Association Analysis And Mechanistic Exploration Of NAFLD With Dietary Fatty Acid Intake Characteristics

Background and Aims:The association between dietary fat composition and nonalcoholic fatty liver disease（NAFLD）in typical diets remains unclear.Additionally,the pathogenesis of NAFLD in individuals carrying risk genes may differ from the general population.This study explores the relationship between dietary patterns characterized by different fatty acid intakes and NAFLD in a typical diet population.It will further confirm the efficacy of beneficial dietary patterns for NAFLD genetic risk groups and their potential mechanisms in reducing liver fat accumulation through observational studies,genome-wide interaction analysis,and single-cell multi-omics techniques.Methods:The observational study and genome-wide interaction analysis（GWIS）used samples from 103,992 unrelated Europeans with nutrient intake information from the UK Biobank（UKB）cohort study.The single-cell analysis used samples from16-week-old C57BL/6J mice fed a high-fat or a control diet for 8 weeks.Using principal component analysis,the study first identified dietary patterns with different fatty acid intakes and assessed the association between dietary patterns and NAFLD using logistic regression and mediation analysis.Subsequently,the effect of beneficial dietary patterns for NAFLD prevention in the typical diet population on NAFLD genetic risk groups was evaluated using genome-wide interaction and genetic marginal effect analysis.The potential mechanisms of action were inferred through pathway and tissue enrichment analysis of interaction loci between the dietary pattern and 11 traits related to NAFLD,blood lipids,and circulating fatty acids.Finally,potential therapeutic targets for NAFLD influenced by the dietary pattern were identified through overlap analysis of interaction loci,and the association between target gene expression in liver cells and NAFLD was validated using single-cell multi-omics analysis to speculate on the mechanism of action of the NAFLD prevention dietary pattern.Results:The observational study revealed that the PUFA-enriched vegetarian dietary pattern（PVD）is negatively correlated with NAFLD risk(OR=0.82,FDR p=2.92×10^-11)and shows BMI differences,while the PUFA-enriched meat dietary pattern（PMD）is not associated with NAFLD risk.Serum fatty acids are significantly related to both PVD and NAFLD,with SFA（27.8%）,PUFA（25.1%）,ω-6 PUFA（14.3%）,LA（15.6%）,and DHA（10%）partially mediating the association between PVD and NAFLD.The genome-wide interaction analysis found interactions between PVD and two major NAFLD risk loci,PNPLA3 I148M（β=-0.12,p=0.003）and TM6SF2 E167K（β=-0.18,p=0.008）,which can improve liver fat accumulation caused by these mutations.PVD also increases serum triglycerides,cholesterol,and LDL levels in TM6SF2 E167K carriers while reducing liver fat accumulation.The 11GWISs identified 37 interaction loci mainly enriched in lipid metabolism pathways,liver,and vascular,with TM6SF2,FADS2,LPL,and LIPC identified as potential therapeutic targets for NAFLD.Single-cell transcriptomics confirmed the association between the expression of these 4 genes in liver cells and hepatic steatosis,but only Fads2 showed significant differences in its high-expression liver cells subgroup(hepatocytes,Chow%=85.9%,HFD%=49.9%,log₂FC=-1.02,adjusted p=4.25×10^-252).In the HFD group,the proportion of hepatocytes highly expressing Fads2 and the expression level of Fads2 were downregulated.Pseudo-time trajectory analysis indicated that the change in the proportion of hepatocytes was related to the trajectory fate of cells transitioning from high to low Fads2 expression.However,the joint analysis of sc ATAC-seq and sc RNA-seq found no significant change in the transcriptional openness of Fads2 in hepatocytes,but the expression levels of its transcription factors（Hnf4a,Yy1,Rxra,and Ppara）were significantly reduced.Conclusion:In summary,this study provides new insights into the dietary prevention and precision nutrition of NAFLD.In the typical diet population,the PUFA-enriched meat dietary pattern（PMD）with high dietary compliance and food diversity does not increase NAFLD risk,while the PUFA-enriched vegetarian dietary pattern（PVD）rich in LA and DHA is beneficial for treating or preventing NAFLD,especially in obese patients.PVD can also slow down liver fat accumulation in NAFLD genetic risk groups,but it may increase blood lipid levels in TM6SF2 E167K carriers,potentially raising their risk of cardiovascular disease.Additionally,TM6SF2,FADS2,LPL,and LIPC,influenced by PVD intake,hold promise as potential therapeutic targets for NAFLD.Among them,the downregulation of Fads2 is associated with NAFLD.The significant decrease in Fads2 expression in hepatocytes of HFD mice is caused by both the reduced proportion of hepatocytes highly expressing Fads2 and the decreased expression of Fads2 transcription factors in these hepatocytes.