Inhibition of contraction-stimulated AMP-activated protein kinase inhibits contraction-stimulated increases in PAS-TBC1D1 and glucose transport without altering PAS-AS160 in rat skeletal muscle

Abstract

Objective: Phosphorylation of two members of the TBC1 domain family of proteins, Akt substrate of 160 kDa (AS160, also known as TBC1D4) and TBC1D1, has been implicated in the regulation of glucose transport in skeletal muscle. Insulin-stimulated phosphorylation (measured using the phospho-Akt substrate [PAS] antibody) of AS160 and TBC1D1 appears to occur in an Akt-dependent manner, but the kinases responsible for contraction-stimulated PAS-AS160 and PAS-TBC1D1 remain unclear. AMP-activated protein kinase (AMPK) and Akt, both activated by contraction, can each phosphorylate AS160 and TBC1D1 in cell-free assays.

Research design and methods: To evaluate the roles of AMPK and Akt on insulin- or contraction-stimulated PAS-AS160, PAS-TBC1D1, and glucose transport, rat epitrochlearis was incubated with and without compound C (inhibitor of AMPK) or Wortmannin (inhibitor of phosphatidylinositol [PI] 3-kinase, which is upstream of Akt) before and during insulin stimulation or contraction.

Results: Insulin-stimulated glucose transport and phosphorylation of both AS160 and TBC1D1 were completely inhibited by Wortmannin. Wortmannin eliminated contraction stimulation of phospho-Ser(21/9)glycogen synthase kinase 3alpha/beta (pGSK3; Akt substrate) and PAS-AS160 but did not significantly alter pAMPK, phospho-Ser79acetyl CoA carboxylase (pACC; AMPK substrate), PAS-TBC1D1, or glucose transport in contraction-stimulated muscle. Compound C completely inhibited contraction-stimulated pACC and PAS-TBC1D1 and partially blocked glucose transport, but it did not significantly alter pAkt, pGSK3, or PAS-AS160.

Conclusions: These data suggest that 1) insulin stimulates glucose transport and phosphorylation of AS160 and TBC1D1 in a PI 3-kinase/Akt-dependent manner, 2) contraction stimulates PAS-AS160 (but not PAS-TBC1D1 or glucose transport) in a PI 3-kinase/Akt-dependent manner, and 3) contraction stimulates PAS-TBC1D1 and glucose transport (but not PAS-AS160) in an AMPK-dependent manner.

FIG. 1.

Abundance of total proteins (Akt, GSK3, AMPK, ACC, CaMKII, AS160, and TBC1D1). There were no statistically significant differences among groups (n = 4 per group) for total protein abundance in muscles with or without insulin and/or Wortmannin (A), contraction and/or Wortmannin (B), and contraction and/or compound C (C). CC, compound C; Wort, Wortmannin.

FIG. 2.

Effects of Wortmannin on insulin-stimulated phosphorylation of Akt^Thr308 (A), GSK3^Ser21/9 (B), PAS-160 (C), PAS-150 (D), and glucose transport (E). Paired isolated rat epitrochlearis muscles were incubated with or without 500 nmol/l of Wortmannin for 30 min. Muscles were then either incubated in identical media (basal) or in solution that contained 2,000 μU/ml of insulin for 20 min, freeze-clamped immediately, and used for immunoblotting or for 3-MG transport measurement. Data are the means ± SE, n = 5–9 per group. Post hoc analysis: *P < 0.05 (effect of insulin); †P < 0.05 (effect of Wortmannin). □, DMSO; ■, Wortmannin. Wort, Wortmannin.

FIG. 3.

Effects of Wortmannin on contraction-stimulated phosphorylation of Akt^Thr308 (A), GSK3^Ser21/9 (B), AMPK^Thr172 (C), ACC^Ser79 (D), CaMKII^Thr286 (E), PAS-AS160 (F), PAS-TBC1D1 (G), and glucose transport (H). Paired isolated rat epitrochlearis muscles were incubated with or without 500 nmol/l of Wortmannin for 30 min. Muscles were then either rested (basal) or stimulated to contract for 20 min, freeze-clamped immediately for immunoprecipitation (IP) and/or immunoblotting (IB), or used for 3-MG transport measurement. Data are the means ± SE, n = 9–17 per group. Post hoc analysis: *P < 0.05 (effect of contraction); †P < 0.05 (effect of Wortmannin). □, DMSO; ■, Wortmannin. Wort, Wortmannin.

FIG. 4.

Effects of compound C on contraction-stimulated phosphorylation of Akt^Thr308 (A), GSK3^Ser21/9 (B), AMPK^Thr172 (C), ACC^Ser79 (D), CaMKII^Thr286 (E), PAS-AS160 (F), PAS-TBC1D1 (G), and glucose transport (H). Paired isolated rat epitrochlearis muscles were incubated with or without 40 μmol/l of compound C for 60 min. Muscles were then either rested (basal) or stimulated to contract for 20 min, freeze-clamped immediately for immunoprecipitation (IP) and/or immunoblotting (IB), or used for 3-MG transport measurement. Data are the means ± SE, n = 6–14 per group. Post hoc analysis: *P < 0.05 (effect of contraction); †P < 0.05 (effect of compound C). □, DMSO; ■, compound C. CC, compound C.

FIG. 5.

Effects of compound C on insulin-stimulated (A) and AICAR-stimulated (B) glucose transport. Paired isolated rat epitrochlearis muscles were incubated with or without 40 μmol/l of compound C for 60 min. Muscles were then either incubated in identical media (basal) or in solution that contained 2,000 μU/ml of insulin (A) for 20 min or in solution that contained 2 mmol/l of AICAR (B) for 40 min and used for 3-MG transport measurement. Data are the means ± SE, n = 6–14 per group. Post hoc analysis: *P < 0.05 (effect of insulin or AICAR); †P < 0.05 (effect of compound C). □, DMSO; ■, compound C.

FIG. 6.

Working model for the roles of PAS-AS160 and PAS-TBC1D1 in insulin- and contraction-stimulated glucose transport. For clarity, the figure focuses on PAS-AS160 and PAS-TBC1D1 and does not depict other possible mechanisms that may influence glucose transport (e.g., calcium-mediated processes with contraction, binding of AS160 or TBC1D1 to 14-3-3 proteins, and/or phosphorylation on sites not recognized by anti-PAS with insulin or contraction). Insulin, by a PI 3-kinase (PI3K)-dependent mechanism, activates Akt, which phosphorylates AS160 (TBC1D4) and TBC1D1 on sites identified using the PAS antibody. The PI 3-kinase inhibitor Wortmannin completely eliminates insulin-stimulated glucose transport and PAS-phosphorylation of AS160 and TBC1D1. Increased PAS-AS160 is required for insulin's full effect on GLUT4 translocation and glucose transport. TBC1D1's role in insulin-stimulated glucose transport is uncertain. Contraction leads to increased phosphorylation of AMPK and Akt; although the ability of Wortmannin to inhibit Akt activation by contraction suggests at PI 3-kinase–dependent process, the precise mechanism is unknown. AMPK inhibition (by compound C) completely eliminates the increased PAS-TBC1D1 without altering PAS-AS160, and Wortmannin completely eliminates the increased PAS-AS160 without altering PAS-TBC1D1. Compound C partially inhibits contraction-stimulated glucose transport, consistent with AMPK-related mechanisms accounting for a portion of contraction-mediated glucose transport. The concomitant AMPK-dependent inhibition of PAS-TBC1D1 suggests it may play a role in contraction-stimulated glucose transport.