Mice Deficient in both Pituitary Adenylyl Cyclase-activating Polypeptide and Vasoactive Intestinal Peptide Survive, but Display Growth Retardation and Sex-dependent Early Death
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- Niewiadomski, P., Coûté-Monvoisin, A., Abad, C. et al. J Mol Neurosci (2008) 36: 200. doi:10.1007/s12031-008-9085-3
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Pituitary adenylyl cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two closely related neuropeptides exhibiting overlapping activities which have actions on almost every organ system of the body. To determine if these peptides exert essential but redundant functions, we interbred VIP- and PACAP-deficient mice to obtain VIP/PACAP double knockout (DKO) mice. DKO mice had normal birth weights and survived to weaning, but exhibited a dramatic postnatal growth rate reduction. Analyses at postnatal day 16 indicated that all organs examined except the brain were reduced in mass by 40–70% compared to mixed background controls, with the thymus and spleen most profoundly affected. Brain size was also significantly reduced, but by only 10%. The reduced growth rate of DKO mice was associated with reduced serum concentrations of insulin-like growth hormone-1 (IGF-1), but unchanged levels of growth hormone. Despite the normal survival of DKO mice up to the weaning stage, many subsequently experienced early sudden death, with only 48% of females and 82% of males surviving past 6 months. The results indicate that a significant percentage of mice deficient in both VIP and PACAP survive to adulthood, but their growth rate is profoundly affected, and that females in particular exhibit high rate of mortality after about 3 months of age.
KeywordsPituitary adenylyl cyclase-activating polypeptidePACAPVasoactive intestinal peptideVIPKnockoutGrowthPostnatalDwarfIGF-1Growth hormone
Pituitary adenylyl cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two closely related neuropeptides in the secretin family (reviewed in Vaudry et al. 2000). VIP and PACAP exist primarily as 28 and 38 amino acid polypeptides, respectively, are 68% identical in the first 28 residues, and exhibit similar high affinity to two mammalian receptor subtypes: VPAC1 and VPAC2. In addition, a closely related receptor, PAC1, preferentially binds PACAP with high affinity, except for a single splice variant which also binds VIP (Lutz et al. 2006). All three receptor subtypes are coupled primarily to Gs-type heterotrimeric G-proteins, and therefore induce cAMP production upon ligand binding. However, there is evidence that other intracellular pathways utilizing PLCβ-PKC, MAP kinase, intracellular Ca2+, and other signaling cascades are in some cases also activated by these peptides. PACAP and VIP have been shown to play important roles in a variety of biological processes, such as inflammation, intestinal function, nervous system development and function, circadian rhythms, and metabolic regulation (reviewed in Delgado et al. 2004; Vaudry et al. 2000). Recent studies have examined the essential functions of these peptides through the use of genetically engineered mice. For example, mice with mutations in the genes encoding PACAP (Colwell et al. 2004; Gray et al. 2001; Hamelink et al. 2002; Hashimoto et al. 2001) and VIP (Colwell et al. 2003) have been generated and characterized. PACAP-deficient knockout (KO) mice exhibit significant postnatal lethality (up to 80%) between the first and second week of age due to an apparent metabolic/thermoregulatory defect, but subsequently appear healthy (Gray et al. 2001, 2002). VIP KO mice do not show early lethality, but a small but significant percentage die in the first year due to a paralytic ileus-like condition (Lelievre et al. 2007). Numerous studies demonstrate that although most surviving VIP and PACAP KO mice show no obvious signs of sickness unless challenged, they show a variety of behavioral defects (reviewed in Abad et al. 2006), and have a reduced capacity to reproduce. Despite the reduced survival and other defects, it remains possible that the phenotypes observed in VIP and PACAP KO mice underestimate the action of the lost peptide due to compensation by PACAP or VIP, respectively. A recent study addressed this question and showed no evidence for upregulation of peptides or receptors in extracts of the developing brain (Girard et al. 2006), providing evidence that molecular compensation does not occur in the CNS of these KO strains during development under basal conditions. In order to further investigate the issue of compensation, and to determine if concurrent loss of both peptides is compatible with life, we crossed the two mutant strains to obtain double VIP/PACAP knockout (DKO) mice. We describe some of the phenotypic features of these mice here, with particular emphasis on postnatal growth and survival.
Materials and Methods
Generation of Mice and Monitoring of Growth and Survival
All studies were performed within the specific pathogen-free Gonda facility at the University of California at Los Angeles (UCLA). All husbandry and experimental procedures were in compliance with the Animal Welfare Act, Institutional Policies and Guidelines, and adhered to all principles stated in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources (U.S.) 1996). The experimental protocol was approved by the UCLA Animal Resource Committee. To facilitate the generation of WT and DKO mice on a similar background, previously generated VIP KO (Colwell et al. 2003) and PACAP KO (Colwell et al. 2004) mice on a C57BL/6Jx129Sv background were used. These mice were crossbred in two stages to obtain sets of WT and DKO breeders. Litters of WT and DKO pups for growth studies were then generated from WT × WT, and DKO × DKO interbreedings, respectively, and weaned at 3 weeks of age. The weights of individual pups were determined on a daily basis beginning at birth for up to 40 days. In some cases, assessment of growth was continued on a weekly basis for up to 15 weeks. The health of all mice was monitored on a daily basis, and the dates of death recorded.
Serum Insulin-like Growth Factor-1 (IGF-1) and Growth Hormone (GH) Determinations
ELISA assay kits from Diagnostic Systems Laboratories, Inc. (Webster, TX, USA) were used for both IGF-1 (DSL-10-2900) and growth hormone (GH) (DSL-10-72100). Assays were performed on serum from postnatal day 16 mice according to the instructions provided with the kits.
Postnatal Growth of VIP/PACAP Double Mutant Mice is Slower than in Wild-type Mice
DKO Mice Show Decreased Levels of Insulin-like Growth Factor, but not Growth Hormone
DKO Mice Die Prematurely in a Sex-Dependent Manner
DKO Mice Show Growth Defects in Multiple Internal Organs
Organ weights and body weight and length of WT and DKO mice at P16
Age = P16
Mean weight (g)
Mean weight (g)
Mean length (cm)
Mean length (cm)
Body length w/tail
Body length no tail
We describe here the phenotype of mice lacking coding regions for the two closely related polypeptides PACAP and VIP. Most of these double knockout mice survived through weaning (at 21 DOA) and were fertile. Given the multiple important putative biological functions of these peptides, it has been a matter of some debate if the reason for relatively mild phenotypes of single PACAP and VIP knockout mice was that, respectively, VIP and PACAP can substitute for the deleted peptide. However, it appears that even lack of both VIP and PACAP does not cause early postnatal lethality in a mixed C57BL/6Jx129Sv genetic background.
Even though DKO mice generally survive until weaning, they have a severe growth defect, especially pronounced for some internal organs. The causes of such growth retardation may be very diverse in view of pleiotropic character of both VIP and PACAP, and in view of their potential mutually redundant functions in the body. We explored two possible non-exclusive reasons for growth retardation: postnatal maternal effect, i.e., provision of inappropriate maternal care or nutrition before weaning, and impaired growth factor production and/or secretion in pups. We found that DKO pups showed growth retardation despite being reared by a surrogate mother, thus most likely due to innate factors. However, WT pups reared by a surrogate DKO dam also showed growth retardation, which, nonetheless, became less pronounced later in life (around P40). These results suggest that both maternal care and innate factors contribute to the observed phenotype. A further possibility not explored here is that the growth impairment is due in part to the loss of a maternal factor during gestation, as predicted by the studies of Brenneman, Hill, Gressens, and colleagues. Those studies showed that ex vivo treatment of whole mouse E9.5 embryos with VIP greatly accelerated embryonic growth over a 4-h period of study (Gressens et al. 1993), and that treatment of pregnant mice with a VIP antagonist on gestational day 9–11 resulted in severe microcephaly at birth, albeit with only a 10% reduction in overall birth body size (Gressens et al. 1994) and no significant delay in postnatal growth (Wu et al. 1997). In our studies, the birth weight of DKO pups born from DKO mothers did not differ from that of WT pups born from WT mothers (Fig. 2a), indicating that maternal contribution of neither VIP nor PACAP during embryogenesis is necessary for proper birth weight. However, we did not determine brain sizes in these mice at birth, leaving open the possibility that the birth weight of this organ could have been affected by loss of either maternal or embryo-derived VIP and/or PACAP during gestation.
We measured serum levels of growth hormone and IGF-1 at P16 to determine if either or both of these were affected by loss of either or both neuropeptides. IGFs are thought to be the primary regulators of body growth, acting in a GH-independent manner in the embryo. IGFs continue to act after birth, but appear to become dependent on GH production in mice around 3 weeks of age (Zhou et al. 1997). IGF-receptor-deficient mice are born with a 55% reduction in size, and die shortly thereafter of respiratory failure. GH receptor knockout mice have normal birth weights and grow normally until about 3 weeks, after which a profound growth rate reduction becomes apparent (Zhou et al. 1997). Mice deficient in both VIP and PACAP exhibit a phenotype distinct from IGF and GH receptor KO mice, with a normal birth weight and a growth reduction becoming apparent 4 days after birth (Fig. 2a). Taken with the fact that mean serum GH levels at P16 were unaffected in any of the mutant strains studied here, it can be concluded that the growth suppression observed in DKO mice has little or nothing to do with regulation of GH synthesis or release. Although it is tempting to speculate that VIP and/or PACAP can directly modulate IGF production, it should be taken into consideration that the IGF signaling lies downstream from many processes. For example, undernutrition can profoundly reduce circulating IGF-I and postnatal growth rates in rodents (Desai et al. 1996; Underwood et al. 1994). It is of interest that rat pups nursed by protein-restricted mothers were more than 50% reduced in weight at 21 days compared to those from mothers fed complete diets, but with only a 10–15% decrease in brain size (Desai et al. 1996). Thus, nutritional deficiencies can profoundly affect growth, but do not seem to grossly affect the growth of the brain. With this in mind, it is of interest that metabolic abnormities have been reported in PACAP (Gray et al. 2001), PAC1 (Jamen et al. 2000; Otto et al. 2004), and VPAC2 (Asnicar et al. 2002) KO mice. These were associated with slightly reduced body weights in adult PAC1-deficient mice (Jamen et al. 2000), and a late onset reduction in growth rate of VPAC2 KO mice, beginning at 8–12 weeks of age (Asnicar et al. 2002). Based on all of the data, metabolic defects impinging on IGF signaling in VIP/PACAP double mutant mice provide a plausible mechanism for their dwarf phenotype.
The dramatically reduced survival of female DKO mice beginning at 3–4 months of age was an unexpected finding. PACAP and PAC1 KO mice are known to have reduced survivals, but only due to deaths between the first and second weeks of life (Gray et al. 2001, 2002; Otto et al. 2004). Despite the fact that mice in our studies were monitored on a daily basis, we were unable to collect carcasses in time to perform studies to document the cause of death. Thus, the mechanism of death in these mice and the profound sex dependence remains unknown.
The present work was supported by the NIH grants CA110384, HD34475, HD04612, and HD06576.