BDK knockout skeletal muscle satellite cells exhibit enhanced protein translation initiation signal in response to BCAA in vitro.

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.

Glucose enhances catecholamine-stimulated lipolysis via increased glycerol-3-phosphate synthesis in 3T3-L1 adipocytes and rat adipose tissue.

BACKGROUND: During lipolysis, triglyceride (TG) are hydrolyzed into a glycerol and fatty acids in adipocyte. A significant portion of the fatty acids are re-esterificated into TG, and this is a critical step in promoting lipolysis. Although glycerol-3-phosphate (G3P) is required for triglyceride synthesis in mammalian cell, the substrate for G3P synthesis during active lipolysis is not known. A recent study showed that the inhibition of glucose uptake reduces catecholamine-stimulated lipolysis, suggesting that glucose availability is important in lipolysis in adipocytes. We hypothesized that glucose might play an essential role in generating G3P and thereby promoting catecholamine-stimulated lipolysis in adipocytes. Therefore, we determined the effect of glucose availability on catecholamine-stimulated lipolysis in 3T3-L1 adipocytes and rat adipose tissue. METHODS AND RESULTS: 3T3-L1 adipocytes and rat epididymal fat pads were cultured in a medium with/without glucose during stimulation by isoproterenol. Glycerol release was higher when adipocytes were cultured in a glucose-containing medium than that in a medium without glucose. Measurement of glucose uptake during catecholamine-stimulated lipolysis showed a slight, but significant increase in glucose uptake. We also compared glucose metabolism-related protein, such as glucose transporter 4, hexokinase, glycerol-3-phosphate dehydrogenase and lipase contents between fat tissues that play a critical role in active lipolysis. Epididymal fat exhibited higher lipolytic activity than inguinal fat because of higher lipase and glucose metabolism-related protein contents. CONCLUSION: We demonstrated that catecholamine-stimulated lipolysis is enhanced in the presence of glucose, and suggests that glucose is one of the primary substrates for G3P in adipocytes during active lipolysis.

Iron deficiency attenuates protein synthesis stimulated by branched-chain amino acids and insulin in myotubes.

Iron deficiency anemia indicates poor nutrition and is a public health problem. Iron deficiency is also associated with muscle weakness. However, the intracellular mechanisms by which iron deficiency induces muscle weakness are obscure. The purpose of the present study was to evaluate the effect of iron deficiency on protein synthesis in basal and branched-amino acids (BCAA)- and insulin-stimulated state in muscle cells. Differentiated C2C12 myotubes were incubated with an iron chelator, deferoxamine mesylate, and then stimulated with BCAA or insulin to activate protein synthesis. This iron deprivation resulted in a significant reduction in the abundance of iron-containing proteins, such as the mitochondrial complex 1 subunit protein, compared to control cells, but not of protein that does not contain iron, such as citrate synthase. Proteins involved in glucose utilization, such as glucose transpoter-1, hexokinase and AMP-activated protein kinase (AMPK), were upregulated under iron deficiency. Additionally, rates of BCAA- and insulin-stimulated protein synthesis, measured by puromycin incorporation, were lower in iron-deficient myotubes than in control cells. We suggest that low iron availability attenuates BCAA- and insulin-stimulated protein synthesis, possibly via activation of AMPK in myotubes. The present findings advance the understanding of the importance of iron to skeletal muscle protein synthesis and, thus, may contribute to the prevention of sarcopenia and frailty.

Low-carbohydrate high-protein diet diminishes the insulin response to glucose load via suppression of SGLT-1 in mice.

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.

Leucine supplementation after mechanical stimulation activates protein synthesis via L-type amino acid transporter 1 in vitro.

Branched-chain amino acid supplements consumed following exercise are widely used to increase muscle mass. Although both exercise (ie, mechanical stimulation) and branched-chain amino acid leucine supplementation have been reported to stimulate muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) signaling pathway independently, the mechanisms underlying their synergistic effects are largely unknown. Utilizing cultured differentiated C2C12 myotubes, we established a combination treatment model in which the cells were subjected to cyclic uniaxial mechanical stretching (4 h, 15%, 1 Hz) followed by stimulation with 2 mM leucine for 45 min. Phosphorylation of p70 S6 kinase (p70S6K), an mTOR-regulated marker of protein translation initiation, was significantly increased following mechanical stretching alone but returned to the baseline after 4 h. Leucine supplementation further increased p70S6K phosphorylation, with a greater increase observed in the stretched cells than in the non-stretched cells. Notably, the expression of L-type amino acid transporter 1 (LAT1), a stimulator of the mTOR pathway, was also increased by mechanical stretching, and siRNA-mediated knockdown partially attenuated leucine-induced p70S6K phosphorylation. These results suggest that mechanical stretching promotes LAT1 expression and, consequently, amino acid uptake, leading to enhanced leucine-induced activation of protein synthesis. LAT1 has been demonstrated to be a point of crosstalk between exercise- and nutrition-induced skeletal muscle growth.

Prenatal myonuclei play a crucial role in skeletal muscle hypertrophy in rodents.

Multinucleated muscle fibers are formed by the fusion of myogenic progenitor cells during embryonic and fetal myogenesis. However, the role of prenatally incorporated myonuclei in the skeletal muscle fibers of adult animals is poorly understood. We demonstrated, using muscle-specific reporter mice, that the prenatal myonuclei remained in the adult soleus muscle, although cardiotoxin injection caused the loss of prenatal myonuclei. Overloading by the tendon transection of synergists failed to induce compensatory hypertrophy in regenerated soleus muscle fibers of adult rats, whereas unloading by tail suspension normally induced the fiber atrophy. Loss of hypertrophying function correlated with the lowered histone acetylation at the transcription start site of Igf1r gene, which was one of the genes that did not respond to the overloading. These parameters were improved by the transplantation of cells harvested from the juvenile soleus muscles of neonatal rats in association with enhanced histone acetylation of Igf1r gene. These results indicated that the presence of prenatal myonuclei was closely related to the status of histone acetylation, which could regulate the responsiveness of muscle fibers to physiological stimuli.

Anti-interleukin-6 receptor antibody (MR16-1) promotes muscle regeneration via modulation of gene expressions in infiltrated macrophages.

BACKGROUND: Although rat anti-mouse IL-6 receptor (IL-6R) antibody (MR16-1) has been reported to effectively ameliorate various tissue damages, its effect on skeletal muscle regeneration has not been determined. Moreover, the localization, persistence and duration of action of this reagent in damaged tissues after systemic administration have not been assessed. METHODS: The MR16-1 was administered i.p. immediately after cardiotoxin (CTX)-induced muscle damage on mice. RESULTS: MR16-1 administered i.p. was observed only to the damaged muscle. This delivered MR16-1 was dramatically decreased from 3 to 7days post-injury concomitantly with a reduction of IL-6R expression. This reduction of the MR16-1 level in the damaged muscle was not rescued by additional administration of MR16-1, suggesting the short half-life of MR16-1 was not the factor for the remaining levels. In addition, a significant inhibitory effect of MR16-1 on phosphorylation of the signal transducer and activator of transcription 3 was observed in the macrophage-enriched area of damaged muscle 3days after injury. Finally, the acceleration of muscle regeneration observed at day 7 post-injury following MR16-1 treatment was associated with reduced expression of fibrosis-related genes, such as interleukin-10 and arginase, in the infiltrated macrophages. CONCLUSIONS: These results suggest that MR16-1 which was found primarily localized in infiltrated macrophages in the damaged muscle might facilitate muscle regeneration via immune modulation. GENERAL SIGNIFICANCE: These findings are deemed to provide further insight into the understanding not only of MR16-1 treatment on muscle regeneration, but also of the other anti-cytokine treatment on the cytokine-related disease.

Mechanical stretch activates mammalian target of rapamycin and AMP-activated protein kinase pathways in skeletal muscle cells.

Cellular protein synthesis is believed to be antagonistically regulated by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways. In the present study, we examined the relationship between mTOR/p70 S6 kinase (p70S6K) and AMPK in response to mechanical stretch. C2C12 myoblasts were grown on a silicone elastomer chamber to confluence and further cultured in differentiation medium for 4 days to form multinucleated myotubes. Cells were subjected to 15% cyclic uniaxial stretch for 4 h at a frequency of 1 Hz. Phosphorylation of p70S6K at threonine 389 and AMPK at threonine 172 of the catalytic α subunit were concomitantly increased by mechanical stretch. Stimulation of the mTOR pathway by adding leucine and insulin increased the phosphorylation of p70S6K without inactivation of AMPK. In contrast, addition of compound C, a pharmacological inhibitor of AMPK, increased the phosphorylation of p70S6K in stretched cells. Activation of AMPK by the addition of 5-amino-4-imidazolecarboxamide ribonucleoside reduced the phosphorylation of p70S6K in response to mechanical stretch. In conclusion, crosstalk between mTOR and AMPK signaling was not tightly regulated in response to physiological stimuli, such as mechanical stress and/or nutrients. However, pharmacological modulation of AMPK influenced the mTOR/p70S6K signaling pathway.

Intermittent fasting reduces mouse body fat while maintaining muscle mass by regulating protein synthesis and autophagy.

OBJECTIVES: The aim of this study is to investigate the effect of intermittent fasting (IF) on the regulation of skeletal muscle protein metabolism in response to nutrient supplementation during fasting. METHODS: Twelve-week-old male C57BL/6J mice were assigned to two groups: ad libitum and IF, with the latter having access to food for only 3 h/d. After 6 wk of experimental periods, an oral glucose tolerance test was performed. One week later, phosphate-buffered saline or a glucose and branched-chain amino acid mixture was administered orally, and blood and tissues were collected 30 min later. RESULTS: The oral glucose tolerance test results revealed that the IF group had better insulin sensitivity. They also had lower body and fat weights while maintaining the same level of skeletal muscle mass as the ad libitum group. The phosphorylation of ribosomal protein S6 in the skeletal muscle, a marker for the activation of protein translation, was greater in the IF group after glucose and branched-chain amino acid mixture administration. Microtubule-associated protein light chain 3-II-to-light chain 3-I ratio, a marker for autophagosome formation, in skeletal muscle during fasting was significantly lower in the IF group than that in the ad libitum group. CONCLUSIONS: Our findings suggest that adaptation to IF regulates protein synthesis and breakdown, leading to the maintenance of skeletal muscle mass while reducing body fat.

Inhibition of the mitochondrial respiratory chain reduces catecholamine­stimulated lipolysis via increasing lactate production in 3T3­L1 adipocytes.

Adipose tissue serves a significant role in the regulation of energy metabolism in the body. The re­esterification of the fatty acids generated during lipolysis is critical for efficient lipolysis. However, the effect of the intracellular energy state on lipolytic activity and fatty acid re­esterification during lipolysis is not yet fully understood. The present study aimed to assess the effect of the intracellular energy state on lipolytic activity and fatty acid re­esterification during lipolysis. 3T3­L1 adipocytes were incubated with mitochondrial respiratory chain inhibitors, oligomycin A or rotenone, during isoproterenol stimulation; and glycerol, glucose and lactate concentrations in the medium were measured. Western blot analysis was performed to examine the phosphorylation levels of cAMP­dependent protein kinase A (PKA). The results showed that inhibition of mitochondrial ATP synthesis decreased catecholamine­stimulated lipolysis without affecting PKA signaling. The inhibition of mitochondrial respiration increased glucose uptake and lactate production, indicating that a large amount of glucose taken up into the cell was preferentially used for ATP production rather than for re­esterification. In conclusion, the results of the present study suggested that the energy state during lipolysis may influence lipolytic activity by suppressing fatty acid re­esterification.