Stimulation Of Glucagon Secretion Research Articles

Lactate and the Mechanism of Hypoglycemia-Associated Autonomic Failure in Diabetes

Iatrogenic hypoglycemia is a problem for many people with diabetes (1). It causes recurrent morbidity in most people with type 1 diabetes and many with advanced type 2 diabetes and is sometimes fatal. It generally precludes maintenance of euglycemia over a lifetime of diabetes and therefore full realization of the benefits of glycemic control. It impairs defenses against subsequent falling plasma glucose concentrations and causes a vicious cycle of recurrent hypoglycemia. Hypoglycemia in diabetes is typically the result of the interplay of therapeutic insulin excess—caused by treatment with insulin, a sulfonylurea, or a glinide—and compromised physiological and behavioral defenses against falling plasma glucose concentrations (1,2). Compromised physiological defenses include loss of the normal decrease in β-cell insulin secretion and increase in α-cell glucagon secretion and attenuation of the normal increase in adrenomedullary epinephrine secretion during hypoglycemia. The compromised behavioral defense is failure to ingest carbohydrates because of loss of symptoms due to attenuation of the normal increase in sympathoadrenal, largely sympathetic neural, activity. The concept of hypoglycemia-associated autonomic failure (HAAF) in diabetes posits that recent antecedent hypoglycemia (or sleep or prior exercise) causes both defective glucose counterregulation (by attenuating the epinephrine response in the setting of absent insulin and glucagon responses) and impaired awareness of hypoglycemia (by attenuating sympathoadrenal and the resulting symptomatic responses) and thus a vicious cycle of recurrent hypoglycemia. Although additional intraislet mechanisms may be involved, it is reasonable to attribute both loss of the insulin and of the glucagon responses to hypoglycemia, the prerequisites to HAAF, to β-cell failure. Insulin normally restrains glucagon secretion and a decrease in insulin normally stimulates glucagon secretion during hypoglycemia. In the setting of absolute endogenous insulin deficiency—β-cell failure—there is …

Study of the Involvement of Pancreastatin in the Physiopathology of Diabetes Mellitus Associated with Nonsecreting Pituitary Adenomas

Pancreastatin, derived from chromogranin A, inhibits insulin and stimulates glucagon secretion in rodents. Immunohistochemistry localised pancreastatin in human pancreatic islet cells and gonadotroph pituitary cells. Nonsecreting pituitary adenomas, frequently associated with diabetes mellitus, arise quasi-constantly from gonadotroph cells. We evaluated the possible involvement of pancreastatin in the physiopathology of diabetes mellitus associated with nonsecreting pituitary adenomas. Plasma pancreastatin levels were measured by radioimmunoassay in 5 groups of subjects: 10 patients with nonsecreting pituitary adenomas associated with diabetes mellitus (group I), 10 patients with nonsecreting pituitary adenomas without diabetes (Group II), 10 patients with ACTH or GH-secreting pituitary adenomas and diabetes mellitus (Group III), 10 diabetic patients without pituitary adenomas (Group IV), and 10 healthy controls (Group V). Kidney and liver functions were normal in all of them and no patient was treated with a proton pump inhibitor. All pituitary adenomas were trans-sphenoidally removed. Immunohistochemistry against pancreastatin was performed in 5 patients of each of the 3 groups of pituitary adenomas. Plasma pancreastatin levels were not different between the different groups: 182±46 pg/ml (Group I), 195±57 pg/ml (Group II), 239±42 pg/ml (Group III), 134±31 pg/ml, (Group IV), and 122±29 pg/ml (Group V). In contrast, they were significantly (p<0.05) higher before (391±65 pg/ml) than after trans-sphenoidal surgery (149±18 pg/ml) without post-surgical change in diabetes. An immunostaining against pancreastatin was found in a majority of pituitary adenomas, associated or not with diabetes mellitus. These results argue against a role of pancreastatin in the pathogenesis of diabetes mellitus associated with nonsecreting pituitary adenomas.

A role for zinc in pancreatic islet β‐cell cross‐talk with the α‐cell during hypoglycaemia

Signalling by intraislet β-cells to neighbouring α-cells was recognized almost 40 years ago, leading to the hypothesis that this is an essential mechanism to regulate the glucagon counterregulatory response to hypoglycaemia. The thesis was that during normoglycaemia or hyperglycaemia insulin secretion from β-cells would enter the islet periportal circulation and travel downstream to α-cells to dampen glucagon secretion. As a corollary, during hypoglycaemia β-cells would stop secreting insulin, which would permit α-cells to release glucagon into the hepatic portal circulation so it could travel to the liver to increase glucose production and thereby correct hypoglycaemia. This mini-review briefly mentions the early work that established this hypothesis and more extensively examines more recent work that has provided direct evidence supporting the hypothesis. A new twist has been introduced based on the fact that zinc is bound to insulin within β-cells and co-secreted with insulin. Zinc is released from insulin when it reaches the higher pH of blood, and zinc has recently been shown to negatively regulate α-cell secretion. It is now suggested that a switch-off signal provided by a sudden cessation of zinc secretion from β-cells during hypoglycaemia may play a critical role in stimulating glucagon secretion that is independent of the effect of insulin.

Ghrelin Directly Stimulates Glucagon Secretion from Pancreatic α-Cells

Previous work has demonstrated that the peptide hormone ghrelin raises blood glucose. Such has been attributed to ghrelin's ability to enhance GH secretion, restrict insulin release, and/or reduce insulin sensitivity. Ghrelin's reported effects on glucagon have been inconsistent. Here, both animal- and cell-based systems were used to determine the role of glucagon in mediating ghrelin's effects on blood glucose. The tissue and cell distribution of ghrelin receptors (GHSR) was evaluated by quantitative PCR and histochemistry. Plasma glucagon levels were determined following acute acyl-ghrelin injections and in pharmacological and/or transgenic mouse models of ghrelin overexpression and GHSR deletion. Isolated mouse islets and the α-cell lines αTC1 and InR1G9 were used to evaluate ghrelin's effects on glucagon secretion and the role of calcium and ERK in this activity. GHSR mRNA was abundantly expressed in mouse islets and colocalized with glucagon in α-cells. Elevation of acyl-ghrelin acutely (after sc administration, such that physiologically relevant plasma ghrelin levels were achieved) and chronically (by slow-releasing osmotic pumps and as observed in transgenic mice harboring ghrelinomas) led to higher plasma glucagon and increased blood glucose. Conversely, genetic GHSR deletion was associated with lower plasma glucagon and reduced fasting blood glucose. Acyl-ghrelin increased glucagon secretion in a dose-dependent manner from mouse islets and α-cell lines, in a manner requiring elevation of intracellular calcium and phosphorylation of ERK. Our study shows that ghrelin's regulation of blood glucose involves direct stimulation of glucagon secretion from α-cells and introduces the ghrelin-glucagon axis as an important mechanism controlling glycemia under fasting conditions.

Acute stimulation of glucagon secretion by linoleic acid results from GPR40 activation and [Ca2+]i increase in pancreatic islet α-cells

The role of free fatty acids (FFAs) in glucagon secretion has not been well established, and the involvement of FFA receptor GPR40 and its downstream signaling pathways in regulating glucagon secretion are rarely demonstrated. In this study, it was found that linoleic acid (LA) acutely stimulated glucagon secretion from primary cultured rat pancreatic islets. LA at 20 and 40 μmol/l dose-dependently increased glucagon secretion both at 3 mmol/l glucose and at 15 mmol/l glucose, although 15 mmol/l glucose reduced basal glucagon levels. LA induced an increase in cytoplasmic free calcium concentrations ([Ca(2)(+)](i)) in identified rat α-cells, which is reflected by increased Fluo-3 intensity under confocal microscopy recording. The increase in [Ca(2)(+)](i) was partly inhibited by removal of extracellular Ca(2)(+) and eliminated overall by further exhaustion of intracellular Ca(2)(+) stores using thapsigargin treatment, suggesting that both Ca(2)(+) release and Ca(2)(+) influx contributed to the LA-stimulated increase in [Ca(2)(+)](i) in α-cells. Double immunocytochemical stainings showed that GPR40 was expressed in glucagon-positive α-cells. LA-stimulated increase in [Ca(2)(+)](i) was blocked by inhibition of GPR40 expression in α-cells after GPR40-specific antisense treatment. The inhibition of phospholipase C activity by U73122 also blocked the increase in [Ca(2)(+)](i) by LA. It is concluded that LA activates GPR40 and phospholipase C (and downstream signaling pathways) to increase Ca(2)(+) release and associated Ca(2)(+) influx through Ca(2)(+) channels, resulting in increase in [Ca(2)(+)](i) and glucagon secretion.

GPCR Ligands Differentially Modulate Islet Metabolism and Ca 2+ Signaling

Glucose homeostasis is a tightly regulated process coordinated by insulin and glucagon secretion. In α- and β-cells, G-protein coupled receptor (GPCR) activation facilitates the optimization of insulin and glucagon secretion. Glucagon-like peptide-1 (GLP-1), a ligand activating the Gs pathway, increases insulin secretion, but inhibits glucagon secretion. Neuropeptide-Y (NPY) and somatostatin (SST), whose receptors are coupled to Gi, inhibit insulin secretion; however, NPY stimulates glucagon release, while SST inhibits glucagon secretion. Here, we question whether and how these GPCR ligands impact cellular metabolism and oscillations of intracellular Ca2+ activity ([Ca2+]i) to alter secretion in the pancreatic islet. We utilized two-photon excitation microscopy to measure the combined autofluorescence of NADH and NADPH (NAD(P)H, a cellular redox state indicator) upon application of NPY, SST, or GLP-1. Under untreated conditions, an increase in extracellular glucose results in elevated NAD(P)H fluorescence intensities. Treatment with NPY and SST produces a supplementary increase in NAD(P)H autofluorescence at glucose concentrations above 5 mM compared to untreated control. NAD(P)H autofluorescence is not impacted significantly (relative to untreated control) by GLP-1 treatment, which is consistent with a previous report that found that GLP-1 does not alter β-cell metabolism. At glucose concentrations greater than ∼7 mM, pancreatic islets display synchronous oscillations in [Ca2+]i, leading to the pulsatile release of insulin. The [Ca2+]i oscillation frequency increases significantly in the presence of NPY. SST treatment decreases the oscillation frequency in β-cells, but does not affect α-cells. Addition of GLP-1 does not significantly alter the [Ca2+]i oscillation frequency. Thus, GLP-1 likely modulates insulin and glucagon secretion downstream of Ca2+ signaling. NPY may inhibit insulin secretion downstream of Ca2+ signaling, but stimulates glucagon secretion upstream of Ca2+ activity. Islet hormone secretion may be inhibited by SST through alteration of pathways up- and downstream of Ca2+ signaling.

Melatonin stimulates glucagon secretion in vitro and in vivo

Recent investigations have demonstrated that melatonin influences carbohydrate metabolism mediated by insulin-inhibiting effects on pancreatic β-cells. This study evaluated whether melatonin has also an effect on pancreatic α-cells and glucagon expression as well as the glucagon secretion in vitro and in vivo. Glucagon-producing pancreatic α-cell line αTC1 clone 9 (αTC1.9) was used, which was characterized as an appropriate model with glucose responsiveness and expression of the melatonin receptors MT1 and MT2. The results demonstrate that melatonin incubation significantly enhanced the expression as well as the secretion of glucagon. These effects appeared to be more pronounced under hyperglycemic conditions compared to basal glucose concentrations. Notably, in vivo studies demonstrated that long-term oral melatonin administration led to significantly elevated plasma glucagon concentrations in Wistar rats. In contrast, plasma glucagon levels were found to be slightly decreased in type 2 diabetic Goto-Kakizaki rats. Moreover, investigations measuring the relative glucagon receptor mRNA expression showed marked differences in the liver of melatonin-substituted rats as well as in melatonin receptor knockout mice. In conclusion, these findings revealed evidence that melatonin influences pancreatic glucagon expression and secretion as well as the peripheral glucagon action.

Regulation of Ca2+-entry in pancreatic α-cell line by transient receptor potential melastatin 4 plays a vital role in glucagon release

Elevation in the intracellular Ca2+ concentration stimulates glucagon secretion from pancreatic α-cells. The Transient Receptor Potential Melastatin 4 channel (TRPM4) is critical for Ca2+ signaling. However, its role in glucagon secreting α-cells has not been investigated. We identified TRPM4 gene expression and protein in the αTC1-6 cell line using RT-PCR and immunocytochemistry. Furthermore, we performed a detailed biophysical characterization of the channel using the patch-clamp technique to confirm that currents typical for TRPM4 were present in αTC1-6 cells. To investigate TRPM4 function, we generated a stable knockdown clone using shRNA and a lentiviral vector. Inhibition of TRPM4 significantly reduced the responses to different agonists during Ca2+ imaging analysis with Fura-2AM. The reduction in the magnitude of Ca2+ signals resulted in decreased glucagon secretion. These results suggested that depolarization by TRPM4 may play an important role in controlling glucagon secretion from α-cells and perhaps glucose homeostasis.

Mechanism of Orexin B-Stimulated Insulin and Glucagon Release From the Pancreas of Normal and Diabetic Rats

To examine the pattern of distribution and effect of orexin B in the islets of normal and diabetic rats. Pancreatic tissue fragments collected from normal and diabetic (4 weeks after the onset of diabetes) rats were either processed for immunohistochemistry or treated with different concentrations (10 to 10 mol/L) of orexin B. Orexin B-positive nerves were observed in the wall of blood vessels of both normal and diabetic rat pancreas. Orexin B is abundant in the islets of normal rats and colocalized with insulin in β cells. The number of orexin B-positive cells decreased after the onset of diabetes. Orexin B evoked significant (P<0.05) increases in insulin release from the pancreas of normal and diabetic rats. Propranolol, a β-adrenergic receptor antagonist, significantly (P<0.04) reduced the stimulatory effect of orexin B on insulin secretion. Orexin B also induced significant (P<0.05) increases in glucagon release from the pancreas of normal rats but failed to stimulate glucagon secretion from the pancreas of diabetic rats. Orexin B stimulated insulin secretion in normal and diabetic rat pancreas through the β-adrenergic pathway. Orexin B may have an important role in the regulation of islet function.

AMP-activated protein kinase regulates glucagon secretion from mouse pancreatic alpha cells

AMP-activated protein kinase (AMPK), encoded by Prkaa genes, is emerging as a key regulator of overall energy homeostasis and the control of insulin secretion and action. We sought here to investigate the role of AMPK in controlling glucagon secretion from pancreatic islet alpha cells. AMPK activity was modulated in vitro in clonal alphaTC1-9 cells and isolated mouse pancreatic islets using pharmacological agents and adenoviruses encoding constitutively active or dominant negative forms of AMPK. Glucagon secretion was measured during static incubation by radioimmunoassay. AMPK activity was assessed by both direct phosphotransfer assay and by western (immuno-)blotting of the phosphorylated AMPK α subunits and the downstream target acetyl-CoA carboxylase 1. Intracellular free [Ca²(+)] was measured using Fura-Red. Increasing glucose concentrations strongly inhibited AMPK activity in clonal pancreatic alpha cells. Forced increases in AMPK activity in alphaTC1-9 cells, achieved through the use of pharmacological agents including metformin, phenformin and A-769662, or via adenoviral transduction, resulted in stimulation of glucagon secretion at both low and high glucose concentrations, whereas AMPK inactivation inhibited both [Ca²(+)](i) increases and glucagon secretion at low glucose. Transduction of isolated mouse islets with an adenovirus encoding AMPK-CA under the control of the preproglucagon promoter increased glucagon secretion selectively at elevated glucose concentrations. AMPK is strongly regulated by glucose in pancreatic alpha cells, and increases in AMPK activity are sufficient and necessary for the stimulation of glucagon release in vitro. Modulation of AMPK activity in alpha cells may therefore provide a novel approach to controlling blood glucose concentrations.

Insulin Reciprocally Regulates Glucagon Secretion in Humans

OBJECTIVEWe tested the hypothesis that an increase in insulin per se, i.e., in the absence of zinc, suppresses glucagon secretion during euglycemia and that a decrease in insulin per se stimulates glucagon secretion during hypoglycemia in humans.RESEARCH DESIGN AND METHODSWe measured plasma glucagon concentrations in patients with type 1 diabetes infused with the zinc-free insulin glulisine on three occasions. Glulisine was infused with clamped euglycemia (∼95 mg/dl [5.3 mmol/l]) from 0 to 60 min on all three occasions. Then, glulisine was discontinued with clamped euglycemia or with clamped hypoglycemia (∼55 mg/dl [3.0 mmol/l]) or continued with clamped hypoglycemia from 60 to 180 min.RESULTSPlasma glucagon concentrations were suppressed by −13 ± 3, −9 ± 3, and −12 ± 2 pg/ml (−3.7 ± 0.9, −2.6 ± 0.9, and −3.4 ± 0.6 pmol/l), respectively, (all P < 0.01) during zinc-free hyperinsulinemic euglycemia over the first 60 min. Glucagon levels remained suppressed following a decrease in zinc-free insulin with euglycemia (−14 ± 3 pg/ml [−4.0 ± 0.9 pmol/l]) and during sustained hyperinsulinemia with hypoglycemia (−14 ± 2 pg/ml [−4.0 ± 0.6 pmol/l]) but increased to −3 ± 3 pg/ml (−0.9 ± 0.9 pmol/l) (P < 0.01) following a decrease in zinc-free insulin with hypoglycemia over the next 120 min.CONCLUSIONSThese data indicate that an increase in insulin per se suppresses glucagon secretion and a decrease in insulin per se, in concert with a low glucose concentration, stimulates glucagon secretion. Thus, they document that insulin is a β-cell secretory product that, in concert with glucose and among other signals, reciprocally regulates α-cell glucagon secretion in humans.

Glucagon-Like Peptide-2 Receptor Modulates Islet Adaptation to Metabolic Stress in the ob/ob Mouse

Glucagon-like peptide-2 (GLP-2) is a gut hormone that increases gut growth, reduces mucosal cell death, and augments mesenteric blood flow and nutrient absorption. Exogenous GLP-2(1-33) also stimulates glucagon secretion and enhances gut barrier function with implications for susceptibility to systemic inflammation and subsequent metabolic dysregulation. We examined the importance of GLP-2 receptor (GLP-2R) signaling for glucose homeostasis in multiple models of metabolic stress, diabetes, and obesity. Body weight, islet function, glucose tolerance, and islet histology were studied in wild-type, high-fat fed, lean diabetic, Glp2r(-/-) and ob/ob:Glp2r(-/-) mice. GLP-2 did not stimulate glucagon secretion from isolated pancreatic islets in vitro, and exogenous GLP-2 had no effect on the glucagon response to insulin-induced hypoglycemia in vivo. Glp2r(-/-) mice exhibit no change in glycemia, and plasma glucagon levels were similar in Glp2r(-/-) and Glp2r(+/+) mice after hypoglycemia or after oral or intraperitoneal glucose challenge. Moreover, glucose homeostasis was comparable in Glp2r(-/-) and Glp2r(+/+) mice fed a high-fat diet for 5 months or after induction of streptozotocin-induced diabetes. In contrast, loss of the GLP-2R leads to increased glucagon secretion and alpha-cell mass, impaired intraperitoneal glucose tolerance and hyperglycemia, reduced beta-cell mass, and decreased islet proliferation in ob/ob:Glp2r(-/-) mice. Our results show that, although the GLP-2R is not critical for the stimulation or suppression of glucagon secretion or glucose homeostasis in normal or lean diabetic mice, elimination of GLP-2R signaling in obese mice impairs the normal islet adaptive response required to maintain glucose homeostasis.

Glucose Suppression of Glucagon Secretion: METABOLIC AND CALCIUM RESPONSES FROM α-CELLS IN INTACT MOUSE PANCREATIC ISLETS

Glucagon is released from alpha-cells present in intact pancreatic islets at glucose concentrations below 4 mm, whereas higher glucose levels inhibit its secretion. The mechanisms underlying the suppression of alpha-cell secretory activity are poorly understood, but two general types of models have been proposed as follows: direct inhibition by glucose or paracrine inhibition from non-alpha-cells within the islet of Langerhans. To identify alpha-cells for analysis, we utilized transgenic mice expressing fluorescent proteins targeted specifically to these cells. Measurements of glucagon secretion from pure populations of flow-sorted alpha-cells show that contrary to its effect on intact islets, glucose does stimulate glucagon secretion from isolated alpha-cells. This observation argues against a direct inhibition of glucagon secretion by glucose and supports the paracrine inhibition model. Imaging of cellular metabolism by two-photon excitation of NAD(P)H autofluorescence indicates that glucose is metabolized in alpha-cells and that glucokinase is the likely rate-limiting step in this process. Imaging calcium dynamics of alpha-cells in intact islets reveals that inhibiting concentrations of glucose increase the intracellular calcium concentration and the frequency of alpha-cell calcium oscillations. Application of candidate paracrine inhibitors leads to reduced glucagon secretion but did not decrease the alpha-cell calcium activity. Taken together, the data suggest that suppression occurs downstream from alpha-cell calcium signaling, presumably at the level of vesicle trafficking or exocytotic machinery.

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCα and PKCδ

Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.

Characterization of Erg K+ Channels in α- and β-Cells of Mouse and Human Islets

Voltage-gated eag-related gene (Erg) K(+) channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in alpha-TC6 and Min6 cells alpha- and beta-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in alpha- and beta-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca(2+)-imaging experiments were performed on isolated alpha- and beta-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca(2+) increase in both alpha- and beta-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of alpha- and beta-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.

Effects of the biguanide synthalin A on the pancreatic A2-cell of the guinea pig.

Synthalin A is a guanidine derivative, chemically related to the antidiabetic drugs of the biguanide group. The effects of this drug have now been studied with respect to pancreatic islet morphology, glucagon release and glucose oxidation of the pancreatic A2-cells using isolated islets from normal and streptozotocin-treated guinea pigs. At 5.5 mmol/l glucose, synthalin A (5-500 micrograms/ml) increased glucagon secretion from isolated normal islets by 2-3 times. The enhanced glucagon release by 5 micrograms/ml of the drug was not suppressed by high concentrations of glucose plus insulin in the medium, whereas exogenous somatostatin markedly counter-acted glucagon release evoked by synthalin A. Addition of synthalin A (5-500 micrograms/ml) to isolated, A2-cells rich islets from streptozotocin-treated animals, suppressed the glucose oxidation rate to less than one third of that obtained in 5.5 mmol/l glucose alone. Glucose oxidation of normal islets was similarly reduced, but to a smaller extent. Light microscopy of islets, cultured for 3 days after exposure (2 hours) to synthalin A, showed vacuolization and other dose-dependent degenerative signs. In conclusion, synthalin A markedly affected islet morphology and stimulated glucagon secretion of isolated guinea pigs islets. Furthermore, the simultaneous inhibition, by synthalin A, of glucose oxidation of the A2-cell rich islets supports the concept of a glucose-mediated regulation of glucagon release, via the energy-yielding metabolism of the A2-cell.

Does high cortisol in uremic patients influence their glucagon levels?

In view of the data reported by Barseghian and Levine (1980) that cortisol exerts a direct stimulatory influence on glucagon secretion, the relationship between plasma cortisol and glucagon levels in a group of 43 patients with chronic renal failure (CRF) was investigated. A positive correlation (r = 0.363 and 0.811) was found with all patients studied or with 10 patients with urine output above 1000 ml/24 h, respectively. The result obtained suggest that the elevated cortisol levels play some role in the stimulation of glucagon secretion in CRF patients.

C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 +/- 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4-23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.

Oral glutamine increases circulating glucagon-like peptide 1, glucagon, and insulin concentrations in lean, obese, and type 2 diabetic subjects

BackgroundIncretin hormones, such as glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play an important role in meal-related insulin secretion. We previously demonstrated that glutamine is a potent stimulus of GLP-1 secretion in vitro. ObjectiveOur objective was to determine whether glutamine increases circulating GLP-1 and GIP concentrations in vivo and, if so, whether this is associated with an increase in plasma insulin. DesignWe recruited 8 healthy normal-weight volunteers (LEAN), 8 obese individuals with type 2 diabetes or impaired glucose tolerance (OB-DIAB) and 8 obese nondiabetic control subjects (OB-CON). Oral glucose (75 g), glutamine (30 g), and water were administered on 3 separate days in random order, and plasma concentrations of GLP-1, GIP, insulin, glucagon, and glucose were measured over 120 min. ResultsOral glucose led to increases in circulating GLP-1 concentrations, which peaked at 30 min in LEAN (31.9 ± 5.7 pmol/L) and OB-CON (24.3 ± 2.1 pmol/L) subjects and at 45 min in OB-DIAB subjects (19.5 ± 1.8 pmol/L). Circulating GLP-1 concentrations increased in all study groups after glutamine ingestion, with peak concentrations at 30 min of 22.5 ± 3.4, 17.9 ± 1.1, and 17.3 ± 3.4 pmol/L in LEAN, OB-CON, and OB-DIAB subjects, respectively. Glutamine also increased plasma GIP concentrations but less effectively than glucose. Consistent with the increases in GLP-1 and GIP, glutamine significantly increased circulating plasma insulin concentrations. Glutamine stimulated glucagon secretion in all 3 study groups. ConclusionGlutamine effectively increases circulating GLP-1, GIP, and insulin concentrations in vivo and may represent a novel therapeutic approach to stimulating insulin secretion in obesity and type 2 diabetes.

Investigation of Transport Mechanisms and Regulation of Intracellular Zn2+ in Pancreatic α-Cells

During insulin secretion, pancreatic alpha-cells are exposed to Zn(2+) released from insulin-containing secretory granules. Although maintenance of Zn(2+) homeostasis is critical for cell survival and glucagon secretion, very little is known about Zn(2+)-transporting pathways and the regulation of Zn(2+) in alpha-cells. To examine the effect of Zn(2+) on glucagon secretion and possible mechanisms controlling the intracellular Zn(2+) level ([Zn(2+)](i)), we employed a glucagon-producing cell line (alpha-TC6) and mouse islets where non-beta-cells were identified using islets expressing green fluorescent protein exclusively in beta-cells. In this study, we first confirmed that Zn(2+) treatment resulted in the inhibition of glucagon secretion in alpha-TC6 cells and mouse islets in vitro. The inhibition of secretion was not likely via activation of K(ATP) channels by Zn(2+). We then determined that Zn(2+) was transported into alpha-cells and was able to accumulate under both low and high glucose conditions, as well as upon depolarization of cells with KCl. The nonselective Ca(2+) channel blocker Gd(3+) partially inhibited Zn(2+) influx in alpha-TC cells, whereas the L-type voltage-gated Ca(2+) channel inhibitor nitrendipine failed to block Zn(2+) accumulation. To investigate Zn(2+) transport further, we profiled alpha-cells for Zn(2+) transporter transcripts from the two families that work in opposite directions, SLC39 (ZIP, Zrt/Irt-like protein) and SLC30 (ZnT, Zn(2+) transporter). We observed that Zip1, Zip10, and Zip14 were the most abundantly expressed Zips and ZnT4, ZnT5, and ZnT8 the dominant ZnTs. Because the redox state of cells is also a major regulator of [Zn(2+)](i), we examined the effects of oxidizing agents on Zn(2+) mobilization within alpha-cells. 2,2'-Dithiodipyridine (-SH group oxidant), menadione (superoxide generator), and SIN-1 (3-morpholinosydnonimine) (peroxynitrite generator) all increased [Zn(2+)](i) in alpha-cells. Together these results demonstrate that Zn(2+) inhibits glucagon secretion, and it is transported into alpha-cells in part through Ca(2+) channels. Zn(2+) transporters and the redox state also modulate [Zn(2+)](i).