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

Pituitary adenylate cyclase–activating polypeptide (PACAP) is a ubiquitous neuropeptide of the vasoactive intestinal peptide (VIP) family that potentiates glucose-stimulated insulin secretion. Pancreatic β 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.

The physiological function of pancreatic β cells is to adapt the rate of insulin secretion to glycemia variations. Insulin release is finely tuned by a complex interplay between blood glucose (the major initiator) and incoming potentiators such as other nutrients, hormones, and neurotransmitters from the autonomic innervation (1). Peptides such as the vasoactive intestinal peptide (VIP), glucagon, and glucagon-like peptide-1 (GLP-1), belonging to the same family, participate in the physiological regulation of insulin secretion (2–4). More recently, pituitary adenylate cyclase–activating polypeptide (PACAP), a member of this family (5), has been demonstrated to potentiate the insulinotropic action of glucose in vitro both in isolated perfused rat pancreas (6–8) and in rat and mouse islets (9, 10), as well as in vivo in mice (11) and humans (12). PACAP exists mostly as a 38-residue peptide (PACAP38), but also as PACAP27, which consists of the 27 NH₂-terminal residues of PACAP38 (13). Both forms of PACAP potently stimulate insulin secretion with similar efficiency in mouse and rat islets (14). The peptide is ubiquitously distributed in central and peripheral nerve cells. PACAP-containing nerve fibers have been localized throughout the exocrine parenchyma and have been shown to innervate the intrapancreatic ganglia as well as the islets of Langerhans in mouse pancreas (15). Three types of high-affinity receptors for PACAP have been cloned: the PACAP-preferring type 1 (PAC1) and two VIP-shared type 2 (VPAC1 and VPAC2) receptors. PAC1 receptor has a much higher affinity for PACAP27 and PACAP38 than for VIP, whereas type 2 receptors recognize all peptides with equal high affinity. Both receptor subtypes are coupled to cAMP production (16–18). PACAP and VIP have been reported to be equipotent on insulin secretion in perfused rat pancreas, mouse islets, and insulinoma cells, suggesting the involvement of VPAC receptors (8, 14, 19). However, Yada et al. (9) have reported that PACAP was more potent than was VIP in stimulating insulin release from rat islets, suggesting that PACAP-selective receptors are expressed in islet β cells. Expression of mRNA encoding PAC1 and VPAC2 receptors has been detected in mouse and rat islets by in situ hybridization (14), and immunoreactivity for the PAC1 receptor has also been observed in rat islet cells (20). Together, these data indicate that PAC1 and VPAC2 receptors are expressed in insulin-producing cells. However, the relative contribution of these PACAP receptor subtypes to endocrine function remains to be established. In mice, PACAP clearly stimulates insulin secretion, but also inhibits insulin sensitivity, as demonstrated by unaltered glucose disposal during an intravenous glucose tolerance test (11).

The aim of this study was to investigate the role of PAC1 receptors in insulin secretion and glucose disposal. A mouse strain deficient in PAC1 receptor (PAC1^–/–) was generated by homologous recombination in embryonic stem cells. PAC1^–/– mice were compared with wild-type counterparts for the effects of glucose and PACAP on insulin secretion and glucose tolerance. This work was performed both in vitro in isolated perfused pancreas, and in vivo.

PAC1 receptor–deficient (PAC1^–/–) mice.

An 18-exon gene (Figure 1a) encodes the mouse PAC1 receptor (21). To produce a null allele, we exchanged sequences in the targeting vector encoding exons 8–11 with a neomycin selection cassette (Figure 1b). After homologous recombination, analysis of genomic DNA was performed using Southern blot, PCR, or both (Figure 1, c and d) to distinguish the targeted allele from the wild type allele. The targeted allele harbors the neomycin cassette in an intronic region between exons 7 and 12, which is spliced out to generate a truncated mRNA that is detectable by RT-PCR analysis (data not shown). Moreover, this deletion leads to a frame shift with a translation stop codon in exon 12. Therefore, the mutated allele was expected to encode a null receptor. This was demonstrated by several things, including the loss of 90% of the ¹²⁵I-PACAP27 binding to PAC1^–/– mouse brain (Figure 2a), a tissue that in the rat contains 10-fold more PACAP binding sites than VIP binding sites (28); and the absence of stimulation by PACAP and VIP of cAMP production in PAC1^–/– cerebellar granule cells (Figure 2b, right panel). The wild-type counterparts displayed the pharmacological profile of the PAC1 receptor mediating cAMP production (Figure 2b, left panel). Additionally, there was a dramatic reduction of PACAP38-induced cAMP production in PAC1^–/– pancreatic islets (Figure 2c), whereas the weak stimulating effect of VIP was not changed.

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.

A mendelian distribution of pup genotypes from heterozygous mating was observed at birth. However, at weaning, a loss of PAC1^–/– mice became very noticeable, with a genotype distribution of 9%, 59%, and 32% for PAC1^–/–, PAC1^+/–, and wild-type mice, respectively (data not shown). This result indicated a 60% loss of PAC1^–/– pups (χ² test, P < 0.005) during the 4 weeks after birth. This mortality remains to be explained. No significant death rate was observed thereafter.

Characteristics of PAC1^–/– mice.

This study was conducted on adult female mice 21–30 weeks of age. Table 1 shows various measurements from wild-type and PAC1^–/– mice. The latter exhibit a weak but significant decrease (∼10%) in body weight. Whereas no significant difference was observed in either plasma glucose or plasma glucagon levels, a mild degree of hyperinsulinemia was recorded in conscious, fed PAC1^–/– mice (P < 0.05). This effect was not observed in fasting conditions. Pancreatic insulin and glucagon were at comparable levels in PAC1^–/– pancreas and wild-type pancreas. Thus, the PAC1 receptor did not appear to be essential for either proinsulin or glucagon biosynthesis.

Table 1

Baseline value characteristics of conscious wild-type and PAC1^–/– mice, fed ad libitum or fasted for 24 hours

In vitro experiments

Insulin response to glucose. To investigate the glucose sensitivity of β cells in PAC1^–/– mice, we measured the effect of increasing the glucose concentration from 4.2 mmol/L to 16.7 mmol/L, on the insulin secretion from isolated perfused pancreas (Figure 3). The glucose stimulation induced an immediate and transient first phase of insulin release, followed by a constant second phase. PAC1^–/– mice displayed a defective insulin response to glucose, particularly during the first phase, when the increment of insulin output over the first 5 minutes was reduced by 55% (P < 0.001) compared with wild-type mice (Figure 3, inset).

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.

Effects of PACAP and VIP on glucose-induced insulin release. PACAP38 and VIP insulinotropic effects were evaluated from isolated pancreas perfused for 20 minutes with high glucose (Figure 4). In the presence of 16.7 mmol/L glucose, 1 nM PACAP38 potentiated insulin secretion, but this effect was about twofold lower in PAC1^–/– mice than in wild-type controls. The increment of insulin output over 20 minutes of peptide infusion was 1.887 ± 0.264% and 3.655 ± 0.646‰ (P < 0.05) of total pancreatic content in PAC1^–/– and wild-type mice, respectively (Figure 4a, inset). In contrast, 1 nM VIP elicited a comparable increase in insulin secretion in PAC1^–/– and control pancreas (3.322 ± 0.808‰ and 3.716 ± 0.740‰ of content; Figure 4b, inset). We and others (8, 29) have previously reported that PACAP and VIP exert a vasodilator effect in the rat whole pancreas by activating VPAC receptors. Both PACAP and VIP elicited a progressive and sustained increase in pancreatic flow rate in a similar manner in PAC1^–/– and wild-type pancreas (data not shown), supporting that VPAC receptors are intact in PAC1^–/– mice.

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.

In vivo experiments

Intravenous glucose tolerance test. In anesthetized PAC1^–/– mice, after an intravenous injection of glucose (1 g/kg), glucose tolerance and insulin response were significantly impaired (Figure 5, left panel). Within 1 minute, the plasma insulin response to glucose was not significantly different among the groups of mice, but the 20-minute (P < 0.05) and 50-minute (P < 0.01) insulin levels, as well as the 50-minute AUC_insulin (P < 0.05) were significantly lower in PAC1^–/– mice than in wild-type mice. Simultaneously, the glucose elimination rate was decreased in PAC1^–/– mice. Thus, the 20- (P < 0.05) and 50-minute (P < 0.01) glucose levels and the 50-minute AUC_glucose (P < 0.01) were significantly higher in PAC1^–/– mice than in wild-type mice (Figure 5, left panel, inset). The impaired insulin response to glucose was also evident when comparing the insulin and glucose data at different timepoints, because the insulinogenic index (the insulin level divided by the glucose level) was lower in PAC1^–/– mice than in wild-type mice both at 20 minutes (7.6 ± 0.3 pmol/mmol vs. 17.7 ± 1.2 pmol/mmol, P < 0.01) and 50 minutes (8.8 ± 0.6 pmol/mmol vs. 26.4 ± 1.9 pmol/mmol, P < 0.001).

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.

In contrast to the impaired insulin response to glucose, the increase in plasma insulin after intravenous administration of arginine was not significantly different between wild-type and PAC1^–/– mice (Figure 5, right panel). However, the plasma glucose levels at 20 (P < 0.05) and 50 (P < 0.01) minutes and 50-minute AUC_glucose (P < 0.01) were significantly higher in PAC1^–/– mice than in wild-type mice.

Insulin and glucose responses to intravenous PACAP and VIP during an intravenous glucose tolerance test. When PACAP27 (1.3 nmol/kg) was administered intravenously together with glucose, a marked increase of plasma insulin was observed in wild-type mice without any discernible effect on the glucose elimination rate (Figure 6a, left panel). In PAC1^–/– mice, however, PACAP27 not only potentiated glucose-induced insulin secretion just slightly, but also clearly decreased the glucose elimination rate (Figure 6a, right panel). Thus, the 50-minute AUC_glucose with PACAP27 stimulation was 0.29 ± 0.04 μmol/L in wild-type mice vs. 0.72 ± 0.05 μmol/L in PAC1^–/– mice (P < 0.001). In contrast, VIP at the same dose (1.3 nmol/kg) exerted a similar stimulatory effect on insulin secretion in PAC1^–/– and wild-type mice (Figure 6b). However, whereas VIP slightly raised the glucose elimination rate in wild-type mice (the 20-minute glucose value was 17.3 ± 1.2 mmol/L in controls vs. 13.5 ± 1.5 mmol/L after administration of VIP, P < 0.05), it did not improve the impaired glucose elimination in PAC1^–/– mice.

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.

Gastric glucose tolerance test. Administration of glucose (150 mg/mouse) through a gastric tube raised insulin and glucose plasma levels in both wild-type and PAC1^–/– mice (Figure 7). Peak insulin levels were observed at 30 minutes after glucose administration. This glucose-induced insulin increase was markedly reduced in PAC1^–/– mice, both at 10 minutes (P < 0.05) and 30 minutes (P < 0.01). The 120-minute AUCinsulin was 44% lower in PAC1^–/– mice than in wild-type mice (P < 0.001). Furthermore, PAC1^–/– mice were markedly glucose intolerant compared with wild-type mice, as judged from elevated 60-minute (P < 0.001) and 120-minute (P < 0.01) glucose levels, and a 55% higher 120-minute AUC_glucose (P < 0.01).

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.

In this study, we explored a role for the PACAP-preferring receptor, PAC1, in the regulation of insulin secretion and glucose homeostasis using mice lacking this receptor. PAC1 receptor deletion resulted in a decrease of PACAP-induced insulin secretion, clearly demonstrating the functional involvement of this receptor in the insulinotropic action of the peptide. More interestingly, these mice exhibited an impairment of glucose-induced insulin secretion and a reduction in glucose tolerance. This suggests that the presence of the PAC1 receptor is required to maintain normal insulin secretory responsiveness to glucose and glucose homeostasis.

Both PAC1 and VPAC2 receptors are expressed in mouse islets (14). These two receptor subtypes show equal affinity for PACAP, but VIP activates only the VPAC2 receptor (17). Therefore, our previous findings that PACAP27, PACAP38, and VIP were equipotent in stimulating insulin secretion (8, 14) suggested that the VPAC2 receptor had the most functional importance for the PACAP insulin secretory effect. However, in the isolated perfused pancreas of PAC1^–/– mice, the insulin secretory response to PACAP was reduced by about twofold. This decrease in responsiveness of PACAP in PAC1^–/– mice is probably not related to a change in pancreatic blood flow, because similar vasodilatory responses to PACAP were recorded in PAC1^–/– and wild-type mice. Moreover, this decrease was not associated with a reduction in pancreatic insulin content. In contrast, in PAC1^–/– mice and wild-type mice, VIP potentiated glucose-induced insulin secretion to the same extent. Therefore, these results suggest that PAC1 receptors are quite important for PACAP-induced insulin secretion, and that the remaining insulinotropic action of PACAP observed in vitro in PAC1^–/– pancreas is caused by activation of VPAC2 receptors, which are intact in these mice.

In the isolated perfused pancreas and in vivo, PAC1^–/– mice exhibited a defect in glucose-stimulated insulin release. Two recent studies have reported the inhibition of glucose-stimulated insulin secretion by immunoneutralization of PACAP in freshly isolated rat and mouse islets (10, 20), suggesting that endogenous PACAP contributes to the insulin secretory response to glucose. Therefore, our data support and extend these previous reports by providing evidence for the involvement of PAC1 receptors in the insulinotropic action of glucose. Moreover, in contrast to glucose, the insulin response to arginine was not impaired in PAC1^–/– mice, showing that the dependence of insulin secretion on the PAC1 receptor is restricted to glucose stimulation and is not a general phenomenon. Although the mechanisms explaining this dependence remain to be studied, one possible explanation could be the fact that cAMP is required for the action of glucose (30). It is indeed known that PACAP, like the intestinal hormone GLP-1, augments the formation of cAMP (19, 31) and increases the glucose competence of pancreatic β cells (32). The strong reduction of islet cAMP production by PACAP in PAC1^–/– mice might be a factor in impaired glucose sensing.

The defect in glucose-induced insulin secretion was associated with impaired glucose tolerance in PAC1^–/– mice, consistent with a role for PAC1 in the control of glucose homeostasis. However, these mice exhibit normal glycemia in both the fasted and fed states. Glucose homeostasis results from a balance between insulin secretion and peripheral tissue sensitivity to insulin, although insulin-independent mechanisms also contribute. Because the fed conscious PAC1^–/– mice had higher insulin levels than wild-type mice, it seems possible that, in addition to the defect in insulin secretion, insulin resistance may also participate in the glucose intolerance. In this context, it has been reported that PAC1 receptors are expressed in adipose tissues (33, 34), and that PACAP potentiates insulin-stimulated glucose uptake in 3T3 L1 adipocytes (34). On the other hand, a previous study in normal mice showed that during an intravenous glucose tolerance test, PACAP, despite its potent insulinotropic action, did not increase glucose elimination (11). Although the mechanism of this action remains to be elucidated, the inhibition of insulin sensitivity by PACAP was thought to be mediated by adrenaline, which is released by PACAP from the adrenal medulla and is known to inhibit insulin action. In our study, the absence of any effect of PACAP on glucose disposal was also observed in wild-type mice, and the peptide was found to induce glucose intolerance in PAC1^–/– mice. This suggests that under in vivo conditions, PACAP counteracts the action of insulin on glucose elimination. The induction of glucose intolerance by PACAP in PAC1^–/– mice may therefore result not only from the defect of the insulinotropic action of the peptide, but also from VPAC receptor activation stimulating glucagon release from pancreatic A cells (8) and hepatic glycogenolysis (35, 36), actions that counteract those of insulin. This is supported by the fact that VIP did not improve glucose tolerance despite its insulinotropic action, preserved in PAC1^–/– mice. However, it must be noted that PACAP was unable to increase plasma glucagon in the presence of high glucose levels during a glucose tolerance test (11). Taken together, these data support the view that PACAP, by activating either PAC1 or VPAC receptors, in addition to its insulinotropic action, could also regulate glucose homeostasis in a complex manner that remains to be fully elucidated.

The impaired insulin secretion and glucose tolerance in PAC1^–/– mice that was recorded after gastric administration of glucose emphasize the physiological importance of the PAC1 receptor for insulin secretion and glycemia control, and suggest its involvement during food intake. This supports a recent demonstration that the PACAP receptor antagonist, PACAP (6–27), inhibits the insulin response to gastric glucose in mice (37), and further suggests that this action is in part mediated by PAC1 receptors. It is known that during the cephalic and intestinal phases of food intake, the parasympathetic nerves supplying pancreatic islets participate in prandial insulin secretion (38). Because PACAP is known to be localized in the islet parasympathetic nerves (15) and released during vagus stimulation (39), our findings suggest that this neuropeptide, through PAC1 receptor activation, could be involved in the parasympathetic control of insulin secretion during food intake.

In conclusion, our data demonstrate, under physiological conditions, that PAC1 receptors are (a) responsible for the insulinotropic action of PACAP in addition to VPAC receptors; (b) involved in the glucose competence of pancreatic β cells; and (c) participate in the control of glucose homeostasis, notably during food intake. PAC1^–/– mice should therefore represent an animal model that may be used to precisely determine the pharmacological role of this family of PACAP/VIP receptors in the regulation of insulin secretion and glucose homeostasis. Furthermore, this may be particularly useful for predicting specific drug actions. Because PACAP is present in pancreas and stimulates insulin secretion in humans (12), it is tempting to speculate that a dysfunction of the PAC1 receptor, associated with a defect of glucose-stimulated insulin secretion and glucose intolerance, may be relevant to the development of the pathogenesis in human noninsulin-dependent diabetes mellitus.

The authors would like to thank A. Dierich and P. Chambon for providing the embryonic stem cells. We are grateful to L. Journot for his help in the project, A. Cohen-Solal, P. Minary, A. Delalbre, G. Fauconnier, L. Kvist, L. Bengtsson, and U. Gustavsson for expert technical assistance, and A. Turner-Madeuf and L. Charvet for manuscript preparation. The study was supported by the Centre National de la Recherche Scientifique, the Institut National de la Santé et la Recherche Médicale, the European Commission (BIO-98-0517), the Swedish Medical Research Council (14X-6834), E. Lundström, A. Påhlsson, the Crafoord Foundation, Novo Nordisk Foundation, the Swedish Diabetes Association, Malmö University Hospital, and the Faculty of Medicine, Lund University.

Nieves Rodriguez-Henche’s present address is: Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Alcalá de Henares, Spain.

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