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Divisions of Endocrine Research (M.A.A., M.L.H., F.T., D.P.S., H.M.H.), Research Technologies and Proteins (A.K., E.G., N.F.), and Gene Regulation, Bone and Inflammatory Diseases (Y.L.M.), Eli Lilly and Co., Indianapolis, Indiana 46285; Eli Lilly and Company, Bad Homburg D-61350, Germany; and University Children’s Hospital (W.F.B.), Giessen D-35392, Germany
Address all correspondence and requests for reprints to: Hansen M. Hsiung, Ph.D., Endocrine Research, DC0424 Lilly Corporate Center, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285. E-mail: hsiung_hansen_m{at}lilly.com.
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Introduction |
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VIP binds to specific receptors on the plasma membrane, initiating a cascade of events involving the cAMP/protein kinase A and phospholipase C pathways (4). The first recombinant VIP receptor was cloned from rat lung (5). This receptor and a highly homologous human VIP receptor (6, 7) were found to bind to VIP and PACAP with equally high affinity and were therefore designated VIP/PACAP (VPAC) receptor 1 (VPAC1R) (8). Subsequently, a second VPAC receptor was identified and was designated VPAC2R (8, 9, 10). Finally, a third homologous VPAC receptor was cloned and was found to bind to PACAP with a 1000-fold higher affinity than VIP; it is generally designated PAC1 receptor (PAC1R) (8, 11).
mRNA encoding VPAC1R is widely distributed in the central nervous system (CNS); in peripheral tissues, including lung, liver, and intestine (5, 12); and in T lymphocytes (9). Likewise, VPAC2R mRNA is found mostly in the thalamus and suprachiasmatic nucleus (SCN) of the CNS (12, 13) and in many peripheral tissues, including pancreas, skeletal muscle, heart, kidney, adipose tissue, testis, stomach, and immune cells (14, 15, 16, 17, 18). PAC1R is expressed predominantly in the CNS and adrenal medulla (11, 19). Finally, VPAC2R expression could be induced by immunological stimuli, such as lipopolysaccharide, whereas VPAC1R are constitutively expressed in immune cells (9).
Both VIP and VPACR were long considered to be important in modulating immune functions. In fact, treatment with VIP significantly reduced the incidence and severity of arthritis in an animal model, preventing joint swelling and cartilage destruction (20). The therapeutic effect of VIP was associated with down-regulation of both inflammatory and autoimmune components of the disease and was mediated through VPAC1R (20).
The functions of VPAC2R or PAC1R are largely unknown. However, PAC1R-deficient mice were shown to display impaired insulinotropic response to glucose (21). On the other hand, overexpression of the human VPAC2R in the suprachiasmatic nucleus alters the circadian rhythm in transgenic mice (22). These mice resynchronized more quickly than wild-type (WT) controls to an advance of 8 h in the light-dark cycle and exhibited a shorter circadian period in constant darkness (22).
As VPAC2R is also expressed at a high level in pancreatic islets, we suspected that VPACR might play a role in energy balance and glucose homeostasis. In this report we generated VPAC2R-deficient mice to study the role of VPAC2R in metabolism and growth.
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Materials and Methods |
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Genomic library screening and mapping of the mouse VPAC2R gene
A 387-bp mouse VPAC2R cDNA fragment (Fig. 2
) that spanned exons VII–XI of the mouse VPAC2R gene was generated by RT-PCR using a mouse brain cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA) as a template. Two oligonucleotide primers, VIPR1F (5'-GTCATGGCYAACTTCTTCTGGCTGCTGCTGGTGGA-3') and VIPR2R (5'-AACATGATGTAGTGYACTCCAAAYAGGGGGAT-3'), designed from the homologous regions of the porcine, turkey, and human VPACR sequences were used in RT-PCR to generate this 387-bp fragment. The radioactively labeled, 387-bp sequence generated by PCR with [32P]deoxy-CTP was used a probe to screen a mouse ES-129/SvJ I genomic bacterial artificial chromosome (BAC)-based library (Genome Systems, St. Louis, MO) to obtain a BAC genomic clone that contained the VPAC2R gene. This BAC genomic clone was mapped using multiple restriction endonucleases, and the restriction fragments containing the VPAC2R gene were subcloned into pZErO-1 (Invitrogen, Carlsbad, CA).
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) was constructed in pZErO-1 by first inserting a 6.6-kb SpeI-Asp718, 5'-VPAC2R gene fragment that included exons VII–X. A synthetic DNA (
70 bp in size) that contained both the loxP site and the BamHI site was then inserted at an NdeI site of the NdeI partially digested vector between exons VII and VIII of the VPAC2R gene (loxP1; Fig. 1A). Subsequently, a 3.0-kb 3' VPAC2R gene fragment (Asp718-HindIII) containing only the intronic sequence between exons X and XI was inserted into the vector. Additionally, a loxP-flanked (loxP2 and loxP3), dual gene cassette (from pBS246neo/tk, LRL, Eli Lilly and Co.) including the pGK-neomycin phosphotransferase (neo) and thymidine kinase (tk) genes (designated lox2 neo/tk) was inserted at the Asp718 site between the 5' and 3' genomic fragments. Finally, the DNA sequence for the A subunit of the diphtheria toxin (DT) gene (from pGT-N29DT, LRL, Eli Lilly and Co.) was inserted into the vector at the PvuI site.
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) was linearized with PvuI and introduced into mouse E14 embryonic stem (ES) cells by electroporation. Selection of stably transfected ES cell clones was initiated after 24 h using G418 (catalog no. 11811-023, Invitrogen, San Diego, CA; 350 µg/ml), and cells were maintained for 8 d before 240 G418-resistant clones were picked and expanded for Southern hybridization analysis. We identified three correctly targeted clones, and one of these was transfected with the plasmid pBS185 (Invitrogen) expressing the cre gene. After 24 h, gancyclovir (2 µM; Syntex, Palo Alto, CA) was added to the transfected ES cell cultures to select the mutant clones that had the lox2 neo/tk cassette evicted from the genome by Cre recombinase. After 8 d of selection, 120 gancyclovir-resistant clones were picked and expanded for Southern hybridization analysis. Southern analysis showed that two clones (D1-28 and D1-78; Fig. 1B) had eviction of the DNA sequence between the 5' loxP site (loxP1) and the 3' loxP site (loxP3), resulting in the deletion of exons VIII–X (Fig. 1A). These two clones were injected into C57BL/6 blastocysts, and the resulting blastocysts were implanted into foster mothers to generate chimeric mice. The resulting 10 male chimeras (5 from each clone) were bred to female C57BL/6 mice to generate F1 mice. The F1 agouti-coated mice were screened by PCR to detect those with the heterozygous genotype. These heterozygous mice were bred together to generate F2 mice. The genotypes of the F2 mice were determined by PCR and then verified by Southern hybridization analysis.
Genotype determination by Southern hybridization and PCR
A 407-bp BamHI-SpeI fragment (Fig. 1A) was used as a 5' external probe to detect correctly targeted ES cells (pre-Cre and post-Cre mutant alleles in Fig. 1A) before and after being transiently transfected with the cre expression plasmid pBS185. This same probe was also used to confirm mouse genotypes. This probe was prepared based on the sequence 5' of the VPAC2R genomic sequence used in construction of the targeting vector. The probe would detect a 9.5-kb BamHI fragment with the wild-type allele(s), a 4.6-kb BamHI fragment with the correctly targeted, mutant VPAC2R allele(s) before Cre excision, and a 6.5-kb BamHI fragment with the correctly targeted, mutant allele(s) post-Cre excision (Fig. 1B).
Genotypes of all F1 or F2 mice were determined by using a multiplex PCR that contained a common forward primer (VIPRcF, 5'-CTGGTTGGTAGTTTAGTCCC-3'), a WT reverse primer (VIPRwtR, 5'-TCATTCTGCCTGGTTTGTCC-3'), and a mutant-specific reverse primer (VIPRmutR, 5'-ATGGCCAGTACTAGTGAACC-3'). The VIPRcF primer sequence was derived from a 20-bp segment of the intron between exons VII and VIII. The VIPRwtR primer sequence was from a 20-bp segment of the intron between exons VIII and IX. The VIPRmutR primer sequence was from a vector sequence carried into the targeting vector when the lox2 neo-tk cassette was inserted. The WT allele produced a 955-bp DNA fragment, whereas the mutant allele produced a 579-bp fragment (Fig. 1C).
Detection of VPAC2R expression in mouse tissues by Northern hybridization
Polyadenylated RNA was extracted from brain and lung of two 20-wk-old male and female WT and knockout (KO) mice using a Poly(A)Pure kit (Ambion, Inc., Austin TX). A sample of 2 or 4 µg of each polyadenylated RNA was electrophoresed on a 1.1% formaldehyde agarose gel at 80 V for 4 h. The RNAs were then transferred by capillary action to Hybond-XL (Amersham Pharmacia Biotech, Zurich, Switzerland) in 10x sodium chloride-sodium citrate (3 M NaCl and 0.3 M sodium citrate) overnight. The RNAs were cross-linked to the membrane by UV irradiation in a Stratalinker (Stratagene, La Jolla, CA). A full-length [32P]VPAC2R cDNA probe (Fig. 2A) generated using a random prime labeling system (Rediprime II, Amersham Pharmacia Biotech) was used to detect the VPAC2R-specific mRNA (Fig. 2B).
Cloning and sequence analysis of VPAC2R cDNA from wild-type and mutant mice
To confirm that mutant VPAC2R KO mice did not express functional WT VPAC2R mRNA in vivo, we used RT-PCR to clone and sequence the VPAC2R mRNAs from WT and mutant mice. The forward primer, VPAC2cDNAf (5'-ACGCTGAGCCCAAGATGAGG-3'; Fig. 2A), containing an initiation codon and the reverse primer, VPAC2cDNAr (5'-ACTGACAGCTCTGTACAAGG-3'; Fig. 2A), containing a segment of the 3'-untranslated region of the VPAC2R gene were used to amplify the WT and KO VPAC2R mRNAs with the Superscript One-Step RT-PCRs and Platinum Taq DNA polymerase kit (Invitrogen). The brain mRNAs from WT and VPAC2R-deficient mice (2 males and 2 females each) were pooled and used as the templates in RT-PCR. The RT-PCR products were electrophoresed on 1.2% agarose gel (Fig. 2C) and then ligated directly to the pcDNA3.1D/V5-His-TOPO vector (Invitrogen). One third of the ligation mixture was used to transform TOP10 electrocompetent Escherichia coli cells (Invitrogen) by electroporation. The plasmid DNA from each of the randomly selected transformants (9 WT and 11 KO) was isolated and sequenced.
Growth, feeding, and pathology studies
After weaning at 3 wk of age, the body weight and food consumption of each group (n = 8–10) of individually housed male and female WT and KO mice were determined weekly or every other week. For food consumption studies preweighed portions of food were dispensed from the wire cage tops. At 32 wk of age mice were anesthetized with isoflurane to obtain length measurements. Mouse lengths were measured from the tip of the nose to the base of the tail.
Full necropsies were performed at 8 and 31 wk of age on both genders of mice (WT and KO, five per group). All mice were screened with full hematology and clinical chemistry panels. The following organs from each mouse were weighed and prepared for histological analysis: kidney, liver, heart, lung, spleen, thyroid, salivary gland, pancreas, lymph node, adrenal, thymus, stomach, duodenum, jejunum, ileum, colon, brain, pituitary, spinal cord, sciatic nerve, ovary/testis with epididymis-prostate-seminal vesicle, skeletal muscle, skin/mammary gland, trachea/esophagus, bone marrow, bone/joint, brown fat, and white fat. Tissues were divided, with one portion flash-frozen for holding at -80 C and the other immersion-fixed in 4% neutral buffered paraformaldehyde for 24 h at 4 C. The fixed tissues were trimmed and processed into paraffin and sectioned at 5 mm for light microscopy.
Oral glucose tolerance (OGTT) and insulin tolerance testing
OGTT was performed on each group of male and female WT and KO mice at 2 months of age and then again at 6 months of age. The mice were fasted overnight, then bled to obtain a specimen for fasting glucose and insulin measurements before they were given a dose (2.5 g/kg) of 50% dextrose solution by oral gavage. Blood specimens for glucose and insulin assays were also obtained 30, 60, and 120 min after the dextrose dose. Blood glucose levels were determined using a Precision·G Blood Glucose Testing System (Abbott Diagnostics, Abbott Park, IL), and insulin levels were determined by RIA (Lilly Research Laboratories, Indianapolis, IN).
Insulin tolerance testing was performed on each group of mice (male or female WT and KO) at 5 months of age. Food was withheld from the mice for 4 h before as well as during the test. After fasting for 4 h initial mouse blood specimens were obtained for glucose determinations, and then Humulin (0.75 U/kg; Eli Lilly and Co.) was administered to each mouse by ip injection. Additional blood specimens for glucose determinations were obtained at 15, 30, 60, and 90 min post-Humulin treatment.
Analysis of body composition by wide-line nuclear magnetic resonance (NMR)
Wide-line NMR, which measures a signal proportional to the total number of hydrogen nuclei, their relaxation times, and diffusion coefficients, was used to quantify tissue mass. Wide-line NMR has been shown to provide similar body composition results as those obtained from more established dual energy x-ray absorptiometry scans, but NMR measurement did not require anesthesia, was much more rapid, and was more precise (23). A Bruker’s Minispec MQ7.5 (Houston, TX) NMR instrument with software modifications developed by Echo Medical Systems (Houston, TX) for Eli Lilly and Co. was used to measure the body composition of mice. A wide-line NMR signal was calibrated for lean mass measurement using chicken breast muscle (fat and skin removed) and for fat mass measurement using canola oil as standards. Water and bone mass account for the difference between body mass and fat plus lean mass. Measurements were performed for 1 min on live mice in triplicate. NMR results reported weights for fat, muscle (lean), and water in each mouse.
Indirect calorimetry
Energy expenditure (EE) and respiratory quotient (RQ) measurements were obtained using an Oxymax (Columbus Instruments International Corp., Columbus, OH) open circuit indirect calorimetry system. These measurements were taken when the mice were 10, 17, and 21 wk of age. After the system was calibrated against standard gas mixtures, the mice were individually placed into acrylic calorimeter chambers with food and water. To measure O2 and CO2 by paramagnetic and spectrophotometric sensors, respectively, the system automatically withdrew gas samples from each chamber hourly for approximately 24 h. The system then calculated the volumes of O2 consumed (vO2, milliliters per kilogram of body weight) and CO2 generated (vCO2; milliliters per kilogram of body weight) by the mouse in 1 h. The RQ was the ratio of the vCO2 to vO2. EE was calculated as the product of the caloric value of oxygen (caloric value of oxygen = 3.815 + 1.232 x RQ) and the vO2. Daily fuel utilization was determined by calculating the total calories expended in 1 d. We used Flatt’s proposal and assumed that protein oxidation and intake were equivalent in adult stable animals to calculate the proportions of protein, fat, and carbohydrate that were used during the 24-h period. Caloric intake in a 24-h period was the product of the mass (grams) of food consumed in 1 d and the nutritional content of the diet (kilocalories per gram). Ambulatory and fine locomotions of each mouse were also detected during the 24-h period. Ambulatory movement was measured by counting the number of times a mouse broke adjacent light beams during the calorimetry measurements. Fine movement was calculated by subtracting the ambulatory movement from the total number of beam breaks (same or adjacent) that occurred over the same period. The first 2 h of measurements were used as a period of adaptation for the mice, and the data obtained during this period were excluded from analysis. The basal metabolic rate was calculated by averaging EE obtained in the 6-h period immediately following the switch to the light period when the mice exhibited the least amount of both ambulatory and fine movements.
Serum leptin, free T3, and IGF-I measurements
Blood samples for serum leptin measurements were taken when the mice were 6 months old, and the leptin measurements, determined by ELISA, were performed by the Clinical Pathology Department at Lilly Research Laboratories. The sensitivity of mouse leptin measurements is 200 pg/ml; the intraassay coefficient of variation (CV) was 5.4%, and the interassay CV was 6.9%. The free T3 RIA was performed on serum samples from 9-month-old mice using a kit from Diagnostic Products (Los Angeles, CA). The sensitivity of the free T3 RIA was 0.2 pg/ml; the intraassay CV was 4–11%, and the interassay CV was 5.0–8.8%. Blood specimens for serum IGF-I determinations were taken at 12 and 24 wk of age. IGF-I levels were determined with an IGF-binding protein (IGFBP)-blocked RIA developed for human IGF-I (24), which shows complete cross-reaction with rodent IGF-I. In brief, serum samples were diluted 1:300 in an acidic phosphate buffer, pH 2.8, to dissociate IGFs from IGFBP. The first antibody was diluted in a phosphate buffer that contained a large excess of IGF-II to block free IGFBP-binding sites. This buffer also neutralized the acidic sample dilution. After the addition of tracer, the reaction mixture was incubated for 2 d, and bound tracer was precipitated using a second antibody method. The intraassay CV for IGF-I determinations was 1.6%, and the interassay CV was 6.4%. The sensitivity of the assay was 3 µg/liter at a sample dilution of 1:300.
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Results |
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). This vector contained a short sequence of synthetic DNA, encompassing a loxP Cre recognition site (loxP1) and a BamHI site, inserted into the intron between exons VII and VIII of the VPAC2R gene. In addition, a lox2 neo-tk cassette was inserted into the intron between exons X and XI in the vector (Fig. 1A). After gene targeting with the vector, we detected, of 240 clones screened, 3 ES cell clones correctly targeted (pre-Cre mutant allele, Fig. 1A) as shown by the presence of 4.6- and 9.5-kb BamHI fragments in the Southern hybridization analysis (lane D1 in Fig. 1B). The Cre recombinase expression vector was then transiently transfected into one of these correctly targeted ES cell clones. The Cre recombinase expression in the transfected ES cells caused eviction of the sequences between loxP sites (loxP1 and loxP3) and generated the VPAC2R null mutation (designated the post-Cre mutant allele; Fig. 1A). Southern hybridization analysis of the DNAs isolated from some of the clones showed the presence of both 6.5- and 9.5-kb BamHI fragments (lanes D1-28 and D1-78 in Fig. 1B), indicating a deletion mutation (loss of exons VIII–X) occurring on one allele of the VPAC2R gene. Two of these ES cell clones were selected, expanded, and injected into blastocysts. Chimeric mice generated from these ES cell mutant clones transmitted the mutation through the germline when mated with C57BL/6 females. The resulting F1 heterozygous mice were intercrossed to generate the F2 WT and KO mice that were later used in all phenotype studies.
We analyzed VPAC2R mRNA expression in WT and KO mouse brain and lung by Northern hybridization using a full-length VPAC2R cDNA probe (Fig. 2A). The hybridization results showed the presence of a shortened approximately 3.6-kb transcript in VPAC2R KO vs. an approximately 3.9-kb transcript in WT mouse tissues (Fig. 2B). Additionally, amplification of the coding region of WT brain VPAC2R mRNA by RT-PCR showed an expected 1.4-kb product, but amplification of VPAC2R-/- mRNAs produced a 1.1-kb product (Fig. 2C). The RT-PCR products from both WT and KO mouse brain tissues were cloned and sequenced. Sequence analysis of 9 WT VPAC2R cDNA clones showed 100% identity with the published mouse VPAC2R sequence (GenBank S82966) (9), whereas sequence analysis of the 11 VPAC2R-/- cDNA clones revealed that all clones had deletions of exons VIII–X and most (8 of 11) were also lacking the exon VII sequence due to alternative splicing of mutant mRNAs (data not shown). The deletion of either exons VIII–X or exons VII–X in KO mice created a frameshift mutation beyond exon VI or exon VII in the VPAC2R gene. As a result, VPAC2R KO mice would generate mRNAs that encode truncated VPAC2R proteins beyond exon VI or exon VII (Fig. 2D).
The VPAC2R KO mice were born at the expected Mendelian frequency, suggesting that no embryonic lethality was associated with the targeted interruption of the VPAC2R gene. Both young adult male and female VPAC2R KO mice appeared healthy and fertile and raised normal sized litters. However, there was physical and clinical evidence of decreased fertility in older male VPAC2R KO mice. In the 31-wk-old mice, but not the 8-wk-old mice, three of the five males had diffuse mild to moderate seminiferous tubular degeneration and associated hypospermia (Fig. 3). The seminiferous tubular degeneration and associated hypospermia may account for the decreased overall fertility observed in older male KO mice. Light microscopy showed no histological differences between WT and KO mice in bone, white fat, brown fat, or skeletal muscle (data not shown).
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, A and B). Data from OGTTs in 6-month-old mice showed that VPAC2R KO mice and their WT siblings had similar blood glucose responses to the challenge (Fig. 4, A and B); however, the insulin response (Fig. 4, C and D) was significantly lower in KO mice relative to that in their WT siblings. These significant differences in serum insulin concentrations were seen in both sexes at all time points, with the exception of the 30 min point in female mice, in which the difference did not reach significance (Fig. 4, C and D). These results suggested that insulin sensitivity was increased in VPAC2R KO mice compared with their WT siblings. To verify this suspected increase in insulin sensitivity in the KO mice, blood glucose levels were measured at different time points (15, 30, 60, and 90 min) after insulin administration (0.75 U/kg, ip). The data showed that blood glucose levels after insulin administration in the male KO mice were significantly lower than those in their male WT siblings (Fig. 4E). However, blood glucose levels after insulin challenge in female WT and KO mice were similar (Fig. 4F).
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), whereas the mean body weight of female KO mice diverged significantly from that of their WT siblings after 15 wk of age (Fig. 5B). At 29 wk of age, KO male and female mice weighed 22% and 25% less, respectively, than their WT siblings. Food consumption (Table 1), measured as average weekly intake from 14–18 wk of age, was decreased by 15% and 19% in the KO male and female mice, respectively, compared with their age- and sex-matched WT controls. When adjusted for differences in body weight, these differences in food consumption between WT and KO mice were no longer statistically significant (data not shown). Male and female KO mice were shorter (-7% in males and -5% in females, respectively) than their WT siblings when measured from nose to base of the tail at 32 wk of age (Table 1).
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) and EE (Fig. 7, A and B) values appeared to be higher in KO mice compared with their gender-matched WT controls, the mean 24-h RQ and EE values did not show a significant difference. However, RQ values immediately following the transition into the dark period were significantly higher in male KO mice than in WT controls (Fig. 6A). Such a spike in RQ values was not seen until later in the 12-h dark period in WT males (Fig. 6A). This difference in RQ values was not seen in females (Fig. 6B). The peak in glucose utilization (increased RQ values; Fig. 6A) at the onset of the dark period was also reflected in the EE values in male KO mice (Fig. 7A). Additionally, no differences in ambulatory (data not shown) or total movement (Fig. 7, C and D) between WT and KO male and female mice were observed. EE values of the KO mice were consistently greater than those of their WT siblings, especially during the first 6 h immediately after the switch from dark to light period. This 6-h period was also the time at which the least amount of total movement was detected (Fig. 7, C and D). We averaged EE values obtained on each mouse during the first 6 h following the switch from the dark to the light period to obtain a basal metabolic rate (Table 1). During this 6-h period, the KO male and female mice exhibited 23% and 10% higher EE, respectively, than their WT siblings. Serum thyroid hormone (free T3) measurements showed that male and female VPAC2R-deficient mice had lower serum T3 levels than their gender-matched WT siblings (Table 1).
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), and fat mass was decreased by 10% (P = 0.02) in female KO mice (Fig. 8). Serum leptin levels in 6-month-old male and female KO mice were 60% and 55% lower (P = 0.003 for KO males and 0.007 for KO females), respectively, than those in their age- and sex-matched WT controls (Table 1).
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). The serum IGF-I levels in male KO mice were 14% lower and approaching significance (P = 0.08) compared with those in male WT mice at 12 wk of age (Fig. 9A). Likewise, IGF-I levels in female KO mice were 24% lower (P < 0.008) than those in female WT controls (Fig. 9B) at 12 wk of age. However, serum IGF-I levels in both male and female KO mice were not different from those in their gender-matched WT siblings at 24 wk of age (Fig. 9, A and B).
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Discussion |
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Furthermore, as overexpression of human VPAC2R in transgenic mice enables them to reentrain more rapidly to changes in the light/dark cycle, with a significantly shorter circadian period in constant darkness, it was suggested that VPAC2R also plays an important role in regulating circadian rhythm (22). Finally, a recent report published while this manuscript was in preparation showed that VPAC2R deficiency in mice caused an increase in delayed-type hypersensitivity and a decrease in immediate-type hypersensitivity, suggesting that VPAC2R also plays an important role in immune functions (28).
In this study, we generated VPAC2R-deficient (KO) mice and studied the effect of VPAC2R deficiency on growth and fuel utilization. Because previous reports showed that the blockade of VIP functions affects brain development and embryonic growth (29, 30), we decided to employ a targeting strategy using the loxP-Cre method that can create either global or tissue-specific knockout in mouse colonies. Using the loxP-Cre method, we created two different mutant ES cell lines resulting from excision of DNA fragments between the loxP sites by Cre recombinase. Eviction of the DNA between the loxP1 site and the loxP3 site created a mutant cell line that harbors the VPAC2R allele with a deletion of exons VIII–X (a heterozygous KO mutant). The second mutant cell line was generated from the eviction of the neo/tk cassette between loxP2 and loxP3, resulting in an allele with loxP sites flanking exons VIII, IX, and X of the VPAC2R gene (results not shown). This second mutant cell line can be used to generate a tissue-specific KO of the VPAC2R gene in mice. The ES cell clones harboring the first mutant allele (VPAC2R global KO) were isolated and injected into C57BL/6 blastocysts. The viable and apparently healthy F2 global VPAC2R KO mice were generated at the expected Mendelian frequency, indicating that VPAC2R is not vital for embryonic growth or brain development.
The VPAC2R gene is complex, containing 13 exons that span approximately 20 kb of the mouse genome. Our targeting strategy was to delete exons VIII–X of the VPAC2R gene and thereby disrupt the function of the VPAC2R. As exons VIII and IX encode the second extracellular (EC2) loop, which contains the conserved amino acids Thr274 and Pro280 that are critical for the structure and function of the receptor (31, 32), we believed that the mutant VPAC2R protein lacking the EC2 loop will also be nonfunctional. Northern hybridization analysis detected a shortened approximately 3.6-kb VPAC2R transcript in lung and brain tissues of the VPAC2R KO mice, suggesting that a predicted deletion mutation occurs in mice. Furthermore, DNA sequence analysis of the VPAC2R cDNAs generated from RT-PCR showed that the exon VII sequence was also spliced out in the majority of the mutant RNAs. Additionally, the deletion of either exons VIII–X or exons VII–X in KO mice created not only a deletion of the EC2 domain but also created a frameshift mutation beyond exon VI or exon VII in the VPAC2R gene. As a result, VPAC2R KO mice would generate mRNAs that encode truncated VPAC2R proteins beyond exon VI or exon VII. Because the truncated proteins would lack several important domains, including transmembrane domains 3–7 or 4–7 as well as EC2, we believe that they will not have VPAC2R functional activity. Furthermore, any truncated mutant VPAC2R proteins that might be expressed would probably not have a dominant negative effect because they could not effectively bind VIP or PACAP even if they were inserted into the membrane.
As both VPAC2R and its homologous PAC1R are expressed in pancreatic ß-cells, and VIP or PACAP is known to enhance glucose-stimulated insulin secretion (25, 26, 27), we decided to investigate the role of VPAC2R in regulating the insulin response to glucose. We performed the OGTTs in which the blood glucose levels were determined in 2- or 6-month-old WT and KO mice in response to an oral glucose challenge (2.5 g/kg mice). Our results showed no detectable differences in glucose tolerance between WT and KO mice. Recently, PAC1R-deficient mice showed a marked intolerance to a glucose challenge because of an impaired insulinotropic response (21). The results indicate that PAC1R, not VPAC2R, is mainly responsible for the insulinotropic response of ß-cells to PACAP.
Insulin levels of 6-month-old mice during a glucose challenge were significantly lower in KO mice than in WT mice. VPAC2R KO mice were able to maintain a normal response to glucose challenge with lower levels of insulin than WT mice, suggesting a significant increase in insulin sensitivity in KO mice. The insulin tolerance results added additional evidence of increased insulin sensitivity in VPAC2R KO mice. Normalized glucose levels, when expressed as a percentage of the fasting glucose level, were decreased more pronouncedly in KO males than in WT males after a 0.75-U/kg dose of recombinant human insulin. This difference in normalized glucose levels between male KO and WT mice was not seen between female KO and WT mice.
Male and female VPAC2R KO mice had reduced body weight and a significant divergence in mean body weight from their WT siblings beginning at 8 wk of age in male mice and at 15 wk of age in female mice. Additionally, body length measurements at 32 wk of age showed that the male and female KO mice were significantly shorter. Serum IGF-I levels at 12 wk of age, but not at 24 wk of age, appeared to show a divergence between VPAC2R-deficient mice and their gender-matched WT controls. We hypothesize that serum IGF-I levels may be partly responsible for impaired growth in KO mice. However, as we had only limiting quantities of mouse serum samples, we were unable to measure serum IGF-I levels over an extended period of time. Further investigations with more serum samples are needed to know whether serum IGF-I levels correlate with growth phenotypes in the studied mice.
Differences in growth rate and body weight between KO and WT mice may also be partly attributed to differences in fuel utilization and basal EE. The abrupt and significant increase in RQ values seen only in male KO mice immediately after the transition to the dark period were indicative of the increased carbohydrate utilization that accompanies eating. Although not seen in female KO mice, male KO mice also appeared to switch more toward using fat as a fuel source during the light period than their WT siblings, because their RQ values during this period were consistently lower than those of male WT mice. The consistently higher EE values seen in KO mice during the period immediately following the switch from the dark to the light cycle were examined closely. During that period the mice also exhibited the least total movement. The EE values obtained during these quiescent periods more accurately reflect the basal metabolic rate (33, 34). We selected the EE values obtained during the first 6 h of the light period to calculate basal metabolic rate. Both male and female KO mice had significantly higher basal EE than their WT siblings. It is likely that the KO mice did not maintain the growth rate seen in their WT siblings, because the KO mice expended more calories without ingesting additional calories. To our surprise, both female and male KO mice had significantly lower thyroid hormone (free T3) levels than their gender-matched WT siblings. We suspected that lower serum thyroid hormone levels were the compensating feedback response to the elevated basal metabolic rate in KO mice.
We performed wide-line NMR analyses on live mice to determine body composition. Wide-line NMR was chosen over the more established noninvasive dual energy x-ray absorptiometry because the NMR analysis can be performed in a very short time period (1 min) on live, nonanesthetized mice, and it provides comparably accurate measurements with far greater precision (23). In proportion to body weight, KO mice tend to be leaner and have reduced body fat as they became older, to the point of becoming significantly leaner than their WT siblings at 22 wk of age. This is consistent with the observed energy deficit in KO mice compared with WT mice. The reduced body fat seen in the VPAC2R KO mice is most likely the cause of the significantly reduced serum leptin levels seen in the 6-month-old KO mice.
Expression of VPAC2R (35) and PAC1R (36) mRNAs in the rat SCN peaks twice (midday and midnight) in a 24-h period. Overexpression of human VPAC2R in the SCN alters the circadian rhythm in transgenic mice (22), suggesting that VPAC2R plays an important role in regulating the circadian clock. In fact, the mutant VPAC2R transgenic mice can synchronize to light-dark cycle changes more quickly (22). Furthermore, PAC1R KO mice showed modest growth retardation (21) and changes in circadian rhythm in response to light-dark cycle changes (37). Our results showed that the VPAC2R KO mice had significant growth retardation, which we suspect might be related to circadian clock changes in KO mice. The circadian clock is responsible for the synchronous and pulsatile release of many hypothalamic releasing factors, resulting in the release of many different pituitary hormones, including GH. Because GH secretion occurs most prominently during rapid eye movement sleep, changes in circadian rhythm may affect GH/IGF-I secretion, resulting in growth retardation. Additionally, circadian rhythm changes may affect TRH release, which can impact thyroid function and increase basal metabolism.
While this manuscript was being prepared, Goetzl et al. (28) reported that VPAC2R-deficient mice exhibited an increase in delayed-type hypersensitivity, but a decrease in immediate-type hypersensitivity; however, the biological effects of VPAC2R-deficiency on growth and metabolism were never reported. Our approach to generate a VPAC2R-deficient mouse colony differed significantly from that described by Goetzl et al. (28), who inserted a lacZ-neoR cassette into the first coding exon of the VPAC2R gene, resulting in a null allele, as confirmed by RT-PCR analysis of T cell-derived RNA. Goetzl et al. (28) described their VPAC2R-deficient mice as "normal in general health, social behavior and breeding pattern," but did not specify whether they checked their growth rates or tried to breed them in old age. The scope of their investigations of the VPAC2R-defiecient mice was very different from that of the present study and focused exclusively on the immune system. It is therefore not clear whether the VPAC2R-deficient mice generated by Goetzl et al. (28) did not show the growth retardation phenotype or whether it was not observed.
Our data showed that VPAC2R-deficient mice exhibited growth retardation, decreased fat mass, and increased lean mass. Furthermore, mutant mice exhibited increased insulin sensitivity and had a higher basal metabolic rate than WT mice. Finally, we also observed diffuse seminiferous tubular degeneration, with hypospermia and a reduced fertility rate in older male mutant mice. In conclusion, our results suggest that VPAC2R plays an important role not only in immune functions, but also in growth, energy homeostasis, and male sexual function.
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Received March 27, 2002.
Accepted for publication June 3, 2002.
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-32P]deoxy-CTP. The RNA blot was subsequently hybridized to an [
, Male and female WT mice (n = 10, each); 
, male and female KO mice (n = 10, each). *, 0.01 < P 
0.05; **, 0.001 < P 
, 0.001 < P 
, dark cycle.
, VPAC2R KO mice (n = 8). *, P 