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In the present study, the effect of long-term fructose feeding on skeletal muscle mitochondrial energetics was investigated. Measurements in isolated tissue were coupled with the determination of whole-body energy expenditure and insulin sensitivity. A significant increase in plasma NEFA, as well as in skeletal muscle TAG and ceramide, was found in fructose-fed rats compared with the controls, together with a significantly higher plasma insulin response to a glucose load, while no significant variation in plasma glucose levels was found. Significantly lower RMR values were found in fructose-fed rats starting from week 4 of the dietary treatment. Skeletal muscle mitochondrial mass and degree of coupling were found to be significantly higher in fructose-fed rats compared with the controls. Significantly higher lipid peroxidation was found in fructose-fed rats, together with a significant decrease in superoxide dismutase activity. Phosphorylated Akt levels normalised to plasma insulin levels were significantly lower in fructose-fed rats compared with the controls. In conclusion, a fructose-rich diet has a deep impact on a metabolically relevant tissue such as skeletal muscle. In this tissue, the consequences of high fructose feeding are altered glucose tolerance, elevated mitochondrial biogenesis and increased mitochondrial coupling. This latter modification could have a detrimental metabolic effect by causing oxidative stress and energy sparing that contribute to the high metabolic efficiency of fructose-fed rats. [ABSTRACT FROM PUBLISHER]