TBC1D1 regulates insulin- and contraction-induced glucose transport in mouse skeletal muscle

Abstract

Objective: TBC1D1 is a member of the TBC1 Rab-GTPase family of proteins and is highly expressed in skeletal muscle. Insulin and contraction increase TBC1D1 phosphorylation on phospho-Akt substrate motifs (PASs), but the function of TBC1D1 in muscle is not known. Genetic linkage analyses show a TBC1D1 R125W missense variant confers risk for severe obesity in humans. The objective of this study was to determine whether TBC1D1 regulates glucose transport in skeletal muscle.

Research design and methods: In vivo gene injection and electroporation were used to overexpress wild-type and several mutant TBC1D1 proteins in mouse tibialis anterior muscles, and glucose transport was measured in vivo.

Results: Expression of the obesity-associated R125W mutant significantly decreased insulin-stimulated glucose transport in the absence of changes in TBC1D1 PAS phosphorylation. Simultaneous expression of an inactive Rab-GTPase (GAP) domain of TBC1D1 in the R125W mutant reversed this decrease in glucose transport caused by the R125W mutant. Surprisingly, expression of TBC1D1 mutated to Ala on four conserved Akt and/or AMP-activated protein kinase predicted phosphorylation sites (4P) had no effect on insulin-stimulated glucose transport. In contrast, expression of the TBC1D1 4P mutant decreased contraction-stimulated glucose transport, an effect prevented by concomitant disruption of TBC1D1 Rab-GAP activity. There was no effect of the R125W mutation on contraction-stimulated glucose transport.

Conclusions: TBC1D1 regulates both insulin- and contraction-stimulated glucose transport, and this occurs via distinct mechanisms. The R125W mutation of TBC1D1 impairs skeletal muscle glucose transport, which could be a mechanism for the obesity associated with this mutation.

FIG. 1.

TBC1D1 expression was significantly increased in mouse tibialis anterior muscles in response to in vivo cDNA injection and electroporation. Empty pCAGGS vector (EV) and TBC1D1 cDNA constructs (wild-type [WT] and 4P, R125W, R/K, 4P/RK, and R125W/RK mutants) were injected into the tibialis anterior muscles of anesthetized mice, followed by in vivo electroporation. The animals were allowed to recover, and protein expression was assessed 1 week after injection. Muscle proteins were separated by SDS-PAGE and immunoblotted with an anti-TBC1D1 antibody. The data are expressed as the means ± SE; n = 8–12/group. #P < 0.05 (vs. empty vector controls).

FIG. 2.

Phosphorylation of TBC1D1 in transfected muscles. To determine whether expression of TBC1D1 altered TBC1D1 PAS phosphorylation, EV and TBC1D1 cDNA constructs were injected into tibialis anterior muscles followed by in vivo electroporation, and mice were studied 1 week later. A: Mice were anesthetized and injected intravenously with saline (−) or glucose (+; 1.0 g of glucose/kg of body wt), and tibialis anterior muscles were obtained 15 min later. B: Mice were anesthetized, one leg was sham treated (−) and the other leg was contracted in situ (+) for 15 min, and tibialis anterior muscles were dissected. Muscle lysates were immunoprecipitated to deplete AS160 and supernatants were separated by SDS-PAGE and immunoblotted with an anti-PAS antibody. White bars represent basal treatment, and black bars represent glucose/insulin (A) or contraction (B) treatment groups. Data are means ± SE, n = 8. *P < 0.05 (vs. basal); #P < 0.05 (vs. empty vector for respective treatment).

FIG. 3.

Overexpression of TBC1D1 altered insulin-stimulated glucose transport in mouse skeletal muscles. To determine whether expression of wild-type TBC1D1 and TBC1D1 mutants altered insulin-stimulated glucose transport, EV or TBC1D1 cDNA constructs were injected into tibialis anterior muscles followed by in vivo electroporation. One week later, mice were anesthetized and administered a saline or 20% glucose bolus (1.0 g of glucose/kg of body wt) through the retro-orbital sinus to stimulate a physiologic insulin response. Insulin-stimulated glucose transport was measured in tibialis anterior muscles using [³H]2-deoxyglucose. A: The effects of expressing the 4P and R125W mutants. B: The effects of the Rab-GAP (R/K) mutants. White bars represent the basal treatment, and black bars represent the glucose/insulin treatment group. Data are means ± SE, n = 8–16 mice/group. *P < 0.05 (vs. basal); #P < 0.05 (vs. empty vector for respective treatment).

FIG. 4.

Overexpression of TBC1D1 altered contraction-stimulated glucose transport in mouse skeletal muscles. To determine whether expression of wild-type TBC1D1 and TBC1D1 mutants altered contraction-stimulated glucose transport, EV or TBC1D1 cDNA constructs were injected into tibialis anterior muscles followed by in vivo electroporation. One week later, mice were anesthetized and contraction was performed by stimulation of the peroneal nerve. One leg was sham treated but unstimulated, and the other leg was contracted for 15 min. Contraction-stimulated glucose transport was measured in tibialis anterior muscles using [³H]2-deoxyglucose. A: The effects of expressing the 4P and R125W mutants. B: The effects of the Rab-GAP (R/K) mutants. White bars represent the basal treatment, and black bars represent the contraction treatment group. Data are means ± SE, n = 8–16 mice/group. *P < 0.05 (vs. basal); #P < 0.05 (vs. empty vector for respective treatment).