RESUMEN
Objective To study the effect of miR-146a on hepatic INSR gene expression in F1 offspring of paternal rats with high-glucose-high-fat diet ( HGFD) . Methods 5-week-old male SD rats were randomly divided into normal diet ( ND) and HGFD groups. Male rats with ND or HGFD feeding for three months mated with normal female ones. Blood glucose concentration, glucose tolerance, and insulin tolerance in newborn male offspring rats were detected respectively. Differential miRNAs between ND and HGFD groups were compared using next generation sequencing and were then confirmed by real-time quantitative PCR ( qPCR) . The relative expression of INSR mRNA and the methylation level of INSR promoter in liver tissues of the offspring newborn rat were detected and the correlations between them and the relative expression of differential microRNA were analyzed respectively. In vitro, effects of miR-146a on expression and methylation of INSR gene in BRL-3A cells were detected using Western-blot assay and qPCR respectively. Results The fasting glucose concentration of different groups were without significant difference, but glucose tolerance and insulin tolerance of neonatal male rats in HGFD group were decreased significantly . Next generation sequencing has revealed 45 up-regulated miRNAs and 15 down-regulated miRNAs in HGFD group. Among them, differences of 8 miRNAs expression in the enlarged samples were confirmed by qPCR. miR-146a was up-regulated for more than 10 times in the liver of the offspring of HGFD group. Expression level of miRNA-146a was negatively correlated with the relative expression of INSR and positively correlated with the methylation level of INSR in livers of neonatal rats in HGFD group. In vitro, miR-146a ( mimics ) promoted the methylation of INSR gene and inhibited the expression of INSR in BRL-3A cells. Conclusion HGFD feeding to male SD rats leads to the inhibition of hepatic INSR gene expression in neonatal offspring via upregulating miR-146a.
RESUMEN
Objective To study the effect of high-glucose-high-fat diet on expression and methylation of insulin receptor ( INSR) gene in F1 offspring. Methods Sixty 5-week-old male SD rats were randomly divided into two groups:normal diet group and high-glucose-high-fat diet group. After rats were fed for three months, all male rats were performed to copulate with normal female rats. The body weight, blood glucose, and blood insulin of neonatal rats of F1 offspring were measured. The genome DNA, total RNA, and total protein were extracted from livers, brains, and muscles of neonatal rats. Relative expression of INSR in both mRNA level and protein level were detected using a realtime PCR test and a Western blot test respectively. Methylation of INSR promoter was analyzed by a methylation specific PCR ( MSP ) . Results Both body weight and fasting glucose were without significant difference in two groups. In high-glucose-high-fat diet group, both the glucose tolerance and insulin tolerance of neonatal rats in F1 offspring were significantly decreased. Except that in brains, the expressions of INSR gene in livers and in muscles of neonatal rats in high-glucose-high-fat diet group were down-regulated in mRNA ( realtime PCR ) and protein levels ( Western blot) compared to the normal diet group. Meanwhile, the methylation of INSR gene in livers and muscles were strengthened in high-glucose-high-fat diet group. Conclusion A high-glucose-high-fat diet fed to male SD rats leads to the decrease in glucose tolerance, insulin tolerance, and the inhibition of expression of hepatic and muscle INSR gene in neonatal offspring. The methylation of INSR gene could be involved in this phenomenon.