucose for oxidation

Abstract

Introduction Fuel sources for skeletal muscle tissue include carbohydrates and fatty acids, and utilization depends upon fiber type, workload, and substrate availability. The use of isotopically labeled substrate tracers combined with nuclear magnetic resonance (NMR) enables a deeper examination of not only utilization of substrates by a given tissue, but also their contribution to tricarboxylic acid (TCA) cycle intermediates. Objectives The goal of this study was to determine the differential utilization of substrates in isolated murine skeletal muscle, and to evaluate how isopotomer anlaysis provided insight into skeletal muscle metabolism. Methods Isolated C57BL/6 mouse hind limb muscles were incubated in oxygenated solution containing uniformly labeled 13C6 glucose, 13C3 pyruvate, or 13C2 acetate at room temperature. Isotopomer analysis of 13C labeled glutamate was performed on pooled extracts of isolated soleus and extensor digitorum longus (EDL) muscles. Results Pyruvate and acetate were more avidly consumed than glucose with resultant increases in glutamate labeling in both muscle groups. Glucose incubation resulted in glutamate labeling, but with high anaplerotic flux in contrast to the labeling by pyruvate. Muscle fiber type distinctions were evident by differences in lactate enrichment and extent of substrate oxidation. Conclusion Isotope tracing experiments in isolated muscles reveal that pyruvate and acetate are avidly oxidized by isolated soleus and EDL muscles, whereas glucose labeling of glutamate is accompanied by high anaplerotic flux. We believe our results may set the stage for future examination of metabolic signatures of skeletal muscles from pre-clinical models of aging, type-2 diabetes and neuromuscular disease.

Phosphorylation of AMPKα at Ser485/491 Is Dependent on Muscle Contraction and Not Muscle-Specific IGF-I Overexpression

“…Therefore, we cannot confirm that the changes observed are due solely to actions in skeletal muscle by IGF-I. More assays, such as uptake measurements of 2-DG glucose [21] or isotopically labeled substrates [22], are needed to validate our findings. From the perspective of exercise capacity, one would predict that an enhancement in glucose uptake afforded by IGF-I would lead to greater running speeds or exercise intensities, but this was not the case.…”

Temporal serum metabolomic and lipidomic analyses distinguish patients with access-related hand disability following arteriovenous fistula creation

“…To remove serum proteins (i.e., albumin) that broaden NMR peaks and complicate metabolite quanti cation, we initially employed a modi ed FOLCH extraction to separate water-soluble and lipid-soluble metabolites to 200µl of serum as previously described 13,14 . Following the modi ed FOLCH extraction, aqueous phase metabolites were subjected to a second acetonitrile:isopropanol:water (3:3:2 vol/vol/vol) extraction as previously described 15 . Following extraction, the aqueous phase metabolites were lyophilized overnight (Labconco Corporation, Kansas, MO, USA) and the lipid phase was dried with inert nitrogen gas.…”

“…All spectra were acquired at room temperature. Detailed methods involved in NMR acquisition and processing have been previously reported 13,15 .…”

Temporal serum metabolomic and lipidomic analyses distinguish patients with access-related hand disability following arteriovenous fistula creation

“…All spectra were acquired at room temperature (25 ± 0.1 °C). Detailed methods involved in NMR acquisition and processing have been previously reported 13 , 18 .…”