The Dynamic Expression Significance Of PACS-2in Non-alcoholic Fatty Liver Disease

BackgroundNonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation andfatty degeneration of the hepatocytes, which is not caused by factors such as viral infectiou,drugs, and alcohol abuse. NAFLD is tightly associated with genetic susceptibility andinsulin resistance and consists of a broad spectrum of diseases, including simple steatosis,nonalcoholic steatohepatitis, and cirrhosis[1]. Althought the pathogenesis of NAFLDremains elusive; the “two-hit” hypothesis is a main theory to account for the progression ofNAFLD. The first hit is initiated by abundant accumulation of lipids such astriglyceriodsaccumulate in the setting of insulin resistance.The second hit includesoxidative stress and endoplasmic reticulum stress (ERS)[2]. Indeed, our previous studieshave demonstrated that ERS plays an important role in the pathogenesis of NAFLD,however, the molecular mechanisms underlying ERS is undefined. Morevoer, the molecularbasis causing abberrent hepatic apoptosis is also not clear in NAFLD.Phosphofurin acidic cluster sorting proteins (PACS) are multifunctional protein family,which include PACS-1and PACS-2.[3]By localization between Golgi apparatus andendoplasmic reticulum for PACS-1and mitochondria and endoplasmic reticulum forPACS-2[4], they are involved in regulating the structure-function association of Golgiapparatus, mitochondria and endoplasmic reticulum. Specifically[5], PACS-2is expressed inthe mitochondria-associated membrane (MAM) and regulates apoptpsis, and ion transport, and lipid metabolism[1]. Up to now, the role of PACS-2in the pathogenesis of NAFLD isunkown, therefore we examined the expression of PACS-2in cellular and animal modelsand disscued its possible function.Methods1. Steatosis of HepG2cells. HepG2cells were cultured with a-MEM mediumcontaining10%fetal bovine serum (FBS). The cells were treated with serum-free mediumovernight and incubated with a-MEM containing1%fatty acid free bonvine serum albumin(FAF-BSA), with or without500um sodium palmitate to induce steatosis for4h,8h,12h,and24h[2]. Thereafter, the cells were stained with Oli red to vadiualized accumulation offatty acid.2. NAFLD model using rats. Male SD rates at4weeks old (weights180g-210g) wererandomely subfractioned, fed with normal food or high fat diet for4,8,12,16, and20weeks. The rates were humanly anesthesized. The livers were harvested for HE staining andWestern blot analysis.3. Real-time PCR assay. Expressions of PACS-2, endoplasmic reticulum stressmarker-glucose regulating protein78(GRP78), Bax, and Caspase-3mRNAs wereexamined by Real-time PCR analysis.4. Western-blot analysis. The protein levels of PACS-2, GRP78, Bax, and Caspase-3were examined by Western-blot analysis.5. Statistic analysis. The data were analyzed by Student’s t-test or ANOVA analysisusing SPSS software13.0. The results were statistically significant when P <0.05.Results1. Sodium palmitate indued steatosis of HepG2cellsOli red staining revealed significant fatty accumulation in HepG2cells that weretreated with sodium palmitate. Moreover, prolonged incubation with sodium palmitate ledto increased amount of fat droplet in HepG2cells. These results demonstated that treatmentwith sodium palmitate induced fatty degeneration in HepG2cells. 2. High fat diet induced NAFLD in SD ratesHE staining and Oli red staining showed progressive steatosis of the livers from the ratsfed with high fat diet. Furthermore, intralobular inflammation and fibrosis were evident inthose rates that were subjected to prolonged feding duration with high fat diet. These datasuggested that high fat diet successfully modeled steatosis, nonaleoholie steatohepatitis, andeven hepatienbrosis in rates.3. Real-time PCR analysis showed that the relative level of PACS-2mRNA weredownregulated in the early stages (4h,8h) but up-regulated in the late stages (12h,24h) oftreatment with sodium palmitate. Moreover, the mRNA levels of GRP78, Bax, andCaspase-3were significantly elevated in HepG2cells that were treated with sodiumpalmitate for12or24h.4. Western blot analysis revealed that PACS-2expression were downregulated in theearly stage but up-regulated in the late stage of fatty degeneration in HepG2cells.Moreover, the levels of GRP78, Bax, and Caspase-3were all elevated in late stage oftreatment with sodium palmitate.In the rats fed with high fat diet, PACS-2expression were also downregulated in theearly stages but were upregulated in the late stage. In addition, the level of GRP78waselevated in early stage of feding with high fat diet and remains upregulated in these rates.However, the levels of Bax and Caspase-3were all increased in the late stage of feed withhigh fat diet.Conclusions1. Dynamic change in PACS-2expression was revealed in both cellular and SD ratsmodels of NAFLD, where its expression was found to be downregulated in the early stagebut upregulated in the late stage of fatty degeneration. Moreover, the level of GRP78wasabberantly expressed in the early stage of feding with high fat diet and remainedupregulated after that. However, the levels of both Bax and Caspase-3were shown to beelevated only in the late stage.2. PACS-2was downregulated in the early stage of NAFLD, which might beassociated with endoplasmic reticulum stress. However, the upregulation of PACS-2in the late stage might be attributable to apopotosis in the late stage of NAFLD.