Intra-islet α-cell Gs signaling promotes glucagon release

Abstract

Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell G_s signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell G_s signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α-cell G_s-coupled A_2A adenosine receptors. Studies with α-cell-specific Gα_s knockout mice showed that α-cell G_s also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched G_s-coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.

Fig. 1. Acute activation of the α-cell GsD designer receptor stimulates glucagon secretion.

Freely fed α-GsD mice and control littermates were injected with saline (a–c) or DCZ (10 μg/kg, i.p.) (d–f). Plasma glucagon (a, d), plasma insulin (b, e), and blood glucose (c, f) levels were measured at the indicated time points. g–j Pancreatic islets prepared from control and α-GsD mice were perifused with the indicated glucose concentrations in the presence of DCZ (10 nM) and (alanine (3 mM)). Glucagon ((g); insert: glucagon from 23 to 70 min) and insulin secretion (i) were measured in the presence of low and high glucose levels (3 mM [G3] and 12 mM [G12], respectively). AOC values were calculated for glucagon (h) and insulin (j) secretion calculated for different stimulation periods. All experiments were carried out with male littermates (12–16 weeks old). Data are given as means ± SEM (in vivo studies: control, n = 9; α-GsD, n = 7; in vitro studies: 3 independent perifusion experiments with 75–100 islets per perifusion chamber). Data were analyzed via two-way repeated measures ANOVA for time with Bonferroni post hoc test for comparison of individual time points (d–f) or two-tailed Student’s t test (h, j). Numbers of above data points or horizontal lines in the bar graphs represent p values. AOC, area of the curve. Source data are provided as a Source Data file.

Fig. 2. Acute activation of the α-cell GsD designer receptor improves glucose tolerance in both lean and obese mice.

α-GsD mice and control littermates consuming regular chow (lean mice) or a high-fat diet (HFD; obese mice) were subjected to a series of metabolic tests. a Glucose tolerance test (ipGTT). Lean mice that had been fasted overnight were co-injected (i.p.) with glucose (2 g/kg) and DCZ (10 μg/kg) (control, n = 8; α-GsKO, n = 6). Changes in plasma glucagon (b) and plasma insulin (c) levels following i.p. co-injection of lean mice with glucose and DCZ (control, n = 7; α-GsKO, n = 8). d Insulin tolerance test (ITT). Lean mice that had been fasted for 4 h after were injected (i.p.) with a mixture of insulin (0.75 U/kg) and DCZ (control, n = 10; α-GsKO, n = 10). e ipGTT. Obese mice that had been fasted overnight were co-injected (i.p.) with glucose (1 g/kg) and DCZ (10 μg/kg) (control, n = 7; α-GsKO, n = 11). Changes in plasma glucagon (f) and plasma insulin (g) levels following i.p. co-injection of obese mice with glucose and DCZ (control, n = 8; α-GsKO, n = 7). h Insulin tolerance test (ITT). Following a 4 h fast, obese mice were injected (i.p.) with a mixture of insulin (1 U/kg) and DCZ (10 μg/kg) (control, n = 8; α-GsKO, n = 11). Blood samples were collected from the tail vein at the indicated time points. All experiments were carried out with male littermates that were at least 14 weeks old. Obese mice consumed the HFD for at least 8 weeks. Data are given as means ± SEM. Data were subjected to two-tailed Student’s t test (AOC bars) or to two-way repeated measures ANOVA for time with Bonferroni post hoc test for comparison of individual time points (a–c, e–g). AOC, area over the curve. Numbers in the bar graphs or next to specific data points data points refer to p values. AOC, area of the curve. Source data are provided as a Source Data file.

Fig. 3. Activation of α-cell A2ARs strongly stimulates glucagon secretion from mouse and human islets.

a, b Measurement of glucagon secretion from perifused mouse WT islets. Experiments were carried out at low and high glucose levels (3 mM [G3] and 12 mM [G12], respectively) either in the presence of UK432097 (A2AR-selective agonist: 50 nM at G3, 10 nM at G12) or SCH442416 (A2AR-selective antagonist, 0.5 μM) alone or in the presence of both ligands (n = 4 mice per group). cAMP production in α-cells from α-CAMPER mice at G3 (c) or G12 (d) in the presence of UK432097 (100 nM) or SCH442416 (0.5 μM) or in the presence of both ligands. e–h Glucagon release studies with islets lacking Gα_s or A2ARs in their α-cells. Islets were prepared from α-GsKO and α-A2AR-KO mice and their corresponding littermates. In (e), islets were treated with ADA (5 U/ml) at G3 to enzymatically remove extracellular adenosine (n = 3 or 4 mice per group). In (f, g), α-GsKO and control islets were treated with UK432097 at G3 and G12 (n = 3 or 4 mice per group). AOC values (h) for glucagon release data shown in (f) and (g) (time period: 8–30 min). i, j A2AR activation stimulates glucagon secretion from human islets. Islets from human donors were perifused with G3 (i) or G12 (j), respectively, either in the presence of vehicle (DMSO), UK432097 or SCH442416 alone, or in the presence of both UK432097 and SCH442416 (n = 3 donors per group). Islets were obtained from male or female mice that were 14–24 weeks old. AOC values were calculated for different stimulation periods. Data are shown as means ± SEM (3 or 4 independent perifusions with 75–100 islets per perifusion chamber). Data were analyzed via two-tailed Student’s t test (AOC values in a, b, e, h–j) or two-way repeated measures ANOVA with time (c, d). Numbers above horizontal lines in the bar graphs represent p values. ADA, adenosine deaminase. AOC, area of the curve. Source data are provided as a Source Data file.

Fig. 4. Activation of α-cell A2ARs in vivo induces glucagon secretion.

a Absence of Adora2a transcript in α-cells of α-A2AR-KO mice. qRT-PCR analysis of Adora2a gene expression (encoded protein: A2AR) in whole islets, α-, β- and δ-cells. The different cell types were isolated via FACS sorting using islets from control and α-A2AR-KO mice (males). b Glucagon secretion from control and α-A2AR-KO islets treated with the A2AR-selective agonist UK432097 (50 nM at G3, 10 nM at G12). c Glucagon secretion from perifused control and α-A2AR-KO islets treated with the A2AR-selective antagonist SCH442416 (0.5 μM) at G3. d Glucagon release from perifused control and α-A2AR-KO islets in the presence of different glucose concentrations and isoproterenol (5 μM), a β-adrenergic receptor agonist. Plasma glucagon (e) and blood glucose (f) levels measured after i.p. injection of control and α-A2AR-KO mice with UK432097 (5 mg/kg). AOC values were calculated for different stimulation periods. All experiments were carried out with male littermates (12–20 weeks old). Data are given as means ± SEM (in vivo studies: (n = 7 per group); in vitro perifusion studies: 3–5 independent perifusions with 75–100 islets per perifusion chamber). Data were analyzed via two-tailed Student’s t test ((a) and AOC values in (b–d)) or two-way repeated measures ANOVA for time with Bonferroni post hoc test for comparison of time (e, f). Numbers in the AOC panels represent p values. AOC, area of the curve. Source data are provided as a Source Data file.

Fig. 5. Selective lack of Gα_s in α-cells leads to impaired glucagon secretion and reduced islet glucagon content and Gcg expression.

a–c Data confirming the absence of Gnas mRNA (encoded protein: Gα_s) or Gα_s protein in α-cells of α-GsKO mice (α-cell Gnas^-/- mice). a Absence of Gnas mRNA in α-cells isolated from α-cell Gnas^-/- mice via FACS (α-GsKO, n = 8 mice; control, n = 5 mice). Gene expression data were obtained via qRT-PCR. b The lack of α-cell Gnas expression does not affect α-cell Gnaq expression (encoded protein: Gα_q) (α-GsKO, n = 8 mice; control, n = 5 mice). c Immunofluorescence staining of pancreatic slices from α-GsKO mice and control littermates. Slices were co-stained with either an anti-Gα_s antibody (Alexa Fluor, red) and an anti-glucagon antibody (Alexa Fluor, green), or an anti-Gα_s antibody (Alexa Fluor, green) and an anti-insulin antibody (Alexa Fluor, red), respectively. The inserts in the upper right of each panel show enlarged islets areas. Scale bar in inserts: 10 μm; scale bars in non-enlarged images: 50 μm. Contrast was adjusted for improved visualization. d, e Glucagon secretion studies carried out with perifused islets prepared from α-GsKO mice and control littermates. While treatment of control islets with 5 μM isoproterenol (β-adrenergic receptor agonist) strongly stimulated glucagon release at both G3 and G12, this response was almost completely abolished in α-GsKO islets (n = 3 or 4 mice per group). f Stimulation of glucagon release by low glucose (G3), a V1b receptor agonist (d[Leu⁴, Lys⁸]VP, 10 nM), and KCl (30 mM) from perifused α-GsKO mice and control islets (n = 4 mice per group). Glucagon content of islets and pancreata from control and α-GsKO mice (g, h: n = 7 and n = 4 mice per group, respectively). Insulin content of islets and pancreata from control and α-GsKO mice (i, j: n = 7 and n = 4 mice per group, respectively). k Expression levels of key α- and β-cell genes determined with RNA prepared from control and α-GsKO islets (n = 4 mice per group). AOC values were calculated for different stimulation periods. The data shown in (g–k) were generated using islets obtained from male mice (age: ~30-weeks). For islet perifusion studies, 75–100 islets per chamber were used. Islets were prepared from male mice (age: 16–20 weeks). Immunofluorescence images are representative of three independent experiments. Data are given as means ± SEM. Data were analyzed via two-tailed Student’s t test (a, g, h, k, and AOC values in d–f). Numbers in the AOC panels represent p values. AOC, area of the curve. Source data are provided as a Source Data file.

Fig. 6. Disruption of α-cell G_s signaling results in reduced plasma glucagon levels.

Plasma hormone ((a), glucagon; (b), insulin) and blood glucose (c) levels in α-GsKO mice and control littermates consuming regular chow (lean mice). Mice had either free access to food (fed) or were fasted overnight. Plasma hormone ((d), glucagon; (e), insulin) and blood glucose (f) levels in α-GsKO mice and control littermates maintained on a HFD (obese mice). Mice had either free access to food (fed) or were fasted overnight. Blood samples were collected from the tail vein. All experiments were carried out with male littermates. At the time of testing, lean mice were 14 weeks old. Obese mice were maintained on the HFD for at least 8 weeks, after having consume regular chow for 18 weeks. Data are given as means ± SEM (lean mice: control, n = 9; α-GsKO, n = 12; obese mice: control, n = 9; α-GsKO, n = 10). Data were analyzed via two-tailed Student’s t test (a, d). Numbers above the horizontal lines in the bar graphs represent p values. Source data are provided as a Source Data file.

Fig. 7. Lack of α-cell G_s signaling leads to impaired glucagon release under different experimental conditions in vivo.

a, b Glucagon release following insulin-induced hypoglycemia. α-GsKO mice and control littermates were injected with insulin (1 U/kg, i.p.), and plasma glucagon (a) and blood glucose (b) levels were measured at the indicated time points (n = 8 per group). c, d Glucagon secretion after 2-DG-induced glucopenia. α-GsKO and control mice were injected with 2-DG (500 mg/kg, i.p.), and plasma glucagon (c) and blood glucose (d) levels were measured at the indicated time points (control, n = 5; α-GsKO, n = 7). e–g Treatment of mice with a mixture of GIP and alanine. α-GsKO and control mice were injected i.p. with a combination of GIP (4 nmol/kg) and alanine (0.325 g/kg), and plasma glucagon (e), plasma insulin (f), and blood glucose (g) levels were measured 15 min later (n = 8 per group). h Treatment of isolated islets with a GIP/alanine mixture to induce glucagon secretion. Perifused islets isolated from control and α-GsKO mice were treated with a combination of GIP (10 nM) and alanine (3 mM) at G12. KCl (30 mM) was added at the end of the experiment (n = 4 mice per group). Note that GIP- and GIP/alanine-induced glucagon secretion was virtually abolished in α-GsKO islets. AOC values were calculated for different stimulation periods. All experiments were carried out with male littermates (14–20 weeks old). Blood was collected from the tail vein. Data are given as means ± SEM (in vitro studies: 4 perifusions with 75–100 islets per perifusion chamber). Data were analyzed via two-way repeated measures ANOVA for time with Bonferroni post hoc test for comparison of time (a, c, d–f) or two-tailed Student’s t test (AOC values in (h)). Numbers above the horizontal lines in the bar graphs represent p values. AOC, area of the curve. Source data are provided as a Source Data file.