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. 2015 Jun 15;156(8):2872–2879. doi: 10.1210/en.2014-1945

IGF-1 Receptor Insufficiency Leads to Age-Dependent Attenuation of Osteoblast Differentiation

Lee-Chuan C Yeh 1, Matthew Wilkerson 1, John C Lee 1, Martin L Adamo 1,✉
PMCID: PMC4511128  PMID: 26076041

Abstract

In the current study, we determined the effects of IGF-1 receptor haploinsufficiency on osteoblast differentiation and bone formation throughout the lifespan. Bone mineral density was significantly decreased in femurs of male and female Igf1r+/− mice compared with wild-type mice. mRNA expression of osteoblast differentiation markers was significantly decreased in femurs and calvariae from Igf1r+/− mice compared with cells from wild-type mice. Bone morphogenetic protein-7-induced ectopic bone in Igf1r+/− mice was significantly smaller with fewer osteoblasts but more lipid droplets and had reduced expression of osteoblast differentiation markers compared with wild-type mice. In bone marrow cells from middle-aged and old wild-type and Igf1r+/− male mice, palmitate inhibited osteoblast markers expression. In cells from young wild-type male mice, palmitate did not inhibit marker expression, but in cells from young male Igf1r+/− mice, palmitate inhibited bone sialoprotein and osterix but not osteocalcin or type I collagen (TIC). In female wild-type mice, palmitate inhibited osteoblast markers expression in cells from young, middle-aged, and old mice except TIC in cells from middle-aged mice. Palmitate inhibited bone sialoprotein expression in cells from middle-aged and old female Igf1r+/− mice and osteocalcin, osterix, and TIC expression in young and middle-aged female Igf1r+/− mice but stimulated expression in cells from old female Igf1r+/− mice. We conclude that IGF-1 receptor haploinsufficiency results in a prolipid accrual phenotype in bone in association with inhibition of growth factor-induced osteoblast differentiation, a situation which may phenocopy age-related decreases in bone formation.

Aging is a major risk factor for osteoporosis and novel strategies for countering age-related bone loss are currently being sought (1). GH and IGF-1 signaling play major roles in aging and maintenance of skeletal integrity. Paradoxically, GH-deficient/resistant mice exhibit robust increases in lifespan (2) but show deficiencies in bone growth (3). GH-deficient/resistant mice show a secondary reduction in serum IGF-1 levels, but the effect of primary IGF-1 deficiency on longevity in mice is controversial (4–6). Nonetheless, mouse models of IGF-1 deficiency uniformly show deficits in bone growth (3, 6).

Complete ablation of the IGF-1 receptor (IGF-1R) in osteoblasts reduces bone mineralization (7), but effects of this genetic manipulation on overall survival or bone formation throughout the lifespan were not reported. Whole-body Igf1r null mice do not survive postnatally, but Igf1r heterozygous (Igf1r+/−) mice do survive, and Igf1r+/− mice on the 129 background were reported to live longer (8) than wild-type littermates. However, we observed little or no lifespan extension of Igf1r+/− mice on either the C57Bl/6J or C57 × 129 F1 hybrid genetic backgrounds (9). Although Igf1r+/− mice on both genetic backgrounds showed reduced body weight (8, 9), they have not been examined for effects on bone formation. None of the genetic models of GH deficiency, resistance, or IGF-1 deficiency or resistance have been studied throughout the lifespan for effects on skeletal integrity.

The reasons for the lack of robust lifespan extension in the models of primary IGF-1 deficiency or resistance are currently unknown but may relate to impaired carbohydrate metabolism and lipotoxicity. Igf1r+/− mice on the C57Bl/6J background exhibited an age-related development of insulin resistance in association with increased adiposity and ectopic lipid accrual in muscle and liver (9 and N. Garg, M.L. Adamo, unpublished data). Female Igf1r+/− mice exhibited an increased susceptibility to high fat diet-induced insulin resistance suggesting aberrant fatty acid metabolism in Igf1r+/− mice (10). Lipotoxicity has been proposed as a contributing factor to osteoporosis (11) and cell culture studies have shown that fatty acids can decrease osteoblast survival and differentiation (12–17). We postulate therefore that Igf1r+/− mice may exhibit increased sensitivity to fatty acid-induced attenuation of osteoblast differentiation.

In addition to mediating the effects of IGF-1 on bone growth, the IGF-1R may mediate the anabolic effects of bone morphogenetic proteins (BMPs). IGF-1 and BMPs synergistically stimulate osteoblast differentiation (18–20), and BMP induces IGF-1 system components in osteoblasts (21). Moreover, osteoblasts from aged mice and rats are resistant to the anabolic effects of IGF-1 and BMP (22, 23). Thus, we further postulate that deficiency of the IGF-1R may inhibit BMP-induced new bone formation. The purpose of the present study was to test these hypotheses by characterizing bone mineral density (BMD) in vivo and osteoblast differentiation marker expression in calvariae and femurs from wild-type and Igf1r+/− mice at various ages. We also characterized BMP-induced ectopic bone formation in wild-type and Igf1r+/− mice. Additionally, we examined osteoblast differentiation of bone marrow progenitor cells from male and female wild-type and Igf1r+/− mice at various ages in response to palmitate. Our results indicate that BMP-7-induced ectopic bone in Igf1r+/− mice is smaller, less differentiated, and contains more lipid than ectopic bone from wild-type mice and support a role for the IGF-1R in growth factor-stimulated bone formation.

Materials and Methods

Materials

Cell culture media and fetal bovine serum were purchased from Invitrogen and Gemini Bio Products, respectively. Other reagents were obtained from Sigma. BMP-7 was obtained from Stryker.

Animals

Mice were handled and killed according to the protocol approved by the Institutional Animal Care and Use Committee of The University of Texas Health Science Center at San Antonio. Mice were maintained in a controlled environment of 12-hour light, 12-hour dark cycles at 22°C. Standard irradiated rodent chow (Harlan Teklad LM-485) and water were provided ad libitum. The Igf1r+/− mice (ie, mice in which 1 copy of the gene coding for the IGF-1R is disrupted) were originally provided by Dr Argiris Efstradiatis (Columbia University College of Physicians and Surgeons, New York, NY) who derived them in a 129Sv background by homologous recombination, which ablated the third exon of the Igf1r gene (24). Mice lacking both copies die shortly after birth but the Igf1r+/− mice were reported to be phenotypically normal. Colony of mice for the present study was established as previously described (9). The genotype of each mouse was determined by PCR mainly as described (24). Age-matched littermates were used as controls. Ages of mice used for the present study were young (5–6 mo), middle-aged (14–15 mo), and old (24 mo).

Dual-energy x-ray absorptiometry

Whole-body densitometry on 12-month-old mice (wild type n = 6, Igf1r+/− n = 6) was performed using the PIXIMUS densitometer (GE Lunar Corp) equipped with software version 2.10. The head was excluded from all regions of interest in the analyses. Femur area BMD values were determined from the whole-body scans using the appropriate regions of interest.

Ectopic bone formation

A total of 8 wild-type and 8 Igf1r+/− male and female mice was administrated a single injection of BMP-7 (40 μg) sc in the dorsal area (23). After 21 days, the animals were terminated following the protocol approved by the Institutional Animal Care and Use Committee of The University of Texas Health Science Center at San Antonio. The mass at the site of injection (ectopic bone) was collected, and frozen in liquid nitrogen immediately until used for RNA isolation.

Bone marrow cell (BMC) culture

Femurs were removed and the proximal and distal ends were cut using sterile technique. BMCs were collected by centrifugation of the femurs and cultured in α Minimum Essential Medium with 15% fetal bovine serum, plus 100 U/mL of penicillin and 100 μg/mL of streptomycin sulfate, at 37°C with 5% CO2 as previously described (17, 25). To study the effect of palmitate, BMC cultures cells were cultured in medium as described above plus 5mM β-glycerol phosphate and 100-μg/mL ascorbic acid and in the absence or presence of palmitate (100μM).

Quantitative RT-PCR (qRT-PCR) analysis

Isolation of total RNA and analysis using qRT-PCR were as published (25, 26). Briefly, total RNA was isolated from cell cultures using the TriReagent (Sigma) according to the manufacturer's instructions. Total RNA from calvariae and femurs were isolated from pulverized tissues which were frozen in liquid N2 immediately after harvesting from the animals and stored in the frozen state until used. Reverse transcription was performed using High Capacity cDNA kit (Applied Biosystems). All reagents and instrumentation for gene expression analysis were from Applied Biosystems. Quantitative PCR was conducted using a 7500 Fast Real-Time PCR System and predesigned TaqMan Gene Expression Assays according to the manufacturer's specifications. Reference numbers for assays are: alkaline phosphatase (AP) (Mm00475834_m1), bone sialoprotein (BSP) (Mm00492555_m1), Runt-related transcription factor 2 (Runx2) (Mm00501584_m1), osteocalcin (OCN) (Mm03413826_mH), osterix (OSX) (Mm04209856_m1), osteopontin (OSP) (Mm01204014_m1), type I collagen (TIC) (Mm00801666_g1), IGF-1 (Mm00439560_m1), IGF-1R (Mm00802831_m1), and β-2 microglobulin (Mm00437762_m1). Expression analysis of genes of interest was measured using the delta delta cycle threshold method of relative quantification. Each cell culture replicate was run in triplicate real-time quantitative PCRs for both genes of interest and β-2 microglobulin.

Statistics

Data are expressed as mean ± SEM. In all instances, P < .05 is considered statistically significant. An ANOVA for repeated measures using mixed models was used to compare all the groups' means on various parameters. Specific pairwise comparisons were done with Bonferroni and Tukey-Kramer methods using the appropriate between and within mouse variability from the analysis. The Statistical Analysis System, version 9.3 for Windows, was used for all these analyses.

Results

General characteristics of the Igf1r+/− male and female mouse

Body weights of male and female Igf1r+/− mice were not significantly different from the respective wild-type mice (Figure 1A). However, both wild-type and Igf1r+/− female mice weighed less (∼20%, P < .05) than their respective male counterparts. Figure 1B shows that the nose to anal lengths (NALs) of both wild-type and Igf1r+/− female were significantly shorter than their respective male counterparts (P < .05). Moreover, NALs of both male and female Igf1r+/− mice are also shorter than the respective wild type. The femur length of the Igf1r+/− male and female mice was significantly less than the wild-type control (P < .05). Moreover, femur length of female Igf1r+/− mice was significantly lower than that of the male Igf1r+/− (Figure 1C). The femoral BMD was significantly lower (P < .05) in male Igf1r+/− mice but not in female Igf1r+/− mice compared with the respective wild-type liter mates (Figure 1D). BMD of wild-type female was significantly lower (P < .05) than wild-type male.

Figure 1.

Figure 1.

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General characteristics of the wild-type and Igf1r+/− young male and female mouse. A, Body weight. B, NAL. C, Femur length. D, Femur BMD. Dual-energy x-ray absorptiometry (DEXA) was used to determine BMD in 12-month-old male (n = 6) and female (n = 6) wild-type and Igf1r+/− mice. *, P < .05 is significant, comparing male vs female of the same genotype; #, P < .05 is significant, comparing the wild-type male or female vs the Igf1r+/−.

Expression of osteoblast differentiation markers and IGF-1/IGF-1R in calvariae and femur of young (4–5 mo) and old (24 mo) male wild-type and Igf1r+/− mice.

mRNA expression of IGF-1, IGF-1R, and 2 osteoblast markers in calvariae of young and old male wild-type and Igf1r+/− mice was measured. A phenotypic reduction in IGF-1R expression in young Igf1r+/− mice was observed (Figure 2). Moreover, old wild-type mice had reduced IGF-1R expression compared with young wild type, whereas old Igf1r+/− mice showed the greatest reduction in IGF-1R expression compared with wild type. Similar regulation of IGF-1 mRNA was observed with a reduction in the young Igf1r+/− mice and further reductions in old wild-type and old Igf1r+/− mice, respectively. Expression of mRNAs for osteoblast differentiation markers OSX and OCN was reduced in calvariae from young and old male Igf1r+/− mice compared with age-matched wild-type mice (Figure 2). The expression of osteoblast differentiation markers in femurs from male and female wild-type and Igf1r+/− mice was determined (Figure 3). In both genders, mRNA expression of Osx, Runx2, AP, OCN, and OSP was significantly reduced in Igf1r+/− mice compared with wild type.

Figure 2.

Figure 2.

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Expression of osteoblast differentiation markers and IGF-1/IGF-1R in calvariae of young and old male wild-type and Igf1r+/− mice. Total RNA was isolated from calvariae as described in Materials and Methods. IGF-1, IGF-1R, OCN, and OSX mRNA levels were measured by qRT-PCR as described in Materials and Methods. For each marker gene, values were normalized to that of the young wild type as 1.0. Values represent the mean ± SEM of n = 6 for young (4–5 mo) and old (24 mo) male wild-type and Igf1r+/− mice. *, P < .05, compared with the wild type, is significant.

Figure 3.

Figure 3.

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Osteoblastic marker mRNA expression in the femur from young (A) male and (B) female wild-type and Igf1r+/− mice. Experimental conditions are similar to those described in Figure 2. Osx, Runx2, AP, OCN, and OSP mRNA expression levels were determined by qRT-PCR as described in Materials and Methods. For each marker gene, values were normalized to that of the wild type as 1.0. Values represent the mean ± SEM of n = 6 for male wild-type and Igf1r+/− mice. *, P < .05, compared with the wild type, is significant.

BMP-7-induced formation of ectopic mineralized bone nodules in male and female wild-type and Igf1r+/− mice

Given our previously published observations, and those of others (18), that BMP-7 and IGF-1 synergistically stimulated osteoblast differentiation and converged on similar signaling pathways, we wished to determine whether BMP-7-induced ectopic bone formation was altered in Igf1r+/− mice. As shown in Figure 4, A and B, the weight and size of bone nodules induced by BMP-7 were significantly smaller in Igf1r+/− mice compared with wild-type mice. Moreover, in the ectopic bone in both male and female Igf1r+/− mice the expression of osteoblast differentiation markers was significantly reduced compared with wild type (Figure 4, C and D). Histologic analysis of the ectopic bone revealed that the nodules from both the wild-type and Igf1r+/− mice exhibited evidence of normal bone formation, including the presence of osteocytes and osteoblasts (Figure 5). However, the BMP-7-induced ectopic bone from Igf1r+/− mice contained greater numbers of lipid droplets than that from wild type.

Figure 4.

Figure 4.

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Characteristics of BMP-7-induced ectopic bone nodules. A, Representative photo of the excised nodules. B, Wet weight of the excised nodules. C and D, Osteoblastic marker mRNA expression levels in ectopic bone nodules from male and female wild-type and Igf1r+/− mice. Ectopic bone nodules at the site of a single BMP-7 injection from 8 male and female wild-type and Igf1r+/− mice were collected. Total RNA was isolated and analyzed by qRT-PCR for expression of OSX, Runx2, AP, OCN, and OSP as described in Materials and Methods. For each marker gene, values were normalized to that of the young wild type as 1.0. Values represent the mean ± SEM. *, P < .05, compared with the wild type, is significant.

Figure 5.

Figure 5.

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Histology of ectopic bone harvested 3 weeks after injection from wild-type and IGF1r+/− mice injected with a single dose of BMP-7. Bone slices were stained with H&E. The arrows point to marrow adipose.

Effects of palmitate on mRNA expression of osteoblastic markers in wild-type and Igf1r+/− young, middle-age, and old mice

To further test the hypothesis that IGF-1R deficiency leads to bone cell lipotoxicity, we examined the effects of palmitate on osteoblast differentiation markers in BMCs cultured from young, middle-age, and old wild-type and Igf1r+/− mice. Figure 6A shows that palmitate either increased (BSP, OSX, and OCN) or had no effect (TIC) on osteoblast marker expression in cells from young male wild-type mice. In cells from male middle-age and old wild-type mice, palmitate reduced the mRNA expression of all 4 osteoblast differentiation markers (Figure 6A). In cells from young male Igf1r+/− mice palmitate attenuated the expression of BSP and OSX but did not alter OCN or TIC (Figure 6B). Palmitate reduced the expression of all osteoblast markers in cells from middle-aged and old male Igf1r+/− mice with the exception of TIC in cells from middle-age mice (Figure 6B). Female wild-type mice at all 3 ages exhibited decreased osteoblast differentiation markers with the exception of type I collagen in the middle-age mice (Figure 7A). In contrast, old female Igf1r+/− mice were protected from reduction in expression of osteoblast differentiation markers with the exception of BSP (Figure 7B).

Figure 6.

Figure 6.

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Effects of palmitate on mRNA expression of osteoblastic markers in (A) wild-type and (B) Igf1r+/− (Het) young (4–5 mo), middle-aged (14–15 mo), and old (24 mo) male mice. BMCs were collected from the femur of male wild-type and Igf1r+/− young, middle-age, and old male mice (n = 6–8 in each group) and cultured in α Minimum Essential Medium plus 15% FBS followed by addition of control (DMSO) and palmitate (100μM). Cultures were terminated 48 hours after initiation of treatment. Total RNA was isolated, and the steady-state mRNA expression levels of osteogenic markers, BSP, OCN, OSX, and TIC were determined by qRT-PCR. The value for TIC of the middle-aged wild-type male was below detection. All data were calculated using the ΔΔCT method and compared with endogenous expression of β-2 microglobulin. To allow comparison of results of different ages, data were plotted as a ratio of palmitate/control. A ratio less than 1.0 indicates inhibition of expression by palmitate. *, P ≤ .01 is significant, when comparing control vs treated; #, P ≤ .05 is significant, when comparing young vs old.

Figure 7.

Figure 7.

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Effects of palmitate on mRNA expression of osteoblastic markers in (A) wild-type and (B) Igf1r+/− (Het) young, middle-age, and old female mice. Experimental conditions are similar to those described in Figure 6.

Discussion

Our results show that IGF-1R heterozygosity leads to IGF-1R haploinsufficiency with respect to osteoblast differentiation and bone formation in vivo. Our results are consistent with previously published findings that IGF-1R deficiency or IGF-1 ligand deficiency compromises bone formation (3, 6). Specifically, osteoblast specific IGF-1R knockout was reported to lead to deficiencies in the ability of osteoblasts to mineralize (7). The published studies used an OCN driving cre delete, which is expressed relatively late in the osteoblastic lineage; thus, our data using germline-directed haploinsufficiency of the IGF-1R are predicted to show a decrease in osteoblast differentiation. We did not observe any significant decrease in body weight of our sample of Igf1r+/− mice. By comparison published studies reported about a 10% decrease in body weight (8, 9). The difference is likely due to a smaller sample size in the present study.

Importantly, in the current study, we observed deficits in bone formation throughout the lifespan of Igf1r+/− mice, confirming that IGF-1 signaling plays important roles in bone maintenance as animals age. These findings along with published data (6) strongly suggest that deficiencies in IGF-1 action lead to deleterious effects on aging bone. We observed that Igf1 mRNA expression but not Igf1r mRNA expression is significantly lower in old wild-type mice than in young mice. To the best of our knowledge this is the first report of age-dependent changes in Igf1 mRNA expression in bone. In published studies (9) we observed the expected approximately 50% reduction in Igf1r mRNA in soft tissues of Igf1r+/− mice. In the current study, we observed a 30%–40% reduction in Igf1r mRNA in cells derived from the calvarial bone of young and old mice, respectively. We do not know the reason for this less-than-expected decrease in Igf1r mRNA. It is possible that expression from the intact Igf1r allele is up-regulated in the calvarial bone of Igf1r+/− mice.

We have previously shown that BMP alters expression of IGF system components in calvarial cells and that IGF-1 and BMP synergistically stimulate calvarial osteoblastic cell differentiation (19, 20). Others have also reported on the synergistic relationship between IGF-1 and BMP with respect to osteoblast differentiation (18). Herein for the first time, we show that the BMP-7-induced new bone formation is attenuated in IGF-1R-deficient mice. Specifically, we used an ectopic bone formation assay to show that ectopic bone induced by BMP-7 was smaller and had reduced expression of osteoblast differentiation markers in Igf1r +/− mice compared with wild type. These results suggest that an intact IGF-1R is required for the full effect of BMP on bone formation. In preliminary studies using calvarial cells we have observed that BMP-7 alone does not significantly stimulate phosphorylation of the IGF-1R, although BMP-7 and IGF-1 produced a greater increase in IGF-1R phosphorylation than did IGF-1 alone (L.C.C. Yeh, M.L. Adamo, J.C. Lee, unpublished results). Moreover, previously published studies did not show any effect on BMP-7 on Igf1 receptor mRNA expression (19). Relevant also is our previous finding that BMP-7-induced osteoblast differentiation requires a rapamycin-sensitive signaling pathway involving phosphorylated 70 kDa ribosomal protein S6 kinase activation (27, 28). Others have reported that rapamycin promotes osteoblast differentiation by blocking mammalian target of rapamycin (mTOR) pathway and stimulating the BMP pathway (29). IGF-1 has been shown to stimulate the mTOR/phosphorylated 70 kDa ribosomal protein S6 kinase signaling system (27). Given our observation that BMP-7 alone does not induce expression or activate phosphorylation of the IGF-1R, it is unclear how the IGF-1R could be permissive for BMP activation of the mTOR signaling pathway. Elucidation of pertinent mechanisms will require extensive further study.

Our findings that the BMP-7-induced ectopic bone in the Igf1r+/− mice showed increased levels of lipid droplets and decreased osteoblasts suggest that the decreased bone formation was due to enhanced adipogenic phenotype. The observations further suggest that deficiencies in IGF-1 action could lead to aberrant lipid metabolism in bone. Similar results have been reported in mice with lower serum IGF-1 levels (30). Moreover, we have observed increased adiposity and ectopic lipid in muscle and liver of Igf1r+/− mice (N. Garg, M.L. Adamo, unpublished data). These results are consistent with the current idea that fatty acids inhibit osteogenesis and that age-related declines in bone formation and increased risk for osteoporosis are due to replacement of bone forming cells with adipose tissue (31, 32). Congruent with this idea is our previous observations that the long chain saturated fatty acid palmitate reduced mRNA expression of Runx2, AP, OCN, and BSP as well as the BMP-7-stimulated mineralized bone formation in fetal rat calvaria cell cultures (17, 27). In the current study, we found that palmitate inhibited expression of osteoblast markers in general in cells from both wild-type and Igf1r+/− male mice. There was some variation in the response to palmitate, which we are currently unable to explain. Except for BSP, the reduction in osteoblast marker expression by palmitate appeared to be similar in cells from old wild-type and old Igf1r+/− mice. This result suggests that the lipotoxic mechanisms were maximal as a function of age in wild-type mice and were not further enhanced by IGF-1R heterozygosity. Female mice showed reduction in osteoblast marker expression in response to palmitate at all 3 ages, although the degree of suppression was variable. In contrast to males however, IGF-1R heterozygosity actually imparted protection from the lipotoxic effects of palmitate on OCN, OSX, and TIC expression in cells from old mice. BSP expression remained significantly suppressed by palmitate in cells from female Igf1r+/− mice at middle and old ages. Reasons for this apparent sexual dimorphism are unclear at the present time and require more extensive investigation.

The mechanisms of aberrant lipid management to decrease osteoblast differentiation are unknown. Palmitate has been reported to interfere with mothers against decapentaplegic signaling (15), but the precise lesion in or the products of fatty acid metabolism causing this defect is currently poorly understood. We have previously observed an increase in lipogenic enzymes in liver and muscle of aging Igf-1r+/− mice and that Igf1r+/− mice are more sensitive to fatty acid-induced insulin resistance than wild type (33, 34). Studies from other laboratories have indicated that the inhibition of triglyceride accumulation may prevent the deleterious effects of fatty acids on bone cells (35, 36). Moreover, the AMP kinase activator 5-aminoimidazole-4-carboxamide-1 beta-riboside prevented palmitate-induced apoptosis in fetal osteoblast cells (17). However, this effect did not appear to require increased fatty acid oxidation. Moreover, the apoptotic effect of palmitate was prevented by an acyl coenzyme A (acyl-CoA) synthetase inhibitor but not by a ceramide synthesis inhibitor. These observations suggest that increased fatty acyl-CoA causes a negative effects of fatty acids on bone cells. Fatty acyl-CoA, diacylglycerol, and ceramide have all been suggested to activate protein kinase C (37). In turn, protein kinase C catalyzes phosphorylation of insulin receptor and insulin receptor substrate 1 resulting in inhibition of insulin signaling (38). Studies are currently underway in our laboratory to determine whether IGF-1 signaling is similarly blocked in osteoblasts concomitant with accumulation of lipid in aging osteoblasts.

In summary, we show that IGF-1R deficiency impairs osteoblast differentiation in both genders and in bone formed via both intramembranous and endochondral pathways as well as BMP-7-induced bone formation. These results could have important implications for the pathogenic effects of age-related lipid accrual on skeletal frailty and increased risk for osteoporosis.

Acknowledgments

This work was supported by National Institute on Aging/National Institutes of Health Grants R03AG037746 and R21AG040612.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
acyl-CoA
acyl coenzyme A
AP
alkaline phosphatase
BMC
bone marrow cell
BMD
bone mineral density
BMP
bone morphogenetic protein
BSP
bone sialoprotein
IGF-1R
IGF-1 receptor
NAL
nose to anal length
OCN
osteocalcin
OSP
osteopontin
OSX
osterix
qRT-PCR
quantitative RT-PCR
Runx2
Runt-related transcription factor 2
TIC
type I collagen.

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