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

Abstract

A variety of neurotransmitters, gastrointestinal hormones, and metabolic signals are known to potentiate insulin secretion through GPCRs. We show here that beta cell-specific inactivation of the genes encoding the G protein alpha-subunits Galphaq and Galpha11 resulted in impaired glucose tolerance and insulin secretion in mice. Interestingly, the defects observed in Galphaq/Galpha11-deficient beta 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 beta cells was impaired in the absence of Galphaq/Galpha11, and closure of KATP channels was inhibited. We provide evidence that this reduced excitability was due to a loss of beta 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 Gq/G11-coupled P2Y6 receptor and extracellular calcium acting through the calcium-sensing receptor. Thus, the Gq/G11-mediated signaling pathway potentiates insulin secretion in response to glucose by integrating systemic as well as autocrine/paracrine mediators.

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.

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.

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.

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.

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.