Previous studies have demonstrated that POMC peptides are present in islets of Langerhans and suggested that ACTH-related peptides may act as paracrine modulators of insulin secretion. In order to shed light on the cellular mechanisms involved in these responses, we have carried out experiments using the MIN6 mouse insulinoma cell-line and synthetic ACTH 1-24. Following the detection of mouse ACTH receptor (MC-2) receptor mRNA by PCR amplification of MIN6 cDNA, perifusion experiments with MIN6 pseudo-islets were performed. These showed that a small, transient increase in insulin secretion was obtained when ACTH 1-24 (1nM) was added to medium containing 2 mM glucose (control) but not when the glucose content was increased to 8mM. Further investigations were performed in 96 well plates using 20 minute incubation with test agents; insulin content of the medium was measured by RIA. Results are expressed as % control secretion (2mM glucose), experiments were repeated 2-3 times. Significant, dose related stimulation of insulin secretion was obtained with doses of ACTH from 0.5 nM to 10 nM (0.5 nM ACTH : 128 plus/minus 9.6 % control ; 10 nM ACTH : 193.5 plus/minus 13 % control); the effects of submaximal doses of ACTH were potentiated in the presence of the phosphodiesterase inhibitor, IBMX (0.1 mM). ACTH (1 & 10 nM ) had no significant effect on insulin secretion in the presence of a competitive inhibitor of cyclic AMP (Rp isomer of 3',5'-cyclic phosphothiorate;1mM). H-89 (100 nM) a blocker of cyclic AMP-dependent protein kinase (PKA) activity also eliminated the stimulatory effects of ACTH. These results indicate that the MC2 receptor is present in MIN6 cells and functionally coupled to insulin secretion through a cyclic AMP / PKA dependent pathway.
08 - 11 Apr 2002
British Endocrine Societies