Sterol Regulatory Element Binding Protein 1c Regulates Oleic Acid-induced Hepatoma Cell Autophagy

Background & aims :It is reported that sterol regulatory element binding protein 1c(SREBP-1c) and autophagy are involved in the development of Non-alcoholic fatty liver disease(NAFLD). But the mechanism is unclear. The purpose of this study was to observe the effect of SREBP-1c on autophagy and apoptosis in oleic acid(OA)/palmitic acid(PA) overloaded H4 IIE cells, and to discern the development of NAFLD by SREBP-1c, probably not only through promoting lipid synthesis, but also its likely involvement in the inhibition of autophagy and lipoptosos. Further, we want to explore whether adenosine monophosphate-activated protein kinase( AMPK) is involved in the development of precancerosis induced by diethylnitrosamine and a high- fat diet by SREBP-1c pathway. This study may provide reference for the prevention and treatment of NAFLD.Methods:In vitro: Cell steatosis was measured by N ile red staining and triglyceride measurement in OA/PA overloaded hepatoma H4 IIE cells. The change of autophagy, apoptosis and SREBP-1c were evaluated by electron microscopy, western blot, real-time PCR and flow cytometry. After SREBP-1c altering by insulin, rapamycin, SREBP-1c bearing plasmids and SREBP-1c-siRNA separately, cellular lipid content and autophagy were measured in OA overloaded H4 IIE cells. After p53 inhibition by Pftα, p53-Bax signaling and cellular apoptosis were measured with or without SREBP-1c interference in OA overloaded H4 IIE cells.In vivo: Pathology change of liver tissue 、SREBP-1c and autophagy flux were assessed using HE staining, western blot and real-time PCR in NAFLD rats induced by a high fat diet.The low dose of diethylnitrosamine(DEN) and high fat diet induced liver precancerosis of rat model in vivo was established to observe the pathological changes of rat livers by HE staining. The expression levels of glutathione S-transferase-π(GST-π)、sterol regulatory element binding protein-1c(SREBP-1c)、fatty acid synthase(FAS) 、 acetyl-CoA carboxylase(ACC) 、 stearoyl-co A desaturase 1(SCD1)、AMPK、p-AMPK were detected by Western blotting, Real-time PCR and immunohistochemistry.Results: 1. OA can elevate autophagy level and autophagy function and decrease SREBP-1c in H4 IIE cells. 2. Insulin can elevate SREBP-1c in mRNA and protein levels in OA overloaded H4 IIE cells, increase cellular lipid and triglyceride content, decrease autophagy level and function; while rapamycin can inhibit SREBP- 1c in mRN A and protein levels in in OA overloaded H4 IIE cells, decrease cellular lipid and triglyceride content, elevate autophagy level and function. 3. Autophagy level and function were decreased after SREBP-1c overexpression in OA overloaded H4 IIE cells, and its triglyceride content was increased. In contrast, autophagy level and function were both increased after SREBP-1c-siRNA transfection in O A induced H4 IIE cells, and its triglyceride content was decreased. 4. High concentration of OA can induce lipoptosis and autophagy-related apoptosis, increase p53、Bax and caspase 3 protein levels in H4 IIE cells. Lipoptosis induced by OA was inhibited by Pftα, indicating that lipoptosis induced by OA was p53 dependent. 5. Cellular apoptosis and caspase 3 protein level were decreased after SREBP-1c overexpression in O A overloaded H4 IIE cells. Meanwhile, cellular apoptosis induced by chloroquine were also decreased; results from SREBP-1c-siRNA transfection verify the conclusion above further, indicating that SREBP-1c can inhibit lipoptosis and autophagy-related apoptosis in O A overloaded H4 IIE cells. 6. The protein levels of p53 and Bax were increased after SREBP-1c-siRN A transfection; lipoptosis and Bax protein level induced by OA were inhibited by Pftα; cellular apoptosis was reversed again after SREBP-1c-siRNA transfection, indicating that the inhibition of SREBP-1c to lipoptosis induced by O A was p53-Bax signaling dependent. 7. SREBP-1c was increased and autophagy-related protein such as LC3 and P62 were also increased in NAFLD rats liver,indicating that autophagy function was decreased in NAFLD rat liver. Furthermore, elevated SREBP-1c expression was positively correlated with the levels of LC3 and P62, indicating that SREBP-1c may inhibit autophagy function in NAFLD rats. 8. Lipid droplets overload evidently in hepatocytes of rats in DEN+HFD group compared with DEN group, some of which behaving ballooning change,necrosis and inflammatory; meanwhile, GST-π was elevated significantly in the livers of rats treated with DEN+HFD group when compared with that of DEN group. Meanwhile, the expression of SREBP-1c and its target genes FAS, ACC, SCD1 were all elevated statistically. Furthermore, p-AMPK level was decreased, indicating that SREBP-1c may involved in the development of NAFLD-related liver precancerosis of rat liver. 9. SREBP-1c, FAS, ACC, SCD1 and TG were all increased in PA treated cells, while treating cells with AIC AR, an activator of AMPK, can reverse the effect of PA. Indicating that AMPK may decrease cellular lipid induced by PA through its inhibition of SREBP-1c. Conclusions: 1. Lipophagy was induced by OA in H4 IIE cells; SREBP-1c can inhibit lipophagy induced by OA. 2. Lipoptosis was induced by OA in H4 IIE cells; SREBP-1c can inhibit lipoptosis and autophagy-related apoptosis induced by OA; furthermore, the inhibition of SREBP-1c to lipoptosis induced by O A was p53-Bax signaling dependent. 3. Autophagy function was decreased in N AFLD rat liver; SREBP-1c might inhibit autophagy function in N AFLD rats. 4. AMPK is involved in liver precancerosis induced by high- fat diet and diethylnitrosamine, and this process may through the inhibition of SREBP-1c.