Magalena Cristobal Garcia

Many factors related to obesity can impact how mitochondria produce ATP, such as the uncoupling of oxidative phosphorylation (OXPHOS) caused by proton leaks from built-up free fatty acids (FFA), the increased levels of uncoupling proteins (UCPs), and changes in the levels of ATPase inhibitory protein factors 1 (IF1). Therefore, the present study aimed to assess the rate of ATP synthesis in mitochondria isolated from skeletal and cardiac muscle from animal models of sucrose diet-induced obesity at different time periods. Short periods of sucrose intake (6 and 12 weeks) are sufficient to induce fat accumulation, hypertriglyceridemia, and high plasma FFA. However, a significant decline in the ATP synthesis rate starts to be obvious in mitochondria from skeletal muscle after 24 weeks of sucrose consumption. This impairment of ATP synthesis is associated with increased FFA in skeletal muscle homogenate. ATP synthesis rates in both skeletal and cardiac muscle were found to be sensitive to oleic acid and GDP, a physiological inhibitor of UCPs that has been shown to increase with aging. In addition, a sucrose diet increases the IF1 content in both skeletal and heart muscle, probably to avoid the hydrolytic activity of ATP synthase. In mitochondria from heart muscle, a decrease in the ATP synthesis rate was only observed according to the age in both groups of rats, and it was not affected by sucrose feeding. Our results suggest that the decline of the ATP synthesis rate in mitochondria from skeletal muscle can be due to the accumulation of FFA in skeletal muscle tissue as uncouplers, and the IF1 overexpression induced by the sucrose diet is a response mechanism to avoid the ATP hydrolysis and to save the energy charge reduced by FFA-uncoupling OXPHOS.

Several factors related to the obesity can affect mitochondrial ATP biosynthesis including the uncoupling of oxidative phosphorylation (OXPHOS) by proton leak induced by accumulated free fatty acids (FFA), the over-expression of uncoupling proteins (UCPs) and altered expression of ATPase inhibitory protein factors 1 (IF1). Therefore, the present work was aimed to evaluate the activity of ATP synthase in isolated mitochondria from skeletal and heart muscle of animal models of obesity induced by sucrose diet at different periods. Short periods of sucrose intake (6 and 12 weeks) are sufficient to induce fat accumulation, hypertriglyceridemia and high plasma FFA. However, a significant decline in the activity of the ATP synthase starts to be obvious in mitochondria from skeletal muscle after 24 weeks of sucrose consumption. This impairment of ATP synthase is associated with increased FFA in skeletal muscle homogenate. Both skeletal and heart muscle ATP synthase activity were found sensitive to oleic acid and to GDP, a physiological inhibitor of UCPs, found enhanced by aging. In addition, sucrose diet increases the IF1 content in both skeletal and heart muscle probably to avoid the hydrolysis activity of ATP synthase. In mitochondria from heart muscle a decrease in the ATP synthase activity was only observed according to the age in both groups of rats and it was not affected by sucrose feeding. Our results suggest that the decline of the ATP synthase activity in mitochondria from skeletal muscle can be due to the accumulation of FFA in skeletal muscle tissue as uncouplers, and the IF1 over-expression induced by sucrose diet is an adaptative mechanism to inhibit the ATP synthase hydrolysis activity.

The aim of this study was to evaluate the effect of leptin on reactive oxygen species’ (ROS) generation of smooth muscle cells (SMCs) from a rat model of obesity and hyperleptinemia. Obesity and hyperleptinemia were induced in rats by a sucrose-based diet for 24 weeks. ROS generation was detected by using dichloro-dihydrofluorescein (DCF), a fluorescent ROS probe in primary SMCs culture. An increase in plasma leptin and oxidative stress markers was observed in sucrose-fed (SF) rats. At baseline SMCs from SF rats showed a more than twofold increase in fluorescence intensity (FI) compared to that obtained in control (C) cells. When the C cells were treated with 20 ng leptin, the FI increased by about 250%, whereas the leptin-induced FI in the SF cells increased only by 28%. In addition, sucrose feeding increased the levels of p22phox and gp91phox, subunits of Nox as an O₂^•− source in SMCs. Treatment of cells with leptin significantly increased p22phox and gp91phox levels in C cells and did not affect SF cells. Regarding STAT3 phosphorylation and the content of PTP1B and SOCS3 as protein markers of leptin resistance, they were found to be significantly increased in SF cells. These results suggest that SF aortic SMCs are partially resistant to leptin-induced ROS generation.