Liver-specific disruption of the murine glucagon receptor produces α-cell hyperplasia: evidence for a circulating α-cell growth factor

Abstract

Glucagon is a critical regulator of glucose homeostasis; however, mechanisms regulating glucagon action and α-cell function and number are incompletely understood. To elucidate the role of the hepatic glucagon receptor (Gcgr) in glucagon action, we generated mice with hepatocyte-specific deletion of the glucagon receptor. Gcgr(Hep)(-/-) mice exhibited reductions in fasting blood glucose and improvements in insulin sensitivity and glucose tolerance compared with wild-type controls, similar in magnitude to changes observed in Gcgr(-/-) mice. Despite preservation of islet Gcgr signaling, Gcgr(Hep)(-/-) mice developed hyperglucagonemia and α-cell hyperplasia. To investigate mechanisms by which signaling through the Gcgr regulates α-cell mass, wild-type islets were transplanted into Gcgr(-/-) or Gcgr(Hep)(-/-) mice. Wild-type islets beneath the renal capsule of Gcgr(-/-) or Gcgr(Hep)(-/-) mice exhibited an increased rate of α-cell proliferation and expansion of α-cell area, consistent with changes exhibited by endogenous α-cells in Gcgr(-/-) and Gcgr(Hep)(-/-) pancreata. These results suggest that a circulating factor generated after disruption of hepatic Gcgr signaling can increase α-cell proliferation independent of direct pancreatic input. Identification of novel factors regulating α-cell proliferation and mass may facilitate the generation and expansion of α-cells for transdifferentiation into β-cells and the treatment of diabetes.

Figures

FIG. 1.

Ablation of the hepatocyte Gcgr leads to hypoglycemia and improved glucose tolerance. A: Glycemia in 12-week-old male GcgrHep−/− and littermate controls fasted for 5 h or 16 h (n = 10–12 mice per group). B: Intraperitoneal (IP) glucose challenge in 9-week-old male GcgrHep−/− and littermate controls fasted for 16 h (n = 8–10 mice). Plasma glucagon (C) and insulin (D) levels measured before and 15 min after IP glucose injection (n = 8–10 mice per group). E: Insulin-to-glucose ratio before and 15 min after IP glucose injection. F: Oral glucose challenge in 8-week-old GcgrHep−/− males and littermate controls fasted for 16 h (n = 10–12 mice per group). Plasma glucagon (G) and insulin (H) levels measured before and 15 min after oral glucose administration (n = 10–12 mice per group). I: Insulin-to-glucose ratio before and 15 min after oral glucose injection. Data are mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. wild-type mice.

FIG. 2.

Ablation of the Gcgr in hepatocytes increases insulin sensitivity. A: Intraperitoneal (IP) insulin tolerance test in 13-week-old male GcgrHep−/− and littermate controls fasted for 5 h (n = 8–10 mice). B: Area under the curve (AUC) glucose from the IP insulin tolerance test shown in A. Hyperinsulinemic euglycemic clamp performed in conscious Gcgr−/− (C, E) or GcgrHep−/− (E, F) males and littermate controls fasted for 5 h (n = 5–7 mice). C and E: Glycemic excursion during stabilization and steady-state phase. D and F: Glucose infusion rates during steady state to maintain euglycemia. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. wild-type mice.

FIG. 3.

Whole-body ablation of the Gcgr increases pancreatic weight, α-cell mass, and α-cell proliferation. A: Histological sections stained for insulin (left panels) or glucagon (right panels). B: Pancreas weight of 20-week-old Gcgr−/− males and littermate controls corrected for body weight (n = 8–10 mice). Pancreatic β-cell mass (C) and α-cell mass (D) per gram of pancreas. Data are mean ± SEM. ***P < 0.001 vs. Gcgr+/+. Representative immunofluorescent sections stained for dapi (blue), glucagon (green), and Ki67 (red) from 22-week-old Gcgr−/− mice (F) and littermate controls (E). F′: Higher magnification of Ki67-positive nuclei from F. White arrows indicate Ki67-positive nuclei of α-cells. G: The α-cell proliferation rate (percentage of glucagon+/Ki67+ cells per total glucagon+ cells, n = 4 per group; 3 depths/pancreas). Data are mean ± SEM. *P < 0.05 vs. Gcgr+/+.

FIG. 4.

Ablation of the Gcgr in hepatocytes results in increased pancreas weight and α-cell hyperplasia. A: Representative histological sections stained for insulin (left panels) or glucagon (right panels). B: Pancreas weight of 20-week-old GcgrHep−/− males and littermate controls corrected for body weight (n = 8–10 mice). Pancreatic β-cell mass (C) and α-cell mass (D) per gram of pancreas. Data are mean ± SEM. ***P < 0.001 vs. wild type. Representative immunofluorescent sections stained for dapi (blue), glucagon (green), and Ki67 (red) from 22-week-old GcgrHep−/− mice (F) and littermate controls (E). E′ and F′: Higher magnification of sections containing Ki67-positive nuclei, denoted by white arrows, in α-cells. Insets are magnifications of selected Ki67-positive nuclei of α-cells (white box). White scale bar represents 50 μm. G: The α-cell proliferation rate (percentage of glucagon+/Ki67+ cells per total glucagon+ cells, n = 4 per group; 3 depths/pancreas). Data are mean ± SEM. ***P < 0.001 vs. Gcgrflox.

FIG. 5.

Gcgr+/+ islets transplanted into Gcgr−/− mice have development of α-cell hyperplasia. A: Experimental schematic showing islets from 14-week-old Gcgr+/+ or Gcgr−/− mice transplanted beneath the right and left kidney capsule of 14-week-old Gcgr+/+ or Gcgr−/− mice, respectively. The kidneys containing the islet graft were removed for analysis 8 weeks later. B: Hormone content of freshly isolated islets of 14-week-old Gcgr+/+ (open bars) and Gcgr−/− (closed bars) mice. Data are mean ± SEM. *P < 0.05, ***P < 0.001 vs. Gcgr+/+ islets. C: Hormone content of Gcgr+/+ grafts removed from 22-week-old mice Gcgr+/+ (open bars) and Gcgr−/− (light grey bars) recipient mice 8 weeks after transplant. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Gcgr+/+ recipients. D: Hormone content of Gcgr−/− grafts removed from 22-week-old mice Gcgr+/+ (closed bars) and Gcgr−/− (dark grey bars) recipient mice 8 weeks after transplantation. Data are mean ± SEM. *P < 0.05, ***P < 0.001 vs. Gcgr−/− recipients. Representative islet graft sections from Gcgr+/+ (donor) to Gcgr+/+ (recipient; E) and Gcgr+/+ (donor) to Gcgr−/− (recipient; F) mice stained for insulin (green) and glucagon (red). E′ and F′: Insets of E and F (white box). Representative islet graft sections from Gcgr−/− (donor) to Gcgr+/+ (recipient; G) and Gcgr−/− (donor) to Gcgr−/− (recipient; H) mice stained for insulin (green) and glucagon (red). G′ and H′: Insets of G and H (white box). White scale bar represents 50 μm. Percentage of islet grafts that stained positive for glucagon for Gcgr+/+ donor (I) and Gcgr−/− donor islets (J) into Gcgr+/+ and Gcgr−/− mice (n = 3/mice, 3 sections/islet graft). Percent glucagon area is defined as the percentage of glucagon area/total insulin + glucagon area. Data are mean ± SEM. ***P < 0.001 vs. Gcgr+/+ recipient grafts.

FIG. 6.

Gcgr+/+ islets transplanted into Gcgr−/− recipients exhibit increased α-cell proliferation. Islet graft sections from Gcgr+/+ (donor) to Gcgr+/+ (recipient; A) and Gcgr+/+ (donor) to Gcgr−/− (recipient; B) mice stained for Ki67 (red), glucagon (green), and dapi (blue). A′ and B′: Insets of A and B (white box from A, B). White arrows indicate Ki67-positive α-cells. Islet graft sections from Gcgr−/− (donor) to Gcgr+/+ (recipient; C) and Gcgr−/− (donor) to Gcgr−/− (recipient; D) mice stained for Ki67 (red), glucagon (green), and dapi (blue). C′ and D′: Insets of C and D (white box from C, D). White arrows indicate Ki67-positive α-cells. White scale bar represents 50 μm. Proliferation rate of α-cells in grafts of islets from 14-week-old Gcgr+/+ (E) or Gcgr−/− (F) mice (donors) transplanted beneath the right and left kidney capsule, respectively, of 14-week-old Gcgr+/+ (recipient) or Gcgr−/− (recipient) mice. The kidneys containing the islet graft were removed for analysis 8 weeks later (n = 3/mice, 3 sections/islet graft). Data are mean ± SEM. **P < 0.01 vs. control grafts (donor and recipient of same genotype). G: Proliferation rate of α-cells in grafts of islets from 14-week-old Gcgr+/+ mice transplanted beneath the right and left kidney capsule of 14-week-old Gcgr+/+ or Gcgr−/− mice. The kidney containing the islet graft was removed for analysis 1 week later (n = 4–6/mice, 3 sections/graft). Data are mean ± SEM. *P < 0.05 vs. Gcgr+/+ recipient grafts.

FIG. 7.

Wild-type islets transplanted into GcgrHep−/− recipients for 4 weeks have increased α-cell area and proliferation. Representative islet graft sections from Gcgr+/+ (donor) to Gcgrflox (recipient; A) and Gcgr+/+ (donor) to GcgrHep−/− (recipient; B) mice stained for insulin (green) and glucagon (red). A′ and B′: Insets of A and B (white box). C: Percentage of islet grafts that stained positive for glucagon for Gcgr+/+ donor islets into Gcgrflox (recipient, open bars) and GcgrHep−/− (recipient, closed bars) mice (n = 4 mice, 3 sections/islet graft). Percent glucagon area is defined as the percentage of glucagon area/total insulin + glucagon area. Data are mean ± SEM. *P < 0.05 vs. Gcgrflox recipient grafts. Islet grafts sections from Gcgr+/+ (donor) to Gcgrflox (recipient; D) and Gcgr+/+ (donor) to GcgrHep−/− (recipient; E) mice stained for Ki67 (red), glucagon (green), and dapi (blue). D′ and E′: Insets of D and E (white box). White arrows indicate the Ki67-positive α-cells. White scale bar represents 25 μm. F: Proliferation rate of α-cells in Gcgr+/+ islet graft (donor) transplanted into Gcgrflox (recipient, open bars) or GcgrHep−/− (recipient, closed bars) mice (n = 4 mice, 3 sections/islet graft). Data are mean ± SEM. **P < 0.01 vs. Gcgrflox recipient grafts.