The Role Of NAMPT In The Mechanism Of Non-alcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease(NAFLD) describes a range of conditions caused by a build-up of fat within liver cells. The excessive fat deposition in liver is the primary caused of NAFLD. The main characteristics of this disease include simple steatosis, fibrosis, cirrhosis and hepatocellular carcinoma. According to recent data, the incidence of NAFLD is 10~20%. Progression to nonalcoholic steatohepatitis(NASH) was seen in 44% of patients with baseline NAFLD. Moreover, the incidence of cirrhosis will be as high as 25% in the following 10 years. At present, NAFLD is considered to the primary cause of end-stage liver disease and liver transplantation over the next decade, replacing viral hepatistis. The most accepted concept of the pathogenesis of NAFLD is multiple ?hits‘. These hits are characterized by the occurrence of parallel events that involve a complex interaction and crosstalk between environmental factors and host genetics. The interaction might promote isolated steatosis, innate immune activation, inflammation, cell death and progressive liver damage.NAMPT(Nicotinamide phosphoribosyltransferase) is the rate-limiting enzymes in NAD(nicotinamide adenine dinucleotide) metabolic pathways in mammalian. Recent studies have pointed out that, NAD is involved in a wide range of important biological functions including apoptosis/survival,, insulin resistance, obesity, inflammation and lipid homeostasis. Since inflammation, insulin resistance, obesity and dyslipidemia contribute to the development of NAFLD, and NAMPT is a crucial regulator of these factors, we perform this project to investigate whether NAMPT affects the progression of NAFLD and try to indentify new molecular mechanism of NAFLD. In this study, we used dominant-negative mutant NAMPT transgenic mice(DN-NAMPT).Research contentsProtein expression of NAMPT in liver and other tissues was studied by western blotting. Then we checkd the expression of the NAMPT in liver and serum after feeding high-fat diet for 12 weeks. The serum concentration of triglyceride(TG), total cholesterol(TC), alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected by automatic biochemical analyser. The pathological changes in liver was detected by haematoxylin and eosin(HE staining), oil red O, Td T-mediated d UTP Nick-End Labeling(TUNEL) staining. Liver triglyceride(TG), total cholesterol(TC), malondialdehyde(MDA) and super dismutase(SOD) content were assayed. The expression of triglycerides and total cholesterol related genes were measured by real-time quantitative fluorescent PCR(Real-time PCR). Macrophage infiltration, TNF-α expression and IL-6 were examined by Immunohistochemistry. Mice were fed high fat diet(HFD) for 12 weeks. The fibrosis in mice liver was detected by Masson‘s staining and a-SMA expression. Moreover, the changes in NLRP3 inflammasome pathway were also determined by immunoblotting.Experimental results1. Western blotting showed that the expression of NAMPT in liver is relatively high. The expression of NAMPT and level of NAD in the liver in HFD-fed mice were lower than feeding normal diet.2. The expression of NAMPT and level of NAD in the liver of DN-NAMPT mice were lower than those in WT mice.3. Compared with WT mice, DN-NAMPT mice displayed higher liver index, higher serum AST and ALT levels. These results suggested that DN-NAMPT mice have a mild liver damage. Meanwhile, the serum lipids of DN-NAMPT mice are slightly lower than those in WT mice, while the lipids in the liver of DN-NAMPT were significantly higher. Oil red O staining showed lipid droplets in liver cells of DN-NAMPT mice. By real-time PCR, we detected the genes expression, related to triglycerides and total cholesterol. we found that triglycerides and total cholesterol associated genes MTTP, ABCG1, ABCG5, ABCG8 and LDL-R were downregulated. Immunohistochemistry of F4/80 and TNF-α showed that DN-NAMPT mice had apparently liver macrophage infiltration and inflammation. There was no difference of IL-6 between two types of mice. We did not observed fibrosis.4. In NAFLD models, we found that WT and DN-NAMPT mice both have lipids droplets. MDA levels of DN-NAMPT mice in the liver were increased while SOD levels were decreased. Immunohistochemical results showed that DN-NAMPT mice liver have more seriously inflammation. We also found that disruption of NAMPT promoted NLRP3 inflammasome activation by western blotting.Immunohistochemical and western blotting demonstrated that DN-NAMPT mice also had more seriously fibrosis. Real-time PCR analyses of TGF-β1, TIMP1, Pro-Col1α1 confirmed this phenotype.Conclusion1. Under normal diet condition, disruption of NAMPT promoted lipid accumulation in the liver and led to mild steatosis.2. Under high fat diet condition, disruption of NAMPT enhanced the inflammation, NLRP3 inflammasome activation, oxidative stress, apoptosis and fibrosis in liver.