Therapeutic Effects Of Nicotinamide Ribose On Non-alcoholic Fatty Liver Disease And The Related Mechanism

Non-alcoholic fatty liver disease(NAFLD)is a kind of clinicopathological syndrome without history of excessive drinking and clear hepatic impairment factors,which is mainly charactered by vast lipoid deposition in parenchymal hepatic cells and hepatic steatosis.NAFLD is an umbrella term for simple hepatic steatesis(SS)with moderate fatty infiltration to non-alcoholic steatohepatitis(NASH),characterized by inflammation and/or fibrosis.A small portion of NAFLD may progress to advanced fibrosis,cirrhosis and eventually hepatocellular carcinoma.With the prevalence of globalization of obesity and related metabolic syndrome,NAFLD has become the most common cause of chronic liver disease in the western developed countries,the prevalence of NAFLD among adults is 10% to 30%.Aging is an important risk factor of NAFLD in line with that the prevalence of NAFLD in humans increases with age.Besides,NAFLD has become the most common cause of liver enzyme abnormalities and chronic liver diseases in addition to hepatitis virus infection in our country.However,there is still no effective drug for the treatment of NAFLD.Nicotinamide phosphoribosyltransferase(NAMPT)is a kind of cytokine secreted by adipocytes,with intracellular and extracellular activity.In mammals,intracellular Nampt is the rate limiting enzyme in the salvage pathway of biosynthesis of nicotinamide adenine dinucleotide(NAD).NAD is a ubiquitous pyridine nucleotide that functions as an essential cofactor in mitochondrial oxidative phosphorylation and plays an important physiological role in the basal life activities of the various cellular.Previous studies showed that the NAMPT-NAD transduction pathway as a critical signaling regulator has been revealed with some pathological and physiological activities,including obesity,insulin resistance,lipid metabolism,inflammation and anti-aging,and these biological functions are closely associated with the NAFLD.Therefore,we proposed whether NAMPT-NAD signaling pathway is involved in the occurrence and the development of NAFLD.Thus,we previously constructed a dominant-negative enzymatic-dead NAMPT transgenic mouse(DN-NAMPT),both the endogenous expressions and activities of NAMPT are inhibited.We explored the relationship between NAMPT-NAD signaling pathway and NAFLD and confirmed that the deficiency of NAMPT-NAD is a critical risk factor NAFLD.Based on this experiment,we furtherinvestigated that the influences of replenishment of nicotinamide riboside(NR),a natural NAD precursor,on the steatesis and steatohepatitis and the casual relationship between the NAD reduction with age and NAFLD development.Methods1.10-weeks old mice were divided into wild type(WT),DN-NAMPT mice and DN-NAMPT+NR treatment of a month,then we compared the plasma AST,ALT,triglyceride and total cholesterol levels among these groups,we also measured hepatic triglyceride(TG)and total cholesterol(TC)levels as well as the lipid peroxidation product malonaldehyde(MDA)levels.The mRNA expressions of lipid transport related genes were detected using Real-time PCR.We further used F4/80 and TNF-αimmunohistochemistry staining to respectively observe the macrophage infiltration and the expression of inflammatory cytokines of the liver,and the body weight and liver weight of mice were also measured.2,10-weeks C57BL/6 mice were divided into normal diet(Chow)group,high fat diet(HFD)group and HFD + NR group.HFD mice were fed with a high-fat diet(45% fat)for 12 weeks to achieve NAFLD model,and HFD + NR group were fed with a high fat diet for 3 months,adding the nicotinamide ribose treatment in the third month.Then we compared the body weight,liver weight,hepatic TG and TC contents among these two groups and normal Chow group.The inflammatory cytokines(TNF-α,IL-1b(8)in liver tissues were also detected.In addition,hepatic lipid deposition was observed by oil red O staining and liver fibrosis was determined by α-SMA immunohistochemistry and Masson staining.3,Hepatic NAD concentration,together with the protein levels of NAMPT and several other critical enzymes(IDO,NADS and NMNAT)regulating NAD biosynthesis pathways,were compared among adult(4 months)middle-aged(12 months)and aged(20 months)mice or middle-aged(<45 years old)and aged(> 60 years old)patients using immunoblotting.In addition,these critical enzymes regulating NAD biosynthesis pathways were also determined by immunohistochemistry staining in middle-aged and aged patients.Results1,Under the normal chow,there is no significant difference about the weight,liver weight,the serum TG and TC levels between the middle-age DN-NAMPT mice and WTmice.While the DN-NAMPT mice displayed moderate NAFLD phenotypes,including hepatic lipid accumulation,enhanced oxidative stress and inflammatory infiltration.Whereas the replenishment of the NAMPT substrate NR successfully improved or partially corrected these NAFLD phenotypes induced by NAMPT deficiency.2,NR replenishment for one month potently blocked the HFD-induced increases of body weight and reduced the marked increases of hepatic triglyceride and total cholesterol levels.NR treatment also reduced liver weight and ratio of live weight to body weight.Oil red O staining confirmed the decline of hepatic lipid content by NR.In addition,a-SMA staining showed NR treatment significantly lowered the hepatic fibrosis.The enhancements of inflammatory cytokines(TNF-α,IL-1b(8)production in liver tissues were also treated by NR treatment.3,Age and the liver NAD contents were negatively correlated.Hepatic NAMPT and NAD were significantly downregulated in aged mice and old patients,whereas the key enzymes in the de novo pathway of NAD biosynthesis IDO,NADS,NMNAT were upregulated.In addition,immunoblotting analysis also demonstrated a significant downregulation of NAMPT in live tissues from old patients.In contrast,IDO,NADS and NMNAT in live tissues from old patients were upregulated.Conclusions1,Replenishment of the NAD precursor NR was able to correct the moderate hepatic steatosis in the mice induced by NAMPT deficiency.2,Replenishment of the NAD precursor NR can improve or partially correct the progress of NAFLD in the mice induced by high fat diet,and supplement of NR may be a promising therapeutic strategy to prevent and treat NAFLD in elderly.3,Hepatic NAMPT and NAD level were significantly decreased in aged mice and the elderly.Impairment of NAMPT-controlled NAD salvage biosynthesis may be a cause for the decline of hepatic NAD content in aged human,as well as a critical risk factor for NAFLD in elderly.