ACTH stimulates insulin secretion from MIN6 cells and primary mouse and human islets of langerhans

Abstract

It has previously been suggested that ACTH and ACTH-related peptides may act as paracrine modulators of insulin secretion in the islets of Langerhans. We have, therefore, examined the expression and function of the ACTH receptor (the melanocortin 2 receptor, MC2-R) in human and mouse primary islet tissue and in the MIN6 mouse insulinoma cell line. Mouse MC2-R mRNA was detected in both MIN6 cells and mouse islet tissue by PCR amplification of cDNA. In perifusion experiments with MIN6 pseudo-islets, a small, transient increase in insulin secretion was obtained when ACTH1-24 (1 nM) was added to medium containing 2 mM glucose (control) but not when the medium glucose content was increased to 8 mM. Further investigations were performed using static incubations of MIN6 cell monolayers; ACTH1-24 (1 pM-10 nM) provoked a concentration-dependent increase in insulin secretion from MIN6 monolayer cells that achieved statistical significance at concentrations of 1 and 10 nM (150 ± 13.6% basal secretion; 187 ± 14.9% basal secretion, P<0.01). Similar responses were obtained with ACTH1-39. The phosphodiesterase inhibitor IBMX (100 μM) potentiated the responses to sub-maximal doses of ACTH1-24. Two inhibitors of the protein kinase A (PKA) signaling pathway, Rp-cAMPS (500 μM) and H-89 (10 μM), abolished the insulin secretory response to ACTH1-24 (0.5-10 nM). Treatment with 1 nM ACTH1-24 caused a small, statistically significant increase in intracellular cAMP levels. Secretory responses of MIN6 cells to ACTH1-24 were also influenced by changes in extracellular Ca2+ levels. Incubation in Ca2+-free buffer supplemented with 0.1 mM EGTA blocked the MIN6 cells' secretory response to 1 and 10 nM ACTH1-24. Similar results were obtained when a Ca2+ channel blocker (nitrendipine, 10 μM) was added to the Ca2+-containing buffer. ACTH1-24 also evoked an insulin secretory response from primary tissues. The addition of ACTH1-24 (0.5 nM) to perifusions of mouse islets induced a transient increase in insulin secretion at 8 mM glucose. Perifused human primary islets also showed a secretory response to ACTH1-24 at basal glucose concentration (2 mM) with a rapid initial spike in insulin secretion followed by a decline to basal levels. Overall the results demonstrate that the MC2-R is expressed in β-cells and suggest that activation of the receptor by ACTH initiates insulin secretion through the activation of PKA in association with Ca2+ influx into β-cells.