Vasoactive Intestinal Polypeptide/Pituitary Adenylate Cyclase ...

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Endocrinology 143(10):3994 – 4006 Copyright © 2002 by The Endocrine Society doi: 10.1210/en.2002-220354

Vasoactive Intestinal Polypeptide/Pituitary Adenylate Cyclase-Activating Peptide Receptor 2 Deficiency in Mice Results in Growth Retardation and Increased Basal Metabolic Rate ¨ STER, MARK L. HEIMAN, FRANK TINSLEY, DENNIS P. SMITH, MARK A. ASNICAR, ANJA KO ELIZABETH GALBREATH, NILES FOX, YANFEI LINDA MA, WERNER F. BLUM, AND HANSEN M. HSIUNG 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 Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are two closely related peptides that bind two homologous G protein-coupled receptors, VIP/PACAP receptor 1 (VPAC1R) and VIP/PACAP receptor II (VPAC2R), with equally high affinity. Recent reports suggest that VPAC2R plays a role in circadian rhythm and T cell functions. To further elucidate the functional activities of VPAC2R, we generated VPAC2R-deficient mice by deleting exons VIII–X of the VPAC2R gene. The VPAC2Rdeficient mice showed retarded growth and had reduced serum IGF-I levels compared with gender-matched, wild-type siblings. The mutant mice appeared healthy and fertile at a young adult age. However, older male mutant mice exhibited diffuse seminiferous tubular degeneration with hypospermia

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ASOACTIVE INTESTINAL peptide (VIP), a 28-amino acid peptide, was first discovered as a smooth muscle relaxant, vasodilator peptide in the lung and small intestine (1, 2). VIP was later found to be present in brain and many other peripheral tissues (3). VIP belongs to the glucagon/ secretin family and members of this peptide family include GHRH, glucagon-like peptide 1, helodermin, peptide histidine isoleucine, peptide histidine methionine, gastric inhibitory polypeptide, and pituitary adenylate cyclase-activating peptide (PACAP) (3). Today, it is well established that VIP is a neuropeptide hormone acting as a neurotransmitter and neuromodulator in practically all tissues (3). 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 Abbreviations: BAC, Bacterial artificial chromosome; CNS, central nervous system; CV, coefficient of variation; DT, diphtheria toxin; EC2, second extracellular; EE, energy expenditure; ES, embryonic stem; IGFBP, IGF-binding protein; KO, knockout; NMR, nuclear magnetic resonance; OGTT, oral glucose tolerance test; PACAP, pituitary adenylate cyclase-activating peptide; RQ, respiratory quotient; SCN, suprachiasmatic nucleus; vCO2, volume of CO2 generated; VIP, vasoactive intestinal polypeptide; vO2, volume of O2 consumed; VPAC1R, vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor 1; WT, wild-type.

and reduced fertility rate. The mutant mice appeared to have an increase in insulin sensitivity. VPAC2R-deficient mice had increased lean mass and decreased fat mass with reduced serum leptin levels. Indirect calorimetry experiments showed that the respiratory quotient values immediately following the transition into the dark cycle were significantly higher in male knockout mice for about 4 h. Additionally, male and female VPAC2R-deficient mice presented an increased basal metabolic rate (23% and 10%, respectively) compared with their wild-type siblings. Our results suggest that VPAC2R plays an important role in growth, basal energy expenditure, and male reproductive functions. (Endocrinology 143: 3994 – 4006, 2002)

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 –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 –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

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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. Materials and Methods Animal care and maintenance The Eli Lilly and Co. Research Laboratories institutional animal care and use committee approved all animal protocols used in these studies. Mice were individually housed in microisolator (Labproducts, Seaford, DE) cages and were maintained on regular chow (11% fat; 5015 Purina Mouse Chow, Ralston Purina Co., St. Louis, MO). Access to both chow and water were allowed ad libitum. Mice were exposed to a 12-h light, 12-h dark cycle, with the dark period beginning at 1000 h.

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).

Construction of a VPAC2R targeting vector The targeting vector (Fig. 1A) 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.

Generation of a targeted mutant VPAC2R⫺/⫺ mouse strain The targeting vector (Fig. 1A) was linearized with PvuI and introduced into mouse E14 embryonic stem (ES) cells by electroporation.

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Selection of stably transfected ES cell clones was initiated after 24 h using G418 (catalog no. 11811-023, Invitrogen, San Diego, CA; 350 ␮g/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 ␮m; 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 ␮g 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 10⫻ 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), con-

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FIG. 1. Generation of VPAC2R KO mice. A, Schematic representations and partial restriction maps: the mouse VPAC2R gene (WT allele), the VPAC2R targeting vector, the mutant allele produced after a homologous targeting event (Pre-Cre mutant allele), and the mutant allele produced after transient cre expression in correctly targeted ES cell clones [PostCre mutant allele (KO)] are shown. Black arrows labeled with Roman numerals indicate exons VII– XIII of the VPAC2R gene. Dark gray arrows labeled 5⬘ arm and 3⬘ arm represent the regions of homology between the WT allele and the targeting construct. Light gray arrows labeled DT, MCl-tk, and neo represent three genes (diphtheria toxin subunit A, herpes simplex virus-thymidine kinase, and the neomycin phosphotransferase genes) used in positive and negative selection of ES cells. Right-angled arrows labeled loxPn indicate the positions of three Cre recombinase recognition sites: the first site (loxP1) is between exons VII and VIII, and the second and third sites (loxP2 and loxP3) flank the neo-tk cassette. The short black bars indicate the 407- and 626-bp probes used in Southern hybridization analyses. The long black bars indicate three genomic fragments [9.5 kb, WT; 4.6 kb, PreCre mutant; and 6.5 kb, PostCre (KO) mutant] detected by the 407-bp probe in Southern hybridization analyses. B, BamHI; S, SpeI; H, HindIII; A, Asp718. B, Southern hybridization analysis of DNA samples isolated from ES cell clones. Genomic DNA samples were digested with BamHI and hybridized with the 407-bp probe depicted in A. The presence of a 4.6-kb mutant fragment hybridized to the probe indicates a correctly targeting event. After transient expression of cre in this ES cell clone, two subclones containing a 6.5-kb mutant fragment were detected, indicating the deletion of exons VIII–X in one allele (lane D1-28 or D1-78 in B). C, PCR analysis of tail DNA samples taken from F2 mice. The 955-bp PCR product indicates the presence of the WT VPAC2R gene, and the 579-bp product indicates the presence of the mutant VPAC2R gene. Het, heterozygous.

taining 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 RTPCRs 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 epididymisprostate-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.



FIG. 2. Detection of VPAC2R mRNA expression in WT and KO mice by Northern hybridization analysis and by RT-PCR. A, Schematic diagram of the mouse VPAC2R mRNA indicating the probes and primers used in relationship to the mRNA sequence. Solid arrows labeled with Roman numerals represent exons. Black bars indicate the 387-bp used for BAC library screening and the random primed, full-length [32P]cDNA probe was used for Northern hybridization analysis. B, Northern analysis of brain and lung mRNAs. Two or 4 ␮g of polyadenylated RNA were electrophoresed in each lane, transferred to Hybond-XL membrane, and probed with the full-length VPAC2R cDNA probe labeled with [␣-32P]deoxy-CTP. The RNA blot was subsequently hybridized to an [␣-32P]deoxy-CTPlabeled ␤-actin probe as an internal control to show that the 1.8-kb mRNA was detected at approximately equal signal intensities in all mRNA samples. C, RT-PCR of mouse VPAC2R mRNA. Primers VPAC2RcDNAf and VPAC2RcDNAr were used in an RT-PCR to amplify the cDNA for the WT and mutant VPAC2R. The RT-PCR product was 1.4 kb in WT mice and approximately 1.1 kb in KO mice. D, Schematic representation of the VPAC2R protein sequence showing the putative structure; Roman numerals indicate the protein domains encoded by exons.

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䡠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 coef-

ficients, 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

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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 ⫻ 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 ␮g/liter at a sample dilution of 1:300.

Results

The VPAC2R gene in mouse ES cells was mutated by homologous recombination with a targeting vector construct (Fig. 1A). 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). As it was suggested that the VPAC2R pathway regulates glucose-stimulated insulin secretion in pancreatic ␤-cells (25–27), an OGTT was performed on 2-month-old mice (data not shown) and then again on 6-month-old mice (Fig. 4, 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



FIG. 3. Micrographs of the mouse testis from 31-wk-old male WT (A) and VPAC2R-deficient (B) mice. Testis were fixed in 4% paraformaldehyde, trimmed, processed into paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin for light microscopy. Note the vacuolization of tubules and lack of mature spermatids in the central lumen of the micrograph taken from VPAC2Rdeficient mouse testis (B).

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).

Although no differences in body weight between the WT and KO mice were detected at weaning, significant differences in body weight were noticed in the interval between the second and sixth month of age. To study the effect of VPAC2R deficiency on growth and energy utilization, a group of mice (n ⫽ 8 KO and 8 WT) was selected to compare body weight, energy intake, energy utilization, and body composition over time. The results showed that male KO mice weighed significantly less than their WT siblings beginning at 8 –9 wk of age (Fig. 5A), whereas the mean body

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FIG. 4. Glucose and insulin tolerance testing of WT and VPAC2R KO mice. A and B, Blood glucose measurements from male and female mice, respectively, obtained in OGTT experiments. The mice were fasted overnight, bled to obtain fasting blood samples, and then given 2.5 g/kg glucose by oral gavage. Blood glucose results are plotted vs. elapsed time (30, 60, and 120 min) after oral glucose treatment. C and D, Serum insulin determinations on the same samples obtained in the OGTT experiment (A and B). E and F, Insulin tolerance tests in male or female WT and KO mice. Mice were fasted for 4 h, bled to obtain a fasting glucose specimen, and then injected with 0.75 U/kg Humulin, ip. The glucose levels in each group of mice were obtained after insulin injection (15, 30, 60, and 90 min) and were plotted as a percentage of the average fasting glucose levels in each group. F, Male and female WT mice (n ⫽ 10, each); E, male and female KO mice (n ⫽ 10, each). *, 0.01 ⬍ P ⱕ 0.05; **, 0.001 ⬍ P ⱕ 0.01; ***, P ⱕ 0.001.

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 sexmatched 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). To study the effects of VPAC2R deficiency on fuel utilization, we performed indirect calorimetry experiments on



TABLE 1. Effects of VPAC2R deficiency on body length, food consumption, serum leptin and T3 levels, and basal energy expenditure in mice Male

Body length (cm)a WT KO P Food consumption (g/wk)b WT KO P Leptin (ng/ml)c WT KO P Basal energy expenditure (kcal/h䡠kg)d WT KO P Free T3 (pg/ml)e WT KO P

Female

10.91 ⫾ 0.06 10.08 ⫾ 0.08 0.0002

10.79 ⫾ 0.13 10.21 ⫾ 0.10 0.0029

29.9 ⫾ 0.61 25.3 ⫾ 0.53 0.0022

29.0 ⫾ 0.87 23.4 ⫾ 1.15 0.0048

54.9 ⫾ 6.4 21.8 ⫾ 2.8 0.003

31.2 ⫾ 3.9 14.1 ⫾ 3.7 0.007

7.38 ⫾ 0.26 9.11 ⫾ 0.32 0.0010

9.25 ⫾ 0.31 10.17 ⫾ 0.28 0.0307

0.98 ⫾ 0.086 0.71 ⫾ 0.058 0.0317

0.77 ⫾ 0.047 0.63 ⫾ 0.037 0.0276

a Body length was measured from nose to base of tail in anesthetized mice at 32 wk of age. b Food consumption was average weekly food intake from 14 –18 wk of age. c Leptin samples were obtained from 6-month-old mice. d Basal energy expenditure was the average energy expenditure of the mice at 21 wk of age obtained from the first 6 h immediately following the switch to the light period when locomotor activity was minimal. e Free T3 samples were obtained from 9-month-old mice.

FIG. 5. Growth curves of WT and VPAC2R KO mice [male (A) and female (B)]. Mice were housed singly and were weighed weekly or every other week. All mice were sustained on 5015 Purina mouse chow ad libitum. F, WT control mice (n ⫽ 8); E, VPAC2R KO mice (n ⫽ 8). *, 0.01 ⬍ P ⱕ 0.05; §, 0.001 ⬍ P ⱕ 0.01.

mice. Although RQ (Fig. 6) 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). To determine the effect of VPAC2R deficiency on body composition, wide-line NMR was used to perform body composition analyses of mice at 12 and 22 wk of age. When we examined the percentage of total body weight that was muscle, lean, or water mass, no significant differences were observed in 12-wk-old KO mice compared with their agematched WT siblings (data not shown). However, at 22 wk of age, lean mass as a percentage of body weight was increased 6 – 8% (P ⬍ 0.02) in KO mice compared with their gender-matched WT siblings (Fig. 8), 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 sexmatched WT controls (Table 1). To investigate a potential cause of the growth retardation seen in the VPAC2R KO mice, blood samples were obtained from two groups of mice (12 and 24 wk of age) to measure

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FIG. 6. RQ of male and female WT and VPAC2R KO mice. Indirect calorimetry was performed using Oxymax chambers. The vO2 and vCO2 were measured hourly on air samples taken from each of the chambers. A, Male mice; B, female mice. F, WT control mice (male, n ⫽ 8; female, n ⫽ 7); E, VPAC2R KO mice (male and female, n ⫽ 8); f, dark cycle.

serum IGF-I levels (Fig. 9). 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). Discussion

VPAC2R is expressed in multiple tissues, including those from CNS, aortic endothelium, heart, renal medulla, adrenal cortex, skeletal muscle, adipose tissue, pancreas, testis, ovary, thymus, and T cells. The specific role that VPAC2R plays as a receptor for both VIP and PACAP is largely unknown. As VPAC2R expression occurs at high levels in


␤-cells, VIP or PACAP might act through this receptor to amplify glucose-stimulated insulin secretion (25–27). 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



FIG. 7. EE and total movement of WT and VPAC2R-deficient mice. Indirect calorimetry was performed using Oxymax chambers. The EE [A and B; EE ⫽ calorific value (CV) of oxygen ⫻ vO2, where CV ⫽ 3.815 ⫹ 1.232 ⫻ RQ] was plotted against the elapsed time (hours) in the chamber. Total movements (C and D), as the number of beam breaks detected in 1 h, were plotted against the elapsed time in the chamber. A and C, Males; B and D, females. F, WT control mice (male, n ⫽ 8; female, n ⫽ 7); E, VPAC2R KO mice (male and female, n ⫽ 8); f, dark cycle.

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 ␤-cells, and VIP or PACAP is known to enhance glucose-stimulated insulin secretion (25–27), 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 ␤-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 0.75U/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

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FIG. 8. Determination of fat and lean masses in 22-wk-old WT and VPAC2R KO mice by wide-line NMR (see Materials and Methods for details). NMR measurements were converted to a percentage of total body mass as fat or lean mass, and the mean results were plotted. f, Matching WT control mice (n ⫽ 8); 䡺, VPAC2R KO mice (n ⫽ 8). *, P ⱕ 0.05.

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

FIG. 9. Serum IGF-I levels in WT and VPAC2R KO male (A) and female (B) mice. Serum specimens for IGF-I determinations were obtained from mice at 12 and 24 wk of age. f, Matching WT control mice (n ⫽ 8 –10); 䡺, VPAC2R KO mice (n ⫽ 8 –10).

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 VPAC2Rdeficient 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 cellderived RNA. Goetzl et al. (28) described their VPAC2Rdeficient 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. Acknowledgments We thank Drs. Paul Burn and Jose Caro for supporting and encouraging this research project. We are indebted to Dr. Derek Yang and Ms. JeAnne Bridwell for helpful suggestions, and to Ms. Min Song, Cindy Shrake, Julie Jacobs, and Qing Zhang for their excellent technical assistance. We are also indebted to Drs. Yuguang Shi, Paul Burn, and Garrett Etgen for critical reading of the manuscript. Received March 27, 2002. Accepted June 3, 2002. 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: [email protected].

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1st Gordon Research Conference on Insulin-Like Growth Factors in Physiology & Disease March 9 –14, 2003, Ventura, California Chairs: Derek LeRoith, Terri Wood Program includes opening addresses by C. R. Kahn, R. Baserga, and presentations on IGFs & Cancer (M. Pollak, S. Hankinson, C. Roberts), IGF signaling (R. O’Connor, D. Clemmons), Clinical Disorders & Mutations (G. van den Bergh, A. Moses, C. Camacho-Hubner), Transgenic & Knock-out Approaches (D. LeRoith, M. Accili), IGFBPs (J. Holly, R. Baxter, C. Connover), Gene Transcription & Regulation (P. Rotwein, H. Werner), Development & Tissue Function (D. Hill, T. Wood, J. Pintar, C. Rosen), Aging & Life Span (R. Garafalo, C. Kenyon). Additional oral presentations by young investigators and poster presentations will be selected from submitted abstracts. Application deadline: November 15, 2002. Website: www.grc.uri.edu/programs/2003/insulin.htm