| BackgroundIt is well recognized that age is a major independent risk factor for morbidity and mortality in ischemic heart disease. The mechanisms responsible for this age-related pathologic change remain elusive. Considerable evidence exist that aging results in systemic insulin resistance which is closely related to the prevalence of cardiovascular disease in aging population. However, the mechanisms of insulin resistance remain unanswered. Recent evidence from studies in aging skeletal muscle indicated that mitochondrial dysfunction in skeletal muscle results in systemic insulin resistance. Whether a similar mitochondrial dysfunction is present in aging myocardium and its relation with aging-associated myocardial insulin resistance are unclear.Myocardial insulin resistance was first reported more than two decades ago in experimental animals and subsequently confirmed by many clinical observations. The mechanism of myocardial insulin resistance remains unclear. Increasing evidence indicates that besides its inimitable function in glucose metabolism, insulin itself protects ischemia/reperfused-induced heart injury via activating endothelial nitric oxide synthase (eNOS) and subsequent nitric oxide (NO) production. However, whether aging-associated myocardial insulin resistance is related to aging-impaired eNOS signaling cascade remains unclear.Exercise is a powerful"tool"in improving aging-associated insulin resistance in skeletal muscle, liver and adipose tissue, and significantly promoting mitochondrial function. Moreover, exercise also significantly reduces the morbidity of cardiovascular diseases. However, whether exercise may alleviate aging-associated myocardial insulin resistance through restoring eNOS signaling and improving mitochondrial function remains unknown.Aims(1) To determine whether mitochondrial dysfunction may contribute to myocardial insulin resistance in aging rats.(2) To investigate whether there is a dysfunctional eNOS signaling cascade in the aging heart, and if so, to determine its role in myocardial insulin resistance in the aging hearts.(3) To test whether aerobic exercise training may alleviate aging-associated myocardial insulin resistance through restoring eNOS signaling cascade and improving mitochondrial function.Methods(1) Aging male SD rats (24 months) were subjected to a 9-week's free-loading swimming exercise training (60 min/d, 5 d/week). (2) Oral Glucose Tolerance Test (OGTT) and Insulin Sensitivity Test (IST) were performed following an 8-9 h fasting to assess whole insulin sensitivity in both aging and exercised aging rats.(3) Glucose uptake stimulated by insulin was measured as the rate of cleavage of D-[2-3H]-glucose from KH's solution.(4) The expressions of eNOS, Cytocheme C (Cyt C), Cytocheme oxidese IV, PGC-1αand the phosphorylation of eNOS were measured using Western blots.(5) The activities of Malate dehydrogenase (MDH), Succinate dehydrogenase (SDH), and Pyruvate kinase (PK) in the heart tissue were determined by detection kits.(6) Myocardium tissue oxygen consumption was tested using Clark-type oxygen electrode.(7) Mitochondrial volume density and the content of mtDNA were measured using transmission electron microscopy and Quantitative real-time PCR, respectively.(8) Whole heart contractile function was assessed using Langendorff system.Results(1) Aerobic exercise training markedly decreased fat content, inhibited the growth of body weight (n=6, P<0.05-0.01), and significantly increased the ratio of left ventricular to body weight in aging rats (n=6, P<0.05). However, aerobic exercise training exerted no effect on food uptake in aging rats (n=6, P>0.05). (2) Compared with adult rats, aging rats significantly appeared a systemic insulin resistance as evidenced by OGTT, IST, fasting plasma insulin level and fasting glucose content (n=6,P<0.05-0.01). While aerobic exercise training significantly improved aging-associated systemic insulin resistance (n=6, P<0.05-0.01).(3) Compared with those in adult rat hearts, myocardial glucose uptake stimulated by insulin and GLUT4 expression were significantly decreased in aging rat hearts (n=6, P<0.05-0.01). These results indicated that aging causes significant myocardial insulin resistance.(4) Compared with those in adult rat hearts, myocardial mitochondrial biogenesis was markedly decreased as evidenced by the ratio of total mitochondrial area to total cytoplasmic area and the expressions of Cyt C, COX IV and PGC-1αin aging rat hearts (n=5, P<0.01). Moreover, mitochondrial function were also significantly decreased as indicated by oxygen consumption in aging rat heart tissue under basal condition and after insulin stimulation (n=6, P<0.01).(5) eNOS expression and eNOS phosphorylation by insulin were significantly reduced in aging rat hearts, compared with those in adult rat hearts, (n=5, P<0.01).(6) Aerobic exercise training significantly improved myocardial insulin sensitivity, enhanced insulin-stimulated mitochondrial function (n=6, P<0.01), restored eNOS signaling initiated by insulin (n=5, P<0.01), and improved insulin's positive inotropic effect in aging heart (n=6, P<0.05-0.01). However, aerobic exercise training exerted no effect on GLUT4 expression (n=6, P>0.05). Pretreatment with L-NANE or Cavtratin, a selective eNOS inhibitor, blocked exercise-improved myocardial insulin sensitivity, abolished exercise-improved mitochondrial response to insulin, and attenuated the positive inotropic response to insulin in the aging hearts.Conclusions:(1) These results demonstrate that dysfunctional eNOS signaling cascade and subsequent mitochondrial depression is a major mechanism contributes to aging-associated myocardial insulin resistance.(2) Aerobic exercise training improves myocardial insulin sensitivity by restoring eNOS signaling cascade and enhancing mitochondrial function.(3) Aging significantly attenuates insulin's positive inotropic effect, while aerobic exercise training can markedly restore aging-impaired insulin's positive inotropic effect via improving insulin sensitivity in the aging hearts.
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