Oscillatory membrane potential response to glucose in islet beta-cells: a comparison of islet-cell electrical activity in mouse and rat

In contrast to mouse, rat islet beta-cell membrane potential is reported not to oscillate in response to elevated glucose despite demonstrated oscillations in calcium and insulin secretion. We aim to clarify the electrical activity of rat islet beta-cells and characterize and compare the electrical activity of both alpha- and beta-cells in rat and mouse islets. We recorded electrical activity from alpha- and beta-cells within intact islets from both mouse and rat using the perforated whole-cell patch clamp technique. Fifty-six percent of both mouse and rat beta-cells exhibited an oscillatory response to 11.1 mm glucose. Responses to both 11.1 mm and 2.8 mm glucose were identical in the two species. Rat beta-cells exhibited incremental depolarization in a glucose concentration-dependent manner. We also demonstrated electrical activity in human islets recorded under the same conditions. In both mouse and rat alpha-cells 11 mm glucose caused hyperpolarization of the membrane potential, whereas 2.8 mm glucose produced action potential firing. No species differences were observed in the response of alpha-cells to glucose. This paper is the first to demonstrate and characterize oscillatory membrane potential fluctuations in the presence of elevated glucose in rat islet beta-cells in comparison with mouse. The findings promote the use of rat islets in future electrophysiological studies, enabling consistency between electrophysiological and insulin secretion studies. An inverse response of alpha-cell membrane potential to glucose furthers our understanding of the mechanisms underlying glucose sensitive glucagon secretion.     

Distribution of melatonin receptors in murine pancreatic islets

Melatonin has multiple receptor-dependent and receptor-independent functions. At the cell membrane, melatonin interacts with its receptors MT1 and MT2, which are expressed in numerous tissues. Genome-wide association studies have recently shown that the MTNR1B/MT2 receptor may be involved in the pathogenesis of type 2 diabetes mellitus. In line with these findings, expression of melatonin receptors has been shown in mouse, rat, and human pancreatic islets. MT1 and MT2 are G-protein-coupled receptors and are proposed to exert inhibitory effects on insulin secretion. Here, we show by immunocytochemistry that these membrane melatonin receptors have distinct locations in the mouse islet. MT1 is expressed in α-cells while MT2 is located to the β-cells. These findings help to unravel the complex machinery underlying melatonin's role in the regulation of islet function.     

Effects of neutral red and imidazole upon insulin secretion

Summary The present communication deals with the possible effect of glucagon released from the alpha-cells upon insulin secretion induced by glucose in the adjacent beta-cells. First, it has been shown that neutral red, a substance which is thought to cause glucagon release from the alpha-cells, did not modify the rate of insulin secretion induced by glucose in pieces of rat pancreatic tissue. Secondly, glucose stimulated insulin secretion under conditions where imidazole, a substance known to activate the phosphodiesterase, completely abolished the stimulant effect of exogenous glucagon upon insulin secretion. It is concluded that glucagon is not necessarily involved in the stimulation of insulin secretion by glucose, and that the effect of locally released glucagon, if any, is only to enhance the stimulant action of glucose.Supported in part by a grant-in-aid from the Lilly Research Laboratories, and by the association contract Euratom-Universities of Pisa and Brussels (026-63-04 BIAC).     

No mantle formation in rodent islets—The prototype of islet revisited

Emerging reports on human islets emphasize distinct differences from the widely accepted prototype of rodent islets, raising questions over their suitability for human studies. Here we aim at elucidating architectural differences and similarities of human versus rodent islets. The cellular composition and architecture of human and rodent islets were compared through three-dimensional (3D) reconstructions. Physiological and pathological changes were examined using islets from various mouse models such as non-obese diabetic (NOD), ob/ob, db/db mice and during pregnancy. A subpopulation of human islets is composed of clusters of alpha-cells within the central beta-cell cores, while the overall proportion of alpha-cells varies among islets. In mouse islets under normal conditions, alpha-cells are localized in the islet periphery, but they do not envelop the entire beta-cell core, so that beta-cells are exposed on the outer layer of the islet, as in most human islets. Also, an increased proportion of alpha-cells within the central core is observed in the pancreas of mouse models exhibiting increased demand for insulin. In summary, human and mouse islets share common architectural features as endocrine micro-organs. Since these may hold a key to better understanding islet plasticity, our concept of the prototypic islet should be revised.     

Melatonin influences somatostatin secretion from human pancreatic δ‐cells via MT1 and MT2 receptors

Melatonin is an effector of the diurnal clock on pancreatic islets. The membrane receptor‐transmitted inhibitory influence of melatonin on insulin secretion is well established and contrasts with the reported stimulation of glucagon release from α‐cells. Virtually, nothing is known concerning the melatonin‐mediated effects on islet δ‐cells. Analysis of a human pancreatic δ‐cell model, the cell line QGP‐1, and the use of a somatostatin‐specific radioimmunoassay showed that melatonin primarily has an inhibitory effect on somatostatin secretion in the physiological concentration range. In the pharmacological range, melatonin elicited slightly increased somatostatin release from δ‐cells. Cyclic adenosine monophosphate (cAMP) is the major second messenger dose‐dependently stimulating somatostatin secretion, in experiments employing the membrane‐permeable 8‐Br‐cAMP. 8‐Br‐cyclic guanosine monophosphate proved to be of only minor relevance to somatostatin release. As the inhibitory effect of 1 nm melatonin was reversed after incubation of QGP‐1 cells with the nonselective melatonin receptor antagonist luzindole, but not with the MT2‐selective antagonist 4‐P‐PDOT (4‐phenyl‐2‐propionamidotetraline), an involvement of the MT1 receptor can be assumed. Somatostatin release from the δ‐cells at low glucose concentrations was significantly inhibited during co‐incubation with 1 nm melatonin, an effect which was less pronounced at higher glucose levels. Transient expression experiments, overexpressing MT1, MT2, or a deletion variant as a control, indicated that the MT1 and not the MT2 receptor was the major transmitter of the inhibitory melatonin effect. These data point to a significant influence of melatonin on pancreatic δ‐cells and on somatostatin release.     

Cannabinoid receptor agonists and antagonists stimulate insulin secretion from isolated human islets of Langerhans

Aims: The role of cannabinoid receptors in human islets of Langerhans has not been investigated in any detail, so the current study examined CB1 and CB2 receptor expression by human islets and the effects of pharmacological cannabinoid receptor agonists and antagonists on insulin secretion. Methods: Human islets were isolated from pancreases retrieved from heart‐beating organ donors. Messenger RNAs encoding human CB1 and CB2 receptors were amplified from human islet RNA by RT‐PCR and receptor localization within islets was identified by immunohistochemistry. Dynamic insulin secretion from human islets perifused with buffers supplemented with CB1 and CB2 receptor agonists and antagonists was quantified by radioimmunoassay. Results: RT‐PCR showed that both CB1 and CB2 receptors are expressed by human islets and immunohistochemistry indicated that receptor expression co‐localized with insulin‐expressing β‐cells. Perifusion experiments using isolated human islets showed that insulin secretion was reversibly stimulated by both CB1 and CB2 receptor agonists, with CB1 receptor activation associated with increased basal secretion whereas CB2 receptors were coupled to initiation and potentiation of insulin secretion. Antagonists at CB1 (N‐(Piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide) and CB2 (N‐(1,3‐Benzodioxol‐5‐ylmethyl)‐1,2‐dihydro‐7‐methoxy‐2‐oxo‐8‐(pentyloxy)‐3‐quinoline carboxamide) receptors failed to inhibit the stimulatory effects of the respective agonists and, unexpectedly, reversibly stimulated insulin secretion. Conclusions: These data confirm the expression of CB1 and CB2 receptors by human islets and indicate that both receptor subtypes are coupled to the stimulation of insulin secretion. They also implicate involvement of CB1/2 receptor‐independent pathways in the antagonist‐induced stimulatory effects.     

Melatonin, endocrine pancreas and diabetes

Melatonin influences insulin secretion both in vivo and in vitro. (i) The effects are MT(1)-and MT(2)-receptor-mediated. (ii) They are specific, high-affinity, pertussis-toxin-sensitive, G(i)-protein-coupled, leading to inhibition of the cAMP-pathway and decrease of insulin release. [Correction added after online publication 4 December 2007: in the preceding sentence, 'increase of insulin release' was changed to 'decrease of insulin release'.] Furthermore, melatonin inhibits the cGMP-pathway, possibly mediated by MT(2) receptors. In this way, melatonin likely inhibits insulin release. A third system, the IP(3)-pathway, is mediated by G(q)-proteins, phospholipase C and IP(3), which mobilize Ca(2+) from intracellular stores, with a resultant increase in insulin. (iii) Insulin secretion in vivo, as well as from isolated islets, exhibits a circadian rhythm. This rhythm, which is apparently generated within the islets, is influenced by melatonin, which induces a phase shift in insulin secretion. (iv) Observation of the circadian expression of clock genes in the pancreas could possibly be an indication of the generation of circadian rhythms in the pancreatic islets themselves. (v) Melatonin influences diabetes and associated metabolic disturbances. The diabetogens, alloxan and streptozotocin, lead to selective destruction of beta-cells through their accumulation in these cells, where they induce the generation of ROS. Beta-cells are very susceptible to oxidative stress because they possess only low-antioxidative capacity. Results suggest that melatonin in pharmacological doses provides protection against ROS. (vi) Finally, melatonin levels in plasma, as well as the arylalkylamine-N-acetyltransferase (AANAT) activity, are lower in diabetic than in nondiabetic rats and humans. In contrast, in the pineal gland, the AANAT mRNA is increased and the insulin receptor mRNA is decreased, which indicates a close interrelationship between insulin and melatonin.     

Melatonin influences insulin secretion primarily via MT(1) receptors in rat insulinoma cells (INS-1) and mouse pancreatic islets

Several studies have revealed that melatonin affects the insulin secretion via MT(1) and MT(2) receptor isoforms. Owing to the lack of selective MT(1) receptor antagonists, we used RNA interference technology to generate an MT(1) knockdown in a clonal β-cell line to evaluate whether melatonin modulates insulin secretion specifically via the MT(1) receptor. Incubation experiments were carried out, and the insulin concentration in supernatants was measured using a radioimmunoassay. Furthermore, the intracellular cAMP was determined using an enzyme-linked immunosorbent assay. Real-time RT-PCR indicated that MT(1) knockdown resulted in a significant increase in the rIns1 mRNA and a significantly elevated basal insulin secretion of INS-1 cells. Incubation with melatonin decreased the amount of glucagon-like peptide 1 or inhibited the glucagon-stimulated insulin release of INS-1 cells, while, in MT(1) -knockdown cells, no melatonin-induced reduction in insulin secretion could be found. No decrease in 3-isobutyl-1-methylxanthine-stimulated intracellular cAMP in rMT(1) -knockdown cells was detectable after treatment with melatonin either, and immunocytochemistry proved that MT(1) knockdown abolished phosphorylation of cAMP-response-element-binding protein. In contrast to the INS-1 cells, preincubation with melatonin did not sensitize the insulin secretion of rMT(1) -knockdown cells. We also monitored insulin secretion from isolated islets of wild-type and melatonin-receptor knockout mice ex vivo. In islets of wild-type mice, melatonin treatment resulted in a decrease in insulin release, whereas melatonin treatment of islets from MT(1) knockout and MT(1/2) double-knockout mice did not show a significant effect. The data indicate that melatonin inhibits insulin secretion, primarily via the MT(1) receptor in rat INS-1 cells and isolated mouse islets.     

Evidence of the receptor‐mediated influence of melatonin on pancreatic glucagon secretion via the Gαq protein‐coupled and PI3K signaling pathways

Abstract: Melatonin has been shown to modulate glucose metabolism by influencing insulin secretion. Recent investigations have also indicated a regulatory function of melatonin on the pancreatic α‐cells. The present in vitro and in vivo studies evaluated whether melatonin mediates its effects via melatonin receptors and which signaling cascade is involved. Incubation experiments using the glucagon‐producing mouse pancreatic α‐cell line αTC1 clone 9 (αTC1.9) as well as isolated pancreatic islets of rats and mice revealed that melatonin increases glucagon secretion. Preincubation of αTC1.9 cells with the melatonin receptor antagonists luzindole and 4P‐PDOT abolished the glucagon‐stimulatory effect of melatonin. In addition, glucagon secretion was lower in the pancreatic islets of melatonin receptor knockout mice than in the islets of the wild‐type (WT) control animals. Investigations of melatonin receptor knockout mice revealed decreased plasma glucagon concentrations and elevated mRNA expression levels of the hepatic glucagon receptor when compared to WT mice. Furthermore, studies using pertussis toxin, as well as measurements of cAMP concentrations, ruled out the involvement of Gαi‐ and Gαs‐coupled signaling cascades in mediating the glucagon increase induced by melatonin. In contrast, inhibition of phospholipase C in αTC1.9 cells prevented the melatonin‐induced effect, indicating the physiological relevance of the Gαq‐coupled pathway. Our data point to the involvement of the phosphatidylinositol 3‐kinase signaling cascade in mediating melatonin effects in pancreatic α‐cells. In conclusion, these findings provide evidence that the glucagon‐stimulatory effect of melatonin in pancreatic α‐cells is melatonin receptor mediated, thus supporting the concept of melatonin‐modulated and diurnal glucagon release.     

Glucose stimulates glucagon release in single rat alpha-cells by mechanisms that mirror the stimulus-secretion coupling in beta-cells

In isolated rat pancreatic alpha-cells, glucose, arginine, and the sulfonylurea tolbutamide stimulated glucagon release. The effect of glucose was abolished by the KATP-channel opener diazoxide as well as by mannoheptulose and azide, inhibitors of glycolysis and mitochondrial metabolism. Glucose inhibited KATP-channel activity by 30% (P<0.05; n=5) and doubled the free cytoplasmic Ca2+ concentration. In cell-attached recordings, azide opened KATP channels. The N-type Ca2+-channel blocker omega-conotoxin and the Na+-channel blocker tetrodotoxin inhibited glucose-induced glucagon release whereas tetraethylammonium, a blocker of delayed rectifying K+ channels, increased secretion. Glucagon release increased monotonically with increasing K+ concentrations. omega-Conotoxin suppressed glucagon release to 15 mM K+, whereas a combination of omega-conotoxin and an L-type Ca2+-channel inhibitor was required to abrogate secretion in 50 mM K+. Recordings of cell capacitance revealed that glucose increased the exocytotic response evoked by membrane depolarization 3-fold. This correlated with a doubling of glucagon secretion by glucose in intact rat islets exposed to diazoxide and high K+. In whole-cell experiments, exocytosis was stimulated by reducing the cytoplasmic ADP concentration, whereas changes of the ATP concentration in the physiological range had little effect. We conclude that glucose stimulates glucagon release from isolated rat alpha-cells by KATP-channel closure and stimulation of Ca2+ influx through N-type Ca2+ channels. Glucose also stimulated exocytosis by an amplifying mechanism, probably involving changes in adenine nucleotides. The stimulatory action of glucose in isolated alpha-cells contrasts with the suppressive effect of the sugar in intact islets and highlights the primary importance of islet paracrine signaling in the regulation of glucagon release.     

Colocalization of somatostatin receptor sst5 and insulin in rat pancreatic beta-cells.

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is secreted by pancreatic delta-cells and inhibits the secretion of both insulin and glucagon. SRIF initiates its actions by binding to a family of six G protein-coupled receptors (sst1, -2A, -2B, -3, -4, and -5) encoded by five genes. Messenger RNA for both sst2 and sst5 have been reported in the rat pancreas, and the sst2A receptor protein has been localized to rat pancreatic alpha and pancreatic polypeptide-secreting cells in the islets as well as to pancreatic acinar cells. In this study we have used double immunostaining to show that the sst5 protein is expressed exclusively in the beta-cells of rat pancreatic islets and localizes with insulin-secreting alpha-cells. The sst5 receptor is not colocalized with sst2A. Thus, in the rat SRIF inhibits pancreatic insulin and glucagon secretion via different sst receptor subtypes.     

Direct regulation of insulin secretion by angiotensin II in human islets of Langerhans

Aims/hypothesis This study aimed to identify the expression of angiotensin II receptors in isolated human islets and beta cells and to examine the functional consequences of their activation. Materials and methods Single-cell RT-PCR was used to identify whether human islet cells express mRNA for type 1 angiotensin II receptors (AT₁), and western blotting was used to determine AT₁ protein expression by human islets and MIN6 beta cells. We measured changes in intracellular calcium by microfluorimetry using Fura 2-loaded MIN6 cells and human islet cells. Dynamic insulin secretory responses were determined by RIA following perifusion of human islets and MIN6 cells. Results Human islets expressed mRNAs for both the angiotensin precursor, angiotensinogen, and for angiotensin-converting enzyme. In addition, human and mouse beta cells expressed AT₁. These were functionally coupled to increases in intracellular calcium, which occurred at least in part through phospholipase-C-sensitive mechanisms and calcium influx through voltage-operated calcium channels. Short-term exposure of human islets and MIN6 cells to angiotensin II caused a rapid, short-lived initiation of insulin secretion at 2 mmol/l glucose and potentiation of insulin secretion induced by glucose (at 8 and 16.7 mmol/l). Conclusions/interpretation These data demonstrate that the AT₁ is expressed by beta cells and that angiotensin II effects a short-lived and direct stimulation of human and mouse beta cells to promote insulin secretion, most probably through elevations in intracellular calcium. Locally produced angiotensin II may be important in regulating a coordinated insulin secretory response from beta cells.     

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.     

Distribution and density of melatonin receptors in human main pancreatic islet cell types

Recent investigations of our group established that melatonin modulates hormone secretion of pancreatic islets via melatonin receptor types MT1 and MT2. Expression of MT1 and MT2 has been shown in mouse, rat, and human pancreatic islets as well as in the β‐, α‐, and δ‐cell lines INS‐1, αTC1.9, and QGP‐1. In view of these earlier investigations, this study was performed to analyze in detail the distribution and density of melatonin receptors on the main islet cell types in human pancreatic tissue obtained from nondiabetic and type 2 diabetic patients. Immunohistochemical analysis established the presence of MT1 and MT2 in β‐, α‐, and δ‐cells, but notably, with differences in receptor density. In general, the lowest MT1 and MT2 receptor density was measured in α‐cells compared to the 2 other cell types. In type 2 diabetic islets, MT1 and MT2 receptor density was increased in δ‐cells compared to normoglycemic controls. In human islets in batch culture of a nondiabetic donor, an increase of somatostatin secretion was observed under melatonin treatment while in islets of a type 2 diabetic donor, an inhibitory influence could be observed, especially in the presence of 5.5 mmol/L glucose. These data suggest the following: i) cell‐type‐specific density of MT1 and MT2 receptors in human pancreatic islets, which should be considered in context of the hormone secretion of islets, ii) the influence of diabetes on density of MT1 and MT2 as well as iii) the differential impact of melatonin on somatostatin secretion of nondiabetic and type 2 diabetic islets.     

Activation of insulin and IGF-1 signaling pathways by melatonin through MT1 receptor in isolated rat pancreatic islets

Melatonin diminishes insulin release through the activation of MT1 receptors and a reduction in cAMP production in isolated pancreatic islets of neonate and adult rats and in INS-1 cells (an insulin-secreting cell line). The pancreas of pinealectomized rats exhibits degenerative pathological changes with low islet density, indicating that melatonin plays a role to ensure the functioning of pancreatic beta cells. By using immunoprecipitation and immunoblotting analysis we demonstrated, in isolated rat pancreatic islets, that melatonin induces insulin growth factor receptor (IGF-R) and insulin receptor (IR) tyrosine phosphorylation and mediates the activities of the PI3K/AKT and MEK/ERKs pathways, which are involved in cell survival and growth, respectively. Thus, the effects of melatonin on pancreatic islets do not involve a reduction in cAMP levels only. This indoleamine may regulate growth and differentiation of pancreatic islets by activating IGF-I and insulin receptor signaling pathways.     

Glucagon plays an important role in the modification of insulin secretion by leptin

Obese people show marked hyerinsulinemia, but the exact mechanism has not been clarified.?Hyperleptinemia is one of possible candidates, although there is an obvious difference in the effect of leptin on insulin secretion between isolated pancreatic islets and β-cell line.?Since glucagon may modulate the effect of leptin on insulin secretion, we determined the influences of glucagon in the leptin effect on insulin secretion.?The influences of glucagon in the leptin effect on insulin secretion for 10 minutes were determined by using isolated mouse islets and HIT-T 15 cells.?The influences of 3-isobutyl-1- methylxanthine (IBMX), forskolin, and dibutyryl cyclic AMP were investigated in the leptin effect on insulin secretion.?Leptin-inhibited insulin and glucagon secretion in isolated mouse pancreatic islets.?In contrast, leptin stimulated insulin secretion in isolated mouse islets previously incubated with monoclonal anti-glucagon antibodies for 18 hours.?In HIT-T 15 cells, leptin dose-dependently increased insulin secretion, but this effect was attenuated by the addition of glucagon.?The stimulatory effect of leptin on insulin secretion was attenuated by 48 hour pre-incubation with glucagon.?In the presence of 100 mM IBMX, leptin decreased insulin secretion from HIT-T 15 cells.?Leptin also reduced insulin secretion in the presence of 1mM forskolin or 1mM dibutyryl cyclic AMP.?The leptin effects on insulin secretion were affected by the existence of glucagon. Intracellular cyclic AMP concentrations may determine the leptin effects on insulin secretion in pancreatic β-cells.     

MIN6 is not a pure beta cell line but a mixed cell line with other pancreatic endocrine hormones

MIN6 cells retains glucose-stimulated insulin secretion (GSIS) as isolated islets. We comprehensively evaluated the gene expression and production of other islet hormones in MIN6 cells. Islet hormones were demonstrated by immunohistochemical staining and measured by ELISA. The gene expression profiles of MIN6 cells were compared with those in the mouse islets obtained by the laser capture micro-dissection (LCM). MIN6 cells excreted insulin, glucagon, somatostatin and ghrelin. They expressed mRNAs of insulin I and II, proglucagon, somatostatin, pancreatic polypeptide (PP) and ghrelin which were shown in the mouse pancreatic islet core and periphery obtained by LCM. A variety of genes closely related to the islet hormone producing cells were expressed in MIN6. Confocal laser scanning microscopy revealed that MIN6 cells included not only insulin positive cells but also insulin and glucagon or somatostin double positive cells. Glucagon, somatostatin and ghrelin were detectable in the culture medium. The present study clearly demonstrated that MIN6 produce pancreatic endocrine cells. It would be possible to use this cell line as a model to research the development, cell differentiation and function of pancreatic islets.     

A role for kisspeptin in islet function

Aims/hypothesis We investigated the production of kisspeptin (KISS1) and the KISS1 receptor, GPR54, in pancreatic islets and determined the effects of exogenous kisspeptin on insulin secretion.Methods RT-PCR and immunohistochemistry were used to detect expression of KISS1 and GPR54 mRNAs and the production of KISS1 and GPR54 in human and mouse islets and in beta (MIN6) and alpha- (alphaTC1) cell lines. The effects of KISS1 on basal and glucose-induced insulin secretion from mouse and human islets were measured in a perifusion system.Results KISS1 and GPR54 mRNAs were both detected in human and mouse islets, and GPR54 mRNA expression was also found in the MIN6 and alphaTC1 endocrine cell lines. In sections of mouse pancreas, KISS1 and GPR54 immunoreactivities were co-localised in both beta and alpha cells within islets, but were not detected in the exocrine pancreas. Exposure of mouse and human islets to KISS1 caused a stimulation of glucose-induced (20 mmol/l) insulin secretion, but had no effect on the basal rate of secretion at a sub-stimulatory concentration of glucose (2 mmol/l). In contrast, KISS1 inhibited insulin secretion from MIN6 cells at both 2 and 20 mmol/l glucose. KISS1 had no significant effect on glucagon secretion from mouse islets.Conclusions/interpretation This is the first report to show that the GPR54/KISS1 system is expressed in the endocrine pancreas, where it influences beta cell secretory function. These observations suggest an important role for this system in the normal regulation of islet function.     

GPR54 peptide agonists stimulate insulin secretion from murine,porcine and human islets

This study was designed to determine the effects of 10 and 13 amino acid forms of kisspeptin on dynamic insulin secretion from mammalian islets since it is not clear from published data whether the shorter peptide is stimulatory while the longer peptide inhibits insulin release. Insulin secretion was measured by radioimmunoassay following perifusion of human, pig, rat and mouse isolated islets with kisspeptin-10 or kisspeptin-13 in the presence of 20 mM glucose. Both peptides stimulated rapid, reversible potentiation of glucose-stimulated insulin secretion from islets of all species tested. These data indicate that both kisspeptin-10 and kisspeptin-13, which is an extension of kisspeptin-10 by three amino acids, act directly at islet β-cells of various species to potentiate insulin secretion, and suggest that inhibitory effects reported in earlier studies may reflect differences in experimental protocols.     

Effects of galanin on insulin and glucagon secretion in the rat

Galanin-like immunoreactivity has been visualized in nerve fibers in the islets of Langerhans, suggesting an involvement of galanin in the neural regulation of islet function. In this study, we investigated the effects of galanin on basal and stimulated insulin and glucagon secretion by infusing the peptide at three different dose rates in rats. We also studied the direct effect of galanin on insulin secretion from freshly isolated rat islets. At 320 pmol/kg/min, but not at 20 or 80 pmol/kg/min, galanin lowered basal plasma insulin levels. In contrast, basal plasma glucagon levels were lowered by galanin already at 20 and 80 pmol/kg/min. Furthermore, galanin inhibited both glucose- and arginine-induced insulin release at all three dose levels, whereas arginine-induced glucagon release was not affected by galanin. Glucose-stimulated insulin secretion from isolated rat islets was dose-dependently suppressed by galanin (10^-6-10^-8M). Therefore, it is concluded that galanin in rats inhibits insulin secretion, both in vivo and in vitro, and that at lower dose levels, the peptide also inhibits basal glucagon release.