Investigation of intracellular signalling cascades mediating stimulatory effect of a Gymnema sylvestre extract on insulin secretion from isolated mouse and human islets of Langerhans.

AIM: Traditional plant-based remedies such as Gymnema sylvestre (GS) extracts have been used to treat diabetes mellitus for many centuries. We have shown previously that a novel GS extract, OSA®, has a direct effect on insulin secretion but its mode of action has not been studied in detail Thus this study investigated the possible underlying mechanism(s) by which OSA® exerts its action. METHODS: The effects of OSA® on [Ca(2+)]i and K(+) conductances were assessed by Ca(2+) microfluorimetry and electrophysiology in dispersed mouse islets and MIN6 ß-cells, respectively. Isolated mouse (from 20 to 25 mice) and human (from 3 donors) islets, and MIN6 ß-cells, were used to investigate whether the stimulatory effect of OSA® on insulin secretion was dependent on the presence of extracellular calcium and protein kinase activation. RESULTS: OSA ®-induced insulin secretion from mouse islets and MIN6 ß-cells was inhibited by nifedipine, a voltage-gated Ca(2+) channel blocker, and by the removal of extracellular Ca(2+), respectively. OSA® did not affect the activities of KATP channels or voltage-dependent K(+) channels in MIN6 ß-cells but it caused an increase in intracellular Ca(2+) ([Ca(2+)]i) concentrations in Fura-2-loaded mouse islet cells. The insulin secretagogue effect of OSA® was dependent, in part, on protein kinase activation since incubating mouse or human islets with staurosporine, a general protein kinase inhibitor, resulted in partial inhibition of OSA®-induced insulin secretion. Experiments using permeabilized, Ca(2+)-clamped MIN6 ß-cells revealed a Ca(2+)-independent component action of OSA® at a late stage in the stimulus-response coupling pathway. OSA®-induced insulin secretion was unexpectedly associated with a decrease in intracellular cAMP levels. CONCLUSIONS: These data indicate that the GS isolate OSA® stimulates insulin secretion from mouse and human islets in vitro, at least in part as a consequence of Ca(2+) influx and protein kinase activation.

A novel Gymnema sylvestre extract stimulates insulin secretion from human islets in vivo and in vitro.

Many plant-based products have been suggested as potential antidiabetic agents, but few have been shown to be effective in treating the symptoms of Type 2 diabetes mellitus (T2DM) in human studies, and little is known of their mechanisms of action. Extracts of Gymnema sylvestre (GS) have been used for the treatment of T2DM in India for centuries. The effects of a novel high molecular weight GS extract, Om Santal Adivasi, (OSA(R)) on plasma insulin, C-peptide and glucose in a small cohort of patients with T2DM are reported here. Oral administration of OSA(R) (1 g/day, 60 days) induced significant increases in circulating insulin and C-peptide, which were associated with significant reductions in fasting and post-prandial blood glucose. In vitro measurements using isolated human islets of Langerhans demonstrated direct stimulatory effects of OSA(R) on insulin secretion from human ß-cells, consistent with an in vivo mode of action through enhancing insulin secretion. These in vivo and in vitro observations suggest that OSA(R) may provide a potential alternative therapy for the hyperglycemia associated with T2DM.

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

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 ACTH(1-24) (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; ACTH(1-24) (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 ACTH(1-39). The phosphodiesterase inhibitor IBMX (100 microM) potentiated the responses to sub-maximal doses of ACTH(1-24). Two inhibitors of the protein kinase A (PKA) signaling pathway, Rp-cAMPS (500 microM) and H-89 (10 microM), abolished the insulin secretory response to ACTH(1-24) (0.5-10 nM). Treatment with 1 nM ACTH(1-24) caused a small, statistically significant increase in intracellular cAMP levels. Secretory responses of MIN6 cells to ACTH(1-24) 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 ACTH(1-24). Similar results were obtained when a Ca2+ channel blocker (nitrendipine, 10 microM) was added to the Ca2+-containing buffer. ACTH(1-24) also evoked an insulin secretory response from primary tIssues. The addition of ACTH(1-24) (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 ACTH(1-24) 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 beta-cells and suggest that activation of the receptor by ACTH initiates insulin secretion through the activation of PKA in association with Ca2+ influx into beta-cells.