Intra-islet glucagon signaling is critical for maintaining glucose homeostasis

Abstract

Glucagon, a hormone released from pancreatic alpha-cells, plays a key role in maintaining proper glucose homeostasis and has been implicated in the pathophysiology of diabetes. In vitro studies suggest that intra-islet glucagon can modulate the function of pancreatic beta-cells. However, because of the lack of suitable experimental tools, the in vivo physiological role of this intra-islet cross-talk has remained elusive. To address this issue, we generated a novel mouse model that selectively expressed an inhibitory designer G protein-coupled receptor (Gi DREADD) in α-cells only. Drug-induced activation of this inhibitory designer receptor almost completely shut off glucagon secretion in vivo, resulting in significantly impaired insulin secretion, hyperglycemia, and glucose intolerance. Additional studies with mouse and human islets indicated that intra-islet glucagon stimulates insulin release primarily by activating β-cell GLP-1 receptors. These new findings strongly suggest that intra-islet glucagon signaling is essential for maintaining proper glucose homeostasis in vivo. Our work may pave the way toward the development of novel classes of antidiabetic drugs that act by modulating intra-islet cross-talk between α- and β-cells.

Keywords: G-protein coupled receptors; Glucose metabolism; Islet cells; Metabolism.

Figure 1. Selective expression of GiD in pancreatic α cells.

Immunofluorescent staining of slices from pancreatic islets of α-GiD and control mice. Note that the GiD receptor carried an N-terminal HA-tag, allowing its localization with an anti-HA antibody. Nuclei were stained blue with DAPI mounting medium. (A) Slices from α-GiD mice (i–iv) and control littermates (v–viii) were stained for HA-GiD (Alexa Fluor, green) and glucagon (Alexa Fluor, red). (B) Slices from α-GiD mice (i–iv) and control littermates (v–viii) were stained for HA-GiD (Alexa Fluor, green) and insulin (Alexa Fluor, red). These representative images show that the GiD receptor is not expressed in control islets but is selectively expressed by α cells of islets from α-GiD mice. Original magnification, ×40.

Figure 2. Acute activation of α cell GiD almost completely shuts down glucagon secretion in vivo.

All mice (α-GiD mice and control littermates) were injected i.p. with CNO (1 mg/kg) at time “0.” (A–C) Studies with fasted mice (overnight fast for 12 hours). (D–F) Studies with freely fed mice. Plasma glucagon (A and D), plasma insulin (B and E), and blood glucose (C and F) levels were measured at the indicated time points using blood collected from the tail vein. All experiments were carried out with male littermates that were at least 12 weeks old. Data are given as mean ± SEM (α-GiD: n = 13; control: n = 6). *P < 0.05; **P < 0.01 (mixed-effects repeated-measures ANOVA for after injection differences).

Figure 3. Acute activation of α cell GiD causes impaired glucose tolerance in α-GiD mice.

(A–D) Mice that had been fasted overnight were injected with glucose (2 g/kg i.p.), either in the absence or presence of CNO (1 mg/kg i.p). (A) i.p. glucose tolerance test (IGTT) in the absence of CNO. (B) IGTT in the presence CNO. (C) Plasma glucagon levels during IGTT in the presence of CNO. (D) Plasma insulin levels during IGTT in the presence of CNO. (E–H) Mice that had been fasted for 4 hours were injected with insulin (0.75 U/kg i.p.), either in the absence or presence of CNO (1 mg/kg i.p). (E) Insulin tolerance test (ITT) in the absence of CNO. (F) ITT in the presence CNO. (G) Plasma glucagon levels during ITT in the presence of CNO. (H) Plasma insulin levels during ITT in the presence of CNO. Blood samples were collected from the tail vein. All experiments were carried out with male littermates that were 20–30 weeks old. Data are given as mean ± SEM (α-GiD: n = 13; control: n = 6). *P < 0.05; **P < 0.01 (mixed-effects repeated-measures ANOVA for after injection differences).

Figure 4. Activation of GiD in perifused α-GiD islets causes strong reductions in glucagon and insulin secretion.

Islets from control and α-GiD mice were perifused with the indicated glucose concentrations (3 mM and 12 mM; G3 and G12, respectively) in the presence (red curve) or absence (black curve) of CNO (10 μM). In the bar graphs, hormone secretion during specific time intervals was expressed as the average of all values measured during a particular perifusion period: G3 ± CNO, 20–40 minutes; G12 ± CNO, 40–60 minutes; G12 + AAM ± CNO, 60–80 minutes. (A) Glucagon secretion from control islets. (B) Insulin secretion from control islets. (C) Glucagon secretion from α-GiD islets. (D) Insulin secretion from α-GiD islets. The amounts of secreted glucagon and insulin were normalized to islet DNA content. All islets were prepared from male littermates that were 14–20 weeks old. Data are given as mean ± SEM (3 or 4 perifusions with 50 islets per perifusion chamber; islets from 2 mice were pooled per perifusion experiment). *P < 0.05; **P < 0.01 (2-tailed Student’s t test). AAM, amino acid mixture (see Methods for details).

Figure 5. Glucagon restores normal insulin release in CNO-treated α-GiD islets.

Islets from α-GiD mice were perifused with the indicated glucose concentrations (3 mM and 12 mM; G3 and G12, respectively) in the presence (red curve) or absence (black curve) of CNO (10 μM). At 45 minutes (15 minutes after the addition of AAM), glucagon (10 nM) was added to the perifusion fluid. In the bar graphs, insulin secretion during specific time intervals was expressed as the average of all values measured during a particular perifusion period: G3, 0–15 minutes; G12, 15–30 minutes; G12+AAM, 30–45 minutes; G12+AAM+glucagon, 45–65 minutes. Insulin secretion was normalized to islet DNA content. All islets were prepared from male littermates that were 20–24 weeks old. Data are given as mean ± SEM (4 perifusions with 50 islets per perifusion chamber; islets from 2 mice were pooled per perifusion experiment). *P < 0.05 (2-tailed Student’s t test). AAM, amino acid mixture (see Methods for details).

Figure 6. Glucagon release strongly promotes insulin secretion from WT mouse and human islets via activation of GLP-1 receptors.

WT mouse pancreatic islets were perifused with 3 and 12 mM of glucose (G3 and G12, respectively) and a physiological amino acid mixture (AAM). Glucagon and insulin secretion were monitored continuously throughout experiments and normalized to islet DNA content. (A and B) Glucagon (A) and insulin (B) secretion in the presence or absence of Ex-9 (1 μM), a selective GLP-1 receptor antagonist. (C and D) Glucagon (C) and insulin (D) secretion in the presence or absence of adomeglivant (Ado; 1 μM), a selective glucagon receptor antagonist. (E and F) Glucagon (E) and insulin (F) secretion in the presence or absence of a mixture of Ex-9 (1 μM) and Ado (1 μM). (G and H) Studies with isolated human islets. Human islets were perifused with 3 and 16.7 mM of glucose (G3 and G16.7, respectively), in the presence of AAM. Glucagon (G) and insulin (H) secretion were monitored continuously throughout experiments. Ex-9 (1 μM) was added 20 minutes before stimulation of islets with G16.7. In the control groups, Ex-9 was omitted from perfusate. The bar graph in H shows insulin release during the G16.7 perifusion period. Data represent mean ± SEM from 3 or 4 perfusions. *P < 0.05 (2-tailed Student’s t test). Mouse islets were prepared from male mice (age 12–20 weeks). Data are mean ± SEM (3 or 4 perifusions with 50 mouse islets per perifusion chamber; islets from 2 mice were pooled per perifusion experiment).

Figure 7. Schematic depicting how glucagon release from α cells stimulates insulin secretion from β cells.

(A) In WT mouse islets, stimulation of α cells (e.g., by an amino acid mixture [AAM]) promotes the release of insulin from adjacent β cells, primarily by activating β cell GLP-1 receptors. Glucagon stimulation of β cell glucagon receptors (GCG receptors) is predicted to make a minor contribution to this response. (B) In islets from α-GiD mice, CNO-mediated activation of the GiD designer receptor inhibits α cell activity, leading to decreased glucagon release, which in turn causes reduced insulin secretion from β cells.