The Effect Of High-Fat Diet On Obesity And Glycolipid Metabolism In Mouse Offsprings And Its Mechanism Exploration

The western-style high-fat diet(HFD)is a major contributor to obesity.If the epigenetic modifications of parental gametes were altered by dietary factors and escaped reprogramming during gametogenesis and embryonic development,the parental adverse phenotypes intergenerational inheritance may occur in offspring.However,obese offspring induced by parental HFD through epigenetic mechanisms(especially DNA methylation)have not been reported.And the relevant molecular mechanism remains to be clarified.In this subject,male mice were exposed to HFD over multiple consecutive generations,which were mated and bred with female mice maintained on a normal diet.Male offspring resulting from these matings were selected as subjects to investigate the impact of HFD on obesity and glycolipid metabolism in mice.Additionally,the study sought to elucidate the underlying mechanism behind these effects,with a particular focus on DNA methylation.1.Paternally multi-generational HFD causes obesity and glycolipid metabolic disorder in offspringF0 male mice were divided into 2 groups(20 mice per group)randomly fed a control diet(CD)or HFD for 9 weeks.Then the F0 males were mated with normal weight independent line females fed with CD to produce F1 offspring.The body weight of mice were recorded weekly.After weaning of F1 mice used for reproduction,they were continued to be fed CD or HFD respectively according to the paternal diet pattern for 9 weeks.Then the even-weight F1 male were also mated with normal weight independent line females fed with CD to obtain F2 offspring.The subsequent generations were fed and bred in the same manner.Mice that were not used for reproduction were included in the phenotypic comparison of each generation,and were kept on a controlled diet(CD)after weaning.The body weight of Con and HFD line male offspring was compared of the same generation,until there was a significant difference between the Con and HFD line group.It was found that there was a tendency for subsequent HFD offspring to incremental weight,and the slightly heavier body weight in HFD line group were observed in the F6 and F7 generation.Notably,until the F8 and F9 generation offspring,the body weight of HFD group mice had a significant increase after weaning,and it was higher about 5%-9%compared to the Con group in adulthood(p<0.01),accompanied by gained liver weight ratio and abdominal fat pad accumulation(p<0.05).The study showed that multi-generational HFD could lead to the intergenerational inheritance of obesity in offspring.Serum and tissue biochemical analysis showed that paternal HFD also produced dyslipidemia in offspring consistent with the trend of body weight increasing especially after F6 generation(p<0.01).Hematoxylin eosin(H&E)and oil red O staining indicated increased adipose vacuoles in liver and significant steatosis in adipose tissue of F9 mice.The mice glucose homeostasis were evaluated by the oral glucose tolerance test(OGTT)and insulin tolerance test(ITT).Paternal HFD induced glucose intolerance and insulin insensitivity in all generations offspring mice.The glucose content in the HFD F1-F9 mice were significantly higher than Con mice at all time points with a slower glucose decline rate(p<0.01).The HFD F9 mice also exhibit insulin production increases and pancreatic damaged.The results showed that paternally multi-generational HFD caused obesity and glycolipid metabolic disorder in offspring.2.Paternally multi-generational HFD alters hepatic gene expression profile in offspringThe hepatic microarray analyse were performed to identify gene(mRNA)expression profile variation in F9 offspring to investigate the molecular mechanism of intergenerational inheritance of obesity and metabolic disorders induced by paternally HFD.Then the expression levels were compared among Con and HFD line groups.Differential gene analysis showed that 876 genes were up-regulated and 1390 genes were down-regulated in the HFD mice compared to the Con group.It is observed that the HFD exposure induced distinct differences of liver expressed mRNAs profile in F9 offspring.Most of the differentially expressed genes(DEGs)were reduced in HFD mice.The signal pathways of DEGs were further investigated by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,it was indicated that paternal HFD mainly interfered canonical pathways associated with obesity and metabolism such as insulin signaling pathway,T2DM,glutathione metabolism,circadian rhythm,cholesterol metabolism,glycolysis/gluconeogenesis,insulin secretion and AMPK signaling pathway,etc.The protein-protein interaction(PPI)network revealed a possible pivotal role of critical genes related to glycolipid metabolism.These results indicate that HFD disrupts gene expression and primarily regulated genes related to glucolipid and lipid metabolism in offspring.3.Paternally multi-generational HFD affects the DNA methylation profile of offspringEpigenetics especially DNA methylation regulate gene expression.Epigenetic modification of gametes genes is essential for intergenerational inheritance.The MeDIP-chip analysis were performed to identify the DNA methylation variation in gene promoter region of F8 offspring sperm,which is the parent generation of F9 used for microarray expression profiling assay.A total of 3847 genes promoter regions were enriched to the peak.After statistics,there were 224 differentially methylated genes(DMGs)hypermethylated by HFD while 100 demethylated.GO and KEGG enrichment were analyzed to investigate further signal pathways of DMGs.Similar to DEGs,many DMGs were enriched to insulin secretion,T2DM,circadian rhythm and cholesterol metabolism pathways etc,associated with obesity and metabolism.These results indicate that HFD primarily leads to genes promoters hypermethylation in offspring gametes related to glucolipid metabolism.4.DEGs and DMGs co-analysis of altered methylation of key genes in offspringThe transcriptional inhibition of genes was positively correlated with promoter methylation degree.The DNA methylation pattern of gametes may avoid reprogramming during organismal development,resulting in altered gene expression levels in offspring and causing phenotypic intergenerational inheritance.Based on DEGs and DMGs,the genes were selected of which expression pattern was consistent with the trend of promoter methylation.There were 16 target genes which were hypermethylated and expression inhibited by paternal HFD,while only 2 with opposite trend.For the F9 offspring,the progeny of sperm for MeDIP-chip analysis,the promoter methylation levels of target genes in liver including Spns2,Lonp1 and Hk1 were detected by bisulfite sequencing.Compared with Con line mice,consistent with MeDIP-chip data,the promoter methylation degree of Spns2,Lonp1 and Hk1 were increased significantly in HFD line group.In addition,the methylation rates of three genes of sperm in F0 HFD group were also significantly enhanced,while the methylation rates of liver tissue(somatic cells)in F1 mice were slightly different between the two groups.The increase of methylation rate caused by HFD in F7 mice was further enhanced than that in F1.Meanwhile,the RT-qPCR and western blot assay confirmed significant downregulation of Spns2,Lonpl and Hk1 transcripts of the HFD line F9 mice liver(p<0.05).The same significant downregulation was observed in the F0 HFD group mice,F1 Spns2 was significantly downregulated(p<0.05),and the different expression in F7 mice was further amplified(p<0.05).The HFD-induced offspring obesity and dysglycolipid metabolism experiment were subjected to repeat,and intervention was performed on HFD mice using the DNA methyltransferase(DNMT)inhibitor RG108.RG108 intervention significantly decreased DNMTs activity and reduced the DNA methylation level of Spns2,Lonp1 and Hk1 promoters in HFD F0 mice sperm or liver.RG108 also reversed the increased promoters DNA methylation rate and decreased expression of Spns2 in HFD F1 and F2 mice(p<0.05),accompanied by its alleviating glucose intolerance and insulin resistance(p<0.05).These results indicated that sustained multigenerational HFD induces target gene hypermethylation,which is transmitted through gametes and alters the gene expression of progeny,causing intergenerational inheritance of parental obesity and glucolipid metabolism disorders.In summary,the present research uniquely performed a paternally multi-generational HFD model and obtained significant stable higher body weight and metabolically disturbed offspring.Mechanism studies showed that paternal HFD induced DNMTs expression and activity increased,and enhanced DNA methylation rate of key genes promoters in glycolipid metabolism,which avoided the reprogramming process and futher accumulated intergenerationally,therefore altered genes expression levels involved in glycolipid metabolism.This study elucidates the main molecular mechanisms underlying intergenerational inheritance of obesity and metabolic disorder phenotypes induced by HFD.Our research extends the previous knowledge of diet and intergenerational inheritance of increased disease risk,which would provide new perspective to healthy diet and lifestyle establishment,meanwhile having profound implications for the human health and progeny.