Effect And Mechanism Of Branched Chain Amino Acids On Insulin Resistance And Inflammatory Aging

ObjectiveBranched chain amino acids(BCAA)are the general term of three essential amino acids-leucine,valine,and isoleucine.In our previous study we observed that the BCAA supplements can increase the ability of gluconeogenesis and promote the lipid accumulation of rats.However,the effect of BCAA on glucose metabolism in type 2 diabetes mellitus is unknown.This study will reveal the effects of BCAA on T2D patients with insulin resistance and the efficacy of metformin,by adding the BCAA to the process of insulin resistance in vivo and in vitro.In addition,this study found that BCAA had a significant improvement effect on the increase of inflammatory factors caused by aging.Therefore,this study explored the influence of BCAA on the inflammatory process of natural aging individuals from the perspective of inflammatory aging,with a view to explaining the mechanism of BCAA in increasing life expectancy from the perspective of inflammation.To provide reasonable guidance for the supplement of BCAA in diabetic patients,and to provide theoretical support for the mechanism of BCAA to delay aging.Materials and MethodsIn vivo experiment:1)KK mice were divided into four groups:model group,BCAA group,metformin group,BCAA combined with metformin group.Basic survival data and life span of mice in each group were measured.Insulin resistance was detected by OGTT,PTT and ITT tests before and after feeding.The changes of glycogen synthesis and gluconeogenesis signaling pathway were detected by Real-time PCR and Western blot.Pathological sections and PAS staining of liver and skeletal muscle were performed to observe the histopathological effect of BCAA on liver and skeletal muscle and glycogen content.Serum free fatty acid(FFAS)metabolism was determined based on GC-MS and serum lipid content was detected by blood biochemical examination.2)Normal middle-aged SD rats were divided into three groups:young group,natural aging group and natural aging BCAA supplement group.The basic survival data and life span of each group were measured.The metabolism of BCAA in rats was determined based on LC-MS.The expressions of inflammatory markers such as IL-6,NF-κB and TNF-α were detected by Real-time PCR and Western blot.The inflammatory infiltration of each tissue was observed by HE staining,and the function of each tissue was detected by blood biochemical examination.In vitro experiment:1)C2C12 cells were induced to differentiate into myotubes,and the insulin resistance model of myotubes was established.The glucose content in the medium before and after induction was detected by colorimetry to assess the ability of cells to intake glucose.Cell proteins were collected and the changes of insulin resistance related pathways before and after induction were detected by Western blot to verify the success of modeling.Gradient concentrations of BCAA were added to the above model cells,and changes in glycogen synthesis and gluconeogenesis signaling pathway before and after addition were detected by Real-time PCR and Western blot to compare the effects of different levels of BCAA on glucose metabolism pathway of C2C12 myotubes with insulin resistance model.2)HUVECs were isolated and cultured.After BCAA treatment,the mRNA expression of inflammatory markers IL-6 and TNF-α was detected by Real-time PCR,and the nuclear accumulation of Nrf2 in HUVECs was detected by nuclear cytoplasmic separation and immunofluorescence staining.ResultsIn vivo experiments:1.BCAA supplementation significantly increased liver weight of KK mice,and significantly reversed the decreace of body weight and liver weight of KK mice induced by metformin;2.BCAA supplementation reduced insulin sensitivity,glucose homeostasis regulation and gluconeogenesis in KK mice,and significantly reduced the efficacy of metformin treatment on insulin sensitivity and gluconeogenesis;3.BCAA supplementation decreased the phosphorylation level of AKT/GSK signaling pathway in the skeletal muscle and liver tissues of KK mice,increased the mRNA levels of G6P,PEPCK and PGC-1a in the liver,and significantly weaken the improvement effect of metformin on AKT/GSK signaling pathway and mRNA levels of G6P,PEPCK and PGC-1A in the liver;4.KK mice fed with BCAA supplementation significantly led the lipid metabolism disorders,such as the higher total cholesterol and triglyceride,and also led to nonalcoholic fatty liver disease and larger adipose tissue in skeletal muscle.Meantime,BCAA significantly damaged the metformin on the improvement of KK mice lipid metabolism;5.BCAA significantly prolonged the median survival of SD rats;6.BCAA could be metabolized to short-chain acylcarnitine quickly after intake in rats,but have no significant effect on blood amino acid level;7.Supplementation of BCAA reduced the expression levels of IL-6 and TNF-α in liver,skeletal muscle and aorta of aged rats,and observed obvious improvement of inflammatory infiltration and morphological.In vitro experiments:1.Numerous bundle myotubes were appeared after C2C12 cells were induced to differentiate,which was consistent with literature reports.After induced by high glucose,the phosphorylation of AKT/GSK signaling pathway was significantly inhibited,and the ability of glucose uptake was significantly reduced,so the insulin resistance model was successfully established.2.BCAA treatment further reduced the glucose uptake capacity of myotubes in insulin resistance model,significantly reduced the expression of PGC-1a mRNA and further inhibited the phosphorylation level of AKT/GSK pathway.3.Compared with the control group,the addition of BCAA resulted in nuclear translocation of Nrf2 in HUVECs.Conclusions 1.BCAA further aggravates insulin resistance and lipid accumulation by inhibiting glycogen synthesis pathways in skeletal muscle and liver,reducing glucose uptake in both,and increasing the expression of hepatic gluconeogenic related genes both in vivo(diabetic KK-Ay mice)and in vitro(C2C12 myotubes).2.BCAA could weaken the therapeutic effect of metformin on KK mice and exacerbate the disease by inhibiting the improvement effect of metformin on skeletal muscle and liver glycogen synthesis pathways and gluconeogenesis related pathways.3.BCAA regulates downstream inflammatory factors by activating NRF2 nuclear translocation and alleviates inflammatory aging.