We have postulated that insulin's actions in vascular cells can be functionally separated into cardio-protective, anti-atherogenic or pro-atherogenic actions. We provided evidence that insulin's cardioprotective, anti-atherogenic actions, such as the expression of eNOS and vascular endothelial growth factor (VEGF), are mediated by the activation of the IRS/PI3K/Akt pathway. In contrast, insulin's pro-atherogenic actions, such as increased expression of PAI-1, matrix protein, and cell growth, are regulated by the Ras-MAPK pathway. We have also shown that in the vascular tissues of insulin resistant animals, there is a selective resistance to insulin's actions via the IRS/PI3K/Akt pathway without diminishing the activation of the MAPK cascade. Further, we have shown that protein kinase C (PKC) ? isoform activation can inhibit not only insulin, but also VEGF's induction of the PI3K/Akt/eNOS pathway. This suggests that metabolic abnormalities, such as hyperglycemia and elevation of free fatty acids, which are reported to activate PKC including the ? isoform, may be inducing the selective insulin resistance in the cardiovascular tissue. Further, mice with IRS 1 or 2 deficiency have decreased angiogenesis in response to hypoxia. Zucker fatty rats exhibited decreased capillary density and VEGF expression in the myocardium in parallel with blunted insulin induced activation of Akt. Thus, these findings suggest that PKC activation can selectively inhibit insulin and VEGF's activation of IRS/PI3/Akt pathway. The resulting decrease in eNOS and VEGF expression may play a role in causing endothelial dysfunction and poor vascular collateral formation in response to hypoxia, leading to poor survival after a myocardial ischemic event. To test this hypothesis, we have proposed to 1) study the effect of activating various PKC isoforms on insulin and VEGF stimulation of IRS/PI3K/Akt and Ras-ERK1/2 and the expression of eNOS and VEGF in vascular and cardiac cells; 2) To identify the phosphorylation sites of PKC on IRS/PI3K/PDK; 3) to characterize changes in insulin and VEGF's biological actions and vascular functions in insulin resistant Zucker rats and genetically altered mice, which include transgenic mice overexpressing PKC ? isoform, IRS2 knockout mice, and endothelial cell specific insulin receptor knockout mice; and 4) to determine whether selective insulin resistance exists in the microvessels of insulin resistant patients and type 2 diabetic patients and whether it can be correlated to PKC activation in monocytes and large vessel endothelial dysfunctions. These studies will provide information on the molecular interactions between PKC activation and insulin and VEGF activation of IRS-PI3 kinase-eNOS pathway in the cardiovascular tissues. ? ?
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