Effects of PTH on osteoblast bioenergetics in response to glucose.

Parathyroid hormone acts through its receptor, PTHR1, expressed on osteoblasts, to control bone remodeling. Metabolic flexibility for energy generation has been demonstrated in several cell types dependent on substrate availability. Recent studies have identified a critical role for PTH in regulating glucose, fatty acid and amino acid metabolism thus stimulating both glycolysis and oxidative phosphorylation. Therefore, we postulated that PTH stimulates increased energetic output by osteoblasts either by increasing glycolysis or oxidative phosphorylation depending on substrate availability. To test this hypothesis, undifferentiated and differentiated MC3T3E1C4 calvarial pre-osteoblasts were treated with PTH to study osteoblast bioenergetics in the presence of exogenous glucose. Significant increases in glycolysis with acute ∼1 h PTH treatment with minimal effects on oxidative phosphorylation in undifferentiated MC3T3E1C4 in the presence of exogenous glucose were observed. In differentiated cells, the increased glycolysis observed with acute PTH was completely blocked by pretreatment with a Glut1 inhibitor (BAY-876) resulting in a compensatory increase in oxidative phosphorylation. We then tested the effect of PTH on the function of complexes I and II of the mitochondrial electron transport chain in the absence of glycolysis. Utilizing a novel cell plasma membrane permeability mitochondrial (PMP) assay, in combination with complex I and II specific substrates, slight but significant increases in basal and maximal oxygen consumption rates with 24 h PTH treatment in undifferentiated MC3T3E1C4 cells were noted. Taken together, our data demonstrate for the first time that PTH stimulates both increases in glycolysis and the function of the electron transport chain, particularly complexes I and II, during high energy demands in osteoblasts.

Development and biophysical characterization of a humanized FSH-blocking monoclonal antibody therapeutic formulated at an ultra-high concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the development of a unique formulation for our first-in-class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, confirmed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) Spectroscopy. Three rapid freeze-thaw cycles at -80 °C/25 °C or -80 °C/37 °C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (Tm) for formulated MS-Hu6 increased by >4.80 °C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Development and Biophysical Characterization of a Humanized FSH-Blocking Monoclonal Antibody Therapeutic Formulated at an Ultra-High Concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the creation of a unique formulation for our first-in- class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, conformed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. Three rapid freeze-thaw cycles at -80°C/25°C or -80°C/37°C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (T m ) for formulated MS-Hu6 increased by >4.80°C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Vitamin D: 100 years of discoveries, yet controversy continues.

Over the past 100 years, many major breakthroughs and discoveries have occurred in relation to vitamin D research. These developments include the cure of rickets in 1919, the discovery of vitamin D compounds, advances in vitamin D molecular biology, and improvements in our understanding of endocrine control of vitamin D metabolism. Furthermore, recommended daily allowances for vitamin D have been established and large clinical trials of vitamin D, aimed at clarifying the effect of Vitamin D in the prevention of multiple diseases, have been completed. However, disappointingly, these clinical trials have not fulfilled the expectations many had 10 years ago. In almost every trial, various doses and routes of administration did not show efficacy of vitamin D in preventing fractures, falls, cancer, cardiovascular diseases, type 2 diabetes, asthma, and respiratory infections. Although concerns about side-effects of long-term high-dose treatments, such as hypercalcaemia and nephrocalcinosis, have been around for four decades, some trials from the past 5 years have had new and unexpected adverse events. These adverse events include increased fractures, falls, and hospitalisations in older people (aged >65 years). Several of these clinical trials were powered appropriately for a primary outcome but did not include dose response studies and were underpowered for secondary analyses. Furthermore, more attention should be paid to the safety of high doses of vitamin D supplementation, particularly in older people. In addition, despite universal recommendations by osteoporosis societies for combining calcium supplements with vitamin D there remains insufficient data about their efficacy and effect on fracture risk in the highest risk groups. More trials are needed for people with severe vitamin D deficiency (ie, serum 25-hydroxyvitamin D <25nmol/L [10ng/mL]). In this Personal View, we summarise and discuss some of the major discoveries and controversies in the field of vitamin D.

Secondary Fracture Prevention: Consensus Clinical Recommendations from a Multistakeholder Coalition.

Osteoporosis-related fractures are undertreated, due in part to misinformation about recommended approaches to patient care and discrepancies among treatment guidelines. To help bridge this gap and improve patient outcomes, the American Society for Bone and Mineral Research assembled a multistakeholder coalition to develop clinical recommendations for the optimal prevention of secondary fractureamong people aged 65 years and older with a hip or vertebral fracture. The coalition developed 13 recommendations (7 primary and 6 secondary) strongly supported by the empirical literature. The coalition recommends increased communication with patients regarding fracture risk, mortality and morbidity outcomes, and fracture risk reduction. Risk assessment (including fall history) should occur at regular intervals with referral to physical and/or occupational therapy as appropriate. Oral, intravenous, andsubcutaneous pharmacotherapies are efficaciousandcanreduce risk of future fracture.Patientsneededucation,however, about thebenefitsandrisks of both treatment and not receiving treatment. Oral bisphosphonates alendronate and risedronate are first-line options and are generally well tolerated; otherwise, intravenous zoledronic acid and subcutaneous denosumab can be considered. Anabolic agents are expensive butmay be beneficial for selected patients at high risk.Optimal duration of pharmacotherapy is unknown but because the risk for second fractures is highest in the earlypost-fractureperiod,prompt treatment is recommended.Adequate dietary or supplemental vitaminDand calciumintake shouldbe assured. Individuals beingtreatedfor osteoporosis shouldbe reevaluated for fracture risk routinely, includingvia patienteducationabout osteoporosisandfracturesandmonitoringfor adverse treatment effects.Patients shouldbestronglyencouraged to avoid tobacco, consume alcohol inmoderation atmost, and engage in regular exercise and fall prevention strategies. Finally, referral to endocrinologists or other osteoporosis specialists may be warranted for individuals who experience repeated fracture or bone loss and those with complicating comorbidities (eg, hyperparathyroidism, chronic kidney disease).

Secondary Fracture Prevention: Consensus Clinical Recommendations from a Multistakeholder Coalition.

Osteoporosis-related fractures are undertreated, due in part to misinformation about recommended approaches to patient care and discrepancies among treatment guidelines. To help bridge this gap and improve patient outcomes, the American Society for Bone and Mineral Research assembled a multistakeholder coalition to develop clinical recommendations for the optimal prevention of secondary fracture among people aged 65 years and older with a hip or vertebral fracture. The coalition developed 13 recommendations (7 primary and 6 secondary) strongly supported by the empirical literature. The coalition recommends increased communication with patients regarding fracture risk, mortality and morbidity outcomes, and fracture risk reduction. Risk assessment (including fall history) should occur at regular intervals with referral to physical and/or occupational therapy as appropriate. Oral, intravenous, and subcutaneous pharmacotherapies are efficacious and can reduce risk of future fracture. Patients need education, however, about the benefits and risks of both treatment and not receiving treatment. Oral bisphosphonates alendronate and risedronate are first-line options and are generally well tolerated; otherwise, intravenous zoledronic acid and subcutaneous denosumab can be considered. Anabolic agents are expensive but may be beneficial for selected patients at high risk. Optimal duration of pharmacotherapy is unknown but because the risk for second fractures is highest in the early post-fracture period, prompt treatment is recommended. Adequate dietary or supplemental vitamin D and calcium intake should be assured. Individuals being treated for osteoporosis should be reevaluated for fracture risk routinely, including via patient education about osteoporosis and fractures and monitoring for adverse treatment effects. Patients should be strongly encouraged to avoid tobacco, consume alcohol in moderation at most, and engage in regular exercise and fall prevention strategies. Finally, referral to endocrinologists or other osteoporosis specialists may be warranted for individuals who experience repeated fracture or bone loss and those with complicating comorbidities (eg, hyperparathyroidism, chronic kidney disease). © 2019 American Society for Bone and Mineral Research.

A peptide containing the receptor binding site of insulin-like growth factor binding protein-2 enhances bone mass in ovariectomized rats.

Male Igfbp2-/- mice have a significant reduction in bone mass and administration of a peptide that contains the insulin-like growth factor binding protein-2(IGFBP-2) receptor-binding domain stimulates bone formation in these animals. Female Igfbp2-/- mice do not have this phenotype but following ovariectomy (OVX) lose more bone than OVX wild-type mice. This suggests that in the absence of estrogen, IGFBP-2 is required to maintain bone mass. Therefore these studies were undertaken to determine if this peptide could stimulate bone acquisition in OVX rats. OVX rats were divided into seven treatment groups: sham animals, OVX animals, OVX animals receiving a control scrambled peptide, or one of three doses of the active peptide termed PEG-HBD-1 (0.7, 2, and 6 mg·kg-1) and an OVX group receiving parathyroid hormone (PTH) (50 µg·kg-1 per day). The peptides were administered for 8 weeks. DXA revealed a significant reduction in femoral and tibial areal bone mineral density (aBMD) after OVX, whereas treatment with the high-dose peptide increased aBMD by 6.2% ± 2.4% (P < 0.01) compared to control peptide; similar to the increase noted with PTH (5.6% ± 3.0%, P < 0.01). Similar increases were noted with two lower doses of the peptide (3.8% ± 1.5%, P < 0.05 for low dose; 3.1% ± 1.6%, P = 0.07 for middle dose). Micro CT showed that the OVX control peptide animals had reductions of 41% and 64% in femoral trabecular BV/TV and trabecular number, respectively. All three doses of the peptide increased bone volume/total volume (BV/TV) significantly, while the low and middle doses increased trabecular number. Cortical BV/TV and thickness at the midshaft increased significantly with each dose of peptide (18.9% ± 9.8%, P < 0.01 and 14.2% ± 7.9%, P < 0.01 for low dose; 23.7% ± 10.7%, P < 0.001 and 15.8% ± 6.1%, P < 0.001 for middle dose; 19.0% ± 6.9%, P < 0.01 and 16.2% ± 9.7%, P < 0.001 for high dose) and with PTH (25.8% ± 9.2%, P < 0.001 and 19.4% ± 8.8%, P < 0.001). Histomorphometry showed that the lowest dose of peptide stimulated BV/TV, trabecular thickness, mineral apposition rate (MAR), bone formation rate/bone surface (BFR/BS), number of osteoblasts/bone perimeter (N.ob/B.pm), and decreased osteoclast surface/bone perimeter (Oc.S/B.Pm). The highest dose stimulated each of these parameters except MAR and BFR/BS. Thus, the heparin-binding domain receptor region of IGFBP-2 accounts for its anabolic activity in bone. Importantly, this peptide enhances bone mass in estrogen-deficient animals.

DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH.

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr knockout (DMP-GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.

Hypothalamic leptin gene therapy reduces body weight without accelerating age-related bone loss.

Excessive weight gain in adults is associated with a variety of negative health outcomes. Unfortunately, dieting, exercise, and pharmacological interventions have had limited long-term success in weight control and can result in detrimental side effects, including accelerating age-related cancellous bone loss. We investigated the efficacy of using hypothalamic leptin gene therapy as an alternative method for reducing weight in skeletally-mature (9 months old) female rats and determined the impact of leptin-induced weight loss on bone mass, density, and microarchitecture, and serum biomarkers of bone turnover (CTx and osteocalcin). Rats were implanted with cannulae in the 3rd ventricle of the hypothalamus and injected with either recombinant adeno-associated virus encoding the gene for rat leptin (rAAV-Leptin, n=7) or a control vector encoding green fluorescent protein (rAAV-GFP, n=10) and sacrificed 18 weeks later. A baseline control group (n=7) was sacrificed at vector administration. rAAV-Leptin-treated rats lost weight (-4±2%) while rAAV-GFP-treated rats gained weight (14±2%) during the study. At study termination, rAAV-Leptin-treated rats weighed 17% less than rAAV-GFP-treated rats and had lower abdominal white adipose tissue weight (-80%), serum leptin (-77%), and serum IGF1 (-34%). Cancellous bone volume fraction in distal femur metaphysis and epiphysis, and in lumbar vertebra tended to be lower (P<0.1) in rAAV-GFP-treated rats (13.5 months old) compared to baseline control rats (9 months old). Significant differences in cancellous bone or biomarkers of bone turnover were not detected between rAAV-Leptin and rAAV-GFP rats. In summary, rAAV-Leptin-treated rats maintained a lower body weight compared to baseline and rAAV-GFP-treated rats with minimal effects on bone mass, density, microarchitecture, or biochemical markers of bone turnover.

Questions About Vitamin D for Primary Care Practice: Input From an NIH Conference.

There is considerable consumer and physician interest in vitamin D as a possible therapeutic agent for a range of clinical conditions and, despite mixed evidence, the interest does not appear to lessen. Some clinicians believe that consumption of vitamin D is inadequate and, in turn, advocate vitamin D supplementation to increase serum levels of the nutrient. However, evidence concerning the role of vitamin D in health and disease is conflicting, and primary care physicians have little time to sort through the data and may find it difficult to advise their patients. To better understand the challenges that primary care physicians face regarding vitamin D, and to help inform those who provide guidance for clinical decision-making, the Office of Dietary Supplements at the National Institutes of Health, with co-sponsorship from other federal health agencies, held a conference titled Vitamin D: Moving Toward Evidence-based Decision Making in Primary Care in December 2014. More than 20 invited presenters and panelists considered laboratory methods for measuring vitamin D status, discussed how clinical studies of vitamin D should be evaluated and used in developing recommendations, noted the role of values and preferences in clinical decision-making, debated the current science related to at-risk groups, and described emerging data about health risks of excessive intakes of vitamin D. Eight questions about vitamin D stem from the Conference presentations as well as other expert sources.

Rationale and design of the Vitamin D and Type 2 Diabetes (D2d) study: a diabetes prevention trial.

OBJECTIVE: Observational studies suggest that vitamin D may lower the risk of type 2 diabetes. However, data from long-term trials are lacking. The Vitamin D and Type 2 Diabetes (D2d) study is a randomized clinical trial designed to examine whether a causal relationship exists between vitamin D supplementation and the development of diabetes in people at high risk for type 2 diabetes. RESEARCH DESIGN AND METHODS: D2d was designed with support from a U34 planning grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The final protocol was approved by the D2d Research Group, the data and safety monitoring board, and NIDDK. Key eligibility criteria are age ≥30 years, BMI of 24 (22.5 for Asian Americans) to 42 kg/m(2), increased risk for diabetes (defined as meeting two of three glycemic criteria for prediabetes established by the American Diabetes Association [fasting glucose 100-125 mg/dL (5.5-6.9 mmol/L), 2-h postload glucose after 75-g glucose load 140-199 mg/dL (7.7-11.0 mmol/L), hemoglobin A1c 5.7-6.4% (39-46 mmol/mol)]), and no hyperparathyroidism, nephrolithiasis, or hypercalcemia. D2d participants are randomized to once-daily vitamin D3 (cholecalciferol 4,000 IU) or placebo and followed for an average of 3 years. The primary end point is time to incident diabetes as assessed by laboratory criteria during the study or by adjudication if diagnosed outside of D2d. Recruitment was initiated at the end of 2013. CONCLUSIONS: D2d will test whether vitamin D supplementation is safe and effective at lowering the risk of progression to diabetes in people at high risk for type 2 diabetes.

Diet and gene interactions influence the skeletal response to polyunsaturated fatty acids.

Diets rich in omega-3s have been thought to prevent both obesity and osteoporosis. However, conflicting findings are reported, probably as a result of gene by nutritional interactions. Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear receptor that improves insulin sensitivity but causes weight gain and bone loss. Fish oil is a natural agonist for PPARγ and thus may exert its actions through the PPARγ pathway. We examined the role of PPARγ in body composition changes induced by a fish or safflower oil diet using two strains of C57BL/6J (B6); i.e. B6.C3H-6T (6T) congenic mice created by backcrossing a small locus on Chr 6 from C3H carrying 'gain of function' polymorphisms in the Pparγ gene onto a B6 background, and C57BL/6J mice. After 9months of feeding both diets to female mice, body weight, percent fat and leptin levels were less in mice fed the fish oil vs those fed safflower oil, independent of genotype. At the skeletal level, fish oil preserved vertebral bone mineral density (BMD) and microstructure in B6 but not in 6T mice. Moreover, fish oil consumption was associated with an increase in bone marrow adiposity and a decrease in BMD, cortical thickness, ultimate force and plastic energy in femur of the 6T but not the B6 mice. These effects paralleled an increase in adipogenic inflammatory and resorption markers in 6T but not B6. Thus, compared to safflower oil, fish oil (high ratio omega-3/-6) prevents weight gain, bone loss, and changes in trabecular microarchitecture in the spine with age. These beneficial effects are absent in mice with polymorphisms in the Pparγ gene (6T), supporting the tenet that the actions of n-3 fatty acids on bone microstructure are likely to be genotype dependent. Thus caution must be used in interpreting dietary intervention trials with skeletal endpoints in mice and in humans.

Vitamin D and calcium: what do we need to know?

This chapter provides a summary of the literature including results from large trials, meta-analyses, and recent major recommendations. Two well-accepted and recent recommendations include those from the Agency for Health Research and Quality on calcium and vitamin D supplementation and from the Institute of Medicine's dietary reference intakes for calcium and vitamin D. This review suggests that there is strong evidence for synergistic roles of calcium and vitamin D in maintaining bone health in postmenopausal women, but the correlation between vitamin D alone and bone health is overall inconclusive.

Common misconceptions about vitamin D--implications for clinicians.

Misconceptions about vitamin D continue to grow despite publications in the past few years that have attempted to clarify risk. We present our perspective, and offer several conclusions. Calcium and vitamin D supplementation can reduce fracture risk by ∼10%. On the other hand, little evidence exists to support a threshold measure for vitamin D status (serum levels of 25-hydroxyvitamin D) above which fractures are reduced. The association of serum concentrations of 25-hydroxyvitamin D with other chronic diseases is confounded by multiple factors and conflicting outcomes that cannot be used to support a causal association. High doses of vitamin D supplements might not be completely harmless and should be avoided until additional data becomes available. Similarly, scant rationale exists for aggressive vitamin D supplementation for pregnant or lactating women. Dispelling misconceptions about vitamin D will ultimately benefit health-care providers and patients alike.

The nonskeletal effects of vitamin D: an Endocrine Society scientific statement.

Significant controversy has emerged over the last decade concerning the effects of vitamin D on skeletal and nonskeletal tissues. The demonstration that the vitamin D receptor is expressed in virtually all cells of the body and the growing body of observational data supporting a relationship of serum 25-hydroxyvitamin D to chronic metabolic, cardiovascular, and neoplastic diseases have led to widespread utilization of vitamin D supplementation for the prevention and treatment of numerous disorders. In this paper, we review both the basic and clinical aspects of vitamin D in relation to nonskeletal organ systems. We begin by focusing on the molecular aspects of vitamin D, primarily by examining the structure and function of the vitamin D receptor. This is followed by a systematic review according to tissue type of the inherent biological plausibility, the strength of the observational data, and the levels of evidence that support or refute an association between vitamin D levels or supplementation and maternal/child health as well as various disease states. Although observational studies support a strong case for an association between vitamin D and musculoskeletal, cardiovascular, neoplastic, and metabolic disorders, there remains a paucity of large-scale and long-term randomized clinical trials. Thus, at this time, more studies are needed to definitively conclude that vitamin D can offer preventive and therapeutic benefits across a wide range of physiological states and chronic nonskeletal disorders.