Somatostatin receptor type 2 antagonism improves glucagon counterregulation in biobreeding diabetic rats

Abstract

Impaired counterregulation during hypoglycemia in type 1 diabetes (T1D) is partly attributable to inadequate glucagon secretion. Intra-islet somatostatin (SST) suppression of hypoglycemia-stimulated α-cell glucagon release plays an important role. We hypothesized that hypoglycemia can be prevented in autoimmune T1D by SST receptor type 2 (SSTR2) antagonism of α-cells, which relieve SSTR2 inhibition, thereby increasing glucagon secretion. Diabetic biobreeding diabetes-prone (BBDP) rats mimic insulin-dependent human autoimmune T1D, whereas nondiabetic BBDP rats mimic prediabetes. Diabetic and nondiabetic rats underwent a 3-h infusion of vehicle compared with SSTR2 antagonist (SSTR2a) during insulin-induced hypoglycemia clamped at 3 ± 0.5 mmol/L. Diabetic rats treated with SSTR2a needed little or no glucose infusion compared with untreated rats. We attribute this effect to SSTR2a restoration of the attenuated glucagon response. Direct effects of SSTR2a on α-cells was assessed by resecting the pancreas, which was cut into fine slices and subjected to perifusion to monitor glucagon release. SSTR2a treatment enhanced low-glucose-stimulated glucagon and corticosterone secretion to normal levels in diabetic rats. SSTR2a had similar effects in vivo in nondiabetic rats and promoted glucagon secretion from nondiabetic rat and human pancreas slices. We conclude that SST contributes to impaired glucagon responsiveness to hypoglycemia in autoimmune T1D. SSTR2a treatment can fully restore hypoglycemia-stimulated glucagon release sufficient to attain normoglycemia in both diabetic and prediabetic stages.

FIG. 1.

A: Plasma glucose levels during hyperinsulinemic hypoglycemic clamp experiments (vehicle or 1,500 nmol/kg/h SSTR2a and 20–50 mU/kg/min insulin). B: Glucose infusion rates during hyperinsulinemic hypoglycemic clamp experiments. ■, n = 8; □, n = 8; ●, n = 6; ○, n = 5. Data are presented as means ± SEM. IV, intravenous.

FIG. 2.

Plasma hormone levels during hyperinsulinemic hypoglycemic clamp experiments (vehicle or 1,500 nmol/kg/h SSTR2a and 20–50 mU/kg/min insulin) and corresponding AUCs. A: Effect of SSTR2a on glucagon response. B: Effect of SSTR2a on corticosterone response. C: Effect of SSTR2a on epinephrine response. D: Plasma SST levels. ■, n = 8; □, n = 8; ●, n = 6; ○, n = 5. AUCs were calculated using Prism software (GraphPad Software, San Diego, CA). Filled bars, n = 8; open bars, n = 8; vertically striped bars, n = 6; horizontally striped bars, n = 5. Data are presented as means ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05. IV, intravenous.

FIG. 3.

Whole pancreatic protein contents of glucagon and SST. A: Nondiabetic and diabetic BBDP rats (with and without insulin pellet implants) that have not undergone clamp experiments. Filled bars, n = 6; open bars, n = 6; horizontally striped bars, n = 5. B: Pancreases were obtained immediately after the hyperinsulinemic hypoglycemic clamp experiments. Diabetic BBDP rats had been treated with insulin pellet implants before the clamp experiments. Filled bars, n = 8; open bars, n = 8; vertically striped bars, n = 6; horizontally striped bars, n = 5. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

FIG. 4.

Effects of SSTR2a on glucagon secretion from pancreatic slices of BBDP diabetic and nondiabetic rats. Immunohistochemistry (brown, SSTR2 labeling [i]) and immunofluorescence (red, SSTR2; green, glucagon [ii] or insulin [iii]; merged images) of the localization of SSTR2 to islet α-cells in BBDP diabetic (A) and nondiabetic (B) rat pancreas slices. Insets in ii are enlarged views showing SSTR2 and glucagon colocalization. In Aiii, the few surviving β-cells in diabetic BBDP rats do not contain SSTR2, as is also true in nondiabetic rats (data not shown). Scale bar, 100 μm. Glucagon secretion from pancreatic slices of diabetic BBDP rats (n = 6) (C) and nondiabetic BBDP rats (n = 6) (D). Relative glucagon secretion was calculated as the glucagon secretion from the slices normalized to the total glucagon content of the slices. Secretion rates were calculated using Prism software (GraphPad Software, San Diego, CA). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. A, arginine; G, glucose.

FIG. 5.

Effects of SSTR2a on glucagon and insulin secretion from normal human pancreatic slices and isolated islets. A: Immunohistochemistry (brown, SSTR2 labeling [i]) and immunofluorescence (red, SSTR2; green, glucagon [ii] or insulin [iii]; merged images) of the localization of SSTR2 to islet α-cells (ii) and β-cells (iii) in normal human pancreas slices. Inset in ii is an enlarged view showing SSTR2 and glucagon colocalization. Scale bar, 100 μm. B: Relative glucagon (top) and insulin (bottom) secretion from pancreatic slices. The filled circles and empty circles represent two sets of perifusion. C: Relative glucagon secretion from isolated pancreatic islets (n = 4). Relative glucagon or insulin secretion was calculated as the glucagon/insulin secretion from the slices/islets normalized to the total glucagon/insulin content of the slices/islets. AUCs were calculated using Prism software (GraphPad Software, San Diego, CA). Data are presented as means ± SEM. **P < 0.01. A, arginine; G, glucose.