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. 2019 Apr 12;160(6):1363–1376. doi: 10.1210/en.2018-00755

Low IGF-I Bioavailability Impairs Growth and Glucose Metabolism in a Mouse Model of Human PAPPA2 p.Ala1033Val Mutation

Masanobu Fujimoto 1,#, Melissa Andrew 1,2,#, Lihong Liao 1,3, Dongsheng Zhang 1, Gozde Yildirim 4, Patrick Sluss 5, Bhanu Kalra 5, Ajay Kumar 5, Shoshana Yakar 4, Vivian Hwa 1,✉, Andrew Dauber 1,2,✉
PMCID: PMC6507901  PMID: 30977789

Abstract

Bioactive free IGF-I is critically important for growth. The bioavailability of IGF-I is modulated by the IGF-binding proteins (IGFBPs) and their proteases, such as pregnancy-associated plasma protein-A2 (PAPP-A2). We have created a mouse model with a specific mutation in PAPPA2 identified in a human with PAPP-A2 deficiency. The human mutation was introduced to the mouse genome via a knock-in strategy, creating knock-in mice with detectable protein levels of Papp-a2 but without protease activities. We found that the Pappa2 mutation led to significant reductions in body length (10%), body weight (10% and 20% in males and females, respectively), and relative lean mass in mice. Micro-CT analyses of Pappa2 knock-in femurs from adult mice showed inhibited periosteal bone expansion leading to more slender bones in both male and female mice. Furthermore, in the Pappa2 knock-in mice, insulin resistance correlated with decreased serum free IGF-I and increased intact IGFBP-3 concentrations. Interestingly, mice heterozygous for the knock-in mutation demonstrated a growth rate for body weight and length as well as a biochemical phenotype that was intermediate between wild-type and homozygous mice. This study models a human PAPPA2 mutation in mice. The mouse phenotype closely resembles that of the human patients, and it provides further evidence that the regulation of IGF-I bioavailability by PAPP-A2 is critical for human growth and for glucose and bone metabolism.

IGF-I plays key roles in multiple biological processes, with its chief action being promotion of cell proliferation and differentiation. IGF-I also has metabolic effects similar to insulin in modulating glucose metabolism. In circulation, IGF-I is sequestered by IGF-binding protein (IGFBP)-3 or IGFBP-5, and the IGF-I–IGFBP binary complexes form stable ternary complexes with IGF acid-labile subunit (ALS), prolonging the half-life of both IGF-I and IGFBPs (1–4). Because IGFBPs have higher affinities for IGFs than do cell surface receptors [IGF-I receptors (IGF1Rs)] on target tissues, IGFBPs act both as carriers of IGFs and as modulators of IGF-I bioavailability (3, 5). We and others have recently identified the first group of human mutations in pregnancy-associated plasma protein-A2 (PAPPA2) associated with decreased IGF-I bioavailability. PAPP-A2, a circulating protease with specificity for IGFBP-3 and IGFBP-5, belongs to the pappalysin family of metzincin metalloproteinases (6–8). These findings suggested that proteolytic cleavage of IGFBP-3 and IGFBP-5 in the ternary complexes is sufficient to release IGF-I for biological activities (5, 9).

We reported the first human homozygous PAPPA2 mutations (p.D643fs*25 and p.A1033V) in patients who presented with short stature (height range, −2.8 to −3.8 SD scores) despite elevated levels of total IGF-I, IGFBP-3, IGFBP-5, and ALS (10). Free IGF-I, however, was low (10) but increased when patient serum samples were exposed to recombinant PAPP-A2 in in vitro assays (11). Additional clinical features include bone manifestations (thin long bones of the fibulae, tibiae, and femurs), variable low bone mineral density (BMD), and, interestingly, insulin resistance (10, 12, 13). Total PAPP-A2 deficiency has been previously modeled in Pappa2 knockout (KO) mice with impaired postnatal growth (14, 15), high serum total IGF-I (10), and altered IGFBPs (14, 16, 17). However, certain aspects of the prior knock-in (KI) mouse models did not completely mirror the human phenotype. To evaluate the effects of the human missense PAPPA2 p.A1033V mutation on somatic growth and carbohydrate metabolism, we generated the first KI Pappa2 mouse model, employing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system (18). Our detailed analyses of the phenotypic and endocrinological features of the Pappa2 KI mouse, carrying the human PAPPA2 p.A1033V mutation, indicate that the human phenotype, including insulin resistance, is remarkably recapitulated in our mouse model, and they suggest that our mouse KI model would be appropriate for future therapeutic testing.

Materials and Methods

Experimental animals

A Pappa2 p.Ala1034Val KI mouse model of the human PAPPA2 p.Ala1033Val missense mutation (10) was generated utilizing the CRISPR/Cas9 system (Transgenic Animal and Genomic Editing Core, Cincinnati Children’s Hospital Medical Center). The methods for the design of single-guide RNAs (sgRNAs) and donor oligonucleotides and the production of animals were described previously (19). The sgRNA, 5′-GAACGAATGGAAATTGATGtTGCACTGCTCACCTCTCGGCCCAATAGTTCCTGG-3′ (t, the mutation site; BtsαI site, underlined), was selected according to the on-target and off-target scores from the Web tool CRISPOR (http://crispor.tefor.net) (20). The selected sgRNA target sequences were cloned, according to the published method (21), into a modified pX458 vector (plasmid no. 48138) (Addgene, Cambridge, MA) that contains an optimized sgRNA scaffold and a high-fidelity Cas9 (22, 23). Their editing activity was validated by the T7 endonuclease 1 assay in mouse mK4 cells (24), compared side by side with a Tet2 sgRNA that was known to work efficiently in mouse embryos (25). The validated sgRNA was transcribed in vitro, using the MEGAshortscript™ T7 kit (Thermo Fisher Scientific, Waltham, MA), purified by the MEGAclear™ kit (Thermo Fisher Scientific), and stored at −80°C. To prepare the injection mix, we incubated sgRNA and Cas9 protein (Thermo Fisher Scientific) at 37°C for 5 minutes to form ribonucleoproteins and added the single-stranded DNA donor oligonucleotide (Integrated DNA Technologies, Skokie, IL). The final concentrations were 50 ng/μL sgRNA, 100 ng/μL Cas9 protein, and 100 ng/μL donor oligonucleotide. The mutant mice were generated by injecting the mix into the cytoplasm of fertilized eggs of B6D2F2 genetic background, using a piezo-driven microinjection technique (26). Injected eggs were transferred into the oviductal ampulla of pseudopregnant CD1 females on the same day. Pups were genotyped by PCR amplification of exon 8 in Pappa2 and Sanger sequencing with the reverse primer, 5′-CTGTGAACTTTCTGTGTGGATGTG-3′, and restriction enzyme digestion with BtsαI (New England Biolabs, Ipswich, MA). Founders (three males and two females) were bred with wild-type (WT) B6D2F2/J mice (The Jackson Laboratory for Genomic Medicine, Farmington, CT). Heterozygous Pappa2 KI F2 mice were bred giving rise to litters containing homozygous and heterozygous KI littermates as well as WT littermates. Pappa2 KI and WT littermates were genotyped at 3 weeks of age and analyzed blinded as indicated. Animals were housed in a controlled environment with a 12-hour light/12-hour dark cycle with free access to water and a standard chow diet. All animal protocols were conducted in accordance with Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital Medical Center.

Assessment of anthropometric data, body composition, and organ weight

The body weight was measured weekly from 3 to 16 weeks of age. Body length was measured as the distance from the nose to anus every other week from 4 to 16 weeks of age under general anesthesia with isoflurane. Body composition measurements, including fat, lean, free water, and total water masses, were measured in duplicate using EchoMRI™ (EchoMRI, Houston, TX) at one time point between 6 and 12 weeks of age. Blood was collected at 16 weeks of age via cardiac puncture prior to euthanizing under general anesthesia. Liver weight was measured. Femurs also were collected as previously described (27).

IP glucose tolerance test and insulin tolerance test

A glucose tolerance test (GTT) was performed at 12 weeks of age. WT and Pappa2 KI mice were fasted for 14 hours (1600 to 0600 hours) and then injected IP with glucose (50% dextrose injection, 1.5 g/kg body weight) (International Medication Systems, South El Monte, CA). Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes after injection with an Ascensia® Breeze™ 2 glucometer (Bayer, Leverkusen, Germany). An insulin tolerance test (ITT) was performed at 13 to 14 weeks of age. WT and Pappa2 KI mice were fasted for 6 hours (0900 to 1500 hours) and then injected IP with insulin (Humulin R®, 0.75 U/kg body weight; Eli Lilly and Company, Indianapolis, IN). Blood glucose sampling times were identical to the GTT protocol.

Serum assays for total IGF-I, free IGF-I, intact IGFBP-3, GH, and insulin

Serum samples for insulin and glucose measurement were collected from WT and Pappa2 KI mice (15 weeks of age) via cheek punch after a 6-hour fast (0900 to 1500 hours). Blood samples for free IGF-I, total IGF-I, and intact IGFBP-3 measurements were collected from WT and Pappa2 KI mice at 16 weeks of age. Blood samples were placed on ice for 30 minutes to allow for clotting. Serum was separated via centrifugation at 8000 rpm for 10 minutes at 4°C and stored at −80°C until analysis. Serum insulin levels were measured by ELISA (catalog no. 80-INSHU-E01.1) from ALPCO (Windham, NH). Serum GH values were determined using a rat/mouse GH ELISA kit (EZRMGH-45K) from EMD Millipore (St. Louis, MO). Serum mouse free IGF-I, mouse total IGF-I, and intact IGFBP-3 values were determined using commercially available ELISA kits (free rat/mouse IGF-I, catalog no. AL-136; total rat/mouse IGF-I, catalog no. AL-137; intact IGFBP-3, catalog no. AL-149) from Ansh Laboratories (Webster, TX). The total and free IGF-I assays use the same pair of monoclonal antibodies. In the two-step sandwich assay configuration, the capture antibody preferentially binds free IGF-I whereas the detection antibody binds either free IGF-I or IGF-I bound to IGFBP-3. Thus, in the absence of preanalytical treatments to release IGF-I from IGFBP-3, only free IGF-I is quantitated using the Ansh Free IGF-I kit (AL-136). Quantitative measurement of total IGF-I requires preanalytical release of IGF-I bound to IGFBP-3 by acidification using reagents supplied as a separate test kit (AL-137). Neither assay, free or total IGF-I, demonstrates cross-reactivity (<0.01%) with IGFBP-2, IGFBP-3, IGFBP-4, of IGFBP-5. The limit of detection for free IGF-I was 0.155 ng/mL and for total IGF-I was 1.60 ng/mL. The coefficient of variation of replicate determinations was <9% for mouse serum specimens with total IGF-I concentrations between 5.20 and 270 ng/mL and <6% for specimens with free IGF-I concentrations between 0.160 and 12 ng/mL. To validate that the free IGF-I ELISA kit (AL-136) is in fact measuring the free hormone level and not IGF-1 bound to IGFBP-3, we added recombinant mouse IGFBP-3 (catalog no. 775-B3-025; R&D Systems, Minneapolis, MN) in excess (100-fold molar ratio) to serum samples from two WT mouse strains. This resulted in complete elimination of measurable free IGF-I as expected (28). The intact IGFBP-3 assay (AL-149) is a double monoclonal antibody sandwich-type assay and uses an acidification and neutralization preanalytical procedure to dissociate intact IGFBP-3 from all of the binding subunits. The assay demonstrates no significant (<0.1%) cross-reactivity with IGF-I, IGF-2, IGFBP-2, IGFBP-4, or IGFBP-5. IGFBP-3 calibrators in intact IGFBP-3 ELISA are traceable to Non WHO Reference Material IGFBP-3 (National Institute for Biological Standards and Control code 93/560; calibrators, 0.34). The antibodies used in the assay were mapped using six IGFBP-3/IGFBP-5 chimeras. The capture antibody mapped to the C-terminal region whereas the detection antibody mapped to the mid-region. Therefore, this assay does not detect cleaved IGFBP-3 fragments. The lower limit of quantitation was 46.5 ng/mL, and the lower limit of detection was 27 ng/mL. The coefficient of variation of replicate measurements was <8% for mouse serum specimens with IGFBP-3 concentrations between 65 and 2018 ng/mL. All assays were performed according to the manufacturers’ instructions.

Immunoblot for ALS and Pappa2

Mouse serum was collected at 16 weeks of age, and three pooled serum samples were prepared for each genotype to assess the concentration of mouse IGFBP ALS (IGFALS) and Pappa2. Each pooled sample included serum from five animals of WT, heterozygous, and homozygous genotypes, respectively. The pooled sample was diluted with PBS (1:50), and 1 μL of diluted serum sample was solubilized in sodium dodecyl sulfate (SDS) sample buffer [0.5 mol/L Tris (pH 6.8), 2% SDS, 10% glycerol, and 0.003% bromophenol blue] with 100 mM dithiothreitol prior to incubation at 70°C for 10 minutes for immunoblotting of mouse ALS (IGFALS). For immunoblotting of Pappa2, 5 μL of pooled serum was also solubilized in SDS sample buffer described above with 5% β-mercaptoethanol prior to incubation at 95°C for 5 minutes. The diluted and nondiluted samples were resolved on 8% SDS–polyacrylamide gels and electroblotted onto a nitrocellulose membrane. The membrane was blocked with 5% (w/v) nonfat milk or 3% (w/v) BSA and 1× Tris-buffered saline. Primary antibodies for immunoblot analysis were goat anti-mouse IGFALS/ALS polyclonal antibody no. AF1436 (dilution, 1:5000; R&D Systems; RRID: AB_2122936) (29) or mouse anti-human PAPPA2 monoclonal antibody no. ab89899 (dilution, 1:3500; Abcam, Cambridge, MA; RRID: AB_2050158) (30). The anti-human PAPPA2 monoclonal antibody cross-reacts with mouse Pappa2 and is able to detect it. Pierce™ reversible protein stain kit for nitrocellulose membranes (Thermo Fisher Scientific) was used to detect the total amount of protein on the blotted membranes. Horseradish peroxidase–linked donkey anti-goat IgG no. sc-2020 (dilution, 1:5000; Santa Cruz Biotechnology, Dallas, TX; RRID: AB_631728) (31) or Amersham ECL horseradish peroxidase–linked donkey anti-rabbit IgG no. NA934 (dilution, 1:5000; GE Healthcare Biosciences, Pittsburgh, PA; RRID: AB_772206) (32) secondary antibodies were applied, and proteins were detected with Amersham ECL Prime western blotting detection reagent (GE Healthcare Biosciences) according to the manufacturer’s protocol. Densitometric analysis of mouse IGFALS and Pappa2 bands was calculated by ImageJ version 1.52d (National Institutes of Health) and the mean intensity of mouse IGFALS or Pappa2 of WT as control and calculated each relative ratio.

Bone morphology and BMD

Micro-CT was performed as previously described (33). The left femora were scanned using a high-resolution SkyScan micro-CT system (SkyScan 1172; Bruker, Kontich, Belgium). Images were acquired using a 10-megapixel digital detector, 10 W of energy (100 kV and 100 mA), and a 0.5-mm aluminum filter with a 9.7-μm image voxel size. A fixed global threshold method was used based on the manufacturer’s recommendations and preliminary studies, which showed that mineral variation between groups was not high enough to warrant adaptive thresholds. The cortical region of interest was selected as the 2.0-mm mid-diaphyseal region directly below the third trochanter, which includes the mid-diaphysis and more proximal cortical regions. The trabecular measurements were taken at the femur distal metaphysis 2.5 mm below the growth plate.

Statistical analysis

Data are shown as a mean ± SEM. Assay measurements reading less than the limit of detection of the method were assigned a value halfway between 0 and the lower limit of detection of the method. We performed all statistical analyses using GraphPad Prism version 7.04 (GraphPad Software, La Jolla, CA). The mouse population and genotype data were analyzed using a binomial test and χ2 test. The mouse anthropometric data and blood glucose levels in GTT and ITT were analyzed using two-way ANOVA followed by a Dunnett test. Mouse body composition, serum total IGF-I, bioactive IGF-I, intact BP-3, the ratio of free IGF-I to total IGF-I, and the relative intensity ratio of Pappa2 and mouse IGFALS were analyzed using one-way ANOVA followed by a Tukey test. Organ weight, bone length, and BMD were compared via an unpaired t test. A P value threshold of <0.05 was considered statistically significant for all experiments.

Results

Pappa2 KI mice express mutant Papp-a2

A Pappa2 p.A1034V KI mutation modeling of human PAPPA2 p.A1033V missense mutation was generated as described above (Fig. 1A). The synonymous oligonucleotide changes introduced a recognition sequence for BtsαI in the same sgRNA, for ease in screening the KI mutation (Fig. 1A). Five homozygous KI founder mice (three males and two females) were confirmed as KI mice based on the results of restriction enzyme digestion with BtsαI and via Sanger sequencing (Fig. 1B and 1C). Serum Pappa2 was detected in Pappa2 KI mice and WT mice. There were no significant differences in serum levels of Pappa2 by immunoblot analysis (P = 0.46) (Fig. 1D). Interestingly, serum Pappa2 levels appear slightly higher in females compared with males in every genotype (data not shown). As confirmation of our prior work demonstrating that this specific human missense mutation lacked proteolytic activity (10), we also overexpressed recombinant mouse WT and p.A1034V Pappa2 in transfected HEK293 cells. As expected, expressed p.A1034V-mutated Pappa2 lacked proteolytic activity for IGFBP-3 (28).

Figure 1.

Figure 1.

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Generation of Pappa2 KI mice. (A) Schematic representation of PAPPA2 protein structure and PAPPA2 mRNA (exons indicated). Human missense PAPPA2 p.A1033V mutation which was identified in our patients (Pt) is shown in red (top). The human mutation, the introduced variant in mouse, and the corresponding amino acid changes are in red (bottom). Synonymous oligonucleotides introduced for restriction enzyme digestion with BtsαI are in green. The recognition sequence of BtsαI is boxed. Protospacer adjacent motif (PAM) is in blue. (B) DNA gel electrophoretic pattern of the PCR products from extracted mouse DNA after restriction enzyme digestion with BtsαI. The size of the amplicon is 387 bp. Digestion with BtsαI results in a 260-bp fragment and a 127-bp fragment when the KI allele is present (arrow). (C) Electropherogram of human PAPPA2 and mouse Pappa2 of WT, human mutation (from our patient), and homozygous KI mice. Mutated oligonucleotides are indicated by arrows and are boxed. The corresponding amino acid changes are in red. (D) Representative Pappa2 immunoblots of the pooled serum samples for each genotype and densitometric analysis of Pappa2 bands. Each pooled sample included serum from five animals of each genotype (16 wk of age). Densitometric analysis of Pappa2 bands (right panel) immune detected in representative Pappa2 immunoblots (left panel) compared with Pappa2 detected in WT mice, which was arbitrarily assigned a value of 1. Each relative ratio was compared with the mean intensity of Pappa2 in WT mice. aa, amino acid; IB immunoblot; ns, not significant.

Pappa2 p.Ala1034Val missense mutation causes growth retardation

Heterozygous Pappa2 KI F2 mice were interbred, yielding a total of 136 offspring (65 males, 71 females) used in our studies. The genotype frequencies of Pappa2 KI littermates were 22.8% WT, 50.0% heterozygous, and 27.2% homozygous. The observed genotype frequencies were consistent with Hardy–Weinberg equilibrium (χ2 test; P = 0.98). There was no difference between the proportion of males and females (P = 0.69).

At 3 weeks of age, the homozygous Pappa2 KI male offspring showed a significant reduction of body weight compared with WT littermates (homozygous 72% of WT weight; P < 0.028). No significant difference was found in female offspring at that age. The homozygous Pappa2 KI male offspring remained significantly smaller than WT males throughout the study (Fig. 2A). Similarly, the homozygous Pappa2 KI female offspring were significantly smaller than WT females starting at 4 weeks of age (Fig. 2B). At the conclusion of the study, when mice reached 16 weeks of age, the mean body weights of homozygous Pappa2 KI male and female offspring were 89.6% and 77.7% of WT littermates, respectively (male, P < 0.01; female, P < 0.001) (Fig. 1A and 1B). Body length at 16 weeks of the homozygous Pappa2 KI littermates (nose to anus) was significantly smaller than that for WT mice (P < 0.001) (Fig. 2C and 2D) in both sexes (male, P < 0.001; female, P < 0.001). Interestingly, heterozygous Pappa2 KI mice showed an intermediate body length in both sexes, whereas intermediate body weight was found only in heterozygous Pappa2 KI females (Fig. 2).

Figure 2.

Figure 2.

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Postnatal growth curves of Pappa2 KI and WT mice. (A and B) Body weights of WT, heterozygous, and homozygous Pappa2 KI (A) male and (B) female mice. Homozygous Pappa2 KI mice were significantly smaller than their WT littermates from 4 wk of age in both males and females. (C and D) Body length (nose to anus) of WT, heterozygous, and homozygous Pappa2 KI (C) male and (D) female mice. Pappa2 KI mice were significantly shorter than WT mice from 4 wk of age in both males and females. The total number of mice in each group is the sum of three independent cohorts of mice. Values are mean ± SEM (error bars). Statistical significance is based on comparison with WT mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA.

Liver weight and mouse IGFALS levels increased in Pappa2 KI mice

Liver weight (expressed as a percentage of total body weight) was increased in homozygous Pappa2 KI females (WT, 5.25 ± 0.14%; homozygous, 6.10 ± 0.12%; P < 0.001) (Fig. 3A). A similar trend was seen in the male mice, although it did not reach significance. Liver size is greatly influenced by GH action. Because IGFALS serves as a surrogate marker of GH activity (4, 34), we assessed serum levels of mouse IGFALS by immunoblot analysis. Distinctly elevated serum mouse IGFALS levels were detected in homozygous male and female Pappa2 KI mice compared with WT mice (P < 0.001) (Fig. 3B and 3C) (28).

Figure 3.

Figure 3.

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Allometric liver weight and serum mouse IGFALS level in Pappa2 KI and WT mice. (A) Wet liver weight of WT and homozygous Pappa2 KI mice. The liver weight was measured when mice were euthanized at 16 wk of age. The number of mice in each group varied between 13 and 21 mice. Values are mean ± SEM (error bars). Statistical significance is based on comparison with WT mice. ****P < 0.0001 by unpaired t test. (B and C) Serum mouse IGFALS concentrations were compared by immunoblot analysis. The pooled serum sample included serum from five animals of each genotype (16 wk of age). (B) Representative mouse IGFALS immunoblots of the pooled serum samples from each genotype. (C) Densitometric analysis of mouse IGFALS bands immune detected in (B) compared with mouse IGFALS detected in WT mice, which was arbitrarily assigned a value of 1. Each relative ratio was compared with the mean intensity of mouse IGFALS in WT mice in respective sex. Results are shown as mean ± SEM. Statistical significance is based on comparison with WT mice. **P < 0.05, ***P < 0.001, ****P < 0.0001 by one-way ANOVA. IB, immunoblot; ns, not significant.

Body composition

Using EchoMRI to assess body composition, we found a significantly increased percentage of fat mass and decreased percentage of lean mass in homozygous Pappa2 KI mice compared with WT littermates in both sexes (Table 1).

Table 1.

Body Composition in Pappa2 KI and WT Mice at 6 to 12 wk of Age by EchoMRI

Genotype Male Female
WT Heterozygous Homozygous WT Heterozygous Homozygous
N 7 17 12 12 21 13
% Lean mass 83.2 ± 1.3 80.5 ± 1.3 75.9 ± 1.2a 82.0 ± 1.1 81.7 ± 0.7 77.5 ± 0.9a
% Fat mass 12.2 ± 1.0 14.4 ± 1.4 18.8 ± 1.4b 11.1 ± 1.3 11.8 ± 0.7 15.0 ± 0.8b
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Values are means ± SEM.

a

P < 0.01 compared with WT group.

b

P < 0.05 compared with WT group.

IGF-I bioavailability was significantly decreased in Pappa2 KI mice

To determine how the Pappa2 p.A1034V mutation affects IGF-I bioavailability, we measured total IGF-I, free IGF-I, and intact IGFBP-3 in Pappa2 KI mice. As expected, total IGF-I and intact IGFBP-3 levels in serum were significantly increased in homozygous Pappa2 KI mice when compared with WT mice (P < 0.0001) (Fig. 4B and 4D). Serum free IGF-I levels, in contrast, were remarkably decreased in Pappa2 KI mice compared with those of WT littermates (P < 0.0001) (Fig. 4A) (28). The ratio of free IGF-I to total IGF-I, a proxy for IGF-I bioavailability, also showed significant decreases in homozygous Pappa2 KI mice compared with WT littermates (P < 0.0001) (Fig. 4C).

Figure 4.

Figure 4.

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Low serum concentration of free IGF-I and high total IGF-I, GH, and intact IGFBP-3 in Pappa2 KI mice. (A‒E) The mean serum concentration of (A) free IGF-I and (B) total IGF-I, (C) the ratio of free to total IGF-I, and the mean serum concentration of intact (D) IGFBP-3 and (E) GH in Pappa2 KI and WT mice at 16 wk of age. Mean free IGF-I values in homozygous Pappa2 KI mice were significantly reduced compared with WT mice. Mean total IGF-I and intact IGFBP-3 values of homozygous Pappa2 KI mice were significantly higher than those of WT mice. Mean GH value of homozygous Pappa2 KI female mice was significantly higher than that of WT female mice, but not significant in males. The number of mice in each group varied between 12 and 21 mice. Values are mean ± SEM (error bars). Red line indicates the lower limit of detection. Statistical significance is based on comparison with WT mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA. NA, not applicable; ns, not significant.

GH levels were significantly increased in homozygous Pappa2 KI female mice compared with WT littermates (P < 0.0001) (Fig. 4E). However, homozygous Pappa2 KI male mice showed slightly higher GH levels than those of WT and heterozygous KI males, but the difference was not significant. Interestingly, heterozygous mice of both sexes demonstrated an intermediate biochemical phenotype.

Insulin resistance is induced by the Pappa2 missense mutation

To assess whether the Pappa2 functional defects were associated with insulin resistance, we performed IP GTTs and ITTS at 12 to 14 weeks of age. Fasting glucose levels did not differ significantly between controls and Pappa2 KI mice. However, homozygous male Pappa2 KI mice showed significantly higher fasting serum insulin levels when compared with WT mice (28). GTTs and ITTs revealed that both male and female Pappa2 KI mice are glucose intolerant and insulin resistant (P < 0.001, two-way ANOVA) (Fig. 5). Area under the curve was significantly elevated in homozygous Pappa2 KI mice as compared with WT littermates (male, P < 0.001; female, P < 0.05; data not shown).

Figure 5.

Figure 5.

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Pappa2 KI GTT and ITT profiles are consistent with insulin resistance. (A and B) GTTs in Pappa2 KI (A) male and (B) female mice and WT mice at 12 wk of age on a normal diet after 14-h fasting. The blood glucose level was significantly higher in homozygous Pappa2 KI males than in WT males. (C and D) ITTs in Pappa2 KI (C) male and (D) female mice and WT mice at 13 to 14 wk of age on a normal diet after 6-h fasting. Values represent the percentage of baseline glucose concentration. The number of mice in each group varied between 5 and 10 mice. Each point represents mean ± SEM (error bars). Statistical significance is based on comparison with WT mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA.

Pappa2 functional defect leads to bone morphological abnormalities

Bone morphology was studied by micro-CT of femurs dissected from 16-week-old male and female mice. Cortical bone traits taken at the femur mid-diaphysis were more affected than were trabecular bone traits in both male and female Pappa2 KI mice (Table 2). We found that both sexes of Pappa2 KI mice showed reduced total cross-sectional area and bone area as compared with controls, indicating that Pappa2 KI bones are more slender than those of controls. Linear bone growth was reduced in both male and female Pappa2 KI mice as evidenced by decreased bone length. However, when bone length was corrected to radial bone growth (total cross-sectional area/length), we found that female Pappa2 KI bones were less robust, whereas in Pappa2 KI males, where bone length was only marginally affected, bone robustness did not vary from controls. Despite bone slenderness in Pappa2 KI mice, cortical bone thickness was not affected, suggesting that periosteal bone growth was inhibited. However, polar moment of inertia was significantly reduced, suggesting that the Pappa2 KI femurs may be mechanically inferior. Cortical BMD did not differ between controls and the Pappa2 KI mice in both sexes. Cancellous bone was not affected significantly in the Pappa2 KI mice. Bone volume/total volume was similar to controls in both sexes. Trabecular BMD was reduced in both male and female Pappa2 KI mice but reached significance only in male mice.

Table 2.

Micro-CT of Femurs Dissected From Pappa2 KI and WT Mice at 16 wk of Age

Bone Morphology by Micro-CT Male Female
WT (N = 13) Homozygous Pappa2 KI (N = 21) P Value WT (N = 17) Homozygous Pappa2 KI (N = 16) P Value
Cortical bone (femur, mid-diaphysis)
 BV/TV (relative cortical area), % 59.690 ± 0.975 60.659 ± 0.610 0.331 60.220 ± 0.779 63.922 ± 0.856 0.003a
 T.Ar, mm2 1.609 ± 0.0472 1.438 ± 0.043 0.018b 1.422 ± 0.024 1.195 ± 0.027 <0.001a
 B.Ar, mm2 0.959 ± 0.0303 0.869 ± 0.023 0.033b 0.856 ± 0.016 0.763 ± 0.017 <0.001a
 MMI (polar), mm4 0.386 ± 0.0219 0.306 ± 0.018 0.011b 0.288 ± 0.009 0.208 ± 0.008 <0.001a
 Cs.Th, mm 0.222 ± 0.0054 0.221 ± 0.003 0.988 0.223 ± 0.003 0.223 ± 0.004 0.938
 BMD, g/cm2 1.285 ± 0.0082 1.295 ± 0.005 0.367 1.299 ± 0.004 1.303 ± 0.006 0.557
 Length, mm 15.340 ± 0.131 14.734 ± 0.200 0.049b 15.476 ± 0.126 14.751 ± 0.114 <0.001a
 M.Ar, mm2 0.649 ± 0.0261 0.569 ± 0.022 0.028b 0.567 ± 0.017 0.433 ± 0.016 <0.001a
 Robustness, T.Ar/length 0.105 ± 0.0026 0.098 ± 0.003 0.184 0.092 ± 0.002 0.081 ± 0.002 <0.001a
Cancellous bone (femur, distal metaphysis)
 BV/TV, % 16.834 ± 1.211 16.130 ± 0.977 0.977 14.598 ± 0.874 12.937 ± 1.215 0.271
 Tb.Th, mm 0.059 ± 0.001 0.062 ± 0.002 0.002a 0.060 ± 0.001 0.057 ± 0.001 0.022b
 Tb.Sp, mm 0.207 ± 0.007 0.218 ± 0.006 0.006a 0.249 ± 0.009 0.249 ± 0.009 0.968
 Tb.N, 1/mm 2.837 ± 0.162 2.710 ± 0.128 0.128 2.401 ± 0.120 2.236 ± 0.174 0.437
 BMD, g/cm2 0.219 ± 0.010 0.215 ± 0.008 0.008a 0.200 ± 0.007 0.193 ± 0.010 0.577
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Cortical and trabecular bone parameters of femurs were taken at the mid-diaphysis and distal metaphysis, respectively. Values are means ± SEM.

Abbreviations: B.Ar, bone area; BV/TV, bone volume/tissue volume; Cs.Th, cortical bone thickness; M.Ar, medullary area; MMI, mean polar moment of inertia; T.Ar, total cross-sectional area; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.

a

P < 0.01 compared with WT group by unpaired t test.

b

P < 0.05 compared with WT group by unpaired t test.

Discussion

Recently, we reported two families with the first identified human mutations in the PAPPA2 gene resulting in progressive postnatal growth retardation due to decreased IGF-I bioavailability (10). One of the families carried the homozygous missense p.Ala1033Val mutation, which was demonstrated in vitro to completely abrogate the proteolytic activity of PAPP-A2 without abrogating expression. In the present study, we modeled this missense mutation in the first mouse KI model of a bona fide human PAPPA2 mutation. Through comprehensive phenotyping, we demonstrate that an Ala1034Val substitution in mouse Pappa2 had as profound effects on growth and metabolism as was observed in the human patients.

Patients with the homozygous p.Ala1033Val mutation presented with height SD scores between −2.8 and −3.8, with the one female patient being most severely affected. We also reported that two of the three affected siblings had birth lengths below −2 SD score (10). These observations in humans are recapitulated in the mouse, as homozygous Pappa2 p.Ala1034Val KI male and female mice also showed a 10% and 20% reduction in body weight, respectively, compared with WT littermates. Body length was also reduced by 5% to 10%. These findings are similar to a previously reported Pappa2 KO mouse model, which showed a 10% and 25% to 30% reduction in body weight in male and female mice, respectively (14), and 5% to 10% reductions in body length when compared with WT mice (14). The similarity in growth patterns between the Pappa2 KO and our Pappa2 KI models supports the hypothesis that not only does this specific missense mutation lead to complete loss of Pappa2 function, but loss of Pappa2 function significantly impacts normal growth. These findings suggest that Pappa2 defects cause postnatal growth retardation. Moreover, it appears that the growth effect is more prominent in females in both mice and humans, although the human data are limited. The explanation for this sexual dimorphism is unknown. Interestingly, our model showed a gene dosage effect, as the heterozygous mice showed significant, although less prominent, defects in growth parameters. Pappa2 was previously identified in a quantitative trait locus for growth in mice (16), and common genetic variants near PAPPA2 were associated with adult human stature in a large genome-wide association study (35). These reports support the concept that moderate modulation of PAPP-A2 activity could affect stature.

The biochemical manifestation of the human missense PAPPA2 mutation is abolishment of the ability to proteolytically cleave IGFBP-3 and IGFBP-5. This is expected to alter the balance between IGF-I bound to its binding proteins and in its free, bioactive form. In vivo, this correlated with marked elevations of total IGF-I, IGFBP-3, and IGFBP-5 levels as well as low free IGF-I levels and serum IGF bioactivity and extremely low free IGF-I/total IGF-I ratios, as circulating IGF-I remains bound in ternary complexes (10). IGF-I negatively regulates GH secretion from the pituitary gland directly and via increased somatostatin secretion from the hypothalamus (36, 37). The loss-of-function PAPPA2 mutation alters the availability of free IGF-I, thereby decreasing inhibition of GH secretion and, consequently, the patients also demonstrated elevated GH levels. Similarly, the homozygous Pappa2 KI mice demonstrated significantly decreased serum free IGF-I owing to increased intact serum IGFBP-3 and significant elevations of serum ALS, a direct proxy for GH secretion. We found that the elevation of GH levels in Pappa2 KI females was significant, but a significant increase was not found in Pappa2 KI males. Unfortunately, we only had a sufficient sample to measure GH at one point in time, which may not accurately reflect GH pulsatility. Although it is possible to measure GH pulsatility in mice, this is technically difficult and was not undertaken in our experiment. Additionally, GH concentration in serum differs depending on mouse strain, sex, and their age (33, 38). As the half-life of GH is as short as ∼1 hour in mouse serum (39), measurement of relatively stable proteins such as IGFBP-3 and ALS can be used as proxies for estimation of GH concentration and were elevated in the KI mice. It is possible that the GH levels are more profoundly affected in females due to a sexually dimorphic role of Pappa2 that is not yet well understood. These data do support the more severe phenotypic presentation in the female mice. Of note, intact IGFBP-5 could not be measured, as currently no reliable ELISA assays are available. In parallel with the growth parameters, the heterozygous KI mice had intermediate levels of free and total IGF-I as compared with the WT and Pappa2 KI mice. Pappa2 KO mice generated by Conover et al. (14) had significantly elevated total and reduced free IGF-I levels compared with WT mice, but IGFBPs levels were not reported (10). A recent independent Pappa2 KO mouse model generated was reported to have increased total serum IGF-I and IGFBP-5 but with decreased serum IGFBP-3 levels (17), which is inconsistent with our present Pappa2 KI mouse data and with the human data. Yakar and colleagues (33) generated a mouse model in which the KI mutant Igf1 had reduced affinity for IGFBPs, thereby shifting the balance of IGF-I, resulting in increased levels of free IGF-I and reduced total IGF-I and IGFBP-3 serum levels. These KI mice had a significant increase in body weight and length when compared with WT mice.

Patients with the missense mutation in PAPPA2 show mild degrees of insulin resistance with fasting hyperinsulinemia or impaired glucose tolerance testing (10, 12). Our Pappa2 KI mice recapitulated this phenotype with elevated glucose levels when assessed by GTT and ITT. Although the exact mechanism underlying the insulin resistance remains to be fully elucidated, we posit that the elevated GH levels, observed in patients and mice, may play a role. IGF-I normally suppresses GH secretion from the pituitary gland as part of a feedback mechanism (40). In PAPP-A2 deficiency, this feedback is clearly diminished, resulting in increased GH secretion. GH has counterregulatory effects on insulin actions such that GH increases glucose production through gluconeogenesis and glycogenolysis from the liver and kidney, suppresses glucose uptake in adipose tissue, and increases free fatty acid concentrations, which induce insulin resistance (41–43). An alternative explanation for the elevation in blood sugars is impaired IGF-I signaling, as there was significant reduction in IGF-I bioavailability. Human IGF-I has ∼50% homology with proinsulin (44, 45) and can bind to both the IGF1R and insulin receptor, which are also highly homologous (46, 47). High concentrations of free IGF-I can signal via IGF1R, insulin receptor, or an insulin/IGF1R hybrid receptor, resulting in increased glucose transport into the muscle (40, 48). It is possible that the low free IGF-I levels in PAPP-A2 deficiency also decrease signaling via the insulin receptor. In Christians et al.’s (17) Pappa2 KO mouse model, they observed no difference in baseline glucose after a 5-hour fast on chow diet nor did they find any difference in glucose levels at any time point after glucose injection as compared with WT mice. Furthermore, their mouse model did not demonstrate any difference in weight gain or adiposity when the mice were placed on a high-fat diet. These findings contrasted with our results where the KI mice had clear differences in glucose tolerance as well as adiposity. Christians et al.’s (17) KO mouse model also had significant decreases in IGFBP-3 levels despite increases in IGFBP-5 levels when compared with WT mice. This difference in binding protein profile may have additional effects on insulin resistance. The reason for the phenotypic differences in this KO model as compared with our Pappa2 KI model is unclear and may include both methodological differences as well as differences in assays, but the findings in our mouse model are more consistent with the phenotype of human patients with PAPP-A2 deficiency. Further study is needed to determine the full effect of Papp-a2 deficiency on glucose metabolism and to understand the mechanisms underlying the insulin resistance.

The two male subjects carrying the homozygous missense mutation in PAPPA2 had an increased fat mass index or decreased lean body mass index when compared with age-matched healthy boys (10, 12, 49). In the homozygous Pappa2 KI mice, there was also significantly increased percentage fat mass and decreased percentage lean mass compared with WT mice. Moreover, we found a gene dosage effect evidenced in the heterozygous Pappa2 KI mice, which showed an intermediate phenotype. The mechanisms for the body composition changes seen in our mice are not completely known. List et al. (50) reported that the liver-specific GH receptor gene-disrupted mice with increased GH and decreased IGF-I showed increased percentage fat mass until 5 months of age in both sexes. Yakar et al. (51) reported that the liver-specific IGF-I deficiency model and triply deficient liver-specific IGF-I–deficient (LID)/ALSKO/BP3 mice with increased GH and decreased IGF-I showed increased percent fat mass at 8 weeks of age. Our Pappa2 KI mice were analyzed for body composition by 12 weeks of age and dissected at 16 weeks of age. Additionally, IGF1R is highly expressed in preadipocytes, whereas insulin receptor is more highly expressed in mature adipocytes compared with IGF1R (52, 53). Fat-specific Igf1r gene KO mice showed a reduction of white and brown adipose tissues (54). These findings might suggest that the effect of GH on body composition is not solely via IGF-I. GH affects insulin action in liver, muscle, and fat. The interplay between GH, IGF, and insulin depends on the age and sex of the mice. The data presenting the body composition was observational and not mechanistic. It is of note that the administration of recombinant human IGF-I for 12 months in one of the subjects improved his body composition with increased lean body mass and decreased percentage body fat and normalize GTT response (12).

The liver is one of the well-recognized target organs of GH. GH administration in WT mice as well as LID mice induced a significant increase in relative liver weight despite low circulating IGF-I levels (55). The LID female mice showed a more robust whole-body growth increase in response to GH administration than did LID males, indicating a sexual dimorphism in GH action in mice. We observed a similar sexual dimorphism in our mice, with the homozygous Pappa2 KI females having a significantly enlarged liver compared with WT females. A similar, but not significant, trend was seen in KI males. Likewise, Pappa2 KO mice had significantly increased liver size (14). GH receptor expression in the liver has been demonstrated to be higher in female rats than in males, and expression in male rats increased with estrogen treatment (56, 57). Thus, it is possible that the difference in liver weights in our mouse model and its sexual dimorphism might be a combination of increased GH levels in Pappa2 KI mice and the differences in GH receptor expression between females and males.

Our study indicates that PAPP-A2 regulates bone growth. Loss-of-function Pappa2 mutation in our KI mouse model was associated with impaired bone architecture. Similar to the patients with the PAPPA2 mutations who presented with thin long bones, we found that the Pappa2 KI mice exhibited slender femurs in both sexes. Additionally, we show decreased linear growth with shorter femurs in both sexes without effects on cortical or trabecular BMD in females, and only a mild decrease in trabecular BMD in male mice. Our data are in line with those observed with the Pappa2 KO mice generated by Conover et al. (14) where adult femur and body length were reduced, but without significant effects on BMD. In Pappa2 KI mice, cortical bone thickness was similar to that in WT mice, suggesting that reduced radial bone growth may be a result of decreased periosteal bone expansion, but this needs to be confirmed. Indeed, most mouse models with decreased serum IGF-I show inhibited radial bone growth (58). We speculate that the impaired IGF-I axis in the Pappa2 KI mice leads to similar consequences.

In conclusion, our results demonstrate that PAPP-A2 deficiency leads to growth retardation as well as insulin resistance, altered body composition, and impaired bone morphology, as consequences of low IGF-I bioavailability. Our KI model recapitulates the human phenotype seen in patients with the homologous missense mutation in PAPPA2 and will be used for evaluation of possible treatment modalities.

Acknowledgments

We thank Takahisa Nakamura and Joan Sánchez-Gurmaches in the Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, for technical advice.

Financial Support: This work was supported by the National Institutes of Health/National Institute of Child Health and Human Development Grant R01HD078592 (to V.H.).

Author Contributions: M.A., V.H., and A.D. conceived of the presented idea. M.F., M.A., D.Z., and S.Y. performed the measurements and the dissection and then processed and analyzed the experimental data. L.L. performed the recombinant Pappa2 and PAPP-A2 proteolytic IGFBP-3 assays. S.Y. and G.Y. performed the measurement for bone and then analyzed them. P.S., B.K., and A.K. performed the validation assays of free IGF-I and intact IGFBP-3 ELISA kits and then analyzed them. A.D., V.H., and S.Y. were involved in planning and supervision of the work. All authors discussed the results and contributed to the final manuscript.

Disclosure Summary: A.D. and V.H. have a patent for the use of recombinant PAPPA2 as a growth-promoting agent. P.S., B.K., and A.K. are employees of Ansh Laboratories. The remaining authors have nothing to disclose.

Glossary

Abbreviations:

ALS

acid-labile subunit

BMD

bone mineral density

Cas9

clustered regularly interspaced short palindromic repeats–associated protein 9

CRISPR

clustered regularly interspaced short palindromic repeats

GTT

glucose tolerance test

IGFALS

IGF-binding protein acid-labile subunit

IGFBP

IGF-binding protein

IGF1R

IGF-I receptor

ITT

insulin tolerance test

KI

knock-in

KO

knockout

LID

liver-specific IGF-I–deficient

PAPP-A2

pregnancy-associated plasma protein-A2

SDS

sodium dodecyl sulfate

sgRNA

single-guide RNA

WT

wild-type

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