Metabotropic glutamate receptor type 4 is involved in autoinhibitory cascade for glucagon secretion by alpha-cells of islet of Langerhans

In islets of Langerhans, L-glutamate is stored in glucagon-containing secretory granules of alpha-cells and cosecreted with glucagon under low-glucose conditions. The L-glutamate triggers secretion of gamma-aminobutyric acid (GABA) from beta-cells, which in turn inhibits glucagon secretion from alpha-cells through the GABAA receptor. In the present study, we tested the working hypothesis that L-glutamate functions as an autocrine/paracrine modulator and inhibits glucagon secretion through a glutamate receptor(s) on alpha-cells. The addition of L-glutamate at 1 mmol/l; (R,S)-phosphonophenylglycine (PPG) and (S)-3,4-dicarboxyphenylglycine (DCPG), specific agonists for class III metabotropic glutamate receptor (mGluR), at 100 micromol/l; and (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I) at 50 micromol/l inhibited the low-glucose-evoked glucagon secretion by 87, 81, 73, and 87%, respectively. This inhibition was dose dependent and was blocked by (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG), a specific antagonist of class III mGluR. Agonists of other glutamate receptors, including kainate and quisqualate, had little effectiveness. RT-PCR and immunological analyses indicated that mGluR4, a class III mGluR, was expressed and localized with alpha- and F cells, whereas no evidence for expression of other mGluRs, including mGluR8, was obtained. L-Glutamate, PPG, and ACPT-I decreased the cAMP content in isolated islets, which was blocked by CPPG. Dibutylyl-cAMP, a nonhydrolyzable cAMP analog, caused the recovery of secretion of glucagon. Pertussis toxin, which uncouples adenylate cyclase and inhibitory G-protein, caused the recovery of both the cAMP content and secretion of glucagon. These results indicate that alpha- and F cells express functional mGluR4, and its stimulation inhibits secretion of glucagon through an inhibitory cAMP cascade. Thus, L-glutamate may directly interact with alpha-cells and inhibit glucagon secretion.     

Different effects of tolbutamide and diazoxide in alpha, beta-, and delta-cells within intact islets of Langerhans

Interaction between the different types of cells within the islet of Langerhans is vital for adequate control of insulin release. Once insulin secretion becomes defective, as in type 2 diabetes, the most useful drugs to increase insulin release are sulfonylureas. It is well-known that sulfonylureas block K(ATP) channels, which results in depolarization of the membrane that provokes calcium influx and increases intracellular calcium concentration ([Ca2+]i), which thereby triggers insulin secretion. The sulfonamide diazoxide produces the opposite effect: it activates K(ATP) channels, resulting in a decreased insulin secretion. Despite such evidence, little is known about the effect of sulfonylureas and sulfonamides in non-beta-cells of the islet of Langerhans. In this article, we describe the effects of tolbutamide and diazoxide on [Ca2+]i in alpha-, beta-, and delta-cells within intact islets of Langerhans. Tolbutamide elicits an increase in [Ca2+li in beta- and delta-cells, regardless of glucose concentrations. Remarkably, tolbutamide is without effect in alpha-cells. When diazoxide is applied, glucose-induced [Ca2+]i oscillations in beta- and delta-cells are abolished, whereas [Ca2+]i oscillations in alpha-cells remain unaltered. Furthermore, the existence of sulfonylurea receptors is demonstrated in beta-cells but not in alpha-cells by using binding of glybenclamide-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) combined with immunostaining for insulin and glucagon.     

Structural and functional considerations of GABA in islets of Langerhans. Beta-cells and nerves

gamma-Aminobutyric acid (GABA), a prominent inhibitory neurotransmitter, is present in high concentrations in beta-cells of islets of Langerhans. The GABA shunt enzymes, glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), have also been localized in islet beta-cells. With the recent demonstration that the 64,000-M, antigen associated with insulin-dependent diabetes mellitus is GAD, there is increased interest in understanding the role of GABA in islet function. Only a small component of beta-cell GABA is contained in insulin secretory granules, making it unlikely that GABA, coreleased with insulin, is physiologically significant. Our immunohistochemical study of GABA in beta-cells of intact islets indicates that GABA is associated with a vesicular compartment distinctly different from insulin secretory granules. Whether this compartment represents a releasable pool of GABA has yet to be determined. GAD in beta-cells is associated with a vesicular compartment, similar to the GABA vesicles. In addition, GAD is found in a unique extensive tubular cisternal complex (GAD complex). It is likely that the GABA-GAD vesicles are derived from this GAD-containing complex. Physiological studies on the effect of extracellular GABA on islet hormonal secretion have had variable results. Effects of GABA on insulin, glucagon, and somatostatin secretion have been proposed. The most compelling evidence for GABA regulation of islet hormone secretion comes from studies on somatostatin secretion, where it has an inhibitory effect. We present new evidence demonstrating the presence of GABAergic nerve cell bodies at the periphery of islets with numerous GABA-containing processes extending into the islet mantle. This close association between GABAergic neurons and islet alpha- and delta-cells strongly suggests that GABA inhibition of somatostatin and glucagon secretion is mediated by these neurons. Intracellular beta-cell GABAA and its metabolism may have a role in beta-cell function. New evidence indicates that GABA shunt activity is involved in regulation of insulin secretion. In addition, GABA or its metabolites may regulate proinsulin synthesis. These new observations provide insight into the complex nature of GABAergic neurons and beta-cell GABA in regulation of islet function.     

Vesicular inhibitory amino acid transporter is present in glucagon-containing secretory granules in alphaTC6 cells,mouse clonal alpha-cells,and alpha-cells of islets of Langerhans

Islets of Langerhans contain gamma-aminobutyrate (GABA) and may use it as an intercellular transmitter. In beta-cells, GABA is stored in synaptic-like microvesicles and secreted through Ca(2+)-dependent exocytosis. Vesicular inhibitory amino acid transporter (VIAAT), which is responsible for the storage of GABA and glycine in neuronal synaptic vesicles, is believed to be responsible for the storage and secretion of GABA in beta-cells. However, a recent study by Chessler et al. indicated that VIAAT is expressed in the mantle region of islets. In the present study, we investigated the precise localization of VIAAT in rat islets of Langerhans and clonal islet cells and found that it is present in alpha-cells, a minor population of F-cells and alphaTC6 cells, and clonal alpha-cells but not in beta-cells, delta-cells, or MIN6 m9-cells (clonal beta-cells). Combined biochemical, immunohistochemical, and electronmicroscopical evidence indicated that VIAAT is specifically localized with glucagon-containing secretory granules in alpha-cells. ATP-dependent uptake of radiolabeled GABA, which is energetically coupled with a vacuolar proton pump, was detected in digitonin-permeabilized alphaTC6 cells as well as in MIN6 m9 cells. These results demonstrate that functional neuronal VIAAT is present in glucagon-containing secretory granules in alpha-cells and suggest that the ATP-dependent GABA transporter in beta-cells is at least immunologically distinct from VIAAT. Because glucagon-containing secretory granules also contain vesicular glutamate transporter and store L-glutamate, as demonstrated by Hayashi et al., the present results suggest more complex features of the GABAergic phenotype of islets than previously supposed.     

Immunohistochemical colocalization of GABA and insulin in beta-cells of rat islet

gamma-Aminobutyric acid (GABA) is found in high concentrations in the pancreatic islet. In addition, enzymes regulating the level of GABA (L-glutamate decarboxylase and GABA-alpha-ketoglutarate transaminase) have been immunohistochemically localized in the medullary cells of the islet. In this study, an immunofluorescence and elution/restaining protocol is used to determine the distribution of GABA and either insulin, glucagon, or somatostatin in a tissue section. GABA was not detected within the islet alpha- or delta-cells but was determined to be localized within the insulin-containing beta-cells.     

Glucose induces opposite intracellular Ca2+ concentration oscillatory patterns in identified alpha- and beta-cells within intact human islets of Langerhans

Homeostasis of blood glucose is mainly regulated by the coordinated secretion of glucagon and insulin from alpha- and beta-cells within the islets of Langerhans. The release of both hormones is Ca(2+) dependent. In the current study, we used confocal microscopy and immunocytochemistry to unequivocally characterize the glucose-induced Ca(2+) signals in alpha- and beta-cells within intact human islets. Extracellular glucose stimulation induced an opposite response in these two cell types. Although the intracellular Ca(2+) concentration ([Ca(2+)](i)) in beta-cells remained stable at low glucose concentrations, alpha-cells exhibited an oscillatory [Ca(2+)](i) response. Conversely, the elevation of extracellular glucose elicited an oscillatory [Ca(2+)](i) pattern in beta-cells but inhibited low-glucose-induced [Ca(2+)](i) signals in alpha-cells. These Ca(2+) signals were synchronic among beta-cells grouped in clusters within the islet, although they were not coordinated among the whole beta-cell population. The response of alpha-cells was totally asynchronic. Therefore, both the alpha- and beta-cell populations within human islets did not work as a syncitium in response to glucose. A deeper knowledge of alpha- and beta-cell behavior within intact human islets is important to better understand the physiology of the human endocrine pancreas and may be useful to select high-quality islets for transplantation.     

Antisomatostatin gamma globulin augments secretion of both insulin and glucagon in vitro: evidence for a physiologic role for endogenous somatostatin in the regulation of pancreatic A- and B-cell function

To assess the effects of endogenous somatostatin on pancreatic islet A- and B-cell function, isolated rat islets were incubated in antisomatostatin gamma-globulin to bind endogenously released somatostatin, and the insulin and glucagon secretion of these islets was compared with that of islets incubated in gamma-globulin isolated from nonimmune serum. Islets incubated in antisomatostatin gamma-globulin released significantly more insulin at 4, 8, 16, and 32 mM glucose and significantly more glucagon at 8, 16, and 32 mM glucose, P < 0.05-0.005. For glucose-stimulated insulin release the threshold was decreased, the Vmax was increased, but the apparent Km was unaltered; for glucose-suppression of glucagon release the threshold was increased, maximal suppression was decreased, but the apparent Ki was unaltered. The augmentative effect of the antisomatostatin gamma-globulin was most prominent at 4 mM glucose for insulin release and at 8mM glucose for glucagon release, but was not limited to glucose since both insulin and glucagon release stimulated by arginine were also augmented by antisomatostatin gamma-globulin. These results provide evidence that endogenous somatostatin may act as a physiologic local regulator of both insulin and glucagon secretion and that its effect on insulin and glucagon secretion is dependent on the prevailing glucose concentration.     

Miniglucagon (glucagon 19-29): a novel regulator of the pancreatic islet physiology.

Miniglucagon, the COOH-terminal (19-29) fragment processed from glucagon, is a potent and efficient inhibitor of insulin secretion from the MIN 6 beta-cell line. Using the rat isolated-perfused pancreas, we investigated the inhibitory effect of miniglucagon on insulin secretion and evaluated the existence of an inhibitory tone exerted by this peptide inside the islet. Miniglucagon dose-dependently inhibited insulin secretion stimulated by 8.3 mol/l glucose, with no change in the perfusion flow rate. A concentration of 1 nmol/l miniglucagon had a significant inhibitory effect on a 1 nmol/l glucagon-like peptide 1 (7-36) amide-potentiated insulin secretion. A decrease in extracellular glucose concentration simultaneously stimulated glucagon and miniglucagon secretion from pancreatic alpha-cells. Using confocal and electron microscopy analysis, we observed that miniglucagon is colocalized with glucagon in mature secretory granules of alpha-cells. Perfusion of an anti-miniglucagon antiserum directed against the biologically active moiety of the peptide resulted in a more pronounced effect of a glucose challenge on insulin secretion, indicating that miniglucagon exerts a local inhibitory tone on beta-cells. We concluded that miniglucagon is a novel local regulator of the pancreatic islet physiology and that any abnormal inhibitory tone exerted by this peptide on the beta-cell would result in an impaired insulin secretion, as observed in type 2 diabetes.     

Age-dependent expression of protein kinase C isoforms in rat islets

The appearance of the biphasic insulin secretory response several days after birth suggests that maturation of a critical step in stimulus-secretion coupling occurs during the early neonatal period. To clarify the role of protein kinase C (PKC) during this time, we examined the pancreatic islets of adult, 3-day neonatal, and 19-day fetal rats for the presence of different PKC isoenzymes. Western-blot analysis of islet extracts showed the presence of PKC isoforms in both adult and neonatal tissues. Immunocytochemistry of adult islets revealed a differential expression in islet cell types. PKC-alpha was found only in beta-cells, PKC-gamma in alpha-cells, and PKC-epsilon in delta-cells and vascular walls. Immunoreactivity for PKC-beta was not detected in any cell type. All three isoenzymes were also present in neonatal islets; however, in contrast to adult tissue, immunoreactivity for either PKC-alpha or PKC-gamma was present in relatively few cells. There was no apparent immunoreactivity for PKC-alpha or PKC-gamma in fetal islets, although these tissues contained strong staining for insulin and glucagon. These data show that three of the PKC isoforms are restricted to a particular islet cell type, where they may play a unique role in the secretion of a specific hormone. Moreover, our results demonstrate that these enzymes, especially PKC-alpha, appear during the early neonatal period. This age-dependent expression may be linked to the development of the biphasic insulin release response.     

Glucose or insulin, but not zinc ions, inhibit glucagon secretion from mouse pancreatic alpha-cells

The mechanisms by which hypoglycemia stimulates glucagon release are still poorly understood. In particular, the relative importance of direct metabolic coupling versus paracrine regulation by beta-cell secretory products is unresolved. Here, we compare the responses to glucose of 1) alpha-cells within the intact mouse islet, 2) dissociated alpha-cells, and 3) clonal alphaTC1-9 cells. Free cytosolic concentrations of ATP ([ATP](c)) or Ca(2+) ([Ca(2+)](c)) were imaged using alpha-cell-targeted firefly luciferase or a green fluorescent protein-based Ca(2+) probe ("pericam"), respectively. Consistent with a direct effect of glucose on alpha-cell oxidative metabolism, an increase in glucose concentration (from 0 or 3 mmol/l to 20 mmol/l) increased [ATP](c) by 7-9% in alpha-cells within the intact islet and by approximately 4% in alphaTC1-9 cells. Moreover, glucose also dose-dependently decreased the frequency of [Ca(2+)](c) oscillations in both dissociated alpha-cells and alphaTC1-9 cells. Although the effects of glucose were mimicked by exogenous insulin, they were preserved when insulin signaling was blocked with wortmannin. Addition of ZnCl(2) slightly increased the frequency of [Ca(2+)](c) oscillations but failed to affect glucagon release from either islets or alphaTC1-9 cells under most conditions. We conclude that glucose and insulin, but not Zn(2+) ions, independently suppress glucagon secretion in the mouse.     

ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/- mouse alpha-cells

Patch-clamp recordings and glucagon release measurements were combined to determine the role of plasma membrane ATP-sensitive K+ channels (KATP channels) in the control of glucagon secretion from mouse pancreatic alpha-cells. In wild-type mouse islets, glucose produced a concentration-dependent (half-maximal inhibitory concentration [IC50]=2.5 mmol/l) reduction of glucagon release. Maximum inhibition (approximately 50%) was attained at glucose concentrations >5 mmol/l. The sulfonylureas tolbutamide (100 micromol/l) and glibenclamide (100 nmol/l) inhibited glucagon secretion to the same extent as a maximally inhibitory concentration of glucose. In mice lacking functional KATP channels (SUR1-/-), glucagon secretion in the absence of glucose was lower than that observed in wild-type islets and both glucose (0-20 mmol/l) and the sulfonylureas failed to inhibit glucagon secretion. Membrane potential recordings revealed that alpha-cells generate action potentials in the absence of glucose. Addition of glucose depolarized the alpha-cell by approximately 7 mV and reduced spike height by 30% Application of tolbutamide likewise depolarized the alpha-cell (approximately 17 mV) and reduced action potential amplitude (43%). Whereas insulin secretion increased monotonically with increasing external K+ concentrations (threshold 25 mmol/l), glucagon secretion was paradoxically suppressed at intermediate concentrations (5.6-15 mmol/l), and stimulation was first detectable at >25 mmol/l K+. In alpha-cells isolated from SUR1-/- mice, both tolbutamide and glucose failed to produce membrane depolarization. These effects correlated with the presence of a small (0.13 nS) sulfonylurea-sensitive conductance in wild-type but not in SUR1-/- alpha-cells. Recordings of the free cytoplasmic Ca2+ concentration ([Ca2+]i) revealed that, whereas glucose lowered [Ca2+]i to the same extent as application of tolbutamide, the Na+ channel blocker tetrodotoxin, or the Ca2+ channel blocker Co2+ in wild-type alpha-cells, the sugar was far less effective on [Ca2+]i in SUR1-/- alpha-cells. We conclude that the KATP channel is involved in the control of glucagon secretion by regulating the membrane potential in the alpha-cell in a way reminiscent of that previously documented in insulin-releasing beta-cells. However, because alpha-cells possess a different complement of voltage-gated ion channels involved in action potential generation than the beta-cell, moderate membrane depolarization in alpha-cells is associated with reduced rather than increased electrical activity and secretion.     

Effect of dynorphin on insulin and somatostatin secretion, calcium uptake, and c-AMP levels in isolated rat islets of Langerhans

Dynorphin-[1-13], at concentrations of 5.8 X 10(-12) to 5.8 X 10(-9) M, stimulated insulin secretion from isolated islets of Langerhans of the rat, in medium containing 6 mM glucose. Higher concentrations of dynorphin had no significant effect on secretion. Dynorphin (5.8 X 10(-9) M) was unable to initiate insulin release from islets in the presence of 2 mM glucose, or to increase insulin secretion further in the presence of 20 mM glucose or 6 and 12 mM glyceraldehyde. Dynorphin-induced insulin secretion from islets was blocked by verapamil (5 microM) or by chlorpropamide (72 microM), but not by a mu opiate receptor antagonist, naloxone (0.11 microM), or by ICI 154129, a specific antagonist for the delta receptor (0.25 microM). Dynorphin had no effect on islet somatostatin secretion, under conditions in which insulin secretion was greatly stimulated. Glucose (20 mM) and glyceraldehyde (6 and 12 mM) significantly increased both insulin and somatostatin secretion. Dynorphin (5.8 X 10(-9) M) increased 45Ca2+ uptake into islets, and also increased intracellular islet c-AMP levels. These changes persisted when higher concentrations of dynorphin were used. These results suggest that (1) dynorphin is a very potent stimulus for insulin secretion; (2) dynorphin does not affect somatostatin secretion in static incubations of islets, in the same way as does glucose and glyceraldehyde; (3) dynorphin's effects may involve increased calcium ion movement and can be blocked by verapamil; (4) dynorphin can also increase islet c-AMP, and could thereby modulate the responsiveness of other secretagogues; (5) the actions of dynorphin on insulin secretion are not mediated by delta or mu opiate receptors in islets.     

Palmitate stimulation of glucagon secretion in mouse pancreatic alpha-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium

We have investigated the short-term effects of the saturated free fatty acid (FFA) palmitate on pancreatic alpha-cells. Palmitate (0.5 or 1 mmol/l bound to fatty acid-free albumin) stimulated glucagon secretion from intact mouse islets 1.5- to 2-fold when added in the presence of 1-15 mmol/l glucose. Palmitate remained stimulatory in islets depolarized with 30 mmol/l extracellular K(+) or exposed to forskolin, but it did not remain stimulatory after treatment with isradipine or triacsin C. The stimulatory action of palmitate on secretion correlated with a 3.5-fold elevation of intracellular free Ca(2+) when applied in the presence of 15 mmol/l glucose, a 40% stimulation of exocytosis (measured as increases in cell capacitance), and a 25% increase in whole-cell Ca(2+) current. The latter effect was abolished by isradipine, suggesting that palmitate selectively modulates l-type Ca(2+) channels. The effect of palmitate on exocytosis was not mediated by palmitoyl-CoA, and intracellular application of this FFA metabolite decreased rather than enhanced Ca(2+)-induced exocytosis. The stimulatory effects of palmitate on glucagon secretion were paralleled by a approximately 50% inhibition of somatostatin release. We conclude that palmitate increases alpha-cell exocytosis principally by enhanced Ca(2+) entry via l-type Ca(2+) channels and, possibly, relief from paracrine inhibition by somatostatin released by neighboring delta-cells.     

Islet transplantation to the renal subcapsular space in streptozotocin-diabetic rats. Long-term effects on insulin and glucagon secretion

In previous studies on streptozotocin-diabetic rats, transplantation of 1,000 (but not of 400) pancreatic islets to the renal subcapsular space was followed within 10 days by near-normalization of the impaired insulin secretion and the hyperglycemia. The long-term effects were now studied by measuring insulin and glucagon secretion 3 months after transplantation of 1,000 collagenase-isolated islets in streptozotocin (70 mg/kg) diabetic rats. At this time, diabetic control rats showed marked hyperglycemia and hyperglucagonemia, whereas the basal glucose and glucagon levels had normalized in the transplanted rats. Furthermore, insulin secretion in response to glucose or arginine stimulation and glucagon secretion following arginine stimulation were normal in all transplant rats, but absent in all diabetic controls. Morphologically the transplanted islets in the renal subcapsular space appeared normal on hematoxylin-eosin staining and immunostaining with antisera directed against insulin, glucagon, somatostatin and chromogranin A/B. Thus the islet transplants normalized basal hyperglycemia and hyperglucagonemia and restored insulin and glucagon secretion on a long-term basis.     

beta----alpha----delta pancreatic islet cellular perfusion in dogs

Intraislet communication between alpha-, beta-, and delta-cells and their secretory products may theoretically occur via the paracrine (interstitial) and/or vascular routes. Recently, we have shown that there is a directed microvascular circulation in the rat islet with a cellular order of perfusion of beta----alpha----delta. The direction of microvascular perfusion of cells within the dog islet has been controversial. Anterograde (arterial) perfusion and retrograde (reversed or venous) perfusion of a segment of isolated dog pancreas with potent insulin antibodies yielded results similar to those found in the rat pancreas (anterograde, 158 +/- 44% increase in glucagon and 65 +/- 20% increase in somatostatin; retrograde, no change in glucagon or somatostatin). Anterograde infusion of glucagon antibody (no change in insulin, -33.5 +/- 3% decrease in somatostatin) or somatostatin antibody (no change in insulin or glucagon) also yielded the same results as in the rat pancreas. Anterograde infusion of 500 pg/ml glucagon caused a larger increase in insulin secretion (245 +/- 10%) than retrograde infusion (45 +/- 4%), whereas somatostatin was stimulated more retrogradely (339 +/- 17%) than anterogradely (121 +/- 9%). Anterograde infusion of somatostatin produced a larger decrease in insulin and glucagon than did retrograde perfusion (P less than .0001 for both comparisons). The retrograde infusion of 0.3 mU/ml insulin caused a decrease in glucagon but was without effect anterogradely. The results from the infusion of exogenous hormones suggest that the sensitivity of the alpha-, beta-, and delta-cells to insulin, glucagon, and somatostatin is determined by the beta----alpha----delta order of perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin and glucagon secretion from rat islets maintained in a tissue culture-perifusion system.

A tissue culture-perifusion system is described that allows for long-term culture of pancreatic islets and study of the dynamics of islet hormone secretion. Islets cultured in this system demonstrate brisk, reproducible biphasic insulin and glucagon release. Glucose-stimulated insulin release is similar after 1 or 14 days in culture. Freshly isolated islets are relatively insensitive to somatostatin, requiring 100 ng/ml to suppress partially the glucose-induced insulin secretion. After 24 h of culture, the same islets demonstrate a marked increase in sensitivity to this hormone. Glucagon secretion from islets maintained in this system occurred in a predictable fashion to arginine stimulation and glucose inhibition.