Action Mechanisms Underlying The Improvement Of Nonalcoholic Fatty Liver Disease And Insulin Resistance By Knockdown Of Selenoprotein S

Selenoprotein S(SelS),is an endoplasmic reticulum(ER)-resident selenoprotein.Some studies have reported the elevation of SelS expression is involved in obesity,insulin resistance(IR)and type 2 diabetes(T2D)rats in the fasted state.Of particular relevance,SelS overexpression in H4IIE cells impairs insulin sensitivity and alters glucose metabolism.In our previous study,we also found that knockdown of SelS could ameliorate IR induced by palmitate in the HepG2 cells through the activation of AMP kinase(AMPK)and inhibition of JNK.Altogether,it still lacks cogent evidence in vivo or in vitro to confirm the relationship between SelS and T2D related disease.Thus,it is necessary to sequentially investigate the effect of SelS on T2D,which will be beneficial to understand the biological function of selenium and SelS in metabolic disease and discover a potential therapeutic target.In this thesis,we previously reviewed the relationship among T2D,IR and nonalcoholic fatty liver disease(NAFLD),hepatic lipid metabolism,the basic situation of AMPK and the relationship between SelS and glucose lipid metabolism.On this basis,we chose db/db mice and diet-induced obese(DIO)mice as our in vivo models,and free fatty acids(FFAs)-induced HepG2 cells as our in vitro model to study the effect of SelS on T2D,and the main results were as follows:(1)Effect of SelS knockdown on glucose lipid metabolism in db/db mice was achieved by small interfering RNA(siRNA),qRT-PCR,western blotting,GC-MS,and HPLC.Our results indicated that hepatic mRNA and protein levels of SelS were increased in db/db mice after a 12 h-fasting.In db/db mice,hepatic knockdown of SelS reduced gradient(%of initiate)of body weight and blood glucose levels in the fasted state,reduced FINS and HOMA-IR,and improved insulin tolerance;hepatic knockdown of SelS increased liver glycogen content and reduced gluconeogenesis through IRS-1/Akt/GSK-3? and FoxO1 pathway;hepatic knockdown of SelS decreased hepatic lipid accumulation presented by oil red O staining,reduced the liver contents of TG and CHO,and decreased serum levels of TG,FFA,ALT and AST;hepatic knockdown of SelS inhibited hepatic mRNA expressions of the key enzymes from the entire lipogenic program,enhanced hepatic mRNA expressions of the genes involved in fatty acid ?-oxidation(FAO),and the hepatic fatty acid composition changed little;hepatic knockdown of SelS significantly increased phosphorylation of AMPK and ACC,increased serum adiponectin levels,decreased phosphorylation of JNK,reduced macrophage infiltration in the liver and decreased serum levels of tumor necrosis factor alpha(TNF-a)and interleukin 6(IL-6);the mechanism for AMPK activation was related to the increase in both AMP/ATP and ADP/ATP ratios.In conclusion,hepatic knockdown of SelS could alleviate hepatic steatosis and IR in db/db mice,the mechanism for which may be related to AMPK activation.(2)Effect of SelS knockdown on glucose lipid metabolism in DIO mice was achieved by siRNA,qRT-PCR and western blotting.Our results indicated that the hepatic protein level of SelS was increased in DIO mice after a 12 h-fasting.In DIO mice,hepatic knockdown of SelS reduced fasting blood glucose levels,FINS and HOMA-IR,and improved glucose tolerance,insulin tolerance and pyruvate tolerance;hepatic knockdown of SelS increased phosphorylation of Akt and decreased phosphorylation of JNK,and decreased serum levels of TNF-a and IL-6;hepatic knockdown of SelS decreased hepatic lipid accumulation presented by oil red O staining,reduced the liver contents of TG and CHO,and decreased serum levels of TG,FFA,ALT and AST;hepatic knockdown of SelS significantly increased phosphorylation of AMPK and ACC,and increased serum adiponectin levels.In conclusion,hepatic knockdown of SelS could alleviate hepatic steatosis and IR in DIO mice,the mechanism for which may be related to AMPK activation.(3)Effect of SelS knockdown on FFA-induced steatosis in HepG2 cells was achieved by siRNA,qRT-PCR,western blotting,and HPLC.Our results suggested that in HepG2 cells with steatosis induced by FFA,knockdown of SelS significantly increased phosphorylation of AMPK and ACC,decreased the lipid accumulation presented by oil red O staining,reduced the TG content,inhibited mRNA expressions of the key enzymes from the lipogenic program,enhanced mRNA expressions of the genes involved in FAO.And Compound C or AMPK siRNA reversed these effects of SelS knockdown.As to the oxygen consumption,AMPK siRNA pretreatment reversed the effect of SelS knockdown.Besides,knockdown of SelS also increased AMP/ATP and ADP/ATP ratios in HepG2 cells with steatosis induced by FFA.In conclusion,in the HepG2 cells model,knockdown of SelS could increase AMP/ATP and ADP/ATP ratios,at least partly,to activate AMPK to inhibit the lipogenesis and promote the FAO for preventing from steatosis.This also provided an evidence to confirm the results that hepatic knockdown of SelS could alleviate hepatic steatosis and IR in DIO mice,the mechanism for which may be related to AMPK activation and that hepatic knockdown of SelS could alleviate hepatic steatosis and IR in db/db mice,the mechanism for which may be related to increased AMP/ATP and ADP/ATP ratios,at least partly,for AMPK activation to inhibit the lipogenesis and promote the FAO.(4)The mRNA expressions of selenoproteins from livers and pancreases in db/db mice and WT mice were measured by qRT-PCR.Our results suggested that twenty-three kinds of selenoproteins were detected in the livers of db/db mice and WT mice,and Dio2 was undetected.In the livers of db/db mice,fourteen kinds of selenoproteins mRNA expressions were increased compared with the WT.And twenty-two kinds of selenoproteins were detected in the pancreases of db/db mice and WT mice,and SelN and Dio2 were undetected.In the pancreases of db/db mice,six kinds of selenoproteins mRNA expressions were increased,and Sell was decreased compared with the WT.In conclusion,the mRNA expressions of many selenoproteins from livers and pancreases in db/db mice were changed compared with the WT.And except for the reported GPx1,SelP,SelT and SelS,the relationship between the selenoproteins of changed contents and T2D related metabolic disease should be further investigated.