Resveratrol enhances GLUT-4 translocation to the caveolar lipid raft fractions through AMPK/Akt/eNOS signalling pathway in diabetic myocardium

S Varma Penumathsa, M Thirunavukkarasu, L Zhan, G Maulik, V P Menon, D Bagchi, N Maulik, S Varma Penumathsa, M Thirunavukkarasu, L Zhan, G Maulik, V P Menon, D Bagchi, N Maulik

Abstract

Homeostasis of blood glucose by insulin involves stimulation of glucose uptake by translocation of glucose transporter Glut-4 from intracellular pool to the caveolar membrane system. In this study we examined resveratrol (RSV)-mediated Glut-4 translocation in the streptozotocin (STZ)-induced diabetic myocardium. The rats were randomized into three groups: Control (Con), Diabetes Mellitus (DM) (STZ 65 mg/kg b.w., i.p.) & DM+RSV (2.5 mg/kg b.wt. for 2 weeks orally) (RSV). Isolated rat hearts were used as per the experimental model. RSV induced glucose uptake was observed in vitro with H9c2 cardiac myoblast cells. Decreased blood glucose level was observed after 30 days (375 mg/dl) in RSV-treated rats when compared to DM (587 mg/dl). Treatment with RSV demonstrated increased Adenosine Mono Phosphate Kinase (AMPK) phosphorylation compared to DM. Lipid raft fractions demonstrated decreased expression of Glut-4, Cav-3 (0.4, 0.6-fold) in DM which was increased to 0.75- and 1.1-fold on RSV treatment as compared to control. Increased Cav-1 expression (1.4-fold) in DM was reduced to 0.7-fold on RSV treatment. Increased phosphorylation of endothelial Nitric Oxide Synthase (eNOS) & Akt was also observed in RSV compared to DM (P<0.05). Confocal microscopy and coimmunoprecipitation studies demonstrated decreased association of Glut-4/Cav-3 and increased association of Cav-1/eNOS in DM as compared to control and converse results were obtained on RSV treatment. Our results suggests that the effect of RSV is non-insulin dependent and triggers some of the similar intracellular insulin signalling components in myocardium such as eNOS, Akt through AMPK pathway and also by regulating the caveolin-1 and caveolin-3 status that might play an essential role in Glut-4 translocation and glucose uptake in STZ- induced type-1 diabetic myocardium.

Figures

Figure 1
Figure 1
(A) Graph represents the resveratrol (RSV) (50 μm) mediated 2-deoxy[3H]glucose uptake in H9c2 cardiac myoblast cells in a time dependent manner. Insulin treatment was used as the positive control. *P< 0.05 represent significant difference in comparison with control or non-treated cells. (B) Graph represents the resveratrol-mediated 2-deoxy[3H]glucose uptake in H9c2 cardiac myoblast cells in a dose dependent manner. 50 μM RSV treatment has shown significant increase when compared to 25 μM but no significant difference was observed between 50 μM and 100 μM. *P< 0.05 represent significant difference in comparison with control or non-treated cells. †p<0.05 represent significant difference in comparison with 25 μM resveratroltreated cells.
Figure 2
Figure 2
(A) Western blot represents the p-Akt protein expression in caveolin/lipid raft fractions in control, diabetes mellitus (DM) and DM + RSV groups. Density value of p-Akt bands were normalized to level of Akt and expressed relative to control. Fractions 4–6 represent the membrane fractions and 8–12 represent the cytosolic fractions. (B) Protein expression levels of p-AMPK in cytosolic fractions of control, DM and DM + RSV groups. Density value of p-AMPK bands were normalized to level of AMPK and expressed relative to control. GAPDH was used as the loading control. n = 4 times repeated experiments with equivalent results. Graphs represent the quantitative expression between the groups. *P<0.05 represent significant difference compared with control. †P< 0.05 represent significant difference compared with DM.
Figure 3
Figure 3
A and B Western blot represents the (A) caveolin-1 protein expression and (B) p-eNOS, eNOS in caveolin/lipid raft fractions in control, DM and DM + RSV groups. Fractions 4–6 represent the membrane fractions and 8–12 represent the cytosolic fractions. Graphs represent the quantitative expression between the groups, n = 6 times repeated experiments with equivalent results. *P<0.05 represent significant difference compared with control. †P<0.05 represent significant difference compared with DM.
Figure 4
Figure 4
A and B Western blot represents the (A) caveolin-3 and (B) Glut-4 protein expression in caveolin/lipid raft fractions in control, DM and DM + RSV groups. Fractions 4–6 represent the membrane fractions and 8–12 represent the cytosolic fractions. Graphs represent the quantitative expression between the groups, n = 6 in each group. *P < 0.05 represent significant difference compared with control. †P<0.05 represent significant difference compared with DM.
Figure 5
Figure 5
(A) Immunoprecipitation with caveolin-3 and re-blot with Glut-4 in control, DM and DM + RSV groups. (B) Immunoprecipitation with caveolin-1 and re-blot with eNOS in control, DM and DM + RSV groups, n = 6 in each group. P< 0.05 represent significant difference compared with control. †P< 0.05 represent significant difference compared with DM.
Figure 6
Figure 6
Represents the rat cardiac paraffin sections labelled with immunofluorescence and visualized using confocal microscopy in control, DM and DM + RSV groups. A, D and G represent red fluorescence labelled caveolin-1. B, E and H represent the green fluorescence labelled caveolin-3. C, Fand I represent the merged photo of Cav-1 and Cav-3. White arrows denote caveolin-1; Yellow arrows denote caveolin-3. The number of arrows represents the extent of expression of either Cav-1 or Cav-3. No significant co-localization (merged) was observed between the caveolin-1 and caveolin-3. n = 6 in each group.
Figure 7
Figure 7
Represents the rat cardiac paraffin sections labelled with immunofluorescence and visualized using confocal microscopy in control, DM and DM + RSV groups. A, D and G represent red fluorescence labelled caveolin-1. B, E and H represent the green fluorescence labelled eNOS. C, F and I represent the merged photo which clearly shows the co-localization of caveolin-1 / eNOS. n = 6 in each group. White arrows denote Caveolin-1, yellow arrows denote eNOS and the white/yellow (merged) arrow denotes the caveolin-1 and eNOS association / co-localization. The number of arrows represents the extent of expression of either cav-1, eNOS or the extent of co-localization. The figure in the box represents the magnified image demonstrating the co-localization.
Figure 8
Figure 8
Represents the rat cardiac paraffin sections labelled with immunofluorescence and visualized using confocal microscopy in control, DM and DM + RSV groups. A, D and G represent red fluorescence labelled Glut-4. B, E and H represent the green fluorescence labelled caveolin-3. C, F and I represent the merged photo that clearly shows the co-localization of caveolin-3 and Glut-4. n = 6 in each group. White arrows denote Glut-4, yellow arrows denote caveolin-3 and the white/yellow (merged) arrow denotes the caveolin-3 and Glut-4 association / co-localization. The number of arrows represents the extent of expression of either cav-3, Glut-4 or the extent of co-localization of cav-3/Glut-4.

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