ABCA6 KO Aggravates Experimental NAFLD In Mice

Objective: Non-alcoholic fatty liver disease(NAFLD)is a kind of clinical pathological syndrome,which is mainly characterized by fatty degeneration of liver cells and lipid accumulation,which excludes the alcohol-induced fat accumulation,and it has frequently been associated with obesity,type 2 diabetes mellitus,hyperlipidemia,and insulin resistance.NAFLD includes from simple fats(hepaticsteatosis)to fatty hepatitis(Nonalcoholic Steatohepatitis)and liver fibrosis(Hepaticfibrosis)and liver cirrhosis(Cirrhosis)and liver cancer(Hepatocarcinoma)and a series of extensive pathological change process.It has become a common chronic liver disease that endangers human health.Its true etiology underlying NAFLD progression remains unknown,but there appears to be a strong relation with accumulation of triglycerides and fatty acid metabolism caused by overnutrition.The ABC binding cassette transporter can obtain energy from ATP hydrolysis and trans-membrane transport of lipids and many other substances.ABCA6 belongs to ABCA subfamily.Our previous studies have shown that ABCA6 may be involved in the transport of intermediate fatty acid oxidation products.At present,the role of ABCA6 in nonalcoholic fatty liver disease has not been reported.In this study,we observed the effect of ABCA6 deficiency on nonalcoholic fatty liver disease in mice,and discussed its mechanism.Methods: The model of chronic nonalcoholic fatty liver disease in mice was constructed with the mice feed on 60% high-fat diets and 30% fruit syrup for 6 months.We use western blot to examinate the ABCA6 protein levels afer the model.Observing the weight changes of mice,we made the exanmination of the gluxose,insulin and pyruvate metabolic tolerance test.Adipose tissue around the kidney and gonad of two groups of mice was collected and the mice were weighed.The levels of serum total cholesterol(T-CHO),triglyceride(TG),blood sugar,ketone body,MDA,alanine transaminase(ALT)and straw transaminase(AST)were detected.The liver tissue of mice was collected for HE staining and observing the morphological changes of the liver.The levels of T-cho and TG in the liver were detected.The expression levels of inflammatory factors,fibrosis factor,fatty acid oxidation,fatty acid production and glycoprotein-related genes in liver were detected by real-time PCR.The expression levels of phosphorylation of P38,Erk and JNK in MAPK signaling pathway and phosphorylation of Ampkα and Akt associated with insulin resistance was detected by Western Blot(WB).In vitro experiments.The primary hepatocytes(PMHs)of the WT mice and ABCA6 KO mice were extracted respectively and then were given PA(palmitic acid)stimulation.We use Real-time PCR to evaluate the expression of inflammatory factors and fibrosis factors in the PMHs afer given PA(palmitic acid)stimulation.Cell viability was detected by MTT assay.Changes of lipid uptake of primary hepatocytes after PA stimulation was observed by oil red O staining and we determined the levels of its TG and T-CHO.For determining effect of ABCA6 deficiency on the activation of hepatic stellate cells,we collect the conditioned medium that primary hepatocytes of WT mice and ABCA6 KO mice which were given the PA stimulation for 72 hours,and then we use it to stimulate human hepatic stellate cells LX-2 cell lines and rat hepatic stellate cells HSC-T6,and determine the changes of inflammation and fibrosis factor by real-time PCR after extracting their RNA.Results: After feeding with high-fat diets and 30% fruit syrup for 6 months,the ABCA6 protein levels of WT mice decreased significantly.The weight of kidney fat/weight and gonad fat/weight of ABCA6 KO mice were significantly higher than that in WT mice.HE staining showed that the total content of the cholesterol and triglyceride of the liver in ABCA6 KO mice increased.The blood sugar,total cholesterol,MDA,ketone body and ALT of in the serum of the ABCA6 KO mice were higher than WT mice.Quantitative RT-PCR showed that the increased expression of IL-1b,COL1A1 and COL1A2 in ABCA6 KO mice,which means more severe inflammation and fibrosisin in ABCA6 KO mice.And the ABCA6 KO mice displayed a decreased expression of antioxidant stress gene SOD and a increased expression of pro-oxidant stress gene NOX2.Western Blot(WB)showed that the expression of α-SMA in ABCA6 KO mice was increased and AMPKα signaling pathway activated.And the levels of MAPK signaling pathway of Erk and JNK phosphorylation were suppressed in ABCA6 KO mice.The survival rate of ABCA6 KO hepatocyte was significantly lower than that of WT after PA treatment.Also,Oil red O staining showed that the lipid deposition of ABCA6 KO liver cells was more obvious after PA treatment.Moreover,RT-PCR was displayed that MCP-1 mRNA level in ABCA6 KO hepatocyte was significantly higher than that of WT after PA treatment.Furthermore,conditioned medium of ABCA6 KO liver cells after PA treatment significantly increased the COL1A2 Mrna expression of LX-2 and HSC-T6 cell lines,which illustrated that ABCA6 depletion liver cells under the stimulus of PA enhanced effect on the activity of hepatic stellate cells.Conclusion: The deficiency of ABCA6 exacerbated NAFLD in mice induced by high-glucose-high-fat diet.ABCA6 KO mice showed elevated liver lipid storage and oxidative stress,lower sensitivity to insulin and increased inflammation and fibrosis in the liver.Moreover,ABCA6 depletion mice also displayed increased lipid toxicity of palmitate to hepatocytes and enhanced activation of hepatocytes to hepatic stellate cells.These changes were associated with hepatic fatty acid oxidation dysfunction due to ABCA6 depletion and increased food intake by AMPKα activation.