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Endocrinology, doi:10.1210/en.2003-0745
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Endocrinology Vol. 144, No. 11 4682-4689
Copyright © 2003 by The Endocrine Society

Lack of Insulin-Like Growth Factor I Exaggerates the Effect of Calcium Deficiency on Bone Accretion in Mice

Yuji Kasukawa, David J. Baylink, Jon E. Wergedal, Yousef Amaar, Apurva K. Srivastava, Rongqing Guo and Subburaman Mohan

Musculoskeletal Disease Center, J. L. Pettis Veterans Affairs Medical Center (Y.K., D.J.B., J.E.W., Y.A., A.K.S., R.G., S.M.), Loma Linda, California 92357; and Departments of Medicine (D.J.B., S.M.), Biochemistry (S.M.), and Physiology (S.M.), Loma Linda University, Loma Linda, California 92350

Address all correspondence and requests for reprints to: Subburaman Mohan, Ph.D., Musculoskeletal Disease Center (151), J. L. Petttis Veterans Administration Medical Center, 11201 Benton Street, Loma Linda, California 92357. E-mail: mohans{at}lom.med.va.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies provide evidence that the GH/IGF-I axis plays a critical role in the regulation of bone accretion that occurs during puberty and that the peak bone mineral density (BMD) is dependent on the amount of dietary calcium intake during the active growth phases. To evaluate whether IGF-I deficiency exaggerates the effect of calcium deficiency on bone accretion during active growth phases, IGF-I knockout (KO) and wild-type (WT) mice were fed with low calcium (0.01%) or normal calcium (0.6%) for 2 wk during the pubertal growth phase and were labeled with tetracycline. The low calcium diet caused significant decreases in endosteal bone formation parameters and a much greater increase in the resorbing surface of both the endosteum and periosteum of the tibia of IGF-I KO mice compared with WT mice. Accordingly, femur BMD measured by dual energy x-ray absorptiometry or peripheral quantitative computed tomography increased significantly in IGF-I WT mice fed the low calcium diet, but not in IGF-I KO mice. IGF-I-deficient mice fed the normal calcium diet showed elevated PTH levels, decreased serum 1,25-dihydroxyvitamin D and serum calcium levels at baseline. Serum calcium changes due to calcium deficiency were greater in IGF-I KO mice compared with WT mice. PTH levels were 7-fold higher in IGF-I KO mice fed normal calcium compared with WT mice, which was further elevated in mice fed the low calcium diet. Treatment of IGF-I-deficient lit/lit mice with GH decreased the serum PTH level by 70% (P < 0.01). Based on these and past findings, we conclude that: 1) IGF-I deficiency exaggerates the negative effects of calcium deficiency on bone accretion; and 2) IGF-I deficiency may lead to 1,25-dihydroxyvitamin D deficiency and elevated PTH levels even under normal calcium diet.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OSTEOPOROSIS is characterized by a low bone mineral density (BMD) leading to higher bone fragility where fractures occur after minimal trauma (1, 2, 3). There are two pathogenic mechanisms involved in the development of both postmenopausal (type I) and senile (type II) osteoporosis: 1) low peak BMD achieved at the end of young adulthood that increases the risk of osteoporosis; and 2) a rapid bone loss rate at the menopause and/or later in life (1, 2, 3).

Puberty is a critical time of life for bone accretion. Past studies in both humans and experimental animal models demonstrate that BMD increases by about 40–50% during the period of sexual maturation (4, 5, 6, 7). In terms of mechanisms that contribute to the rapid increase in bone mass during puberty, it is generally accepted that about 70% variation in peak BMD can be attributed to genetic differences (8, 9, 10). Regarding potential regulatory molecules that contribute to the rapid acquisition of BMD during postnatal growth, sex steroid hormones and the GH/IGF axis have received much attention in the past. In this regard, recent studies using mice deficient in IGF-I, IGF-II, and GH provide direct experimental evidence that some aspects of the GH/IGF system are involved throughout prepubertal, pubertal, and postpubertal growth phases in regulating the raise in peak BMD and bone size, two important determinants of bone strength (11, 12).

Besides genetic influence, other variables that influence bone accretion during active growth phases include physical activity, calcium intake, diet, vitamin D intake, and interaction between environmental and genetic factors (8, 9, 10). Of the various dietary factors, calcium intake has an important impact on the increase in bone mass that occurs during the postnatal growth period (13, 14, 15, 16, 17). In this regard, several clinical studies have demonstrated that calcium supplementation enhanced bone mineral status in adolescent girls (13, 14, 15, 16, 17). The aim of this study was to test the hypothesis that the combination of IGF-I deficiency and calcium deficiency exerts a greater negative impact on bone accretion during the pubertal growth period compared with IGF-I deficiency alone. To examine this hypothesis, we tested the effect of calcium deficiency on bone metabolism in mice lacking functional IGF-I and corresponding control mice during the period of sexual maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The calcium-deficient diet (TD95027; <0.01% calcium by weight) and the control diet (TD97191; containing 0.6% calcium by weight) were purchased from Harlan Teklad (Madison, WI). Both diet formulations include 0.4% phosphorus, 17.5% protein (17.4% from casein and 0.1% from corn starch), and 6.3% fat (6% from soybean oil, 0.2% from casein, and 0.1% from corn starch) by weight. Tetracycline hydrochloride and demeclocycline were purchased from Sigma-Aldrich Corp. (St. Louis, MO). {alpha}MEM, fetal bovine serum, and DMEM were purchased from Life Technologies, Inc. (Gaithersburg, MD). BSA was purchased from Fluka (Buchs, Switzerland). Recombinant human IGF-I (tissue culture grade) was purchased from GroPep (Adelaide, Australia). All other chemicals used were at least reagent grade.

Animals
Breeder mice heterozygous for the IGF-I knockout (KO) allele were supplied by A. Efstratiadis (Columbia University, New York, NY). The surviving IGF-I-null pups (~25%) were identified by characteristic small size at birth and a failure to grow after birth (18), and this was confirmed by PCR analysis using the IGF-I forward primer (5'-CCACAGGCTATGGCTCCAGCA TTC-3'), the IGF-I reverse primer (5'-GTCAGTGTGGCGCTCGGCAC-3'), and the neo reverse primer (5'-ATCCATCTTGTTCAATGGCCGATCCC-3'), yielding a 450-bp product for the disrupted IGF-I gene and a 160-bp product for the wild-type (WT) gene (19). Heterozygous littermates were genotyped and used for breeding, and the WT littermates were used as control animals in this study. The animals were housed in a controlled environment with 12-h light, 12-h dark cycles at 70 F. The mice were killed by CO2 inhalation, followed by cervical dislocation, at which time blood, bilateral femurs and tibiae, lumbar vertebrae, and calvaria were collected. The experimental procedures performed in this study were in compliance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the animal studies subcommittee at the J. L. Pettis Veterans Administration Medical Center (Loma Linda, CA).

In vivo experiments
Experiment 1.
IGF-I KO and WT mice were weaned at 3 wk of age. At 4 wk of age, IGF-I KO and WT mice were divided into three treatment groups (n = 8 mice of each type/group): 1) the baseline control group (killed at the start of study); 2) the low calcium group, which received the low calcium diet (0.01% calcium) for 2 wk; and 3) the normal calcium group, which received the normal calcium diet (0.6% calcium) for 2 wk. With the exception of the baseline group, all mice were double-labeled with tetracycline and demeclocycline. The fluorescent labels were injected ip 3 and 8 d before death. Body weight was measured with an electronic balance (Scout SC2020, OHAUS, Florham Park, NJ) at the beginning of the experiment and at death. Serum was collected for measurements of calcium and PTH. The left tibiae were dissected and cleaned of soft tissue for quantitative bone histomorphometry. The right femora and tibiae were dissected and used for bone density measurement and geometric parameter determination. The femur length was measured using a caliper (Dial Caliper, Fisher Scientific, Hampton, NH) before bone density measurement.

Experiment 2.
Serum samples were available from a separate study in which GH-deficient lit/lit (mutation in the GHRH receptor) mice were treated with vehicle or GH for 2 wk (20). The lit/lit mouse is a dwarf mouse characterized by no detectable levels of circulating GH and, as a consequence, by low serum and bone IGF-I levels (20). GH was given three times a day at a cumulative dose of 4 mg/kg body weight. Serum samples collected at the end of vehicle or GH treatment were used for PTH measurements in this study.

Preparation of bone samples
In each mouse the left tibia was defleshed and embedded in glycol methacrylate. A thin cross-section (0.5 mm thickness) was removed just proximal to the fibular junction with a diamond wire Histo-saw (Delaware Diamond Knives, Wilmington, DE). The cross-sections were lightly ground and stained for tartrate-resistant acid phosphatase (TRAP). After measurement of TRAP-covered surfaces, the cross-sections were further ground to 50 µm and mounted in aqueous Flourmount-G (Fisher Scientific, Pittsburgh, PA) for measurements of bone areas and tetracycline labels.

Histomorphometry
All bone histomorphometric parameters were measured with the OsteoMeasure system equipped with a digitizing tablet (Osteo-Metrics, Atlanta, GA) and a color video camera (Sony, Kansas City, MO) as described previously (21). The total medullary area (square millimeters), periosteal perimeter, and endosteal perimeter (millimeters) were determined from the ground sections. To evaluate the activity of bone resorption, the sections were stained for TRAP detection and then coded for quantitative measurement. We measured the endosteal or periosteal TRAP-positive surface as the absolute length of the TRAP-positive surface and calculated the percent TRAP-positive surface to total endosteal and periosteal surface (%Ec. TRAP and %Ps. TRAP). We also measured the periosteal and endosteal bone-forming parameters (the exception being the basal control group, which was not measured). The mineralizing surface (MS; millimeters) was calculated as the sum of the length of double labels (dL) plus half the length of single labels (sL) along the entire endosteal or periosteal bone surfaces: MS = dL + 0.5sL. The mineral apposition rate at the endosteal or periosteal surface (microns per day) was calculated by dividing the mean width of double-fluorescent labels by the interval (5 d). The bone formation rate at the endosteal or periosteal surface was calculated by multiplying the mineral apposition rate by tetracycline-labeled surface and was expressed as square millimeters x 10-3 per day. Because there was a large difference in the size of bone between the KO and WT mice, the bone formation data were presented in two ways: 1) using the endosteal or periosteal surface referent, and 2) using a total cross-sectional referent. Histomorphometric indexes were based on nomenclature recommended by the American Society of Bone and Mineral Research (22).

Measurement of BMD
The BMD of right femur and tibia were measured by dual energy x-ray absorptiometry (DEXA; PIXImus instrument, Lunar Corp., Madison, WI). The precision was ±1% coefficient of variation in vitro (12).

The volumetric bone density and geometric parameters were determined by peripheral quantitative computed tomography (pQCT; Stratec XCT 960M, Norland Medical Systems, Fort Atkinson, WI). Previously, pQCT has been validated as a reliable method for determining volumetric bone density and geometric parameters in mice (23, 24). Routine calibration was performed daily with a defined standard (cone phantom) containing hydroxyapatite embedded in Lucite (Norland Medical Systems, Inc.). Analysis of the scans was performed using the manufacturer-supplied software program (Stratec Medizintechnic, Bone Density Software, version 5.40 C, Norland, Madison, WI). Volumetric bone density and geometric parameters were estimated with Loop analysis. The thresholds were set at 230–630 mg/cm3 for total BMD measurement. The voxel size was set at 0.07 mm, and a 0.5-mm-thick slice was scanned through the entire length of bone. The reference line as a center of scanning was set at the midpoint of femur; thereafter, the nine slices were scanned symmetrically from the reference line. The data for each of the three slices from distal, middle, or proximal were taken as an average and were expressed as distal metaphysis, middiaphysis, or proximal metaphysis. The coefficients of variation for total BMD, periosteal circumference, and endosteal circumference for repeat measurements of four femurs (two to five measurements) were 3%, 1%, and 2%, respectively (12, 23, 24).

Biochemical assays
Serum calcium.
Serum calcium levels were measured by a colorimetric method using O-CPC as substrate (Wako Chemicals, Richmond, VA). Calcium standards from Sigma-Aldrich Corp. were used as the calibrator (25). All specimens were diluted 1:4 to 1:8 before calcium measurements. The linear range of the assay was 1–15 mg/dl. The intraassay variation (n = 20) for the control sample was less than 5%.

Serum PTH.
PTH levels were determined by a sandwich ELISA (Immunotopics, Inc., San Clemente, CA) designed for measurement of mouse PTH with slight modifications. We used 50 µl serum from a pool made by combining serum samples from two or three animals, instead of the 25 µl serum recommended by manufacturer (Immunotopics, Inc.). In addition, the lowest standard was diluted 2-fold with zero standard and used as an additional calibrator to allow measurements at lower PTH concentration. All samples were analyzed in one assay. The intra- and interassay coefficients of variation were less than 14%.

Serum 1,25-dihydroxyvitamin D3[1,25-(OH)2D3].
1,25-Dihydroxyvitamin D measurements were made using an RIA kit (DiaSorin, Inc., Stillwater, MN), which requires extraction of serum samples on a C18 column (provided by the same manufacturer). The extraction procedure removes lipids, protein, salts, pigments, 25-hydroxyvitamin D [25(OH)D], 24,25-(OH)2D3, and 25,26-(OH)2D3. In brief, pooled serum from five to eight animals (400–500 µl) were treated with acetonitrile, precipitated proteins were removed by centrifugation, and supernatant was applied to a C18 column primed with methanol. After washing the column with methanol/water, hexane/methylene, and hexane/isoporpanol (99:1) the purified 1,25-(OH)2D is eluted with hexane-isopropanol (92:8), dried, reconstituted in a buffer, and used for RIA. All samples were analyzed in one assay to minimize variation. According to the package insert provided with the kit, the sensitivity of the RIA procedure is 4 pg/ml, and the cross-reactivity of the antiserum is 100% with 1,25-(OH)2D3 and 1,25-(OH)2D2, 0.002% with 25-(OH)D3, 0.012% with 24,25-(OH)2D3, and 0.003% with 25,26-(OH)2D3. The intraassay variation is less than 16%.

Vitamin D receptor (VDR) gene expression
Total RNA was isolated from pooled kidneys from IGF-I KO and corresponding control mice using the TRIzol reagent (Invitrogen, Carlsbad, CA). One to 2 µg total RNA were used as a template for RT-PCR. The first strand cDNA synthesis was prepared using oligo(deoxythymidine) primer as outlined in the Omniscript RT Kit (QIAGEN, Valencia, CA). The first cDNA strand synthesized was used as a template for amplifying cDNA fragments using specific gene primers for VDR and ß-actin: for 273-bp VDR cDNA amplification, the forward primer 5'-ATCGCCATCCTGCTCGATGC-3' and the reverse primer (5'-CAGCATGGAGAGCGGAGA-3'; for 154-bp ß-actin cDNA amplification, the forward primer 5'-CAGGCATTGCTGACAGGATG-3' and the reverse primer 5'-TGCTGATCCACATCTGCTGG-3' were used. The number of PCR cycles chosen was based on preliminary experiments, such that the amounts of PCR products were within the linear range of amplified products under the conditions used in this study. The PCR products obtained were electrophoresed on a 2% agarose gel under standard conditions and stained with ethidium bromide. The intensity of the band was measured using ChemiImager 4400 Low Light Imaging System (Alpha Innotech Corp., San Leandro, CA).

Statistical analysis
The data are expressed as the mean ± SEM. Statistical analysis of the data was performed by unpaired t test or Fisher’s protected least significant difference method (post hoc test) for multiple comparisons in a two-way ANOVA as appropriate. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of calcium deficiency on body weight and femoral bone length
To determine whether calcium depletion influences overall growth, we measured initial and final body weights and femur length in IGF-I KO and WT mice. The initial body weights of the three different groups in IGF-I KO or WT mice were similar (Table 1Go). Body weight as well as femur length increased similarly in IGF-I KO mice fed with low calcium diet compared with the normal calcium diet. Similar results were obtained with WT mice (Table 1Go). Thus, calcium deficiency did not affect body weight or femur length in either IGF-I KO or WT mice.


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  		TABLE 1. Initial and final body weight and femoral bone length in IGF-I KO or WT mice

 
Effects of calcium deficiency on bone resorption parameters
Calcium depletion for 2 wk caused significant increases in the medullary area and endosteal perimeter in both IGF-I KO and WT mice (P < 0.05 and P < 0.001, respectively) compared with corresponding control mice fed a normal calcium diet (Table 2Go). Treatment with the low calcium diet caused a much greater increase in the %Ec. TRAP (resorbing surface) in IGF-I KO mice (144% of normal calcium-treated KO mice; P < 0.01) compared with WT mice (46% of normal calcium-treated WT mice, P < 0.05; Fig. 1AGo). Comparison of bone resorption parameters in IGF-I KO vs. WT mice revealed that %Ec. TRAP was significantly lower in IGF-I KO mice fed a normal calcium diet compared with WT mice. On the other hand, although TRAP-positive surface was absent at the periosteum of WT mice, approximately 10% of the surface was covered with TRAP-positive osteoclasts in IGF-I KO mice (Table 2Go). Although the TRAP-positive surface at the periosteum was increased by 65% in IGF-I KO mice fed the low calcium diet compared with the normal calcium diet, this increase was not statistically significant (Fig. 1BGo).


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  		TABLE 2. Bone histomorphometry of tibia in IGF-I KO or WT mice before and during 2 wk of low or normal calcium diet

 


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  		FIG. 1. Effects of dietary calcium deficiency on bone resorption and formation parameters at the endosteal (A) or periosteal (B) surface measured by bone histomorphometry. Bone histomorphometry was performed in tibia at the fibular junction. The endosteal and periosteal bone parameters in IGF-I KO or WT mice fed the low calcium diet are compared with those in corresponding control mice fed the normal calcium diet and are expressed as the mean ± SEM (n = 5–8). A, P < 0.05 vs. WT mice, by t test.

 
Effects of calcium deficiency on bone formation parameters
To evaluate the effects of calcium depletion on bone formation, the endosteal and periosteal bone formation parameters were evaluated in low or normal calcium-treated IGF-I KO or WT mice (Table 2Go). The 2 wk of calcium depletion significantly decreased endosteal bone formation parameters in IGF-I KO mice (60%; P < 0.05), but not in IGF-I WT mice (7%; P = 0.68), compared with the normal calcium-treated mice (Fig. 1AGo). Although calcium depletion decreased endosteal bone formation parameters in IGF-I KO mice, it had no effect on periosteal bone formation parameters (Fig. 1BGo). The periosteal bone formation rates, but not the periosteal mineral apposition rate, were significantly decreased in IGF-I KO mice compared with WT mice under normal dietary calcium conditions (Table 2Go).

Effects of calcium deficiency on femur BMD and related parameters measured by DEXA and pQCT
The period (4–6 wk) chosen for calcium depletion represents an active growth period for BMD accretion in mice (7). Based on the findings that bone formation and bone resorption are uncoupled in IGF-I KO mice fed the low calcium diet, we predicted impaired bone accretion in these mice. DEXA measurements revealed that 2 wk of calcium depletion caused significant decreases in the femoral BMD, bone mineral content (BMC), and bone area in IGF-I KO (16%, 51%, and 44%, respectively) and WT (21%, 30%, and 10%, respectively) mice compared with the corresponding control mice fed a normal calcium diet. BMC and bone area increased significantly during calcium deficiency in WT mice, but not in IGF-I KO mice. BMD, BMC, and bone area in IGF-I KO mice treated with the low or normal calcium diet were significantly lower (P < 0.0001) than those in corresponding IGF-I WT mice (by post hoc test). These parameters in tibia showed a similar trend as in femur (Table 3Go).


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  		TABLE 3. BMD, BMC, and bone area by DEXA in IGF-I KO or WT mice

 
The 2 wk of calcium depletion caused significant decreases in the total volumetric BMD (vBMD) measured by pQCT at the distal metaphysis, middiaphysis, and proximal metaphysis in both IGF-I KO mice (31%; P < 0.001) and WT (23–26%; P < 0.001) mice compared with the corresponding control mice fed the normal calcium diet, except at the middiaphysis in IGF-I KO mice (Table 4Go). Under normal dietary calcium conditions, significant increases in total vBMD occurred at all three sites in both IGF-I KO and WT mice compared with the corresponding baseline control mice. The total vBMDs of femur in IGF-I KO mice were significantly lower than those in IGF-I WT mice (P < 0.0001) in both low and normal calcium-treated groups (by post hoc test).


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  		TABLE 4. Femur BMD and geometrical parameters by pQCT in IGF-I KO or WT mice

 
Although the periosteal circumferences in IGF-I KO mice were increased by 2 wk of treatment with a normal calcium diet (9–18%), the dietary calcium deficiency for 2 wk in IGF-I KO mice did not increase the periosteal circumferences at all three sites of femur (Table 4Go). On the other hand, in IGF-I WT mice, the 2 wk of treatment with a low calcium diet as well as the normal calcium diet significantly increased the periosteal circumference at the middiaphysis (12–15%) and proximal metaphysis (10–12%). Although 2 wk of dietary calcium deficiency in IGF-I WT mice significantly increased the endosteal circumference at the middiaphysis and proximal metaphysis, the calcium deficiency in IGF-I KO mice did not affect the endosteal circumference at all three sites of femur (Table 4Go). Both periosteal and endosteal circumferences in IGF-I KO mice were significantly lower than those in IGF-I WT mice (P < 0.0001).

Effects of calcium deficiency on serum calcium and PTH levels
Serum calcium levels were significantly lower in IGF-I KO mice compared with WT mice at baseline (9.92 ± 0.48 vs. 10.59 ± 0.49 mg/dl; P = 0.02). Figure 2Go shows that serum calcium levels were significantly reduced in both IGF-I KO and WT mice fed the low calcium diet compared with mice fed the normal calcium diet. However, the change in serum calcium due to calcium deficiency was significantly greater in IGF-I KO mice compared with WT mice.



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  		FIG. 2. Serum calcium deficit in IGF-I KO and WT mice fed a low calcium diet. Four-week-old mice were treated with normal calcium or low calcium diet for 2 wk, and serum calcium levels were measured at the end of the treatment period. The values are expressed as the percent change from the corresponding group of mice fed the normal calcium diet and are the mean ± SEM (n = 7–8). A, P < 0.01 compared with the normal calcium diet, by t test. B, P < 0.05 compared with WT mice, by t test.

 
We next measured serum PTH levels in IGF-I KO and WT mice fed low or normal calcium diets to determine the cause of decreased serum calcium levels in low calcium diet-treated IGF-I KO mice. Dietary calcium deficiency caused significant increases in serum PTH level in both IGF-I KO and WT mice compared with corresponding control mice fed the normal calcium diet (Fig. 3Go). Serum PTH levels were significantly higher in IGF-I KO mice fed the low calcium diet compared with WT mice. Furthermore, serum PTH levels in IGF-I KO mice fed the normal calcium diet were seven times higher than those in IGF-I WT mice fed a similar diet (Fig. 3Go).



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  		FIG. 3. Serum PTH levels in IGF-I KO and WT mice fed a low calcium or normal calcium diet. Serum PTH levels were measured after 2 wk of treatment with different diets. Serum from two or three IGF-I KO mice were pooled before analysis. The values are expressed as the mean ± SEM (n = 4–8). A, P < 0.01 compared with the corresponding control group fed the normal calcium diet, by post hoc test. B, P < 0.01 compared with the corresponding WT mice, by post hoc test.

 
Effects of IGF-I deficiency on serum 1,25-(OH)D levels and VDR expression in kidney
To determine whether the increased PTH levels in IGF-I KO mice fed the normal calcium diet can be explained on the basis of 1,25-(OH)D deficiency, we measured serum levels of 1,25(OH)D in IGF-I KO and WT mice at baseline. Figure 4Go shows that the serum 1,25-(OH)D level in IGF-I KO was significantly lower (24%; P < 0.05) than that in IGF-I WT mice.



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  		FIG. 4. Serum 1,25-(OH)2D3 levels in IGF-I KO and WT mice at baseline. Serum samples were collected from 4-wk-old IGF-I KO and WT mice and were pooled from five to eight animals. The values are expressed as the mean ± SEM (n = 4). A, P < 0.05 compared with the WT mice, by t test.

 
In previous studies we and others found that IGF-I treatment increased the expression of VDR in a number of cell types in vitro (26, 27, 28, 29). The reduction in VDR expression would lead to corresponding decreases in the responsiveness of target cell types to 1,25-(OH)D. We therefore evaluated VDR gene expression in the kidneys of IGF-I KO and corresponding age-matched control mice. Figure 5Go shows that the expression level of VDR was reduced by 70% in IGF-I KO mice compared with control mice. In contrast, the expression of the ß-actin control gene was not reduced in IGF-I KO mice.



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  		FIG. 5. VDR mRNA expression is reduced in the kidneys of IGF-I KO mice compared with corresponding age-matched control mice. Two micrograms of total RNA pooled from IGF-I KO (lanes 3 and 4) and corresponding age-matched control mice (lanes 1 and 2) were used for RT-PCR using VDR-specific (lanes 1 and 3) and ß-actin-specific (lanes 2 and 4) primers. An aliquot of PCR product after 36 cycles was run on 2% agarose and stained with ethidium bromide. The data shown here were confirmed in a separate independent experiment.

 
Effects of GH treatment on serum PTH levels in GH-deficient lit/lit mice
If increases in serum PTH levels in IGF-I KO mice fed a normal calcium diet are due to dysregulation of mechanisms involved in the maintenance of serum calcium caused by 1,25-(OH)D deficiency, then correcting the IGF-I deficiency should eliminate hyperparathyroidism in IGF-I KO mice. To evaluate this possibility, we measured serum PTH levels in GH-deficient lit/lit mice treated with vehicle or GH. Because of a mutation in the GHRH receptor, lit/lit mice exhibit a severe deficiency in both endocrine and local IGF-I actions (20). As expected, serum PTH levels were elevated in GH-deficient lit/lit mice (55.1 vs. 13.1 pg/ml; P < 0.01) compared with corresponding control mice. PTH levels were significantly lower in lit/lit mice treated with GH for 2 wk compared with vehicle-treated lit/lit mice (Fig. 6Go).



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  		FIG. 6. Serum PTH levels in GH-deficient lit/lit mice treated with vehicle () or GH ({square}). Postpubertal (7-wk-old) and adult (29-wk-old) GH-deficient lit/lit mice were treated for 2 wk with vehicle or GH (4 mg/kg·d). At the end of treatment, blood was removed and used for PTH measurements. The values are expressed as the mean ± SEM (n = 4). A, P < 0.05 compared with the GH-treated mice, by t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies show that dietary calcium deficiency during the pubertal growth period causes an impairment in bone accretion and that this effect is exaggerated during IGF-I deficiency. During 2 wk of study, femur BMD, measured by DEXA, increased by 35% and 7%, respectively, in WT mice fed normal calcium and low calcium diets, suggesting that bone accretion is impaired during calcium deficiency. Bone accretion measured by DEXA is 17% and 0%, respectively, in the femur of IGF-I KO mice fed normal calcium and low calcium diets. Consistent with these data, total volumetric BMD increased significantly in the middiaphysis of femur of WT, but not IGF-I KO, mice during 2 wk of the low calcium diet. The effects of a low calcium diet on bone accretion in IGF-I KO mice are specific to bone, as the low calcium diet did not affect the increase in body weight in IGF-I KO mice.

In terms of the mechanisms that contribute to impaired bone accretion in IGF-I KO mice during dietary calcium deficiency, our histomorphometric data provide evidence for increased bone resorption and decreased bone formation to be the causes. Our studies demonstrate that the endosteal bone resorption rate is increased to a greater extent in IGF-I KO mice compared with control mice during calcium deficiency (144% vs. 46%; P < 0.01). Furthermore, periosteal bone surface covered with active osteoclasts is seen in IGF-I KO, but not in control, mice during calcium deficiency. Based on previous findings that GH/IGF-I treatment increased the formation and activity of osteoclasts in vitro (30, 31, 32) and that GH/IGF-I treatment increased bone resorption parameters besides bone formation parameters in vivo, we had predicted a role for IGF-I in the regulation of osteoclastic bone resorption. However, there was no evidence of impaired bone resorption in IGF-I KO mice fed the low calcium diet, suggesting that mechanisms other than IGF-I are involved in regulating the formation and activation of osteoclasts during calcium deficiency.

Interestingly, bone-resorbing surface at the endosteum, as measured by %Ec.TRAP, was significantly lower in the baseline IGF-I KO group compared with WT mice despite elevated PTH levels. The reduced bone resorption in IGF-I KO mice at baseline may reflect PTH resistance caused by IGF-I deficiency-induced 1,25-(OH)D deficiency. Further studies are needed to evaluate whether correcting 1,25-(OH)D deficiency would increase bone resorption and correct PTH levels in IGF-I KO mice fed a normal calcium diet at baseline.

One of the key findings of our study is that the bone formation rate is severely compromised in IGF-I KO mice compared with control mice during calcium deficiency. Endosteal mineral apposition rate and bone formation rates were both reduced by 40% after 2 wk of dietary calcium deficiency in IGF-I KO mice but not in control mice. The mechanisms that contribute to decreased bone formation in IGF-I KO mice fed the low calcium diet can only be speculated at this time. In this regard, we have found that serum hypocalcemia was significantly greater in IGF-I KO mice fed with low calcium diet compared with control mice. Furthermore, PTH levels were significantly greater in IGF-I KO mice fed the low calcium diet compared with control mice. Thus, the greater serum calcium deficiency along with elevated PTH levels could contribute to the greater impairment in bone formation in IGF-I KO mice fed the low calcium diet compared with control mice (33, 34).

Surprisingly, PTH levels were elevated 7-fold in IGF-I KO mice fed the normal calcium diet compared with corresponding control mice. In terms of the causes of the elevated PTH levels in IGF-I KO mice fed the normal calcium diet, we found that the serum 1,25-(OH)D level was significantly lower in IGF-I KO mice at baseline compared with control mice despite elevated PTH levels. Consistent with these data, several studies have shown evidence that IGF-I regulates 1,25-(OH)D levels in vitro and in vivo (35, 36, 37, 38, 39, 40, 41, 42, 43). The reduction in the expression of VDR seen in the kidneys of IGF-I KO mice would further reduce the activity of 1,25-(OH)D. If VDR expression is also reduced in other tissues, such as intestine, of IGF-I KO mice, we could then predict that the 1,25-(OH)D-deficient state caused by IGF-I deficiency would lead to reduced calcium absorption in the intestine, decreased serum calcium level, and, consequently, increased PTH production to correct serum calcium levels. Alternatively, elevated PTH levels in IGF-I KO mice fed the normal calcium diet could be due to direct actions of 1,25-(OH)D deficiency and/or IGF-I deficiency on parathyroid glands or could be due to PTH resistance.

If IGF-I deficiency is the cause of the elevated PTH levels, we should be able to correct PTH levels by eliminating IGF-I deficiency. We therefore evaluated this prediction by raising IGF-I levels in GH-deficient lit/lit mice by correcting GH deficiency. We used lit/lit mice rather than IGF-I KO mice for this study because homozygous IGF-I KO are difficult to generate due to poor reproduction of heterozygous IGF-I KO mice and poor survival (20%) of homozygous IGF-I KO after birth. We found that GH treatment for 2 wk decreased serum PTH levels by 70–80% compared with vehicle treatment in lit/lit mice. Consistent with these data, several clinical studies have shown that GH administration decreased serum PTH levels in both GH-deficient and normal subjects (38, 44, 45). Furthermore, Fatayerji et al. (46) recently demonstrated that serum IGF-I levels showed significant negative correlation with serum PTH levels in 178 healthy men, aged 20–79 yr. Although these data provide evidence that IGF-I deficiency could lead to secondary hyperparathyroidism, the exact mechanism by which IGF-I deficiency leads to an increase in serum PTH levels cannot be discerned without further studies.

In conclusion, our studies using mice deficient in IGF-I demonstrate that the impairment in bone accretion during calcium deficiency is exaggerated due to IGF-I deficiency and that PTH levels are elevated during IGF-I deficiency caused in part by 1,25-(OH)D deficiency. The confirmation that IGF-I treatment could correct the 1,25-(OH)D and calcium deficit in IGF-I KO mice fed a normal calcium diet and restore normal PTH levels would provide further support to the hypothesis that IGF-I is important for normal 1,25-(OH)D action and that secondary hyperparathyroidism in senile osteoporosis is due in part to IGF-I deficiency.


   Acknowledgments
 
We acknowledge the expert technical assistance provided by Jann Smallwood, Alice Kramer, Joe Rung-Aroon, Nancy Lowen, and Aurora Petrila, and the secretarial assistance of Sean Belcher.


   Footnotes
 
This work was supported by funds from the NIH, National Research Service Award AR-31062 from the NIAMS, and the Department of Veterans Affairs. All work was also performed with facilities provided by the Department of Veterans Affairs.

Abbreviations: BMC, Bone mineral content; BMD, bone mineral density; DEXA, dual energy x-ray absorptiometry; %Ec. TRAP and %Ps. TRAP, percent TRAP-positive surface to total endosteal and periosteal surface; KO, knockout; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; 25(OH)D, 25-hydroxyvitamin D; pQCT, peripheral quantitative computed tomography; TRAP, tartrate-resistant acid phosphatase; VDR, vitamin D receptor; vBMD, volumetric BMD; WT, wild-type.

Received June 13, 2003.

Accepted for publication July 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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