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In vivo effects of vanadium on glucose transporter translocation in cardiac tissue Li, Shi Hong

Abstract

The effects of vanadium treatment on insulin-stimulated glucose transporter type 4 (GLUT4) translocation were studied in cardiac tissue of streptozotocin (STZ)-induced diabetic Wistar rats by determining the subcellular distribution of GLUT4. Four groups of rats were examined: control and diabetic, with or without bis(maltolato)oxovanadium(IV) (BMOV, an organic form of vanadium) treatment for 8 weeks. Plasma membrane and intracellular membrane fractions were purified from heart ventricles isolated from rats either in the basal state or injected with insulin. GLUT4 content in the membrane fractions was quantified with a competitive enzyme-linked immunosorbant assay (ELISA) and also with enhanced chemiluminescence (ECL) Western blot. A time-course study was first conducted in control rats to determine the points at which insulin stimulated an initial GLUT4 translocation and a maximal GLUT4 translocation. It was shown that after insulin injection plasma membrane GLUT4 level increased about 60% at 15 minutes and intracellular GLUT4 decreased about 40% at 5 minutes and remained at this level throughout the remaining 25 minutes. We therefore decided to study the effects of vanadium on insulin-induced GLUT4 translocation at 5 minutes as the initial insulin response and at 15 minutes after insulin injection as the maximal insulin response. Towards this goal, 50% of animals in each group at the time of termination received an intravenous (iv) dose of insulin (5 U/kg) and hearts were removed 5 minutes later. In another experiment using a second set of animals divided into groups as stated above, the same dose of insulin was administrated and the hearts were taken at 15 minutes after insulin injection. At 5 minutes after insulin injection, plasma membrane GLUT4 levels in the control group under basal and insulin-stimulated states were not significantly different from their respective control-treated groups. However, insulin stimulation caused a significant increase in plasma membrane GLUT4 level in the control-vanadium-treated group (~30%) but not in the control group (~12%). Basal plasma membrane GLUT4 level was significantly lower in the diabetic group (50%) when compared to the control and control-vanadium-treated groups. No significant difference in basal plasma membrane GLUT4 was detected between the diabetic and the diabetic-vanadium-treated groups. There was a significant increase in plasma membrane GLUT4 after insulin stimulation in these two groups. The magnitude of insulin-induced GLUT4 translocation was about 2.1-fold in the diabetic and 3.6-fold in the diabetic-vanadium-treated groups. On the one hand, plasma membrane GLUT4 levels following insulin administration in the diabetic group was still significantly lower than that of the corresponding control groups. On the other hand, plasma membrane GLUT4 level after insulin injection in the diabetic-vanadium-treated group was not different from the control groups and was significantly higher than that of the insulin-stimulated diabetic group, indicating an enhancement of insulin response on GLUT4 translocation brought about by vanadium treatment. At 15 minutes after insulin injection, all the groups had a significant increase in plasma membrane GLUT4 when compared to their corresponding non-insulin treated groups. The magnitude of insulin-mediated GLUT4 translocation was about 60% in the control and control-vanadium- treated groups, which was consistent with that from the time course study whereas the degree of insulin-induced GLUT4 mobilization was about 2.8-fold in the diabetic and 2.4-fold in the diabetic-vanadium-treated groups. Consistent with what we found at the 5-minute time point, basal plasma membrane GLUT4 levels were significantly lower in the diabetic and diabetic-vanadium- treated groups (50%) when compared to the control groups. In contrast to that at 5 minutes after insulin injection, no significant difference in the plasma membrane GLUT4 level was observed between the diabetic and the diabetic-vanadium-treated groups at this time point. GLUT4 mobilization from the intracellular pool in response to insulin, as indicated by a decrease in GLUT4 level from the basal level, was also investigated at 15 minutes after insulin injection. All the groups had a significant decrease in intracellular membrane GLUT4 when compared to their corresponding non-insulin treated groups. Intracellular GLUT4 dropped by 44-57% after insulin stimulation in the control and control-vanadium-treated groups, which was again consistent with that from the time course study. In the diabetic and diabetic-vanadium-treated groups, insulin stimulation resulted in 60% and 45% decrease, respectively, in intracellular GLUT4 pool. Intracellular GLUT4 content under basal and insulin-stimulated conditions was significantly higher in the diabetic-vanadium-treated group when compared to the diabetic group under the same condition. Both basal and insulin-stimulated levels of intracellular GLUT4 in the diabetic-vanadium-treated group were comparable to their corresponding control groups. Taken together, these results show that firstly, insulin stimulation significantly increased plasma membrane GLUT4 content which correlated to the decrease in the intracellular GLUT4 pool at 15 minutes post-insulin injection in the control and control-vanadium-treated groups. Secondly, long-term insulin deficiency (9-week STZ diabetes) resulted in a decrease of total cellular GLUT4 content, but hearts from long term STZ-diabetic animals were still responsive to supraphysiological dose of insulin in terms of GLUT4 translocation. Thirdly, the observation that basal intracellular GLUT4 level was significantly higher in the diabetic-vanadium-treated group when compared to that of the diabetic group indicated that vanadium treatment may restore total cellular GLUT4 content in the diabetic group. However, increased basal intracellular GLUT4 in the diabetic-vanadium-treated group did not result in more insulin-mediated GLUT4 translocation at 15 minutes after insulin injection. Vanadium treatment did, however, enhance the insulin response in terms of GLUT4 translocation at 5 minutes in the cardiac tissue of diabetic rats. Finally, the finding that plasma membrane GLUT4 in the diabetic-treated group was significantly higher than that of the diabetic group at 5 minutes but not at 15 minutes post-insulin injection indicated that vanadium treatment enhances insulin-mediated GLUT4 translocation by enhancing its early response and by accelerating this process. This was further supported by data showing that insulin stimulation significantly increases plasma membrane GLUT4 in the control-vanadium-treated but not in the control group at 5 minutes after insulin administration. We also verified the ELISA method, which was developed in our laboratory. The data from the ELISA assay showed a pattern similar to that from the ECL Western blot, indicating that the ELISA assay was reliable.

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