Effects Of N-3 Polyunsaturated Fatty Acids On Non-Alcoholic Fatty Liver Disease And Metabolic Syndrome And Related Mechanisms

Non-alcoholic fatty liver disease(NAFLD)is a state of glucose and lipid metabolism disorders caused by interactions of genetics and environmental factors.NAFLD consists of a series of pathologic processes ranging from simple fatty liver to non-alcoholic steatohepatitis,cirrhosis and hepatocellular carcinoma.As a kind of chronic liver injured disease,the prevalence of NAFLD has become the second most chronic liver disease following viral hepatitis in China.Metabolic syndrome is a state with multiple risk factors in individuals,including central obesity,elevated triglyceride(TAG),low-density lipoprotein cholesterol(LDL-C)and hypertension,and low high-density lipoprotein cholesterol(HDL-C),and impaired glucose tolerance.The patients with metabolic syndrome,who are generally complicated with type 2 diabetes mellitus and cardiovascular disease threaten the quality of life,health and lives greatly.The present thesis aimed to investigate the effects of n-3 polyunsaturated fatty acids(PUFA)on NAFLD and metabolic syndrome via evidence-based nutritional studies.Based on animal models,we explored the mechanisms why dietary n-3 PUFA could protect against NAFLD and metabolic syndrome.In addition,the effects of EPA(20:5n-3),DPA(22:5n-3)and DHA(22:6n-3)on blood lipid metabolism and cardio-metabolic risks were also investigated using cross-over study.Whether dietary n-3 PUFA could prevent NAFLD and metabolic syndrome are still inconclusive.The associations of n-3 PUFA with NAFLD and metabolic syndrome were investigated via evidence-based nutritional studies.The pooled estimates of randomized controlled trials showed that dietary intake of n-3 PUFA significantly reduced circulating alanine aminotransferase(ALT),aspartate aminotransferase(AST),and TAG concentrations.The summary effects of cross-sectional and case-control studies indicated that the circulating n-3 PUFA were inversely associated with metabolic syndrome risk.Furthermore,the compositions of circulating DPA and DHA were significantly higher in the controls compared with cases.Based on above findings,we established NAFLD model with high-fat and high-sugar(fructose,glucose and sucrose)diet to investigate whether fat-1 transgenic mice with endogenously synthesized n-3 PUFA could protect against NAFLD.The fat-1 and C57/BL6J wild-type mice(n=8 per group,3 month-old)were conducted high-fat and high-sugar intervention for 8 weeks.Compared with wild-type mice,the serum levels of ALT,AST,glucose,insulin,homeostasis model assessment of insulin resistance(HOMA-IR),total cholesterol,LDL-C and TAG were significantly decreased in fat-1 mice.Histological analysis showed that fat-1 mice inhibited TAG accumulation and steatosis in the liver,compared with wild-type mice.The endogenously synthesized n-3 PUFA improved TAG metabolism in the liver through suppressing the mRNA expression levels related fatty acid synthesis(sterol regulatory element-binding protein(SREBP)-1C,acetyl-CoA carboxylase(ACC)1,and fatty acid synthase(Fasn))and activating fatty acid ?-oxidation(peroxisome proliferator activated receptor(PPAR)-a and acyl-CoA oxidase(AOX)).In addition,n-3 PUFA inhibited the key genes(TLR-4 and myeloid differentiation primary response gene 88)and protein(TLR-4)expression levels of toll-like receptor(TLR)-4/nuclear factor-kappa B(NF-?B)signaling pathway,and mRNA expression levels of pro-inflammatory factors(interleukin(IL)-1,IL-6 and monocyte chemoattractant protein(MCP)-1)to prevent the progress of NAFLD and metabolic syndrome.Aforementioned evidence indicated that n-3 PUFA could inhibit the accumulation of TAG in the liver,improve hepatic liver function,and risk factors of metabolic syndrome.However,the biological effects of individual n-3 PUFA(EPA,DPA and DHA)are unclear.The aim of this part was to investigate and compare the effects of dietary supplementation with EPA,DPA and DHA on liver function and metabolic risk factors with C57/BL6J wild-type mice(n = 10 per group,3 month-old).Compared with high-fat group,supplemental EPA,DPA and DHA suppressed expression levels of key genes and proteins related fatty acid synthesis and TLR-4/NF-?B signaling pathway,inhabited TAG accumulation in the liver,and reduced serum glucose,LDL-C,total cholesterol and ALT concentrations.Supplementation with DPA significantly improved serum HOMA-IR levels,compared with high-fat group.Supplementation with DPA was the most effective in reducing serum levels of glucose and HOMA-IR,compared with EPA and DHA.Supplementation with DPA and DHA both prevented the decreased serum adiponectin levels,and prevented the increased serum ALT levels compared with EPA group.Based on the results of liver function and histological analysis,DPA and DHA seem to exert similar effects in protection against NAFLD and these effects seem to be superior compared with EPA.To explore the metabolisms of n-3 PUFA monomer,the effects of supplementation with EPA,DPA and DHA on blood lipid metabolism were conducted systematically.Twelve female healthy subjects consumed 12 ml of EPA-olive oil(00)(1:1,W/W),DPA-OO(1:1,W/W),and DHA-00(1:1,W/W),respectively,in a cross-over double-blind study over a 6-day period.The placebo treatment was OO.The fasting venous blood was extracted at days zero,three and six,and the red blood cell(RBC)phospholipids(PL)and plasma lipid fractions were separated for fatty acid determination using gas chromatography.Supplemental EPA significantly increased the concentration of EPA in RBC PL(days 3 and 6).In the DPA group,the concentrations of EPA and DPA were significantly increased in RBC PL over a 6-day period,respectively.For plasma PL fraction,EPA and DPA supplementation significantly increased the concentrations of EPA and DPA at both days 3 and 6,respectively.Supplemental DHA significantly increased the concentration of DHA over a 6-day period.For plasma TAG fraction,supplementation with EPA significantly increased the concentrations of EPA and DPA at both days 3 and 6,respectively.Significant increase in the concentration of DHA was found relative to baseline at both days 3 and 6 after subjects consuming DHA.DPA could be metabolized into DHA and retro-conversed back to EPA.For plasma CE fraction,EPA supplementation significantly increased the concentration of EPA(days 3 and day 6),and the concentration of DPA(day 6).Additionally,the concentration of DHA was significantly increased over a 6-day period of intervention.However,supplementation with EPA,DPA and DHAhad no significant difference on serum lipids and glucose concentrations.In conclusion,evidence-based nutritional studies indicated that dietary intake of n-3 PUFA significantly reduced the concentrations of ALT,AST and TAG in patients with NAFLD.Circulating n-3 PUFA were inversely associated with metabolic syndrome risk.Animal models showed that n-3 PUFA suppressed TAG accumulation in the liver,and improved risk factors of metabolic syndrome through inhabiting expression levels of genes and proteins related fatty acid synthesis and TLR-4/NF-?B signaling pathway,and activating expression levels of key genes regarding fatty acid ?-oxidation.DPA as well as DHA exerted similar effects in improving liver function and risk factors of metabolic syndrome and these effects seem to be different in comparison with EPA.Cross-over study demonstrated that supplemental EPA could be metabolized into DPA.The retro-conversion from DHA to DPA was not apparent.As a reservoir of n-3 PUFA,DPA could be incorporated into blood lipid fractions,metabolized into DHA,and retro-conversed back to EPA.The present study provides substantial evidence that dietary supplemental n-3 PUFA may be effective in the prevention and treatment of NAFLD and metabolic syndrome.In order to establish bases for clinical application,well-designed randomized controlled trials with a large sample-size and multi-center should be conducted to obtain the dose-response relationship of EPA,DPA and DHA associated with biomarkers of NAFLD and risk factors of metabolic syndrome.