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.

Aims/hypothesis Rapamycin impaired glucose tolerance and insulin sensitivity. Our previous study demonstrated that rapamycin significantly increases the expression of gastric ghrelin, which is critical in the regulation of glucose metabolism. Here, we investigated whether ghrelin contributes to derangements of glucose metabolism induced by rapamycin. Methods The effects of rapamycin on glucose metabolism were examined in mice receiving ghrelin receptor antagonist or with ghrelin receptor gene deletion. Changes in Glut4, JNK, and pS6 were investigated by immnuofluorescent staining or Western. Related hormones were detected by radioimmuno-assay kits. Results Rapamycin impaired glucose metabolism and insulin sensitivity not only in normal C57BL/6J mice but also in both obese mice induced by high fat diet and db/db mice. This was accompanied by elevation of plasma acylated ghrelin. Rapamycin significantly increased the levels of plasma acylated ghrelin in normal C57BL/6J mice, high fat diet induced obese mice, and db/db mice. Elevation in plasma acylated ghrelin and derangements of glucose metabolism upon administration of rapamycin was significantly correlated. The deterioration in glucose homeostasis induced by rapamycin was blocked by D-Lys3-GHRP-6, a ghrelin receptor antagonist, or by deletion of ghrelin receptor gene. Ghrelin receptor antagonism and ghrelin receptor gene deletion blocked the up-regulation of JNK activity, and GLUT4 expression and translocation in the gastrocnemius muscle induced by rapamycin. Conclusions The current study demonstrates that ghrelin contributes to derangements of glucose metabolism induced by rapamycin via altering the expression and translocation of GLUT4 in muscles.