Gut-specific Knockout Of Endoplasmic Reticulum Transmembrane Protein TM6SF2 On The Occurrence And Development Of NAFLD

Background and Objective:The development and progression of non-alcoholic fatty liver disease(NAFLD)is closely related to genetic susceptibility and endoplasmic reticulum stress.The latest international epidemiological and genetic studies have found that endoplasmic reticulum transmembrane protein TM6SF2 is an important risk factor for NAFLD,and the E167 K polymorphism of TM6SF2 is significantly associated with the progression of NAFLD.TM6SF2 is highly expressed in intestinal tissues and is localized across the endoplasmic reticulum in cells.However,the physiological function of TM6SF2 in the intestinal,and its role on lipid metabolism,the development and progression of NAFLD are still unclear.In this study,a TM6SF2gut-specific knockout mouse model was constructed to investigate the effect of intestinal TM6SF2 protein on liver lipid metabolism and the development and progression of NAFLD at the animal level.Methods:TM6SF2 gut-specific knockout(TM6SF2 GKO)mouse model was established based on the CRISPR/Cas9 technology.The mRNA and protein expressions of TM6SF2 in the small intestine and other tissues of TM6SF2 GKO mice were detected by q RT-PCR and Western-Blot to confirm the model was established successfully.Wild type(WT)mice and TM6SF2 GKO mice were fed with normal diet and high-fat diet(NAFLD model)for 12 weeks.The glucose metabolism and lipid metabolism related indexes of the above mice were detected.The body weight and oral glucose tolerance test(OGTT)were recorded during the growth of the mice.The content of triglyceride(TG)in the liver of the above mouse model and normal control mice was determined by ELISA kit.Plasma TG,high density lipoprotein(HDL),aspartate aminotransferase(AST),alanine aminotransferase(ALT)and other biochemical indexes and insulin resistance(IR)were also detected by ELISA kit.Oil red staining and HE staining were used to observe the lipid content and pathological changes in liver tissue and small intestine of the above mouse model and normal control mice.The intestinal epithelial cells of TM6SF2 GKO mice and WT mice were used to qualitatively detect lipid accumulation in intestinal epithelial cells to explore the role of intestinal TM6SF2 protein on intestinal lipid absorption and transport,liver fat metabolism and the occurrence and development of NAFLD in mice.Results:We successfully constructed the TM6SF2 GKO mouse model.The body weight,liver weight and liver index of TM6SF2 GKO mice were higher than those of WT mice at aged 7 weeks.After 12 weeks of normal diet,a significant increase in blood glucose levels was detected in TM6SF2 GKO mice compared with normal mice at 30 min by OGTT assay(P=0.022).Moreover,after 12 weeks of high-fat diet,the OGTT assay detected higher blood glucose levels in TM6SF2 GKO mice at 30 and 60 min than in normal mice.In addition,the OGTT-AUC of TM6SF2 GKO mice was higher than that of WT mice at 7 weeks of age,12 weeks of normal diet and high fat diet.After 12 weeks of high-fat diet feeding,the content and area of lipid droplets in hepatocytes of TM6SF2 GKO mice were significantly higher than those of WT mice.HE staining showed that the liver tissue of TM6SF2 GKO mice possessed the macrovesicular steatosis after 12 weeks of high-fat diet feeding,and the pathological damage was more severe than that of WT mice.In addition,the area and number of lipid droplets in the small intestine of TM6SF2 GKO mice were significantly increased by Oil red staining and HE staining compared with that of normal mice after12 weeks of high-fat diet feeding.Conclusion:The TM6SF2 GKO mouse model was constructed successfully for the first time.TM6SF2 intestinal specific knockout resulted in the significantly increase of body weight,liver weight,and liver index in adult mice compared with WT mice.TM6SF2 intestinal specific knockout impaired glucose tolerance in adult mice fed a high-fat diet and a normal diet for 12 weeks.TM6SF2 intestinal specific knockout can increase the degree of steatosis and liver inflammation in mice,and promote the development and progression of NAFLD.TM6SF2 intestinal specific knockout can increase lipid absorption capacity of small intestinal epithelial cells to a certain extent.