Branched Chain Amino Acids Induced Downregulation Of AKT2 Protein In Hepatocytes, Promoted High Fat Diet Induced Hepatic Insulin Resistance

BackgroundWith the development of society and the prevalence of Western diet, the number of patients with diabetes increased year by year. According to the International Diabetes Federation published data, the total number of diabetes in the world has been close to 400 million. Diabetes, type 2 diabetes in particular, with high morbidity and mortality, has become one of the chief culprits that threaten human health. Insulin resistance, characterized by decreased insulin sensitivity of the peripheral tissues including liver, skeletal muscle and fat tissue and impaired insulin secretion [1], is the main pathogenesis of type 2 diabetes mellitus. The mechanism of insulin resistance is complex, and studies have shown that, in addition to the "sugar toxicity" and "fat toxicity", the disorders of protein and amino acid metabolism may also contribute to the development of insulin resistance. A series of literatures reported that elevated serum levels of BCAA were found in obesity and metabolic syndrome patients[2,3], elevated BCAA may contribute to the onset and development of insulin resistance [4,5]. But there are also some reports foud that BCAA can improve the insulin resistance and the survival rate of middle aged mice, and promote the biogenesis of mitochondria in mouse heart and skeletal muscle[6]. A literature published in Cell Metabolism in 2007 pointed out that there are significantly increased serum BCAA concentration in the branched chain amino acid aminotransferase knockout mice, which improved the insulin sensitivity of the mice [7]. Therefore, studies on the relationship between BCAA and insulin resistance is controversial. AKT is a key molecule in insulin signaling pathway, insulin regulates metabolism, cell survival, growth, development, proliferation, migration and many other functions Through activation of AKT.Mammalian cells express three AKT isoforms(AKT1-3). AKT2 is expressed at the highest level in insulin-responsive tissues, and is the main isoforms in regulating glucose and lipid metabolism. Whether BCAA affects the protein level of AKT and its subtypes, AKT2 in particular, and then affecting insulin resistance has not been reported. Due to the complex relationship between BCAA and insulin resistance, this study focuses on studying the relationship between BCAA and insulin sensitivity, as well as whether BCAA interacts with high fat to promote the development of insulin resistance and the mechanism. Aims To investigate the effect of BCAA on liver insulin sensitivity, and to investigate whether BCAA contributes to the development of liver insulin resistance through changing the protein level of AKT and the isoforms.Methods1. Cell experiments 1) Experimental grouping and treatment:(1) Hep G2 cells were divided into control group, 0.5 m M palmitate treatment group, 3 m M BCAA+0.5 m M palmitate treatment group, the effect of BCAA on palmitate induced insulin resistance in Hep G2 cells were detected;(2) Hep G2 cells were divided into different concentrations(0, 0.3, 1, 3, 10, 30 m M) ofBCAA treatment groups and 10 m M BCAA+40?M BT2 treatment group, the effect of of BCAA on the protein level and m RNA level of AKT and AKT1, AKT2 and AKT3 Hep G2 cells were detected;(3) Hep G2 cells were divided into different concentrations(0, 0.3, 1, 3, 10, 30 m M) of BCAA treatment groups and 10 m M BCAA+40?M BT2 treatment group, the effect of BCAA on the protein level of the the main components of m TORC2 were detected;(4) Hep G2 cells were divided into control group, 10 m M BCAA treatment group, 100 n M rapamycin treatment group, 10 m M BCAA+100 n M rapamycin treatment group, the effect of long time(48 h) 100 n M rapamycin incubation on BCAA induced downregulation of AKT protein level was detected;(5) Hep G2 cells were divided into control group, 10 m M BCAA treatment group, 10 ?M PYR41/1?M MG132 treatment group, and 10 m M BCAA+10 ?M PYR41/1?M MG132 treatment group, the effect of ubiquitin-proteasome inhibitor on BCAA induced downregulation of AKT protein level was detected. 2) The protein level of IRS1, p-IRS1(ser307), AKT, p-AKT(ser473), AKT1, AKT2, AKT3, Rictor, Raptor and m TOR in Hep G2 cells were detected in different treatment groups by Western blot. 3) AKT1, AKT2, and AKT3 m RNA level in different concentrations of BCAA incubated Hep G2 cells were detected by RT-PCR. 2. Animal experiments 1) Experimental grouping and treatment: 60 healthy C57BL/6 male mice of 8 weeks old were divided into four groups, 15 mice in each group, and were offered a choice of normal diet+tap water(ND group), normal diet+tap water containing 5% BCAA(ND/BCAA group), high fat diet+tap water(HD group), high fat diet+tap water containing 5%BCAA(HD/BCAA group). The body weight of mice were Weekly monitored and recorded until 18 weeks after feeding. 2) Intraperitoneal glucose tolerance(IPGTT) and intraperitoneal insulin tolerance(ITT) were used to detect the level of mice insulin resistance. 3) BCAA detection kit was used to detect the mice serum BCAA level.4) Insulin detection kit was used to detect mice serum insulin levels. 5) Western blot was used to detect the hepatic insulin signaling pathway, the level of insulin resistance, AKT and AKT2 protein levels in mice.Results1. BCAA induced downregulation of AKT/AKT2 protein level in Hep G2 cells, promoted the development of insulin resistance in Hep G2 cells. 1) BCAA induced downregulation of AKT and p-AKT(ser473) protein level in Hep G2 cells, promoted the development of insulin resistance in Hep G2 cells. BCAA treatment alone can induced downregulation of AKT and p-AKT(ser473) protein levels in Hep G2 cells. BCAA promoted palmitate induced insulin resistance in Hep G2 cells. 2) BCAA induced downregulation of AKT1, AKT2, and AKT3 protein levels in Hep G2 cells, of which AKT2 is the most sensitive one. Compared with contol group, 10 m M BCAA induced Hep G2 cells AKT1 and Akt3 protein level downregulated, but 1-3 m M BCAA can significantly induce AKT2 downregulation; the effect of downregulation of three isoforms of AKT protein induced by BCAA can be reversed by BT2. 3) BCAA induced degradation of AKT by ubiquitin-Proteasome dependent pathway through down-regulation of m TORC2 in Hep G2 cell. Rictor, the major component of m TORC2, were significantly down-regulated in Hep G2 cells after BCAA incubation for 24 h, and this effect could be reversed by BT2, which indicated that BCAA can induced down-regulation of m TORC2 in Hep G2 cell. The function of m TORC2 was significantly inhibited after 100 n M rapamycin incubated for 48 h in Hep G2 cells, which promoted BCAA induced downregulation of AKT protein in Hep G2 cells, indicating that BCAA may induce down-regulation of AKT in Hep G2 cells through down-regulation of m TORC2. The effect of downregulation of AKT protein induced by BCAA in Hep G2 cells can be inhibited by ubiquitin activating enzyme E1 inhibitor PYR41 and proteasome inhibitor MG132, showed that the BCAA induced protein degradation of AKT in Hep G2 cells may through ubiquitin dependent degradation pathway. 2. BCAA promoted high fat diet induced hepatic insulin resistance in mice by down regulating AKT2 protein. 1) BCAA inhibited high fat diet induced body weight gain in mice. Compared with the HD group, the body weight gain in HD/BCAA group was significantly slower since 2 weeks after feeding. 2) The mice serum BCAA concentration significantly increased after high fat diet feeding for 18 weeks, and the serum BCAA concentration in HD/BCAA group increased further. Compared with the ND group, the serum BCAA concentration in HD group was significantly higher, and the serum BCAA concentration in HD/BCAA group was higher than that in HD group. 3) The serum insulin concentration increased significantly after high fat diet feeding for 18 weeks. Compared with HD group, serum insulin concentration in HD/BCAA group increased further, hepatic insulin resistance were significantly exacerbated. Compared with the ND group, the serum insulin level in HD group was significantly higher. Compared with HD group, the serum insulin concentration in the HD/BCAA group was higher, the glucose tolerance and insulin resistance were significantly impaired, hepatic insulin signaling pathway was significantly inhibited. 4) BCAA promoted high fat diet induced mice liver insulin resistance through down-regulation of AKT2. Serum BCAA concentration gradually increased in WT mice ND group, HD group, HD/BCAA group and PP2 Cm knockout mice ND group mice, ND/BCAA, HD group, HD/BCAA group; compared with WT mice ND group, mice liver AKT2 protein level decreased in turn in WT mice HD/BCAA group, PP2 Cm knockout mice ND group, ND/BCAA, HD group and HD/BCAA group.Conclusions1. This study confirmed that BCAA induced AKT protein degradation by down-regulation of m TORC2 through the ubiquitin dependent degradation pathway.And AKT2 is the most sensitive one among the three AKT isoforms. BCAA and palmitate work together to promote the development of Hep G2 cells insulin resistance. 2. Normal diet feeding with or without BCAA did not affect the liver insulin sensitivity, but BCAA promoted the high fat diet induced hepatic insulin resistance in C57BL/6 mice. The mechanism may be that BCAA induced down-regulation of AKT2 in mice liver, exacerbating the development of insulin resistance.