The Role And Mechanism Of NAD+ Biosynthesis Rate-limiting Enzymes NRK1 And NAMPT In Fatty Liver Disease

BackgroundLiver is the main places of fat metabolism,fatty liver disease is a serious threat to people’s health,and it becomes the second to the viral hepatitis liver disease,the incidence of the disease is improving,also the increasingly younger age.Nicotinamide adenine dinucleotide(NAD+)serves as a coenzyme in cellular redox reactions and is also a substrate for multiple enzymes,including sirtuins and poly(ADP-ribose)polymerases(PARPs).Dietary supplementation of NAD+ precursors nicotinamide mononucleotide(NMN)or nicotinamide riboside(NR)protects against metabolic disease,neurodegenerative disorders and age-related physiological decline in mammals.Recent evidence has shown that NRK1 is not only rate-limiting for NR metabolism in mammalian cells,but also necessary for the conversion of extracellular NMN to NR for cellular uptake and NAD+ synthesis.Liver,the organ mediating many of the metabolic effects of NR,displays high NRK1 levels.However,whether NRK1 indeed mediates the increase in hepatic NAD+ pool in vivo is still largely unknown.Nicotinamide phosphoribosyltransferase(NAMPT)is the rate-limiting enzyme in the NAD+ salvage pathway converting NAM to the intermediate nicotinamide mononucleoti-de(NMN),which is further converted to NAD+ by NMN adenylyltransferases(NMNATs).NAMPT influences the activity of NAD+-dependent enzymes,such as sirtuins,and thereby regulates cellular metabolism.Disturbances in NAMPT and NAMPT-mediated NAD+ biosynthesis have been reported during the development of non-alcoholic fatty liver disease(NAFLD)and NAMPT is implicated in the regulation of hepatic lipid metabolism.However,whether NAMPT modulates the processes involved in the pathogenesis of alcoholic liver disease(ALD)still remains unknown.ObjectivesEstablish the mouse model of nonalcoholic fatty liver disease(NAFLD)or alcoholic fatty liver disease(AFLD),in vivo and in vitro,to study the roles of NRK1 and NAMPT plays in development of regulating hepatic NAD+ biosynthesis and lipid metabolism,respectively.Methods1.The roles and mechanisms of NRK1 plays in development of nonalcoholic fatty liver disease(NAFLD).(1)Cells were cultured to test the infection efficiency of NRK1 and the expression levels of NAD +.(2)Adenoviruses were injected through the tail vein into mice to detect NRK1 infection efficiency and the effections of changes on phenotypic in mice.(3)The animal models have been fed with high fit diet(HFD),qRT-PCR were used to examined the expression levels of NRK1 gene.(4)Adenoviruses were injected through the tail vein into mice which have been fed with HFD,to assess glucose tolerance and insulin sensitivity and phenotypic changes,respectively.(5)Adenoviruses were injected through the tail vein into aged mice to check the effect of hepatic NRK1 overexpression in age-induced metabolic disorders.2.The roles and mechanisms of NAMPT plays in development of alcoholic fatty liver disease(AFLD).(1)Cells were cultured to test the infection efficiency of Nampt and whether ethanol treatment has an effect on Nampt expression in hepatocytes.(2)The mouse model of chronic plus binge ethanol feeding test whether ethanol treatment has an effect on Nampt expression in mice liver.Adenoviruses were injected through the tail vein in a mouse model of chronic plus binge ethanol feeding to exam the effect of hepatic Nampt overexpression in alcohol-induced metabolic disorders in mice.(3)Whether SIRT1 has the effects of NAMPT on ethanol-induced hepatic steatosis.Results1.The roles and mechanisms of NRK1 plays in development of nonalcoholic fatty liver disease(NAFLD).(1)Exposure to Ad-NRK1 resulted in ~10-fold increase over endogenous levels of Nmrk1 mRNA in primary hepatocytes.(2)Adenovirus-mediated overexpression of NRK1 is sufficient to boost NR-driven NAD+ synthesis in all the cells tested(p<0.05).(3)Adenovirus-mediated overexpression/knockdown NRK1 expression would increase/decrease hepatic NAD+ levels and decrease/increase triglyceride content(p<0.05),but serum triglyceride levels were unaffected by hepatic Nmrk1 overexpression or knockdown.(4)Nmrk1 mRNA levels were significantly decreased in the liver samples of HFD treatment mice(p<0.05).However overexpression of NRK1 in liver will ameliorate HFDinduced glucose intolerance and insulin resistance,also fatty liver including reduced expression of lipogenic genes while increased expression of genes involved in fatty acid boxidation by upregulating NAD+ biosynthesis.Hepatic and serum triglyceride levels were decreased in Ad-NRK1 treated mice by 30% and 20% relative to the Ad-GFP treated mice,respectively(p<0.05).(5)We have observed a significant reduction of Nmrk1 mRNA levels in liver samples of aged mice(p<0.05).Since hepatic NRK1 overexpression also be effective in ageinduced metabolic disorders,firstly,glucose tolerance and insulin sensitivity were improved in mice receiving Ad-NRK1,secondly,hepatic overexpression of NRK1 upregulated liver NAD+ levels while reduced hepatic triglycride levels in aged mice(p<0.05).2.The roles and mechanisms of NAMPT plays in development of alcoholic fatty liver disease(AFLD).(1)Ethanol exposure significantly increased intracellular triglyceride(TG)levels whereas reduced NAD+ levels,also mRNA and protein levels of Nampt were both markedly reduced after ethanol treatment(p<0.05).Overexpression of NAMPT led to an increase in intracellular NAD+ concentrations and resulted in a significant reduction of intracellular TG concentrations in hepatocytes of ethanol treated group(p<0.05).(2)A mouse model of chronic plus binge ethanol feeding finds that ethanol can reduce Nampt expression and NAD+ concentrations in vivo,ethanol feeding induced significantly higher levels of serum alanine aminotransferase(ALT)and glutamic oxaloacetic transaminase(AST)(p<0.05).Hepatic and serum levels of TG were much higher in ethanol-fed group than pair-fed group,whereas liver and serum levels of cholesterol(TC)were comparable between these two groups.NAMPT overexpression alleviates ethanolinduced liver steatosis for hepatic TG concentrations and serum ALT,AST level(p<0.05).(3)Chronic ethanol feeding reduces the expression of SIRT1 and SIRT6 in liver(p<0.05).Increased NAD+ production and SIRT1 activity caused by NAMPT overexpression contributed to alleviation of ethanol-induced hepatic steatosis.overexpression of hepatic NAMPT led to reduced TG concentrations and serum ALT,AST levels,but this effect was abolished when SIRT1 was knocked down(p<0.05).Conclusions1.Overexpression of NRK1 ameliorates diet-and age-induced hepatic steatosis and insulin resistance.2.NAMPT overexpression alleviates alcohol-induced hepatic steatosis in mice.