Complex regulation of PKCβ2 and PDK-1/AKT by ROCK2 in diabetic heart.

The RhoA/ROCK pathway contributes to diabetic cardiomyopathy in part by promoting the sustained activation of PKCβ2 but the details of their interaction are unclear. The purpose of this study was to investigate if over-activation of ROCK in the diabetic heart leads to direct phosphorylation and activation of PKCβ2, and to determine if their interaction affects PDK-1/Akt signaling.Regulation by ROCK of PKCβ2 and related kinases was investigated by Western blotting and co-immunoprecipitation in whole hearts and isolated cardiomyocytes from 12 to 14-week diabetic rats. Direct ROCK2 phosphorylation of PKCβ2 was examined in vitro. siRNA silencing was used to confirm role of ROCK2 in PKCβ2 phosphorylation in vascular smooth muscle cells cultured in high glucose. Furthermore, the effect of ROCK inhibition on GLUT4 translocation was determined in isolated cardiomyocytes by confocal microscopy.Expression of ROCK2 and expression and phosphorylation of PKCβ2 were increased in diabetic hearts. A physical interaction between the two kinases was demonstrated by reciprocal immunoprecipitation, while ROCK2 directly phosphorylated PKCβ2 at T641 in vitro. ROCK2 siRNA in vascular smooth muscle cells or inhibition of ROCK in diabetic hearts reduced PKCβ2 T641 phosphorylation, and this was associated with attenuation of PKCβ2 activity. PKCβ2 also formed a complex with PDK-1 and its target AKT, and ROCK inhibition resulted in upregulation of the phosphorylation of PDK-1 and AKT, and increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in diabetic hearts.This study demonstrates that over-activation of ROCK2 contributes to diabetic cardiomyopathy by multiple mechanisms, including direct phosphorylation and activation of PKCβ2 and interference with the PDK-1-mediated phosphorylation and activation of AKT and translocation of GLUT4. This suggests that ROCK2 is a critical node in the development of diabetic cardiomyopathy and may be an effective target to improve cardiac function in diabetes

Mechanisms by which bis(maltolato)oxovanadium(IV) normalizes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression in streptozotocin-diabetic rats in vivo

Vanadium treatment normalizes plasma glucose levels in streptozotocin-diabetic rats in vivo, but the mechanism(s) involved are still unclear. Here, we tested the hypothesis that the in vivo effects of vanadium are mediated by changes in gluconeogenesis. Diabetic rats were treated with bis(maltolato)oxovanadium(IV) (BMOV) in the drinking water (0.75-1 mg/ml, 4 wk) or, for comparison, with insulin implants (4 U/d) for the final week of study. As with insulin, BMOV lowered plasma glucose and normalized phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) mRNA in the liver and kidney of diabetic rats. To determine the importance of reducing hyperglycemia per se, diabetic rats were treated either with a single ED dose of BMOV (0.1 mmol/kg, ip) or with phlorizin (900 mg/kg·d, 5 d). BMOV rapidly restored PEPCK and G-6-Pase mRNA and normalized plasma glucose in responsive (50%) diabetic rats but had no effect on the nonresponsive hyperglycemic rats. Phlorizin corrected plasma glucose but had no effect on PEPCK mRNA and only partially normalized G-6-Pase mRNA. In conclusion, 1) BMOV inhibits PEPCK mRNA expression and activity by rapid mechanisms that are not reproduced simply by correction of hyperglycemia; and 2) BMOV inhibits G-6-Pase expression by complex mechanisms that depend, in part, on correction of hyperglycemia. 5

Effect of ROCK inhibition on phosphorylation of PKCβ2 and its targets.

<p>A. Phosphorylation of PKCβ2 in cardiomyocytes isolated from control (C) and diabetic (D) hearts. Cardiomyocytes were either untreated (C,D) or incubated with 1 µM Y-27632 (C+Y, D+Y) or 1 µM H-1152 (C+H, D+H). ***, P<0.001 compared to corresponding control groups; #, P<0.05 compared to corresponding untreated diabetic group, n = 4 in each group. B. Representive immunoblot showing phosphorylation of PKCβ2 immunoprecipitated from untreated and Y-27632-treated control and diabetic hearts. C. Co-immunoprecipitation of PKCβ2 binding proteins with anti-PKCβ2 antibody coupled to Dynal beads, followed by detection by Coomassie Brilliant Blue staining (left). ROCK-dependent PKCβ2 phosphorylation targets were detected by anti-PKC phospho substrate antibody (right). Arrows indicate the two protein bands as PKCβ2 downstream targets.</p

Effect of ROCK inhibition on phosphorylation of PKCβ2 and its targets.

<p>A. Phosphorylation of PKCβ2 in cardiomyocytes isolated from control (C) and diabetic (D) hearts. Cardiomyocytes were either untreated (C,D) or incubated with 1 µM Y-27632 (C+Y, D+Y) or 1 µM H-1152 (C+H, D+H). ***, P<0.001 compared to corresponding control groups; #, P<0.05 compared to corresponding untreated diabetic group, n = 4 in each group. B. Representive immunoblot showing phosphorylation of PKCβ2 immunoprecipitated from untreated and Y-27632-treated control and diabetic hearts. C. Co-immunoprecipitation of PKCβ2 binding proteins with anti-PKCβ2 antibody coupled to Dynal beads, followed by detection by Coomassie Brilliant Blue staining (left). ROCK-dependent PKCβ2 phosphorylation targets were detected by anti-PKC phospho substrate antibody (right). Arrows indicate the two protein bands as PKCβ2 downstream targets.</p