Abstract 16655: Role of PKCβII/p66Shc Pathway As A Determinant Of Metabolic Memory In Human Endothelial Cells
Purpose: The concept that persistence of hyperglycaemic stress even after blood glucose normalization sustains vascular damage has recently emerged and defined as “metabolic memory”. Reactive oxygen species (ROS) may be involved in this phenomenon. The adaptor protein p66Shc is a downstream target of protein kinase CβII involved in mitochondrial ROS generation. We investigated whether a PKCβII/p66Shc-dependent mechanism maintains metabolic memory in diabetes.
Methods: Human aortic endothelial cells (HAEC) were exposed for 1 week either to normal glucose (NG, 5 mmol/L) or high glucose (HG, 25 mmol/L) as well as to HG for 4 days followed by NG for the remaining 3 days (HN) with and without PKCβII and p66Shc specific siRNA- mediated knockdown by electroporation. Scrambled siRNA was used as control. Protein expression was assessed by WB. Superoxide anion (O2-) and nitric oxide (NO) levels were measured by electron spin resonance spectroscopy. Apoptosis was assessed by Annexin V staining. Data are shown as percentages of NG.
Results: HG increased both phospho-PKCβII and p66Shc (177±79 and 207±53%, respectively; n=6, p<0.01). Interestingly, phosphorylation of PKCβII and its downstream target p66Shc remained significantly elevated after glucose normalization (158±37 and 181±58%, respectively; n=6, p<0.05). After HG exposure O2- was markedly increased and remained high even after glucose normalization (panel a, n=6, *p<0.01). As expected, a persistence of reduced NO release after glucose normalization was observed (panel b, n= 4 *p<0.01). Similar findings were observed for cellular apoptosis (panel c, n=4 *p<0.01). By contrast, glucose normalization with silencing of PKCβII and its downstream target p66Shc abolished ROS production (#p<0.01), restored NO availability (#p<0.01) and blunted cellular death (#p<0.01).
Conclusions: These results demonstrate that PKCβ/p66Shc pathway may play a crucial role in triggering hyperglycaemia-induced metabolic memory.
- © 2010 by American Heart Association, Inc.