Evaluation of bone marrow glucose uptake and adiposity in male rats after diet and exercise interventions.

Objectives: Obesity impairs bone marrow (BM) glucose metabolism. Adult BM constitutes mostly of adipocytes that respond to changes in energy metabolism by modulating their morphology and number. Here we evaluated whether diet or exercise intervention could improve the high-fat diet (HFD) associated impairment in BM glucose uptake (BMGU) and whether this associates with the morphology of BM adipocytes (BMAds) in rats. Methods: Eight-week-old male Sprague-Dawley rats were fed ad libitum either HFD or chow diet for 24 weeks. Additionally after 12 weeks, HFD-fed rats switched either to chow diet, voluntary intermittent running exercise, or both for another 12 weeks. BMAd morphology was assessed by perilipin-1 immunofluorescence staining in formalin-fixed paraffin-embedded tibial sections. Insulin-stimulated sternal and humeral BMGU were measured using [18F]FDG-PET/CT. Tibial microarchitecture and mineral density were measured with microCT. Results: HFD rats had significantly higher whole-body fat percentage compared to the chow group (17% vs 13%, respectively; p = 0.004) and larger median size of BMAds in the proximal tibia (815 µm2 vs 592 µm2, respectively; p = 0.03) but not in the distal tibia. Switch to chow diet combined with running exercise normalized whole-body fat percentage (p < 0.001) but not the BMAd size. At 32 weeks of age, there was no significant difference in insulin-stimulated BMGU between the study groups. However, BMGU was significantly higher in sternum compared to humerus (p < 0.001) and higher in 8-week-old compared to 32-week-old rats (p < 0.001). BMAd size in proximal tibia correlated positively with whole-body fat percentage (r = 0.48, p = 0.005) and negatively with humeral BMGU (r = -0.63, p = 0.02). HFD significantly reduced trabecular number (p < 0.001) compared to the chow group. Switch to chow diet reversed this as the trabecular number was significantly higher (p = 0.008) than in the HFD group. Conclusion: In this study we showed that insulin-stimulated BMGU is age- and site-dependent. BMGU was not affected by the study interventions. HFD increased whole-body fat percentage and the size of BMAds in proximal tibia. Switching from HFD to a chow diet and running exercise improved glucose homeostasis and normalized the HFD-induced increase in body fat but not the hypertrophy of BMAds.

The complex role of Rcor2: Regulates mesenchymal stromal cell differentiation in vitro but is dispensable in vivo.

Recent research has revealed several important pathways of epigenetic regulation leading to transcriptional changes in bone cells. Rest Corepressor 2 (Rcor2) is a coregulator of Lysine-specific histone demethylase 1 (Lsd1), a demethylase linked to osteoblast activity, hematopoietic stem cell differentiation and malignancy of different neoplasms. However, the role of Rcor2 in osteoblast differentiation has not yet been examined in detail. We have previously shown that Rcor2 is highly expressed in mesenchymal stromal cells (MSC) and particularly in the osteoblastic lineage. The role of Rcor2 in osteoblastic differentiation in vitro was further characterized and we demonstrate here that lentiviral silencing of Rcor2 in MC3T3-E1 cells led to a decrease in osteoblast differentiation. This was indicated by decreased alkaline phosphatase and von Kossa stainings as well as by decreased expression of several osteoblast-related marker genes. RNA-sequencing of the Rcor2-downregulated MC3T3-E1 cells showed decreased repression of Rcor2 target genes, as well as significant upregulation of majority of the differentially expressed genes. While the heterozygous, global loss of Rcor2 in vivo did not lead to a detectable bone phenotype, conditional deletion of Rcor2 in limb-bud mesenchymal cells led to a moderate decrease in cortical bone volume. These findings were not accentuated by challenging bone formation by ovariectomy or tibial fracture. Furthermore, a global deletion of Rcor2 led to decreased white adipose tissue in vivo and decreased the capacity of primary cells to differentiate into adipocytes in vitro. The conditional deletion of Rcor2 led to decreased adiposity in fracture callus. Taken together, these results suggest that epigenetic regulation of mesenchymal stromal cell differentiation is mediated by Rcor2, which could thus play an important role in defining the MSC fate.

Short- and long-term exposure to high glucose induces unique transcriptional changes in osteoblasts in vitro.

Bone is increasingly recognized as a target for diabetic complications. In order to evaluate the direct effects of high glucose on bone, we investigated the global transcriptional changes induced by hyperglycemia in osteoblasts in vitro. Rat bone marrow-derived mesenchymal stromal cells were differentiated into osteoblasts for 10 days, and prior to analysis, they were exposed to hyperglycemia (25 mM) for the short-term (1 or 3 days) or long-term (10 days). Genes and pathways regulated by hyperglycemia were identified using mRNA sequencing and verified with qPCR. Genes upregulated by 1-day hyperglycemia were, for example, related to extracellular matrix organization, collagen synthesis and bone formation. This stimulatory effect was attenuated by 3 days. Long-term exposure impaired osteoblast viability, and downregulated, for example, extracellular matrix organization and lysosomal pathways, and increased intracellular oxidative stress. Interestingly, transcriptional changes by different exposure times were mostly unique and only 89 common genes responding to glucose were identified. In conclusion, short-term hyperglycemia had a stimulatory effect on osteoblasts and bone formation, whereas long-term hyperglycemia had a negative effect on intracellular redox balance, osteoblast viability and function.

Bone marrow metabolism is affected by body weight and response to exercise training varies according to anatomical location.

AIM: High body weight is a protective factor against osteoporosis, but obesity also suppresses bone metabolism and whole-body insulin sensitivity. However, the impact of body weight and regular training on bone marrow (BM) glucose metabolism is unclear. We studied the effects of regular exercise training on bone and BM metabolism in monozygotic twin pairs discordant for body weight. METHODS: We recruited 12 monozygotic twin pairs (mean ± SD age 40.4 ± 4.5 years; body mass index 32.9 ± 7.6, mean difference between co-twins 7.6 kg/m2 ; eight female pairs). Ten pairs completed the 6-month long training intervention. We measured lumbar vertebral and femoral BM insulin-stimulated glucose uptake (GU) using 18 F-FDG positron emission tomography, lumbar spine bone mineral density and bone turnover markers. RESULTS: At baseline, heavier co-twins had higher lumbar vertebral BM GU (p < .001) and lower bone turnover markers (all p < .01) compared with leaner co-twins but there was no significant difference in femoral BM GU, or bone mineral density. Training improved whole-body insulin sensitivity, aerobic capacity (both p < .05) and femoral BM GU (p = .008). The training response in lumbar vertebral BM GU was different between the groups (time × group, p = .02), as GU tended to decrease in heavier co-twins (p = .06) while there was no change in leaner co-twins. CONCLUSIONS: In this study, regular exercise training increases femoral BM GU regardless of weight and genetics. Interestingly, lumbar vertebral BM GU is higher in participants with higher body weight, and training counteracts this effect in heavier co-twins even without reduction in weight. These data suggest that BM metabolism is altered by physical activity.

Perilipin-1 immunostaining improves semi-automated digital quantitation of bone marrow adipocytes in histological bone sections.

Bone marrow adipocytes (BMAds) are not just passive fillers inside the bone marrow compartment but respond to various metabolic changes. Assessment of those responses requires evaluation of the number of BMAds and their morphology for which laborious and error-prone manual histological analysis remains the most widely used method. Here, we report an alternative image analysis strategy to semi-automatically quantitate and analyse the morphology of BMAds in histological bone sections. Decalcified, formalin-fixed paraffin-embedded histological sections of long bones of Sprague-Dawley rats were stained with either haematoxylin and eosin (HE) or by immunofluorescent staining for adipocyte-specific protein perilipin-1 (PLIN1). ImageJ-based commands were constructed to detect BMAds sized 200 µm2 or larger from standardized 1 mm2 analysis regions by either classifying the background colour (HE) or the positive and circular PLIN1 fluorescent signal. Semi-automated quantitation strongly correlated with independent, single-blinded manual counts regardless of the staining method (HE-based: r=0.85, p<0.001; PLIN1 based: r=0.95, p<0.001). The detection error was higher in HE-stained sections than in PLIN1-stained sections (14% versus 5%, respectively; p<0.001), which was due to false-positive detections of unstained adipocyte-like circular structures. In our dataset, the total adiposity area from standardised ROIs in PLIN-1-stained sections correlated with that in whole-bone sections (r=0.60, p=0.02).

The evolution of the adolescent growth spurt: Urinary biomarkers of bone turnover in wild chimpanzees (Pan troglodytes).

Life history theory addresses how organisms balance development and reproduction. Mammals usually invest considerable energy into growth in infancy, and they do so incrementally less until reaching adult body size, when they shift energy to reproduction. Humans are unusual in having a long adolescence when energy is invested in both reproduction and growth, including rapid skeletal growth around puberty. Although many primates, especially in captivity, experience accelerated growth in mass around puberty, it remains unclear whether this represents skeletal growth. Without data on skeletal growth in nonhuman primates, anthropologists have often assumed the adolescent growth spurt is uniquely human, and hypotheses for its evolution have focused on other uniquely human traits. The lack of data is largely due to methodological difficulties of assessing skeletal growth in wild primates. Here, we use two urinary markers of bone turnover-osteocalcin and collagen-to study skeletal growth in a large, cross-sectional sample of wild chimpanzees (Pan troglodytes) at Ngogo, Kibale National Park, Uganda. For both bone turnover markers, we found a nonlinear effect of age, which was largely driven by males. For male chimpanzees, values for osteocalcin and collagen peaked at age 9.4 years and 10.8 years, respectively, which corresponds to early and middle adolescence. Notably, collagen values increased from 4.5 to 9 years, suggesting faster growth during early adolescence compared to late infancy. Biomarker levels plateaued at 20 years in both sexes, suggesting skeletal growth continues until then. Additional data, notably on females and infants of both sexes, are needed, as are longitudinal samples. However, our cross-sectional analysis suggests an adolescent growth spurt in the skeleton of chimpanzees, especially for males. Biologists should avoid claiming that the adolescent growth spurt is uniquely human, and hypotheses for the patterns of human growth should consider variation in our primate relatives.

Glucose transporters GLUT1, GLUT3, and GLUT4 have different effects on osteoblast proliferation and metabolism.

Bone is an active tissue that undergoes constant remodeling. Bone formation requires energy and one of the energy sources of bone-forming osteoblasts is glucose, which is transported inside the cells via glucose transporters. However, the role of class I glucose transporters in the differentiation and metabolism of osteoblasts and their precursors, bone marrow mesenchymal stromal cells (BMSCs) remains inconclusive. Our aim was to characterize the expression and contribution of main class I glucose transporters, GLUT1, GLUT3, and GLUT4, during osteoblast proliferation and differentiation. To investigate the role of each GLUT, we downregulated GLUTs with siRNA technology in primary rat BMSCs. Live-cell imaging and RNA-seq analysis was used to evaluate downstream pathways in silenced osteoblasts. Glucose transporters GLUT1, GLUT3, and GLUT4 had distinct expression patterns in osteoblasts. GLUT1 was abundant in BMSCs, but rapidly and significantly downregulated during osteoblast differentiation by up to 80% (p < 0.001). Similar downregulation was observed for GLUT4 (p < 0.001). In contrast, expression levels of GLUT3 remained stable during differentiation. Osteoblasts lacked GLUT2. Silencing of GLUT4 resulted in a significant decrease in proliferation and differentiation of preosteoblasts (p < 0.001) and several pathways related to carbohydrate metabolism and cell signaling were suppressed. However, silencing of GLUT3 resulted in increased proliferation (p < 0.001), despite suppression of several pathways involved in cellular metabolism, biosynthesis and actin organization. Silencing of GLUT1 had no effect on proliferation and less changes in the transcriptome. RNA-seq dataset further revealed that osteoblasts express also class II and III glucose transporters, except for GLUT7. In conclusion, GLUT1, -3 and -4 may all contribute to glucose uptake in differentiating osteoblasts. GLUT4 expression was clearly required for osteoblast proliferation and differentiation. GLUT1 appears to be abundant in early precursors, but stable expression of GLUT3 suggest also a role for GLUT3 in osteoblasts. Presence of other GLUT members may further contribute to fine-tuning of glucose uptake. Together, glucose uptake in osteoblast lineage appears to rely on several glucose transporters to ensure sufficient energy for new bone formation.

Bone Turnover Marker Profiling and Fracture Risk in Older Women: Fracture Risk from Age 75 to 90.

PURPOSE: A major challenge in osteoporosis is to identify individuals at high fracture risk. We investigated six bone turnover markers (BTMs) to determine association with specific fracture types; the time-frame for risk prediction and whether these are influenced by age at assessment. METHODS: Population-based OPRA cohort (n = 1044) was assessed at ages 75, 80, 85 and fractures documented for up to 15 years. Six BTMs were analyzed at each time-point (N-terminal propeptide of type I collagen, PINP; total osteocalcin, OC; bone-specific alkaline phosphatase, BALP; C-terminal telopeptide of type I collagen, CTX; tartrate-resistant acid phosphatase 5b, TRAcP5b; urinary osteocalcin). Hazard ratios (HR) for any, major osteoporotic, vertebral and hip fractures were calculated as short (1, 2, 3 years) and long-term risk (5, 10, 15 years). RESULTS: At 75 year, high CTX levels were associated with an increased risk of all fractures, including major osteoporotic fractures, across most time-frames (HRs ranging: 1.28 to 2.28). PINP was not consistently associated. Urinary osteocalcin was consistently associated with elevated short-term risk (HRs ranging: 1.83-2.72). Other BTMs were directionally in accordance, though not all statistically significant. BTMs were not predictive for hip fractures. Association of all BTMs attenuated over time; at 80 year none were associated with an increased fracture risk. CONCLUSION: CTX, urinary OC and TRAcP5b are predictive for fracture in a 1 to 3 year, perspective, whereas in the long-term or above age 80 years, BTMs appear less valuable. Resorption markers, particularly CTX, were more consistently associated with fracture risk than formation markers in the very elderly.

Age-Progressive and Gender-Dependent Bone Phenotype in Mice Lacking Both Ebf1 and Ebf2 in Prrx1-Expressing Mesenchymal Cells.

Ebfs are a family of transcription factors regulating the differentiation of multiple cell types of mesenchymal origin, including osteoblasts. Global deletion of Ebf1 results in increased bone formation and bone mass, while global loss of Ebf2 leads to enhanced bone resorption and decreased bone mass. Targeted deletion of Ebf1 in early committed osteoblasts leads to increased bone formation, whereas deletion in mature osteoblasts has no effect. To study the effects of Ebf2 specifically on long bone development, we created a limb bud mesenchyme targeted Ebf2 knockout mouse model by using paired related homeobox gene 1 (Prrx1) Cre. To investigate the possible interplay between Ebf1 and Ebf2, we deleted both Ebf1 and Ebf2 in the cells expressing Prrx1. Mice with Prrx1-targeted deletion of Ebf2 had a very mild bone phenotype. However, deletion of both Ebf1 and Ebf2 in mesenchymal lineage cells lead to significant, age progressive increase in bone volume. The phenotype was to some extent gender dependent, leading to an increase in both trabecular and cortical bone in females, while in males a mild cortical bone phenotype and a growth plate defect was observed. The phenotype was observed at both 6 and 12 weeks of age, but it was more pronounced in older female mice. Our data suggest that Ebfs modulate bone homeostasis and they are likely able to compensate for the lack of each other. The roles of Ebfs in bone formation appear to be complex and affected by multiple factors, such as age and gender.

Guidelines for Biobanking of Bone Marrow Adipose Tissue and Related Cell Types: Report of the Biobanking Working Group of the International Bone Marrow Adiposity Society.

Over the last two decades, increased interest of scientists to study bone marrow adiposity (BMA) in relation to bone and adipose tissue physiology has expanded the number of publications using different sources of bone marrow adipose tissue (BMAT). However, each source of BMAT has its limitations in the number of downstream analyses for which it can be used. Based on this increased scientific demand, the International Bone Marrow Adiposity Society (BMAS) established a Biobanking Working Group to identify the challenges of biobanking for human BMA-related samples and to develop guidelines to advance establishment of biobanks for BMA research. BMA is a young, growing field with increased interest among many diverse scientific communities. These bring new perspectives and important biological questions on how to improve and build an international community with biobank databases that can be used and shared all over the world. However, to create internationally accessible biobanks, several practical and legislative issues must be addressed to create a general ethical protocol used in all institutes, to allow for exchange of biological material internationally. In this position paper, the BMAS Biobanking Working Group describes similarities and differences of patient information (PIF) and consent forms from different institutes and addresses a possibility to create uniform documents for BMA biobanking purposes. Further, based on discussion among Working Group members, we report an overview of the current isolation protocols for human bone marrow adipocytes (BMAds) and bone marrow stromal cells (BMSCs, formerly mesenchymal), highlighting the specific points crucial for effective isolation. Although we remain far from a unified BMAd isolation protocol and PIF, we have summarized all of these important aspects, which are needed to build a BMA biobank. In conclusion, we believe that harmonizing isolation protocols and PIF globally will help to build international collaborations and improve the quality and interpretation of BMA research outcomes.

Serum and Urinary Osteocalcin in Healthy 7- to 19-Year-Old Finnish Children and Adolescents.

Children and adolescents have high bone turnover marker (BTM) levels due to high growth velocity and rapid bone turnover. Pediatric normative values for BTMs reflecting bone formation and resorption are vital for timely assessment of healthy bone turnover, investigating skeletal diseases, or monitoring treatment outcomes. Optimally, clinically feasible measurement protocols for BTMs would be validated and measurable in both urine and serum. We aimed to (a) establish sex- and age-specific reference intervals for urinary and serum total and carboxylated osteocalcin (OC) in 7- to 19-year-old healthy Finnish children and adolescents (n = 172), (b) validate these against standardized serum and urinary BTMs, and (c) assess the impact of anthropometry, pubertal status, and body composition on the OC values. All OC values in addition to other BTMs increased with puberty and correlated with pubertal growth, which occurred and declined earlier in girls than in boys. The mean serum total and carboxylated OC and urinary OC values and percentiles for sex-specific age categories and pubertal stages were established. Correlation between serum and urinary OC was weak, especially in younger boys, but improved with increasing age. The independent determinants for OC varied, the urinary OC being the most robust while age, height, weight, and plasma parathyroid hormone (PTH) influenced serum total and carboxylated OC values. Body composition parameters had no influence on any of the OC values. In children and adolescents, circulating and urinary OC reflect more accurately growth status than bone mineral density (BMD) or body composition. Thus, validity of OC, similar to other BTMs, as a single marker of bone turnover, remains limited. Yet, serum and urinary OC similarly to other BTMs provide a valuable supplementary tool when assessing longitudinal changes in bone health with repeat measurements, in combination with other clinically relevant parameters.

Recombinant Antibodies with Unique Specificities Allow for Sensitive and Specific Detection of Uncarboxylated Osteocalcin in Human Circulation.

Osteocalcin is a bone-specific protein which contains three glutamic acid residues (Glu) that undergo post-translational gamma-carboxylation. Uncarboxylated osteocalcin (ucOC) may participate in the regulation of glucose metabolism, thus measurement of ucOC could be useful in evaluating interactions between bone and glucose metabolism. We developed recombinant antibodies and immunoassay to specifically detect ucOC in human blood samples. ucOC-specific recombinant antibodies were selected from an antibody library by phage display. Four candidates were characterized, and one (Fab-AP13) was used to set up an immunoassay with a pre-existing MAb. Plasma ucOC levels were measured in subjects with normal fasting blood glucose (≤ 6 mmol/l, N = 46) or with hyperglycemia (≥ 7 mmol/l, N = 29). Further, we analyzed ucOC in age- and gender-matched patients with diagnosed type 2 diabetes (T2D, N = 49). Antibodies recognized ucOC without cross-reaction to carboxylated osteocalcin. Antibodies had unique binding sites at the carboxylation region, with Glu17 included in all epitopes. Immunoassay was set up and characterized. Immunoassay detected ucOC in serum and plasma, with on average 1.6-fold higher levels in plasma. ucOC concentrations were significantly lower in subjects with hyperglycemia (median 0.58 ng/ml, p = 0.008) or with T2D diagnosis (0.68 ng/ml, p = 0.015) than in subjects with normal blood glucose (1.01 ng/ml). ucOC negatively correlated with fasting plasma glucose in subjects without T2D (r = - 0.24, p = 0.035) but not in T2D patients (p = 0.41). Our immunoassay, based on the novel recombinant antibody, allows for specific and sensitive detection of ucOC in human circulation. Correlation between ucOC and plasma glucose suggests interactions between osteocalcin and glucose metabolism in humans.

Bone Marrow Metabolism Is Impaired in Insulin Resistance and Improves After Exercise Training.

CONTEXT: Exercise training improves bone mineral density, but little is known about the effects of training on bone marrow (BM) metabolism. BM insulin sensitivity has been suggested to play an important role in bone health and whole-body insulin sensitivity. OBJECTIVE: To study the effects of exercise training on BM metabolism. DESIGN: Randomized controlled trial. SETTING: Clinical research center. PARTICIPANTS: Sedentary healthy (n = 28, 40-55 years, all males) and insulin resistant (IR) subjects (n = 26, 43-55 years, males/females 16/10). INTERVENTION: Two weeks of sprint interval training or moderate-intensity continuous training. MAIN OUTCOME MEASURES: We measured femoral, lumbar, and thoracic BM insulin-stimulated glucose uptake (GU) and fasting free fatty acid uptake (FFAU) using positron-emission tomography and bone turnover markers from plasma. RESULTS: At baseline, GU was highest in lumbar, followed by thoracic, and lowest in femoral BM (all Ps < 0.0001). FFAU was higher in lumbar and thoracic than femoral BM (both Ps < 0.0001). BM FFAU and femoral BM GU were higher in healthy compared to IR men and in females compared to males (all Ps < 0.05). Training increased femoral BM GU similarly in all groups and decreased lumbar BM FFAU in males (all Ps < 0.05). Osteocalcin and PINP were lower in IR than healthy men and correlated positively with femoral BM GU and glycemic status (all Ps < 0.05). CONCLUSIONS: BM metabolism differs regarding anatomical location. Short-term training improves BM GU and FFAU in healthy and IR subjects. Bone turnover rate is decreased in insulin resistance and associates positively with BM metabolism and glycemic control. CLINICAL TRIAL REGISTRATION NUMBER: NCT01344928.

Human Bone Marrow Adipose Tissue is a Metabolically Active and Insulin-Sensitive Distinct Fat Depot.

CONTEXT: Bone marrow (BM) in adult long bones is rich in adipose tissue, but the functions of BM adipocytes are largely unknown. We set out to elucidate the metabolic and molecular characteristics of BM adipose tissue (BMAT) in humans. OBJECTIVE: Our aim was to determine if BMAT is an insulin-sensitive tissue, and whether the insulin sensitivity is altered in obesity or type 2 diabetes (T2DM). DESIGN: This was a cross-sectional and longitudinal study. SETTING: The study was conducted in a clinical research center. PATIENTS OR OTHER PARTICIPANTS: Bone marrow adipose tissue glucose uptake (GU) was assessed in 23 morbidly obese subjects (9 with T2DM) and 9 healthy controls with normal body weight. In addition, GU was assessed in another 11 controls during cold exposure. Bone marrow adipose tissue samples for molecular analyses were collected from non-DM patients undergoing knee arthroplasty. INTERVENTION(S): Obese subjects were assessed before and 6 months after bariatric surgery and controls at 1 time point. MAIN OUTCOME MEASURE: We used positron emission tomography imaging with 2-[18F]fluoro-2-deoxy-D-glucose tracer to characterize GU in femoral and vertebral BMAT. Bone marrow adipose tissue molecular profile was assessed using quantitative RT-PCR. RESULTS: Insulin enhances GU in human BMAT. Femoral BMAT insulin sensitivity was impaired in obese patients with T2DM compared to controls, but it improved after bariatric surgery. Furthermore, gene expression analysis revealed that BMAT was distinct from brown and white adipose tissue. CONCLUSIONS: Bone marrow adipose tissue is a metabolically active, insulin-sensitive and molecularly distinct fat depot that may play a role in whole body energy metabolism.

Dovitinib dilactic acid reduces tumor growth and tumor-induced bone changes in an experimental breast cancer bone growth model.

Advanced breast cancer has a high incidence of bone metastases. In bone, breast cancer cells induce osteolytic or mixed bone lesions by inducing an imbalance in bone formation and resorption. Activated fibroblast growth factor receptors (FGFRs) are important in regulation of tumor growth and bone remodeling. In this study we used FGFR1 and FGFR2 gene amplifications containing human MFM223 breast cancer cells in an experimental xenograft model of breast cancer bone growth using intratibial inoculation technique. This model mimics bone metastases in breast cancer patients. The effects of an FGFR inhibitor, dovitinib dilactic acid (TKI258) on tumor growth and tumor-induced bone changes were evaluated. Cancer-induced bone lesions were smaller in dovitinib-treated mice as evaluated by X-ray imaging. Peripheral quantitative computed tomography imaging showed higher total and cortical bone mineral content and cortical bone mineral density in dovitinib-treated mice, suggesting better preserved bone mass. CatWalk gait analysis indicated that dovitinib-treated mice experienced less cancer-induced bone pain in the tumor-bearing leg. A trend towards decreased tumor growth and metabolic activity was observed in dovitinib-treated mice quantified by positron emission tomography imaging with 2-[18F]fluoro-2-deoxy-D-glucose at the endpoint. We conclude that dovitinib treatment decreased tumor burden, cancer-induced changes in bone, and bone pain. The results suggest that targeting FGFRs could be beneficial in breast cancer patients with bone metastases.

Testicular Function and Bone in Young Men with Severe Childhood-Onset Obesity.

BACKGROUND: Previous studies suggest increased risk for hypoandrogenism and fractures in men with obesity. We aimed to describe the effects of severe childhood-onset obesity on the cross talk between metabolic state, testes, and skeleton at late puberty. METHODS: A cohort of adolescent and young adult males with severe childhood-onset obesity (n = 21, mean age 18.5 years) and an age-matched control group were assessed for testicular hormones and X-ray absorptiometry-derived bone mass. RESULTS: Current median body mass indexes for the obese and control subjects were 37.4 and 22.9. Severe early-onset obesity manifested with lower free testosterone (median [interquartile range] 244 [194-332] vs. 403 [293-463] pmol/L, p = 0.002). Lower insulin-like 3 (1.02 [0.82-1.23] vs. 1.22 [1.01-1.46] ng/mL, p = 0.045) and lower ratio of testosterone to luteinizing hormone (2.81 [1.96-3.98] vs. 4.10 [3.03-5.83] nmol/IU, p = 0.008) suggested disrupted Leydig cell function. The degree of current obesity inversely correlated with free testosterone (τ = -0.516, p = 0.003), which in turn correlated positively with bone area at all measurement sites in males with childhood-onset obesity. CONCLUSIONS: Severe childhood-onset obesity is associated with impaired Leydig cell function in young men and lower free testosterone may contribute to impaired skeletal characteristics.

Low free 25-hydroxyvitamin D and high vitamin D binding protein and parathyroid hormone in obese Caucasians. A complex association with bone?

BACKGROUND: Studies have shown altered vitamin D metabolism in obesity. We assessed differences between obese and normal-weight subjects in total, free, and bioavailable 25-hydroxyvitamin D (25(OH)D, 25(OH)DFree, and 25(OH)DBio, respectively), vitamin D binding protein (DBP), parathyroid hormone (PTH) and bone traits. METHODS: 595 37-47-year-old healthy Finnish men and women stratified by BMI were examined in this cross-sectional study. Background characteristic and intakes of vitamin D and calcium were collected. The concentrations of 25(OH)D, PTH, DBP, albumin and bone turnover markers were determined from blood. 25(OH)DFree and 25(OH)DBio were calculated. pQCT was performed at radius and tibia. RESULTS: Mean±SE (ANCOVA) 25(OH)DFree (10.8±0.6 vs 12.9±0.4 nmol/L; P = 0.008) and 25(OH)DBio (4.1±0.3 vs 5.1±0.1 nmol/L; P = 0.003) were lower in obese than in normal-weight women. In men, 25(OH)D (48.0±2.4 vs 56.4±2.0 nmol/L, P = 0.003), 25(OH)DFree (10.3±0.7 vs 12.5±0.6 pmol/L; P = 0.044) and 25(OH)DBio (4.2±0.3 vs 5.1±0.2 nmol/L; P = 0.032) were lower in obese. Similarly in all subjects, 25(OH)D, 25(OH)DFree and 25(OH)DBio were lower in obese (P<0.001). DBP (399±12 vs 356±7mg/L, P = 0.008) and PTH (62.2±3.0 vs 53.3±1.9 ng/L; P = 0.045) were higher in obese than in normal-weight women. In all subjects, PTH and DBP were higher in obese (P = 0.047and P = 0.004, respectively). In obese women, 25(OH)D was negatively associated with distal radius trabecular density (R2 = 0.089, P = 0.009) and tibial shaft cortical strength index (CSI) (R2 = 0.146, P = 0.004). 25(OH)DFree was negatively associated with distal radius CSI (R2 = 0.070, P = 0.049), radial shaft cortical density (CorD) (R2 = 0.050, P = 0.045), and tibial shaft CSI (R2 = 0.113, P = 0.012). 25(OH)DBio was negatively associated with distal radius CSI (R2 = 0.072, P = 0.045), radial shaft CorD (R2 = 0.059, P = 0.032), and tibial shaft CSI (R2 = 0.093, P = 0.024). CONCLUSIONS: The associations between BMI and 25(OH)D, 25(OH)DFree, and 25(OH)DBio, DBP, and PTH suggest that obese subjects may differ from normal-weight subjects in vitamin D metabolism. BMI associated positively with trabecular bone traits and CSI in our study, and slightly negatively with cortical bone traits. Surprisingly, there was a negative association of free and bioavailable 25(OH)D and some of the bone traits in obese women.

Changes in bone metabolism after bariatric surgery by gastric bypass or sleeve gastrectomy.

Bariatric surgery results in rapid weight loss and beneficial metabolic effects, but may have negative effects on the skeleton. The objective of this prospective study was to evaluate changes in bone metabolism in response to bariatric surgery with two surgical techniques. 46 morbidly obese subjects (mean 44.9years, BMI 42.1) with (n=19) or without (n=27) type 2 diabetes (T2DM) at baseline underwent either Roux-en-Y gastric bypass (RYGB, n=21) or sleeve gastrectomy (SG, n=25). Bone turnover markers (CTX, PINP, TRAcP5b, TotalOC and ucOC) were measured before and six months after surgery. Volumetric bone mineral density (vBMD) at lumbar spine and vertebral bone marrow (VBM) fat were measured in 21 subjects (7 RYGB and 14 SG) with three-dimensional quantitative computer tomography and 1H MR spectroscopy, respectively. 25 non-obese subjects were recruited as controls (mean 45.8years, BMI 23.0) and assessed at a single cross-sectional visit. Obese subjects had significantly lower bone turnover at baseline when compared to non-obese controls. Bone metabolic markers markedly increased post-operatively (p<0.0001 for all). The activation of bone remodeling was significantly higher after RYGB than after SG and was particularly observed in patients, whose type 2 diabetes was in remission after weight loss. There was no change in volumetric BMD or marrow fat at lumbar spine six months after surgery in our sample. In conclusion, severe obesity decreases bone remodeling, which is activated after bariatric surgery. The increase in bone turnover after surgery is affected by the choice of surgical technique and by the post-surgery remission of T2DM.

Bone mineral density is increased after a 16-week resistance training intervention in elderly women with decreased muscle strength.

OBJECTIVE: Non-pharmacological interventions are important in reducing risk for osteoporotic fractures. We investigated the effects of a 16-week individualized resistance training intervention on bone mineral density (BMD), bone turnover markers and 10-year relative risk (RR) for osteoporotic fracture. DESIGN: Interventional study with a follow-up. METHODS: In total, 37 elderly women (mean age 71.9 ± 3.1 years) with decreased muscle strength participated in the resistance training intervention three times per week with 60 min per session for 16 weeks under the supervision of a licensed physiotherapist. Total hip BMD with quantitative CT, bone markers (sclerostin, osteocalcin, CTX, PINP, IGF-1, 25(OH)-D) and 10-year RR for osteoporotic fracture were measured at baseline, post-intervention and at 1-year follow-up after the end of the intervention. Eleven age- and sex-matched controls did not participate in the intervention but were studied at baseline and at 1-year follow-up. RESULTS: Resistance training seemed to increase total hip BMD by 6% (P = 0.005). Sclerostin (P < 0.001) and total osteocalcin (P = 0.04) increased while other bone markers remained unchanged. A 10-year RR for major osteoporotic and hip fracture remained unchanged. At follow-up total hip BMD (P < 0.001) decreased back to the baseline level with a simultaneous decrease in serum sclerostin (P = 0.045), CTX (P < 0.001) and an increase in 25(OH)-D (P < 0.001), 10-year RR for major osteoporotic (P = 0.002) and hip fracture (P = 0.01). CONCLUSIONS: Our findings suggest an important role of continuous supervised resistance training for the prevention of osteoporotic fractures in elderly women with decreased muscle strength.

Obese young adults exhibit lower total and lower free serum 25-hydroxycholecalciferol in a randomized vitamin D intervention.

OBJECTIVE: Although obesity is a risk factor for vitamin D insufficiency, its impact on vitamin D-binding protein (DBP) concentration, and thereby possibly also on free 25OHD, is less well known. Our aim was to compare total and free serum 25OHD, and DBP concentrations between obese and normal-weight young adults at baseline and their responses to cholecalciferol supplementation. DESIGN: A 12-week randomized, double-blinded clinical trial. PATIENTS: Obese subjects N = 18 (BMI = 38, 67% men) with severe childhood-onset obesity and 24 normal-weight subjects (BMI = 23, 46% men), age between 15 and 25 years, were randomized into two groups to receive either placebo or cholecalciferol 50 µg (2000 IU) daily. MEASUREMENTS: At baseline, 6-week and 12-week blood samples and anthropometric measurements were collected; baseline body composition was assessed by dual-energy X-ray absorptiometry. RESULTS: At baseline, obese subjects had, compared with normal-weight, lower total and free serum 25OHD (49 vs 62 nmol/l, P = 0·041; 2·8 vs 4·7 pg/ml, P = 0·001), without differences in DBP concentrations (309 vs 346 µg/ml, P = 0·212). Cholecalciferol 50 µg per day increased both total and free 25OHD (ancova P < 0·001 and P = 0·021). The response of total 25OHD to supplementation was inferior in the obese compared with normal-weight subjects (P = 0·027). On the contrary, the change in free 25OHD concentration was similar in groups (P = 0·487). CONCLUSIONS: Obese young adults exhibit lower total and free 25OHD concentration, which is not directly explained by differences in DBP status. The response of free 25OHD to supplementation did not differ between obese and normal-weight subjects.