Urocortin 3 regulates glucose-stimulated insulin secretion and energy homeostasis

Abstract

Urocortin 3 (Ucn 3), a member of the corticotropin-releasing factor (CRF) family of peptides, is strongly expressed in mammalian pancreatic beta cells and has been shown to stimulate insulin secretion. Here we report the investigation of the hypothesis that endogenous Ucn 3 regulates insulin secretion, particularly in the presence of nutrient excess. Secretion of Ucn 3-like immunoreactivity from cultured beta cells was stimulated by high glucose and insulin secretagogs such as GLP-1; furthermore, 5 pancreatic Ucn 3 mRNA levels in vivo were increased during the positive energy balance caused by high-fat diet and by the absence of leptin. Immunoneutralization of Ucn 3 or pharmacologic blockade of its receptor, the type 2 CRF receptor (CRFR2), attenuated high but not low glucose-induced insulin secretion from isolated islets in vitro. Cultured islets isolated from Ucn 3-null mice also secreted less insulin in response to high glucose concentrations. Consistently, peripheral injection of a selective CRFR2 antagonist before the administration of a glucose challenge significantly attenuated glucose-induced insulin secretion in vivo. Ucn 3-null mice were relatively protected from the hyperinsulinemia, hyperglycemia, glucose intolerance, hepatic steatosis, and hypertriglyceridemia induced by high-fat diet. Additionally, we found that aged Ucn 3-null mice maintained better glucose tolerance than age-matched wild-type littermates. These results suggest that endogenous Ucn 3 in the pancreas is induced under excessive caloric conditions and acts locally to augment insulin production, which in the long-term may contribute to reduced insulin sensitivity and harmful metabolic consequences.

Fig. 1.

Elevation of pancreatic Ucn 3 expression in diabetic rodent models and stimulation of Ucn 3 release from β cells by glucose. (a) Representative agarose gel electrophoresis showing Ucn 3 and S16 PCR products. (b) Summary of Ucn 3 expression in ob/ob obese mice and WT littermates. *, P < 0.05 vs. WT. (c) Ucn 3 mRNA levels in the pancreas of high-fat-fed rats (HF) and chow-fed controls (Ctrl). *, P < 0.05 vs. Ctrl. (d) Ucn 3 secretion from MIN6 cells in response to various glucose concentrations. (e) Ucn 3 release from MIN6 cells in response to glucose and/or insulin secretagogs. Values with different superscripts are significantly different with P < 0.01.

Fig. 2.

Blocking Ucn 3 or CRFR2 attenuates high glucose-induced insulin secretion. (a) Insulin secretion from isolated rat islets induced by various concentrations of glucose with (filled bars) or without (open bars) concurrent treatment of a CRFR2 selective antagonist, Ast₂-B (100 nM). *, P < 0.05 vs. 16.8 mM glucose alone. (b) Insulin secretion induced by 16.8 mM glucose is attenuated by Ast₂-B in a dose-dependent manner. **, P < 0.01 vs. 16.8 mM glucose alone. (c) Exendin-4 (10 nM) stimulated insulin release in the presence of 8.4 mM glucose, and the effect was attenuated by Ast₂-B. *, P < 0.05 vs. exendin-4 + 8.4 mM glucose alone. (d) Anti-Ucn 3 antibody, but not normal rabbit IgG, dose-dependently attenuated 16.8 mM glucose-induced insulin secretion. *, P < 0.05; **, P < 0.01 vs. 16.8 mM glucose alone.

Fig. 3.

Blocking peripheral CRFR2 in vivo attenuates glucose-induced insulin secretion. Plasma insulin (a) and glucose (b) levels in male rats treated with vehicle or Ast₂-B (0.09–9 nmol/kg) are shown. *, P < 0.05 vs. vehicle.

Fig. 4.

Generation of Ucn 3-null mice and insulin secretion in islets isolated from Ucn 3 WT and null (KO) mice. (a) Schematic representation of Ucn 3 WT and KO allele and targeting construct. The position of the 3′ probe used in genomic Southern blot analysis is shown by a black bar. (b) Southern blot analysis of SphI-digested genomic DNA from WT, +/−, and KO mice. (c and d) In situ hybridization (c) and immunohistochemistry (d) of adult male WT and KO mouse brain sections for Ucn 3 mRNA and peptide, respectively. (Scale bar: 200 μm.) (e) Insulin secretion of islets isolated from Ucn 3-null (KO) and WT mice in response to various concentrations of glucose. *, P < 0.05 vs. WT.

Fig. 5.

Phenotypes of Ucn 3-null mice in glucose homeostasis. (a and b) Basal plasma glucose (a) and insulin (b) levels of Ucn 3 KO and WT mice after 16 weeks of HFD treatment. (c and d) Glucose (c) and insulin (d) tolerance tests in male Ucn 3 KO and WT mice (n = 8 per genotype). *, P < 0.05; **, P < 0.01 vs. WT. (e) Livers of male Ucn 3 KO and WT mice after HFD. (f) Histology of livers from Ucn 3 KO and WT mice fed HFD. (Scale bar: 40 μm.) (g) Liver triglyceride levels of Ucn 3 KO and WT mice. *, P < 0.05 vs. WT. (h) Glucose tolerance tests in Ucn 3 KO and WT mice at 10–12 weeks (young) and 12–13 months (old) of age. *, P < 0.05; **, P < 0.01 for old WT vs. old KO.