Modulation Of Insulin Release Research Articles

GPR119, a novel G protein‐coupled receptor target for the treatment of type 2 diabetes and obesity

GPR119 is a G protein-coupled receptor expressed predominantly in the pancreas (beta-cells) and gastrointestinal tract (enteroendocrine cells) in humans. De-orphanization of GPR119 has revealed two classes of possible endogenous ligands, viz., phospholipids and fatty acid amides. Of these, oleoylethanolamide (OEA) is one of the most active ligands tested so far. This fatty acid ethanolamide is of particular interest because of its known effects of reducing food intake and body weight gain when administered to rodents. Agonists at the GPR119 receptor cause an increase in intracellular cAMP levels via G(alphas) coupling to adenylate cyclase. In vitro studies have indicated a role for GPR119 in the modulation of insulin release by pancreatic beta-cells and of GLP-1 secretion by gut enteroendocrine cells. The effects of GPR119 agonists in animal models of diabetes and obesity are reviewed, and the potential value of such compounds in future therapies for these conditions is discussed.

Endocannabinoid Dysregulation in the Pancreas and Adipose Tissue of Mice Fed With a High‐fat Diet

In mice, endocannabinoids (ECs) modulate insulin release from pancreatic beta-cells and adipokine expression in adipocytes through cannabinoid receptors. Their pancreatic and adipose tissue levels are elevated during hyperglycemia and obesity, but the mechanisms underlying these alterations are not understood. We assessed in mice fed for up to 14 weeks with a standard or high-fat diet (HFD): (i) the expression of cannabinoid receptors and EC biosynthesizing enzymes (N-acyl-phosphatidyl-ethanolamine-selective phospholipase D (NAPE-PLD) and DAGLalpha) and degrading enzymes (fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)) in pancreatic and adipose tissue sections by immunohistochemical staining; (ii) the amounts, measured by liquid chromatography-mass spectrometry, of the ECs, 2-AG, and anandamide (AEA). Although CB(1) receptors and biosynthetic enzymes were found mostly in alpha-cells, degrading enzymes were identified in beta-cells. Following HFD, staining for biosynthetic enzymes in beta-cells and lower staining for FAAH were observed together with an increase of EC pancreatic levels. While we observed no diet-induced change in the intensity of the staining of EC metabolic enzymes in the mesenteric visceral fat, a decrease in EC concentrations was accompanied by lower and higher staining of biosynthesizing enzymes and FAAH, respectively, in the subcutaneous fat. No change in cannabinoid receptor staining was observed following HFD in any of the analyzed tissues. We provide unprecedented information on the distribution of EC metabolic enzymes in the pancreas and adipose organ, where their aberrant expression during hyperglycemia and obesity contribute to dysregulated EC levels.

Receptor-mediated Regulation of Tomosyn-Syntaxin 1A Interactions in Bovine Adrenal Chromaffin Cells

Tomosyn, a soluble R-SNARE protein identified as a binding partner of the Q-SNARE syntaxin 1A, is thought to be critical in setting the level of fusion-competent SNARE complexes for neurosecretion. To date, there has been no direct evaluation of the dynamics in which tomosyn transits through tomosyn-SNARE complexes or of the extent to which tomosyn-SNARE complexes are regulated by secretory demand. Here, we employed biochemical and optical approaches to characterize the dynamic properties of tomosyn-syntaxin 1A complexes in live adrenal chromaffin cells. We demonstrate that secretagogue stimulation results in the rapid translocation of tomosyn from the cytosol to plasma membrane regions and that this translocation is associated with an increase in the tomosyn-syntaxin 1A interaction, including increased cycling of tomosyn into tomosyn-SNARE complexes. The secretagogue-induced interaction was strongly reduced by pharmacological inhibition of the Rho-associated coiled-coil forming kinase, a result consistent with findings demonstrating secretagogue-induced activation of RhoA. Stimulation of chromaffin cells with lysophosphatidic acid, a nonsecretory stimulus that strongly activates RhoA, resulted in effects on tomosyn similar to that of application of the secretagogue. In PC-12 cells overexpressing tomosyn, secretagogue stimulation in the presence of lysophosphatidic acid resulted in reduced evoked secretory responses, an effect that was eliminated upon inhibition of Rho-associated coiled-coil forming kinase. Moreover, this effect required an intact interaction between tomosyn and syntaxin 1A. Thus, modulation of the tomosyn-syntaxin 1A interaction in response to secretagogue activation is an important mechanism allowing for dynamic regulation of the secretory response.

Mechanisms for the coupling of cannabinoid receptors to intracellular calcium mobilization in rat insulinoma β-cells

In RIN m5F rat insulinoma β-cells, agonists at cannabinoid CB 1 receptors modulate insulin release. Here we investigated in these cells the effect of the activation of cannabinoid CB 1 and CB 2 receptors on intracellular Ca 2+ ([Ca 2+] i). The CB 1 agonist arachidonoyl-chloro-ethanolamide (ACEA), and the CB 2 agonist JWH133, elevated [Ca 2+] i in a way sensitive to the inhibitor of phosphoinositide-specific phospholipase C (PI-PLC), U73122 (but not to pertussis toxin and forskolin), and independently from extracellular Ca 2+. PI-PLC-dependent Ca 2+ mobilization by ACEA was entirely accounted for by activation of inositol-1,3,4-phosphate (IP 3) receptors on the endoplasmic reticulum (ER), whereas the effect of JWH133 was not sensitive to all tested inhibitors of IP 3 and ryanodine receptors. ACEA, but not JWH133, significantly inhibited the effect on [Ca 2+] i of bombesin, which acts via G q/11- and PI-PLC-coupled receptors in insulinoma cells. The endogenous CB 1 agonists, anandamide and N-arachidonoyldopamine, which also activate transient receptor potential vanilloid type 1 (TRPV1) receptors expressed in RIN m5F cells, elevated [Ca 2+] i in the presence of extracellular Ca 2+ in a way sensitive to both CB 1 and TRPV1 antagonists. These results suggest that, in RIN m5F cells, CB 1 receptors are coupled to PI-PLC-mediated mobilization of [Ca 2+] i and might inhibit bombesin signaling.

Dopamine D2-like Receptors Are Expressed in Pancreatic Beta Cells and Mediate Inhibition of Insulin Secretion

Dopamine signaling is mediated by five cloned receptors, grouped into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families. We identified by reverse transcription-PCR the presence of dopamine receptors from both families in INS-1E insulin-secreting cells as well as in rodent and human isolated islets. D2 receptor expression was confirmed by immunodetection revealing localization on insulin secretory granules of INS-1E and primary rodent and human beta cells. We then tested potential effects mediated by the identified receptors on beta cell function. Dopamine (10 microM) and the D2-like receptor agonist quinpirole (5 microM) inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E beta cells, fluorescence-activated cell-sorted primary rat beta cells, and pancreatic islets of rat, mouse, and human origin. Insulin exocytosis is controlled by metabolism coupled to cytosolic calcium changes. Measurements of glucose-induced mitochondrial hyperpolarization and ATP generation showed that dopamine and D2-like agonists did not inhibit glucose metabolism. On the other hand, dopamine decreased cell membrane depolarization as well as cytosolic calcium increases evoked by glucose stimulation in INS-1E beta cells. These results show for the first time that dopamine receptors are expressed in pancreatic beta cells. Dopamine inhibited glucose-stimulated insulin secretion, an effect that could be ascribed to D2-like receptors. Regarding the molecular mechanisms implicated in dopamine-mediated inhibition of insulin release, our results point to distal steps in metabolism-secretion coupling. Thus, the role played by dopamine in glucose homeostasis might involve dopamine receptors, expressed in pancreatic beta cells, modulating insulin release.

A Highly Ca2+-sensitive Pool of Granules Is Regulated by Glucose and Protein Kinases in Insulin-secreting INS-1 Cells

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca2+ to directly measure the Ca2+ sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca2+ sensitivity of release in that a small proportion of granules makes up a highly Ca2+-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca2+. A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca2+ are not located close to Ca2+ channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca2+-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca2+, suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca2+ sensor that is poised to respond to modest, global elevations of [Ca2+]i.

Synthesis and evaluation of 7-substituted-3-cyclobutylamino-4H-1,2,4-benzothiadiazine-1,1-dioxide derivatives as KATP channel agonists

A series of 7-substituted-3-cyclobutylamino-4H-1,2,4-benzothiadiazine-1,1-dioxide derivatives has been synthesized and evaluated as KATP channel agonists using the inside-out excised patch clamp technique. The most active compounds were ∼20-fold more potent than diazoxide in opening KATP channels. A linear relationship exists between the potency of the compound and the sigma value of the 7-substituent with electron-withdrawing groups exhibiting higher activity. These compounds may be useful in modulating insulin release from pancreatic β-cells and in diseases associated with hyperinsulinemia.

COMPARISON OF THE SECRETORY PROPERTIES OF FOUR INSULIN-SECRETING CELL LINES

The insulin-secretory responsiveness of four popular and widely used insulin-secreting cells lines (RINm5F, HIT-T15, INS-1 and BRIN-BD11cells) to a range of stimuli including glucose, amino acids, neurotransmitters, peptide hormones and sulphonylureas was studied. Differences were seen in the pattern of responsiveness of the cell lines to the various modulators of insulin release. While these studies revealed that INS-1 cells had the highest insulin content, only BRIN-BD11 cells exhibited a significant step-wise insulin secretory response to increasing glucose concentrations. BRIN-BD11 cells also showed pronounced insulin responses to leucine, KIC, L-arginine, L-alanine and palmitic acid. All the cell lines tested gave significant responses to the neurotransmitters carbachol and glibenclamide with increased insulin release. A comparison was made between the functional characteristics of the various cell lines with those of freshly isolated rat islets. This illustrated the general value of each cell line as a model for studies of insulin secretion. Electrofusion-derived BRIN-BD11 cells appeared to closely mimic the glucose sensitivity and overall secretory performance of normal rat islets.

Possible modulatory effect of endogenous islet catecholamines on insulin secretion.

BackgroundThe possible participation of endogenous islet catecholamines (CAs) in the control of insulin secretion was tested.MethodsGlucose-induced insulin secretion was measured in the presence of 3-Iodo-L-Tyrosine (MIT), a specific inhibitor of tyrosine-hydroxylase activity, in fresh and precultured islets isolated from normal rats. Incubated islets were also used to measure CAs release in the presence of low and high glucose, and the effect of α2-(yohimbine [Y] and idazoxan [I]) and α1-adrenergic antagonists (prazosin [P] and terazosin [T]) upon insulin secretion elicited by high glucose.ResultsFresh islets incubated with 16.7 mM glucose released significantly more insulin in the presence of 1 μM MIT (6.66 ± 0.39 vs 5.01 ± 0.43 ng/islet/h, p < 0.02), but did not affect significantly the insulin response to low glucose. A similar enhancing effect of MIT upon insulin secretion was obtained using precultured islets devoid of neural cells, but absolute values were lower than those from fresh islets, suggesting that MIT inhibits islet rather than neural tyrosine hydroxylase. CAs concentration in the incubation media of fresh isolated islets was significantly higher in the presence of 16.7 than 3.3 mM glucose: dopamine 1.67 ± 0.13 vs 0.69 ± 0.13 pg/islet/h, p < 0.001, and noradrenaline 1.25 ± 0.17 vs 0.49 ± 0.04 pg/islet/h, p < 0.02. Y and I enhanced the release of insulin elicited by 16.7 mM glucose while P and T decreased such secretion.ConclusionOur results suggest that islet-originated CAs directly modulate insulin release in a paracrine manner.

Role of protein kinase C, PI3-kinase and tyrosine kinase in activation of MAP kinase by glucose and agonists of G-protein coupled receptors in INS-1 cells.

MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and []ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 (=51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). []Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but not for its insulin secretory response with respect to major initiators and modulators of insulin release. The data indicate that MAP kinase is active and under the control of MAP kinase. PKC is upstream of a genisteinsensitive tyrosine kinase and probably downstream of a PI3-kinase in INS-1 cells.

Possible modulatory effect of endogenous islet catecholamines on insulin secretion

The possible participation of endogenous islet catecholamines (CAs) in the control of insulin secretion was tested. Glucose-induced insulin secretion was measured in the presence of 3-Iodo-L-Tyrosine (MIT), a specific inhibitor of tyrosine-hydroxylase activity, in fresh and precultured islets isolated from normal rats. Incubated islets were also used to measure CAs release in the presence of low and high glucose, and the effect of α2-(yohimbine [Y] and idazoxan [I]) and α1-adrenergic antagonists (prazosin [P] and terazosin [T]) upon insulin secretion elicited by high glucose. Fresh islets incubated with 16.7 mM glucose released significantly more insulin in the presence of 1 μM MIT (6.66 ± 0.39 vs 5.01 ± 0.43 ng/islet/h, p < 0.02), but did not affect significantly the insulin response to low glucose. A similar enhancing effect of MIT upon insulin secretion was obtained using precultured islets devoid of neural cells, but absolute values were lower than those from fresh islets, suggesting that MIT inhibits islet rather than neural tyrosine hydroxylase. CAs concentration in the incubation media of fresh isolated islets was significantly higher in the presence of 16.7 than 3.3 mM glucose: dopamine 1.67 ± 0.13 vs 0.69 ± 0.13 pg/islet/h, p < 0.001, and noradrenaline 1.25 ± 0.17 vs 0.49 ± 0.04 pg/islet/h, p < 0.02. Y and I enhanced the release of insulin elicited by 16.7 mM glucose while P and T decreased such secretion. Our results suggest that islet-originated CAs directly modulate insulin release in a paracrine manner.

Metallothionein induction in islets of Langerhans and insulinoma cells

Isolated pancreatic islets from rat and mouse and the insulinoma cell lines, βHC9 and RINm5F, were investigated to determine the regulation of metallothionein (MT). Dexamethasone (DEX) increased rat and mouse islet and insulinoma cell MT levels in a time- and concentration-dependent manner. Rat islet MT expression was increased with interleukin-1β (IL-1β), but not tumor necrosis factor-α (TNF). However, MT induction by IL-1β and TNF was synergistic with DEX in rat islets and insulinoma cells. Mouse islet MT failed to respond to IL-1β alone, although IL-1β and TNF were synergistic. IL-1β and TNF did not synergize with DEX for mouse islet MT induction. Zinc sulfate induced MT in rat islets but not mouse islets. MT messenger RNA levels were significantly increased in rat islets in response to DEX and IL-1β plus DEX. The inducible nitric oxide synthase inhibitors N G-monomethyl- l-arginine and aminoguanidine failed to inhibit IL-1β induced MT levels in insulinoma cells, and the nitric oxide generating agent sodium nitroprusside failed to significantly affect MT levels. Phorbol dibutyrate increased MT levels in rat islets and βHC9 cells, but phorbol dibutyrate and IL-1β effects were not additive. Transgenic MT-null and wild-type mouse islets had similar insulin contents, but basal and glucose-stimulated insulin release from MT-null islets were significantly lower than in wild-type islets. Blood glucose levels in MT-null mice were, however, slightly lower than those in wild-type mice. Thus, MT induction in pancreatic islets and β-cells is regulated by cytokines and DEX, and protein kinase C activation may play a role. However, regulation of MT induction in mouse and rat islets differs. MT also appears to modulate insulin release from pancreatic islets.

Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells.

In this article, we show that glucagon-like peptide 1 (GLP-1) can induce AR42J cells to differentiate into insulin, pancreatic polypeptide, and glucagon-positive cells. In their natural state, these cells, which are derived from a chemically induced pancreatic tumor, possess exocrine and neuroendocrine properties but are negative for islet hormones and their mRNAs. We found that when these cells were exposed to GLP-1 (1 or 10 nmol), a peptide normally released from the gut in response to food and a modulator of insulin release, intracellular cAMP levels were increased, and proliferation of cells was increased for the first 24 h, followed by inhibition. Up to 50% of the cells became positive for islet hormones. The mRNAs for glucose transporter 2 and glucokinase were detected in the GLP-1-treated cells. Insulin was detected by radioimmunoassay (RIA) in the medium of GLP-1-treated cells, and the cells were capable of releasing insulin in a glucose-mediated fashion. Exendin-4, an analog of GLP-1, in some critical experiments performed in a similar manner to GLP-1, with the exception of it being 10-fold more potent. We therefore propose that GLP-1 and exendin-4 are capable of causing pancreatic precursor cells to differentiate into islet cells.

Miniglucagon (Glucagon 19–29), a Potent and Efficient Inhibitor of Secretagogue-induced Insulin Release through a Ca2+Pathway

Using the MIN6 B-cell line, we investigated the hypothesis that miniglucagon, the C-terminal () fragment processed from glucagon and present in pancreatic A cells, modulates insulin release, and we analyzed its cellular mode of action. We show that, at concentrations ranging from 0.01 to 1000 pM, miniglucagon dose-dependently (ID50 = 1 pM) inhibited by 80-100% the insulin release triggered by glucose, glucagon, glucagon-like peptide-1-(7-36) amide (tGLP-1), or glibenclamide, but not that induced by carbachol. Miniglucagon had no significant effects on cellular cAMP levels. The increase in 45Ca2+ uptake induced by depolarizing agents (glucose or extracellular K+), by glucagon, or by the Ca2+channel agonist Bay K-8644 was blocked by miniglucagon at the doses active on insulin release. Electrophysiological experiments indicated that miniglucagon induces membrane hyperpolarization, probably by opening potassium channels, which terminated glucose-induced electrical activity. Pretreatment with pertussis toxin abolished the effects of miniglucagon on insulin release. It is concluded that miniglucagon is a highly potent and efficient inhibitor of insulin release by closing, via hyperpolarization, voltage-dependent Ca2+ channels linked to a pathway involving a pertussis toxin-sensitive G protein.

Roles of intracellular Ca2+ receptors in the pancreatic beta-cell in insulin secretion.

Ca2+ is the central second messenger in the regulation of insulin release from the pancreatic beta-cell; and intracellular Ca2+ -binding proteins, classified into two groups, the EF hand proteins and the Ca2+/phospholipid binding proteins, are considered to mediate Ca2+ signaling. A number of Ca binding proteins have been suggested to participate in the secretory machinery in the beta-cell. Calmodulin, the ubiquitous EF hand protein, is the predominant intracellular Ca2+ receptor that modulates insulin release via the multiplicity of its binding to target proteins including protein kinases. Other Ca binding proteins such as calcyclin and the Ca2+/phospholipid binding proteins may also be suggested to be involved. Ca2+ influx from the extracellular space appears to be responsible for exocytosis of insulin via Ca2+ -dependent protein/protein interactions. On the other hand, intracellular Ca2+ mobilization resulting in secretory granule movement may be controlled by Ca2+/calmodulin-dependent protein phosphorylation. Thus, Ca2+ exerts versatile effects on the secretory cascade via binding to specific binding proteins in the pancreactic beta-cells.

Role of potassium channels in the modulation of insulin release.

Role of potassium channels in the modulation of insulin release.

GCAP I as a paracrine peptide in pancreatic islets modulates insulin release from B-cells

GCAP I as a paracrine peptide in pancreatic islets modulates insulin release from B-cells

Modulation of GABA Transmission by Diazoxide and Cromakalim in the Globus Pallidus: Implications for the Treatment of Parkinson's Disease

An ATP-sensitive potassium channel (KATP) is known to modulate insulin release from pancreatic beta cells. It has been proposed that potassium channels related to KATPin the nervous system might similarly modulate neurotransmitter release. We have therefore investigated the effects of KATPopening agents on GABA release in the globus pallidus. Diazoxide and cromakalim decreased the K+-evoked release of [3H]GABA from pallidal slices. The maximum inhibition observed for diazoxide (59%) and cromakalim (66%) was achieved at a concentration of 100 μM.The effects of both cromakalim and diazoxide were significantly antagonized by the concurrent application of the sulfonylurea glibenclamide (100 μM). Intrapallidal injections of diazoxide in the reserpine-treated rat model of Parkinson's disease reduced akinesia in a dose-dependent manner. These data suggest that manipulation of neuronal potassium channels with pharmacological properties similar to KATPmay prove useful in the treatment of Parkinson's disease.

Modulation of islet G-proteins, α-glucosidehydrolase inhibition and insulin release stimulated by various secretagogues

Guanine nucleotide-binding proteins (G-proteins) are known to act as important modulators of insulin release from the islets of Langerhans. We have recently found that the deoxynojirimycin-derivative emiglitate, a recognized inhibitor of intestinal alpha-glucosidehydrolase activity, is a powerful inhibitor of glucose-induced insulin release. With the use of isolated mouse islets the present investigation was performed in a primary attempt to elucidate whether this inhibitory mechanism in some way was linked to the beta-cell G-protein system. Treatment of freshly isolated islets with pertussis toxin (PTX), which is known to inactivate the G (i)-proteins, abolished the inhibitory effect of the alpha(2)-adrenoceptor agonist clonidine on insulin release stimulated by the phosphodiesterase inhibitor IBMX in the presence of the protein kinase C activator TPA and even changed it into an increase. Emiglitate did not display any inhibitory action on insulin release induced by these secretagogues. Similarly, clonidine-induced inhibition of glucose stimulated insulin release was reversed by PTX. However, PTX did not influence the suppressive action of emiglitate on glucose-induced insulin secretion. In contrast, the adenylate cyclase activator forskolin totally abolished the inhibitory effect of emiglitate, but not that of the glucose analogue mannoheptulose, on glucose-induced insulin release. Moreover, the stimulatory effect of forskolin and cholera toxin (CTX) (activator of G (s)-proteins) on the secretion of insulin was markedly enhanced in the presence of emiglitate. In conclusion, our results suggest that the inhibitory effect of emiglitate on glucose-induced insulin release is not directly related to the G(s)-proteins, but most likely exerted solely through the selective suppression of lysosomal aglucosidehydrolase activity, a step in between the proximal and the distal G(i)-proteins, in glucose induced stimulus-secretion mechanisms. Our data also suggests that the inhibitory action of emiglitate on glucose stimulated insulin release can be compensated for by an increased sensitivity of the cyclic AMP-protein kinase A pathway. Hence, emiglitate might indirectly elicit an increased activity of the G(s)-proteins to facilitate the secretory process.

An ATP-sensitive Cl- channel current that is activated by cell swelling, cAMP, and glyburide in insulin-secreting cells.

Although chloride ions are known to modulate insulin release and islet electrical activity, the mechanism or mechanisms mediating these effects are unclear. However, numerous studies of islet Cl- fluxes have suggested that Cl- movements and glucose and sulfonylurea sensitive and are blocked by stilbene-derivative Cl- channel blockers. We now show for the first time that insulin-secreting cells have a Cl- channel current, which we term ICl,islet. The current is activated by hypotonic conditions, 1-10 mumol/l glyburide and 0.5 mmol/l 8-bromoadenosine 3':5'-cyclic monophosphate sodium. ICl,islet is mediated by Cl- channels, since replacing [Cl-]o with less permeant aspartate reduces current amplitude and depolarizes its reversal potential. In addition, 100 mumol/l 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or glyburide, which blocks the Cl- channels of other cell types, block ICl,islet. Reducing [ATP]i reduces the amplitude of the current, suggesting that it may be under metabolic control. The current is time-independent and shows strong outward-rectification beyond approximately 0 mV. At potentials associated with the silent phase of islet electrical activity (approximately -65 mV), ICl,islet mediates a large inward current, which would be expected to depolarize islet membrane potential. Thus, activation of this novel current by increased intracellular cAMP, sulfonylureas, or ATP may contribute to the well-known depolarizing effects of these agents.