SND1 Affects Insulin Signaling Pathway In Hepatocytes

Objective: Type 2 diabetes is one of the most serious metabolic diseases in the world, the pathogenesis of which is the insulin resistance of peripheral tissue and the dysfunction of insulin secretion in β Cell of islet. The study shows that one of the main molecular mechanisms is the impaired insulin receptor signaling in the liver, muscle, fat and other insulin target organs, resulting in the occurrence of diabetes. SND1 is a kind of widespread multifunctional protein, which is highly conservative. Recent studies have indicated that SND1 protein can be directly combined with PPARγ, and it’s specifically combined with the PPRE area of PPARγ target gene in the adipocyte, promoting the process of adipocyte as a transcription co-activating factor. In our study, the changes of hepatic steatosis and insulin sensitivity were found in the SND1 overexpression mice with high fat diet. These results suggested that SND1 was involved in the insulin resistance of liver and fat, which could affect the response of insulin in the tissues sensitive to insulin such as liver and adipose tissue. Based on the existing research, we aimed at further discussing the effect of SND1 on insulin receptor signaling in liver cells and its possible molecular mechanism.Methods:To discuss the role of SND1 in liver cells, we chose Hep G2 cells and primary hepatocytes of mice as our study model. We simulate high fat condition by stimulating the Hep G2 cells of knock down SND1(KD) and Control(Ctrl) with palmitate(Palm), and determine whether SND1 can affect the fatty degeneration of cultured liver cells by observing the lipid accumulation of two kinds of cells after staining the cells with oil red; collect cell lysis solution of Hep G2 KD and Ctrl cells with instantaneous stimulation of insulin, then test the expression levels of insulin receptor(IRβ)、Akt/p-Akt by western-blot, in order to observe the effect of SND1 on insulin signaling pathway; observe the changes of IRβ after treating Hep G2 KD and Ctrl cells with cyclheximide(CHX) and protease inhibitor MG132 and insulin stimulation for a long time, and then analyzes the mechanism of SND1 affecting insulin receptor signal; in order to verify the concrete mechanisms of SND1 mediating IRβ ubiquitin proteasome-mediated degradation, detect the expression levels of SND1 gene and Smurf1 gene with real-time quantitative PCR(RT-PCR), verify SND1 protein combined with IRβ、E3 ubiquitin ligase Smurf1 with Co-IP experiments; in Hep G2 KD and Ctrl cells, as well as primary hepatocytes extracted from SND1 over expression mice(Transgenosis,TG) and wild-type mice(WT), simulate high fat condition by Palm for 24 h, observe the expression level of related protein in the insulin signaling pathway and activation of downstream signaling pathways as given short phase insulin stimulation.Results:1. SND1 reduced the lipid accumulation in the primary hepatocytes of mice and human hepatocellular carcinoma cell line Hep G2 which were in high-fat condition with a higher intracellular level of p-IRS1、p-Akt and enhanced insulin sensitivity of hepatocytes.The liver cells with long time stimulation of high fat accelerated the degradation of IRβ, which required the participation of SND1.2. The liver cells with long time stimulation of high fat accelerated the degradation of IRβ, which required the participation of SND1.3. The use of MG132 and CHX confirmed that SND1 affected the degradation of IRβ through ubiquitin-proteasome pathway.4. The protein expression and m RNA level of E3 ubiquitin ligase Smurf1 of HACT class and SND1 were consistent under different types of stimulation, but Smurf1 didn’t combined with SND1 directly. The Co-IP experiments of IRβ and SND1 showed that SND1 didn’t combined with IRβ directly, but Smurf1 could combine with IRβ directly, thereby mediating the degradation of IRβ.Conclusion:1. SND1 reduced the lipid accumulation in the hepatocytes and enhanced insulin sensitivity of hepatocytes.2. SND1 mediated the ubiquitin proteasome-mediated degradation of IRβ, resulting in an impact in the insulin signaling pathway. Further more, high level of SND1 increased the intracellular level of p-IRS1、p-Akt and promoted the insulin sensitivity of hepatocytes in turn.3. The mRNA and protein expression levels of SND1 are consistent with Smurf1, but it can’t regulate the ubiquitin degradation of IRβ through a confidant of protein combination with Smurf1 and IRβ.