Paradoxical Stimulation of Glucagon Secretion by High Glucose Concentrations
http://www.100md.com
《糖尿病学杂志》
1 Department of Clinical Science, Clinical Research Center, Malm University Hospital, Malm, Sweden
2 Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
[Ca2+]i, cytoplasmic Ca2+ concentration; KATP channel, ATP-sensitive K+ channel
ABSTRACT
Hypersecretion of glucagon contributes to the dysregulation of glucose homeostasis in diabetes. To clarify the underlying mechanism, glucose-regulated glucagon secretion was studied in mouse pancreatic islets and clonal hamster In-R1-G9 glucagon-releasing cells. Apart from the well-known inhibition of secretion with maximal effect around 7 mmol/l glucose, we discovered that mouse islets showed paradoxical stimulation of glucagon release at 25–30 mmol/l and In-R1-G9 cells at 12–20 mmol/l sugar. Whereas glucagon secretion in the absence of glucose was inhibited by hyperpolarization with diazoxide, this agent tended to further enhance secretion stimulated by high concentrations of the sugar. Because U-shaped dose-response relationships for glucose-regulated glucagon secretion were observed in normal islets and in clonal glucagon-releasing cells, both the inhibitory and stimulatory components probably reflect direct effects on the -cells. Studies of isolated mouse -cells indicated that glucose inhibited glucagon secretion by lowering the cytoplasmic Ca2+ concentration. However, stimulation of glucagon release by high glucose concentrations did not require elevation of Ca2+, indicating involvement of novel mechanisms in glucose regulation of glucagon secretion. A U-shaped dose-response relationship for glucose-regulated glucagon secretion may explain why diabetic patients with pronounced hyperglycemia display paradoxical hyperglucagonemia.
Hyperglycemia in diabetes is primarily due to inappropriate or no secretion of insulin. However, there is ample evidence that hypersecretion of glucagon plays an important role in initiating and maintaining hyperglycemic conditions in diabetic animals and humans (1,2). Considerable attention is therefore given to glucagon signaling as a target in the treatment of diabetic hyperglycemia (2,3). The elevated glucagon concentrations in diabetes can be reduced but not normalized by insulin treatment (4,5), and the mechanisms underlying the hyperglucagonemia have not been fully elucidated.
Glucagon has an important role in glucose counter-regulation (6). Secretion occurs in response to hypoglycemia and is inhibited already when the glucose concentration reaches 4–6 mmol/l (7,8). Because such concentrations are at or below the threshold for stimulation of insulin secretion, it is apparent that release of insulin or other -cell factors does not suffice to explain inhibition of glucagon secretion. We now discovered that the dose-response relationships for glucose-regulated glucagon secretion from mouse pancreatic islets and clonal hamster glucagon-releasing In-R1-G9 cells are U-shaped with inhibition at 7–8 mmol/l glucose followed by stimulation at high concentrations of the sugar. Studying the mechanism underlying these effects in mouse -cells, we found that inhibition of secretion was associated with a lowering of the cytoplasmic Ca2+ concentration ([Ca2+]i), whereas the stimulation at high glucose concentrations occurred independently of [Ca2+]i elevation. In the mouse islets, the stimulatory component was most pronounced above 20 mmol/l glucose when insulin secretion is maximally stimulated. A similar regulation of human -cells may explain why diabetic patients with hyperglycemia display paradoxical hyperglucagonemia and why a further glucose challenge has been found to stimulate glucagon release (9–11).
RESEARCH DESIGN AND METHODS
Preparation and culture of pancreatic islets and cells and clonal glucagon-releasing cells.
Islets of Langerhans were isolated with collagenase (Boehringer Mannheim, Mannheim, Germany) from C57 BL/6 mice. Local ethics committees approved the experimental procedures. The animals were killed by decapitation under anesthesia with CO2. The peritoneal cavity was opened, and a collagenase solution was injected into the bile-pancreatic duct to expand the pancreas (glucagon secretion experiments). The pancreas was excised and cut into small pieces, which were digested with collagenase to obtain free islets of Langerhans. The lower duodenal part of the pancreas was rejected to avoid islets with cells producing pancreatic polypeptide (12). The freshly isolated islets were used for studies of glucagon and insulin secretion or preparation of free cells. Free cells were obtained by incubating the islets for 4 min at 37°C in Ca2+-deficient medium containing 0.5 mmol/l EDTA and 0.05% trypsin (Invitrogen, Carlsbad, CA) followed by brief shaking. The cells were suspended in RPMI 1640 medium with 5.5 mmol/l glucose (Gibco, Paisley, Scotland) supplemented with 10% FCS (Gibco), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 30 μg/ml gentamicin. Small samples of this suspension (15 μl) were applied to the centers of poly-L-lysine-coated (Sigma Chemical, St. Louis, MO) circular 25-mm coverslips. The coverslips were then kept for 60 min in a culture incubator at 37°C with a humidified atmosphere of 5% CO2 to allow cells to settle and begin attachment. More medium was then cautiously added, and the cells were cultured for 1–3 days.
Glucagon-secreting clonal hamster In-R1-G9 cells (13) were provided by Dr. Bjrn Olde (Lund University) with permission from Dr. Jacques Philippe (University of Geneva, Geneva, Switzerland). The cells were cultured on plastic dishes or culture bottles in GlutaMAX-containing RPMI 1640 medium with 11 mmol/l glucose (Gibco) supplemented with 10% FCS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. For secretion studies, the In-R1-G9 cells were seeded at a density of 100,000 cells/well in 48-well plates and cultured as described above for 3 days.
Measurements of [Ca2+]i.
The cells were loaded with the Ca2+ indicator fura-2 during a 40-min incubation at 37°C in a buffer containing 0.5 mg/ml BSA (Sigma), 125 mmol/l NaCl, 4.8 mmol/l KCl, 1.2 mmol/l MgCl2, 1.28 mmol/l CaCl2, 3 mmol/l glucose, 1 μmol/l fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) (0.1% dimethylsulfoxide; Sigma), and 25 mmol/l HEPES (Sigma) with pH adjusted to 7.4 with 13 mmol/l NaOH. The coverslips with the attached cells were used as exchangeable bottoms of an open chamber. The chamber volume was 0.16 ml, and the cells were superfused at a rate of 1 ml/min. The superfusion chamber was placed on the stage of an inverted Nikon Diaphot microscope equipped with an epifluorescence illuminator and a 40x oil immersion fluorescence objective. The chamber holder and the objective were maintained at 37°C by custom-built thermostats. The epifluorescence illuminator was connected through a 5-mm-diameter liquid light guide to an Optoscan monochromator (Cairn Research, Faversham, U.K.) with rapid grating and slit width adjustment and a 150 W xenon arc lamp. The monochromator provided excitation light at 340 nm (2.8-nm half-bandwidth) and 380 nm (2.5-nm half-bandwidth), and emission was measured at >515 nm by an intensified CCD camera. The Metafluor software (Universal Imaging, Downingtown, PA) controlled the monochromator acquiring fluorescence images of 30 accumulated frames at 340 and 380 nm every 4 s. [Ca2+]i images were calculated from 340-to-380 nm ratio images as previously described (14).
Identification of -cells.
The -cells were initially selected by their small size and [Ca2+]i response to epinephrine (Sigma) (14,15), which is not shared by -cells (14) and -cells (16). Only -cells confirmed by immunostaining were included in the analyses (14).
Hormone secretion.
Batches of 8–12 freshly isolated islets were preincubated for 30 min at 37°C in 1 ml Krebs-Ringer buffer (pH 7.4) supplemented with 10 mmol/l HEPES, 0.1% BSA, and 1 mmol/l glucose. Each incubation vial was gassed with 95% O2 and 5% CO2 to obtain constant pH and oxygenation. The islets were then incubated for 1 h at 37°C in the Krebs-Ringer buffer containing different glucose or 3-o-methyl glucose concentrations and 4.8 mmol/l K or 30 mmol/l K+ plus 250 μmol/l diazoxide. At the end of the incubation, aliquots of the medium were removed and frozen pending the radioimmunoassay of glucagon and insulin (17).
The 3-day cultured In-R1-G9 cells were carefully washed and preincubated for 30 min at 37°C in the Krebs-Ringer buffer containing 1 mmol/l glucose (1 ml/well). The medium was then replaced by one containing different glucose concentrations and incubated for 60 min at 37°C. Aliquots of the medium were removed and frozen pending assay of glucagon, insulin, and somatostatin (17,18).
Statistical analysis.
Dose-response relationships for the effect of glucose on glucagon and insulin secretion were analyzed with ANOVA and paired Student’s t test. The reaction patterns in individual -cells were analyzed with two-tailed Fisher’s exact test. All calculations were made by SigmaStat software (Systat Software, Erkrath, Germany).
RESULTS
Rise of [Ca2+]i is probably the most important trigger of pancreatic islet hormone secretion. Accordingly, the inhibition of glucagon secretion obtained with elevation of glucose was associated with lowering of [Ca2+]i. In 57% (8 of 14; P < 0.01) of individual mouse -cells selected for showing [Ca2+]i oscillations at 1 mmol/l glucose, [Ca2+]i was reduced to basal levels at 20 mmol/l sugar (Fig. 2). After further elevation to 30 mmol/l glucose, 86% (12 of 14; P < 0.001) of the -cells were inhibited.
To clarify the role of [Ca2+]i in glucose-regulated glucagon secretion, mouse islets were studied also under hyperpolarizing conditions, preventing voltage-dependent influx of the ion. This was achieved by exposure to 250 μmol/l diazoxide, which clamps the membrane potential close to the equilibrium potential for K+ by activating the ATP-sensitive K+ channels (KATP channels) that are present in all types of islet cells (12,14,16,19). Hyperpolarization in the absence of glucose had no effect on [Ca2+]i of resting -cells but lowered [Ca2+]i of oscillating -cells (not shown). In the presence of diazoxide, introduction of 7 mmol/l glucose resulted in a small reduction of basal [Ca2+]i in 78% of the -cells (14 of 18; P < 0.001) and all of 12 -cells (P < 0.001; Fig. 3). Further elevation of glucose to 30 mmol/l had no effect on the -cells but induced a more pronounced additional reduction of [Ca2+]i in 23 of 25 -cells (P < 0.001). These data are consistent with stimulated sequestration and outward transport of Ca2+ (14,20,21) being saturated at lower glucose concentrations in -cells (14) than in -cells (21). Measurements of hormone secretion under similar conditions demonstrated that hyperpolarization with diazoxide reduced glucagon release at 0–1 mmol/l glucose by 70–80% of the maximal glucose inhibition (Fig. 4, top panel). Nevertheless, the additional inhibition obtained by raising the sugar concentration to 7 mmol/l remained statistically significant. Glucagon secretion at higher glucose concentrations was not diminished during hyperpolarizing conditions and even slightly exceeded control secretion in the 12–30 mmol/l range. Basal insulin secretion at 0–7 mmol/l glucose was unaffected by the hyperpolarization (Fig. 4, bottom panel), whereas insulin secretion at 12–30 mmol/l glucose was reduced by 70% compared with control.
Glucose initiates insulin release by raising [Ca2+]i in -cells. The sugar also has an amplifying effect on secretion, which is apparent when [Ca2+]i is held constant at an elevated level (22). This situation can be achieved by clamping the membrane potential at a depolarized level (30 mmol/l K+ plus diazoxide) to activate voltage-dependent Ca2+ influx. We used this approach to clarify whether glucose also has an amplifying effect on glucagon secretion. Figure 5 shows that depolarization by combining diazoxide with 30 mmol/l K+ resulted in rapid increases of [Ca2+]i in both - and -cells. Subsequent introduction of 3 mmol/l glucose caused a small reduction of [Ca2+]i in 3 of 19 -cells, but there was no change of [Ca2+]i when increasing the glucose concentration from 0 to 3, 7, and 30 mmol/l in the remaining 16 -cells. However, 14 of 16 -cells responded with temporary reduction of [Ca2+]i after introducing at the highest glucose concentration (P < 0.001), supporting previous observations that stimulated Ca2+ sequestration and outward transport can transiently affect [Ca2+]i in this cell type also when voltage-dependent influx is stimulated (23). Under these depolarizing conditions, basal insulin secretion increased 3.5-fold at 0–7 mmol/l (Fig. 6, bottom panel). Glucose did not affect the depolarization-induced insulin secretion in the 0–7 mmol/l range, but higher concentrations of the sugar had a pronounced amplifying effect. The corresponding glucagon data demonstrated that 30 mmol/l K+ plus diazoxide markedly stimulated glucagon secretion in the 0–30 mmol/l range (Fig. 6, top panel). Glucose elevation to 7 mmol/l failed to induce significant inhibition of secretion. Higher concentrations of the sugar still stimulated glucagon release, but this effect was not more pronounced than under control or hyperpolarizing conditions.
DISCUSSION
It is generally accepted that glucose is an inhibitor of glucagon secretion (1,2). However, its was evident from the present study of mouse pancreatic islets and clonal hamster In-R1-G9 glucagon-releasing cells that inhibition of glucagon secretion by intermediate glucose concentrations is followed by reversal of this effect and even stimulation at higher concentrations of the sugar. The U-shaped dose-response patterns are different from those obtained with perfused rat pancreas (7) and batch-incubated rat islets (8), which only show glucose inhibition of glucagon secretion. A recent observation that glucose stimulates glucagon secretion from purified rat -cells was taken to indicate that this secretion is regulated in a -cell–like manner and that the inhibitory effect of glucose requires release of paracrine factors from non–-cells within the islets (8). The present observation of glucose-stimulated glucagon secretion from mouse islet is principally different from that in purified rat -cells. Whereas the latter is attributed to a -cell–like pathway with closure of KATP channels leading to depolarization and voltage-dependent Ca2+ influx, glucose stimulation of glucagon secretion from mouse islets occurred independent of KATP channels and did not require elevation of [Ca2+]i. A reason for the different [Ca2+]i responses is apparently that rat -cells have a high density of KATP channels, exceeding that in rat -cells (24), whereas mouse -cells show little KATP channel expression (25,26), corresponding to 2% of that in mouse -cells (27). Depolarization by tolbutamide-induced closure of the KATP channels thus increases the electrical activity (24), [Ca2+]i (8), exocytosis (28), and secretion (8) in rat -cells, but tolbutamide-induced elevation of [Ca2+]i is observed only in a small minority of mouse -cells (14). Moreover, whereas glucose increases the electrical activity and [Ca2+]i of rat -cells (24), the sugar hyperpolarizes mouse -cells (14,27,29) and, as now confirmed, lowers [Ca2+]i (14).
The present study of mouse -cells and islets and clonal hamster glucagon-releasing cells provides arguments that glucose has direct effects on the -cells in terms of inhibition and stimulation of glucagon release. Although the In-R1-G9 cells are derived from an insulin-secreting cell line also producing glucagon (13), the presently used cells did not release detectable amounts of insulin. Considering dilutions and detection limits of the assays, this means that maximal theoretical insulin secretion on a molar basis is lower than or similar to glucagon secretion compared with an 40-fold excess of insulin secretion in the mouse islets. Also, somatostatin secretion was undetectable, indicating that maximal theoretical secretion of this hormone corresponded to 0.5–4% of glucagon secretion, which is much lower than in the mouse islets (data not shown). It seems unlikely that phenotypic diversity among the In-R1-G9 cells explains inhibitory or stimulatory effects of glucose by paracrine routes, but an inhibitory effect of insulin is not entirely excluded. The observation that most inhibition of glucagon secretion from mouse islets is obtained at glucose concentrations, which fail to stimulate insulin release argues against the involvement of the proposed -cell factors insulin (30), Zn2+ (30,31), and -aminobutyric acid (32,33). Moreover, glucose stimulation of glucagon secretion was not diminished, and some glucose inhibition of glucagon secretion remained under conditions expected to hyperpolarize the islet cells and reduce release of paracrine factors.
Three alternative mechanisms have been proposed to mediate a direct glucose inhibition of the -cell. In conflict with the hyperpolarizing effect of glucose (14,27,29), one of these proposals assumes that glucose paradoxically inhibits glucagon secretion by depolarizing mouse -cells (26,34). The other two alternatives involve glucose-induced hyperpolarization by activation of the electrogenic Na,K-ATPase (35) or inhibition of a depolarizing store-operated cation influx by glucose-stimulated Ca2+ sequestration in the endoplasmic reticulum (14).
Because the present and previous data (14) show that glucose lowers [Ca2+]i in mouse -cells, the question arises how high concentrations of the sugar stimulate glucagon secretion. In the insulin-releasing -cells, glucose seems to stimulate insulin secretion by at least three mechanisms. Initiation of secretion is generally attributed to depolarization with increase of [Ca2+]i after closure of KATP channels (19). Glucose also has an amplifying effect, which does not require further elevation of [Ca2+]i (22). In addition, there is evidence that increased metabolism can stimulate insulin release independent of [Ca2+]i elevation (36,37). We now confirm all of these actions by showing 1) stimulated insulin secretion after [Ca2+]i elevation due to depolarization with 30 mmol/l K+ in the presence of diazoxide; 2) glucose amplification of this secretory response, although the sugar temporarily lowered [Ca2+]i in isolated -cells; and 3) maintenance of some glucose-stimulated insulin secretion under hyperpolarizing conditions when the sugar lowered [Ca2+]i below the basal level.
The Ca2+-dependent and -independent pathways for stimulated secretion were present also in mouse -cells. 1) Elevation of [Ca2+]i in isolated -cells correlated to enhanced glucagon release from pancreatic islets. 2) High glucose concentrations stimulated glucagon secretion under hyperpolarizing conditions, although [Ca2+]i of the -cells was lowered below basal levels. It was recently reported that glucose amplifies Ca2+-initiated glucagon secretion from purified rat -cells (8). However, the presently observed stimulatory effect of glucose on glucagon secretion from mouse islets did not depend on the [Ca2+]i level of the -cells, indicating that glucose initiates rather than amplifies secretion. It may seem surprising that the -cells can sense high concentrations of glucose because they express low Km glucose transporter GLUT1 but lack high Km GLUT2 (38). The situation is reminiscent of that in human -cells, which, unlike the GLUT2-expressing rodent -cells, predominantly express GLUT1 (39). Apparently glucose transport via GLUT1 is not rate limiting in these cases, and the sensing is explained by the presence of the high Km glucokinase (39,40). The signal transduction of the intriguing glucose stimulation of glucagon secretion remains to be elucidated.
Hypersecretion of glucagon is an important feature of diabetes contributing to glucose dysregulation (1,2). The mechanism underlying this phenomenon is unclear. The present study of mouse islets and clonal hamster glucagon-releasing cells indicated that paradoxical hypersecretion of glucagon in hyperglycemia may depend on a U-shaped dose-response relationship for glucose regulation of secretion. A similar regulation of human -cells may explain why diabetic patients with hyperglycemia display paradoxical hyperglucagonemia and why a further glucose challenge has been found to stimulate glucagon release (9–11).
ACKNOWLEDGMENTS
This work has received support from the Swedish Medical Research Council (grant 12X-6240), the Swedish Diabetes Association, the Carl Trygger Foundation, Family Ernfors Foundation, Albert Phlsson’s Foundation, the Crafoord Foundation, and the Scandinavian Physiological Society.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Gerich JE, Charles A, Grodsky GM: Regulation of pancreatic insulin and glucagon secretion. Annu Rev Physiol 38:353–388, 1976
Jiang G, Zhang BB: Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab 284:E671–E678, 2003
Sloop KW, Michael MD, Moyers JS: Glucagon as a target for the treatment of type 2 diabetes. Expert Opin Ther Targets 9:593–600, 2005
Unger RH: Diabetes and the cell. Diabetes 25:136–151, 1976
Gossain VV, Rovner DR: Pancreatic glucagon: possible implications of the hyperglycemic hormone in diabetes control. Postgrad Med 72:87–88, 91–93, 96, 1982
Cryer PE, Davis SN, Shamoon H: Hypoglycemia in diabetes. Diabetes Care 26:1902–1912, 2003
Gerich JE, Charles A, Grodsky GM: Characterization of the effects of arginine and glucose on glucagon and insulin release from the perfused rat pancreas. J Clin Invest 54:833–841, 1974
Olsen HL, Theander S, Bokvist K, Buschard K, Wollheim CB, Gromada J: Glucose stimulates glucagon release in single rat -cells by mechanisms that mirror the stimulus-secretion coupling in -cells. Endocrinology 146:4861–4870, 2005
Buchanan KD, McCarroll AM: Abnormalities of glucagon metabolism in untreated diabetes mellitus. Lancet 300:1394–1395, 1972
Ohneda A, Watanabe K, Horigome K, Sakai T, Kai Y, Oikawa S: Abnormal response of pancreatic glucagon to glycemic changes in diabetes mellitus. J Clin Endocrinol Metab 46:504–510, 1978
Mitrakou A, Kelley D, Veneman T, Jenssen T, Pangburn T, Reilly J, Gerich J: Contribution of abnormal muscle and liver glucose metabolism to postprandial hyperglycemia in NIDDM. Diabetes 39:1381–1390, 1990
Liu YJ, Hellman B, Gylfe E: Ca2+ signaling in mouse pancreatic polypeptide cells. Endocrinology 140:5524–5529, 1999
Takaki R, Ono J, Nakamura M, Yokogawa Y, Kumae S, Hiraoka T, Yamaguchi K, Hamaguchi K, Uchida S: Isolation of glucagon-secreting cell lines by cloning insulinoma cells. In Vitro Cell Dev Biol 22:120–126, 1986
Liu YJ, Vieira E, Gylfe E: A store-operated mechanism determines the activity of the electrically excitable glucagon-secreting pancreatic -cell. Cell Calcium 35:357–365, 2004
Johansson H, Gylfe E, Hellman B: Cyclic AMP raises cytoplasmic calcium in pancreatic 2-cells by mobilizing calcium incorporated in response to glucose. Cell Calcium 10:205–211, 1989
Berts A, Ball A, Dryselius S, Gylfe E, Hellman B: Glucose stimulation of somatostatin-producing islet cells involves oscillatory Ca2+ signalling. Endocrinology 137:693–697, 1996
Panagiotidis G, Salehi AA, Westermark P, Lundquist I: Homologous islet amyloid polypeptide: effects on plasma levels of glucagon, insulin and glucose in the mouse. Diabetes Res Clin Pract 18:167–171, 1992
Etzrodt H, Rosenthal J, Schroder KE, Pfeiffer EF: Radioimmunoassay of somatostatin in human plasma. Clin Chim Acta 133:241–251, 1983
Ashcroft FM, Rorsman P: ATP-sensitive K+ channels: a link between B-cell metabolism and insulin secretion. Biochem Soc Trans 18:109–111, 1990
Johansson H, Gylfe E, Hellman B: The actions of arginine and glucose on glucagon secretion are mediated by opposite effects on cytoplasmic Ca2+. Biochem Biophys Res Commun 147:309–314, 1987
Gylfe E: Nutrient secretagogues induce bimodal early changes in cytoplasmic calcium of insulin-releasing ob/ob mouse -cells. J Biol Chem 263:13750–13754, 1988
Gembal M, Gilon P, Henquin JC: Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells. J Clin Invest 89:1288–1295, 1992
Gylfe E: Glucose-induced buffering of cytoplasmic Ca2+ in the pancreatic -cell: an artifact or a physiological phenomenon Biochem Biophys Res Commun 159:907–912, 1989
Bokvist K, Olsen HL, Hy M, Gotfredsen CF, Holmes WF, Buschard K, Rorsman P, Gromada J: Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells. Pflügers Arch 438:428–436, 1999
Quesada I, Nadal A, Soria B: Different effects of tolbutamide and diazoxide in -, -, and -cells within intact islets of Langerhans. Diabetes 48:2390–2397, 1999
Gpel SO, Kanno T, Barg S, Weng XG, Gromada J, Rorsman P: Regulation of glucagon secretion in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels. J Physiol (Lond) 528:509–520, 2000
Barg S, Galvanovskis J, Gpel SO, Rorsman P, Eliasson L: Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting -cells. Diabetes 49:1500–1510, 2000
Hy M, Olsen HL, Bokvist K, Buschard K, Barg S, Rorsman P, Gromada J: Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells. J Physiol (Lond) 527:109–120, 2000
Hjortoe GM, Hagel GM, Terry BR, Thastrup O, Arkhammar PO: Functional identification and monitoring of individual and cells in cultured mouse islets of Langerhans. Acta Diabetol 41:185–193, 2004
Franklin I, Gromada J, Gjinovci A, Theander S, Wollheim CB: -Cell secretory products activate -cell ATP-dependent potassium channels to inhibit glucagon release. Diabetes 54:1808–1815, 2005
Ishihara H, Maechler P, Gjinovci A, Herrera PL, Wollheim CB: Islet -cell secretion determines glucagon release from neighbouring -cells. Nat Cell Biol 5:330–335, 2003
Wendt A, Birnir B, Buschard K, Gromada J, Salehi A, Sewing S, Rorsman P, Braun M: Glucose inhibition of glucagon secretion from rat -cells is mediated by GABA released from neighboring -cells. Diabetes 53:1038–1045, 2004
Xu E, Kumar M, Zhang Y, Ju W, Obata T, Zhang N, Liu S, Wendt A, Deng S, Ebina Y, Wheeler MB, Braun M, Wang Q: Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system. Cell Metab 3:47–58, 2006
Gromada J, Ma X, Hoy M, Bokvist K, Salehi A, Berggren PO, Rorsman P: ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1–/– mouse -cells. Diabetes 53 (Suppl. 3):S181–S189, 2004
Bode HP, Weber S, Fehmann HC, Gke B: A nutrient-regulated cytosolic calcium oscillator in endocrine pancreatic glucagon-secreting cells. Pflügers Arch 437:324–334, 1999
Westerlund J, Hellman B, Bergsten P: Pulsatile insulin release from mouse islets occurs in the absence of stimulated entry of Ca2+. J Clin Invest 97:1860–1863, 1996
Ravier MA, Gilon P, Henquin JC: Oscillations of insulin secretion can be triggered by imposed oscillations of cytoplasmic Ca2+ or metabolism in normal mouse islets. Diabetes 48:2374–2382, 1999
Heimberg H, De Vos A, Pipeleers DG, Thorens B, Schuit FC: Differences in glucose transporter gene expression between rat pancreatic - and -cells are correlated to differences in glucose transport but not in glucose utilization. J Biol Chem 270:8971–8975, 1995
De Vos A, Heimberg H, Quartier E, Huypens P, Bouwens L, Pipeleers D, Schuit F: Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression. J Clin Invest 96:2489–2495, 1995
Heimberg H, De Vos A, Moens K, Quartier E, Bouwens L, Pipeleers D, Van Schaftingen E, Madsen O, Schuit F: The glucose sensor protein glucokinase is expressed in glucagon-producing -cells. Proc Natl Acad Sci U S A 93:7036–7041, 1996(Albert Salehi, Elaine Vie)
2 Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
[Ca2+]i, cytoplasmic Ca2+ concentration; KATP channel, ATP-sensitive K+ channel
ABSTRACT
Hypersecretion of glucagon contributes to the dysregulation of glucose homeostasis in diabetes. To clarify the underlying mechanism, glucose-regulated glucagon secretion was studied in mouse pancreatic islets and clonal hamster In-R1-G9 glucagon-releasing cells. Apart from the well-known inhibition of secretion with maximal effect around 7 mmol/l glucose, we discovered that mouse islets showed paradoxical stimulation of glucagon release at 25–30 mmol/l and In-R1-G9 cells at 12–20 mmol/l sugar. Whereas glucagon secretion in the absence of glucose was inhibited by hyperpolarization with diazoxide, this agent tended to further enhance secretion stimulated by high concentrations of the sugar. Because U-shaped dose-response relationships for glucose-regulated glucagon secretion were observed in normal islets and in clonal glucagon-releasing cells, both the inhibitory and stimulatory components probably reflect direct effects on the -cells. Studies of isolated mouse -cells indicated that glucose inhibited glucagon secretion by lowering the cytoplasmic Ca2+ concentration. However, stimulation of glucagon release by high glucose concentrations did not require elevation of Ca2+, indicating involvement of novel mechanisms in glucose regulation of glucagon secretion. A U-shaped dose-response relationship for glucose-regulated glucagon secretion may explain why diabetic patients with pronounced hyperglycemia display paradoxical hyperglucagonemia.
Hyperglycemia in diabetes is primarily due to inappropriate or no secretion of insulin. However, there is ample evidence that hypersecretion of glucagon plays an important role in initiating and maintaining hyperglycemic conditions in diabetic animals and humans (1,2). Considerable attention is therefore given to glucagon signaling as a target in the treatment of diabetic hyperglycemia (2,3). The elevated glucagon concentrations in diabetes can be reduced but not normalized by insulin treatment (4,5), and the mechanisms underlying the hyperglucagonemia have not been fully elucidated.
Glucagon has an important role in glucose counter-regulation (6). Secretion occurs in response to hypoglycemia and is inhibited already when the glucose concentration reaches 4–6 mmol/l (7,8). Because such concentrations are at or below the threshold for stimulation of insulin secretion, it is apparent that release of insulin or other -cell factors does not suffice to explain inhibition of glucagon secretion. We now discovered that the dose-response relationships for glucose-regulated glucagon secretion from mouse pancreatic islets and clonal hamster glucagon-releasing In-R1-G9 cells are U-shaped with inhibition at 7–8 mmol/l glucose followed by stimulation at high concentrations of the sugar. Studying the mechanism underlying these effects in mouse -cells, we found that inhibition of secretion was associated with a lowering of the cytoplasmic Ca2+ concentration ([Ca2+]i), whereas the stimulation at high glucose concentrations occurred independently of [Ca2+]i elevation. In the mouse islets, the stimulatory component was most pronounced above 20 mmol/l glucose when insulin secretion is maximally stimulated. A similar regulation of human -cells may explain why diabetic patients with hyperglycemia display paradoxical hyperglucagonemia and why a further glucose challenge has been found to stimulate glucagon release (9–11).
RESEARCH DESIGN AND METHODS
Preparation and culture of pancreatic islets and cells and clonal glucagon-releasing cells.
Islets of Langerhans were isolated with collagenase (Boehringer Mannheim, Mannheim, Germany) from C57 BL/6 mice. Local ethics committees approved the experimental procedures. The animals were killed by decapitation under anesthesia with CO2. The peritoneal cavity was opened, and a collagenase solution was injected into the bile-pancreatic duct to expand the pancreas (glucagon secretion experiments). The pancreas was excised and cut into small pieces, which were digested with collagenase to obtain free islets of Langerhans. The lower duodenal part of the pancreas was rejected to avoid islets with cells producing pancreatic polypeptide (12). The freshly isolated islets were used for studies of glucagon and insulin secretion or preparation of free cells. Free cells were obtained by incubating the islets for 4 min at 37°C in Ca2+-deficient medium containing 0.5 mmol/l EDTA and 0.05% trypsin (Invitrogen, Carlsbad, CA) followed by brief shaking. The cells were suspended in RPMI 1640 medium with 5.5 mmol/l glucose (Gibco, Paisley, Scotland) supplemented with 10% FCS (Gibco), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 30 μg/ml gentamicin. Small samples of this suspension (15 μl) were applied to the centers of poly-L-lysine-coated (Sigma Chemical, St. Louis, MO) circular 25-mm coverslips. The coverslips were then kept for 60 min in a culture incubator at 37°C with a humidified atmosphere of 5% CO2 to allow cells to settle and begin attachment. More medium was then cautiously added, and the cells were cultured for 1–3 days.
Glucagon-secreting clonal hamster In-R1-G9 cells (13) were provided by Dr. Bjrn Olde (Lund University) with permission from Dr. Jacques Philippe (University of Geneva, Geneva, Switzerland). The cells were cultured on plastic dishes or culture bottles in GlutaMAX-containing RPMI 1640 medium with 11 mmol/l glucose (Gibco) supplemented with 10% FCS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. For secretion studies, the In-R1-G9 cells were seeded at a density of 100,000 cells/well in 48-well plates and cultured as described above for 3 days.
Measurements of [Ca2+]i.
The cells were loaded with the Ca2+ indicator fura-2 during a 40-min incubation at 37°C in a buffer containing 0.5 mg/ml BSA (Sigma), 125 mmol/l NaCl, 4.8 mmol/l KCl, 1.2 mmol/l MgCl2, 1.28 mmol/l CaCl2, 3 mmol/l glucose, 1 μmol/l fura-2 acetoxymethyl ester (Molecular Probes, Eugene, OR) (0.1% dimethylsulfoxide; Sigma), and 25 mmol/l HEPES (Sigma) with pH adjusted to 7.4 with 13 mmol/l NaOH. The coverslips with the attached cells were used as exchangeable bottoms of an open chamber. The chamber volume was 0.16 ml, and the cells were superfused at a rate of 1 ml/min. The superfusion chamber was placed on the stage of an inverted Nikon Diaphot microscope equipped with an epifluorescence illuminator and a 40x oil immersion fluorescence objective. The chamber holder and the objective were maintained at 37°C by custom-built thermostats. The epifluorescence illuminator was connected through a 5-mm-diameter liquid light guide to an Optoscan monochromator (Cairn Research, Faversham, U.K.) with rapid grating and slit width adjustment and a 150 W xenon arc lamp. The monochromator provided excitation light at 340 nm (2.8-nm half-bandwidth) and 380 nm (2.5-nm half-bandwidth), and emission was measured at >515 nm by an intensified CCD camera. The Metafluor software (Universal Imaging, Downingtown, PA) controlled the monochromator acquiring fluorescence images of 30 accumulated frames at 340 and 380 nm every 4 s. [Ca2+]i images were calculated from 340-to-380 nm ratio images as previously described (14).
Identification of -cells.
The -cells were initially selected by their small size and [Ca2+]i response to epinephrine (Sigma) (14,15), which is not shared by -cells (14) and -cells (16). Only -cells confirmed by immunostaining were included in the analyses (14).
Hormone secretion.
Batches of 8–12 freshly isolated islets were preincubated for 30 min at 37°C in 1 ml Krebs-Ringer buffer (pH 7.4) supplemented with 10 mmol/l HEPES, 0.1% BSA, and 1 mmol/l glucose. Each incubation vial was gassed with 95% O2 and 5% CO2 to obtain constant pH and oxygenation. The islets were then incubated for 1 h at 37°C in the Krebs-Ringer buffer containing different glucose or 3-o-methyl glucose concentrations and 4.8 mmol/l K or 30 mmol/l K+ plus 250 μmol/l diazoxide. At the end of the incubation, aliquots of the medium were removed and frozen pending the radioimmunoassay of glucagon and insulin (17).
The 3-day cultured In-R1-G9 cells were carefully washed and preincubated for 30 min at 37°C in the Krebs-Ringer buffer containing 1 mmol/l glucose (1 ml/well). The medium was then replaced by one containing different glucose concentrations and incubated for 60 min at 37°C. Aliquots of the medium were removed and frozen pending assay of glucagon, insulin, and somatostatin (17,18).
Statistical analysis.
Dose-response relationships for the effect of glucose on glucagon and insulin secretion were analyzed with ANOVA and paired Student’s t test. The reaction patterns in individual -cells were analyzed with two-tailed Fisher’s exact test. All calculations were made by SigmaStat software (Systat Software, Erkrath, Germany).
RESULTS
Rise of [Ca2+]i is probably the most important trigger of pancreatic islet hormone secretion. Accordingly, the inhibition of glucagon secretion obtained with elevation of glucose was associated with lowering of [Ca2+]i. In 57% (8 of 14; P < 0.01) of individual mouse -cells selected for showing [Ca2+]i oscillations at 1 mmol/l glucose, [Ca2+]i was reduced to basal levels at 20 mmol/l sugar (Fig. 2). After further elevation to 30 mmol/l glucose, 86% (12 of 14; P < 0.001) of the -cells were inhibited.
To clarify the role of [Ca2+]i in glucose-regulated glucagon secretion, mouse islets were studied also under hyperpolarizing conditions, preventing voltage-dependent influx of the ion. This was achieved by exposure to 250 μmol/l diazoxide, which clamps the membrane potential close to the equilibrium potential for K+ by activating the ATP-sensitive K+ channels (KATP channels) that are present in all types of islet cells (12,14,16,19). Hyperpolarization in the absence of glucose had no effect on [Ca2+]i of resting -cells but lowered [Ca2+]i of oscillating -cells (not shown). In the presence of diazoxide, introduction of 7 mmol/l glucose resulted in a small reduction of basal [Ca2+]i in 78% of the -cells (14 of 18; P < 0.001) and all of 12 -cells (P < 0.001; Fig. 3). Further elevation of glucose to 30 mmol/l had no effect on the -cells but induced a more pronounced additional reduction of [Ca2+]i in 23 of 25 -cells (P < 0.001). These data are consistent with stimulated sequestration and outward transport of Ca2+ (14,20,21) being saturated at lower glucose concentrations in -cells (14) than in -cells (21). Measurements of hormone secretion under similar conditions demonstrated that hyperpolarization with diazoxide reduced glucagon release at 0–1 mmol/l glucose by 70–80% of the maximal glucose inhibition (Fig. 4, top panel). Nevertheless, the additional inhibition obtained by raising the sugar concentration to 7 mmol/l remained statistically significant. Glucagon secretion at higher glucose concentrations was not diminished during hyperpolarizing conditions and even slightly exceeded control secretion in the 12–30 mmol/l range. Basal insulin secretion at 0–7 mmol/l glucose was unaffected by the hyperpolarization (Fig. 4, bottom panel), whereas insulin secretion at 12–30 mmol/l glucose was reduced by 70% compared with control.
Glucose initiates insulin release by raising [Ca2+]i in -cells. The sugar also has an amplifying effect on secretion, which is apparent when [Ca2+]i is held constant at an elevated level (22). This situation can be achieved by clamping the membrane potential at a depolarized level (30 mmol/l K+ plus diazoxide) to activate voltage-dependent Ca2+ influx. We used this approach to clarify whether glucose also has an amplifying effect on glucagon secretion. Figure 5 shows that depolarization by combining diazoxide with 30 mmol/l K+ resulted in rapid increases of [Ca2+]i in both - and -cells. Subsequent introduction of 3 mmol/l glucose caused a small reduction of [Ca2+]i in 3 of 19 -cells, but there was no change of [Ca2+]i when increasing the glucose concentration from 0 to 3, 7, and 30 mmol/l in the remaining 16 -cells. However, 14 of 16 -cells responded with temporary reduction of [Ca2+]i after introducing at the highest glucose concentration (P < 0.001), supporting previous observations that stimulated Ca2+ sequestration and outward transport can transiently affect [Ca2+]i in this cell type also when voltage-dependent influx is stimulated (23). Under these depolarizing conditions, basal insulin secretion increased 3.5-fold at 0–7 mmol/l (Fig. 6, bottom panel). Glucose did not affect the depolarization-induced insulin secretion in the 0–7 mmol/l range, but higher concentrations of the sugar had a pronounced amplifying effect. The corresponding glucagon data demonstrated that 30 mmol/l K+ plus diazoxide markedly stimulated glucagon secretion in the 0–30 mmol/l range (Fig. 6, top panel). Glucose elevation to 7 mmol/l failed to induce significant inhibition of secretion. Higher concentrations of the sugar still stimulated glucagon release, but this effect was not more pronounced than under control or hyperpolarizing conditions.
DISCUSSION
It is generally accepted that glucose is an inhibitor of glucagon secretion (1,2). However, its was evident from the present study of mouse pancreatic islets and clonal hamster In-R1-G9 glucagon-releasing cells that inhibition of glucagon secretion by intermediate glucose concentrations is followed by reversal of this effect and even stimulation at higher concentrations of the sugar. The U-shaped dose-response patterns are different from those obtained with perfused rat pancreas (7) and batch-incubated rat islets (8), which only show glucose inhibition of glucagon secretion. A recent observation that glucose stimulates glucagon secretion from purified rat -cells was taken to indicate that this secretion is regulated in a -cell–like manner and that the inhibitory effect of glucose requires release of paracrine factors from non–-cells within the islets (8). The present observation of glucose-stimulated glucagon secretion from mouse islet is principally different from that in purified rat -cells. Whereas the latter is attributed to a -cell–like pathway with closure of KATP channels leading to depolarization and voltage-dependent Ca2+ influx, glucose stimulation of glucagon secretion from mouse islets occurred independent of KATP channels and did not require elevation of [Ca2+]i. A reason for the different [Ca2+]i responses is apparently that rat -cells have a high density of KATP channels, exceeding that in rat -cells (24), whereas mouse -cells show little KATP channel expression (25,26), corresponding to 2% of that in mouse -cells (27). Depolarization by tolbutamide-induced closure of the KATP channels thus increases the electrical activity (24), [Ca2+]i (8), exocytosis (28), and secretion (8) in rat -cells, but tolbutamide-induced elevation of [Ca2+]i is observed only in a small minority of mouse -cells (14). Moreover, whereas glucose increases the electrical activity and [Ca2+]i of rat -cells (24), the sugar hyperpolarizes mouse -cells (14,27,29) and, as now confirmed, lowers [Ca2+]i (14).
The present study of mouse -cells and islets and clonal hamster glucagon-releasing cells provides arguments that glucose has direct effects on the -cells in terms of inhibition and stimulation of glucagon release. Although the In-R1-G9 cells are derived from an insulin-secreting cell line also producing glucagon (13), the presently used cells did not release detectable amounts of insulin. Considering dilutions and detection limits of the assays, this means that maximal theoretical insulin secretion on a molar basis is lower than or similar to glucagon secretion compared with an 40-fold excess of insulin secretion in the mouse islets. Also, somatostatin secretion was undetectable, indicating that maximal theoretical secretion of this hormone corresponded to 0.5–4% of glucagon secretion, which is much lower than in the mouse islets (data not shown). It seems unlikely that phenotypic diversity among the In-R1-G9 cells explains inhibitory or stimulatory effects of glucose by paracrine routes, but an inhibitory effect of insulin is not entirely excluded. The observation that most inhibition of glucagon secretion from mouse islets is obtained at glucose concentrations, which fail to stimulate insulin release argues against the involvement of the proposed -cell factors insulin (30), Zn2+ (30,31), and -aminobutyric acid (32,33). Moreover, glucose stimulation of glucagon secretion was not diminished, and some glucose inhibition of glucagon secretion remained under conditions expected to hyperpolarize the islet cells and reduce release of paracrine factors.
Three alternative mechanisms have been proposed to mediate a direct glucose inhibition of the -cell. In conflict with the hyperpolarizing effect of glucose (14,27,29), one of these proposals assumes that glucose paradoxically inhibits glucagon secretion by depolarizing mouse -cells (26,34). The other two alternatives involve glucose-induced hyperpolarization by activation of the electrogenic Na,K-ATPase (35) or inhibition of a depolarizing store-operated cation influx by glucose-stimulated Ca2+ sequestration in the endoplasmic reticulum (14).
Because the present and previous data (14) show that glucose lowers [Ca2+]i in mouse -cells, the question arises how high concentrations of the sugar stimulate glucagon secretion. In the insulin-releasing -cells, glucose seems to stimulate insulin secretion by at least three mechanisms. Initiation of secretion is generally attributed to depolarization with increase of [Ca2+]i after closure of KATP channels (19). Glucose also has an amplifying effect, which does not require further elevation of [Ca2+]i (22). In addition, there is evidence that increased metabolism can stimulate insulin release independent of [Ca2+]i elevation (36,37). We now confirm all of these actions by showing 1) stimulated insulin secretion after [Ca2+]i elevation due to depolarization with 30 mmol/l K+ in the presence of diazoxide; 2) glucose amplification of this secretory response, although the sugar temporarily lowered [Ca2+]i in isolated -cells; and 3) maintenance of some glucose-stimulated insulin secretion under hyperpolarizing conditions when the sugar lowered [Ca2+]i below the basal level.
The Ca2+-dependent and -independent pathways for stimulated secretion were present also in mouse -cells. 1) Elevation of [Ca2+]i in isolated -cells correlated to enhanced glucagon release from pancreatic islets. 2) High glucose concentrations stimulated glucagon secretion under hyperpolarizing conditions, although [Ca2+]i of the -cells was lowered below basal levels. It was recently reported that glucose amplifies Ca2+-initiated glucagon secretion from purified rat -cells (8). However, the presently observed stimulatory effect of glucose on glucagon secretion from mouse islets did not depend on the [Ca2+]i level of the -cells, indicating that glucose initiates rather than amplifies secretion. It may seem surprising that the -cells can sense high concentrations of glucose because they express low Km glucose transporter GLUT1 but lack high Km GLUT2 (38). The situation is reminiscent of that in human -cells, which, unlike the GLUT2-expressing rodent -cells, predominantly express GLUT1 (39). Apparently glucose transport via GLUT1 is not rate limiting in these cases, and the sensing is explained by the presence of the high Km glucokinase (39,40). The signal transduction of the intriguing glucose stimulation of glucagon secretion remains to be elucidated.
Hypersecretion of glucagon is an important feature of diabetes contributing to glucose dysregulation (1,2). The mechanism underlying this phenomenon is unclear. The present study of mouse islets and clonal hamster glucagon-releasing cells indicated that paradoxical hypersecretion of glucagon in hyperglycemia may depend on a U-shaped dose-response relationship for glucose regulation of secretion. A similar regulation of human -cells may explain why diabetic patients with hyperglycemia display paradoxical hyperglucagonemia and why a further glucose challenge has been found to stimulate glucagon release (9–11).
ACKNOWLEDGMENTS
This work has received support from the Swedish Medical Research Council (grant 12X-6240), the Swedish Diabetes Association, the Carl Trygger Foundation, Family Ernfors Foundation, Albert Phlsson’s Foundation, the Crafoord Foundation, and the Scandinavian Physiological Society.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Gerich JE, Charles A, Grodsky GM: Regulation of pancreatic insulin and glucagon secretion. Annu Rev Physiol 38:353–388, 1976
Jiang G, Zhang BB: Glucagon and regulation of glucose metabolism. Am J Physiol Endocrinol Metab 284:E671–E678, 2003
Sloop KW, Michael MD, Moyers JS: Glucagon as a target for the treatment of type 2 diabetes. Expert Opin Ther Targets 9:593–600, 2005
Unger RH: Diabetes and the cell. Diabetes 25:136–151, 1976
Gossain VV, Rovner DR: Pancreatic glucagon: possible implications of the hyperglycemic hormone in diabetes control. Postgrad Med 72:87–88, 91–93, 96, 1982
Cryer PE, Davis SN, Shamoon H: Hypoglycemia in diabetes. Diabetes Care 26:1902–1912, 2003
Gerich JE, Charles A, Grodsky GM: Characterization of the effects of arginine and glucose on glucagon and insulin release from the perfused rat pancreas. J Clin Invest 54:833–841, 1974
Olsen HL, Theander S, Bokvist K, Buschard K, Wollheim CB, Gromada J: Glucose stimulates glucagon release in single rat -cells by mechanisms that mirror the stimulus-secretion coupling in -cells. Endocrinology 146:4861–4870, 2005
Buchanan KD, McCarroll AM: Abnormalities of glucagon metabolism in untreated diabetes mellitus. Lancet 300:1394–1395, 1972
Ohneda A, Watanabe K, Horigome K, Sakai T, Kai Y, Oikawa S: Abnormal response of pancreatic glucagon to glycemic changes in diabetes mellitus. J Clin Endocrinol Metab 46:504–510, 1978
Mitrakou A, Kelley D, Veneman T, Jenssen T, Pangburn T, Reilly J, Gerich J: Contribution of abnormal muscle and liver glucose metabolism to postprandial hyperglycemia in NIDDM. Diabetes 39:1381–1390, 1990
Liu YJ, Hellman B, Gylfe E: Ca2+ signaling in mouse pancreatic polypeptide cells. Endocrinology 140:5524–5529, 1999
Takaki R, Ono J, Nakamura M, Yokogawa Y, Kumae S, Hiraoka T, Yamaguchi K, Hamaguchi K, Uchida S: Isolation of glucagon-secreting cell lines by cloning insulinoma cells. In Vitro Cell Dev Biol 22:120–126, 1986
Liu YJ, Vieira E, Gylfe E: A store-operated mechanism determines the activity of the electrically excitable glucagon-secreting pancreatic -cell. Cell Calcium 35:357–365, 2004
Johansson H, Gylfe E, Hellman B: Cyclic AMP raises cytoplasmic calcium in pancreatic 2-cells by mobilizing calcium incorporated in response to glucose. Cell Calcium 10:205–211, 1989
Berts A, Ball A, Dryselius S, Gylfe E, Hellman B: Glucose stimulation of somatostatin-producing islet cells involves oscillatory Ca2+ signalling. Endocrinology 137:693–697, 1996
Panagiotidis G, Salehi AA, Westermark P, Lundquist I: Homologous islet amyloid polypeptide: effects on plasma levels of glucagon, insulin and glucose in the mouse. Diabetes Res Clin Pract 18:167–171, 1992
Etzrodt H, Rosenthal J, Schroder KE, Pfeiffer EF: Radioimmunoassay of somatostatin in human plasma. Clin Chim Acta 133:241–251, 1983
Ashcroft FM, Rorsman P: ATP-sensitive K+ channels: a link between B-cell metabolism and insulin secretion. Biochem Soc Trans 18:109–111, 1990
Johansson H, Gylfe E, Hellman B: The actions of arginine and glucose on glucagon secretion are mediated by opposite effects on cytoplasmic Ca2+. Biochem Biophys Res Commun 147:309–314, 1987
Gylfe E: Nutrient secretagogues induce bimodal early changes in cytoplasmic calcium of insulin-releasing ob/ob mouse -cells. J Biol Chem 263:13750–13754, 1988
Gembal M, Gilon P, Henquin JC: Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells. J Clin Invest 89:1288–1295, 1992
Gylfe E: Glucose-induced buffering of cytoplasmic Ca2+ in the pancreatic -cell: an artifact or a physiological phenomenon Biochem Biophys Res Commun 159:907–912, 1989
Bokvist K, Olsen HL, Hy M, Gotfredsen CF, Holmes WF, Buschard K, Rorsman P, Gromada J: Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells. Pflügers Arch 438:428–436, 1999
Quesada I, Nadal A, Soria B: Different effects of tolbutamide and diazoxide in -, -, and -cells within intact islets of Langerhans. Diabetes 48:2390–2397, 1999
Gpel SO, Kanno T, Barg S, Weng XG, Gromada J, Rorsman P: Regulation of glucagon secretion in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels. J Physiol (Lond) 528:509–520, 2000
Barg S, Galvanovskis J, Gpel SO, Rorsman P, Eliasson L: Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting -cells. Diabetes 49:1500–1510, 2000
Hy M, Olsen HL, Bokvist K, Buschard K, Barg S, Rorsman P, Gromada J: Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells. J Physiol (Lond) 527:109–120, 2000
Hjortoe GM, Hagel GM, Terry BR, Thastrup O, Arkhammar PO: Functional identification and monitoring of individual and cells in cultured mouse islets of Langerhans. Acta Diabetol 41:185–193, 2004
Franklin I, Gromada J, Gjinovci A, Theander S, Wollheim CB: -Cell secretory products activate -cell ATP-dependent potassium channels to inhibit glucagon release. Diabetes 54:1808–1815, 2005
Ishihara H, Maechler P, Gjinovci A, Herrera PL, Wollheim CB: Islet -cell secretion determines glucagon release from neighbouring -cells. Nat Cell Biol 5:330–335, 2003
Wendt A, Birnir B, Buschard K, Gromada J, Salehi A, Sewing S, Rorsman P, Braun M: Glucose inhibition of glucagon secretion from rat -cells is mediated by GABA released from neighboring -cells. Diabetes 53:1038–1045, 2004
Xu E, Kumar M, Zhang Y, Ju W, Obata T, Zhang N, Liu S, Wendt A, Deng S, Ebina Y, Wheeler MB, Braun M, Wang Q: Intra-islet insulin suppresses glucagon release via GABA-GABAA receptor system. Cell Metab 3:47–58, 2006
Gromada J, Ma X, Hoy M, Bokvist K, Salehi A, Berggren PO, Rorsman P: ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1–/– mouse -cells. Diabetes 53 (Suppl. 3):S181–S189, 2004
Bode HP, Weber S, Fehmann HC, Gke B: A nutrient-regulated cytosolic calcium oscillator in endocrine pancreatic glucagon-secreting cells. Pflügers Arch 437:324–334, 1999
Westerlund J, Hellman B, Bergsten P: Pulsatile insulin release from mouse islets occurs in the absence of stimulated entry of Ca2+. J Clin Invest 97:1860–1863, 1996
Ravier MA, Gilon P, Henquin JC: Oscillations of insulin secretion can be triggered by imposed oscillations of cytoplasmic Ca2+ or metabolism in normal mouse islets. Diabetes 48:2374–2382, 1999
Heimberg H, De Vos A, Pipeleers DG, Thorens B, Schuit FC: Differences in glucose transporter gene expression between rat pancreatic - and -cells are correlated to differences in glucose transport but not in glucose utilization. J Biol Chem 270:8971–8975, 1995
De Vos A, Heimberg H, Quartier E, Huypens P, Bouwens L, Pipeleers D, Schuit F: Human and rat beta cells differ in glucose transporter but not in glucokinase gene expression. J Clin Invest 96:2489–2495, 1995
Heimberg H, De Vos A, Moens K, Quartier E, Bouwens L, Pipeleers D, Van Schaftingen E, Madsen O, Schuit F: The glucose sensor protein glucokinase is expressed in glucagon-producing -cells. Proc Natl Acad Sci U S A 93:7036–7041, 1996(Albert Salehi, Elaine Vie)