Gyslaine Bertrand

Evidence for a glutamate receptor of the AMPA subtype which mediates insulin release from rat perfused pancreas

1 The effect of l‐glutamate has been studied on insulin secretion by the isolated perfused pancreas of the rat. The glutamate receptor subtype involved has been characterized. 2 In the presence of a slightly stimulating glucose concentration (8.3 mm), l‐glutamate (5 × 10−5−4 × 10−3 m) induced an immediate, transient and concentration‐dependent insulin response. On the other hand, in the presence of a non stimulating glucose concentration (2.8 mm), l‐glutamate (10−3 m) did not modify the basal insulin secretion. 3 The three non‐NMDA receptor agonists, kainate (10−4−10−3 m), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA, 5 × 10−5−10−4 m) and quisqualate (5 × 10−6−5 × 10−5 m) all provoked a transient and concentration‐dependent insulin response from pancreas perfused with 8.3 mm glucose. Compared with glutamate, kainate exhibited a similar efficacy, whereas AMPA and quisqualate elicited only a 3 fold lower maximal insulin response. In contrast, NMDA (10−4−10−3 m) was ineffective. 4 An antagonist of non‐NMDA receptors, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 5 × 10−5 m) totally prevented the stimulatory effect of l‐glutamate (4 × 10−4 m) and kainate (2 × 10−4 m). In contrast, the NMDA receptor antagonist, (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine ((+) MK801) was without effect. 5 The insulin secretory effect of glutamate (4 × 10−4 m) was not affected by atropine (3 × 10−7 m) or tetrodotoxin (3 × 10−6 m). 6 Quisqualate at a high maximally effective concentration (4 × 10−4 m) inhibited glutamate (10−3 m) or kainate (4 × 10−4 m)‐induced insulin release. 7 This study shows that l‐glutamate stimulates insulin secretion in rat pancreas, by acting on an excitatory amino acid receptor of the AMPA subtype.

Glutamate stimulates glucagon secretion via an excitatory amino acid receptor of the AMPA subtype in rat pancreas.

The effect of L-glutamate was studied on glucagon secretion from rat isolated pancreas perfused with 2.8 mM glucose. L-Glutamate (3.10(-5)-10(-4)M) induced an immediate, transient and concentration-dependent glucagon release. The three non-N-methyl-D-aspartate (NMDA) receptor agonists, kainate (3.10(-5)-3.10(-3)M), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (3.10(-5)-10(-4)M) and quisqualate (3.10(-6)-10(-5)M), all elicited a peak-shaped glucagon response. Compared to glutamate, AMPA and quisqualate exhibited a similar efficacy, whereas kainate caused a 4-fold higher maximal glucagon response. In contrast, NMDA (10(-3)M) was ineffective. The selective antagonist of non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5.10(-5)M), totally prevented the glucagon response to 10(-4) M glutamate (IC50 congruent to 0.8 +/- 0.3 10(-6)M) and 3.10(-4)M kainate. Furthermore, quisqualate at a maximal effective concentration (3.10(-4)M) inhibited the response to kainate (10(-3)M). This study showed that L-glutamate stimulates glucagon release in rat pancreas by activating a receptor of the AMPA subtype.

The NALCN ion channel is activated by M3 muscarinic receptors in a pancreatic β-cell line

A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non‐selective), has been recently shown to be responsible for the tetrodotoxin (TTX)‐resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic β‐cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co‐expression of NALCN and M3R in human embryonic kidney‐293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I–II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic‐activated inward sodium current that is independent of G‐protein activation, and provide new insights into the properties of NALCN channels.

Comparative effects of PACAP and VIP on pancreatic endocrine secretions and vascular resistance in rat.

1 The effects of pituitary adenylate cyclase‐activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and secretin on pancreatic endocrine secretions and vascular resistance were investigated and compared in the isolated perfused pancreas of the rat. The PACAP/VIP receptor types involved have been characterized. 2 On insulin secretion, in the range 10−11 to 10−8 m, PACAP and VIP elicited a concentration‐dependent biphasic response from pancreas perfused with 8.3 mM glucose; the peptides were equipotent. In contrast, secretin was ineffective in the range 10−11 to 10−9 m; at 10−8 and 10−7 m, it induced only low and transient insulin responses. On the other hand, the peptides did not modify the basal insulin release in the presence of a non stimulating glucose concentration (2.8 mM). 3 On glucagon secretion, PACAP and VIP (10−11 to 10−8 m) but also secretin (10−9 to 10−7 m) caused a concentration‐dependent peak shaped response from pancreas perfused with 2.8 mM glucose; PACAP and VIP were equipotent and 20 times more potent than secretin. On the other hand, the peptides did not affect the glucagon release in the presence of 8.3 mM glucose. 4 On pancreatic vessels, in the range 10−11 to 10−9 m, the three peptides were equipotent in inducing a concentration‐dependent sustained increase in pancreatic flow rate. On the other hand, at the high concentration of 10−7 m PACAP but not VIP provoked a transient decrease of flow rate. 5 This study provides evidence for PACAP/VIP type II receptors mediating insulin and glucagon secretion as well as vasodilatation in rat pancreas. In addition, the different efficacies of secretin suggest that these effects are mediated by different PACAP/VIP type II receptor subtypes.

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells.

Quercetin is a natural polyphenolic flavonoid that displays anti‐diabetic properties in vivo. Its mechanism of action on insulin‐secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca2+]i) in beta cells, in the absence of any co‐stimulating factor.

Effects of imidazolines and derivatives on insulin secretion and vascular resistance in perfused rat pancreas

The effects of imidazolines and derivatives were studied on insulin secretion and vascular resistance in the isolated perfused rat pancreas. On insulin secretion, two imidazoline alpha 2-adrenoceptor antagonists, efaroxan (1-100 microM) and RX821002 (10 microM), had a stimulating response; however, idazoxan, like the non-imidazoline alpha 2-adrenoceptor antagonist yohimbine, was ineffective at 10 microM. The oxazoline rilmenidine with alpha 2-adrenergic activity at 10 microM), an imidazoline devoid of alpha 2-adrenergic activity, also had an insulin-releasing effect. On pancreatic vessels, all imidazolines tested (efaroxan, RX821002, antazoline and idazoxan), in contrast to yohimbine, induced vasoconstriction. Rilmenidine did not have a vasoconstrictor effect after blockade of alpha 2-adrenoceptors. Furthermore, the efaroxan-induced insulin release or vasoconstriction was not affected by the blockade of alpha 2- and alpha 1-adrenoceptors. This study shows that imidazolines and derivatives are able to stimulate insulin release and induce vasoconstriction in the rat pancreas. These effects cannot be ascribed to an interaction with alpha-adrenoceptors but may involve different types of imidazoline sites.

β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1

Background: FFAR1/GPR40 is a potential target to enhance insulin secretion in type 2 diabetes, yet knowledge of the pharmacobiology of GPR40 remains incomplete. Results: GPR40 functions via both G protein-mediated and β-arrestin-mediated mechanisms; endogenous and synthetic ligands differentially engage these pathways to promote insulin secretion. Conclusion: GPR40 is subject to functionally relevant biased agonism. Significance: Biased agonism at GPR40 could be exploited for therapeutic purposes. FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.

β-Arrestin2 plays a key role in the modulation of the pancreatic beta cell mass in mice

Aims/hypothesisBeta cell failure due to progressive secretory dysfunction and limited expansion of beta cell mass is a key feature of type 2 diabetes. Beta cell function and mass are controlled by glucose and hormones/neurotransmitters that activate G protein-coupled receptors or receptor tyrosine kinases. We have investigated the role of β-arrestin (ARRB)2, a scaffold protein known to modulate such receptor signalling, in the modulation of beta cell function and mass, with a specific interest in glucagon-like peptide-1 (GLP-1), muscarinic and insulin receptors.Methodsβ-arrestin2-knockout mice and their wild-type littermates were fed a normal or a high-fat diet (HFD). Glucose tolerance, insulin sensitivity and insulin secretion were assessed in vivo. Beta cell mass was evaluated in pancreatic sections. Free cytosolic [Ca2+] and insulin secretion were determined using perifused islets. The insulin signalling pathway was evaluated by western blotting.ResultsArrb2-knockout mice exhibited impaired glucose tolerance and insulin secretion in vivo, but normal insulin sensitivity compared with wild type. Surprisingly, the absence of ARRB2 did not affect glucose-stimulated insulin secretion or GLP-1- and acetylcholine-mediated amplifications from perifused islets, but it decreased the islet insulin content and beta cell mass. Additionally, there was no compensatory beta cell mass expansion through proliferation in response to the HFD. Furthermore, Arrb2 deletion altered the islet insulin signalling pathway.Conclusions/interpretationARRB2 is unlikely to be involved in the regulation of insulin secretion, but it is required for beta cell mass plasticity. Additionally, we provide new insights into the mechanisms involved in insulin signalling in beta cells.

Evidence for a direct stimulatory effect of cibenzoline on insulin secretion in rats

The effect of cibenzoline succinate, a new antiarrhythmic agent, was studied on insulin secretion in rats. Experiments were performed both in vivo and in vitro using two preparations: the isolated perfused pancreas and isolated islets. In anaesthetized rats, cibenzoline was able to increase plasma insulin levels and to reduce glycaemia. These effects were observed at 1 mg/kg i.v. in fed rats and at 3 mg/kg i.v. in fasted rats. In the isolated pancreas perfused in the presence of a slightly stimulating glucose concentration (8.3 mM), cibenzoline (2 and 6 microM) elicited a progressive and sustained insulin response in a concentration-dependent manner. In the presence of a non-stimulating glucose concentration (4.2 mM), cibenzoline was ineffective at 2 microM and slightly increased basal insulin release at 6 microM. In isolated islets incubated with 8.3 mM glucose, cibenzoline (6 and 20 microM) caused a concentration-dependent stimulation of insulin release. It is concluded that cibenzoline stimulates insulin secretion by a direct action on pancreatic B cells in rats.

Antazoline increases insulin secretion and improves glucose tolerance in rats and dogs

In vivo effects of an imidazoline devoid of alpha2-adrenoceptor antagonistic properties, antazoline, on insulin secretion and glycemia were investigated both in fasted rats and dogs. In both species, antazoline (1.5 mg/kg i.v.) transiently increased insulinemia without affecting basal plasma glucose levels. In contrast, during an i.v. glucose tolerance test, antazoline markedly potentiated insulin release and thus increased the glucose disappearance rate. In rats, during an oral glucose tolerance test, the intragastric administration of antazoline (1.5 mg/kg) clearly enhanced insulin secretion and reduced hyperglycemia. In dogs provided with a venous pancreatico-duodenal bypass, antazoline (0.5 mg/kg i.v.) induced an immediate and transient increase in insulin and somatostatin but not in glucagon pancreatico-duodenal outputs. In conclusion, intravenously and orally administered, the imidazoline antazoline is able to stimulate insulin secretion in vivo and improve glucose tolerance. The imidazoline compounds could therefore have a potential therapeutic relevance as new antihyperglycemic insulinotropic agents.