The Effects And The Underlying Mechanisms Of Chronic Dietary Leucine Supplementation On Insulin Resistance And Insulin Signaling Pathway In Insulin-target Tissues Of Rats On High-fat Diet

Objective: To explore the effects of leucine on insulin sensitivity in rats onnormal chow diet or high-fat diet rats and to explore the underlying molecularmechanisms.Methods: SD rats were randomly divided into four groups, each group have10rats. Differerrnt groups were fed with differernt diet: Normal chow diet (ND); Normalchow diet with1.5%leucine (ND+1.5%Leu); High fat diet (HFD) and high fat dietwith1.5%leucine (HFD+1.5%Leu). At the end of24week, rats were fasted for10hours, then the insulin tolerance test was performed. At the end of experiment, theserum levels of blood glucose and insulin were determined and HOMA index wascalculated; levels of IRS-1and phosphor-IRS1(pSer302and pTyr632), Akt andphosphor-Akt(pSer473), mTOR and phosphor-mTOR (pSer2448) expression inskeletal muscle, liver and adiopse tissue were determined by western blotting.Results:1. Metabolic changes:(1) Food intake in rats on HFD was greatly lower than that in rats on ND (P <0.05) from the beginning of the3rdweek. Leucine supplementation tends to decreasefood intake in rats on ND and to increase food intake in rats on HFD.(2) The calorie intake was only increased significantly by HFD during the first2weeks. Chronic leucine supplementation had no significant effect on calorie intake inrats.(3) In the6thweek, the body weight of HFD and HFD+1.5%leu groups began to rise. HFD increased body weight gain significantly in comparison to ND at thebeginning of7thweek (P<0.05). Leucine supplementation significantly increasedbody weight gain in rats on HFD after the11thweek (P<0.05). Leucinesupplementation did not obviously change body weight gain in animals on ND.2. Insulin sensitivity:(1) The level of the fasting blood glucose was not significantly different amongall groups. Compared with ND group, the levels of the fasting plasma insulin andHOMA-IR index in rats on HFD were significantly increased (P<0.05); comparedwith HFD group, chronic supplementation of leucine significantly decreased levels ofinsulin and HOMA-IR index (P<0.05), but had no obvious effects on insulinsensitivity in rats on ND group.(2) The results of ITT showed that:, the glucose levels at15thminute and30thminute and AUC of HFD group were significantly increased(P<0.05) when comparedwith ND group; the glucose levels at15thminute and30thminute and AUC ofHFD+1.5%Leu group were significantly decreased (P<0.05) when compared withHFD group.3. Insulin signaling pathway in skeletal muscle, liver and adipose tissues(1) The protein levels of IRS-1and phosphorylated IRS-1in skeletal muscle、liver and adiposeInsulin stimulation induced IRS-1phosphorylation at serine302in adipose tissueof rats on ND. HFD induced IRS-1phosphorylation at serine302both at basal leveland after insulin stimulation in skeletal muscle, liver, and adipose tissue; but leucinesupplementation had no effect on it.Leucine supplementation increased IRS-1phosphorylation at tyrosine632atbasal level in skeletal muscle, liver, and adipose tissue of rats on HFD, but had noeffect on the acute insulin-induced IRS-1phosphorylation at tyrosine632.(2) The protein levels of Akt and pSer473Akt in skeletal muscle, liver andadipose tissues HFD increased basal level of Akt phosphorylation at residue473in skeletalmuscle, liver, and adipose tissue of rats. Acute insulin stimulation elevated Aktphosphorylation in skeletal muscle, liver and adipose tissue of rats on ND, but not inthat of rats on HFD. Chronic leucine supplementation did not alter the basal level ofAkt phosphorylation in skeletal muscle and adipose tissue in animals on either ND orHFD, but enhanced the insulin-stimulated Akt phosphorylation in skeletal muscle andadipose tissue in rats on HFD. Chronic leucine supplementation tended to increaseAkt phosphorylation both at basal level and after insulin stimulation in liver, andespecially enhanced Akt phosphorylation in liver of rats on HFD after insulinstimulation.(3) The protein levels of mTOR and pSer2448mTOR in skeletal muscle、liverand adiposeThe result showed that, HFD increased basal mTOR phosphorylation level atresidue2448in liver and adipose tissue, not in skeletal muscle. Acute insulinstimulation elevated mTOR phosphorylation in skeletal muscle and liver of rats onND, but not in that of rats on HFD. Chronic leucine supplementation did not alter thebasal level of mTOR phosphorylation in all tissues of ND or HFD, but enhancedinsulin-stimulated mTOR phosphorylation in liver of rats on ND or HFD and inskeletal muscle and adipose tissuse of rats on HFD.Conclusion:1. High-fat diet induced insulin resistance; Dietary leucine supplementationcould significantly increase the insulin sensitivity in rats on high-fat diet, but had nosignificant effect for rats on normal diet.2. Leucine supplementation increase insulin sensitivity and improve insulinresistance likely by facilitation insulin signaling in insulin-target tissues...