IRS2ATHERO · Molecular basis of atherosclerosis induced by hypercholesterolemia and type 2 diabetes

Final Activity Report Summary - IRS2ATHERO (Molecular basis of atherosclerosis induced by hypercholesterolemia and type 2 diabetes)

Atherosclerosis and cardiovascular disease are the major cause of mortality in Western societies. Type 2 diabetes mellitus and the metabolic syndrome (T2DM/MetS, also referred as insulin-resistant states) are among the most relevant cardiovascular risk factors due to their high incidence among the population (between 20-30%). The mechanisms underlying the atherosclerosis accelerated by T2DM/MetS remain poorly understood. The goal of the initial project was to generate and characterise a murine model of atherosclerosis accelerated by T2DM/MetS. The fellow has completed both main objectives of the proposed research. Mice double-deficient in apoE and Irs2 genes (apoE-/-Irs2-/- mice) were generated and characterised as described in objective 1 of the original proposal. Double-deficient apoE-/-Irs2-/- mice displayed hypercholesterolemia compared to IRS2-/- and WT control mice. As hypothesised, lesion characterisation and analysis demonstrated accelerated atherosclerosis in apoE-/-Irs2-/- compared to apoE-/- mice, in spite of the development of comparable hypercholesterolemia. In addition, the hyperinsulinemia found in apoE-/-Irs2-/- but not in apoE-/- mice correlated positively with the development of atherosclerosis. These results identify IRS2 as a modulator of atherosclerosis and let us speculate that subtle changes in IRS2 might account for accelerated atherosclerosis in T2DM/MetS-insulin resistant states. The results of these studies are reported in González-Navarro et al., 2007, Front. Biosc. 12, 2291-8. During the execution of the project we also found that mice with only one allele of Irs2 inactivated (apoE-/-Irs2+/-) exhibit increased glucose intolerance and enhanced atherosclerosis compared to apoE-/- mice. To carry out the objective 2 of the initial project, which consisted of the identification of alterations in gene expression in T2DM/MetS-accelerated atherosclerosis, apoE-/-Irs2+/- mice were used for gene expression studies. RNA from aortic tissue of fat-fed apoE-/-Irs2+/- and apoE-/- mice was isolated to perform real time qPCR array analysis (insulin signalling, Superarray). Comparison between fat-fed apoE-/-Irs2+/- and apoE-/- mice led to the identification of changes in 14 genes related to the insulin-signalling pathway, among which Akt2 and Ras, two downstream effectors of Irs2, were diminished in aorta. In addition, the pro-atherogenic cytokine Mcp1 was significantly increased in aorta and primary vascular smooth muscle cells (VSMCs) and macrophages from apoE-/-Irs2+/- mice. Inhibition of AKT or ERK1/2 (Ras-regulated kinase) similarly upregulated Mcp1 in VSMCs. Therefore, enhanced levels of MCP1 resulting from reduced IRS2 expression and accompanying defects in AKT2 and Ras/ERK1/2 signalling pathways may contribute to accelerated atherosclerosis in MetS states. Finally, although not part of the original proposal, we also investigated the expression of Irs2 and its downstream effectors in circulating leucocytes from MetS patients. qPCR analysis in circulating leukocytes from insulin-resistant MetS patients, showed that mRNA levels of Irs2 and Akt2 are reduced compared to insulin-sensitive MetS patients, suggesting that our findings in apoE-/-Irs2+/- mice might be clinically relevant in the setting of MetS-dependent acceleration of atherosclerosis (new objective of the project, objective 3). All this second part of the project has been recently submitted for publication to the journal Arteriosclerosis, Thrombosis and Vascular Biology (González-Navarro et al. "Molecular basis of atherosclerosis in metabolic syndrome: Role of reduced IRS2/AKT2 and IRS2/ERK1/2 signaling"). The major accomplishments can be summarised as: 1) the establishment of a murine model of atherosclerosis accelerated by T2DM/MetS; and 2) the findings that enhanced levels of MCP1 resulting from reduced IRS2 expression and accompanying defects in AKT2 and Ras/ERK1/2 signaling pathways may be a potential mechanism promoting atherosclerosis in T2DM/MetS states.

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