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 p , 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 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

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 (= 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

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

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-[ 18 F]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.</p

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.</p

Total and Carboxylated Osteocalcin Associate with Insulin Levels in Young Adults Born with Normal or Very Low Birth Weight

Peer reviewe

Bone turnover markers are correlated with total skeletal uptake of 99mTc-methylene diphosphonate (99mTc-MDP)

ABSTRACT: BACKGROUND: Skeletal uptake of 99mTc labelled methylene diphosphonate (99mTc-MDP) is used for producing images of pathological bone uptake due to its incorporation to the sites of active bone turnover. This study was done to validate bone turnover markers using total skeletal uptake (TSU) of 99mTc-MDP. METHODS: 22 postmenopausal women (52-80 years) volunteered to participate. Scintigraphy was performed by injecting 520 MBq of 99mTc-MDP and taking whole body images after 3 minutes, and 5 hours. TSU was calculated from these two images by taking into account the urinary loss and soft tissue uptake. Bone turnover markers used were bone specific alkaline phosphatase (S-Bone ALP), three different assays for serum osteocalcin (OC), tartrate resistant acid phosphatase 5b (S-TRACP5b), serum C-terminal cross-linked telopeptides of type I collagen (S-CTX-I) and three assays for urinary osteocalcin (U-OC). RESULTS: The median TSU of 99mTc-MDP was 23% of the administered activity. All bone turnover markers were significantly correlated with TSU with r-values from 0.52 (p = 0.013) to 0.90 (p < 0.001). The two resorption markers had numerically higher correlations (S-TRACP5b r = 0.90, S-CTX-I r = 0.80) than the formation markers (S-Total OC r = 0.72, S-Bone ALP r = 0.66), but the difference was not statistically significant. TSU did not correlate with age, weight, body mass index or bone mineral density. CONCLUSION: In conclusion, bone turnover markers are strongly correlated with total skeletal uptake of 99mTc-MDP. There were no significant differences in correlations for bone formation and resorption markers. This should be due to the coupling between formation and resorption

The effects of acute hyperinsulinemia on bone metabolism

Peer reviewe

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

ContextBone 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.ObjectiveOur 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).DesignThis was a cross-sectional and longitudinal study.SettingThe study was conducted in a clinical research center.Patients or Other ParticipantsBone 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 MeasureWe 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.ResultsInsulin 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.ConclusionsBone marrow adipose tissue is a metabolically active, insulin-sensitive and molecularly distinct fat depot that may play a role in whole body energy metabolism.</p