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

An D, Toyoda T, Taylor EB, Yu H, Fujii N, Hirshman MF, Goodyear LJ - Diabetes (2010)

Bottom Line: Expression of the obesity-associated R125W mutant significantly decreased insulin-stimulated glucose transport in the absence of changes in TBC1D1 PAS phosphorylation.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.

View Article: PubMed Central - PubMed

Affiliation: Research Division, Joslin Diabetes Center, Boston, Massachusetts, USA.

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.

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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).
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Figure 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).

Mentions: We examined whether TBC1D1 regulates contraction-stimulated glucose transport. The contraction protocol used was maximal, and therefore the increase in glucose transport with contraction was greater than with glucose-induced physiological insulin (Figs. 3 and 4). Similar to the effects of insulin, expression of wild-type TBC1D1 did not alter contraction-stimulated glucose transport (Fig. 4A). However, in contrast to what was observed with glucose/insulin, muscles overexpressing the TBC1D1 4P mutant exhibited a significant decrease in contraction-stimulated glucose transport (Fig. 4A). Furthermore, although the expression of the TBC1D1 R125W mutation decreased insulin-induced glucose transport, it did not alter contraction-induced glucose transport (Fig. 4A).


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

An D, Toyoda T, Taylor EB, Yu H, Fujii N, Hirshman MF, Goodyear LJ - Diabetes (2010)

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).
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Figure 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).
Mentions: We examined whether TBC1D1 regulates contraction-stimulated glucose transport. The contraction protocol used was maximal, and therefore the increase in glucose transport with contraction was greater than with glucose-induced physiological insulin (Figs. 3 and 4). Similar to the effects of insulin, expression of wild-type TBC1D1 did not alter contraction-stimulated glucose transport (Fig. 4A). However, in contrast to what was observed with glucose/insulin, muscles overexpressing the TBC1D1 4P mutant exhibited a significant decrease in contraction-stimulated glucose transport (Fig. 4A). Furthermore, although the expression of the TBC1D1 R125W mutation decreased insulin-induced glucose transport, it did not alter contraction-induced glucose transport (Fig. 4A).

Bottom Line: Expression of the obesity-associated R125W mutant significantly decreased insulin-stimulated glucose transport in the absence of changes in TBC1D1 PAS phosphorylation.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.

View Article: PubMed Central - PubMed

Affiliation: Research Division, Joslin Diabetes Center, Boston, Massachusetts, USA.

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.

Show MeSH
Related in: MedlinePlus