The Role Of AMPK In Inflammation Induced Liver Lipid Deposition

Objective:Clinical studies have found that inflammation may be involved in fat deposition process of early nonalcoholic fatty liver disease (NAFLD), but its mechanism has not yet been elucidated. Therefore, in this part of the study, we aim to establish inflammation induced liver lipid deposition of animal and cell models, preparing for further mechanism research.Main methods: ① Male C57BL/6J mice were randomly divided into inflammation group (Casein, n=7) and control group (NC, n=7). Inflammation group mice were subcutaneous injected of 0.5 ml 10% casein, control group mice were injected corresponding dose of saline for total 18 weeks. Serum were collected and inflammatory factor including MCP-1、 TNF-α and IL-6 were detected. Liver lipid deposition and the expression of key gene expression about lipid metabolism were detected by oil red O staining, quantitative test of TG and qRT-PCR. ② Kupffer cells were intervened with LPS100ng/mL for 24h and then the supernatant were collected. The content of inflammation factors were examined. HepG2 cells were treated with supernatant, simulating of growing environment about hepatocytes and Kupffer cells in the body. Cells lipid deposition and the expression of key gene expression about lipid metabolism were detected by oil red O staining, quantitative test of TG and qRT-PCR. ③ Exogenous TNF-a 20 ng/mL intervention HepG2 cells for 24 h. Lipid accumulation was detected by oil red O staining and quantitative test of TG. The expression of lipogenesis genes were detected by qRT-PCR and Western blot.Results:①Animal models:Compared with control group, the levels of inflammatory factors were increased significantly in C57BL/6J with casein injection. The lipid deposition and lipid metabolic disorder were more obviously, ②Cell model 1:Including HepG2 cells treated with supernatant from Kupffer cells and HepG2 cells intervened with TNF-a. We observed that the content of inflammatory cytokines was significantly increased in LPS stimulated Kupffer cells. Lipid accumulation was significantly increased and lipid metabolic was abnormal of HepG2 cells treated with the supernatant. ③Cell model 2:A significant increment of TG content in HepG2 cells was observed after TNF-a treatment. The mRNA and proteins levels of lipogenesis genes were increased.Conclusion:Establishment inflammation-induced liver lipid deposition of animal and cell models successfully.Objective:AMP-activated protein kinase (AMPK) is known as a critical regulator of energy homeostasis in metabolic processes. In the first part of the study, we have observed that inflammation induced liver lipid accumulation. However, it is still unknown whether AMPK is involved in the inflammation-induced lipid deposition process in the liver. Therefore, we first tested examined the AMPK and the downstream ACC phosphorylation levels in vivo and vitro, and then we investigated the role of TNF-α on lipid deposition of HepG2 cells and examined the modification of AMPK pathway.Main methods: ①The levels of AMPK and ACC phosphorylation were detected in inflammation-induced lipid deposition in vivo and vitro by Western blotting. ②To explore the mechanism:An AMPK agonist (metformin lmM or AICAR 1mM) or antagonist (Compound C 40μM) was co-administrated with TNF-α in HepG2 cells for 24 h to investigate its effect on TNF-α induced lipid deposition. Lipid accumulation was analyzed by Oil Red O staining and quantitative test of TG AMPK and its pathway (including mTOR and SREBP1) were determined by Western blotting and qRT-PCR.Results:① Compared with control group, the levels of AMPK and downstream ACC phosphorylation were decreased in inflammation induced lipid accumulation in liver tissue; The expression of AMPK and ACC phosphorylation were decreased significantly in liver cells, which treated with supernatant from LPS stimulated Kupffer cells; AMPK and ACC phosphorylation were suppressed obviously in TNF-α induced lipid deposition in HepG2 cells. ② Co-treatment with metformin or AICAR decreased the TNF-α-induced intracellular TQ accompanied by significantly enhanced AMPK and ACC phosphorylation, suppressed mTOR and p70S6K phosphorylation, and reduced SREBP expressions. On the contrary, while co-incubated with Compound C, AMPK and ACC phosphorylation were suppressed and the inhibitory effect of metformin on HepG2 cell lipid deposition was also attenuated.Conclusion:Our results suggest AMPK pathway is involved inflammation induced liver lipid deposition and AMPK/mTOR/SREBP1 pathway is inhibited in TNF-a induced lipid deposition in HepG2 cells.