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 AMPKα2 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.

Fig. 1.

Muscle biopsies were taken before (open bars) and after a 2-wk intervention with an isocaloric diet with a very low carbohydrate content (closed bars). The protein expression of TBC1D1 in these biopsies was analyzed by Western blotting. A: a long exposure of TBC1D1 immunoblots revealed 3 splice variants of TBC1D1, with 1 prominent band that migrated above the 150-kDa marker (long form) and 2 additional bands below 150 kDa. (medium and short form). B and C: there were no differences in the protein expression of the long form of TBC1D1 and AS160 after diet intervention.

Fig. 2.

A and B: AMP-activated protein kinase (AMPK)α2 and -α1 activity were measured in muscle biopsies taken before (open bars) and after 30 min of exercise on a cycle ergometer at 70% of maximal oxygen uptake (V̇o_{2 max}; closed bars). Exercise increased AMPKα2 activity 3-fold before and after diet intervention, whereas AMPKα1 activity remained at basal level after exercise. C and D: phosphorylation of AMPK (pAMPK) and the downstream target acetyl-CoA carboxylase (ACC) was assessed by Western blotting, and the increase in AMPKα2 activity was associated with a significant increase in AMPK phosphorylation and phosphorylation of ACC. E–H: there were no differences in expression of the AMPKα2 and -α1 isoforms, ACC, or the AMPK kinase LKB1 before (open bars) and after diet intervention (closed bars). †P < 0.01.

Fig. 3.

TBC1D1 phosphorylation at sites Ser²³¹, Ser⁶⁶⁰, Ser⁷⁰⁰, and Thr⁵⁹⁰, phosphorylation of AS160 at Ser⁷¹¹, and phospho-Akt substrate (PAS) phosphorylation were measured by Western blot. Open bars are before exercise and closed bars after 30 min of exercise on a cycle ergometer at 70% of V̇o_{2 max}. A: exercise increased phosphorylation of TBC1D1 at Ser²³¹, and there was no diet effect. B: exercise increased the phosphorylation of TBC1D1 at Ser⁶⁶⁰. There was a main effect of diet intervention on the phosphorylation level of Ser⁶⁶⁰. C and D: TBC1D1 phosphorylation at Ser⁷⁰⁰ and Thr⁵⁹⁰ was not changed after exercise or diet intervention. E: AS160 phosphorylation at Ser⁷¹¹ increased after exercise, and this exercise response was not affected by diet intervention. F: both AS160 and TBC1D1 contain phosphorylation sites that can be expected to be detected using the PAS antibody, but we did not see changes in the PAS signal after exercise or diet intervention. †P < 0.01; *P < 0.05.

Fig. 4.

A: glycogen content was measured in biopsies taken before (open bars) and after 30 min exercise on a cycle ergometer at 70% of V̇o_{2 max} (closed bars) in the pre- and postdiet conditions. Postdiet glycogen levels were reduced compared with the prediet levels (*P = 0.01 for main effect of diet), and 30 min of exercise reduced muscle glycogen content (†P < 0.01). B: expression of the glucose transporter GLUT4 in skeletal muscle was not changed by diet intervention.