Effects Of Leucine And Calorie Restriction On Hepatic Mitochondrial Biogenesis In Rats On High-Fat Diet

Objectives : To clarify the effects of leucine and calorie restriction on insulin resistance and mitochondrial biogenesis of rats on high-fat diet, to explore the molecular mechanism by which leucine and calorie restriction improve insulin sensitivity and mitochondrial function, and provide a new basis for the prevention and treatment of type 2 diabetes.Methods:Adult male SD rats, weighing 170-220 g, were randomly distributed to 5groups(12 rats/group) and fed with different diets: normal chow(ND); high fat diet(HFD); high fat diet with 20% calorie-restricted(HFD+CR); high fat diet with 1.5%leucine(HFD+Leu) and high fat diet supplemented with 1.5% leucine and 20%calorie-restricted(HFD+Leu+CR) for 24 weeks. At the finis of the experiment, ITT and GTT were performed in animals after fasting for 10 hours. The rats were killed;blood samples and liver tissues were gathered. Liver TC, TG content and serum levels of glucose, insulin, TC, LDL,TG, HDL and were measured. The mt NDA content and the expression of mitochondrial biogenesis correlated genes, such as PGC-1α, SIRT1,TFAM and NRF-1 were detected by real-time PCR. HE staining, oil red O staining and electron microscope of liver tissue were performed.Results :1. Food intake, calorie intake, body weight and organ coefficient(1) Food intake in rats on HFD was greatly lower than that in rats on ND during2-16 weeks(P<0.05). Food intake in rats on HFD+Leu was significantly lower than that in rats on HFD during 1-4 weeks and 17-24weeks(P<0.05). Food intake in rats on HFD+Leu+CR was greatly lower than that in HFD+CR at the beginning of the 3rd week(P<0.05).(2) The calorie intake in rats on HFD+CR and HFD+Leu+CR was 20% restricted compared with HFD and HFD+Leu. HFD elevated significantly the calorie intake in contrast with ND from the first week(P<0.05); the calorie intake in rats on HFD+Leu was greatly higher than that in rats on HFD+CR from the 2nd week(P<0.05); the calorie intake in rats on HFD+Leu+CR was greatly lower than that in rats on HFD+CR from the 2nd week(P<0.05).(3) The body weight of HFD was significantly higher than that of ND from the5 th week(P<0.05); the body weight of HFD+CR was greatly lower than that of HFD from the 3rd week(P<0.05); the body weight of HFD+Leu was greatly lower than that of HFD from the 13 th week(P<0.05); the body weight of HFD+Leu+CR was significantly lower than that of HFD+CR after the 15 th week and that of HFD+Leu from the 3rd week(P<0.05).(4) Fat coefficient of HFD was significantly higher than that of ND(P<0.05);kidney, liver, testis, fat coefficient of HFD+CR were greatly lower than those of HFD(P<0.05); fat weight in percentage of body weight of HFD+Leu+CR was significantly lower than that of HFD+CR(P<0.05); kidney, testis, fat coefficient of HFD+Leu+CR were significantly lower than HFD+Leu(P<0.05).2. Insulin sensitivity:(1) The level of fasting plasma insulin of HFD was significantly higher than that of ND(P<0.05); in comparison to HFD and HFD+Leu, the plasma insulin levels of HFD+CR and HFD+Leu+CR were greatly decreased(P<0.05). Fasting blood glucose was not of great significant different among all groups.(2) GTT showed that the glucose levels and AUC of HFD were greatly elevated when compared with ND(P<0.05); the glucose levels and AUC of HFD+CR and HFD+Leu were greatly lower than those of HFD(P<0.05); the glucose levels and AUC of HFD+Leu+CR were greatly lower than those of HFD+Leu(P<0.05).(3) ITT showed that the glucose levels and AUC of HFD were greatly elevated when compared with ND(P<0.05); the glucose levels of HFD+CR and HFD+Leu were greatly lower than those of HFD(P<0.05); the glucose levels of HFD+Leu+CR were significantly decreased when compared with HFD+Leu(P<0.05).3. Morphology of liver tissues(1) Results revealed in HE staining, oil red O staining: the structure of liver tissue of ND was complete, the liver cells were arranged in order, lipid droplets were not discovered in the cytoplasm. The structure of liver cell of HFD was damaged, a large number of lipid droplets emerged which containing single large lipid droplets and uniform small lipid droplets, and the nuclear was squeezed to the edge of the cell.Compared with HFD, the structures of liver tissues of HFD+CR, HFD+Leu and HFD+Leu+CR were more complete, lipid droplets were decreased.(2) Results revealed in electron microscope: a large number of structurecomplete mitochondria were seen in the liver cell of ND without lipid droplets. In the liver cell of HFD, the mitochondria became smaller, mitochondria membrane and cristae were fused, rough ER was increased and a large number of lipid droplets could be seen. Compared with HFD, the lipid droplets of HFD+CR, HFD+Leu and HFD+Leu+CR decreased significantly, the numbers of mitochondria of the three groups were increased and mitochondrial structure were improved, the membrane and cristae of mitochondria were clear, the dilated degree of rough ER were reduced.4.Lipid metabolism(1) The levels of TC and TG of HFD were significantly higher than those of ND(P<0.05); TC, TG and LDL-C levels of HFD+CR were significantly lower than thoseof HFD(P<0.05); the level of TG of HFD+Leu was greatly lower than that of HFD(P<0.05); TC, TG, LDL-C levels of HFD+Leu+CR were decreased in contrast with HFD, and TC level was greatly lower than that of HFD+Leu, LDL-C level was greatly lower than that of HFD+CR(P<0.05).(2) The levels of liver TC, TG of HFD were increased in comparison with ND,and there was significant difference in liver TC(P<0.05). The levels of liver TG of HFD+CR, HFD+Leu and HFD+Leu+CR were greatly lower than those of HFD(P<0.05).5.Liver mitochondria biogenesis related genes(1) The m RNA levels of NRF-1, PGC-1α, SIRT1, TFAM in livers of HFD were decreased in comparison with ND, and the m RNA expression of TFAM had great difference(P<0.05); the m RNA levels of NRF-1, TFAM in HFD+CR and HFD+Leu were greatly higher than those in HFD(P<0.05); the m RNA levels of NRF-1,PGC-1α and TFAM in livers of HFD+Leu+CR were significantly higher than those in HFD(P<0.05).(2) The mt DNA content in liver of HFD was lower than that of ND; the mt DNA content in livers of HFD+CR and HFD+Leu were higher than those of HFD.Compared with HFD, the mt DNA content of HFD+Leu+CR were significantly increased(P<0.05).ConclusionsHigh-fat diet leads to insulin resistance. Leucine and calorie restriction can improve IR and lipid metabolism. Leucine and calorie restriction promote liver mitochondrial biogenesis in rats on high-fat diet. The outcomes of the study are of great significance to provide new clues of dietary preventive strategies against insulin resistance and type 2 diabetes mellitus.