Liver-derived IGF-I is permissive for ovariectomy-induced trabecular bone loss

INTRODUCTION: Estrogen deficiency results in trabecular bone loss, associated with T-cell proliferation in the bone marrow. Insulin-like growth factor I (IGF-I) is involved in the regulation of both bone metabolism and lymphopoiesis. A major part of serum IGF-I is derived from the liver. The aim of the present study was to investigate the role of liver-derived IGF-I for ovariectomy (ovx)-induced trabecular bone loss. MATERIALS AND METHODS: Mice with adult liver-specific IGF-I inactivation (LI-IGF-I-/-) and wild type mice (WT) were either ovx or sham operated. After 5 weeks, the skeletal phenotype was analyzed by pQCT and microCT. The bone marrow cellularity was analyzed using FACS technique, and mRNA levels were quantified using real-time PCR. RESULTS: Ovx resulted in a pronounced reduction in trabecular bone mineral density (-52%, P < 0.001), number (-45%, P < 0.01) and thickness (-13%, P < 0.01) in WT mice while these bone parameters were unaffected by ovx in LI-IGF-I-/- mice. Furthermore, ovx increased the number of T-cells in the bone marrow of the femur in WT but not in LI-IGF-I-/- mice. Interleukin 7 (IL-7) has been reported to stimulate the formation and function of osteoclasts by inducing the expression of receptor activator of NF-kappaB ligand (RANKL) on T-cells. IL-7 mRNA levels and the RANKL/osteoprotegerin ratio in bone were increased by ovx in WT but not in LI-IGF-I-/- mice. CONCLUSIONS: Liver-derived IGF-I is permissive for ovx-induced trabecular bone loss. Our studies indicate that IGF-I might exert this permissive action by modulation of the number of T-cells and the expression of IL-7, which in turn is of importance for the RANKL/OPG ratio and consequently osteoclastogenesis in the bone marrow.     

Impact of androgens, growth hormone, and IGF-I on bone and muscle in male mice during puberty.

The interaction between androgens and GH/IGF-I was studied in male GHR gene disrupted or GHRKO and WT mice during puberty. Androgens stimulate trabecular and cortical bone modeling and increase muscle mass even in the absence of a functional GHR. GHR activation seems to be the main determinant of radial bone expansion, although GH and androgens are both necessary for optimal stimulation of periosteal growth during puberty. INTRODUCTION: Growth hormone (GH) is considered to be a major regulator of postnatal skeletal growth, whereas androgens are considered to be a key regulator of male periosteal bone expansion. Moreover, both androgens and GH are essential for the increase in muscle mass during male puberty. Deficiency or resistance to either GH or androgens impairs bone modeling and decreases muscle mass. The aim of the study was to investigate androgen action on bone and muscle during puberty in the presence and absence of a functional GH/insulin-like growth factor (IGF)-I axis. MATERIALS AND METHODS: Dihydrotestosterone (DHT) or testosterone (T) were administered to orchidectomized (ORX) male GH receptor gene knockout (GHRKO) and corresponding wildtype (WT) mice during late puberty (6-10 weeks of age). Trabecular and cortical bone modeling, cortical strength, body composition, IGF-I in serum, and its expression in liver, muscle, and bone were studied by histomorphometry, pQCT, DXA, radioimmunoassay and RT-PCR, respectively. RESULTS: GH receptor (GHR) inactivation and low serum IGF-I did not affect trabecular bone modeling, because trabecular BMD, bone volume, number, width, and bone turnover were similar in GHRKO and WT mice. The normal trabecular phenotype in GHRKO mice was paralleled by a normal expression of skeletal IGF-I mRNA. ORX decreased trabecular bone volume significantly and to a similar extent in GHRKO and WT mice, whereas DHT and T administration fully prevented trabecular bone loss. Moreover, DHT and T stimulated periosteal bone formation, not only in WT (+100% and +100%, respectively, versus ORX + vehicle [V]; p < 0.05), but also in GHRKO mice (+58% and +89%, respectively, versus ORX + V; p < 0.05), initially characterized by very low periosteal growth. This stimulatory action on periosteal bone resulted in an increase in cortical thickness and occurred without any treatment effect on serum IGF-I or skeletal IGF-I expression. GHRKO mice also had reduced lean body mass and quadriceps muscle weight, along with significantly decreased IGF-I mRNA expression in quadriceps muscle. DHT and T equally stimulated muscle mass in GHRKO and WT mice, without any effect on muscle IGF-I expression. CONCLUSIONS: Androgens stimulate trabecular and cortical bone modeling and increase muscle weight independently from either systemic or local IGF-I production. GHR activation seems to be the main determinant of radial bone expansion, although GHR signaling and androgens are both necessary for optimal stimulation of periosteal growth during puberty.     

Relative impact of androgen and estrogen receptor activation in the effects of androgens on trabecular and cortical bone in growing male mice: a study in the androgen receptor knockout mouse model.

The relative importance of AR and ER activation has been studied in pubertal male AR knockout and WT mice after orchidectomy and androgen replacement therapy, either with or without an aromatase inhibitor. AR activation dominates normal trabecular bone development and cortical bone modeling in male mice. Moreover, optimal periosteal bone expansion is only observed in the presence of both AR and ER activation. INTRODUCTION: Androgen receptor (AR)-mediated androgen action has traditionally been considered a key determinant of male skeletal growth. Increasing evidence, however, suggests that estrogens are also essential for normal male bone growth. Therefore, the relative importance of AR-mediated and estrogen receptor (ER)-mediated androgen action after aromatization remains to be clarified. MATERIALS AND METHODS: Trabecular and cortical bone was studied in intact or orchidectomized pubertal AR knockout (ARKO) and male wildtype (WT) mice, with or without replacement therapy (3-8 weeks of age). Nonaromatizable (dihydrotestosterone [DHT]) and aromatizable (testosterone [T]) androgens and T plus an aromatase inhibitor (anastrazole) were administered to orchidectomized ARKO and WT mice. Trabecular and cortical bone modeling were evaluated by static and dynamic histomorphometry, respectively. RESULTS: AR inactivation or orchidectomy induced a similar degree of trabecular bone loss (-68% and -71%, respectively). Both DHT and T prevented orchidectomy-induced bone loss in WT mice but not in ARKO mice. Administration of an aromatase inhibitor did not affect T action on trabecular bone. AR inactivation and orchidectomy had similar negative effects on cortical thickness (-13% and -8%, respectively) and periosteal bone formation (-50% and -26%, respectively). In orchidectomized WT mice, both DHT and T were found to stimulate periosteal bone formation and, as a result, to increase cortical thickness. In contrast, the periosteum of ARKO mice remained unresponsive to either DHT or T. Interestingly, administration of an aromatase inhibitor partly reduced T action on periosteal bone formation in orchidectomized WT mice (-34% versus orchidectomized WT mice on T), but not in ARKO mice. This effect was associated with a significant decrease in serum IGF-I (-21% versus orchidectomized WT mice on T). CONCLUSIONS: These findings suggest a major role for AR activation in normal development of trabecular bone and periosteal bone growth in male mice. Moreover, optimal stimulation of periosteal growth is only obtained in the presence of both AR and ER activation.     

Postnatal growth and bone mass in mice with IGF-I haploinsufficiency

We examined the influence of IGF-I haploinsufficiency on growth, bone mass and osteoblast differentiation in Igf1 heterozygous knockout (HET) mice. Cohorts of male and female wild type (WT) and HET mice in the outbred CD-1 background were analyzed at 1, 2, 4, 8, 12, 15 and 18 months of age for body weight, serum IGF-I and bone morphometry. Compared to WT mice, HET mice had 20–30% lower serum IGF-I levels in both genders and in all age groups. Female HET mice showed significant reductions in body weight (10–20%), femur length (4–6%) and femoral bone mineral density (BMD) (7–12%) before 15 months of age. Male HET mice showed significant differences in all parameters at 2 months and thereafter. At 8 and 12 months, WT mice also showed a significant gender effect: despite their lower body weight, female mice had higher femoral BMD and femur length compared to males. Microcomputed tomography showed a significant reduction in cortical bone area (7–20%) and periosteal circumference (5–13%) with no consistent pattern of change in trabecular bone measurements in 2- and 8-month old HET mice in both genders. HET primary osteoblast cultures showed a 40% reduction in IGF-I protein expression and a 50% decrease in IGF-I mRNA expression. Cell growth and proliferation were decreased in HET cultures. Thus, IGF-I haploinsufficiency in outbred male and female mice resulted in reduced body weight, femur length and areal BMD at most ages. Serum IGF-I levels showed a high level of positive correlation with body weight and skeletal morphometry. These studies show that IGF-I is a determinant of bone size and mass in postnatal life. We speculate that impaired osteoblast proliferation may contribute to the skeletal phenotype of mice with IGF-I haploinsufficiency.     

beta-Arrestin2 regulates the differential response of cortical and trabecular bone to intermittent PTH in female mice.

Cytoplasmic arrestins regulate PTH signaling in vitro. We show that female beta-arrestin2(-/-) mice have decreased bone mass and altered bone architecture. The effects of intermittent PTH administration on bone microarchitecture differed in beta-arrestin2(-/-) and wildtype mice. These data indicate that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH at endosteal and periosteal bone surfaces. INTRODUCTION: The effects of PTH differ at endosteal and periosteal surfaces, suggesting that PTH activity in these compartments may depend on some yet unidentified mechanism(s) of regulation. The action of PTH in bone is mediated primarily by intracellular cAMP, and the cytoplasmic molecule beta-arrestin2 plays a central role in this signaling regulation. Thus, we hypothesized that arrestins would modulate the effects of PTH on bone in vivo. MATERIALS AND METHODS: We used pDXA, muCT, histomorphometry, and serum markers of bone turnover to assess the skeletal response to intermittent PTH (0, 20, 40, or 80 mug/kg/day) in adult female mice null for beta-arrestin2 (beta-arr2(-/-)) and wildtype (WT) littermates (7-11/group). RESULTS AND CONCLUSIONS: beta-arr2(-/-) mice had significantly lower total body BMD, trabecular bone volume fraction (BV/TV), and femoral cross-sectional area compared with WT. In WT females, PTH increased total body BMD, trabecular bone parameters, and cortical thickness, with a trend toward decreased midfemoral medullary area. In beta-arr2(-/-) mice, PTH not only improved total body BMD, trabecular bone architecture, and cortical thickness, but also dose-dependently increased femoral cross-sectional area and medullary area. Histomorphometry showed that PTH-stimulated periosteal bone formation was 2-fold higher in beta-arr2(-/-) compared with WT. Osteocalcin levels were significantly lower in beta-arr2(-/-) mice, but increased dose-dependently with PTH in both beta-arr2(-/-) and WT. In contrast, whereas the resorption marker TRACP5B increased dose-dependently in WT, 20-80 mug/kg/day of PTH was equipotent with regard to stimulation of TRACP5B in beta-arr2(-/-). In summary, beta-arrestin2 plays an important role in bone mass acquisition and remodeling. In estrogen-replete female mice, the ability of intermittent PTH to stimulate periosteal bone apposition and endosteal resorption is inhibited by arrestins. We therefore infer that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH on cancellous and cortical bone.     

Liver-derived IGF-I is of importance for normal carbohydrate and lipid metabolism.

IGF-I is important for postnatal body growth and exhibits insulin-like effects on carbohydrate metabolism. The function of liver-derived IGF-I is still not established, although we previously demonstrated that liver-derived IGF-I is not required for postnatal body growth. Mice whose IGF-I gene in the liver was inactivated at 24 days of age were used to investigate the long-term role of liver-derived IGF-I for carbohydrate and lipid metabolism. Serum levels of leptin in these mice were increased by >100% at 3 months of age, whereas the fat mass of the mice was decreased by 25% at 13 months of age. The mice became markedly hyperinsulinemic and yet normoglycemic, indicating an adequately compensated insulin resistance. Furthermore, they had increased serum levels of cholesterol. We conclude that liver-derived IGF-I is of importance for carbohydrate and lipid metabolism.     

The Role of GH/IGF-I-Mediated Mechanisms in Sex Differences in Cortical Bone Size in Mice

Cortical bone dimensions are important determinants of bone strength. Gender differences in cortical bone size caused by greater periosteal expansion in males than in females during the pubertal growth spurt are well established both in humans and in experimental animal models. However, the mechanism by which gender influences cortical bone size is still a matter of investigation. The role of androgens and estrogen in pubertal bone growth has been examined in human disorders as well as animal models, such as gonadectomized or sex steroid receptor knockout mice. Based on the findings that growth hormone (GH) and insulin-like growth factor I (IGF-I) are major regulators of postnatal skeletal growth, we and others have predicted that sex hormones interact with the GH/IGF-I axis to regulate cortical bone size. However, studies conflict as to whether estrogen and androgens impact cortical bone size through the canonical pathway, through GH without IGF-I mediation, through IGF-I without GH stimulation, or independent of GH/IGF-I. We review recent data on the impact of sex steroids and components of the GH/IGF axis on sexual dimorphism in bone size. While the GH/IGF-I axis is a major player in regulating peak bone size, the relative contribution of GH/IGF-dependent mechanisms to sex differences in cortical bone size remains to be established.     

Insulin-like growth factor I is required for the anabolic actions of parathyroid hormone on mouse bone.

Parathyroid hormone (PTH) is a potent anabolic agent for bone, but the mechanism(s) by which it works remains imperfectly understood. Previous studies have indicated that PTH stimulates insulin-like growth factor (IGF) I production, but it remains uncertain whether IGF-I mediates some or all of the skeletal actions of PTH. To address this question, we examined the skeletal response to PTH in IGF-I-deficient (knockout [k/o]) mice. These mice and their normal littermates (NLMs) were given daily injections of PTH (80 microg/kg) or vehicle for 2 weeks after which their tibias were examined for fat-free weight (FFW), bone mineral content, bone structure, and bone formation rate (BFR), and their femurs were assessed for mRNA levels of osteoblast differentiation markers. In wild-type mice, PTH increased FFW, periosteal BFR, and cortical thickness (C.Th) of the proximal tibia while reducing trabecular bone volume (BV); these responses were not seen in the k/o mice. The k/o mice had normal mRNA levels of the PTH receptor and increased mRNA levels of the IGF-I receptor but markedly reduced basal mRNA levels of the osteoblast markers. Surprisingly, these mRNAs in the k/o bones increased several-fold more in response to PTH than the mRNAs in the bones from their wild-type littermates. These results indicate that IGF-I is required for the anabolic actions of PTH on bone formation, but the defect lies distal to the initial response of the osteoblast to PTH.     

Bone Mineral Content and Density in the Humerus of Adult Myostatin-Deficient Mice

Myostatin (GDF-8), a member of the transforming growth factor-b superfamily of secreted growth and differentiation factors, is a negative regulator of skeletal muscle growth. We investigated the effects of increased muscle mass on bone morphology by examining bone mineral content and density in the humeri of myostatin-deficient mice. We compared the humeri of 11 mixed-gender, adult mice homozygous for the disrupted myostatin sequence with those from 11 mixed-gender, adult wild-type mice. Body mass, deltoid mass, and triceps mass were recorded from each animal and densitometric and geometric parameters were collected from the humerus using peripheral quantitative computed tomography (pQCT). Cross-sectional slices were scanned at four different positions along the humerus corresponding to 15%, 40%, 60%, and 85% of total humerus length. Results show that the myostatin- deficient mice weigh more than controls and have significantly larger triceps and deltoid muscles. The myostatin-deficient animals also have significantly (P < 0.05) higher trabecular area and trabecular bone mineral content (BMC) in the proximal humerus (15% length) and significantly (P < 0.01) higher cortical BMC, cortical area, and periosteal circumference in the region of the deltoid crest (40% length). The myostatin knockouts otherwise do not differ from controls in cortical BMC. Moreover, experimental and control mice do not differ significantly from one another in cortical bone mineral density (BMD) at any of the sites examined. These results suggest that the effects of increased muscle mass on the mouse humerus are localized to regions where muscles attach; furthermore, these effects include increased mineral content of both trabecular and cortical bone.     

Age-Associated Decline in Human Femoral Neck Cortical and Trabecular Content of Insulin-Like Growth Factor I: Potential Implications for Age-Related (Type II) Osteoporotic Fracture Occurrence

Recent evidence suggests that regulatory peptides such as insulin-like growth factor-I (IGF-I) are released locally from bone during resorption, and may then act in a sequential manner to regulate the cellular events required for the coupling of bone formation to resorption. Among other factors, a decrease in bone-associated IGF-I levels could therefore result in remodeling imbalance and contribute to the gradual loss of bone that occurs with age. As the femoral neck region is of primary concern for the clinical manifestations of osteoporosis, the current study was intended to assess the IGF-I contents in femoral neck cortical and trabecular bone from aging individuals. Bone samples from the neck region were obtained at postmortem from 39 females and 35 males, aged 23–92 years. Concentrations of IGF-I and osteocalcin were measured by radioimmunoassay in the supernatants obtained after EDTA and guanidine hydrochloride extraction. The total amount of protein present in the extracts was determined by spectrophotometry. IGF-I levels were significantly lower in trabecular compared with cortical bone. Though femoral neck total protein did not vary with donor age, both IGF-I and osteocalcin were found to decline markedly. Between the ages of 23 and 92 years, average yearly rates of loss of 0.30 and 0.21 ng IGF-I/mg protein were observed in cortical and trabecular bone, respectively, corresponding with net losses of nearly 35% of the cortical skeletal content of IGF-I and 41% of the trabecular skeletal content of IGF-I. These changes in bone-associated IGF-I paralleled those of osteocalcin, consistent with an overall decrease in osteoblast function with aging. In women, the rate of decline was significantly faster for trabecular than for cortical IGF-I, however in men, age-dependent changes in cortical and trabecular IGF-I were similar. These findings support the hypothesis that changes in the local IGF regulatory system over time could be a pathophysiologic component of the age-related (type II) femoral neck osteoporotic syndrome. Received: 12 December 1996 / Accepted: 23 April 1997     

Mice lacking the plasminogen activator inhibitor 1 are protected from trabecular bone loss induced by estrogen deficiency.

Bone turnover requires the interaction of several proteases during the resorption phase. Indirect evidence suggests that the plasminogen activator/plasmin pathway is involved in bone resorption and turnover, and recently we have shown that this cascade plays a role in the degradation of nonmineralized bone matrix in vitro. To elucidate the role of the plasminogen activator inhibitor 1 (PAI-1) in bone turnover in vivo, bone metabolism was analyzed in mice deficient in the expression of PAI-1 gene (PAI-1-/-) at baseline (8-week-old mice) and 4 weeks after ovariectomy (OVX) or sham operation (Sham) and compared with wild-type (WT) mice. PAI-1 inactivation was without any effect on bone metabolism at baseline or in Sham mice. However, significant differences were observed in the response of WT and PAI-1-/- mice to ovariectomy. The OVX WT mice showed, as expected, decreased trabecular bone volume (BV/TV) and increased osteoid surface (OS/BS) and bone formation rate (BFR), as assessed by histomorphometric analysis of the proximal tibial metaphysis. In contrast, no significant change in any of the histomorphometric variables studied was detected in PAI-1-/- mice after ovariectomy. As a result, the OVX PAI-1-/- had a significantly higher BV/TV, lower OS/BS, lower mineral apposition rate (MAR) and BFR when compared with the OVX WT mice. However, a comparable decrease in the cortical thickness was observed in OVX PAI-1-/- and WT mice. In addition, the cortical mineral content and density assessed in the distal femoral metaphysis by peripheral quantitative computed tomography (pQCT), decreased significantly after ovariectomy, without difference between PAI-1-/- mice and WT mice. In conclusion, basal bone turnover and bone mass are only minimally affected by PAI-1 inactivation. In conditions of estrogen deficiency, PAI-1 inactivation protects against trabecular bone loss but does not affect cortical bone loss, suggesting a site-specific role for PAI-1 in bone turnover.     

Role of the androgen receptor in skeletal homeostasis: the androgen-resistant testicular feminized male mouse model.

The role of androgen receptor-mediated androgen action on bone was investigated in testicular feminized male (Tfm) mice. Cortical bone was found to be unresponsive to testosterone (T) in orchidectomized Tfm mice, whereas cortical thickness as well as trabecular BMD and structure were fully maintained by T in the corresponding Tabby control mice. These data show an essential role for androgen receptor-mediated androgen action in periosteal bone formation. INTRODUCTION: Androgens can affect the male skeleton both directly-through activation of the androgen receptor (AR)-and indirectly-through stimulation of estrogen receptors after aromatization. We assessed the importance of AR-mediated androgen action on bone in a mouse model of androgen resistance. MATERIALS AND METHODS: Eight-week-old androgen-resistant testicular feminized male (Tfm) and Tabby control mice were orchidectomized (ORX) and treated for 4 weeks with a slow-release testosterone (T) pellet (delivering 167 microg/day) or a placebo pellet. A comprehensive analysis of the skeletal effects of androgen deficiency and replacement was performed using histomorphometry, QCT, and biochemical assessment of bone turnover. RESULTS: As expected, T increased trabecular BMD, volume, number, and width in ORX Tabby mice. In ORX Tfm mice, however, T had less effect on trabecular BMD and no effect on trabecular bone structure. T action on trabecular bone was associated with opposite changes in bone turnover: trabecular and endocortical bone turnover and serum levels of osteocalcin were all reduced by T in ORX Tabby mice, but not in ORX Tfm mice. T also increased cortical thickness (+16%), area, and density in ORX Tabby mice, but not in Tfm mice, resulting in greater bone strength in the Tabby control strain. The positive effects of T on cortical bone reflected a stimulatory effect on periosteal bone formation (+137%), which was again absent in Tfm mice. CONCLUSIONS: These data show that, in male mice, AR-mediated T action is essential for periosteal bone formation and contributes to trabecular bone maintenance.     

Reduced cortical bone mass in mice with inactivation of interleukin-4 and interleukin-13.

The aim of the present study was to study the in vivo role of IL-4 and IL-13 on bone metabolism. The skeletal phenotypes of male and female IL-13(-/-) (n = 7+7), IL-4(-/-)IL-13(-/-) (n = 7+7), and WT (n = 7+7) mice were compared. Analysis was made at 6 weeks of age (juvenile) by pQCT, and at 20 weeks of age (adult) by pQCT, biomechanical testing, and by S-IGF-1 and S-Osteocalcin measurements. The skeletal phenotype was affected only in adult male IL-4(-/-)IL-13(-/-) mice. These animals displayed a reduction in cortical bone mineral content (BMC) of both the tibia and the femur, as measured by mid-diaphyseal pQCT scans, compared with WT mice (tibia -8.2%; femur -8.5%; p < 0.01). This reduction in cortical BMC was due to a decreased cross-sectional area as a result of a reduced cortical thickness. The mechanical strength of the cortical bone, tested by three-point-bending at the mid-diaphyseal region of the femurs, demonstrated a significant reduction of displacement at failure (-11.4%), maximal load at failure (-10.6%), and total energy until failure (-29.4%). S-IGF-1 and S-Osteocalcin levels as well as trabecular bone mineral density (tvBMD) were unaffected in adult male IL-4(-/-)IL-13(-/-) mice. IL-4(-/-)IL-13(-/-) male mice show adult onset reduction of cortical bone mass and strength, indicating that the two anti-inflammatory Th(2) cytokines IL-4 and IL-13 are involved in the regulation of bone remodeling.     

Ovariectomy-induced bone loss varies among inbred strains of mice.

There is a subset of women who experience particularly rapid bone loss during and after the menopause. However, the factors that lead to this enhanced bone loss remain obscure. We show that patterns of bone loss after ovariectomy vary among inbred strains of mice, providing evidence that there may be genetic regulation of bone loss induced by estrogen deficiency. INTRODUCTION: Both low BMD and increased rate of bone loss are risk factors for fracture. Bone loss during and after the menopause is influenced by multiple hormonal factors. However, specific determinants of the rate of bone loss are poorly understood, although it has been suggested that genetic factors may play a role. We tested whether genetic factors may modulate bone loss subsequent to estrogen deficiency by comparing the skeletal response to ovariectomy in inbred strains of mice. MATERIALS AND METHODS: Four-month-old mice from five inbred mouse strains (C3H/HeJ, BALB/cByJ, CAST/EiJ, DBA2/J, and C57BL/6J) underwent ovariectomy (OVX) or sham-OVX surgery (n = 6-9/group). After 1 month, mice were killed, and microCT was used to compare cortical and trabecular bone response to OVX. RESULTS: The effect of OVX on trabecular bone varied with mouse strain and skeletal site. Vertebral trabecular bone volume (BV/TV) declined after OVX in all strains (-15 to -24%), except for C3H/HeJ. In contrast, at the proximal tibia, C3H/HeJ mice had a greater decline in trabecular BV/TV (-39%) than C57BL/6J (-18%), DBA2/J (-23%), and CAST/EiJ mice (-21%). OVX induced declines in cortical bone properties, but in contrast to trabecular bone, the effect of OVX did not vary by mouse strain. The extent of trabecular bone loss was greatest in those mice with highest trabecular BV/TV at baseline, whereas cortical bone loss was lowest among those with high cortical bone parameters at baseline. CONCLUSIONS: We found that the skeletal response to OVX varies in a site- and compartment-specific fashion among inbred mouse strains, providing support for the hypothesis that bone loss during and after the menopause is partly genetically regulated.     

Diminished response to in vivo mechanical loading in trabecular and not cortical bone in adulthood of female C57Bl/6 mice coincides with a reduction in deformation to load

Bone loss occurs during adulthood in both women and men and affects trabecular bone more than cortical bone. The mechanism responsible for trabecular bone loss during adulthood remains unexplained, but may be due at least in part to a reduced mechanoresponsiveness. We hypothesized that trabecular and cortical bone would respond anabolically to loading and that the bone response to mechanical loading would be reduced and the onset delayed in adult compared to postpubescent mice. We evaluated the longitudinal adaptive response of trabecular and cortical bone in postpubescent, young (10 week old) and adult (26 week old) female C57Bl/6J mice to axial tibial compression using in vivo microCT (days 0, 5, 10, and 15) and dynamic histomorphometry (day 15). Loading elicited an anabolic response in both trabecular and cortical bone in young and adult mice. As hypothesized, trabecular bone in adult mice exhibited a reduced and delayed response to loading compared to the young mice, apparent in trabecular bone volume fraction and architecture after 10 days. No difference in mechanoresponsiveness of the cortical bone was observed between young and adult mice. Finite element analysis showed that load-induced strain was reduced with age. Our results suggest that trabecular bone loss that occurs in adulthood may in part be due to a reduced mechanoresponsiveness in this tissue and/or a reduction in the induced tissue deformation which occurs during habitual loading. Therapeutic approaches that address the mechanoresponsiveness of the bone tissue may be a promising and alternate strategy to maintain trabecular bone mass during aging.     

Generation of a new congenic mouse strain to test the relationships among serum insulin-like growth factor I, bone mineral density, and skeletal morphology in vivo.

Insulin-like growth factor (IGF) I is a critical peptide for skeletal growth and consolidation. However, its regulation is complex and, in part, heritable. We previously indicated that changes in both serum and skeletal IGF-I were related to strain-specific differences in total femoral bone mineral density (BMD) in mice. In addition, we defined four quantitative trait loci (QTLs) that contribute to the heritable determinants of the serum IGF-I phenotype in F2 mice derived from progenitor crosses between C3H/HeJ (C3H; high total femoral BMD and high IGF-I) and C57BL/6J (B6; low total femoral BMD and low IGF-I) strains. The strongest QTL, IGF-I serum level 1 (Igflsl-1; log10 of the odds ratio [LOD] score, approximately 9.0), is located on the middle portion of chromosome (Chr) 6. For this locus, C3H alleles are associated with a significant reduction in serum IGF-I. To test the effect of this QTL in vivo, we generated a new congenic strain (B6.C3H-6T [6T]) by placing the Chr 6 QTL region (D6Mit93 to D6Mit150) from C3H onto the B6 background. We then compared serum and skeletal IGF-I levels, body weight, and several skeletal phenotypes from the N9 generation of 6T congenic mice against B6 control mice. Female 6T congenic mice had 11-21% lower serum IGF-I levels at 6, 8, and 16 weeks of age compared with B6 (p < 0.05 for all). In males, serum IGF-I levels were similar in 6T congenics and B6 controls at 6 weeks and 8 weeks but were lower in 6T congenic mice at 16 weeks (p < 0.02). In vitro, there was a 40% reduction in secreted IGF-I in the conditioned media (CMs) from 6T calvaria osteoblasts compared with B6 cells (p < 0.01). Total femoral BMD as measured by peripheral quantitative computed tomography (pQCT) was lower in both 6T male (-4.8%, p < 0.01) and 6T female (-2.3%, p = 0.06) congenic mice. Geometric features of middiaphyseal cortical bone were reduced in 6T congenic mice compared with control mice. Femoral cancellous bone volume (BV) density and trabecular number (Tb.N) were 50% lower, whereas trabecular separation (Tb.Sp) was 90% higher in 8-week-old female 6T congenic mice compared with B6 control mice (p < 0.01 for all). Similarly, vertebral cancellous BV density and Tb.N were lower (-29% and -19%, respectively), whereas Tb.Sp was higher (+29%) in 16-week-old female 6T congenic mice compared with B6 control mice (p < 0.001 for all). Histomorphometric evaluation of the proximal tibia indicated that 6T congenics had reduced BV fraction, labeled surface, and bone formation rates compared with B6 congenic mice. In summary, we have developed a new congenic mouse strain that confirms the Chr 6 QTL as a major genetic regulatory determinant for serum IGF-I. This locus also influences bone density and morphology, with more dramatic effects in cancellous bone than in cortical bone.     

Allelic differences in a quantitative trait locus affecting insulin-like growth factor-I impact skeletal acquisition and body composition

Insulin-like growth factor-I (IGF-I) is critical for optimal skeletal growth and maintenance. Knockout and transgenic models have provided significant insights into the role of IGF-I in bone modeling and remodeling. Congenic mice demonstrate allelic differences in particular quantitative trait loci (QTL). One such model is congenic 6T, which contains a QTL for reduced serum IGF-I donated from C3H/HeJ on a pure C57Bl/6 J (B6) background. In this study we found a 30%–50% reduction in IGF-I expression in bone, liver, and fat of the congenic 6T mouse, as well as lower circulating IGF-I compared with control B6. 6T mice also had a greater percentage body fat, but reduced serum leptin. These changes were associated with reduced cortical and trabecular bone mineral density, impaired bone formation but no change in bone resorption. Moreover, the anabolic skeletal response to intermittent parathyroid hormone (PTH) therapy was blunted in 6T compared with B6, potentially in response to greater programmed cell death in osteocytes and osteoblasts of 6T. In summary, allelic differences in IGF-I expression impact peak bone acquisition and body composition, as well as the skeletal response to PTH. Lifelong changes in circulating and skeletal IGF-I may be relevant for the pathophysiology of several diseases, including chronic renal failure.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

IGF-I receptor is required for the anabolic actions of parathyroid hormone on bone.

We showed that the IGF-IR-null mutation in mature osteoblasts leads to less bone and decreased periosteal bone formation and impaired the stimulatory effects of PTH on osteoprogenitor cell proliferation and differentiation. INTRODUCTION: This study was carried out to examine the role of IGF-I signaling in mediating the actions of PTH on bone. MATERIALS AND METHODS: Three-month-old mice with an osteoblast-specific IGF-I receptor null mutation (IGF-IR OBKO) and their normal littermates were treated with vehicle or PTH (80 microg/kg body weight/d for 2 wk). Structural measurements of the proximal and midshaft of the tibia were made by microCT. Trabecular and cortical bone formation was measured by bone histomorphometry. Bone marrow stromal cells (BMSCs) were obtained to assess the effects of PTH on osteoprogenitor number and differentiation. RESULTS: The fat-free weight of bone normalized to body weight (FFW/BW), bone volume (BV/TV), and cortical thickness (C.Th) in both proximal tibia and shaft were all less in the IGF-IR OBKO mice compared with controls. PTH decreased FFW/BW of the proximal tibia more substantially in controls than in IGF-IR OBKO mice. The increase in C.Th after PTH in the proximal tibia was comparable in both control and IGF-IR OBKO mice. Although trabecular and periosteal bone formation was markedly lower in the IGF-IR OBKO mice than in the control mice, endosteal bone formation was comparable in control and IGF-IR OBKO mice. PTH stimulated endosteal bone formation only in the control animals. Compared with BMSCs from control mice, BMSCs from IGF-IR OBKO mice showed equal alkaline phosphatase (ALP)(+) colonies on day 14, but fewer mineralized nodules on day 28. Administration of PTH increased the number of ALP(+) colonies and mineralized nodules on days 14 and 28 in BMSCs from control mice, but not in BMSCs from IGF-IR OBKO mice. CONCLUSIONS: Our results indicate that the IGF-IR null mutation in mature osteoblasts leads to less bone and decreased bone formation, in part because of the requirement for the IGF-IR in mature osteoblasts to enable PTH to stimulate osteoprogenitor cell proliferation and differentiation.