Effects Of Chronic Dietary Leucine Supplementation On Mitochondrial Biogenesis In Liver Of Rats On High-fat Diet

Objectives To explore the effects of leucine on mitochondrial biogenesis in liver of rats on high-fat diet, to reveal the molecular mechanism by which leucine supplementation improves mitochondrial function, and provide evidence for the prevention and management of type2diabetes by dietary modification.Methods Adult male Sprague-Dawley rats were randomly divided into4groups and fed with different diets:Normal chow diet (ND); Normal chow diet supplemented with1.5%leucine (ND+1.5%Leu); High fat diet (HFD) and high fat supplemented with1.5%leucine (HFD+1.5%Leu) for24weeks. Before the end of the experiment, rats were fasted for10hours, the insulin tolerance test (ITT) was performed and the area under the glucose curve was calculated. At the end of experiment, blood, liver and skeletal muscles were collected, serum levels of MDA, fasting glucose and insulin were determined and HOMA index was calculated; levels of GSH/GSSG in liver was measured with enzymatic method. Real-time PCR was used to detect the expression of MnSOD, mtDNA content and the expression of mitochondrial biogenesis related genes, such as YY1, NRF-1, TFAM, PGC-la, SIRT3and SIRT1. Levels of mTOR protein and phosphorylated mTOR (pSer2448) were determined by western blotting.Results1. Food intake in rats on HFD was significantly lower than that in rats on ND from the beginning of the3rd week (P<0.05). The calorie intake was only increased significantly by HFD during the first2weeks. Chronic leucine supplementation had no significant effect on calorie intake in rats. Since the6th week, the body weight of HFD group and HFD+1.5%leu began to rise. HFD increased body weight gain significantly in comparison to ND at the beginning of7th week (P<0.05).Leucine supplementation significantly increased body weight in rats on HFD after the11th week (P<0.05). Leucine supplementation did not obviously alter body weight gain in animals on ND.2. Compared with ND group,fasting serum glucose level of HFD group was not altered while fasting serum insulin level and the HOMA index of HFD group was significantly increased(P<0.05); Compared with HFD group, the level of insulin and the HOMA index of HFD+1.5%Leu group was significantly decreased(P<0.05). The results of ITT showed that, compared with ND group,the glucose levels at15th minute and30th minute and AUC of HFD group were significantly increased(P<0.05); Compared with HFD group, glucose levels at15th minute and30th minute and AUC of HFD+1.5%Leu group were significantly decreased(P<0.05).3. Chronic leucine supplementation had no significant effects on GSH/GSSG in liver. The mRNA level of MnSOD in liver of rats on HFD was significantly higher than that of rats on ND group (P<0.05). Leucine supplementation decreased the mRNA level of MnSOD in liver of rats on HFD (P<0.05). The level of MDA of rats on HFD+1.5%leu was significantly lower than that of rats on HFD group (P<0.05).4. Compared with ND group, the mtDNA content of HFD group were significantly decreased (P<0.05).1.5%Leucine supplementation significantly increased the mtDNA content in liver of rats on HFD (P<0.05). Compared with that in ND group, the expressions of TFAM and SIRT3in liver of HFD group were significantly decreased(P<0.05); the expressions of YY1, NRF-1, TFAM, PGC-1α and SIRT1of ND+1.5%Leu group were significantly increased(P<0.05). Compared with HFD group, leucine supplementation increased the expression of YY1, NRF-1, TFAM, PGC-la significantly (P<0.05).5. Compared with the level at basal state, insulin stimulation increased mTOR phosphorylation at Ser2448residue in the liver of rats on ND, but not in the liver of rats on HFD, indicating that HFD induced insulin resistance. Leucine supplementation could significantly increase the insulin-stimulated pSer2448mTOR in the liver of rats on HFD (P<0.05).ConclusionsHigh-fat diet induced insulin resistance. Leucine supplementation is effective to improve insulin sensitivity in rats on high-fat diet. Leucine supplementation can promote mitochondrial biogenesis in rats on both normal diet and high-fat diet, which implicates leucine supplementation may meliorate insulin resistance and oxidative stress. The underling mechanisms may involve in the activation of YY1-PGC1α via mTOR pathway. Based on the results, leucine supplementation may thus be helpful to prevent insulin resistance and type2diabetes by promotion mitochondrial biogenesis.