ABSTRACT
We examined the effects of branched-chain amino acids (BCAAs) and electrical pulse stimulation (EPS) on the mTORC1 pathway in muscle satellite cells (MSCs) isolated from branched-chain α-keto acid dehydrogenase kinase (BDK) knockout (KO) mice in vitro. MSCs were isolated from BDK KO and wild-type (WT) mice, proliferated, and differentiated into myotubes. BCAA stimulation increased the phosphorylation of p70 S6 kinase (p70S6K), a marker of protein translation initiation, in MSCs from WT and BDK KO mice, but the rate of the increase was higher in MSCs isolated from BDK KO mice. Contrarily, there was no difference in the increase in p70S6K phosphorylation by EPS. Acute BDK knockdown in MSCs from WT mice using shRNA decreased p70S6K phosphorylation in response to BCAA stimulation. Collectively, the susceptibility of mTORC1 to BCAA stimulation was elevated by chronic, but not acute, enhancement of BCAA catabolism.
Subject(s)
Satellite Cells, Skeletal Muscle , Amino Acids, Branched-Chain/metabolism , Animals , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Knockout , Muscle, Skeletal/metabolism , Peptide Chain Initiation, Translational , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Satellite Cells, Skeletal Muscle/metabolismABSTRACT
Proteolysis is known to play a crucial role in maintaining skeletal muscle mass and function. Autophagy is a conserved intracellular process for the bulk degradation of proteins in lysosomes. Although nutrient starvation is known to induce autophagy, the effect of nutrient repletion following starvation on the mTOR pathway-mediated protein translation remains unclear. In the present study, we examined the effect of glucose starvation on the initiation of protein translation in response to glucose re-addition in C2C12 myotubes. Glucose starvation decreased the phosphorylation of p70 S6 kinase (p70S6K), a bonafide marker for protein translation initiation. Following re-addition of glucose, phosphorylation of p70S6K markedly increased only in glucose-starved cells. Inhibiting autophagy using pharmacological inhibitors diminished the effect of glucose re-addition on the phosphorylation of p70S6K, whereas inhibition of the ubiquitin-proteasome system did not exert any effect. In conclusion, autophagy under glucose starvation partially accounts for the activation of translation initiation by re-addition of glucose.
Subject(s)
Autophagy/physiology , Muscle Fibers, Skeletal/metabolism , Peptide Chain Initiation, Translational/physiology , Animals , Autophagy/genetics , Cell Line , Glucose/metabolism , Lysosomes/metabolism , Mice , Muscle, Skeletal/metabolism , Peptide Chain Initiation, Translational/genetics , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/pharmacology , Proteolysis , Ribosomal Protein S6 Kinases, 70-kDa/analysis , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , UbiquitinABSTRACT
Mouse myoblast C2C12 cells are commonly used as a model system for investigating the metabolic regulation of skeletal muscle. As it is therefore important to understand the metabolic features of C2C12 cells, we examined the effect of glucose starvation on autophagy in C2C12 myotubes. After culture of C2C12 myotubes with high (HG, 25.0 mM) or low (LG, 5.6 mM) glucose concentrations, the concentration of glucose in the LG group had decreased to 0 mM after 24 h of culture and was around 17 mM after 48 h of culture in the HG group. The concentration of lactate increased from 0 to approximately 9 mM at 24 h and then dropped slightly in the LG group, while it increased linearly to 21 mM in the HG group at 48 h. The phosphorylation of p70 S6 kinase, marker for the protein translation initiation was significantly lower and the ratio of LC3-II/LC3-I, marker for the induction of autophagy was significantly higher in the LG group. GLUT1 and hexokinase II expression were significantly higher in the LG group. Together, these changes in glucose and lactate concentrations in the culture media suggest that C2C12 myotubes depend on anaerobic glycolysis. Our findings also suggest that glucose depletion stimulates the expression of key molecules involved in glycolysis and that cellular autophagy is also activated in C2C12 myotubes.
Subject(s)
Autophagy/physiology , Glucose/metabolism , Muscle Fibers, Skeletal/metabolism , Amino Acids, Branched-Chain/metabolism , Animals , Cell Line , Glycolysis , Hexokinase/metabolism , Lactates/metabolism , Mice , Muscle Fibers, Skeletal/cytology , Myoblasts/metabolismABSTRACT
The purpose of this study was to examine the effects of a low-carbohydrate high-protein (LCHP) diet on the expression of glucose transporters and their relationships to glucose metabolism. Male C57BL/6 mice were fed a normal control or LCHP diet for 2 weeks. An oral glucose tolerance test and insulin tolerance test (ITT) were performed, and the expression of glucose transporters was determined in the gastrocnemius muscle, jejunum and pancreas. The increase in plasma insulin concentrations after glucose administration was reduced in the LCHP group. However, LCHP diet had no effects on peripheral insulin sensitivity or glucose transporters expression in the gastrocnemius and pancreas. Soluble glucose transporter (SGLT)-1 protein content in jejunum was lower in the LCHP group. Taken together, these results suggest that the blunted insulin response after glucose administration in LCHP diet-fed mice might be due to decreased SGLT-1 expression, but not to an increase in peripheral insulin sensitivity. Abbreviations: LCHP: low-carbohydrate high-protein; ITT: insulin tolerance test; GLUT: glucose transporter; SGLT: soluble glucose transporter; OGTT: oral glucose tolerance test; AUC: area under the curve.
