PAC1 receptor-deficient mice display impaired insulinotropic response to glucose and reduced glucose tolerance

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic beta cells express two PACAP receptor subtypes, a PACAP-preferring (PAC1) and a VIP-shared (VPAC2) receptor. We have applied a gene targeting approach to create a mouse lacking the PAC1 receptor (PAC1(-/-)). These mice were viable and normoglycemic, but exhibited a slight feeding hyperinsulinemia. In vitro, in the isolated perfused pancreas, the insulin secretory response to PACAP was reduced by 50% in PAC1(-/-) mice, whereas the response to VIP was unaffected. In vivo, the insulinotropic action of PACAP was also acutely reduced, and the peptide induced impairment of glucose tolerance after an intravenous glucose injection. This demonstrates that PAC1 receptor is involved in the insulinotropic action of the peptide. Moreover, PAC1(-/-) mice exhibited reduced glucose-stimulated insulin secretion in vitro and in vivo, showing that the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose. The defective insulinotropic action of glucose was associated with marked glucose intolerance after both intravenous and gastric glucose administration. Thus, these results are consistent with a physiological role for the PAC1 receptor in glucose homeostasis, notably during food intake.

Figure 1

Genetic inactivation of the PAC1 gene. (a) Genomic organization of the mouse PAC1 receptor gene. Exons are represented by boxes, with open boxes for untranslated sequences and filled boxes for translated sequences. The numbers beneath indicate exon numbering. (b) Genomic structure of wild-type and targeted PAC1 alleles and the targeting vector. Primers (p7, p8, and pNeo) used for PCR analysis, 5′ probe used for Southern blot analysis, and expected NcoI restricted fragment sizes of the wild-type and mutated alleles are indicated. (c) Southern blot analysis of the NcoI-digested DNA from tail biopsies of wild-type (PAC1^+/+) mice, PAC1^+/– mice, and PAC1^–/– mice. (d) PCR analysis of DNA from tail biopsies of wild-type mice, PAC1^+/– mice, and PAC1^–/– mice. wt, wild-type; mut, mutant.

Figure 2

Absence of a functional PAC1 receptor. (a) Scatchard representation of ¹²⁵I-PACAP27 binding on brain membranes from wild-type, PAC1^+/–, and PAC1^–/– adult mice. (b) measurement of cAMP production in 7-day cultures of cerebellar granule cells from 8-day-old pups after stimulation with PACAP27, PACAP38, or VIP. Data are expressed as the percentage of cAMP production induced by 100 nM forskolin, and are the mean ± SEM of at least three independent experiments performed in triplicate. (c) Measurement of cAMP production in freshly isolated pancreatic islets from adult mice after 16.7 mM glucose alone (control), with PACAP38 or VIP stimulation. Data are expressed as a percentage of cAMP formation induced by glucose alone. Values are mean ± SEM from at least five independent experiments. B, bound; F, free; Kd, dissociation constant.

Figure 3

Insulin response to glucose in vitro. Effect of a glucose increase on insulin secretion from the isolated perfused pancreas of PAC1^–/– and wild-type mice. Inset shows the increment of insulin output over the first 5 minutes of glucose stimulation. Values are expressed as mean ± SEM, n = 8. ^AP < 0.001.

Figure 4

Insulin responses to PACAP and VIP in vitro. Effects of PACAP38 (a) or VIP (b) on 16.7 mmol/L glucose–induced insulin secretion from the isolated perfused pancreas of PAC1^–/– and wild-type mice. Insets show the increment of insulin output over the 20 minutes of peptide infusion. Values are expressed as mean ± SEM (n = 4–6). ^AP < 0.05.

Figure 5

Intravenous glucose tolerance test. Plasma insulin and glucose levels immediately before and after an intravenous injection of glucose (1 g/kg) or arginine (0.25 g/kg) in anesthetized wild-type mice (n = 27 in left panel; n = 7 in right panel) and PAC1^–/– mice (n = 24 in left panel; n = 7 in right panel). Insets are AUC_insulin and AUC_glucose during the 50 minutes after glucose or arginine injection in wild-type mice (open bars) and in PAC1^–/– mice (filled bars). Data presented as mean ± SEM. ^AP < 0.05; ^BP < 0.01. iv, intravenous.

Figure 6

Effects of intravenous PACAP and VIP on glucose-induced insulin secretion and glucose disappearance. Plasma insulin and glucose levels immediately before and after an intravenous injection of 1 g/kg glucose alone (n = 9–11 in a and n = 5–7 in b), glucose with 1.3 nmol/kg PACAP27 (a, n = 10–11), or glucose with 1.3 nmol/kg VIP (b, n = 6–7) in anesthetized wild-type mice and PAC1^–/– mice. Insets show AUC_insulin and AUC_glucose during the 50 minutes after administration of glucose alone (open bars), glucose with PACAP27 (filled bars in a), or glucose with VIP (filled bars in b) in wild-type and PAC1^–/– mice. Data are presented as mean ± SEM. ^AP < 0.05; ^BP < 0.01; ^CP < 0.001.

Figure 7

Gastric glucose tolerance test. Plasma insulin (a) and glucose (b) immediately before and at 10, 30, 60, and 120 minutes after gastric administration of glucose (150 mg/mouse) in 2-hour fasted anesthetized wild-type mice (n = 12) and PAC1^–/– mice (n = 12). Insets are 120-minute AUC_insulin and AUC_glucose (areas under the 120-minute insulin and glucose curves, respectively), in wild-type mice (open bars) and in PAC1^–/– mice (filled bars). Data are presented as mean ± SEM. ^AP < 0.05; ^BP < 0.01; ^CP < 0.001.