Hypermethylation of hepatic Gck promoter in ageing rats contributes to diabetogenic potential

Abstract

Aims/hypothesis: Hepatic glucokinase (GCK) is a key enzyme in glucose utilisation. Downregulation of its activity is associated with insulin resistance and type 2 diabetes mellitus. However, it is unknown whether hepatic Gck expression is influenced by age and is involved in ageing-mediated diabetes, and whether the degree of methylation of the hepatic Gck promoter is correlated with the transcription of Gck. To address the question, we evaluated hepatic Gck transcription and promoter methylation in young (14 weeks), adult (40 weeks) and aged (80 weeks) rats. Methods: Hepatic glycogen, Gck expression and the kinase activity of GCK were measured in three age groups. The CpG methylation status was determined by both bisulphite direct sequencing and clone sequencing of the PCR amplificates of Gck promoter. The causal relationship between Gck methylation and mRNA expression was confirmed by treating rat primary hepatocytes with 5-aza-2′-deoxycytidine (5-Aza-CdR). Results: We have shown an age-associated decline in hepatic glycogen, Gck expression levels and the kinase activity of hepatic GCK. The eleven CpG sites studied displayed age-related progressive methylation changes in hepatic Gck promoter, which were confirmed by two methods: direct and clone sequencing. After 5-Aza-CdR treatment of rat primary hepatocytes, there was a fourfold increase in Gck expression. Conclusions/interpretation: Our results demonstrate that an age-related increase in methylation is negatively associated with hepatic Gck expression, suggesting that DNA methylation could be involved in increasing age-dependent susceptibility to hepatic insulin resistance and diabetes. Thus, the epigenetic modification of the hepatic Gck promoter may represent an important marker for diabetogenic potential during the ageing process. © 2008 Springer-Verlag.