The risk for heart failure and death after myocardial infarction is abnormally high in diabetic subjects. We and others have shown previously that mitochondrial uncoupling protein 3 (UCP3) improves functional recovery of the rodent heart during reperfusion. Here, we demonstrate that pharmacological induction of hyperinsulinemia in mice down-regulates myocardial UCP3. Decreased UCP3 expression was linked to the development of selective insulin resistance in the heart, characterized by decreased basal activity of Akt but preserved activity of the p44/42 mitogen-activated protein kinase, and overactivation of the sterol regulatory element-binding protein (SREBP)-1-mediated lipogenic program. In cultured myocytes, insulin treatment and SREBP-1 overexpression decreased, whereas SREBP-1 interference increased, peroxisome proliferator-activated receptor-stimulated expression of UCP3. Promoter deletion and site-directed mutagenesis identified three functional sterol regulatory elements in the vicinity of a known complex intronic enhancer. Increased binding of SREBP-1 to this DNA region was confirmed in the heart of hyperinsulinemic mice. In conclusion, we describe a hitherto unknown regulatory mechanism by which insulin inhibits cardiac UCP3 expression through activation of the lipogenic factor SREBP-1. Sustained down-regulation of cardiac UCP3 by hyperinsulinemia may partly explain the poor prognosis of type 2 diabetic patients after myocardial infarction.Type 2 diabetes represents 95% of all diabetes cases, and its incidence is expected to soar in the next decades as a consequence of the obesity epidemic (1). The insulin resistance and hyperinsulinemia that characterize this category of patients are both strong predictors of ischemic heart disease (2, 3). In addition to increasing the incidence of myocardial infarction, diabetes is also associated with increased cardiac morbidity and mortality following revascularization interventions (4 -7). Following pharmacological and surgical revascularization techniques, type 2 diabetic patients treated with insulin have more postoperative complications and have a mortality rate 2-3-fold higher than type 2 diabetic patients who are not on insulin (8,9). This suggests that insulin treatment may unexpectedly worsen prognosis in type 2 diabetic patients following an ischemic insult, and the defect may reside in the myocardium.Uncoupling protein 3 (UCP3) 2 is an inner mitochondrial membrane protein predominantly expressed in brown adipocytes, skeletal muscle, and the heart (10, 11). Uncoupling proteins have a tripartite structure, which has been compared with that of the ADP/ATP carrier of the mitochondrial inner membrane (12). Despite its high sequence homology with the archetypal uncoupling protein 1 and the fact that it can mediate mitochondrial proton leak, the physiological function of UCP3 remains unknown. There is compelling evidence that UCP3 protects the heart from ischemia-reperfusion injury. For example, we and others have recently shown both ex vivo and in vivo that the ...