Abstract 1390: Enhanced Leptin Expression in the Hypothalamus Reverses p66ShcA Dependent Oxidative Stress and Myocyte Apoptosis in Akita Mice
A point mutation of the Insulin 2 gene causes early onset of pancreatic β- cell dysfunction, hyperglycemia (HG) and diabetes mellitus (DM) in Akita mice. A primary cardiomyopathy complicates DM characterized by oxidative stress (OS), apoptosis and LV pump dysfunction. P66ShcA controls mitochondrial metabolism and cell response to OS, apoptosis and aging. We have shown that enhancing leptin, a hormone secreted by adipocytes and involved in the hypothalamic integration of energy homeostasis, with the aid of gene therapy in the hypothalamus restored normoglycemia in Akita mice. We have now tested the hypothesis that leptin gene therapy will attenuate p66ShcA redox activity and prevent OS-induced myocyte apoptosis in hearts of Akita mice. Six to eight week old insulin-deficient nonobese (D) Akita mice were obtained from Jackson Laboratory and injected (1.5ul) intracerebroventricularly (icv) a non-immunogenic, nonpathogenic and replicative deficient recombinant adeno-associated virus vector encoding either leptin (2.4 × 1010 particles; lep-D group) or green fluorescence protein (1.8 × 1010 particles; GFP-D) and blood glucose (BG) were monitored on weekly basis. Mice were euthanized 8–10 weeks post-injection and hearts excised for molecular and immunochemical analyses. Male C57BL/6J wild type (WT) age-matched mice served as control. BG levels at sacrifice were; WT 150±20; GFP-D 568±40 and Lep-D 96±21 mg/dl; (n=3–5). Phospho p66 was significantly enhanced (1.9 fold; p<0.05) in GFP-D compared with WT or lep-D mice. Phospho Akt (thr308 and ser473) expression was significantly increased (2.0 –2.5 fold, p<0.05) in GFP-D compared with WT or lep-D mice. Catalase and superoxide dismutase (SOD) expression was significantly higher (p<0.05) in GFP- D compared with WT or lep-D mice. Caspase3 and caspase9 were 2-fold higher in GFP-D (p<0.05) compared with WT or lep-D mice. Phospho-p53 and bax/bcl2 expression were increased 2.1 and 2.6 fold (p<0.05) and cytosolic cytochrome c was 1.8 fold higher in GFP-D compared with WT or lep-D mice. These results indicate that activation of hypothalamic signaling pathways by leptin normalizes BG levels, decreases the markers of oxidative stress and protects the heart from apoptosis in diabetic Akita mice.