The G_q/G₁₁-mediated signaling pathway is critical for autocrine potentiation of insulin secretion in mice

A variety of neurotransmitters, gastrointestinal hormones, and metabolic signals are known to potentiate insulin secretion through GPCRs. We show here that β cell–specific inactivation of the genes encoding the G protein α-subunits Gα_q and Gα₁₁ resulted in impaired glucose tolerance and insulin secretion in mice. Interestingly, the defects observed in Gα_q/Gα₁₁-deficient β cells were not restricted to loss of muscarinic or metabolic potentiation of insulin release; the response to glucose per se was also diminished. Electrophysiological recordings revealed that glucose-induced depolarization of isolated β cells was impaired in the absence of Gα_q/Gα₁₁, and closure of K_ATP channels was inhibited. We provide evidence that this reduced excitability was due to a loss of β cell–autonomous potentiation of insulin secretion through factors cosecreted with insulin. We identified as autocrine mediators involved in this process extracellular nucleotides such as uridine diphosphate acting through the G_q/G₁₁-coupled P2Y6 receptor and extracellular calcium acting through the calcium-sensing receptor. Thus, the G_q/G₁₁-mediated signaling pathway potentiates insulin secretion in response to glucose by integrating systemic as well as autocrine/paracrine mediators.

The adequate secretion of insulin from pancreatic β cells is essential for the maintenance of normoglycemia; impaired insulin secretion results in diabetes mellitus with hyperglycemia, dyslipidemia, and consequent long-term tissue damage (1). The on-demand release of insulin from β cells is mainly regulated by blood glucose levels: high concentrations of glucose result in enhanced intracellular glucose metabolism with accumulation of ATP and consecutive closure of ATP-sensitive K⁺ channels, leading to the opening of voltage-operated Ca²⁺ channels and Ca²⁺-mediated exocytosis of insulin-containing vesicles (2, 3).

While the ATP-dependent mechanism is clearly the master regulator of insulin release, various mediators potentiate insulin release in response to glucose. For example, gastrointestinal hormones such as glucose-dependent insulinotropic polypeptide (GIP) or glucagon-like peptide–1 (GLP-1) potentiate insulin secretion by activation of GPCRs, which signal through the G_s family of heterotrimeric G proteins (4–6). The potentiating effect of G_s on glucose-induced insulin release depends on activation of adenylyl cyclase and consecutive phosphorylation of voltage-operated Ca²⁺ channels (7, 8) or opening of nonselective cation channels (9).

Another important group of modulators are neurotransmitters and neuropeptides (10, 11), most prominent among them being the neurotransmitter acetylcholine, which is released from vagal nerve terminals and potentiates insulin secretion through the muscarinic receptor subtype M₃ (12–15). In contrast to the receptors for GIP and GLP-1, the M₃ receptor does not elicit G_s-mediated adenylyl cyclase activation, but was shown to signal through the G_q/G₁₁ family of heterotrimeric G proteins. The two main members of the G_q/G₁₁ family, G_q and G₁₁, are ubiquitously expressed (16, 17); their activation results in stimulation of phospholipase C β (PLC β) isoforms and consequent inositol 1,4,5-trisphosphate–mediated (IP₃-mediated) intracellular calcium mobilization and PKC activation (18). Interestingly, β cells express in addition to M₃ a wide variety of other potentially G_q/G₁₁-coupled receptors (19–21), and most of these receptors have been shown to be involved in the potentiation of insulin secretion, such as receptors for fatty acids (22), cholecystokinin (23), arginine vasopressin (24, 25), endothelin (26), extracellular nucleotides (27, 28), calcium (29), or zinc (30). Though for many of these receptors, the physiological relevance in the regulation of insulin secretion is unclear, the sheer number of potentially G_q/G₁₁-coupled receptors expressed in β cells suggests an important role of this G protein family in regulation of β cell function. However, due to the lack of β cell–specific inhibitors of G_q/G₁₁, and due to the embryonic lethality of mice that lack the α-subunits of G_q and G₁₁, Gα_q and Gα₁₁ (31), the in vivo functions of G_q/G₁₁ in insulin secretion have not been studied so far.

In order to investigate the role of the G_q/G₁₁-mediated signaling pathway in the regulation of insulin secretion in vivo, we generated and studied mice with β cell–specific knockout of the genes encoding Gα_q and Gα₁₁. We show here that, in addition to their role in vagal and metabolic potentiation, Gα_q and Gα₁₁ are required for a β cell–autonomous feedback loop in which cosecreted factors such as nucleotides or calcium potentiate glucose-induced insulin secretion through G_q/G₁₁-coupled receptors.

Characterization of β cell–specific Gα_q/Gα₁₁-deficient mice. To generate β cell–specific Gα_q/Gα₁₁-deficient mice, we crossed the RipCre mouse line (32) with mice carrying floxed alleles of the gene coding for Gα_q, Gnaq (Gnaq^fl/fl) (33), and constitutive Gα₁₁-deficient animals (Gna11^–/–) (31). Such β cell–specific Gα_q/Gα₁₁-double-deficient mice (RipCre^+/–Gnaq^fl/flGna11^–/–, henceforth termed β-Gα_q/Gα₁₁ DKOs) were born at expected numbers and were viable and fertile. Their body weight did not differ from that of C57BL/6 wild-type mice or wild-type mice carrying the RipCre transgene alone (RipCre^+/–Gnaq^WT/WTGna11^WT/WT) (data not shown). Since expression of the RipCre transgene has been reported to have metabolic effects per se (34), RipCre-positive animals were used as controls throughout the study. Western blotting of cholate extracts from controls and β-Gα_q/Gα₁₁ DKOs revealed only a mild decrease in Gα_q/Gα₁₁ immunoreactivity in whole pancreata but a strong reduction in Gα_q/Gα₁₁ in isolated islets (Figure 1A). The remaining immunoreactivity was most likely due to other endocrine cell types, for example, α, δ, or PP cells, or to endothelial cells. Since PLCβ-mediated hydrolysis of phosphatidyl inositol-3,4-diphosphate (PIP₂) to IP₃ and diacylglycerol (DAG) is the major cellular effect of Gα_q/Gα₁₁ activation, we investigated inositol phosphate (IP) production and intracellular calcium mobilization in Gα_q/Gα₁₁-deficient β cells (Figure 1, B and C). Control islets responded to stimulation of muscarinic M₃ receptors with oxotremorine M (OxoM) with a 2.4-fold increase in IP production, which was almost abrogated in islets of β-Gα_q/Gα₁₁ DKOs (Figure 1B). As in the immunoblotting experiments, the remaining 1.2-fold increase in IP production was most likely due to other non-recombined islet cell types. Fura-2–based imaging of intracellular calcium responses showed that calcium mobilization elicited by OxoM was abrogated in mutant β cells (Figure 1C).

Figure 1

Characterization of β cell–specific Gα_q/Gα₁₁-deficient mice. (A) Western blots of extracts of whole pancreas or isolated islets probed with antibodies directed against Gα_q/Gα₁₁ or against α-tubulin as loading control. (B) OxoM-induced (50 μM) production of IPs in control (white) and Gα_q/Gα₁₁-deficient (black) β cells (n = 3 independent experiments). (C) Intracellular calcium mobilization in response to OxoM (50 μM) in Fura-2/AM–loaded control and mutant β cells. (D) Exemplary microphotographs of histological (original magnification, ×100) and immunohistochemical stainings (×200) as well as electron microscopic sections (EM, ×3,000) of pancreatic islets or individual β cells from control and β-Gα_q/Gα₁₁–deficient mice. (E and F) Quantification of the number (E) and size (F) of control and mutant islets (4 animals per group, 20 sections per animal). *P ≤ 0.05.

In order to investigate whether β cell–specific inactivation of Gα_q/Gα₁₁ led to developmental abnormalities of pancreatic islets, we performed histological and immunohistochemical analyses. In neither H&E-stained sections nor sections stained with antibodies directed against insulin, glucagon, or glucose transporter 2 did we detect differences between the genotypes (Figure 1D and data not shown). Also, using transmission electron microscopy, we did not find differences in α and β cells, with β cells showing characteristic granules with crystalline cores (Figure 1D). The average number of islets per section or the average size of islets also did not differ (Figure 1, E and F). The expression of islet-specific genes such as insulin, uncoupling protein 2, glucose transporter 2, glucokinase, or different transcription factors was not altered in mutant β cells (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI41541DS1).

β Cell–specific Gα_q/Gα₁₁-deficient mice show impaired glucose tolerance and are diabetic. To assess the physiological role of Gα_q/Gα₁₁ in β cells, we determined blood glucose levels after oral and i.v. glucose challenge. β-Gα_q/Gα₁₁ DKOs showed in both tests significantly increased blood glucose levels throughout the test period (Figure 2, A and C). We analyzed insulin levels during oral and i.v. glucose tolerance tests and found that the first peak of glucose-induced insulin secretion was significantly reduced in mutant animals (Figure 2, B and D). The first peak of insulin secretion is known to be crucial for a rapid suppression of hepatic glucose production (35); we therefore hypothesized that the impaired early insulin surge in β-Gα_q/Gα₁₁ DKOs is the basis of prolonged hyperglycemia in later phases. Since insulin-mediated reduction of hepatic glucose output is, at least partly, due to insulin-dependent suppression of glucagon secretion from pancreatic α cells (36, 37), we investigated glucagon serum levels after i.v. glucose challenge. These experiments showed that suppression of glucagon levels was impaired in β-Gα_q/Gα₁₁ DKOs (Supplemental Figure 2), suggesting that uncurbed hepatic glucose output contributes to prolonged hyperglycemia in these mice. Interestingly, the impairment of insulin secretion in mutant mice was only observed during the early phase of glucose tolerance tests; at later time points it was enhanced, most likely in response to the prolonged hyperglycemia. In order to exclude peripheral insulin resistance as a contributing factor, we performed insulin tolerance tests but did not detect any differences between the genotypes (Figure 2E).

Figure 2

β Cell–specific Gα_q/Gα₁₁-deficient mice show impaired glucose tolerance and are diabetic. (A and C) Blood glucose levels in control mice and β-Gα_q/Gα₁₁ DKOs following oral (2 mg/g body weight) (A) or i.v. (1 mg/g) (C) application of glucose (n = 8–10 animals per group). (B and D) Serum insulin levels during oral (B) and i.v. (D) glucose tolerance testing (n = 10 animals per group for oral tests, n = 4–6 for i.v. tests). (E) Blood glucose levels in control mice (gray triangles) and β-Gα_q/Gα₁₁ DKOs (black squares) after application of 0.5 U/kg body weight insulin intraperitoneally (insulin tolerance test) (n = 5 animals per group). (F and G) Blood glucose levels (F) and serum insulin levels (G) in fasted control mice and β-Gα_q/Gα₁₁ DKOs at different ages (n = 4–6 animals per group). *P ≤ 0.05, **P ≤ 0.005.

We next investigated whether hyperglycemia was observed only after glucose challenge or also in the basal state. We found that at all ages investigated, β-Gα_q/Gα₁₁ DKOs were hyperglycemic compared with control animals after overnight fasting (Figure 2F). Basal insulin levels after overnight fasting were mildly but not significantly increased in β-Gα_q/Gα₁₁ DKOs (Figure 2G). Blood glucose levels and insulin sensitivity of Gα₁₁-single-deficient mice did not differ from those of wild-type animals (data not shown).

Loss of Gα_q/Gα₁₁ impairs glucose-induced insulin secretion in isolated islets. To investigate the underlying mechanisms of impaired glucose tolerance and reduced insulin secretion in β-Gα_q/Gα₁₁ DKOs, we studied insulin secretion in perifused islets in vitro. We found that the potentiating effect of OxoM on glucose-induced insulin secretion was almost completely abrogated (Figure 3A). In addition, the potentiating effect of palmitic acid, an agonist of the receptor GPR40, was also strongly impaired (Figure 3A). Other mediators that have been suggested to contribute to insulin secretion through potentially G_q/G₁₁-coupled receptors, such as cholecystokinin or endothelin, did not significantly affect glucose-induced insulin secretion in control or mutant β cells. The effects of agonists of G_s-coupled receptors, for example, pituitary adenylate cyclase–activating polypeptide (PACAP) or GLP-1, were not altered in Gα_q/Gα₁₁-deficient β cells (Figure 3A).

Figure 3

Loss of Gα_q/Gα₁₁ impairs not only agonist-induced potentiation but also glucose-induced insulin secretion per se. (A) Potentiation of glucose-induced insulin secretion from perifused control islets and islets from β-Gα_q/Gα₁₁ DKOs by OxoM (50 μM), palmitic acid (PA), cholecystokinin-8 (CCK), endothelin-1 (ET), GLP-1 (100 μM each), or PACAP (10 nM) (n = 3–5 independent experiments). Data are expressed as x-fold above insulin secretion induced by 16.7 mM glucose alone. (B) Example of insulin secretion from perifused islets of control mice and β-Gα_q/Gα₁₁ DKOs in response to 16.7 mM glucose (G16.7). (C) Quantification of the maximal stimulatory effect of 16.7 mM glucose or 40 mM KCl on insulin secretion from perifused control and mutant islets (n = 5–6 independent experiments). Data are expressed as x-fold above insulin secretion at 2.8 mM glucose. *P ≤ 0.05.

Surprisingly, glucose-induced insulin secretion per se was significantly reduced in perifused islets from β-Gα_q/Gα₁₁ DKOs (Figure 3, B and C). This was not due to a general reduction in the secretory ability of Gα_q/Gα₁₁-deficient cells, since potassium chloride–mediated depolarization resulted in normal insulin secretion (Figure 3C). These data show that Gα_q/Gα₁₁ is required for full glucose-induced insulin secretion independent of potentiating signals produced by other cell types.

Impaired glucose-induced depolarization and K_ATP channel closure in Gα_q/Gα₁₁-deficient β cells. In order to study the mechanisms that underlie impaired glucose-induced insulin release, we investigated the electrophysiological properties of isolated β cells from control and mutant animals. We compared the responses of control and Gα_q/Gα₁₁-deficient β cells to two glucose concentrations around the threshold (6 and 8 mM glucose) and found that at both concentrations, the percentage of depolarized cells that elicited action potentials was lower in the Gα_q/Gα₁₁-deficient cell population (Figure 4A). Also, at high glucose concentrations, the frequency of action potentials was reduced in mutant cells (Figure 4B).

Figure 4

Impaired glucose-induced depolarization and K_ATP channel closure in Gα_q/Gα₁₁-deficient β cells. (A) Percentage of depolarized cells that elicited action potentials at 2 glucose concentrations (6 and 8 mM glucose) around the threshold. For A and B, membrane potential was recorded in the current-clamp mode with the perforated-patch technique. (B) Exemplary recording (left) and statistical evaluation (right) of action potential frequency at 15 mM glucose in control and mutant β cells. (C and D) Exemplary recordings (C) and statistical evaluation (D) of the effect of an augmentation of the glucose concentration from 0.5 mM to 8 mM on K_ATP activity in control and mutant β cells. Single-channel recordings were performed in the cell-attached mode. Data in D are expressed as percentage of open probability in 0.5 mM glucose (100%) for each genotype. *P ≤ 0.05.

Since β cell depolarization in response to glucose is mainly mediated by ATP-dependent closure of ATP-sensitive K⁺ (K_ATP) channels (2, 3), we studied the electrophysiological properties of K_ATP in control and mutant cells. While we did not find significant differences in K_ATP channel open probability at low glucose concentrations (0.5 mM), the responses at 8 mM glucose differed significantly between the genotypes: control cells responded to the elevation of glucose concentration with a significant reduction in open probability of K_ATP channels, and this response was strongly impaired in Gα_q/Gα₁₁-deficient β cells (Figure 4, C and D). These findings show that glucose/ATP-dependent closure of K_ATP channels is impaired in the absence of Gα_q/Gα₁₁, resulting in a reduced responsiveness toward glucose.

Cosecreted factors potentiate insulin secretion in a Gα_q/Gα₁₁-dependent manner. PLCβ-mediated hydrolysis of PIP₂ is the major effector pathway of activated G_q/G₁₁ (38), and PIP₂ has been suggested to take part in the regulation of K_ATP channels (39, 40). We therefore hypothesized that glucose application activates by a yet-to-be defined pathway, Gα_q/Gα₁₁, leading to PIP₂ hydrolysis and facilitation of K_ATP channel closure. To test this hypothesis, we determined glucose-induced IP production in the presence and absence of Gα_q/Gα₁₁. We found that glucose induced a 1.85-fold increase in IP production in isolated control islets and that this effect was abrogated in islets of β-Gα_q/Gα₁₁ DKOs (Figure 5A). Glucose-induced, Gα_q/Gα₁₁-mediated PIP₂ hydrolysis might be mediated by different mechanisms: direct activation of a G_q/G₁₁-coupled receptor by glucose; or glucose-induced release of an autocrine mediator, which in turn acts on a G_q/G₁₁-coupled receptor. To test the possibility that glucose directly activates a G_q/G₁₁-coupled receptor, we compared the kinetics of intracellular calcium mobilization in response to OxoM with the calcium mobilization elicited by glucose. While OxoM induced the fast and short-lived calcium response typically seen after activation of G_q/G₁₁-coupled receptors, the glucose-induced response was slower and longer-lived (Figure 5B). What is more, this calcium response depended on intracellular glucose metabolism, since the non-metabolizable glucose derivative 2-deoxyglucose did not evoke any response (Figure 5B). We concluded from these experiments that glucose does not directly activate a G_q/G₁₁-coupled receptor and instead focused on the second possibility, the release of a soluble mediator from glucose-stimulated β cells.

Figure 5

Cosecreted factors potentiate insulin secretion in a Gα_q/Gα₁₁-dependent manner. (A) IP production in control (white) and Gα_q/Gα₁₁-deficient (black) islets in response to an increase in glucose from 2.8 mM to 16.7 mM (n = 4 independent experiments). (B) Intracellular calcium mobilization in control β cells elicited by 16.7 mM glucose, 50 μM OxoM, or 16.7 mM 2-deoxyglucose. The arrow indicates the time point of application; before application, cells were kept at 5 mM glucose. (C) Calcium mobilization in isolated Fura-2/AM–loaded control β cells in response to different mediators known to be released from glucose-stimulated β cells (ADP and ATP, 100 μM each; serotonin [5-HT], amylin, and prostaglandin E₂ [PGE₂], 1 μM each; UDP, 200 μM). (D) Calcium mobilization in isolated Fura-2/AM–loaded control and mutant β cells in response to the nucleotides UDP, ADP, and ATP. Data are presented as the 340/380 nm fluorescence ratio (Ratio F₃₄₀/F₃₈₀). (E) Stimulation of insulin secretion in control (white) and mutant (black) β cells in response to UDP and ADP (data expressed as x-fold of basal secretion) (n = 5–6 per group). (F) Insulin secretion of control islets in response to 16.7 mM glucose in the absence or presence of antagonists directed against different GPCR subtypes (data represent insulin concentration in the supernatant of 20 islets after 30 minutes of static incubation) (n = 4 independent experiments). *P ≤ 0.05.

β Cells are known to cosecrete with insulin a variety of substances that can act as agonists of GPCRs, for example, serotonin, nucleotides, amylin, or bivalent cations (41–43); in addition, they produce and release prostaglandins upon glucose stimulation (44). In order to investigate which of these mediators are able to potentiate β cell activation in a Gα_q/Gα₁₁-dependent manner, we studied calcium mobilization in isolated β cells at sub-threshold glucose levels (Figure 5, C and D). We found that the nucleotides adenosine diphosphate (ADP), uridine diphosphate (UDP), or ATP induced calcium mobilization in control β cells, while other substances, such as serotonin or prostaglandin E₂ had no effect (Figure 5C). In Gα_q/Gα₁₁-deficient islets, calcium mobilization in response to UDP and ADP was abrogated, while the effect of ATP was preserved (Figure 5D). The normal calcium response to ATP is most likely due to the presence ATP-gated P2X ion channels (45). We next investigated whether UDP- and ADP-induced calcium mobilization was sufficient to trigger insulin release at threshold glucose levels and found that these nucleotides increased insulin secretion 1.75-fold and 1.5-fold in control islets, while their effects were abrogated in islets of β-Gα_q/Gα₁₁ DKOs (Figure 5E).

Having established that UDP and ADP enhance calcium mobilization and insulin secretion in a Gα_q/Gα₁₁-dependent manner, we investigated whether this putative autocrine potentiation relevantly contributes to glucose-induced insulin secretion. To do so, and also to identify the GPCR subtypes involved in this feedback loop, we studied glucose-induced insulin secretion in isolated islets in the absence and presence of different GPCR antagonists (Figure 5F). Since murine β cells have been shown to express the UDP receptor P2Y₆ as well as the ADP/ATP receptor P2Y₁ (19, 27), we used the P2Y₁ receptor antagonist MRS2179 and the P2Y₆ antagonist MRS2578 for this purpose. In vehicle-treated control islets, elevation of glucose concentration from 2.8 mM to 16.7 mM resulted in a significant increase in insulin secretion. In the presence of the P2Y₁ receptor antagonist MRS2179, glucose-induced insulin secretion was slightly but not significantly reduced, while blockade of P2Y₆ by MRS2578 significantly reduced insulin secretion in response to glucose (Figure 5F). To study the potential involvement of coreleased calcium or prostaglandins, we also applied antagonists of the calcium-sensing receptor and of prostaglandin E₂ receptor subtypes, which have been shown to be expressed in murine β cells and islets, respectively (19, 46). We found that blockade of the calcium-sensing receptor with antagonist NVP-CAR16 resulted in a significant reduction of insulin secretion, while blockade of prostaglandin receptors with AH 6809 had no effects. Neither NVP-CAR16 nor MRS2578 affected basal insulin secretion (data not shown), suggesting that these antagonists did not disturb islet function per se. We conclude from these experiments that nucleotides and calcium released from glucose-stimulated β cells potentiate insulin release through the G_q/G₁₁-coupled receptors P2Y₁, P2Y₆, and CaR, thereby contributing to adequate on-demand secretion of insulin.

We show in this study that β cell–specific inactivation of Gα_q/Gα₁₁ leads to hyperglycemia with a significant decrease in the early phase of insulin secretion in both oral and i.v. glucose tolerance tests. Since the first peak of insulin secretion is crucial for the efficient suppression of hepatic glucose production (35), the observed reduction of early insulin secretion most likely explains the prolonged hyperglycemia observed in mutant mice.

The impairment of oral glucose tolerance is probably partly due to the loss of parasympathetic potentiation of insulin secretion, which is known to be mainly mediated by the G_q/G₁₁-coupled muscarinic acetylcholine receptor M₃ (10–13, 47). In addition to abrogation of M₃-mediated effects, impaired signaling through the fatty acid receptor GPR40 (48) might contribute to the impaired oral glucose tolerance in β-Gα_q/Gα₁₁ DKOs, though the relevance of GPR40 in glucose-induced insulin secretion in vivo is still under debate (49–51).

In contrast to previous studies performed in isolated rat islets (52–54), our study did not reveal facilitation of insulin secretion in response to cholecystokinin-8, which might be partly due to species-specific differences in gene expression: The cholecystokinin receptor CCK-A has been shown to be highly expressed in rat islets (55), while expression in murine islets is — according to our own analyses (data not shown) and published data — weak to undetectable (19, 46, 56). However, a mild facilitating effect of CCK at 8.3 mM glucose has been described by others (57), and we therefore hypothesize that CCK mediates a weak facilitation at low to intermediate glucose levels, which is masked by the high glucose concentrations used in our experiments. In contrast to cholecystokinin-8, endothelin-1 elicited a weak (1.3-fold) facilitation of secretion at 16.7 mM glucose, which was not impaired in islets from β-Gα_q/Gα₁₁ DKOs. This finding might point to an involvement of other G protein families besides G_q/G₁₁, for example, G₁₂/G₁₃, a G protein family that has in other cell types been suggested to couple to the endothelin-1 receptor ET-A (58) and been implicated in regulation of secretion (59).

Interestingly, our results show that glucose-induced β cell activation is disturbed even under conditions that exclude potentiating signals produced by other cell types, suggesting that inactivation of Gα_q/Gα₁₁ impairs glucose sensitivity of β cells per se. One possible explanation might be a disturbance of β cell development as described in β cell–specific Gα_s-deficient mice (60), but we did not find differences in islet mass, number, histology, immunohistochemistry, electron microscopy, or the expression pattern of β cell marker genes. What is more, insulin secretion in response to agonists of G_s-coupled receptors such as GLP-1 or PACAP did not differ between the genotypes, nor did the response to direct depolarization by potassium chloride. Our findings therefore show that Gα_q/Gα₁₁ is not essential for β cell development but makes a major contribution to the β cell–autonomous regulation of glucose-induced insulin secretion.

Our electrophysiological data revealed an impaired responsiveness of mutant cells at threshold glucose concentrations, and even at maximal stimulation, the frequency of action potentials was reduced. As to the potential mechanism by which Gα_q/Gα₁₁ modulates β cell excitability, our electrophysiological recordings suggest an altered glucose-dependent closure of the K_ATP channel. A possible connection between Gα_q/Gα₁₁ and K_ATP is local membrane phosphoinositide concentrations, especially those of PIP₂, the most abundant phosphoinositide (61). Membrane-bound PIP₂ was shown to increase the open probability and reduce the ATP sensitivity of K_ATP channels (39, 40), thereby contributing to β cell hyperpolarization. Upon activation of G_q/G₁₁-coupled receptors, PIP₂ is hydrolyzed to IP₃ and DAG (38), and the local reduction of membrane PIP₂ was suggested to facilitate K_ATP channel closure and cellular depolarization (62). Reduced Gα_q/Gα₁₁-mediated PIP₂ hydrolysis might accordingly result in impaired K_ATP channel closure and reduced β cell depolarization. However, in addition to PIP₂-mediated modulation of K_ATP channel open probability, other G_q/G₁₁-mediated effects such as intracellular Ca²⁺ mobilization (63–65) or PKC activation (66) might contribute to Gα_q/Gα₁₁-mediated potentiation of insulin secretion.

An important question is certainly by which mechanism does glucose induce Gα_q/Gα₁₁-mediated phosphoinositide hydrolysis. It has long been known that glucose can stimulate IP production in pancreatic islets (67, 68), but neither we nor others (69) have found evidence for an involvement of a glucose-activated plasma membrane receptor. Instead, it was suggested that the glucose-induced phosphoinositide response might be triggered by the accompanying increase in calcium influx (70, 71). Our data, however, show that glucose-induced production of IP₃ depends on Gα_q/Gα₁₁, which led us to the hypothesis that mediators cosecreted with insulin might activate Gα_q/Gα₁₁ and potentiate insulin release in an autocrine feedback loop.

A variety of substances are known to be cosecreted with insulin or to be released upon glucose stimulation, such as nucleotides, bivalent cations, C-peptide, amylin, or prostaglandins (41). For example, β cell vesicles contain 3.5 mM ATP and 5 mM ADP (41), and glucose stimulation releases ATP from a single pancreatic β cell to a local extracellular ATP concentration exceeding 25 μM (72). In addition, the pyrimidines UTP and UDP can be released in a controlled exocytotic fashion from various cell types (73–75). As to the effect of nucleotides on insulin secretion, both stimulation and inhibition have been described, depending on the nucleotide, tissue, and species tested (27, 45, 76–81). Our own data clearly show that ADP and UDP potentiate insulin secretion in a Gα_q/Gα₁₁-dependent manner, while ATP-induced calcium responses are independent of Gα_q/Gα₁₁, most likely mediated by ATP-gated P2X ion channels (45). With respect to the receptors involved in the autocrine potentiation of insulin secretion, we identified the UDP receptor P2Y₆ as a major player, while the contribution of the P2Y₁ receptor, which is activated by ADP and ATP, seems less prominent. In addition to P2Y receptor subtypes, our data show an important role of the extracellular calcium-sensing receptor CaR in the potentiation of glucose-induced insulin secretion from isolated β cells. In line with this finding, the calcimimetic R-568, a substance that induces CaR activation already at calcium concentrations as low as 0.2 mM, was shown to increase insulin release from human islets and the murine β cell line MIN6 (29), and shRNA-mediated knockdown of CaR in MIN6 pseudoislets impaired glucose-induced insulin secretion (82). Interestingly, combined application of NVP-CAR16 and MRS2578 did not result in further reduction of glucose-induced insulin secretion, suggesting that P2Y₆- and CaR-mediated signaling are not interchangeable and that concomitant activation of both receptors is required for full facilitation of insulin release.

Taken together, our data show that the G_q/G₁₁ family is crucial not only for neural and metabolic facilitation, but also for the β cell–autonomous potentiation of glucose-induced insulin secretion by nucleotides and calcium cosecreted with insulin. Thus, given the multitude of potentiating signals that converge on G_q/G₁₁, a direct pharmacological modulation of this signaling pathway in β cells might in the future prove more efficient in enhancing insulin secretion than targeting individual GPCRs.

View Supplemental data

We thank Mark A. Magnuson for the RipCre line, Klaus Seuwen for the CaR antagonist NVP-CAR16, and Barbara Zimmermann for expert technical assistance.

Address correspondence to: Nina Wettschureck, Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Ludwigstr. 43, 61231 Bad Nauheim, Germany. Phone: 49.6032.705.1214; Fax: 49.6032.705.1204; E-mail: Nina.Wettschureck@mpi-bn.mpg.de.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2010;120(6):2184–2193. doi:10.1172/JCI41541.

1. Kahn CR. Banting Lecture. Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes. 1994;43(8):1066–1084.
   View this article via: PubMed Google Scholar
2. Ashcroft FM, Harrison DE, Ashcroft SJ. Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells. Nature. 1984;312(5993):446–448.
   View this article via: PubMed Google Scholar
3. Cook DL, Hales CN. Intracellular ATP directly blocks K+ channels in pancreatic B-cells. Nature. 1984;311(5983):271–273.
   View this article via: PubMed CrossRef Google Scholar
4. Robertson RP, Seaquist ER, Walseth TF. G proteins and modulation of insulin secretion. Diabetes. 1991;40(1):1–6.
   View this article via: PubMed CrossRef Google Scholar
5. Emami S, et al. Stimulatory transducing systems in pancreatic islet cells. Ann N Y Acad Sci. 1998;865:118–131.
   View this article via: PubMed Google Scholar
6. Marie JC, Rosselin G, Skoglund G. Pancreatic beta-cell receptors and G proteins coupled to adenylyl cyclase. Ann N Y Acad Sci. 1996;805:122–131; discussion 132.
   View this article via: PubMed Google Scholar
7. Islam MS, Larsson O, Nilsson T, Berggren PO. Effects of caffeine on cytoplasmic free Ca2+ concentration in pancreatic beta-cells are mediated by interaction with ATP-sensitive K+ channels and L-type voltage-gated Ca2+ channels but not the ryanodine receptor. Biochem J. 1995;306(pt 3):679–686.
   View this article via: PubMed Google Scholar
8. Ammala C, Ashcroft FM, Rorsman P. Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells. Nature. 1993;363(6427):356–358.
   View this article via: PubMed Google Scholar
9. Holz GG 4th, Leech CA, Habener JF. Activation of a cAMP–regulated Ca(2+)-signaling pathway in pancreatic beta–cells by the insulinotropic hormone glucagon–like peptide-1. J Biol Chem. 1995;270(30):17749–17757.
   View this article via: PubMed Google Scholar
10. Miller RE. Pancreatic neuroendocrinology: peripheral neural mechanisms in the regulation of the Islets of Langerhans. Endocr Rev. 1981;2(4):471–494.
    View this article via: PubMed CrossRef Google Scholar
11. Ahren B, Taborsky GJ Jr, Porte D Jr. Neuropeptidergic versus cholinergic and adrenergic regulation of islet hormone secretion. Diabetologia. 1986;29(12):827–836.
    View this article via: PubMed CrossRef Google Scholar
12. Boschero AC, et al. Oxotremorine-m potentiation of glucose-induced insulin release from rat islets involves M3 muscarinic receptors. Am J Physiol. 1995;268(2 pt 1):E336–E342.
    View this article via: PubMed Google Scholar
13. Duttaroy A, Zimliki CL, Gautam D, Cui Y, Mears D, Wess J. Muscarinic stimulation of pancreatic insulin and glucagon release is abolished in m3 muscarinic acetylcholine receptor–deficient mice. Diabetes. 2004;53(7):1714–1720.
    View this article via: PubMed CrossRef Google Scholar
14. Gautam D, et al. A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo. Cell Metab. 2006;3(6):449–461.
    View this article via: PubMed CrossRef Google Scholar
15. Gilon P, Henquin JC. Mechanisms and physiological significance of the cholinergic control of pancreatic beta-cell function. Endocr Rev. 2001;22(5):565–604.
    View this article via: PubMed Google Scholar
16. Strathmann M, Simon MI. G protein diversity: a distinct class of alpha subunits is present in vertebrates and invertebrates. Proc Natl Acad Sci U S A. 1990;87(23):9113–9117.
    View this article via: PubMed CrossRef Google Scholar
17. Wilkie TM, Scherle PA, Strathmann MP, Slepak VZ, Simon MI. Characterization of G-protein alpha subunits in the Gq class: expression in murine tissues and in stromal and hematopoietic cell lines. Proc Natl Acad Sci U S A. 1991;88(22):10049–10053.
    View this article via: PubMed CrossRef Google Scholar
18. Exton JH. Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other agonists linked to G proteins. Annu Rev Pharmacol Toxicol. 1996;36:481–509.
    View this article via: PubMed CrossRef Google Scholar
19. Regard JB, et al. Probing cell type–specific functions of Gi in vivo identifies GPCR regulators of insulin secretion. J Clin Invest. 2007;117(12):4034–4043.
    View this article via: JCI PubMed Google Scholar
20. Wettschureck N, Offermanns S. Mammalian G proteins and their cell type specific functions. Physiol Rev. 2005;85(4):1159–1204.
    View this article via: PubMed CrossRef Google Scholar
21. Winzell MS, Ahren B. G-protein–coupled receptors and islet function-implications for treatment of type 2 diabetes. Pharmacol Ther. 2007;116(3):437–448.
    View this article via: PubMed CrossRef Google Scholar
22. Itoh Y, et al. Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature. 2003;422(6928):173–176.
    View this article via: PubMed CrossRef Google Scholar
23. Verspohl EJ, Herrmann K. Involvement of G proteins in the effect of carbachol and cholecystokinin in rat pancreatic islets. Am J Physiol. 1996;271(1 pt 1):E65–E72.
    View this article via: PubMed Google Scholar
24. Richardson SB, Laya T, VanOoy M. Similarities between hamster pancreatic islet beta (HIT) cell vasopressin receptors and V1b receptors. J Endocrinol. 1995;147(1):59–65.
    View this article via: PubMed CrossRef Google Scholar
25. Lee B, Yang C, Chen TH, al-Azawi N, Hsu WH. Effect of AVP and oxytocin on insulin release: involvement of V1b receptors. Am J Physiol. 1995;269(6 pt 1):E1095–E1100.
    View this article via: PubMed Google Scholar
26. Gregersen S, Thomsen JL, Hermansen K. Endothelin-1 (ET-1)-potentiated insulin secretion: involvement of protein kinase C and the ET(A) receptor subtype. Metabolism. 2000;49(2):264–269.
    View this article via: PubMed CrossRef Google Scholar
27. Parandeh F, Abaraviciene SM, Amisten S, Erlinge D, Salehi A. Uridine diphosphate (UDP) stimulates insulin secretion by activation of P2Y6 receptors. Biochem Biophys Res Commun. 2008;370(3):499–503.
    View this article via: PubMed CrossRef Google Scholar
28. Farret A, et al. P2Y receptor mediated modulation of insulin release by a novel generation of 2-substituted-5′-O-(1-boranotriphosphate)-adenosine analogues. Pharm Res. 2006;23(11):2665–2671.
    View this article via: PubMed CrossRef Google Scholar
29. Gray E, et al. Activation of the extracellular calcium-sensing receptor initiates insulin secretion from human islets of Langerhans: involvement of protein kinases. J Endocrinol. 2006;190(3):703–710.
    View this article via: PubMed CrossRef Google Scholar
30. Tremblay F, et al. Disruption of G protein–coupled receptor 39 impairs insulin secretion in vivo. Endocrinology. 2009;150(6):2586–2595.
    View this article via: PubMed CrossRef Google Scholar
31. Offermanns S, Zhao LP, Gohla A, Sarosi I, Simon MI, Wilkie TM. Embryonic cardiomyocyte hypoplasia and craniofacial defects in G alpha q/G alpha 11-mutant mice. EMBO J. 1998;17(15):4304–4312.
    View this article via: PubMed CrossRef Google Scholar
32. Postic C, et al. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem. 1999;274(1):305–315.
    View this article via: PubMed CrossRef Google Scholar
33. Wettschureck N, et al. Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Galphaq/Galpha11 in cardiomyocytes. Nat Med. 2001;7(11):1236–1240.
    View this article via: PubMed CrossRef Google Scholar
34. Lee JY, Ristow M, Lin X, White MF, Magnuson MA, Hennighausen L. RIP-Cre revisited, evidence for impairments of pancreatic beta–cell function. J Biol Chem. 2006;281(5):2649–2653.
    View this article via: PubMed CrossRef Google Scholar
35. Del Prato S. Loss of early insulin secretion leads to postprandial hyperglycaemia. Diabetologia. 2003;46(suppl 1):M2–M8.
    View this article via: PubMed Google Scholar
36. Xu E, et al. Intra–islet insulin suppresses glucagon release via GABA-GABAA receptor system. Cell Metab. 2006;3(1):47–58.
    View this article via: PubMed CrossRef Google Scholar
37. Maruyama H, Hisatomi A, Orci L, Grodsky GM, Unger RH. Insulin within islets is a physiologic glucagon release inhibitor. J Clin Invest. 1984;74(6):2296–2299.
    View this article via: JCI PubMed Google Scholar
38. Taylor SJ, Chae HZ, Rhee SG, Exton JH. Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. Nature. 1991;350(6318):516–518.
    View this article via: PubMed Google Scholar
39. Baukrowitz T, et al. PIP2 and PIP as determinants for ATP inhibition of KATP channels. Science. 1998;282(5391):1141–1144.
    View this article via: PubMed Google Scholar
40. Shyng SL, Nichols CG. Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science. 1998;282(5391):1138–1141.
    View this article via: PubMed CrossRef Google Scholar
41. Hutton JC, Penn EJ, Peshavaria M. Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate. Biochem J. 1983;210(2):297–305.
    View this article via: PubMed Google Scholar
42. Gylfe E. Association between 5-hydroxytryptamine release and insulin secretion. J Endocrinol. 1978;78(2):239–248.
    View this article via: PubMed CrossRef Google Scholar
43. Fehmann HC, Weber V, Goke R, Goke B, Arnold R. Cosecretion of amylin and insulin from isolated rat pancreas. FEBS Lett. 1990;262(2):279–281.
    View this article via: PubMed CrossRef Google Scholar
44. Moran M. Prostaglandins and the release of insulin. A review and a proposal. Prostaglandins Leukot Essent Fatty Acids. 1988;32(2):95–99.
    View this article via: PubMed CrossRef Google Scholar
45. Petit P, Lajoix AD, Gross R. P2 purinergic signalling in the pancreatic beta-cell: control of insulin secretion and pharmacology. Eur J Pharm Sci. 2009;37(2):67–75.
    View this article via: PubMed Google Scholar
46. Regard JB, Sato IT, Coughlin SR. Anatomical profiling of G protein-coupled receptor expression. Cell. 2008;135(3):561–571.
    View this article via: PubMed CrossRef Google Scholar
47. Wollheim CB, Sharp GW. Regulation of insulin release by calcium. Physiol Rev. 1981;61(4):914–973.
    View this article via: PubMed Google Scholar
48. Briscoe CP, et al. The orphan G protein–coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol Chem. 2003;278(13):11303–11311.
    View this article via: PubMed CrossRef Google Scholar
49. Steneberg P, Rubins N, Bartoov-Shifman R, Walker MD, Edlund H. The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse. Cell Metab. 2005;1(4):245–258.
    View this article via: PubMed CrossRef Google Scholar
50. Latour MG, et al. GPR40 is necessary but not sufficient for fatty acid stimulation of insulin secretion in vivo. Diabetes. 2007;56(4):1087–1094.
    View this article via: PubMed CrossRef Google Scholar
51. Alquier T, et al. Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets. Diabetes. 2009;58(11):2607–2615.
    View this article via: PubMed CrossRef Google Scholar
52. Verspohl EJ, Ammon HP, Williams JA, Goldfine ID. Evidence that cholecystokinin interacts with specific receptors and regulates insulin release in isolated rat islets of Langerhans. Diabetes. 1986;35(1):38–43.
    View this article via: PubMed CrossRef Google Scholar
53. Zawalich WS, Cote SB, Diaz VA. Influence of cholecystokinin on insulin output from isolated perifused pancreatic islets. Endocrinology. 1986;119(2):616–621.
    View this article via: PubMed Google Scholar
54. Karlsson S, Ahren B. CCKA receptor antagonism inhibits mechanisms underlying CCK-8-stimulated insulin release in isolated rat islets. Eur J Pharmacol. 1991;202(2):253–257.
    View this article via: PubMed CrossRef Google Scholar
55. Julien S, Laine J, Morisset J. The rat pancreatic islets: a reliable tool to study islet responses to cholecystokinin receptor occupation. Regul Pept. 2004;121(1–3):73–81.
    View this article via: PubMed Google Scholar
56. Bourassa J, Laine J, Kruse ML, Gagnon MC, Calvo E, Morisset J. Ontogeny and species differences in the pancreatic expression and localization of the CCK(A) receptors. Biochem Biophys Res Commun. 1999;260(3):820–828.
    View this article via: PubMed Google Scholar
57. Simonsson E, Karlsson S, Ahren B. Islet phospholipase A(2) activation is potentiated in insulin resistant mice. Biochem Biophys Res Commun. 2000;272(2):539–543.
    View this article via: PubMed Google Scholar
58. Kilts JD, Lin SS, Lowe JE, Kwatra MM. Selective activation of human atrial Galpha12 and Galpha13 by Galphaq-coupled angiotensin and endothelin receptors. J Cardiovasc Pharmacol. 2007;50(3):299–303.
    View this article via: PubMed CrossRef Google Scholar
59. Sabbatini ME, Bi Y, Ji B, Ernst SA, Williams JA. CCK activates RhoA and Rac1 differentially through G{alpha}13 and G{alpha}q in mouse pancreatic acini. Am J Physiol Cell Physiol. 2009;298(3):C592–C601.
    View this article via: PubMed CrossRef Google Scholar
60. Xie T, Chen M, Zhang QH, Ma Z, Weinstein LS. Beta cell-specific deficiency of the stimulatory G protein alpha-subunit Gsalpha leads to reduced beta cell mass and insulin-deficient diabetes. Proc Natl Acad Sci U S A. 2007;104(49):19601–19606.
    View this article via: PubMed CrossRef Google Scholar
61. Fruman DA, Meyers RE, Cantley LC. Phosphoinositide kinases. Annu Rev Biochem. 1998;67:481–507.
    View this article via: PubMed CrossRef Google Scholar
62. Xie LH, Horie M, Takano M. Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism. Proc Natl Acad Sci U S A. 1999;96(26):15292–15297.
    View this article via: PubMed CrossRef Google Scholar
63. Best L, Malaisse WJ. Stimulation of phosphoinositide breakdown in rat pancreatic islets by glucose and carbamylcholine. Biochem Biophys Res Commun. 1983;116(1):9–16.
    View this article via: PubMed Google Scholar
64. Zawalich WS, Zawalich KC, Rasmussen H. Cholinergic agonists prime the beta–cell to glucose stimulation. Endocrinology. 1989;125(5):2400–2406.
    View this article via: PubMed CrossRef Google Scholar
65. Nakano K, et al. Intracellular Ca(2+) modulation of ATP-sensitive K(+) channel activity in acetylcholine-induced activation of rat pancreatic beta-cells. Endocrinology. 2002;143(2):569–576.
    View this article via: PubMed CrossRef Google Scholar
66. Arkhammar P, et al. Protein kinase C modulates the insulin secretory process by maintaining a proper function of the beta-cell voltage–activated Ca2+ channels. J Biol Chem. 1994;269(4):2743–2749.
    View this article via: PubMed Google Scholar
67. Wolf BA, Florholmen J, Turk J, McDaniel ML. Studies of the Ca2+ requirements for glucose- and carbachol-induced augmentation of inositol trisphosphate and inositol tetrakisphosphate accumulation in digitonin-permeabilized islets. Evidence for a glucose recognition site in insulin secretion. J Biol Chem. 1988;263(8):3565–3575.
    View this article via: PubMed Google Scholar
68. Biden TJ, Peter-Riesch B, Schlegel W, Wollheim CB. Ca2+-mediated generation of inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate in pancreatic islets. Studies with K+, glucose, and carbamylcholine. J Biol Chem. 1987;262(8):3567–3571.
    View this article via: PubMed Google Scholar
69. Montague W, Morgan NG, Rumford GM, Prince CA. Effect of glucose on polyphosphoinositide metabolism in isolated rat islets of Langerhans. Biochem J. 1985;227(2):483–489.
    View this article via: PubMed Google Scholar
70. Prentki M, Matschinsky FM. Ca2+, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev. 1987;67(4):1185–1248.
    View this article via: PubMed Google Scholar
71. Hedeskov CJ, Thams P, Gembal M, Malik T, Capito K. Characteristics of phosphoinositide-specific phospholipase C activity from mouse pancreatic islets. Mol Cell Endocrinol. 1991;78(3):187–195.
    View this article via: PubMed CrossRef Google Scholar
72. Hazama A, Hayashi S, Okada Y. Cell surface measurements of ATP release from single pancreatic beta cells using a novel biosensor technique. Pflugers Arch. 1998;437(1):31–35.
    View this article via: PubMed CrossRef Google Scholar
73. Kreda SM, et al. Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells. J Physiol. 2007;584(pt 1):245–259.
    View this article via: PubMed CrossRef Google Scholar
74. Kreda SM, Seminario-Vidal L, Heusden C, Lazarowski ER. Thrombin–promoted release of UDP-glucose from human astrocytoma cells. Br J Pharmacol. 2008;153(7):1528–1537.
    View this article via: PubMed CrossRef Google Scholar
75. Tatur S, Groulx N, Orlov SN, Grygorczyk R. Ca2+-dependent ATP release from A549 cells involves synergistic autocrine stimulation by coreleased uridine nucleotides. J Physiol. 2007;584(pt 2):419–435.
    View this article via: PubMed CrossRef Google Scholar
76. Farret A, Vignaud M, Dietz S, Vignon J, Petit P, Gross R. P2Y purinergic potentiation of glucose-induced insulin secretion and pancreatic beta-cell metabolism. Diabetes. 2004;53(suppl 3):S63–S66.
    View this article via: PubMed CrossRef Google Scholar
77. Chevassus H, et al. P2Y receptor activation enhances insulin release from pancreatic beta-cells by triggering the cyclic AMP/protein kinase A pathway. Naunyn Schmiedebergs Arch Pharmacol. 2002;366(5):464–469.
    View this article via: PubMed CrossRef Google Scholar
78. Fischer B, et al. 2-thioether 5′-O-(1-thiotriphosphate)adenosine derivatives as new insulin secretagogues acting through P2Y-Receptors. J Med Chem. 1999;42(18):3636–3646.
    View this article via: PubMed CrossRef Google Scholar
79. Fernandez-Alvarez J, Hillaire-Buys D, Loubatieres-Mariani MM, Gomis R, Petit P. P2 receptor agonists stimulate insulin release from human pancreatic islets. Pancreas. 2001;22(1):69–71.
    View this article via: PubMed CrossRef Google Scholar
80. Petit P, Bertrand G, Schmeer W, Henquin JC. Effects of extracellular adenine nucleotides on the electrical, ionic and secretory events in mouse pancreatic beta-cells. Br J Pharmacol. 1989;98(3):875–882.
    View this article via: PubMed Google Scholar
81. Poulsen CR, et al. Multiple sites of purinergic control of insulin secretion in mouse pancreatic beta–cells. Diabetes. 1999;48(11):2171–2181.
    View this article via: PubMed CrossRef Google Scholar
82. Kitsou-Mylona I, Burns CJ, Squires PE, Persaud SJ, Jones PM. A role for the extracellular calcium–sensing receptor in cell–cell communication in pancreatic islets of langerhans. Cell Physiol Biochem. 2008;22(5–6):557–566.
    View this article via: PubMed CrossRef Google Scholar
83. Leung YM, et al. Electrophysiological characterization of pancreatic islet cells in the mouse insulin promoter–green fluorescent protein mouse. Endocrinology. 2005;146(11):4766–4775.
    View this article via: PubMed CrossRef Google Scholar
84. Asada N, Shibuya I, Iwanaga T, Niwa K, Kanno T. Identification of alpha– and beta–cells in intact isolated islets of Langerhans by their characteristic cytoplasmic Ca2+ concentration dynamics and immunocytochemical staining. Diabetes. 1998;47(5):751–757.
    View this article via: PubMed Google Scholar