Abstract 647: Mild Whole Body Insulin Resistance and Accelerated Endothelial Dysfunction: Increased Reactive Oxygen Species Derived from Mitochondria During Ageing Despite Preserved Glycaemic Control
Mice heterozygous for a knockout of the insulin receptor gene (IRKO) are a model of mild insulin resistance. We examined the effect of mild whole body insulin resistance on endothelial function with ageing. We studied metabolic function & blood pressure in vivo, and vascular function in aortic rings ex vivo in young (2months) and adult (6 months) male IRKO and their wild type (WT) littermates. Data presented as mean±SEM, n=6 – 8 per group, p<0.05 taken as significant, student t test and ANOVA used where appropriate. Newman-Keuls used for post hoc analysis. Adult IRKO mice were non-obese, had preserved glycaemic control (maintained through compensatory hyperinsulinaemia), normal fasting triglyceride and free fatty acid levels. Despite this they showed impaired acetylcholine (Ach) mediated relaxation (measured as a percentage of maximal constriction to phenylepherine) compared to age matched WT (66.0%±5 vs 87.3%±4; p=0.003). IRKO Ach responses were normalised by the SOD mimetic MnTMPyP (10μM; Emax 84.6±5 vs 85.8±5) but not the BH4 analogue sepiapterin (10μM; Emax 74.6±6 vs 93.2±8). FACS analysis of coronary microvascular endothelial cells (CMVEC) exposed to dihydroethidium (DHE, 2μM) confirmed increased superoxide in IRKO endothelium compared to WT, as did DHE staining of thoracic aorta (173±4 vs 140±5 units of fluorescence, p=0.001). NADPH dependent superoxide generation was greater in IRKO CMVEC than WT (assessed by Lucigenin enhanced chemiluminescence, 3.1±0.2 vs 1.6±0.3 integrated light units, p=0.02) and was inhibited by the flavoprotein inhibitor diphenyleneiodonium (DPI, 10μM, p<0.001), the superoxide scavenger tiron (20mM, p<0.001) and the mitochondrial inhibitors rotenone (20μM, p<0.01) and thenoyltrifluoroacetone (TTFA, 10μM, p<0.01). Suggesting mitochondria to be the principal source of increased superoxide. Inhibitors of eNOS (l-NAME, 100μM) and Xanthine oxidase (allopurinol, 100μM) had no effect. Real time PCR examination of thoracic aortae from IRKO and WTs showed similar levels of expression of eNOS, NOX2 and NOX4 mRNA. In conclusion, mild insulin resistance promotes endothelial dysfunction with ageing. Moreover, our data supports a role for mitochondrial derived superoxide in this process in the absence of hyperglycaemia.