| Background:The prevalence of diabetes is steadily increase global wide,and it has been revealed by epidemiology studies that family history as well as previous gestational diabetes are known factors contributing to increased risk of developing metabolic disease.In the past,numerous studies have highlighted mother-infant interactions,whereas fathers’ legacy in shaping metabolic landscape of offspring drawn less attention.Notably,paternal nutritional status,exposure to drugs and toxins and even unpleasant social experience could mediate transgenerational effect through epigenetic reprogramming,namely DNA methylation and histone modification.In previous studies,to address the transgenerational inheritance of metabolic disorders from fathers with hyperglycemia,we produced a hyperglycemic rat model by single injection of low dose of streptozocin(STZ),and then bred with healthy female rats to generate offspring from STZ fathers(STZ-O),in comparison with those from citrate buffer(CB)treated euglycemic fathers(CB-O).Metabolic derangement has been observed in STZ-O with increased body weight,hyperlipidemia,as well as impaired regulation of hypothalamus mediated food intake and energy expenditure.However,the detailed molecular mechanism and physiological implication of the gene and life-stage-specific changes in DNA methylation in father’slineage has not been fully addressed.In the present study,using the previous animal model,we address how paternal hyperglycemia exerts an intergenerational effect in mammals,and we further explore the underlying mechanisms.Purpose:This study was designed to investigate how paternal hyperglycemia exerts an intergenerational effect in offspring causing metabolic derangement,and the underlying mechanisms.Methods:1 Male Sprague Dawley(SD)rats were randomly received two different treatments: streptozocin(STZ,S0130,Sigma)and citrate buffer(CB).After one-week acclimatization,hyperglycemic model was induced by intraperitoneal injection of STZ(35 mg/kg body weight)and control rats received an equal volume of CB after 16-h of fasting.Rats with glucose levels were persistently higher than 16.7 m M at the 3rd,6th and9 th day after STZ injection and immediately before mating were considered as hyperglycemia and used for subsequent experiments.STZ treated hyperglycemic rats and CB rats were mated with age-matched healthy female rats.The offspring were labeled according to their fathers,forming two groups: STZ-O(STZ-offspring)and CB-O(CB-offspring).2.To investigate the consequences of paternal hyperglycemia on the epigenetic profile of offspring,we examined the DNA methylation alterations of Cp G islands,promoter and genebody in the offspring liver using Me DIP-Sequencing.Combining GO and Pathway Analysis,differentially methylated regions were analyzed.Methylation changes of genes relating to lipid metabolism were selected as the focus of the study.3.To obtain molecular insight into the impact of paternal hyperglycemia on liver lipid accumulation in offspring,liver tissues weresubjected to microarray gene expression profiling.Differentially expressed genes enriched in GO and KEGG pathway analysis were investigated by quantitative real-time PCR analysis and western blot for m RNA and protein expression.4.We further explored the methylation patterns of Cp G islands in the PPARα promoter of adult offspring liver using bisulfite sequencing PCR and region-852 to-601 was amplified.To determine whether the altered methylation patterns of the PPARα locus were already existed during early life stage,we determined the CGI methylation in the fetal liver(ED16.5)using the same methods.5.Using Genomatix Matinspector(Genomatix Software Gmb H),we investigated transcriptional factor binding motifs in the amplified 252 bp sequence.Sequential of putative binding site deletions were generated and inserted into a luciferase reporter construct.The luciferase reporter assay was conducted with or without the presence of plasmids subcloned with the rats SP1 c DNAs.To investigate whether DNA methylation of Cp G located within the SP1 binding site can directly affect the PPARαexpression,M.HPII was used to generate site specific methylation of PPARa promoter sequence,and subcloned into the p GL3-basic.Luciferase reporter assay was performed after transfection into 293 T cells with methylated or unmethylated PPARa-p GL3-basic.Results:1.The liver DNA Cp G islands were susceptible to changes in methylation status as a result of paternal hyperglycemia.GO and KEGG analysis of differentially methylated genes showed lipid metabolic process(BP),lipid digestion(BP),lipid binding(MF),lipase activator activity(MF),bile secretion,Fatty acid elongation were among the top ontology terms with the highest enrichment score.It is also noticeable thatdiscrepancy in CGIs methylation level were also evidenced by the Me DIP analysis in four nuclear receptor family members,PPARg,PPARa,Fxr2 and RXRa2.Under paternal hyperglycemic conditions,3381 genes were differentially expressed.KEGG pathway analysis indicated fatty acid metabolism was highly enriched with differently expressed genes acyl-Co A dehydrogenase(Acadsb),carnitine palmitoyltransferase 1(Cpt1),alcohol dehydrogenase1b1,6 and 7(Adh1b1,Adh6 and Adh7)and PPARα.It was confirmed by PCR and Western Blot the m RNA and protein level were significantly reduced for PPARα,ACOX1,CPT1α and CD36.3.DNA methylation analysis by pyrosequencing showed the methylation profile of the CGI-A in PPARα promoter of STZ-O rat liver(-852 to-601 bp)was significantly changed.Of the 23 Cp G sites identified,the methylation level of Cp G sites 9,12,13,15 were statistically increased in liver samples from the STZ-O rats,and Cp G sites2,11,16,17,18,21 were decreased in methylation level.CGI-A methylation in the fetal liver(ED16.5)from the STZ-O rats was studied.Variations in epigenetic signature were detected,but interestingly,Cp G site 13 was significantly hypermehylated as in adult offspring liver,Cp G sites 21 were also significantly hypomehylated.4.Using Genomatix Matinspector(Genomatix Software Gmb H),3motifs were significantly enriched in the Cp G site 13 that gained DNA methylation in adult liver(V$CTCF,V$SP1F,V$ZF5F).We generated sequential deletions of the two SP1 binding sites individually and in combination,then those sequences were inserted into a luciferase reporter construct.Deletion of the any of the SP1 putative binding sites caused a significant decrease in PPARα promoter activity,but could be rescued by cotransfection with SP1 plasmids.5.Methylase M.HPII was used to generate methylated Cp G site 13.PPARa promoter activity was significantly repressed after methylation.Cotransfection with SP1 expression plasmids increased activity of methylated PPARα promoter constructs compared with vector controls.Conclusion:The present study reveals paternal hyperglycemia induces epigenetic modifications in the liver cells of offspring,namely PPARa,and this signatureis maintained from early life to adulthood.Hypermethylation of Cp G 13 abrogated binding of SP1 to the PPARa promoter,and caused down regulation of PPARa as well as other genes which controls the lipid metabolism.This mechanism contributes partly to the development of lipid dysregulation in the second generation offspring. |
