The E3 Ubiquitin Ligase TRIM8 Targets TAK1 To Promote Insulin Resistance And Steatohepatitis

Nonalcoholic fatty liver disease(NAFLD)is characterized by lipid accumulation in the hepatocytes,which is mainly caused by the metabolic disorders.NAFLD is considered as the hepatic manifestation of the metabolic syndrome.Patients with NAFLD show simple steatosis in early stage.Once NAFLD progresses to NASH,it may irreversibly advance to fibrosis,cirrhosis and even hepatocellular carcinoma.Multiple factors including environment,genetics,and metabolic stress contribute to NAFLD.Particularly,insulin resistance and inflammatory response are two critical players that synergistically affect the pathogenesis of NAFLD.Controlling steatosis transition to NASH has been proposed to be the key interfering point for these diseases.Understanding the molecular mechanisms,especially the role of insulin resistance and inflammatory response in this process,are essential for development of novel therapeutic agents to treat these diseases.Our preliminary study shows E3 ligase TRIM8 is tightly associated with hepatic steatosis.Given that inflammation is critical for NASH and TRIM gene family plays important roles in inflammatory response,we hypothesize that TRIM8 contributes to the pathogenesis of the NAFLD.We propose to test its function in NAFLD and explore the underlying mechanisms.Firstly,we verify the correlation of TRIM8 expression level and NAFLD.The TRIM8 protein level dramatically increases in the livers from patients with NAFLD compared with non-steatotic controls and the TRIM8 expression level positively correlates to pathological degree.Increase of TRIM8 protein is confirmed in high-fat diet(HFD)-induced hepatic steatosis mouse model and ob/ob genetic mouse model.In primary hepatocytes,palmitate significantly upregulates TRIM8 protein expression.All these results suggest that TRIM8 upregulation is tightly associated with hepatic steatosis and may be a potential regulator of NAFLD.Secondly,we reveal that TRIM8 aggravates the hepatic insulin resistance.We have generated TRIM8 transgenic mice with hepatocyte-specific over-expression(TRIM8-HTG)and hepatocyte-specific TRIM8 knockout mice(TRIM8-HKO),which are subjected to HFD treatment.HFD treatment rapidly increases body weights,fasting blood glucose and insulin levels of NTG and TRIM8-HTG mice compared to the normal chowl treated mice.TRIM8-HTG group show much higher level of fasting blood glucose and insulin than that of NTG littermates although body weight are comparable in TRIM8-HTG and NTG mice.In IPGTT and IPITT assays,TRIM8-HTG mice exhibit significantly higher glucose concentrations.Consistently,TRIM8 significantly reduces the phosphorylation of IRSTyr608,AKTSer473,and GSK3?.Additionally,the expression of key enzyme of gluconeogenesis PEPCK as well as glycogen synthesis G9Pase increases inTRIM8-HTG mice.As expected,mice with TRIM8 deficiency shows an opposite phenotype.These findings demonstrate that TRIM8 upregulation aggravates HFD-induced hepatic insulin resistance.Thirdly,we demonstrate that TRIM8 exacerbates hepatic steatosis,inflammation and fibrosis.Sustained HFD elicits dramatic increases in liver weight,which is further augmented by TRIM8 upregulation but blunted by TRIM8-deficiency.Oil red O staining and measurement of TG,TC,NEFA and liver function index shows increased lipid accumulation and liver injury in TRIM8-HTG mice.RT-PCR analysis indicates that inflammatory factors and chemokines such as IL-6,TNF-a and MCP1 are dramatically elevated and IL-10 mRNA levels are reduced in TRIM8-HTG mice compared with the levels in NTG controls.Consistently,immunofluorescence results show increased infiltration of F4/80-positive cells in the liver and hepatic fibrosis is aggravated by TRIM8 overexpression.Interestingly,mice with TRIM8 deficiency show an opposite phenotype in terms of lipid accumulation,liver function,inflammation and fibrosis.Together,these results suggest that TRIM8 function as a positive regulator of hepatic steatosis,inflammation and fibrosis in response to HFDadministration and TRIM8 upregulation may be an important factor to promote transition from hepatic steatosis to NASH.Fourthly,we further dissect the mechanism of TRIM8 to promote insulin resistance and NASH development.JNK and p38 phosphorylation,but not ERK phosphorylation,is significantly increased by the TRIM8 overexpression.We further identify TAK1 as the upstream kinase.Indeed,a specific inhibitor of TAK1,5Z-7-OX,almost completely abolishes the positive regulation of TRIM8 on liver weight,lipid accumulation and liver dysfunction.Our findings indicated that TRIM8 activated JNK,p38 by TAK1 to promote HFD induced insulin resistance and NASH development.Finally,we elucidate the mechanisms by which TRIM8 interacts with and ubiquitinates TAK1.Palmitate treatment induces TRIM8 to translocate into cytoplasm and co-localize with TAK1.Co-immunoprecipitation experiment confirms that palmitate treatment enhances interaction of TRIM8 and TAK1.Mapping experiments shows that 59-182aa segment of TRIM8 and 301-480aa segment of TAK1 mediates their interaction.More important,interfering interaction can block TAK1 ubiquitination and completely reverse the effect of TRIM8 in HFD-induced insulin resistance and NASH phenotype.Collectively,we unveil the mechanism by which TRIM8 ubiquitinates TAK1 to promote insulin resistance and NASH.Taken together,TRIM8,a member of TRIM family of E3 ligase,is not only an important regulator in the innate immunity,but also plays a vital role in regulating NAFLD and related metabolic disease by ubiquitinating TAK1,which further alters the activity of downstream signal molecular JNK and p38.Our study not only help understand the pathogenesis of NAFLD,but also provide a potential target for the diagnosis and therapy.