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Exercise increases TBC1D1 phosphorylation in human skeletal muscle.

ABSTRACT: Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% Vo(2 max)). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser(711) (AMPK), TBC1D1 Ser(231) (AMPK), TBC1D1 Ser(660) (AMPK), TBC1D1 Ser(700) (AMPK), and TBC1D1 Thr(590) (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPK?2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser(711), TBC1D1 Ser(231), and TBC1D1 Ser(660) but had no effect on TBC1D1 Ser(700). Exercise did not increase TBC1D1 Thr(590) phosphorylation or TBC1D1/AS160 PAS phosphorylation, consistent with the lack of Akt activation. These data demonstrate that a single bout of exercise regulates TBC1D1 and AS160 phosphorylation on multiple sites in human skeletal muscle.

SUBMITTER: Jessen N

PROVIDER: S-EPMC3129834 | biostudies-other | 2011 Jul

REPOSITORIES: biostudies-other

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Exercise increases TBC1D1 phosphorylation in human skeletal muscle.

Jessen Niels N An Ding D Lihn Aina S AS Nygren Jonas J Hirshman Michael F MF Thorell Anders A Goodyear Laurie J LJ

American journal of physiology. Endocrinology and metabolism 20110419 1

Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating t ...[more]

PMID: 21505148

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Project description:Introduction: Amino acid transporters are essential for cellular amino acid transport and promoting protein synthesis. While previous literature has demonstrated the association of amino acid transporters and protein synthesis following acute resistance exercise and amino acid supplementation, the chronic effect of resistance exercise and supplementation on amino acid transporters is unknown. The purpose herein was to determine if amino acid transporters and amino acid metabolic enzymes were related to skeletal muscle hypertrophy following resistance exercise training with different nutritional supplementation strategies. Methods: 43 college-aged males were separated into a maltodextrin placebo (PLA, n = 12), leucine (LEU, n = 14), or whey protein concentrate (WPC, n = 17) group and underwent 12 weeks of total-body resistance exercise training. Each group's supplement was standardized for total energy and fat, and LEU and WPC supplements were standardized for total leucine (6 g/d). Skeletal muscle biopsies were obtained prior to training and ~72 h following each subject's last training session. Results: All groups increased type I and II fiber cross-sectional area (fCSA) following training (p < 0.050). LAT1 protein increased following training (p < 0.001) and increased more in PLA than LEU and WPC (p < 0.050). BCKDHα protein increased and ATF4 protein decreased following training (p < 0.001). Immunohistochemistry indicated total LAT1/fiber, but not membrane LAT1/fiber, increased with training (p = 0.003). Utilizing all groups, the change in ATF4 protein, but no other marker, trended to correlate with the change in fCSA (r = 0.314; p = 0.055); however, when regression analysis was used to delineate groups, the change in ATF4 protein best predicted the change in fCSA only in LEU (r 2 = 0.322; p = 0.043). In C2C12 myoblasts, LAT1 protein overexpression caused a paradoxical decrease in protein synthesis levels (p = 0.002) and decrease in BCKDHα protein (p = 0.001). Conclusions: Amino acid transporters and metabolic enzymes are affected by resistance exercise training, but do not appear to dictate muscle fiber hypertrophy. In fact, overexpression of LAT1 in vitro decreased protein synthesis.

Project description:Immobilization of the lower limbs promotes a catabolic state that reduces muscle mass, whereas physical training promotes an anabolic state that increases muscle mass. Understanding the molecular mechanisms underlying this is of clinical interest, as loss of muscle mass is a major complication to critical illness in humans. To determine the molecular regulation of protein synthesis and degradation during muscle loss and hypertrophy, we examined skeletal muscle biopsies from healthy human subjects after 2 weeks unilateral immobilization of a lower limb and during 6 weeks of physical rehabilitation. We have previously shown that cross-sectional area of the knee muscle-extensors decreased by ∼10% during immobilization and was completely restored during rehabilitation. Here we provide novel data to suggest that autophagy is an important underlying mechanism involved in regulation of muscle mass. Protein expression of MuRF1 and ATROGIN-1 did not change during the study, indicating that the recruitment of substrates to the proteasomes was unaltered. Phosphorylation of mTORat Ser2448 did not change during the study, and neither did phosphorylation of the mTORC1 substrates 4EBP1 Thr37/46 and p70S6K Thr389, suggesting that this pathway does not suppress protein synthesis during muscle wasting. Protein levels of p62 and ULK1 increased during immobilization and returned to baseline levels during rehabilitation. Same pattern was observed for FOXO3a phosphorylation at Ser318/321, suggesting transcriptional activation during immobilization and inactivation during rehabilitation. To investigate this further, we analyzed mRNA expression of seven autophagy-related genes controlled by FOXO3a. Five of these (p62, LC3B, BECLIN-1, ATG12, and BNIP3) increased during immobilization and returned to baseline during rehabilitation. In conclusion, immobilization of a lower limb increases autophagy-related gene and protein expression in human skeletal muscle in a pattern that mirrors FOXO3a phosphorylation. These findings could imply that FOXO3a dependent transcriptional regulation of autophagy is involved in the regulation of muscle mass in humans.Clinical trial registrationThe study was approved by the Ethics Committee of Copenhagen (j.no. H-1-2010-016).

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Exercise increases TBC1D1 phosphorylation in human skeletal muscle.

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Exercise increases TBC1D1 phosphorylation in human skeletal muscle.

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