Epigenetic Study Of Genes Related To Hepatocyte Glucose Metabolism In Aging And Obese Rats

Background and AimsAging has been posing a global challenge to mankind, which is concomitant with many health issues. When bodies grow old, obesity accompanies, and these two factors make bodies susceptible to Type 2 diabetes mellitus(T2DM) and a number of other metabolic diseases. With the progress in the research of Metabolic Syndrome (MS), researchers have pinned the common pathophysiological basis on insulin resistance(IR). During The Second International Congress on Prediabetes and the Metabolic Syndrome" hosted by International Diabetes Federation in 2007, the etiology of prediabetes was attributed to insulin resistance, which reflected the central importance of insulin resistance in the development of diabetes.Aging and obesity are two independent risk factors in insulin resistance. With the increase of human lifespan, the elucidation of the effect of aging and obesity on insulin resistance will provide valuable information for the health and prevention of diseases in older population. The molecular mechanism of insulin resistance remains unclear up to now. Epigenetic changes link environmental factors, age and disease risk, and has been indicated to play an important role in cellular and individual aging. Epigenetic marks are easier to change than DNA sequence, under the influence of environmental factors. A slight change of epigenetic marks may activate or silence gene expression and produce a series of effects on the development of insulin resistance and diabetes. In this project, we sought to dissect the epigenetic mechanism of insulin resistance from two aspects—aging and obesity, using glucose-metabolic genes.Methods and results:1. The epigenetic study of aging rat hepatic glucose-metabolism-related genes.1) Determination of expression of hepatic glucose-metabolism-related genes.(1) Physiological indices of rats used in the experiments. Age: 14 wks (young), 40 wks (adult), 80 wks (aged). While fasting blood glucose levels in the three groups are normal, blood insulin increase with age, and the increase was significant in adult and aged rats, compared to the young group, indicating that rats were insulin resistant in the former two groups.(2) Transcription level of hepatic glucose-metabolism-related genes: hepatic glycogen decrease with increasing age, and the expression of hepatic glucose-metabolism-related genes Glut2, Gck and L-PK decrease with increasing age. What's more, the degree of reduction of the activity and transcription of Gck and L-PK were similar.2). Epigenetic study of hepatic glucose-metabolism-related genes in aging rats: the methylation levels of Glut2,Gck and L-PK promoters were measured(1) The methylation degree of each of the 12 CpG sites in Glut2 promoter is showed no difference among the three groups. This result indicated that the methylation degree of Glut2 promoter methylation is not related to aging, ruling out the regulatory effect of DNA methylation on transcription.(2) The methylation levels of Gck and L-PK promoter increased with age in the three groups of rat livers, showing an age-dependent manner, which indicated a negative correlation between Gck and L-PK promoter methylation and their transcriptions.2. Epigenetic study of hepatic glucose-metabolism-related genes in obesity rats.(1) Establishment of obese rat model: the rats were fed high-fat food beginning at the age of 4 wks. Body weights of these rats increase quickly with time, and after 8 wks of high-fat feeding we confirmed the successful establishment of obese rat model.(2) Detection of transcription of glucose-metabolism-related genes in obese rat livers: glycogen contents of obese rats were higher than normal rats; Real time PCR detection of glycolysis-Gck and L-PK-is significantly lower than normal rats.(3) The epigenetic study of obese rat hepatic Gck and L-PK genes: the methylation level of both Gck and L-PK promoters are significantly higher than normal-weighted rats, indicating a negative correlation between the methylation levels of the these promoters and transcription, which hinted that obesity is probably a primary factor in the hypermethylation of hepatic Gck and L-PK promoter.3. Epigenetic study of insulin resistant hepatocytes induced by free fatty acids. 1). Establishment of insulin resistant hepatic cell line. Glucose utilization the "gold standard" glucose uptake were significantly altered after 24 hrs induction of normal rat hepatocytes with 0.1 mM PA, confirmed that the model was insulin resistant cell model (BRL/IR for short).3). Changes in the expression of glucose-metabolism-related genes in BRL/IR cells: hepatic glycogen content of BRL/IR cells ware significantly reduced; transcription, translation and enzymatic activity of Gck was significantly reduced.4). Effect of BBR on the treatment of BRL/IR: 20μM BBR signifieantly increase glucose uptake and hepatic glycogen content in BRL/IR cells; significantly increase Gck transcription and translation and enzymatic activity. What's more, BBR significantly reduced hepatic TG content in BRL/IR, and reversed hepatic steatosis.5). Epigenetic study of rat hepatocytes-BRL cell line Gck promoter(1) Determination of BRL cells Gck promoter methylation revealed a minor and variable increase in almost every CpG site after 24 hrs PA induction and showed a negative correlation.(2) Transcription of Gck in BRL/IR was elevated by demethylating agent 5-Aza-CdR. The decrease of methylation in hypomethylated CpG sites is significant, but not significant in hypermethylated CpG sites.(3) The methylation level of hypomethylated CpG sites in BRL/IR cells Gck promoter were significantly reduced by BBR treatment, but the reduction is not significant in hypermethylated CpG sites. However, the demethylating effect of BBR is higher than 5-Aza-CdR, indicating that BBR might activate Gck expression through demethylation of Gck promoter.(4) Construction of Gck-expressing and Gck-siRNA-expressing adenovirus and their effects on insulin resistant cells: Adenoviruses expressing Gck (Ad-Gck) and Gck siRNA (Ad-Gck-siRNA) were contructed. Our experiments showed that overexpression of Ad-Gck can significantly improve glucose uptake and hepatic glycogen in BRL/IR cells; Ad-Gck-siRNA can signifieantly repress hepatic Gck expression and decrease hepatic glucose uptake and glycogen. The role of Gck in hepatic insulin resistance was evidenced by overexpression or repression of Gck, paving the way for functional study of Gck in vivo. Conclusions:Insulin resistance develops gradually, and aging and obesity are two primary factors inducing insulin resistance, which was intimately related with glucose tolerance. In this project, we analyzed the molecular mechanism of aging and obese rats' susceptibility to diabetes from the epigenetic point of view, and discovered the reverse relationship between gene transcription and the promoter methylation status of two hepatic glucose metabolizing genes: Gck and L-PK. The hypermethylation of these promoters impaired insulin's effect and glucose metabolism, and contributes to the susceptibility to diabetes. In the cell model of insulin resistance, the reverse relationship between Gck transcription and promoter methylation is confirmed, and we found that BBR increase Gck transcription through promoter demethylation. This study is a primary exploration of DNA methylation in insulin resistant cell and animal models, and will shed a new light on further study of the epigenetics of diabetes, and provide foundation for epigenetic therapy of metabolic diseases by regulating DNA methylation..