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Glucose‐dependent insulinotropic polypeptide (GIP) and glucagon‐like peptide‐2 (GLP‐2) are gut hormones secreted postprandially. In healthy humans, both hormones decrease bone resorption accompanied by a rapid reduction in parathyroid hormone (PTH). The aim of this study was to investigate whether the changes in bone turnover after meal intake and after GIP‐ and GLP‐2 injections, respectively, are mediated via a reduction in PTH secretion. This was tested in female patients with hypoparathyroidism given a standardized liquid mixed‐meal test (n = 7) followed by a peptide injection test (n = 4) using a randomized crossover design. We observed that the meal‐ and GIP‐ but not the GLP‐2‐induced changes in bone turnover markers were preserved in the patients with hypoparathyroidism. To understand the underlying mechanisms, we examined the expression of the GIP receptor (GIPR) and the GLP‐2 receptor (GLP‐2R) in human osteoblasts and osteoclasts as well as in parathyroid tissue. The GIPR was expressed in both human osteoclasts and osteoblasts, whereas the GLP‐2R was absent or only weakly expressed in osteoclasts. Furthermore, both GIPR and GLP‐2R were expressed in parathyroid tissue. Our findings suggest that the GIP‐induced effect on bone turnover may be mediated directly via GIPR expressed in osteoblasts and osteoclasts and that this may occur independent of PTH. In contrast, the effect of GLP‐2 on bone turnover seems to depend on changes in PTH and may be mediated through GLP‐2R in the parathyroid gland. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Denosumab reduced bone resorption, increased bone mineral density (BMD), and decreased new vertebral, hip, and nonvertebral fracture risk in postmenopausal women with osteoporosis in the FREEDOM trial. Consistent with its mechanism of action, transiliac crest bone biopsies from subjects treated with denosumab for 1 to 3 years demonstrated reduced bone turnover that was reversible upon treatment cessation. Long-term denosumab treatment for up to 6 years in the FREEDOM extension provides sustained bone turnover reduction and continued low fracture incidence. Here, we evaluate 5 years of denosumab treatment on bone remodeling at the tissue level. Transiliac crest bone biopsies were obtained from 41 subjects (13 cross-over and 28 long-term from the FREEDOM placebo and denosumab groups, respectively) at year 2 of the FREEDOM extension, representing up to 5 years of denosumab treatment. Demographics for this subset were comparable to the overall extension cohort. The mean (SD) duration from the last denosumab dose to the first dose of tetracycline was 5.7 (0.5) months. Qualitative bone histology assessed in all biopsy samples was unremarkable, showing normally mineralized lamellar bone. Structural indices, including trabecular bone volume, number, and surface, were similar between cross-over and long-term groups. Bone resorption was decreased as reflected by eroded surface in cross-over and long-term subjects. A total of 11 of 13 (85%) cross-over subjects and 20 of 28 (71%) long-term subjects had specimens with double or single tetracycline label in trabecular and/or cortical compartments; specimens from 5 cross-over subjects and 10 long-term subjects were evaluable for dynamic trabecular bone parameters. Dynamic remodeling indices were low for both groups and consistent with reduced bone turnover with denosumab. In conclusion, denosumab treatment through 5 years resulted in normal bone quality with reduced bone turnover. These observations are consistent with its mechanism of action and associated with continued BMD increases and low fracture incidence.

Parathyroid hormone (PTH) and PTH(1-34) have been shown to promote bone healing in several animal studies. It is known that the mechanical environment is important in fracture healing. Furthermore, PTH and mechanical loading has been suggested to have synergistic effects on intact bone. The aim of the present study was to investigate whether the effect of PTH(1-34) on fracture healing in rats was influenced by reduced mechanical loading. For this purpose, we used female, 25-week-old ovariectomized rats. Animals were subjected to closed midshaft fracture of the right tibia 10 weeks after ovariectomy. Five days before fracture, half of the animals received Botulinum Toxin A injections in the muscles of the fractured leg to induce muscle paralysis (unloaded group), whereas the other half received saline injections (control group). For the following 8 weeks, half of the animals in each group received injections of hPTH(1-34) (20 µg/kg/day) and the other half received vehicle treatment. Fracture healing was assessed by radiology, dual-energy X-ray absorptiometry (DXA), histology, and bone strength analysis. We found that unloading reduced callus area significantly, whereas no effects of PTH(1-34) on callus area were seen in neither normally nor unloaded animals. PTH(1-34) increased callus bone mineral density (BMD) and bone mineral content (BMC) significantly, whereas unloading decreased callus BMD and BMC significantly. PTH(1-34) treatment increased bone volume of the callus in both unloaded and control animals. PTH(1-34) treatment increased ultimate force of the fracture by 63% in both control and unloaded animals and no interaction of the two interventions could be detected. PTH(1-34) was able to stimulate bone formation in normally loaded as well as unloaded intact bone. In conclusion, the study confirms the stimulatory effect of PTH(1-34) on fracture healing, and our data suggest that PTH(1-34) is able to promote fracture healing, as well as intact bone formation during conditions of reduced mechanical loading. © 2013 American Society for Bone and Mineral Research.

Osteoporosis is a chronic disease and requires long-term treatment with pharmacologic therapy to ensure sustained antifracture benefit. Denosumab reduced the risk for new vertebral, nonvertebral, and hip fractures over 36 months in the Fracture Reduction Evaluation of Denosumab in Osteoporosis Every 6 Months (FREEDOM) trial. Whereas discontinuation of denosumab has been associated with transient increases in bone remodeling and declines in bone mineral density (BMD), the effect on fracture risk during treatment cessation is not as well characterized. To understand the fracture incidence between treatment groups after cessation of investigational product, we evaluated subjects in FREEDOM who discontinued treatment after receiving two to five doses of denosumab or placebo, and continued study participation for ≥7 months. The off-treatment observation period for each individual subject began 7 months after the last dose and lasted until the end of the study. This subgroup of 797 subjects (470 placebo, 327 denosumab), who were evaluable during the off-treatment period, showed similar baseline characteristics for age, prevalent fracture, and lumbar spine and total hip BMD T-scores. During treatment, more placebo-treated subjects as compared with denosumab-treated subjects sustained a fracture and had significant decreases in BMD. During the off-treatment period (median 0.8 years per subject), 42% versus 28% of placebo- and denosumab-treated subjects, respectively, initiated other therapy. Following discontinuation, similar percentages of subjects in both groups sustained a new fracture (9% placebo, 7% denosumab), resulting in a fracture rate per 100 subject-years of 13.5 for placebo and 9.7 for denosumab (hazard ratio [HR] 0.82; 95% confidence interval [CI], 0.49–1.38), adjusted for age and total hip BMD T-score at baseline. There was no apparent difference in fracture occurrence pattern between the groups during the off-treatment period. In summary, there does not appear to be an excess in fracture risk after treatment cessation with denosumab compared with placebo during the off-treatment period for up to 24 months. © 2013 American Society for Bone and Mineral Research.

Low bone mass is reported in growth-retarded patients harboring mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene causing Rett syndrome (RTT). We present the first study addressing both bone mineral density (BMD) and bone size in RTT. Our object was to determine whether patients with RTT do have low BMD when correcting for smaller bones by examination with dual-energy X-ray absorptiometry (DXA). We compared areal BMD (aBMDspine and aBMDtotal hip) and volumetric bone mineral apparent density (vBMADspine and vBMADneck) in 61 patients and 122 matched healthy controls. Further, spine and hip aBMD and vBMAD of patients were associated with clinical risk factors of low BMD, low-energy fractures, MECP2 mutation groups, and X chromosome inactivation (XCI). Patients with RTT had reduced bone size on the order of 10% and showed lower values of spine and hip aBMD and vBMAD ( p < .001) adjusted for age, pubertal status, and body mass index (BMI). aBMDspine, vBMADspine, and aBMDtotal hip were associated with low-energy fractures ( p < .05). Walking was significantly associated to aBMDtotal hip and vBMADneck adjusted for age and body mass index (BMI). Further, vBMADneck was significantly associated to a diagnosis of epilepsy, antiepileptic treatment, and MECP2 mutation group, but none of the associations with vBMADneck remained clinically significant in a multiple adjusted model including age and BMI. Neither aBMDspine, vBMADspine, nor aBMDtotal hip were significantly associated with epilepsy, antiepileptic treatment, MECP2 mutation group, XCI, or vitamin D status. Low bone mass and small bones are evident in RTT, indicating an apparent low-bone-formation phenotype. 2011 American Society for Bone and Mineral Research.

The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganization in mature osteoclasts. Introduction: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express uPAR and produce urokinase (uPA). The purpose of this study was to investigate the role of uPAR in bone remodeling. Materials and Methods: In vivo studies were performed in uPAR knockout (KO) and wildtype (WT) mice on a C57Bl6/SV129 (75:25) background. Bone mass was analyzed by pQCT. Excised tibias were subjected to mechanical tests. UPAR KO calvaria osteoblasts were characterized by proliferation assays, RT-PCR for important proteins secreted during differentiation, and immunoblot for activator protein 1 (AP-1) family members. In vitro osteoclast formation was tested with uPAR KO bone marrow monocytes in the presence of macrophage-colony stimulating factor (M-CSF) and RANKL. Phalloidin staining in osteoclasts served to study actin ring and podosome formation. Results: pQCT revealed increased bone mass in uPAR-null mice. Mechanical tests showed reduced load- sustaining capability in uPAR KO tibias. uPAR KO osteoblasts showed a proliferative advantage with no difference in apoptosis, higher matrix mineralization, and earlier appearance of alkaline phosphatase (ALP). Surface RANKL expression at different stages of differentiation was not altered. AP-1 components, such as JunB and Fra-1, were upregulated in uPAR KO osteoblasts, along with other osteoblasts markers. On the resorptive side, the number of osteoclasts formed in vitro from uPAR KO monocytes was decreased. Podo- some imaging in uPAR KO osteoclasts revealed a defect in actin ring formation. Conclusions: The defective proliferation and differentiation of bone cells, coincident with both aberrant expression of transcription factors and cytoskeletal organization, are typical uPAR-dependent molecular phenotypes, and we have now shown their function in osteoblasts and osteoclasts function in vivo. J Bone Miner Res 2007;22:1387-1396. Published online on May 29, 2007; doi: 10.1359/JBMR.070516

In the non-HRT arms of the DOPS study, 10-year fracture risk was higher at each level of T score than predicted by the Kanis algorithm. Under-reporting of fractures in registers and inclusion of HRT users are probable explanations for inappropriately low fracture risk estimates for younger women. Introduction: International recommendations highlight the importance of absolute fracture risk in establishing intervention thresholds. The available estimates of long-term risk have been derived by combining relative risks from meta-analyses with U.S. normative BMD data and Swedish fracture incidence records. We validated the 2001 Kanis risk algorithm using incident fractures observed in untreated women in the first 10 years of the Danish Osteoporosis Prevention Study (DOPS). Comparisons were also made with the relative risks derived from a recent meta-analysis of 12 cohort studies. Materials and Methods: We analyzed DXA of the spine and hip from 872 women who were enrolled in the non–hormone replacement therapy (HRT) arms of the study and had not received HRT, bisphosphonates, or raloxifene. We collected verified reports of fractures at each visit. We focused on fractures of the hip, spine, shoulder, and forearm to provide risks comparable with the Kanis algorithm. Accordingly, asymptomatic radiographic vertebral fractures were not included. Results: Seventy-eight women (9%) sustained relevant fractures. The risk of fracture increased by 1.32 (95% CI, 1.02; 1.70) for each unit decrease in femoral neck T score and by 1.30 (95% CI, 1.06; 1.58) for each unit decrease in lumbar spine T score at baseline. Absolute fracture risk was higher than expected from the Kanis algorithm at all T score levels. The difference was greatest for participants in the higher range of T scores. At T = −1, the observed risk was 10.9% as opposed to an expected risk of 5.7%. Relative risk gradients were similar to those of the recent meta-analysis. Conclusions: In healthy women, examined in the first year or two after menopause, 10-year fracture risk was higher at each level of BMD T score than expected from the model by Kanis et al. Inclusion of HRT users in the cohorts used may have led to higher BMD values and lower absolute fracture risk in the Kanis model. These longitudinal data can be used directly in estimating absolute fracture risk in untreated north European women from BMD at menopause.

The significance of an interrelation between nongenetic factors and genotype effects in the regulation of bone mass is not clear. In this prospective study of 429 healthy early postmenopausal Danish women, we investigated the association between bone mineral density (BMD) and the FokI and BsmI polymorphisms in the vitamin D receptor (VDR) gene. Participants were allocated to either hormone-replacement therapy (HRT) or no treatment by randomization or personal choice. After 5 years, 332 women with unchanged treatment status were available for analyses, 98 of these women were still on HRT. No association with initial BMD or 5-year change in BMD was found for either polymorphism. In women with body mass index (BMI) < 25 (n = 282), the f allele was associated with lower BMD of the hip (p < 0.001) and forearm (p = 0.001), and the b allele was associated with lower spine BMD (p = 0.02). Comparing thin/normal weight women with overweight/ obese women of the same genotype, FF women had similar BMD at all measured sites in contrast to Ff and ff women in whom BMD, as expected, was higher in the overweight/obese women. Similar results were found for the BsmI polymorphism with no difference in BMD between BMI groups in BB women. Segregation into groups according to dietary calcium intake did not reveal any genotype association with BMD. These results provide some evidence of a modifying effect of nongenetic factors, specifically BMI, on the association between VDR genotype and BMD. High BMI may protect against lower BMD seen in association with thef or b alleles. In some genotypes (FF and BB), BMI had relatively little effect on BMD.