Exercise increases TBC1D1 phosphorylation in human skeletal muscle

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.