- Email: [email protected]
Functional roles of the neuropeptide PACAP in brain and pancreas
Life Sciences 74 (2003) 337 – 343 www.elsevier.com/locate/lifescie
Functional roles of the neuropeptide PACAP in brain and pancreas Norihito Shintani a, Shuhei Tomimoto a, Hitoshi Hashimoto a, Chihiro Kawaguchi a, Akemichi Baba a,b,* a
Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan b Laboratory of Molecular Pharmacology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
Abstract Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide implicated in a broad variety of physiological processes. To assess PACAP’s function in vivo, we recently generated PACAP knockout mice (PACAP / ) and transgenic mice overexpressing PACAP specifically in the pancreas (PACAP-Tg). In PACAP / mice, we have demonstrated a marked phenotypic changes including a high early mortality rate, increased novelty-seeking behavior and abnormal explosive jumping in a novel environment, as well as reduced female fertility. In this paper, we reevaluated these phenotypes in terms of the genetic background of the mice. Genetic background appears to modulate critically the magnitude but not the general nature of the PACAP-null phenotype. In PACAP-Tg mice, we have recently demonstrated that enhanced glucose-induced insulin secretion with normal glucose tolerance, amelioration of streptozotocin-induced diabetes with increased h-cell proliferation, and a trend towards an increase in total islet mass with age. Here we show that PACAP / mice exhibit significantly impaired glucose-induced insulin secretion but still have normal glucose tolerance. These observations suggest that PACAP may play important roles in and beyond the regulation of insulin release. Taken together, the mutant phenotypes revealed both expected and unexpected roles of PACAP in the brain and pancreatic functions. D 2003 Elsevier Inc. All rights reserved. Keywords: Pituitary adenylate cyclase-activating polypeptide (PACAP); Gene knockout mice; Transgenic mice; Genetic background; Psychomotor behavior; Insulin secretion
* Corresponding author. Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81-6-6879-8180; fax: +81-6-6879-8184. E-mail address: [email protected] (A. Baba). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.021
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Introduction PACAP is 27- or 38-amino acid neuropeptide that was first isolated from ovine hypothalamic extracts on the basis of its ability to stimulate cyclic AMP formation in anterior pituitary cells (Arimura, 1998; Miyata et al., 1989). PACAP belongs to the VIP/secretin/glucagon family of neuropeptides and is distributed not only in the central nervous system, including the hypothalamus, hippocampus and many other brain regions, but also in peripheral tissues such as the testis, adrenal medulla, pancreatic ganglia, and enteric nerves (Arimura, 1998; Vaudry et al., 2000). PACAP-mediated signaling has been implicated in a broad variety of physiological processes including neuronal differentiation, hypophysiotropic functions, synaptic plasticity, fertility, circadian photoentrainment, and insulin release (Arimura, 1998; Hashimoto et al., 2002; Vaudry et al., 2000). These pleiotropic functions of PACAP are mediated through three hepthahelical G-protein-linked receptors, PACAP-preferring type 1 (PAC1) and VIP-shared type 2 (VPAC1 and VPAC2) receptors. However, the physiological and pathophysiological roles of PACAP as well as those of each receptor subtype have not been fully addressed because specific low-molecular-weight ligands have not been developed (Vaudry et al., 2000). We previously cloned PAC1 receptor cDNA (Hashimoto et al., 1996a) and the genes for PAC1 receptor (Aino et al., 1995), VPAC1 receptor (Hashimoto et al., 1999) and PACAP (Yamamoto et al., 1998) and analyzed their localization in the nervous system (Hashimoto et al., 1996b; Nogi et al., 1997a,b). Recently we have generated several lines of mutant mice which lack PAC1 receptor exon 2 (Hashimoto et al., 2000) or PACAP (Hashimoto et al., 2001), and which overexpressing PACAP specifically in the pancreas (Yamamoto et al., 2003). In this paper the phenotypic alterations in the mutant mice are reviewed based on the results obtained in our laboratory (Hashimoto et al., 2001; Shintani et al., 2002; Yamamoto et al., 2003), mainly with regard to the genetic background of the mice. In addition, we describe here impaired glucose-induced insulin secretion in PACAP / mice, and also discuss the roles of PACAP signaling in glucose homeostasis. Methods All animal care and handling procedures were approved by the institutional animal care and use committee of Osaka University. PACAP / mice were generated by gene targeting (Hashimoto et al., 2001). The intraperitoneal glucose tolerance test was conducted with F2-F3 generation PACAP / mice on a mixed genetic background of C57BL/6 and 129/Ola. Mice were fasted for 14 h with free access to water. Each mouse was then injected intraperitoneally with glucose (1 mg/g body weight), and blood samples were taken from the tail vein before glucose injection and at identical time points after the injection. The plasma glucose and insulin concentrations were measured as described previously (Yamamoto et al., 2003). Results and discussion Role of PACAP in brain function PACAP as well as PAC1 receptor have been shown to be expressed predominantly in the central nervous system (Arimura, 1998; Hashimoto et al., 1996b; Nogi et al., 1997a,b; Vaudry et
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al., 2000). To examine whether PACAP–PAC1 signaling is involved in specific brain functions, we recently generated mice lacking PAC1 exon 2 (Hashimoto et al., 2000) or PACAP (Hashimoto et al., 2001) and unveiled a previously unknown roles of PACAP in brain function (the regulation of psychomotor behaviors). PACAP / mice display remarkable behavioral changes including locomotor hyperactivity and explosive jumping behaviors in an open field, increased exploratory behavior and less anxiety in the elevated plus maze, emergence, and novel-object tests (Hashimoto et al., 2001, 2002). The hyperactivity of PACAP / mice may be related to altered habituation to novelty. Indeed, PACAP / mice show minimal habituation to the novel open-field over a 60 min period, by which time wild-type mice are significantly habituated, and exhibit hyperactivity (increased explorative behavior) specifically in response to novel stimuli in a novel object test (Hashimoto et al., 2001). PACAP / mice thus exhibit altered psychiatric brain function. Recently, we also demonstrated reduced fertility in female PACAP / mice, partly due to reduced mating frequency (Shintani et al., 2002). PACAP is known to stimulate release of several sexual hormones from pituitary (Arimura, 1998; Vaudry et al., 2000), and these reproductive defects might also result from abnormalities in brain neuroendocrine function. Interactions between the effects of multiple genes are responsible for determining phenotype in animals and are likely to be major contributors to disease susceptibility in humans. We thus evaluated the contribution of genetic background to the knockout phenotypes; the null allele of PACAP was backcrossed onto an ICR background. Results are summarized in Table 1. Less than 50% of PACAP / mice are viable at postnatal day 21 in mixed C57BL/6 129/Ola background. However, this high early mortality rate tends to be normalized in the ICR background: approximately 50–60% of PACAP / mice survived, and in specific environmental conditions, nearly 100% of PACAP / mice survived until at least 12 weeks-old (unpublished observation). Fertility and litter size of PACAP / female mice are also affected by their genetic background (Shintani et al., 2002). However, the psychomotor abnormalities, especially jumping behavior, are prominent in PACAP / mice regardless of genetic background (Hashimoto et al., 2001). These results further suggest the importance of PACAP especially in the psychiatric function and imply the possibility of genetic interactions between PACAP and its modifier genes. In wild-type females, ICR mice have a larger litter size (about 1.4 fold) compared with those in mixed C57BL/6 129/Ola background (Shintani et al., 2002). Thus, these observations indicate that greater numbers of PACAP / mice can be obtained in the ICR background compared with mixed C57BL/6 129/Ola background, because of the larger litter size (about 1.4 fold) and improved postnatal mortality (1–2 fold) in the ICR background. Role of PACAP in pancreatic function In the pancreas, PACAP as low as 10 13 M stimulates insulin secretion from rat isolated islets in a glucose-dependent manner, indicating that PACAP has the most potent insulinotropic effect of the factors so far investigated (Yada et al., 1994). Daily intraperitoneal injections of PACAP have been shown to reduce circulating glycemia in diabetic GK (Goto-Kakizaki) rats and high-fat-fed mice with impaired glucose tolerance (Yada et al., 2000). To understand the in vivo function of PACAP in pancreatic islet h-cells, we recently generated transgenic mice overexpressing PACAP in pancreas (PACAP-Tg), specifically under the control of the h-cell-specific insulin promoter. In PACAP-Tg
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Table 1 Phenotypic differences between adult PACAP-mutants Mutants
PACAP /
Genetic background
ICR
Mixed C57BL/6 129/Ola
C57BL/6
Postnatal mortality
# (!)
###a
!
Psychomotor function Activity in an open-field Number of jumps in an open-field
za zzza
zzza zzza
! N.D.
Female reproductive function Fertilityd Litter size
##b #b
###b !b
N.D. N.D.
Pancreatic (metabolic) function Serum glucose level on fed Serum insulin level on fed Serum triglycerid level on fed Glucose disposal on GTT Insulin secretion on GTT STZ-induced diabetes Age-related changes
! # ! z N.D. ! N.D.
N.D. N.D. N.D. ! # N.D. N.D.
!c !c N.D. !c zc # (ameliorated)c Increase in h-cell massc
PACAP-Tg
N.D. not determined. a Ref. Hashimoto et al., 2001. b Ref. Shintani et al., 2002. c Ref. Yamamoto et al., 2003. d Fertility was determined from the ratio of the number of parturitions relative to the number of pairings in which the male and female were together for 7 days.
mice, plasma glucose and glucagon levels during a glucose tolerance test are not different from those in nontransgenic littermates; however, plasma insulin levels in PACAP-Tg mice are slightly higher after glucose loading (Yamamoto et al., 2003). In this paper, we analyzed the glucose-stimulated insulin secretion in PACAP / mice on mixed C57BL/6 129/Ola to determine the function of endogenous PACAP in the pancreas. An intraperitoneal glucose tolerance test showed no difference in plasma glucose levels between PACAP / mice and their littermate wild-types at all time points (Fig. 1A). However, PACAP / mice showed lower plasma insulin level in response to glucose injection (Fig. 1B). As summarized in Table 1, results obtained from PACAP / as well as PACAP-Tg mice indicate that PACAP is required for insulin secretory responsiveness to glucose in vivo; however, the reason why the plasma glucose levels were not different between PACAP / mice, PACAP-Tg mice, and the wild-types is currently unknown. Using PACAP-Tg mice, recently we also reported that the PACAP transgene ameliorates streptozotocin (STZ)-induced increase in plasma glucose and increases 5-bromo-2-deoxyuridine-positive h-cells in the STZ-treated mice (Yamamoto et al., 2003). In addition, morphometric analysis revealed that total islet mass tends to increase in 12-month-old PACAP-Tg mice (Yamamoto et al., 2003). These results suggest that PACAP may play an important role in the proliferation of h-cells. Future studies using
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Fig. 1. Intraperitoneal glucose tolerance tests of PACAP / mice. Blood samples were taken from the tail vain of PACAP / mice (closed circles; n = 9) and littermate wild-types (open circles; n = 12) immediately before and at 15, 30, 60, 90, 120 min after an intraperitoneal injection of glucose (1 mg/g body weight). The samples were centrifuged, and plasma glucose (A) and insulin (B) concentrations were measured. Values are expressed as means F SE. Statistical significance of differences was assessed by ANOVA with repeated measure analysis and student’s t-test.
PACAP / mice will provide further insight into the involvement of PACAP signaling in the growth and differentiation (neogenesis) of h-cells. Phenotypic comparison with other PACAP-targeting mice lines As mentioned above, we recently demonstrated that endogenous PACAP is implicated in psychomotor function, female reproductive function, and insulinotropic response. Two different PAC1 receptor-deficient mouse strains have recently been developed separately from our colony (Otto et al., 2001a; Jamen et al., 2000a), and reported to exhibit locomotor hyperactivity in an open field (Otto et al., 2001b), reduced female fertility (Jamen et al., 2000b), and impaired glucoseinduced insulin release (Jamen et al., 2000a). In parallel to our study, two different PACAP / mouse strains have also been reported (Gray et al., 2001; Hamelink et al., 2002). Gray et al. reported an extremely high mortality rate (only 9% of PACAP / mouse survives until postnatal day 12) associated with dysfunction of lipid and carbohydrate metabolism, including higher serum ketones, triglycerides and cholesterol. Serum corticosterone levels of their PACAP / mice showed two distinct populations; some mice have normal levels, whereas others have extremely high levels (Gray et al., 2001). Hamelink et al. revealed that PACAP is a neurotransmitter involved in catecholamine regulation and glucose homeostasis. Serum corticosterone levels of PACAP / mice were almost identical to that of wild-type mice (Hamelink et al., 2002). Thus, the variable phenotypic expression has been shown especially in peripheral metabolic parameters (Table 1; Gray et al., 2001, Hamelink et al., 2002). It has been reported that early mortality rate of PACAP / mice was markedly affected by breeding temperature (Gray et al., 2003). The phenotypic expression due to lack of the PACAP gene may be highly affected by the genetic and/or environmental backgrounds.
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Conclusion The results from PACAP / mice put forward previously uncharacterized roles for PACAP in the regulation of brain psychomotor functions and in pancreatic h-cell proliferation and/or differentiation. Genetic background appears to modulate critically the magnitude but not the general nature of the PACAP-null phenotype, although definitive conclusions await further study. Finally, environmental factors will need to be carefully considered in the phenotypic expression of PACAP / mice.
Acknowledgements We would like to thank Tatsuya Ojika and Kazuhiro Tanaka for expert technical assistance. This research was supported, in part, by Grant-in-Aid for Scientific Research and Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by grants from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, Taisho Pharmaceutical Co. Ltd., AstraZeneca, and The Naito Foundation.
References Aino, H., Hashimoto, H., Ogawa, N., Nishino, A., Yamamoto, K., Nogi, H., Nagata, S., Baba, A., 1995. Structure of the gene encoding the mouse pituitary adenylate cyclase-activating polypeptide receptor. Gene 164 (2), 301 – 304. Arimura, A., 1998. Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems. Japanese Journal of Physiology 48, 301 – 331. Gray, S.L., Cummings, K.J., Jirik, F.R., Sherwood, N.M., 2001. Targeted disruption of the pituitary adenylate cyclase-activating polypeptide gene results in early postnatal death associated with dysfunction of lipid and carbohydrate metabolism. Molecular Endocrinology 15, 1739 – 1747. Gray, S.L., Yamaguchi, N., Vencova, P., Sherwood, N.M., 2003. Temperature-sensitive phenotype in mice lacking pituitary adenylate cyclase-activating polypeptide. Endocrinology 143 (10), 3946 – 3954. Hamelink, C., Tjurmina, O., Damadzic, R., Young, W.S., Weihe, E., Lee, H.W., Eiden, L.E., 2002. Pituitary adenylate cyclaseactivating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation. Proceedings of the National Academy of Sciences of the United States of America 98, 13355 – 13360. Hashimoto, H., Yamamoto, K., Hagigara, N., Ogawa, N., Nishino, A., Aino, H., Nogi, H., Imanishi, K., Matsuda, T., Baba, A., 1996a. cDNA cloning of a mouse pituitary adenylate cyclase-activating polypeptide receptor. Biochimica et Biophysica Acta 1281 (2), 129 – 133. Hashimoto, H., Nogi, H., Mori, K., Ohishi, H., Shigemoto, R., Yamamoto, K., Matsuda, T., Mizuno, N., Nagata, S., Baba, A., 1996b. Distribution of the mRNA for a pituitary adenylate cyclase-activating polypeptide receptor in the rat brain: an in situ hybridization study. Journal of Comparative Neurology 371 (4), 567 – 577. Hashimoto, H., Nishino, A., Shintani, N., Hagihara, N., Copeland, N.G., Jenkins, N.A., Yamamoto, K., Matsuda, T., Ishihara, T., Nagata, S., Baba, A., 1999. Genomic organization and chromosomal location of the mouse vasoactive intestinal polypeptide 1 (VPAC1) receptor. Genomics 58 (1), 90 – 93. Hashimoto, H., Shintani, N., Nishino, A., Okabe, M., Ikawa, M., Matsuyama, S., Itoh, K., Yamamoto, K., Tomimoto, S., Fujita, T., Hagihara, N., Mori, W., Koyama, Y., Matsuda, T., Nagata, S., Baba, A., 2000. Mice with markedly reduced PACAP (PAC(1)) receptor expression by targeted deletion of the signal peptide. Journal of Neurochemistry 75, 1810 – 1817. Hashimoto, H., Shintani, N., Tanaka, K., Mori, W., Hirose, M., Matsuda, T., Sakaue, M., Miyazaki, J., Niwa, H., Tashiro, F., Yamamoto, K., Koga, K., Tomimoto, S., Kunugi, A., Suetake, S., Baba, A., 2001. Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP). Proceedings of the National Academy of Sciences of the United States of America 98, 13355 – 13360.
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Hashimoto, H., Shintani, N., Baba, A., 2002. Higher brain functions of PACAP and a homologous Drosophila memory gene amnesiac; insights from knockouts and mutants. Biochemical and biophysical research communications 297, 427 – 432. Jamen, F., Persson, K., Bertrand, G., Rodriguez-Henche, N., Puech, R., Bockaert, J., Ahren, B., Brabet, P., 2000a. PAC1 receptor-deficient mice display impaired insulinotropic response to glucose and reduced glucose tolerance. Journal of Clinical Investigation 105, 1307 – 1315. Jamen, F., Rodriguez-Henche, N., Pralong, F., Jegou, B., Gaillard, R., Bockaert, J., Brabet, P., 2000b. PAC1 null females display decreased fertility. Annals of the New York Academy of Sciences 921, 400 – 404. Miyata, A., Jian, L., Dahl, R.R., Minamino, N., Uehara, A., Jiang, L., Culler, M.D., Coy, D.H., 1989. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochemical and biophysical research communications 164, 567 – 574. Nogi, H., Hashimoto, H., Hagihara, N., Shimada, S., Yamamoto, K., Matsuda, T., Tohyama, M., Baba, A., 1997a. Distribution of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP receptor, vasoactive intestinal polypeptide (VIP), and VIP receptors in the rat superior cervical ganglion. Neuroscience Letters 227 (1), 37 – 40. Nogi, H., Hashimoto, H., Fujita, T., Hagihara, N., Matsuda, T., Baba, A., 1997b. Pituitary adenylate cyclase-activating polypeptide (PACAP) receptor mRNA in the rat adrenal gland: localization by in situ hybridization and identification of splice variants. Japanese Journal of Pharmacology 75 (2), 203 – 207. Otto, C., Kovalchuk, Y., Wolfer, D.P., Gass, P., Martin, M., Zuschratter, W., Grone, H.J., Kellendonk, C., Tronche, F., Maldonado, R., Lipp, H.P., Konnerth, A., Schutz, G., 2001a. Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient mice. Journal of Neuroscience 21 (15), 5520 – 5527. Otto, C., Martin, M., Wolfer, D.P., Lipp, H.P., Maldonado, R., Schutz, G., 2001b. Altered emotional behavior in PACAP-type-Ireceptor-deficient mice. Brain research. Molecular brain research 92 (1 – 2), 78 – 84. Shintani, N., Mori, W., Hashimoto, H., Imai, M., Tanaka, K., Tomimoto, S., Hirose, M., Kawaguchi, C., Baba, A., 2002. Defects in reproductive functions in PACAP-deficient female mice. Regulatory peptides 109 (1 – 3), 45 – 48. Yada, T., Sakurada, M., Ihida, K., Nakata, M., Murata, F., Arimura, A., Kikuchi, M., 1994. Pituitary adenylate cyclase activating polypeptide is an extraordinarily potent intra-pancreatic regulator of insulin secretion from islet beta-cells. Journal of biological chemistry 269, 1290 – 1293. Yada, T., Sakurada, M., Filipsson, K., Kikuchi, M., Ahren, B., 2000. Intraperitoneal PACAP administration decreases blood glucose in GK rats, and in normal and high fat diet mice. Annals of the New York Academy of Sciences 921, 259 – 263. Yamamoto, K., Hashimoto, H., Hagihara, N., Nishino, A., Fujita, T., Matsuda, T., Baba, A., 1998. Cloning and characterization of the mouse pituitary adenylate cyclase-activating polypeptide (PACAP) gene. Gene 211 (1), 63 – 69. Yamamoto, K., Hashimoto, H., Tomimoto, S., Shintani, N., Miyazaki, J., Tashiro, F., Aihara, H., Fukuyama, Y., Koga, K., Mori, W., Tanaka, K., Matsuda, T., Baba, A., 2003. Overexpression of PACAP in transgenic mouse pancreatic beta-cells enhances insulin secretion and ameliorates streptozotocin-induced diabetes. Diabetes 52 (5), 1155 – 1162. Vaudry, D., Gonzalez, B.J., Basille, M., Yon, L., Fournier, A., Vaudry, H., 2000. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Pharmacological reviews 52, 269 – 324.
Functional roles of the neuropeptide PACAP in brain and pancreas Norihito Shintani a, Shuhei Tomimoto a, Hitoshi Hashimoto a, Chihiro Kawaguchi a, Akemichi Baba a,b,* a
Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan b Laboratory of Molecular Pharmacology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan
Abstract Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide implicated in a broad variety of physiological processes. To assess PACAP’s function in vivo, we recently generated PACAP knockout mice (PACAP / ) and transgenic mice overexpressing PACAP specifically in the pancreas (PACAP-Tg). In PACAP / mice, we have demonstrated a marked phenotypic changes including a high early mortality rate, increased novelty-seeking behavior and abnormal explosive jumping in a novel environment, as well as reduced female fertility. In this paper, we reevaluated these phenotypes in terms of the genetic background of the mice. Genetic background appears to modulate critically the magnitude but not the general nature of the PACAP-null phenotype. In PACAP-Tg mice, we have recently demonstrated that enhanced glucose-induced insulin secretion with normal glucose tolerance, amelioration of streptozotocin-induced diabetes with increased h-cell proliferation, and a trend towards an increase in total islet mass with age. Here we show that PACAP / mice exhibit significantly impaired glucose-induced insulin secretion but still have normal glucose tolerance. These observations suggest that PACAP may play important roles in and beyond the regulation of insulin release. Taken together, the mutant phenotypes revealed both expected and unexpected roles of PACAP in the brain and pancreatic functions. D 2003 Elsevier Inc. All rights reserved. Keywords: Pituitary adenylate cyclase-activating polypeptide (PACAP); Gene knockout mice; Transgenic mice; Genetic background; Psychomotor behavior; Insulin secretion
* Corresponding author. Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: +81-6-6879-8180; fax: +81-6-6879-8184. E-mail address: [email protected] (A. Baba). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.021
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Introduction PACAP is 27- or 38-amino acid neuropeptide that was first isolated from ovine hypothalamic extracts on the basis of its ability to stimulate cyclic AMP formation in anterior pituitary cells (Arimura, 1998; Miyata et al., 1989). PACAP belongs to the VIP/secretin/glucagon family of neuropeptides and is distributed not only in the central nervous system, including the hypothalamus, hippocampus and many other brain regions, but also in peripheral tissues such as the testis, adrenal medulla, pancreatic ganglia, and enteric nerves (Arimura, 1998; Vaudry et al., 2000). PACAP-mediated signaling has been implicated in a broad variety of physiological processes including neuronal differentiation, hypophysiotropic functions, synaptic plasticity, fertility, circadian photoentrainment, and insulin release (Arimura, 1998; Hashimoto et al., 2002; Vaudry et al., 2000). These pleiotropic functions of PACAP are mediated through three hepthahelical G-protein-linked receptors, PACAP-preferring type 1 (PAC1) and VIP-shared type 2 (VPAC1 and VPAC2) receptors. However, the physiological and pathophysiological roles of PACAP as well as those of each receptor subtype have not been fully addressed because specific low-molecular-weight ligands have not been developed (Vaudry et al., 2000). We previously cloned PAC1 receptor cDNA (Hashimoto et al., 1996a) and the genes for PAC1 receptor (Aino et al., 1995), VPAC1 receptor (Hashimoto et al., 1999) and PACAP (Yamamoto et al., 1998) and analyzed their localization in the nervous system (Hashimoto et al., 1996b; Nogi et al., 1997a,b). Recently we have generated several lines of mutant mice which lack PAC1 receptor exon 2 (Hashimoto et al., 2000) or PACAP (Hashimoto et al., 2001), and which overexpressing PACAP specifically in the pancreas (Yamamoto et al., 2003). In this paper the phenotypic alterations in the mutant mice are reviewed based on the results obtained in our laboratory (Hashimoto et al., 2001; Shintani et al., 2002; Yamamoto et al., 2003), mainly with regard to the genetic background of the mice. In addition, we describe here impaired glucose-induced insulin secretion in PACAP / mice, and also discuss the roles of PACAP signaling in glucose homeostasis. Methods All animal care and handling procedures were approved by the institutional animal care and use committee of Osaka University. PACAP / mice were generated by gene targeting (Hashimoto et al., 2001). The intraperitoneal glucose tolerance test was conducted with F2-F3 generation PACAP / mice on a mixed genetic background of C57BL/6 and 129/Ola. Mice were fasted for 14 h with free access to water. Each mouse was then injected intraperitoneally with glucose (1 mg/g body weight), and blood samples were taken from the tail vein before glucose injection and at identical time points after the injection. The plasma glucose and insulin concentrations were measured as described previously (Yamamoto et al., 2003). Results and discussion Role of PACAP in brain function PACAP as well as PAC1 receptor have been shown to be expressed predominantly in the central nervous system (Arimura, 1998; Hashimoto et al., 1996b; Nogi et al., 1997a,b; Vaudry et
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al., 2000). To examine whether PACAP–PAC1 signaling is involved in specific brain functions, we recently generated mice lacking PAC1 exon 2 (Hashimoto et al., 2000) or PACAP (Hashimoto et al., 2001) and unveiled a previously unknown roles of PACAP in brain function (the regulation of psychomotor behaviors). PACAP / mice display remarkable behavioral changes including locomotor hyperactivity and explosive jumping behaviors in an open field, increased exploratory behavior and less anxiety in the elevated plus maze, emergence, and novel-object tests (Hashimoto et al., 2001, 2002). The hyperactivity of PACAP / mice may be related to altered habituation to novelty. Indeed, PACAP / mice show minimal habituation to the novel open-field over a 60 min period, by which time wild-type mice are significantly habituated, and exhibit hyperactivity (increased explorative behavior) specifically in response to novel stimuli in a novel object test (Hashimoto et al., 2001). PACAP / mice thus exhibit altered psychiatric brain function. Recently, we also demonstrated reduced fertility in female PACAP / mice, partly due to reduced mating frequency (Shintani et al., 2002). PACAP is known to stimulate release of several sexual hormones from pituitary (Arimura, 1998; Vaudry et al., 2000), and these reproductive defects might also result from abnormalities in brain neuroendocrine function. Interactions between the effects of multiple genes are responsible for determining phenotype in animals and are likely to be major contributors to disease susceptibility in humans. We thus evaluated the contribution of genetic background to the knockout phenotypes; the null allele of PACAP was backcrossed onto an ICR background. Results are summarized in Table 1. Less than 50% of PACAP / mice are viable at postnatal day 21 in mixed C57BL/6 129/Ola background. However, this high early mortality rate tends to be normalized in the ICR background: approximately 50–60% of PACAP / mice survived, and in specific environmental conditions, nearly 100% of PACAP / mice survived until at least 12 weeks-old (unpublished observation). Fertility and litter size of PACAP / female mice are also affected by their genetic background (Shintani et al., 2002). However, the psychomotor abnormalities, especially jumping behavior, are prominent in PACAP / mice regardless of genetic background (Hashimoto et al., 2001). These results further suggest the importance of PACAP especially in the psychiatric function and imply the possibility of genetic interactions between PACAP and its modifier genes. In wild-type females, ICR mice have a larger litter size (about 1.4 fold) compared with those in mixed C57BL/6 129/Ola background (Shintani et al., 2002). Thus, these observations indicate that greater numbers of PACAP / mice can be obtained in the ICR background compared with mixed C57BL/6 129/Ola background, because of the larger litter size (about 1.4 fold) and improved postnatal mortality (1–2 fold) in the ICR background. Role of PACAP in pancreatic function In the pancreas, PACAP as low as 10 13 M stimulates insulin secretion from rat isolated islets in a glucose-dependent manner, indicating that PACAP has the most potent insulinotropic effect of the factors so far investigated (Yada et al., 1994). Daily intraperitoneal injections of PACAP have been shown to reduce circulating glycemia in diabetic GK (Goto-Kakizaki) rats and high-fat-fed mice with impaired glucose tolerance (Yada et al., 2000). To understand the in vivo function of PACAP in pancreatic islet h-cells, we recently generated transgenic mice overexpressing PACAP in pancreas (PACAP-Tg), specifically under the control of the h-cell-specific insulin promoter. In PACAP-Tg
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Table 1 Phenotypic differences between adult PACAP-mutants Mutants
PACAP /
Genetic background
ICR
Mixed C57BL/6 129/Ola
C57BL/6
Postnatal mortality
# (!)
###a
!
Psychomotor function Activity in an open-field Number of jumps in an open-field
za zzza
zzza zzza
! N.D.
Female reproductive function Fertilityd Litter size
##b #b
###b !b
N.D. N.D.
Pancreatic (metabolic) function Serum glucose level on fed Serum insulin level on fed Serum triglycerid level on fed Glucose disposal on GTT Insulin secretion on GTT STZ-induced diabetes Age-related changes
! # ! z N.D. ! N.D.
N.D. N.D. N.D. ! # N.D. N.D.
!c !c N.D. !c zc # (ameliorated)c Increase in h-cell massc
PACAP-Tg
N.D. not determined. a Ref. Hashimoto et al., 2001. b Ref. Shintani et al., 2002. c Ref. Yamamoto et al., 2003. d Fertility was determined from the ratio of the number of parturitions relative to the number of pairings in which the male and female were together for 7 days.
mice, plasma glucose and glucagon levels during a glucose tolerance test are not different from those in nontransgenic littermates; however, plasma insulin levels in PACAP-Tg mice are slightly higher after glucose loading (Yamamoto et al., 2003). In this paper, we analyzed the glucose-stimulated insulin secretion in PACAP / mice on mixed C57BL/6 129/Ola to determine the function of endogenous PACAP in the pancreas. An intraperitoneal glucose tolerance test showed no difference in plasma glucose levels between PACAP / mice and their littermate wild-types at all time points (Fig. 1A). However, PACAP / mice showed lower plasma insulin level in response to glucose injection (Fig. 1B). As summarized in Table 1, results obtained from PACAP / as well as PACAP-Tg mice indicate that PACAP is required for insulin secretory responsiveness to glucose in vivo; however, the reason why the plasma glucose levels were not different between PACAP / mice, PACAP-Tg mice, and the wild-types is currently unknown. Using PACAP-Tg mice, recently we also reported that the PACAP transgene ameliorates streptozotocin (STZ)-induced increase in plasma glucose and increases 5-bromo-2-deoxyuridine-positive h-cells in the STZ-treated mice (Yamamoto et al., 2003). In addition, morphometric analysis revealed that total islet mass tends to increase in 12-month-old PACAP-Tg mice (Yamamoto et al., 2003). These results suggest that PACAP may play an important role in the proliferation of h-cells. Future studies using
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Fig. 1. Intraperitoneal glucose tolerance tests of PACAP / mice. Blood samples were taken from the tail vain of PACAP / mice (closed circles; n = 9) and littermate wild-types (open circles; n = 12) immediately before and at 15, 30, 60, 90, 120 min after an intraperitoneal injection of glucose (1 mg/g body weight). The samples were centrifuged, and plasma glucose (A) and insulin (B) concentrations were measured. Values are expressed as means F SE. Statistical significance of differences was assessed by ANOVA with repeated measure analysis and student’s t-test.
PACAP / mice will provide further insight into the involvement of PACAP signaling in the growth and differentiation (neogenesis) of h-cells. Phenotypic comparison with other PACAP-targeting mice lines As mentioned above, we recently demonstrated that endogenous PACAP is implicated in psychomotor function, female reproductive function, and insulinotropic response. Two different PAC1 receptor-deficient mouse strains have recently been developed separately from our colony (Otto et al., 2001a; Jamen et al., 2000a), and reported to exhibit locomotor hyperactivity in an open field (Otto et al., 2001b), reduced female fertility (Jamen et al., 2000b), and impaired glucoseinduced insulin release (Jamen et al., 2000a). In parallel to our study, two different PACAP / mouse strains have also been reported (Gray et al., 2001; Hamelink et al., 2002). Gray et al. reported an extremely high mortality rate (only 9% of PACAP / mouse survives until postnatal day 12) associated with dysfunction of lipid and carbohydrate metabolism, including higher serum ketones, triglycerides and cholesterol. Serum corticosterone levels of their PACAP / mice showed two distinct populations; some mice have normal levels, whereas others have extremely high levels (Gray et al., 2001). Hamelink et al. revealed that PACAP is a neurotransmitter involved in catecholamine regulation and glucose homeostasis. Serum corticosterone levels of PACAP / mice were almost identical to that of wild-type mice (Hamelink et al., 2002). Thus, the variable phenotypic expression has been shown especially in peripheral metabolic parameters (Table 1; Gray et al., 2001, Hamelink et al., 2002). It has been reported that early mortality rate of PACAP / mice was markedly affected by breeding temperature (Gray et al., 2003). The phenotypic expression due to lack of the PACAP gene may be highly affected by the genetic and/or environmental backgrounds.
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Conclusion The results from PACAP / mice put forward previously uncharacterized roles for PACAP in the regulation of brain psychomotor functions and in pancreatic h-cell proliferation and/or differentiation. Genetic background appears to modulate critically the magnitude but not the general nature of the PACAP-null phenotype, although definitive conclusions await further study. Finally, environmental factors will need to be carefully considered in the phenotypic expression of PACAP / mice.
Acknowledgements We would like to thank Tatsuya Ojika and Kazuhiro Tanaka for expert technical assistance. This research was supported, in part, by Grant-in-Aid for Scientific Research and Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by grants from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, Taisho Pharmaceutical Co. Ltd., AstraZeneca, and The Naito Foundation.
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