The Protection And Mechanisms Of NAMPT In High Fat Diet-induced Hepatic Steatosisin Mice

Background and Aims: Nonalcoholic fatty liver disease(NAFLD)refers to the clinical pathological syndrome,accompanied by excessive accumulation of fat in liver cells which caused by non-alcohol factors and it is an acquired metabolic stress induced liver injury which is closely related with insulin resistance and genetic susceptibility.In recent years,with the changes of people's diet and lifestyle,the incidence of NAFLD is getting higher and higher,and the risk of developing cirrhosis and hepatocellular carcinoma is also increased.Nicotinamide phosphate ribosyltransferase(NAMPT)is a key enzyme for nicotinamide adenine dinucleotide(NAD+)biosynthesis and plays an important role in cellular metabolism of various organs of mammals.It has been reported that the decrease of NAD+ is closely related to many metabolic diseases.However,the specific roles and mechanisms of NAMPT in the NAFLD are still unclear.The purpose of this study was to investigate the effects of NAMPT on NAFLD induced by high fat diet,and to provide an important target and experimental basis for the prevention and treatment of NAFLD.Methods: 1.Preparation and analysis of hepaticsteatosis induced by high fat diet: six to eight weeks old male wild-type mice were randomly assigned to four groups:(1)Normal diet(ND)group;(2)ND+FK866(2 mg/kg/day for one week,IP);(3)High fat diet(HFD)group;(4)HFD+FK866(2 mg/kg/day for one week,IP).The mice were first fed either ND or HFD(60% fat)with or without FK866(2 mg/kg/day,IP)for one week.Then,the mice were continually fed with ND or HFD for 12 weeks.Liver paraffin-embedded tissue sections were stained with haematoxylin and eosin(H&E)to examine lipid accumulation.Total RNA and protein was extracted from livers to detect the expressions of NAMPT,SREBP1 and FASN in ND and HFD groups.In addition,the expressions of lipogenic genes such as SREBP1,FASN and ACC was detected in livers from HFD and HFD+FK866 groups.2.Preparation and analysis of hepaticsteatosis induced by oleic acid in hepatocyte: 1)The Hep G2 cells were treated with NAMPT inhibitor FK866(20 n M)or combined with NAD or NMN under the condition with or without oleic acid(0.5 m M)for 24 h.Oil red staining was used to examine the lipid accumulation.The intracellular TG content was detected by Triglyceride assay kit.The expression of lipogenic genes was detected by RT-QPCR and Western blot.2)Hep G2 and murine liver cell lines Hep1-6 cells were transfected with NAMPT expression plasmid for 24 h and then treated with oleic acid(0.5 m M)for 24 h.Another method was that the cells were treated with oleic acid(0.5 m M)for 24 h and then transfected with NAMPT expression plasmid for 24 h.Then the lipid accumulation and the intracellular TG content were detected and the m RNA and protein expressions of lipogenic genes were examined by Q-PCR and Western blot analysis,respectively.3.Mechanism analysis: 1)Sirt1 and phosphorylation of AMPK? were examined in livers from HFD and HFD+FK866 mice.2)The phosphorylation of Akt was detected in Hep G2 cells treated with FK866 and insulin stimulation.3)The expressions of lipogenic genes such as FASN and ACC were detected in Hep G2 cells treated with Sirt1 activator Res or specific inhibitor EX527.Results: 1.The expression of NAMPT was downregulated in livers from mice under HFD compared with ND,while the expressions of SREBP1 and FASN were upregulated.The H&E staining showed that there was a significant increase of hepaticlipid deposition in the mice fed with HFD compared with control mice.2.FK866,the inhibitor of NAMPT,significantly promoted lipid accumulation and the expressions of the lipogenic genes such as SREBP1,FASN,ACC and SCD1 in liver of the mice fed with HFD.3.Oil Red O staining revealed that there was a significant increase of the lipid accumulation after treated with FK866 with or without oleic acid.The intracellular TG content was increased in cells treated with FK866.FK866 also markedly promoted the expressions of the lipogenic genes including SREBP1,FASN,ACC and SCD1.NAD and NMN significantly rescued the effects of FK866.4.Overexpression of NAMPT in HepG2 and Hep1-6 cell lines significantly reduced lipid accumulation and the intracellular TG content under oleic acid treatment and attenuated the expressions of SREBP1,FASN and ACC.5.FK866 significantly reduced the Sirt1,phosphorylation of AMPK? and insulin-stimulated Akt phosphorylation protein levels in liver of the mice fed with HFD.The combination of FK866 and Resveratrol significantly reduced lipogenic gene ACC expressions compared with normal group.The expression levels of FASN and ACC were decreased in cells treated with oleic acid and resveratrol compared with cells treated with OA alone,while EX527 displayed the opposite influences.In addition,NMN could reverse the effects of FK866 that promoted OA-mediated lipid synthesis.Conclusion: 1.The expressions of NAMPT were downregulated in liver from mice under HFD and in hepatocytes treated with oleic acid.2.Hepatic lipid accumulations were increased by inhibition of NAMPT in vitro and in vivo.NAD and NMN could rescue the effects of FK866,an inhibitor of NAMPT and overexpression of NAMPT improved oleic acid induced hepatic lipid synthesis.3.NAMPT inhibited liver lipid synthesis through activating the Sirt1/SREBP1/AMPK? signaling pathway.