Regulation of gluconeogenesis in type 2 diabetes mellitus: An investigation of the role of corticosteroids?

Abstract

Type 2 diabetes mellitus (T2DM) is a complex disease involving various physiological factors, hormones and metabolites. Over expression of key gluconeogenic enzymes, such as PEPCK, cause features of T2DM including obesity and insulin resistance. Previous studies showed intravenous administration of corticotropin releasing factor (CRF) and sauvagine in rats cause marked hyperglycaemia, which is adrenal dependent. The 11-β hydroxylase inhibitor metyrapone augmented this hyperglycaemia raising important questions about the role of known and unknown corticosteroids in hepatic carbohydrate metabolism. The goal of this project was to identify and characterise the gluconeogenic activity of adrenal corticosteroids secreted under basal and stimulated conditions. This was accomplished by establishing a rapid, sensitive and robust multi-well assay for measurement of PEPCK activity in the hepatocyte cell line H4-II-E-C3. A sensitive fluorescent method for assay of glucose production by these cells was also developed. Extracts of media samples from adrenal glands incubated with sauvagine and metyrapone significantly increased hepatocyte glucose production (HGP) despite low corticosterone concentrations. HPLC characterisation of these extracts revealed increases in 11-deoxycorticosterone (DOC) and other unidentified peaks. However, none of these individual fractions significantly affected HGP in culture. Commercially available corticosterone, which contains DOC as an impurity, had greater gluconeogenic effect compared to purified corticosterone alone. Based on these observations and discrepancies in the literature, the effect of DOC on hepatic carbohydrate metabolism was characterised. Surprisingly, DOC suppressed the activity of gluconeogenic enzyme PEPCK in fed rat hepatocytes and enhanced insulin 4 stimulated glycogen stores in cultured hepatocytes at higher glucose concentrations (25 mM) over a 24 hour period. In serum-starved, fasted hepatocytes these effects were not significant, suggesting the need for a detailed investigation of the signalling pathways and regulatory control of DOC on GK, GS, G6Pase and PEPCK.