| ObjectiveNonalcoholic fatty liver disease (NAFLD) is the performance of metabolic syndrome in the liver, which is one of the most common chronic liver diseases. The pathogenesis of NAFLD is complex, many aspects of which have not yet been fully understood.The main pathological change of NAFLD is the abnormal accumulation of triglycerides in hepatocytes. The current study suggests that endoplasmic reticulum stress (ERS) has been involved in the development and progression of NAFLD, which is the hot issue to study the pathogenesis of NAFLD.Effect of ERS and its specific mechanism on lipid deposition in hepatocytes are for further study.Sterol regulatory element binding proteins(SREBPs) are zinc lipoproteins which bind with sterol regulatory element of the gene regulatory region of lipogenesis, play a regulatory role in maintaining the balance of lipid metabolism. SREBP-lc is the key transcription factor which regulates the activity and expression of enzymes of lipogenesis, promotes fatty acid synthesis metabolism in hepatocytes.SREBP cleavage-activating protein(SCAP) is the membrane protein whose main function is to transport SREBP-1c to Golgi where hydrolysis is activated. Insulin induced gene 1(Insig-1) could prevent the shift of SCAP/ SREBP-lc compound and retain it in ER by combining SCAP, resulting in inhibition of hydrolysis activation of SREBP-lc. Therefore, Insig-1/SCAP /SREBP-1c pathway plays an important role on balance of lipid metabolism. The changes of this signaling pathway and their impacts on lipid deposition in hepatocytes under ERS are not entirely clear.Ubiquitin-proteasome pathway of protein is a common way in the degradation of endogenous proteins. Autocrine motility factor receptor(AMFR) is a ubiquitin ligase, which causes that insig-1 becomes the target for proteasome degradation through linking ubiquitin-protein with insig-1,which participates in the regulation of the protein level of Insig-1.In this study, thapsigargin(Tg) is applied to construct the model of ERS-induced hepatic steatosis in vitro hepatocytes. On this basis,by inhibiting endoplasmic reticulum stress and interfering the expressions of SCAP and AMFR transiently, the aim is to investigate the regulation of ERS on lipid metabolism through Insig-1 ubiquitination and SCAP in hepatocytes and its specific mechanism.Methods1 L02 and HepG2 cells were treated with thapsigargin, real-time quantitative PCR and Western blot were applied to test the mRNA and protein expressions of glucose regulated protein78 (GRP78) which is the molecular marker of endoplasmic reticulum stress. Triglycerides levels and oil red O staining, from the quantitative and qualitative perspection respectively, were used to detect the lipid levels in hepatocytes. The mRNA and protein expressions of Insig-1, SCAP and SREBP-1c in hepatocytes were determined by q-PCR and Western blot analysis.Hepatocytes were pretreated with 4-phenyl butyric acid(PBA) to inhibit ERS, then the changes of the above indexes were explored.2 Construction of miRNA interference plasmids of SCAP, triglycerides and oil red O staining were measured to detect the changes of lipid levels. Western blot and q-PCR were used to determine the effect of SCAP-miRNA on SREBP-lc and its downstream lipid metabolism related genes fatty acid synthase (FAS) and Acetyl coenzyme A carboxylase (ACC1)3 Establishment of stably overexpressing Insig-1 gene in HepG2LV-5 hepatocytes through human Insig-1 overexpression lentivirus. Western blot and q-PCR were applied to explore the mRNA and protein levels of AMFR under ERS. Construction of siRNAs of AMFR, triglycerides and oil red O staining were used to measure the changes of lipid levels.Western blot and q-PCR were applied to determine the effect of AMFR-siRNA on the mRNA and protein levels of Insig-1 in hepatocytes.Results1 Effect of ERS on hepatic lipid metabolism in L02 and HepG2 cells and the molecular mechanism1.1 Tg group was compared with the control group, the mRNA and protein expressions of GRP78 in hepatocytes were significantly increased(P<0.05); Tg+PBA group was compared with Tg group, which were apparently reduced(P<0.05).1.2 Tg group was compared with the control group, the triglyceride content and lipid droplets in hepatocytes were increased significantly(P<0.05); Tg+PBA group was compared with Tg group, those were apparently lower and reduced(P<0.05); while the PBA group and the control group had no obvious difference (P> 0.05).1.3 The mRNA and protein expressions of Insig-1 in HepG2 hepatocytes were not detected. In L02 hepatocytes, Tg group was compared with the control group, the mRNA level of Insig-1 was significantly upregulated, but the protein expression was apparently reduced (P<0.05); Tg+PBA group was compared with Tg group, the mRNA expression of Insig-1 was significantly reduced, but the protein level was apparently increased (P <0.05); there was no obvious difference between PBA group and the control group (P> 0.05).In L02 and HepG2 hepatocytes, Tg group was compared with the control group, the mRNA and protein expressions of SCAP and SREBP-lc were significantly increased (P<0.05); Tg+PBA group was compared with Tg group, those were decreased apparently(P<0.05); there was no obvious change between PBA group and the control group (P> 0.05).2 ERS regulates lipid synthesis metabolism in L02 and HepG2 hepatocytes through SCAP/SREBP-1c2.1 Tg group was compared with the control group, the triglyceride content and lipid droplets in hepatocytes were increased significantly(P<0.05); the negative plasmid group was compared with Tg group, those did not change obviously (P> 0.05); SCAP interference plasmid group was compared with Tg group, those were apparently reduced (P<0.05).2.2 Tg group was compared with the control group, the expressions of SREBP-1c, FAS and ACC1 were significantly increased (P<0.05); SCAP interference plasmid group was compared with Tg group, those were apparently reduced (P<0.05); the negative control group was compared with Tg group, those did not change obviously(P> 0.05).3 Regulation of ERS on lipid metabolism in L02 and HepG2LV-5 hepatocytes by Insig-1 ubiquitination3.1 A lot of green fluorescent protein expressions could be seen under fluorescence microscope, the protein expression of Insig-1 in HepG2LV-5 hepatocytes was detected by Western blot, successful establishment of HepG2LV-5 hepatocytes which stably overexpress Insig-1 gene.3.2 Tg group was compared with the control group, the expression of AMFR was significantly increased (P<0.05); Tg+PBA group was compared with Tg group, which was apparently reduced (P<0.05); the difference of PBA and the control group was not statistically significant (P> 0.05).3.3 Tg group was compared with the control group, the triglyceride content and lipid droplets in hepatocytes were increased significantly(P<0.05); the negative control group was compared with Tg group, those did not change obviously (P> 0.05); AMFR-1siRNA group was compared with Tg group, those were decreased apparently (P<0.05).3.4 Tg, the negative control and AMFR-1siRNA group were compared with the control group, the relative mRNA expressions of Insig-1 were significantly increased (P<0.05), but two groups of the three sets showed no statistical significance (P>0.05). Tg group was compared with the control group, the protein expression of Insig-1 was significantly decreased (P<0.05); AMFR-1siRNA group was compared with Tg group, which was apparently increased(P<0.05); the negative control group was compared with Tg group,which did not obviously change (P> 0.05).Conclusion1 ERS induced hepatic steatosis. The reduced protein expression of Insig-1 and the elevated mRNA and protein levels of SCAP and SREBP-lc were important mechanisms to ERS-induced lipid deposition in hepatocytes.The protein level of Insig-1 was down, but the gene expression of Insig-1 was upregulated under ERS.2 The high expression of SCAP lead to the enhanced transportion and activation of SREBP-lc precursor and the increased lipogenesis under ERS, which was one of the important mechanisms of ERS-induced lipid deposition.3 The gene and protein levels of Insig-1 were inconsistent resulting from AMFR-mediated Insig-1 ubiquitination under ERS. Regulation of ERS on hepatic lipid metabolism through Insig-1 ubiquitination in hepatocytes. |
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