Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets.

Insulin secretion has been studied in isolated rat pancreatic islets under stringent Ca(2+)-depleted, Ca(2+)-free conditions. Under these conditions, the effect of 16.7 mM glucose to stimulate insulin release was abolished. Forskolin, which activates adenylyl cyclase, also failed to stimulate release in the presence of either low or high glucose concentrations. A phorbol ester (phorbol 12-myristate 13-acetate; PMA) increased the release rate slightly and this was further increased by 16.7 mM glucose. Remarkably, in the presence of both forskolin and PMA, 16.7 mM glucose strongly augmented insulin release. The augmentation was concentration dependent and monophasic and had a temporal profile similar to the "second phase" of glucose-stimulated insulin release, which is seen under normal conditions when Ca2+ is present. Metabolism is required for the effect because mannoheptulose abolished the glucose response. Other nutrient secretagogues, alpha-ketoisocaproate, and the combination of leucine and glutamine augmented release under the same conditions. Norepinephrine, a physiological inhibitor of insulin secretion, totally blocked the stimulation of release by forskolin and PMA and the augmentation of release by glucose. Thus, under the stringent Ca(2+)-free conditions imposed, the stimulation of insulin release by forskolin and PMA, as well as the augmentation of release by glucose, is under normal physiological control. As no increase in intracellular [Ca2+] was observed, the results demonstrate that glucose can increase the rate of exocytosis and insulin release by pancreatic islets in a Ca(2+)-independent manner. This interesting pathway of stimulus-secretion coupling for glucose appears to exert its effect at a site beyond the usual elevation of intracellular [Ca2+] and is not due to an activation by glucose of protein kinase A or C.

Ascorbic acid is essential for the release of insulin from scorbutic guinea pig pancreatic islets.

Pancreatic islets from young normal and scorbutic male guinea pigs were examined for their ability to release insulin when stimulated with elevated D-glucose. Islets from normal guinea pigs released insulin in a D-glucose-dependent manner showing a rapid initial secretion phase and three secondary secretion waves during a 120-min period. Islets from scorbutic guinea pigs failed to release insulin during the immediate period, and only delayed and decreased responses were observed over the 40-60 min after D-glucose elevation. Insulin release from scorbutic islets was greatly elevated if 5 mM L-ascorbic acid 2-phosphate was supplemented in the perifusion medium during the last 60 min of perifusion. When 5 mM L-ascorbic acid 2-phosphate was added to the perifusion medium concurrently with elevation of medium D-glucose, islets from scorbutic guinea pigs released insulin as rapidly as control guinea pig islets and to a somewhat greater extent. L-Ascorbic acid 2-phosphate without elevated D-glucose had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, suggesting that the decreased insulin release from the scorbutic islets was not due to decreased insulin synthesis but due to abnormal insulin secretion.

Identification, isolation, and characterization of daintain (allograft inflammatory factor 1), a macrophage polypeptide with effects on insulin secretion and abundantly present in the pancreas of prediabetic BB rats

A bioactive macrophage factor, the polypeptide daintain/allograft inflammatory factor 1 (AIF1), has been isolated from porcine intestine. It was discovered when searching for intestinal peptides with effects on insulin release, and its purification was monitored by the influence of the peptide fractions on pancreatic glucose-induced insulin secretion. Daintain/AIF1 is a 146-aa residue polypeptide with a mass of 16,603 Da and an acetylated N terminus. An internal 44-residue segment with the sequence pattern –KR–KK–GKR– has a motif typical of peptide hormone precursors, i.e., dibasic sites for potential activation cleavages and at the sequentially last such site, the structure GKR. The latter is a signal for C-terminal amide formation in the processing of peptide hormones. Daintain/AIF1 is immunohistochemically localized to microglial cells in the central nervous system and to dendritic cells and macrophages in several organs. A particularly dense accumulation of daintain/AIF1-immunoreactive macrophages was observed in the insulitis affecting the pancreatic islets of prediabetic BB rats. When injected intravenously in mice, daintain/AIF1 at 75 pmol/kg inhibited glucose (1 g/kg)-stimulated insulin secretion, with a concomitant impairment of the glucose elimination, whereas at higher doses (7.5 and 75 nmol/kg), daintain/AIF1 potentiated glucose-stimulated insulin secretion and enhanced the glucose elimination. Its dual influence on insulin secretion in vivo at different peptide concentrations, and the abundance of macrophages expressing daintain/AIF1 in the pancreatic islets of prediabetic rats, suggest that daintain/AIF1 may have a role in connection with the pathogenesis of insulin-dependent diabetes mellitus.

Dissociation between changes in cytoplasmic free Ca2+ concentration and insulin secretion as evidenced from measurements in mouse single pancreatic islets.

Simultaneous measurements of cytosolic free Ca2+ concentration and insulin release, in mouse single pancreatic islets, revealed a direct correlation only initially after stimulation with glucose or K+. Later, there is an apparent dissociation between these two parameters, with translocation of alpha and epsilon isoenzymes of protein kinase C to membranes and simultaneous desensitization of insulin release in response to glucose. Recovery of insulin release, without any concomitant changes in cytosolic free Ca2+ concentration, after addition of phorbol 12-myristate 13-acetate, okadaic acid, and forskolin supports the notion that the desensitization process is accounted for by dephosphorylation of key regulatory sites of the insulin exocytotic machinery.

Pancreatic β cells synthesize and secrete nerve growth factor

Differentiation and function of pancreatic β cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic β cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in β cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic β-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

MgATP activates the β cell KATP channel by interaction with its SUR1 subunit

ATP-sensitive potassium (KATP) channels in the pancreatic β cell membrane mediate insulin release in response to elevation of plasma glucose levels. They are open at rest but close in response to glucose metabolism, producing a depolarization that stimulates Ca2+ influx and exocytosis. Metabolic regulation of KATP channel activity currently is believed to be mediated by changes in the intracellular concentrations of ATP and MgADP, which inhibit and activate the channel, respectively. The β cell KATP channel is a complex of four Kir6.2 pore-forming subunits and four SUR1 regulatory subunits: Kir6.2 mediates channel inhibition by ATP, whereas the potentiatory action of MgADP involves the nucleotide-binding domains (NBDs) of SUR1. We show here that MgATP (like MgADP) is able to stimulate KATP channel activity, but that this effect normally is masked by the potent inhibitory effect of the nucleotide. Mg2+ caused an apparent reduction in the inhibitory action of ATP on wild-type KATP channels, and MgATP actually activated KATP channels containing a mutation in the Kir6.2 subunit that impairs nucleotide inhibition (R50G). Both of these effects were abolished when mutations were made in the NBDs of SUR1 that are predicted to abolish MgATP binding and/or hydrolysis (D853N, D1505N, K719A, or K1384M). These results suggest that, like MgADP, MgATP stimulates KATP channel activity by interaction with the NBDs of SUR1. Further support for this idea is that the ATP sensitivity of a truncated form of Kir6.2, which shows functional expression in the absence of SUR1, is unaffected by Mg2+.

Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits

The ATP-sensitive potassium channel (K-ATP channel) plays a key role in insulin secretion from pancreatic β-cells. It is closed by glucose metabolism, which stimulates secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and MgADP concentration, which inhibit and potentiate channel activity, respectively. The β-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. The site at which ATP mediates channel inhibition lies on Kir6.2, while the potentiatory action of MgADP involves the nucleotide-binding domains of SUR1. K-ATP channels are also activated by MgGTP and MgGDP. Furthermore, both nucleotides support the stimulatory actions of diazoxide. It is not known, however, whether guanine nucleotides mediate their effects by direct interaction with one or more of the K-ATP channel subunits or indirectly via a GTP-binding protein. We used a truncated form of Kir6.2, which expresses independently of SUR1, to show that GTP blocks K-ATP currents by interaction with Kir6.2 and that the potentiatory effects of GTP are endowed by SUR1. We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. This argues that the stimulatory effects of guanine nucleotides require the presence of both Walker A lysines.

Phentolamine block of KATP channels is mediated by Kir6.2

The ATP-sensitive K+-channel (KATP channel) plays a key role in insulin secretion from pancreatic β cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca2+ entry and insulin release. The β cell KATP channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K+-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit KATP channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. KATP channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac KATP channel.

A common muscarinic pathway for diapause recovery in the distantly related nematode species Caenorhabditis elegans and Ancylostoma caninum

Converging TGF-β and insulin-like neuroendocrine signaling pathways regulate whether Caenorhabditis elegans develops reproductively or arrests at the dauer larval stage. We examined whether neurotransmitters act in the dauer entry or recovery pathways. Muscarinic agonists promote recovery from dauer arrest induced by pheromone as well as by mutations in the TGF-β pathway. Dauer recovery in these animals is inhibited by the muscarinic antagonist atropine. Muscarinic agonists do not induce dauer recovery of either daf-2 or age-1 mutant animals, which have defects in the insulin-like signaling pathway. These data suggest that a metabotropic acetylcholine signaling pathway activates an insulin-like signal during C. elegans dauer recovery. Analogous and perhaps homologous cholinergic regulation of mammalian insulin release by the autonomic nervous system has been noted. In the parasitic nematode Ancylostoma caninum, the dauer larval stage is the infective stage, and recovery to the reproductive stage normally is induced by host factors. Muscarinic agonists also induce and atropine potently inhibits in vitro recovery of A. caninum dauer arrest. We suggest that host or parasite insulin-like signals may regulate recovery of A. caninum and could be potential targets for antihelminthic drugs.

The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.

Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).

Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine release from the rat hippocampal formation

Insulin-like growth factors-I and -II (IGF-I and -II) are structurally related mitogenic polypeptides with potent growth promoting effects. These peptides and their corresponding IGF-I and -II receptors are selectively localized in the brain. To date, most of the effects of IGFs are believed to be mediated by IGF-I receptors whereas the significance of IGF-II receptor in mediating biological responses remains unclear. In the present study, we characterized the distribution of IGF-I and IGF-II receptor sites and investigated the effects of both factors on endogenous acetylcholine (ACh) release in adult rat hippocampus. [125I]IGF-I receptor binding sites are recognized by IGF-I> IGF-II> insulin, whereas [125I]IGF-II binding was competed potently by IGF-II> IGF-I but not by insulin. At the cellular level, IGF-I receptor sites were primarily noted in the molecular layer of the dentate gyrus and the CA2-CA3 subfields of the Ammon’s horn whereas IGF-II sites were localized predominantly in the pyramidal cell layer of the CA1-CA3 subfields and in the granular cell layer of the dentate gyrus. IGF-I (10−14–10−8 M) and des(1–3) IGF-I (10−10–10−8 M) were found to inhibit whereas IGF-II (10−14–10−8 M) potentiated K+-evoked ACh release from hippocampal slices. Tetrodotoxin altered the effects of IGF-I but not those of IGF-II suggesting that IGF-I acts indirectly via the release of other modulators whereas IGF-II acts directly on or in close proximity to the cholinergic terminals. The inhibitory effects of IGF-I were also observed in the frontal cortex but not in the striatum. In contrast, the stimulatory effects of IGF-II were evident both in the frontal cortex and striatum. Taken together, these results reveal the differential localization of IGF-I and IGF-II receptor sites in the hippocampal formation and the opposite role for these growth factors in the acute regulation of ACh release likely via two distinct mechanisms. Additionally, these data provide the first evidence for a direct role for IGF-II and its receptors in the regulation of transmitter release in the central nervous system.

Defects in inositol 1,4,5-trisphosphate receptor expression, Ca2+ signaling, and insulin secretion in the anx7(+/−) knockout mouse

The mammalian anx7 gene codes for a Ca2+-activated GTPase, which supports Ca2+/GTP-dependent secretion events and Ca2+ channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca2+ signaling in secreting pancreatic β cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the β cells. The nullizygous anx7 (−/−) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/−) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/−) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca2+ channel functions are normal. However, electrooptical recordings indicate that the (+/−) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP3)-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP3 receptor expression and function in pancreatic islets. The profound increase in islets, β cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic β cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca2+ signaling through IP3-sensitive Ca2+ stores.