Effect Of Cellular Repressor Of E1A Stimulated Genes On Phenotype And Mechanism Of Hepatic Fat Metabolism

The way of "high accumulation and low consumption" in modern society become a major threat to human health.Metabolic syndrome(MS),which is characterized by disturbance of metabolism in human body,attracted more and more attention in recent years.As aggregation of risk factors,MS has a positive correlation with cardiovascular disease.Liver is an important organ for the metabolism of the three nutrients,internal equilibrium and stability of liver played important role in MS.Non-alcoholic fatty liver disease(NAFLD)including hepatic steatosis,non-alcoholic steatohepatitis and cirrhosis,which mechanism is about lipid deposition and inflammation in hepatocytes,were closely related to obesity,insulin resistance and MS.Recently,NAFLD has become the most common chronic liver disease in western developed countries.With the gradually controlling of hepatitis B virus and the spread of unhealthy modern lifestyles,NAFLD has also become the main disease threatening public health in China.However,the detailed mechanisms,including the complex molecular interactions and related cellular behaviors,involved in the process and initiation of hepatic steatosis and metabolic disorder are not yet fully understood,thus leading to a lack of effective treatment strategies or reagents that may alleviate NAFLD.Therefore,it is necessary to find an effective drug to alleviate NAFLD and clarify its mechanism.Cellular repressor of E1A-stimulated genes(CREG)participates in inducing cell maturation,promoting cell differentiation and maintenance of differentiation.CREG displayed extensive biological functions in cardiovascular system,including relieving myocardial ischemia;myocardial hypertrophy,inflammatory response,apoptosis and fibrosis.These effects above is happened to be closely related to MS and NAFLD,thus we presumed a cytoprotection effect of CREG.Results from preliminary study confirmed mild glucose and lipid metabolism disorder in CREG+/-mice.Based on evidence above,CREG may play a key role in glucose and lipid metabolism of NAFLD.Here,we applied gain and loss of function approaches to study phenotype and mechanism of CREG regulating NAFLD.In our study,methods of molecular biology,morphology and cell biology were used.We studied roles of CREG and roles of ASK1-JNK1 from mitogen activated protein kinases(MAPKs)family on hepatic gluco-lipid metabolism,which supports CREG as a promising target for this disease.The major methods and results are listed in the follow:1.The correlation of CREG with NAFLD.First,changes of CREG expression in hepatocyte and hepatic tissue from NAFLD model were studied.Data shown that mRNA and protein level were significantly decreased in NAFLD animal model.(HFD 0.43 ± 0.04 vs NC 1.00 ± 0.21,P* < 0.05 vs NC;HFD 2.68 ± 1.04 vs NC 5.88 ± 0.92,P* < 0.05 vs NC)To directly investigate whether CREG levels changed in liver hepatocytes exposed to a dynamic change,primary hepatocytes were exposed to concentration gradients of palmitate,and CREG expression was significantly decreased in PA-treated primary hepatocytes in a dose-dependent manner compared with the CREG expression in the control group.Considering the multiple pathogenic signs of NAFLD,a classical feed/fasted model was established,and CREG expression was measured.In addition to changes to PEPCK and G6 Pase,lipid metabolism-related proteins,CREG expression levels were significantly decreased after 24 h of fasting.(P* < 0.05 vs feed)Above data suggested a negative correlation between CREG expression level and lipid accumulation.Furthermore,an examination of CREG protein levels in human samples was performed by DAB staining(Dako REALTM En VisionTM Detection System).The obtained images showed that CREG was mainly distributed in the cytoplasm of hepatocytes and was dramatically reduced in livers of NAFLD patients compared with normal group subjects.Next,western blotting confirmed decreased CREG levels in the NAFLD group(NAFLD 1.00 ± 0.25 vs normal 2.77 ± 0.34,P < 0.05).In summary,CREG m RNA and protein expression decreased as fat accumulation increased,thus suggesting a potential role of CREG in metabolism disorde r.2.Phenotypic study of CREG regulating gluco-lipid metabolismAfter confirming the dramatic downregulation of CREG in fatty livers,we sought to assess the role of CREG in NAFLD and associated gluco-lipid metabolism complications.Therefore,CREG CKO mice were generated.CREG knockout in the liver led to significantly higher body weight,liver weight/body weight and liver weight in mice administered an HFD for 12 weeks compared with the wild-type(WT)controls.In the following glucometabolism studies,CREG-CKO mice exhibited higher fasting blood glucose and fasting serum insulin levels,more severe glucose tolerance and insulin sensitivity,thus leading to a higher value in HOMA-IR.To further study the influence of CREG deletion on insulin resistance,insulin signaling(Irs1/Akt,GSK3?,FOXO1)was analyzed by using western blotting.The phosphorylation levels of all the tested signaling components,for which higher levels indicate higher glucometabolism ability,were markedly lower in the KO-HFD group than in the controls.Periodic acid-Schiff staining(PAS)showed that the glycogen content in the liver was impaired by CREG deletion.In summary,this loss of function study suggests that CREG deletion specifically in the liver aggravates glucometabolism disorder.Meanwhile,gain-of-function study suggested that CREG overexpression in the liver reversed all the index above,which means CREG overexpression improved glucometabolism disorders.Next,we evaluated the effect of CREG on perturbation of lipid metabolism.Liver ultrasound demonstrated that at the same position and detection angle,the liver was thicker in the KO-HFD group than in the WT-HFD group(thickness: 4.97 ± 0.25 mm vs 3.53 ± 0.35 mm,P = 0.05).Increased lipid accumulation in the liver demonstrated by H&E and oil Red O staining accompanied increased NEFA,total cholesterol,triglyceride and liver function levels in the KO-HFD group.According to the above glucometabolism studies,CREG overexpression reversed above changes.Meanwhile,in vitro Oil red O staining and BODIPY-C16 fluorescence staining showed that lipid accumulation increased after CREG knockout and decreased after CREG overexpression.Next,the lipid metabolism-related AMPK,ACC,m TOR,p70S6 K signaling pathways were analyzed,and AMPK and ACC phosphorylation were positively correlated with CREG protein expression,whereas m TOR and p70S6 K were negatively correlated.In addition,the m RNA levels of lipid metabolism-and inflammation-related genes were analyzed.After chronic HFD administration,CREG-TG mouse livers showed decreased expression of mRNAs(HMGCR,SREBP-1C,FAS,ACCa,CD36,FATP1,FABP1,PPAR-?,PEPCK,G6 PC,IL-1b,IL-6,TNF-?,MCP-1)and increased levels of m RNAs(ABCG1,CYP7A1,PPAR-?,CPT-1a,ACOX-1,UCP2,IL-10,PDK4),whereas CREG-KO mouse livers exhibited expression changes that were opposite from those observed in the CREG-TG mice.Data in this part showed that CREG overexpression significantly improved hepatic steatosis and inflammatory reaction.It is environmental factor in above HFD treatment study.To study the effect of CREG on genetic NAFLD,we used genetically obese ob/ob mice.CREG protein expression decreased with increased feeding time(2w,4w,8w,12w)in the liver of ob/ob mice.Lipid accumulation in the liver demonstrated by H&E and oil Red O staining also decreased in the ob/ob-Ad CREG group.However,the body weight and liver weight/body weight ratio did not change with CREG overexpression in ob/ob mice,although CREG overexpression largely improved hepatic steatosis and metabolic disorder and insulin resistance.Collectively,CREG overexpression alleviated hepatic steatosis and metabolic disorder in hepatic steatosis animal model.3.Molecular mechanism study of CREG regulating gluco-lipid metabolismTo explore the underlying CREG mechanism,the phosphorylation levels of MEK,ERK,JNK and P38 were analyzed by western blotting in vivo and in vitro.In the tested proteins,only JNK phosphorylation increased after hepatic steatosis,and decrease after CREG overexpression.In the gluco-lipid phenotypic study,we found that SP600125 successfully reversed the adverse effect of CREG knockout on body weight gain,liver weight/body weight,blood glucose,serum insulin,IPGTT and IPITT response.Importantly,immunohistochemistry results from human samples showed that the expression was negatively correlated with P-JNK.These data indicated that CREG mediates hepatic steatosis and metabolic disorder via the JNK signaling pathway.We next investigated the precise contribution of these two major isoforms JNK1 a nd JNK2 to CREG-mediated hepatic steatosis and metabolic disorders.CREG-JNK1-DKO and CREG-JNK2-DKO mice were generated,HE and oil red O staining showed that lipid accumulation was decreased in the CREG-JNK1-DKO group but not the CREGJNK2-DKO group.To further confirm the reversal of this effect in CREG-JNK1-DKO mice,the body weight gain,liver weight/body weight,blood glucose,and HOMA-IR were analyzed.The adverse effect of CREG deletion was reversed by knocking out JNK1.However,JNK2 deletion did not reverse the effect of CREG knock out.Therefore,our findings clearly demonstrate that the role of CREG in NAFLD pathologies is largely dependent on inactivation of JNK1 but not JNK2 activation.We next explored the link between CREG and JNK1.The physical interactions of CREG with components in this cascade were examined.Notably,CREG was strongly bound to ASK1.As expected,CREG inhibited ASK1 phosphorylation in a dose-dependent manner.Collectively,these data demonstrated that CREG alleviates NAFLD pathologies by directly binding to ASK1 and inhibiting ASK1-JNK1 signaling.In the present study,we provided the first demonstration of a protective role of CREG and detailed CREG-ASK1-JNK mechanism in gluco-lipid metabolism.First,we observed that changing rule of CREG in hepatic steatosis in vivo and in vitro,putting forward the scientific hypothesis that CREG may be correlated with gluco-lipid metabolism.Second,loss of function and gain of function study were performed,and data showed that CREG overexpression alleviated hepatic steatosis and metabolic disorder in hepatic steatosis animal model.Finally,CREG alleviates NAFLD pathologies by directly binding to ASK1 and inhibiting its phosphorylation,which may subsequently inhibit downstream JNK1 signaling pathway and then alleviating NAFLD and metabolism syndrome.Our findings of novel binding signals ASK1 extends the biological functions and clinical application of CREG in NAFLD.The specific CREG regulation of ASK1 enables the maintenance of CREG as a therapeutic strategy for NAFLD treatment.