Endoplasmic Reticulum Stress Involved In The Course Of Lipogenesis In Fatty Acids Induced Hepatic Steatosis

ObjectiveWith the improvement of living standards and lifestyle increasingly Westernized, nonalcoholic fatty liver disease (NAFLD) incidence is rising gradually. NAFLD ranges from fat accumulation in the liver (steatosis), to steatosis accompanied by inflammation and necrosis with or without fibrosis (nonalcoholic steatohepatitis or NASH), to end-stage liver disease and even hepatocellular carcinoma. NAFLD has become a potential impact on our health risks of major diseases. And more and more data indicate that NAFLD is independently associated with cardiovascular and cerebrovascular events, and obesity-related deaths. Currently considered, NAFLD is closely related to obesity, diabetes, hyperlipidemia and other metabolic syndrome, and high-fat diet is an important element for high incidence of obesity and the complication of obesity. Besides,the high-fat diet is often complicated with elevated plasma free fatty acids that can induce hepatic steatosis, however, its exact pathogenesis is uncertain. Endoplasmic reticulum stress has been identified involved in many diseases, including obesity and type 2 diabetes, the latter two are the risk factors for NAFLD. It is important to detect whether endoplasmic reticulum stress involved in the course of lipogenesis in free fatty acids induced hepatic steatosis and explore the drugs such as metformin's potentially beneficial effects on preventing endoplasmic reticulum stress in NAFLD.MethodsCell culture and treatmentHuman hepatoblastoma cell line, HepG2 cell, was cultured in Minimum Essential Medium containing 10% fetal bovine serum. Cell cultures were maintained in 5% CO2 atmosphere at 37℃. After cultured 72h, HepG2 cells at 70-80% confluency were exposed to defferrent types of culture media,that is conventional culture with no treatment (control group),2mM metformin (MET group), 1mM free fatty acids mixture (2:loleate:palmitate)(FFA group), 1mM fatty acids mixture+2mM metformin (FFA+MET group).After incubation for another 24 hours, cells were harvested to evaluate cell survival rate and lipid level. Thereafter, reverse transcriptase-polymerase chain reaction for glucose-regulated protein-78(GRP78),sterol response element-binding protein-lc (SREBPlc) and fatty acid synthase (FAS) was exerted. Moreover, Western blot for GRP78 and SREBP1 was also applied.RT-PCRTotal RNA was isolated from HepG2 cells using the Trizol reagent. Aliquots of 6.0 ul of total RNA were used for cDNA reverse transcription with a two-step RT-PCR kit and finally recovered as 20ul of cDNA solution. GRP78, SREBPlc, fatty acid synthase (FAS) and P-actin cDNAs were simultaneously amplified. Products were analyzed by 1.5% agarose gel electrophoresis. Band intensities were compared by imaging of ethidium bromide staining and relative expression levels of PCR products compared to those of P-actin quantified using the PDQuestTM(Bio-Rad Laboratories). Western blotSamples protein in each sample was denatured at 100℃for 5 minutes and then loaded on a 10% SDS polyacrylamide gel. After electrophoresis, proteins were electroblotted on Immobilon P membrane. The membrane was blocked by incubation in blocking buffer, incubated with polyclonal antibody against anti-goat-GRP78 or anti-mouse-SREBP1 antibodies, washed and incubated with secondary antibody. GRP78 and SREBP1 protein expressions were detected using an enhanced chemilluminescence detection system. Signals were visualized by autoradiography and determined by quantitative analysis of digital photographs of gels using Labworks software. The integral optical density (IOD) values of GRP78, SREBP1 and GAPDH were measured. The ratio of the integral optical density (IOD) GRP78/GAPDH or SREBP1/GAPDH was used to express the relative level of GRP78 or SREBP1 protein.ResultsThe levels of triglyceride (TG), mRNA of FAS, mRNA and protein of GRP78 and SREBP1c significantly increased in free fatty acids(FFA) induced hepatic steatosis group significantly. Then, HepG2 cells with hepatic steatosis induced by FFA were treated by metformin, levels of TG, GRP78 mRNA, SREBPlc mRNA and FAS mRNA as well as GRP78 and SREBP1 protein levels were partially decreased but without significant differences. It is worth noting, however, compared with those in FFA group, relative levels of GRP78 mRNA and protein in FFA+MET group decreased in some degree with P value approaching to 0.05 (P=0.053,0.054 respectively).Conclusion:Endoplasmic reticulum stress might be involved in lipogenesis in fatty acids induced hepatic steatosis. Therefore, endoplasmic reticulum stress might serve as a novel target in the pathogenesis and therapy of non-alcoholic fatty liver disease.