A high-fat diet leads to an accumulation of lipid in skeletal muscle, and the development of both mitochondrial dysfunction and insulin resistance. Recently, our lab reported that lipid overload leads to elevated H[subscript]2O[subscript]2 emission from muscle mitochondria, and that mitochondrial-targeted scavenging of H[subscript]2O[subscript]2 completely prevents the development of high fat diet-induced insulin resistance. These findings raise the possibility that interventions which acutely restore cellular metabolic balance in muscle may also acutely restore insulin sensitivity. We hypothesized that mitochondrial function and insulin sensitivity can be restored in skeletal muscle of high-fat fed rats by creating an acute deficit in metabolic balance via 2 h low-intensity treadmill exercise or 16 h fasting. Male Sprague-Dawley rats (125-150g) were either maintained on a standard high carbohydrate- diet or fed a high-fat (60%) diet for 6 weeks and divided into three groups the day before the study: one group was maintained on the normal high-fat diet, another group was fasted overnight (16 h), and a third group completed a single 2 h bout of low-intensity treadmill exercise (10 m/min) and then were given normal overnight ad libitum access to the high-fat diet. Oral glucose tolerance tests were administrated to assess insulin action. Red gastrocnemius muscles were harvested and permeabilized fibers prepared for determination of mitochondrial respiratory function and H[subscript]2O[subscript]2 emission. A single 16 h fast significantly (P<0.05) improved insulin sensitivity in rats maintained on a high-fat diet (P<0.05). Oxygen consumption rate in permeabilized fibers in response to submaximal and maximal ADP concentration when supported exclusively with complex I substrates were not different among groups. However, when respiration was supported by fatty acids (palmitoylcarnitine plus malate, complex I + II substrates), high-fat diet plus exercise group showed higher (P<0.05) rates compared with high-fat diet group. There were no significant differences in H[subscript]2O[subscript]2 emission among the 4 groups. In conclusion, a single 16 h overnight fast is sufficient to restore insulin sensitivity in high fat diet-induced insulin resistant rats, providing evidence that insulin action in muscle is acutely sensitive to the metabolic state of cells. A single bout of low-intensity treadmill exercise in high-fat fed rats failed to restore insulin action but increased ADP-stimulated respiratory capacity, providing evidence of an as yet unidentified regulatory mechanism of the respiratory system. Somewhat surprisingly however, neither fasting nor exercise altered the H[subscript]2O[subscript]2 emitting potential in permeabilized fibers, suggesting that further work is required to better understand the factors influencing mitochondrial function and their potential link to insulin sensitivity.  M.S
