Study Of N-Glycosylation On Cyclic AMP Responsive Element Binding Protein H In Nonalcoholic Fatty Liver Disease

Objective To analyze the effects of N-glycosylation cyclic AMP responsive element binding protein H(CREBH)in proteolytic activation,and to investigate the changes of CREBH glycosylation on lipid metabolism,sterol metabolism,lipotoxicity,lipid peroxidation and inflammation in PA-induced hepatic steatosis cell model.Further,to explore the significance of CREBH glycosylation in nonalcoholic fatty liver disease(NAFLD).Methods 1.Establishment of a PA-induced hepatic steatosis cell model.AML-12 cells were treated with palmitic acid(PA)to induce hepatic steatosis cell model.Intracellular lipid levels were measured by oil red O staining and the optimal concentration and time were screened.2.Effect of N-glycosylation on molecular weight and protein activation of CREBH.The expression plasmids of mouse CREBH glycosylation site mutants as well as the expression plasmids of CREBH wide-type(CREBH WT)were constructed.Glycosylation of CREBH was detected in the lysates through PNGase F treatment by Western blotting.We also compared the subcellular localization of CREBH protein between CREBH WT and CREBH m1+m2+m3 groups in AML-12 cells treated with PA by immunofluorescence.3.Effects of CREBH N-glycosylation in PPARα and SCD-1.The plasmids of CREBH WT and the mutant plasmids were transfected into AML-12 cells,respectively,and then treated with PA to induce hepatic steatosis.Glycosylation of CREBH was detected in the lysates through PNGase F treatment by western blot and the protein expression of PPARα and SCD-1 were observed.The expression of mRNA levels was detected by qRT-PCR.4.Comparison of glycosylation,un-glycosylation and deglycosylation in CREBH.We used Tm of different doses and different time duration in AML-12 cells transfected with CREBH WT and CREBH m1+m2+m3 plasmids to compare the different roles of Tm in inducing deglycosylation and endoplasmic reticulum stress(ERs)and analysis the effect on CREBH protein expression.In addition,differences between CREBH WT and un-glycosylation in PA-induced glycosylation and in Tm-induced deglycosylation and ERs were compared.5.Regulation of CREBH glycosylation on key factors of in hepatic steatosis.The effects of CREBH glycosylation,un-glycosylation,deglycosylation,and hyperglycosylation to CREBH,PPARα and SCD-1 protein expression were analyzed by western blot,and the localization of CREBH,PPARα and SCD-1 in cells were observed by IF.In addition,qRT-PCR was used to detect the gene expression,and chemical enzyme method was used to detect the changes of TG and TC.The changes of inflammatory factors TNF-α and IL-6 as well as lipid peroxidation factors MDA and SOD were detected by ELISA.6.Regulation mechanism of CREBH glycosylation on PPARα and SCD-1.The molecular mechanism of CREBH glycosylation regulating PPARα and SCD-1 was elucidated.The dual luciferase reporter assay was used to measure the luciferase activity.To identify the interaction of CREBH with PPARα/SCD-1,co-immunoprecipitation using different tag antibodies and target gene antibodies was performed.Results 1.After staining by oil red O,Significant lipid droplet deposition(>70%)in the cytoplasm of hepatocytes was observed by intervention of 200 μM PA for 48 h,and there was no significant necrosis of hepatocytes.2.The expression plasmids of mouse CREBH glycosylation site mutants with single(m1,m2,m3),double(m1+m2,m1+m3,m2+m3)and triple(m1+m2+m3),as well as the expression plasmids of CREBH WT were constructed.After treatment with PNGase F,the molecular weight of CREBH was reduced significantly(P<0.01)in CREBH WT group,while no significant difference was observed in CREBH m1+m2+m3 group(P>0.05).The molecular weight of CREBH in single and double mutants was intermediate.Upon PA treatment,CREBH was susceptible to induce proteolysis and the active fragment was accumulated in the nucleus in CREBH WT group and the expression of CREBH was decreased(P<0.05).In contrast,CREBH might not be activated by PA and so the majority of CREBH proteins were found in the cytoplasm in CREBH m1+m2+m3 group and there was no change of protein(P>0.05).3.After PA treatment for 48 h,the expression of CREBH protein was reduced most dramatically in CREBH WT group(P<0.01),but showed no significant change in CREBH m1+m2+m3 group(P>0.05).The expression of CREBH with single and double mutants was intermediate.After further treatment with PNGase F,we found that the molecular weight and protein expression of CREBH were all dramatically decreased in CREBH WT group(P<0.01).On the contrary,the expression of CREBH protein and molecular weight was not declined in CREBH m1+m2+m3 group(P>0.05).The others were intermediate.In addition,after PA intervention,PPARα protein and gene expression were decreased in the CREBH WT group(P<0.05),and SCD-1 were increased(P<0.01).When CREBH was hypoglycosylated or un-glycosylated,the production of PPARα was consumed in large amounts,and the expression of SCD-1 was abundantly produced because of its disorder by CREBH(P<0.05).4.After treatment with different doses(2 μg/mL,5 μg/mL and 10 μg/mL)of Tm for 6 h before harvesting,we found that the molecular weight of CREBH in CREBH WT group was gradually decreased(P<0.05).The molecular weight and expression of CREBH were both decreased in CREBH WT group after treatment with Tm(10 μg/mL)for 6 h(P<0.05).Upon treatment with Tm for 24 h,the molecular weight and expression of CREBH were slightly reduced(P<0.05).At 48 h after treatment,the expression of CREBH was significantly reduced(P<0.01),and the molecular weight was decreased but showed no significant difference as compared to those at 6 h(P<0.05).However,in CREBH m1+m2+m3 group,Tm showed no significant effects on glycosylation of CREBH,leading to no significant change in the molecular weight and protein expression of CREBH at different doses and time points(P>0.05).5.After treatment with PA(200 μM)for 48 h,the expression of CREBH were both decreased in CREBH WT group(P<0.01).In the same group,the molecular weight and the expression of CREBH were decreased after treatment with Tm(P<0.05).There was no significant difference in CREBH WT group after treatment with PA in combination with Tm,and similar results were obtained in the CREBH m1+m2+m3 group(P>0.05).The expression of PPARα was decreased and SCD-1 was increased in CREBH WT group(P<0.05),while these changes were aggravated due to Tm-induced deglycosylation.The most significant change was observed in the CREBH m1+m2+m3 group(P<0.05)Similarly,when CREBH was modified by un-glycosylation,the changes of key factors in lipid and sterol metabolism,inflammation,lipid toxicity and lipid peroxidation were more significant than CREBH WT(P<0.05).These changes were significantly reversed when GnT-V induced hyperglycosylation of CREBH(P<0.05).6.The luciferase activity of PPARα promoter was elevated,whereas SCD-1 promoter was significantly inhibited in CREBH WT group(P<0.05).In sharp contrast,CREBH m1+m2+m3 could not promote significant increase or decrease in the luciferase activities of PPARα and SCD-1 promoter(P>0.05).The results demonstrated that only CREBH WT could interaction with PPARα or SCD-1,while CREBH m1+m2+m3 did not.Conclusion 1.The sites of N-glycosylation of CREBH were all in the luminal domain and mutations of these three sites simultaneously destroyed the complete glycosylation of CREBH.2.The N-glycosylation of CREBH is essential for proteolytic activation.The N-glycosylation of CREBH was susceptible to induce proteolysis and the active fragment was accumulated in the nucleus,and the protein expression of CREBH were all dramatically decreased.In contrast,un-glycosylated CREBH did not translocate into the nucleus and was not activated by PA so that there was no change in the protein expression of CREBH.3.Tm had a dual effect with different treatment time durations,that is,Tm played a role of glycosylation inhibition of CREBH after treatment for 6 h and was used for ER stress activation at 48 h to promote proteolysis of CREBH.In addition,CREBH WT was more susceptible to proteolytic cleavage induced by PA or Tm than that of un-glycosylation.The deglycosylation of CREBH induced by Tm might inhibit the proteolysis of CREBH induced by PA.4.Both un-glycosylation and deglycosylation of CREBH caused dysregulation of lipid and sterol metabolism,inflammation and lipid peroxidation,which aggravates the development of hepatocyte steatosis.GnT-V played a protective role in hepatocyte steatosis by promoting high glycosylation of CREBH.5.The glycosylation of CREBH could affect the expression of PPARα and SCD-1 by regulating their promoter-driven transcription activity and interaction.N-glycosylation of CREBH played a key regulatory role in lipid metabolism,lipotoxicity and inflammation in NAFLD.Objective To analyze the effects of N-glycosylation of CREBH on lipid metabolism,sterol metabolism,lipotoxicity,lipid peroxidation and inflammation in animal models of NAFLD.The mechanism of N-glycosylation of CREBH in NAFLD was further clarified.Methods 1.Fed with high fat diet(HFD)for 16 weeks or methionine and choline-deficient diet(MCD)for 4 weeks,male C57BL/6 mice were injected with LV-CREBH m1+m2+m3 and LV-Gn T-V via the tail vein to overexpress the endogenous hepatic CREBH m1+m2+m3 and Gn T-V gene.The negative control(LV-NC)was used.2.The liver weight of mice was weighed after modeling,and the morphological changes of liver tissue and the formation of lipid droplets in liver cells were observed by H&E staining and oil red O staining.3.Western blot and IF were used to detect and analyze the expression of CREBH and PPARα and SCD-1 proteins.4.The expression of F4/80 in liver tissue was detected by IHC.5.The expression of CREBH mRNA、PPARα mRNA、SCD-1 mRNA、Apo A-I mRNA、SREBP-1c mRNA and FGF21 mRNA was detected by qRT-PCR.6.Changes of liver function ALT,AST,TG and TC were detected by automatic biochemical analyzer.7.The plasma concentrations of TNF-α,IL-6,MDA and SOD were measured by ELISA.Results 1.Changes of liver morphology and liver weight.The liver weight of the mice injected with LV-CREBH m1+m2+m3 was the lightest in the MCD models and the heaviest in the HFD models(P<0.01).Compared with the model group,the liver morphology and liver weight were significantly improved in LV-Gn T-V group(P<0.05).2.Identification of NAFLD mice models.H&E staining showed that more than 80% of the model mice had lipid changes and inflammatory infiltration after the end of the model.Oil red O staining showed that the appearance of orange-red fat droplets of different sizes was in the liver cells of model mice.The above results all suggestd that the NAFLD model was successful.Liver lipids in LV-CREBH m1+m2+m3 group were significantly worse than LV-NC(P<0.05).LV-Gn T-V improved significantly compared with LV-NC(P<0.05).3.The expression of F4/80 in liver tissue was detected by IHC.The expression of F4/80 in liver tissue of model mice was significantly higher than that in normal mice(P<0.01),and the MCD model group was more obvious than HFD model(P<0.05).In the same model group,compared with LV-NC group,the expression of F4/80 was significantly increased after LV-CREBH m1+m2+m3 treatment(P<0.05),while LV-Gn T-V significantly improved this change(P<0.05).4.Expression of CREBH,PPARα and SCD-1 proteins in liver tissue.The results from western blot indicated that the expression of CREBH protein was decreased significantly in NAFLD mice models(P<0.05).After injection with LV-CREBH m1+m2+m3,the expression of CREBH showed no obvious reduction as compared with LV-NC group(P<0.05).On the contrary,the expression of hyperglycosylated CREBH in LV-Gn T-V group was significantly increased as compared with that in LV-NC group(P<0.01),but it was still lower than that in LV-CREBH m1+m2+m3 group(P<0.05).In LV-CREBH m1+m2+m3 group,the expression of PPARα was the lowest and SCD-1 was the highest in the two NAFLD models(P<0.01).However,in LV-Gn T-V group,the expression of PPARα was significantly higher than that in LV-NC group(P<0.05),and the expression of SCD-1 was significantly lower than that in LV-NC group(P<0.05).The discrepancies of PPARα were more pronounced in the HFD models than in the MCD models,whereas the differences of SCD-1 were just the opposite.Similar results were demonstrated by immunofluorescence 5.Effects of N-glycosylation of CREBH on key factors of NAFLD.The results of qRT-PCR showed that the expression of CREBH mRNA,PPARα mRNA and FGF21 mRNA in the liver tissues of the two animal models was lower than that in the normal group(P<0.05),and the expression of SCD-1 mRNA,Apo A-I mRNA and SREBP-1c mRNA was higher than that in normal group(P<0.05).In LV-CREBH m1+m2+m3 group,this change was significantly higher than that in LV-NC group(P<0.01),while LV-Gn T-V improved this phenomenon and the difference was statistically significant(P<0.05).Similarly,the levels of ALT,AST,TG,TC and the expression of TNF-α,IL-6 and MDA were highest in LV-CREBH m1+m2+m3 group(P<0.01),and the expression of SOD was the lowest(P<0.01).LV-Gn T-V was able to reverse this change(P<0.05).Conclusion 1.In the two NAFLD models,the HFD models was more related to lipid metabolism and sterol metabolism,while the MCD models was more related to lipotoxicity,lipid peroxidation and inflammation.2.The un-glycosylated of CREBH caused the imbalance of lipids and sterols metabolism,lipotoxicity,inflammation,lipid peroxidation,and aggravated the development of NAFLD,while Gn T-V improved these imbalances and played a protective role in NAFLD by promoting the glycosylation of CREBH.3.N-glycosylation of CREBH affected the development of NAFLD in lipid metabolism,sterol metabolism,lipotoxicity,lipid peroxidation,and inflammation by modulating PPARα and SCD-1.