Decreased insulin-dependent glucose transport by chronic ethanol feeding is associated with dysregulation of the Cbl/TC10 pathway in rat adipocytes

Abstract

Heavy alcohol consumption is an independent risk factor for type 2 diabetes. Although the exact mechanism by which alcohol contributes to the increased risk is unknown, impaired glucose disposal is a likely target. Insulin-stimulated glucose disposal in adipocytes is regulated by two separate and independent pathways, the PI3K pathway and the Cbl/TC10 pathway. Previous studies suggest that chronic ethanol feeding impairs insulin-stimulated glucose transport in adipocytes in a PI3K-independent manner. In search of potential targets of ethanol that would affect insulin-stimulated glucose transport, we investigated the effects of 4-wk ethanol feeding to male Wistar rats on the Cbl/TC10 pathway in isolated adipocytes. Chronic ethanol feeding inhibited insulin-stimulated cCbl phosphorylation compared with pair feeding. Insulin receptor and Akt/PKB phosphorylation were not affected by ethanol feeding. Chronic ethanol exposure also impaired cCbl and TC10 recruitment to a lipid raft fraction isolated from adipocytes by detergent extraction. Furthermore, chronic ethanol feeding increased the amount of activated TC10 and filamentous actin in adipocytes at baseline and abrogated the ability of insulin to further activate TC10 or polymerize actin. These results demonstrate that the impairment in insulin-stimulated glucose transport observed in adipocytes after chronic ethanol feeding to rats is associated with a disruption of insulin-mediated Cbl/TC10 signaling and actin polymerization.

Fig. 1.

Chronic ethanol exposure impairs insulin-stimulated cCbl tyrosine phosphorylation. A. Isolated rat adipocytes were stimulated with or without 10 nM insulin for 0-1.5 min and lysed. Lysates were subjected to immunoprecipitation with a polyclonal antibody directed against cCbl. Samples were probed with an anti-phosphotyrosine (PY100) or anti-cCbl antibody. Representative blots are shown. Graph represents mean values ± SEM, n = 4. Open bars, unstimulated; solid bars, insulin-stimulated. Values with different letters are significantly different, p < 0.05. B. Isolated rat adipocytes were stimulated with or without 10 nM insulin for 0-1.5 min and lysed. Lysates were separated by SDS-PAGE and analyzed with either anti-IRβ (insulin receptor β subunit), anti-phosphotyrosine antibody (4G10; for phospho-IRβ), anti-phosphospecific-Akt/PKB, or anti-Akt/PKB.

Fig. 2.

Insulin-stimulated cCbl translocation to lipid raft is inhibited by chronic ethanol feeding. Isolated adipocytes from pair- and ethanol-fed rats were incubated with or with 10 nM insulin for 0-5 min and lysed. Lysates were subjected to detergent extraction (1% Triton X-100) and Triton-insoluble fractions were solubilized with 60 mM octyl β-D-thioglucopyranoside (OTG) as described in Experimental Procedures. Fractions were separated by SDS-PAGE and transferred to PVDF membrane for Western blot analysis using a polyclonal anti-cCbl antibody. Representative blots are shown. Graph represents mean values ± SEM, n = 4. Open bars, unstimulated; checkered bars, 2 min insulin; lined bars, 5 min insulin. Values with different letters are significantly different, p < 0.01.

Fig. 3.

The distribution of proteins to lipid raft and non-lipid raft plasma membrane domains is disrupted by chronic ethanol feeding. Isolated adipocytes from pair- and ethanol (EtOH) fed rats, stimulated with or without 10 nM insulin for 0-5 min were lysed and Triton-soluble and —insoluble fractions were obtained as described in Experimental Procedures, and separated by SDS-PAGE for Western blot analysis. Antibodies directed against the beta subunit of the insulin receptor (IRβ), APS, TC10, transferrin receptor (TfR), or caveolin were used for ECL detection. Representative blots are shown (IRβ and APS, n = 5; TfR, and caveolin, n = 4; and TC10, n = 3).

Fig. 4.

TC10 recruitment to lipid raft domains of the plasma membrane is inhibited after chronic ethanol exposure. Triton-soluble and —insoluble fractions were obtained as described in Experimental Procedures from plasma membrane fractions of adipocytes isolated from pair- and ethanol- (EtOH) fed rats, stimulated with or without 10 nM insulin for 0-5 min. Samples were separated by SDS-PAGE for Western analysis. Antibodies directed against the beta subunit of the insulin receptor (IRβ), TC10 or caveolin were used for ECL detection. Representative blots of three repeats are shown. Graph values are represented as the mean ± SEM of n = 3. Open bars, unstimulated; solid bars, insulin stimulated. *p = 0.010.

Fig. 5.

Chronic ethanol feeding interferes with TC10 activation. Adipocytes from pair- and ethanol-fed rats were isolated and incubated with or without 10 nM insulin for 0-5 min, lysed and TC10 activation was assessed using a PAK-1 PBD pull-down assay kit (Upstate) and a polyclonal antibody directed against TC10. Samples were subjected to SDS-PAGE and transferred for western analysis. Blots are representative of n = 3, graph shown display mean values ± SEM, n = 3. Open bars, unstimulated; solid bars, insulin stimulated. Values with different letters are significantly different, p < 0.05.

Fig. 6.

Insulin-stimulated F-actin formation is disrupted in adipocytes from chronic ethanol-fed animals. Adipocytes from pair- and ethanol-fed rats were isolated and treated with or without 10 nM insulin for 0-5 min, lysed and the relative amounts of filamentous actin assessed using a G-actin/F-actin In Vivo Assay Kit (Cytoskeleton, Inc.) as described in Experimental Procedures. Samples were separated by SDS-PAGE and analyzed by immunoblotting using an antibody directed against actin. Whole cell lysates were used to determine total actin. Blots are representative of six repeats and graph shown represents mean values ± SEM, n = 5-6. Open bars, unstimulated; solid bars, insulin stimulated. Values with different letters are significantly different, p < 0.05.