Adipogenic Changes Of Hepatocytes In The Pathogenesis Of Non-alcoholic Fatty Liver Disease

Objective:To establish high fat diet induced non-alcoholic fatty liver disease (NAFLD) model in C57BL/6J mice and evaluates the pathophysiological changes of this mice model.Methods:Sixty male C57BL/6J mice were randomly divided into two groups. One group was fed on a high fat diet (HFD) containing42%fat, the other was fed on a normal laboratory chow. Continuous feeding for24weeks, each group was randomly selected five mice to sacrifice at the12,16,20,24weeks of the feeding period. The changes of body weight, liver weight and liver pathology were observed. The serum ALT, albumin, triglyceride and fasting plasma glucose were also tested. SABC immuohistochemical technique was used to detect CD68expression in the liver tissue, and RT-PCR technique was used to detect the expression of c-Jun NH2-terminal kinase1(JNK1).Results:After feeding for12weeks, more than70%of the HFD-fed mice had steatosis by HE staining. All the HFD-fed mice had steatosis at the end of20-week, and the grade of steatosis did not aggravate with the feeding time proceed. Oil red O staining showed varying sizes of orange lipid droplets in the hepatocytes from the HFD-fed mice. Body weights and liver weights in HFD-fed mice were much heavier than the control mice after12-week feeding period (P<0.05). Serum ALT, triglyceride and fasting plasma glucose increased gradually in HFD-fed mice while serum albumin decreased, and the significant difference was found after16weeks comparing to the control (P<0.05). Immuohistochemistry showed increased expression of CD68in the liver of HFD-fed mice comparing to the control. The mRNA level of JNK1was increased3-7-fold in HFD-fed mice after24weeks. Conclusion:The NAFLD model in mice was constructed successfully by the HFD for12to24weeks. The model was characterized by overweight, hyperlipidemia, insulin resistance and hepatic steatosis. The HFD feeding can activate the expression of JNKl in liver and induce the hepatic insulin resistance. Objective:To investigate the meanings of the expression of adipocyte-specific genes in the liver tissue of non-alcoholic fatty liver disease (NAFLD) mice model and NAFLD patients.Methods:The markers for adipocytes such as aP2, PPARγ, C/EBPa and adiponectin in the liver of C57BL/6J mice in both groups were detected by real time-PCR and western blot analysis at12,16,20and24weeks of feeding. The expression of epithelial cell marker E-Cadherin was detected by western blot analysis. Paraffin-embedded liver specimens of sixteen NALFD patients and ten normal people were analyzed by SABC immunohistochemical staining of PPARy. Double immunofluorescence was used to locate aP2in the liver tissue of NAFLD patients.Results:Real time-PCR analysis of the total RNA of mice liver tissue of both groups, during24weeks of feeding, hepatic aP2mRNA levels were increased2-3-fold in HFD-fed mice in comparison to the control. Similarly there was a greatly increase in hepatic expression of PPARy from4-fold at12weeks to9-fold at24weeks in HFD-fed mice. Western blot analysis also showed the same changes. However, the mRNA and protein expression of C/EBPa and adiponectin was decreased in HFD-fed mice in comparison to the control, except that C/EBPa mRNA level was increased4-fold at24weeks. The expression of E-cadherin in the liver was gradually decreased as the HFD feeding proceeded. Increased expression of PPARy was found in the hepatocytes of NAFLD patients, mainly in the nucleus by immunohistochemical staining. FITC-labeled adipocyte marker aP2and rhodamine-labeled hepatocyte marker albumin were both dyed in the cytoplasm of hepatocytes in NAFLD patients by confocal laser scanning microscopy. Conclusion:In the liver of HFD-fed mice model and NAFLD patients, there is overexpression of some adipocyte-specific genes in the hepatocytes, indicating the adipogenic changes of the hepatocytes. While their secretory function and epithelial phenotype was impaired in the pathogenesis of NAFLD. Objective:To study the influence of steatotic primary hepatocytes on the activation of kupffer cell line RAW264.7.Methods:When the mice were feeding for20weeks, we used the collagenase perfusion in situ by a low-speed, iso-density percoll centrifugation method to separate primary hepatocytes. Oil Red O staining of hepatocytes showed the grade of steatosis. Immunofluorescence was used to detect the changes of adipocyte markers aP2and epithelial cell marker E-cadherin in the hepatocytes. Co-culture began24hours after mouse primary hepatocytes were harvested in the transwell inserts, and the primary hepatocytes and RAW264.7cells shared the same medium. After co-culture for48hours, the culture supernatant was collected for ELISA analyses for TNF-a, MCP-1, IL-6and IL-18. The proliferation of RAW264.7cells were detected by flow cytometry and western blot.Results:Oil red O staining showed a lot of orange lipid droplets in the pimary hepatocytes from the HFD-fed mice. The co-localization of aP2and albumin in the steatotic pimary hepatocytes was found by double immunofluorescence staining. The expression of albumin and E-cadherin was reduced in HFD-fed mice comparing to the control. We found a significant increase of the content of inflammatory cytokines IL-6, IL-18, MCP-1and TNF-α in the culture supernatant in co-cultured HFD group compared to the other groups (P<0.05). Steatotic hepatocytes cultured alone could also release these inflammatory cytokines. Furthermore, the proliferation of kupffer cells was significantly faster than those of control groups by flow cytometry using CFSE staining of RAW264.7after co-culture for48hours. Extraction the total protein of kupffer cells and western blot analysis showed that CD68expression from co-cultured HFD group was significantly higher than that of control groups (P<0.05).Conclusion:Steatotic hepatocytes undergo adipogenic changes in the HFD induced mice model. These kinds of steatotic hepatocytes can not only express and secrete inflammatory cytokines and chemokines, but also promote macrophage proliferation, while their secretory function and epithelial phenotype are reduced. Therefore, we speculate that these changes may be involved in the pathogenesis of NAFLD.