No Evidence of Association Between Undercarboxylated Osteocalcin and Incident Type 2 Diabetes.

Mouse models suggest that undercarboxylated osteocalcin (ucOC), produced by the skeleton, protects against type 2 diabetes development, whereas human studies have been inconclusive. We aimed to determine if ucOC or total OC is associated with incident type 2 diabetes or changes in fasting glucose, insulin resistance (HOMA-IR), or beta-cell function (HOMA-Beta). A subcohort (n = 338; 50% women; 36% black) was identified from participants without diabetes at baseline in the Health, Aging, and Body Composition Study. Cases of incident type 2 diabetes (n = 137) were defined as self-report at an annual follow-up visit, use of diabetes medication, or elevated fasting glucose during 8 years of follow-up. ucOC and total OC were measured in baseline serum. Using a case-cohort design, the association between biomarkers and incident type 2 diabetes was assessed using robust weighted Cox regression. In the subcohort, linear regression models analyzed the associations between biomarkers and changes in fasting glucose, HOMA-IR, and HOMA-Beta over 9 years. Higher levels of ucOC were not statistically associated with increased risk of incident type 2 diabetes (adjusted hazard ratio = 1.06 [95% confidence interval, 0.84-1.34] per 1 standard deviation [SD] increase in ucOC). Results for %ucOC and total OC were similar. Adjusted associations of ucOC, %ucOC, and total OC with changes in fasting glucose, HOMA-IR, and HOMA-Beta were modest and not statistically significant. We did not find evidence of an association of baseline undercarboxylated or total osteocalcin with risk of incident type 2 diabetes or with changes in glucose metabolism in older adults. © 2022 American Society for Bone and Mineral Research (ASBMR).

Biomolecular regulation, composition and nanoarchitecture of bone mineral.

Tough natural nanocomposites like bone, nacre and sea sponges contain within their hierarchy, a mineral (phosphate, silicate or carbonate) phase that interacts with an organic phase. In bone, the role of mineral ultrastructure (organization, morphology, composition) is crucial to the mechanical and biological properties of the tissue. Better understanding of mineral interaction with the organic matrix, in particular non-collagenous proteins, osteocalcin (OC) and osteopontin (OPN), can lead to better design of biomimetic materials. Using small angle x-ray scattering (SAXS) and wavelength dispersive spectroscopy (WDS) on single (OC-/- and OPN-/-) and double (OC-OPN-/-;-/-) genetic knockout mice bones, we demonstrate that both osteocalcin and osteopontin have specific roles in the biomolecular regulation of mineral in bone and together they are major determinants of the quality of bone mineral. Specifically, for the first time, we show that proteins osteocalcin and osteopontin regulate bone mineral crystal size and organization in a codependent manner, while they independently determine crystal shape. We found that OC is more dominant in the regulation of the physical properties of bone mineral, while OPN is more dominant in the regulation of the mineral composition.

Osteocalcin and osteopontin influence bone morphology and mechanical properties.

Osteocalcin (OC) and osteopontin (OPN) are major non-collagenous proteins (NCPs) involved in bone matrix organization and deposition. In spite of this, it is currently unknown whether OC and OPN alter bone morphology and consequently affect bone fracture resistance. The goal of this study is to establish the role of OC and OPN in the determination of cortical bone size, shape, and mechanical properties. Our results show that Oc-/- and Opn-/- mice were no different from each other or wild type (WT) with respect to bone morphology (P > 0.1). Bones from mice lacking both NCPs (Oc-/- Opn-/- ) were shorter, with thicker cortices and larger cortical areas, compared with the WT, Oc-/- , and Opn-/- groups (P < 0.05), suggesting a synergistic role for NCPs in the determination of bone morphology. Maximum bending load was significantly different among the groups (P = 0.024), while tissue mineral density and measures of stiffness and strength were not different (P > 0.1). We conclude that the removal of both OC and OPN from bone matrix induces morphological adaptation at the structural level to maintain bone strength.

Identification and characterization of glycation adducts on osteocalcin.

Osteocalcin is an important extracellular matrix bone protein that contributes to the structural properties of bone through its interactions with hydroxyapatite mineral and with collagen I. This role may be affected by glycation, a labile modification the levels of which has been shown to correlate with bone fragility. Glycation starts with the spontaneous addition of a sugar onto a free amine group on a protein, forming an Amadori product, and then proceeds through several environment-dependent stages resulting in the formation of an advanced glycation end product. Here, we induce the first step of this modification on synthetic osteocalcin, and then use multiple mass spectrometry fragmentation techniques to determine the location of this modification. Collision-induced dissociation resulted in spectra dominated by neutral loss, and was unable to identify Amadori products. Electron-transfer dissociation showed that the Amadori product formed solely on osteocalcin's N-terminus. This suggests that the glycation of osteocalcin is unlikely to interfere with osteocalcin's interaction with hydroxyapatite. Instead, glycation may interfere with its interaction with collagen I or another bone protein, osteopontin. Potentially, the levels of glycated osteocalcin fragments released from bone during bone resorption could be used to assess bone quality, should the N-terminal fragments be targeted.

Reducing Undercarboxylated Osteocalcin With Vitamin K Supplementation Does Not Promote Lean Tissue Loss or Fat Gain Over 3 Years in Older Women and Men: A Randomized Controlled Trial.

Osteocalcin (OC) is a vitamin K-dependent protein synthesized during bone formation. Mice injected with the undercarboxylated form of OC (ucOC) had more skeletal muscle mass and less fat mass than sham-treated controls, suggesting a unique metabolic role for ucOC. UcOC decreases in response to vitamin K supplementation. Our objective was to determine the effect of reducing ucOC on change in lean tissue and fat mass in older community-dwelling adults (n = 401, mean ± SD 69 ± 6 years) using data from a randomized controlled trial of vitamin K supplementation. Over 3 years, serum ucOC was reduced by 58% in women and by 61% in men randomized to vitamin K, whereas in the control group, ucOC decreased by 1% in women and 4% in men (supplementation*time p < 0.001 in men and women). However, there were no differences in the change in appendicular lean mass (calculated as arm lean mass + leg lean mass) or total body fat mass between women randomized to vitamin K and control over 3 years (supplementation*time p values all ≥ 0.18) or between men randomized to vitamin K and control (supplementation*time p values all ≥ 0.54). Consistent with these findings, ucOC was not associated cross-sectionally with appendicular lean mass or fat mass in men or women after adjustment for total OC at baseline (all p ≥ 0.12). These findings indicate the undercarboxylated form of OC is not implicated in age-related changes in skeletal muscle or adipose tissue mass in older community-dwelling adults. © 2016 American Society for Bone and Mineral Research.

Influence of carboxylation on osteocalcin detection by mass spectrometry.

RATIONALE: Osteocalcin is a small, abundant bone protein that is difficult to detect using high-throughput tandem mass spectrometry (MS/MS) proteomic approaches from bone protein extracts, and is predominantly detected by non-MS immunological methods. Here, we analyze bovine osteocalcin and its post-translational modifications to determine why a protein of this size goes undetected. METHODS: Osteocalcin was purified from cow bone using well-established methods. Intact osteocalcin or trypsin-digested osteocalcin were separated using an Agilent 1200 series high-performance liquid chromatography (HPLC) system and analyzed using a ThermoScientific LTQ-Orbitrap XL after fragmentation with higher-energy collision dissociation. Data were analyzed using Mascot or Prosight Lite. RESULTS: Our results support previous findings that the cow osteocalcin has up to three carboxylations using both intact osteocalcin and digested forms. Using Mascot, we were able to detect osteocalcin peptides, but no fragments that localized the carboxylations. Full annotation using Prosight Lite of the intact (three carboxylations), N-terminal peptide (one carboxylation), and middle peptide (two carboxylations) showed complete fragmentation was present, but complete neutral loss was observed. CONCLUSIONS: Osteocalcin carboxylation, and its associated neutral losses, makes high-throughput detection of this protein challenging; however, alternative fragmentation or limited purification can overcome these challenges. Copyright © 2016 John Wiley & Sons, Ltd.

Osteocalcin Is Not Associated with the Risk of Type 2 Diabetes: Findings from the EPIC-NL Study.

OBJECTIVE: To investigate whether total osteocalcin (tOC), uncarboxylated osteocalcin (ucOC) and percentage of uncarboxylated osteocalcin (%ucOC) are associated with the risk of type 2 diabetes. METHODS: This nested case control study included 1,635 participants, 833 incident diabetes cases and 802 non-diabetic control participants, aged 21-70 years from the EPIC-NL cohort. Baseline concentrations of tOC, ucOC and %ucOC were assessed. During 10 years of follow-up, diabetes cases were self-reported and verified against information from general practitioners or pharmacists. The association between the different forms of osteocalcin and diabetes risk was assessed with logistic regression adjusted for diabetes risk factors (waist circumference, age, sex, cohort, smoking status, family history of diabetes, hypertension, alcohol intake, physical activity and education) and dietary factors (total energy intake and energy adjusted intake of fat, fiber, protein and calcium). RESULTS: TOC concentration was not associated with diabetes risk, with an odds ratio (OR) of 0.97 (0.91-1.03) for each ng/ml increment after adjustment for diabetes risk factors and dietary factors. No association between ucOC and %ucOC and the risk of diabetes was observed either. In sex stratified analyses (P interaction = 0.07), higher %ucOC tended to be associated with an increased risk of type 2 diabetes in a multivariable model in women (OR 1.05 for each increment of 5% ucOC (1.00-1.11), Ptrend = 0.08), but not in men (OR 0.96 for each increment of 5% ucOC (0.88-1.04)). When waist circumference was replaced by body mass index, none of the osteocalcin forms were associated with the risk of type 2 diabetes in the final model among both women and men. CONCLUSIONS: Available evidence suggests that tOC, ucOC and %ucOC are each not associated with the risk of type 2 diabetes. However, more large-scale cohort studies are needed to clarify the presence of any association between the different forms of osteocalcin and the risk of type 2 diabetes.

Osteocalcin carboxylation is not associated with body weight or percent fat changes during weight loss in post-menopausal women.

Osteocalcin (OC) is a vitamin K-dependent bone protein used as a marker of bone formation. Mouse models have demonstrated a role for the uncarboxylated form of OC (ucOC) in energy metabolism, including energy expenditure and adiposity, but human data are equivocal. The purpose of this study was to determine the associations between changes in measures of OC and changes in body weight and percent body fat in obese, but otherwise healthy post-menopausal women undergoing a 20-week weight loss program. All participants received supplemental vitamins K and D and calcium. Body weight and body fat percentage (%BF) were assessed before and after the intervention. Serum OC [(total (tOC), ucOC, percent uncarboxylated (%ucOC)], and procollagen type 1N-terminal propeptide (P1NP; a measure of bone formation) were measured. Women lost an average of 10.9 ± 3.9 kg and 4 %BF. Serum concentrations of tOC, ucOC, %ucOC, and P1NP did not significantly change over the twenty-week intervention, nor were these measures associated with changes in weight (all p > 0.27) or %BF (all p > 0.54). Our data do not support an association between any serum measure of OC and weight or %BF loss in post-menopausal women supplemented with nutrients implicated in bone health.

Gamma-carboxylation and fragmentation of osteocalcin in human serum defined by mass spectrometry.

Serum osteocalcin (Oc) concentration is a highly specific measure of bone turnover, but its circulating proteoform(s) have not been well defined. Based on immunological methods, the major forms are thought to be the intact polypeptide and a large N-terminal-mid molecule fragment for which there is no consensus on the precise sequence. Vitamin K-dependent gamma (γ)-carboxylated variants of Oc are also found in circulation but there have been no methods that can define how many of the three potential γ-carboxyglutamic acid (Gla) residues are γ-carboxylated or provide their relative abundances. Recent reports that uncarboxylated and partially γ-carboxylated Oc forms have hormonal function underscore the need for precise evaluation of Oc at all three potential γ-carboxylation sites. Herein, mass spectrometric immunoassay (MSIA) was used to provide qualitative and semiquantitative (relative percent abundance) information on Oc molecular variants as they exist in individual plasma and serum samples. Following verification that observable Oc proteoforms were accurately assigned and not simply ex vivo artifacts, MALDI-MSIA and ESI-MSIA were used to assess the relative abundance of Oc truncation and γ-carboxylation, respectively, in plasma from 130 patients enrolled in vitamin K supplementation trials. Human Oc was found to circulate in over a dozen truncated forms with each of these displaying anywhere from 0-3 Gla residues. The relative abundance of truncated forms was consistent and unaffected by vitamin K supplementation. In contrast, when compared with placebo, vitamin K supplementation dramatically increased the fractional abundance of Oc with three Gla residues, corresponding to a decrease in the fractional abundance of Oc with zero Gla residues. These findings unequivocally document that increased vitamin K intake reduces the uncarboxylated form of Oc. Several reports of a positive effect of vitamin K intake on insulin sensitivity in humans have shown that un- or undercarboxylation of Oc, unlike in mice, is not associated with insulin resistance. Analyses similar to those described here will be useful to understand the functional significance of Oc γ-carboxylation in human health and disease.

Bone as an endocrine organ relevant to diabetes.

There are well-established associations between diabetes and fracture risk and yet the mechanism underlying these associations are controversial. Guided by a series of mouse studies, a specific form of the bone protein, osteocalcin, was proposed to be the mechanistic link between these two chronic diseases. Translation to humans initially appeared elusive in part because serum concentrations of osteocalcin are a biomarker of bone turnover and not necessarily specific to the biology of this protein. The suitability of the mouse model for the study of osteocalcin as a therapeutic target also appears ambiguous. With greater discrimination of the different forms of osteocalcin present in circulation and inclusion of multiple measures of bone turnover, evidence currently does not support osteocalcin as a protein critical to the diabetes and fracture association in humans.

Effect of paricalcitol on circulating parathyroid hormone in X-linked hypophosphatemia: a randomized, double-blind, placebo-controlled study.

CONTEXT: Hyperparathyroidism occurs frequently in X-linked hypophosphatemia (XLH) and may exacerbate phosphaturia, potentially affecting skeletal abnormalities. OBJECTIVE: The objective of the study was to suppress elevated PTH levels in XLH patients. DESIGN: This was a prospective, randomized, placebo-controlled, double-blind, 1-year trial of paricalcitol, with outcomes measured at entry and 1 year later. SETTING: PATIENTS were recruited from the investigators' clinics or referred from throughout the United States. Data were collected in an in-patient hospital research unit. PATIENTS: Subjects with a clinical diagnosis of XLH and hyperparathyroidism were offered participation and were eligible if they were 9 years old or older and not pregnant, and their serum calcium level was less than 10.7 mg/dL, their 25-hydroxyvitamin D level was 20 ng/mL or greater, and their creatinine level was 1.5 mg/dL or less. INTERVENTION: The intervention for this study was the use of paricalcitol or placebo for 1 year. MAIN OUTCOME MEASURES: Determined prior to trial onset was the change in PTH area under the curve. Secondary outcomes included renal phosphate threshold per glomerular filtration rate, serum phosphorus, serum alkaline phosphatase activity, and (99m)Tc-methylenediphosphonate bone scans. RESULTS: PTH area under the curve decreased 17% with paricalcitol, differing (P = .007) from the 20% increase with placebo. The renal phosphate threshold per glomerular filtration rate increased 17% with paricalcitol and decreased 21% with placebo (P = .05). Serum phosphorus increased 12% with paricalcitol but did not differ from placebo. Paricalcitol decreased alkaline phosphatase activity in adults by 21% (no change with placebo, P = .04). Bone scans improved in 6 of 17 paricalcitol subjects, whereas no placebo-treated subject improved. Hypercalciuria developed in six paricalcitol subjects and persisted from baseline in one placebo subject. CONCLUSIONS: Suppression of PTH may be a useful strategy for skeletal improvement in XLH patients with hyperparathyroidism, and paricalcitol appears to be an effective adjunct to standard therapy in this setting. Although paricalcitol was well tolerated, urinary calcium and serum calcium and creatinine should be monitored closely with its use.

NMR investigation of the role of osteocalcin and osteopontin at the organic-inorganic interface in bone.

Mechanical resilience of bone tissue decreases with age. The ability to comprehensively probe and understand bone properties could help alleviate this problem. One important aspect of bone quality that has recently been made evident is the presence of dilatational bands formed by osteocalcin (OC) and osteopontin (OPN), which contribute to fracture toughness. However, experimental evidence of the structural role of these two proteins at the organic-mineral interface in bone is still needed. Solid state nuclear magnetic resonance (SSNMR) is emerging as a useful technique in probing molecular level aspects of bone. Here, we present the first SSNMR study of bone tissue from genetically modified mice lacking OC and/or OPN. Probing the mineral phase, the organic matrix and their interface revealed that, despite the absence of OC and OPN, the organic matrix and mineral were well preserved, and the overall exposure of collagen to hydroxyapatite (HA) nanoparticles was hardly affected. However, the proximity to the HA surface was slightly increased for a number of bone components including less abundant amino acids like lysine, suggesting that this is how the tissue compensates for the lack of OC and OPN. Taken together, the NMR data supports the recently proposed model, in which the contribution of OC-OPN to fracture toughness is related to their presence at the extrafibrillar organic-mineral interfaces, where they reinforce the network of mineralized fibrils and form dilatational bands. In an effort toward further understanding the structural role of individual amino acids of low abundance in bone, we then explored the possibility of specific (13)C enrichment of mouse bone, and report the first SSNMR spectra of 97% (13)C lysine-enriched tissue. Results show that such isotopic enrichment allows valuable molecular-level structural information to be extracted, and sheds light on post-translational modifications undergone by specific amino acids in vivo.

A comparison of parathyroid hormone-related protein (1-36) and parathyroid hormone (1-34) on markers of bone turnover and bone density in postmenopausal women: the PrOP study.

Parathyroid hormone-related protein (PTHrP)(1-36) increases lumbar spine (LS) bone mineral density (BMD), acting as an anabolic agent when injected intermittently, but it has not been directly compared with parathyroid hormone (PTH)(1-34). We performed a 3-month randomized, prospective study in 105 postmenopausal women with low bone density or osteoporosis, comparing daily subcutaneous injections of PTHrP(1-36) to PTH(1-34). Thirty-five women were randomized to each of three groups: PTHrP(1-36) 400 µg/day; PTHrP(1-36) 600 µg/day; and PTH(1-34) 20 µg/day. The primary outcome measures were changes in amino-terminal telopeptides of procollagen 1 (PINP) and carboxy-terminal telopeptides of collagen 1 (CTX). Secondary measures included safety parameters, 1,25(OH)2 vitamin D, and BMD. The increase in bone resorption (CTX) by PTH(1-34) (92%) (p < 0.005) was greater than for PTHrP(1-36) (30%) (p < 0.05). PTH(1-34) also increased bone formation (PINP) (171%) (p < 0.0005) more than either dose of PTHrP(1-36) (46% and 87%). The increase in PINP was earlier (day 15) and greater than the increase in CTX for all three groups. LS BMD increased equivalently in each group (p < 0.05 for all). Total hip (TH) and femoral neck (FN) BMD increased equivalently in each group but were only significant for the two doses of PTHrP(1-36) (p < 0.05) at the TH and for PTHrP(1-36) 400 (p < 0.05) at the FN. PTHrP(1-36) 400 induced mild, transient (day 15) hypercalcemia. PTHrP(1-36) 600 required a dose reduction for hypercalcemia in three subjects. PTH(1-34) was not associated with hypercalcemia. Each peptide induced a marked biphasic increase in 1,25(OH)2 D. Adverse events (AE) were similar among the three groups. This study demonstrates that PTHrP(1-36) and PTH(1-34) cause similar increases in LS BMD. PTHrP(1-36) also increased hip BMD. PTH(1-34) induced greater changes in bone turnover than PTHrP(1-36). PTHrP(1-36) was associated with mild transient hypercalcemia. Longer-term studies using lower doses of PTHrP(1-36) are needed to define both the optimal dose and full clinical benefits of PTHrP. © 2013 American Society for Bone and Mineral Research.

The effects of GATA-1 and NF-E2 deficiency on bone biomechanical, biochemical, and mineral properties.

Mice deficient in GATA-1 or NF-E2, transcription factors required for normal megakaryocyte (MK) development, have increased numbers of MKs, reduced numbers of platelets, and a striking high bone mass phenotype. Here, we show the bone geometry, microarchitecture, biomechanical, biochemical, and mineral properties from these mutant mice. We found that the outer geometry of the mutant bones was similar to controls, but that both mutants had a striking increase in total bone area (up to a 35% increase) and trabecular bone area (up to a 19% increase). Interestingly, only the NF-E2 deficient mice had a significant increase in cortical bone area (21%) and cortical thickness (27%), which is consistent with the increase in bone mineral density (BMD) seen only in the NF-E2 deficient femurs. Both mutant femurs exhibited significant increases in several biomechanical properties including peak load (up to a 32% increase) and stiffness (up to a 13% increase). Importantly, the data also demonstrate differences between the two mutant mice. GATA-1 deficient femurs break in a ductile manner, whereas NF-E2 deficient femurs are brittle in nature. To better understand these differences, we examined the mineral properties of these bones. Although none of the parameters measured were different between the NF-E2 deficient and control mice, an increase in calcium (21%) and an increase in the mineral/matrix ratio (32%) was observed in GATA-1 deficient mice. These findings appear to contradict biomechanical findings, suggesting the need for further research into the mechanisms by which GATA-1 and NF-E2 deficiency alter the material properties of bone.

Evaluation of markers of bone turnover during lactation in African-Americans: a comparison with Caucasian lactation.

CONTEXT: The African-American skeleton is resistant to PTH; whether it is also resistant to PTHrP and the hormonal milieu of lactation is unknown. OBJECTIVES: The objective of the study was to assess bone turnover markers in African-Americans during lactation vs Caucasians. DESIGN AND PARTICIPANTS: A prospective cohort study with repeated measures of markers of bone turnover in 60 African-American women (3 groups of 20: lactating, bottle feeding, and healthy controls), compared with historic Caucasian women. SETTING: The study was conducted at a university medical center. OUTCOME MEASURES: Biochemical markers of bone turnover and calcium metabolism were measured. RESULTS: 25-Hydroxyvitamin D (25-OHD) and PTH were similar among all 3 African-American groups, but 25-OHD was 30%-50% lower and PTH 2-fold higher compared with Caucasians (P < .001, P < .002), with similar 1,25 dihydroxyvitamin D [1,25(OH)(2)D] values. Formation markers [amino-terminal telopeptide of procollagen-1 (P1NP) and bone-specific alkaline phosphatase (BSAP)] increased significantly (2- to 3-fold) in lactating and bottle-feeding African-American women (P1NP, P < .001; BSAP, P < .001), as did resorption [carboxy-terminal telopeptide of collagen-1 (CTX) and serum amino-terminal telopeptide of collagen 1 (NTX), both P < .001]. P1NP and BSAP were comparable in African-American and Caucasian controls, but CTX and NTX were lower in African-American vs Caucasian controls. African-American lactating mothers displayed quantitatively similar increases in markers of bone formation but slightly lower increases in markers of resorption vs Caucasians (P = .036). CONCLUSIONS: Despite reported resistance to PTH, lactating African-American women have a significant increase in markers of bone resorption and formation in response the hormonal milieu of lactation. This response is similar to that reported in Caucasian women despite racial differences in 25-OHD and PTH. Whether this is associated with similar bone loss in African-Americans as in Caucasians during lactation is unknown and requires further study.

The role of osteocalcin in human glucose metabolism: marker or mediator?

Increasing evidence supports an association between the skeleton and energy metabolism. These interactions are mediated by a variety of hormones, cytokines and nutrients. Here, the evidence for a role of osteocalcin in the regulation of glucose metabolism in humans is reviewed. Osteocalcin is a bone matrix protein that regulates hydroxyapatite size and shape through its vitamin-K-dependent, γ-carboxylated form. The concentration of osteocalcin in the circulation is a measure of bone formation. The undercarboxylated form of osteocalcin is active in glucose metabolism in mice. Total serum osteocalcin concentrations in humans are inversely associated with measures of glucose metabolism; however, human data are inconclusive with regard to the role of uncarboxylated osteocalcin in glucose metabolism because most studies do not account for the influence of vitamin K on the proportion of undercarboxylated osteocalcin or differentiate between the total and uncarboxylated forms of osteocalcin. Furthermore, most human studies do not concomitantly measure other bone turnover markers to isolate the role of osteocalcin as a measure of bone formation from its effect on glucose metabolism. Carefully designed studies are required to define the role of osteocalcin and its carboxylated or undercarboxylated forms in the regulation of glucose metabolism in humans.

LIM kinase 1 deficient mice have reduced bone mass.

The cytoskeleton determines cell shape and is involved in cell motility. It also plays a role in differentiation and in modulating specialized cellular functions. LIM kinase 1 (LIMK1) participates in cytoskeletal remodeling by phosphorylating and inactivating the actin-severing protein, cofilin. Severing F-actin to release G-actin monomers is required for actin cytoskeletal remodeling. Although less well established, LIMK1 may also influence the cell cycle and modulate metalloproteinase activity. Since the role of LIMK1 in bone cell biology has not been reported, the skeletal phenotype of LIMK1(-/-) mice was examined. LIMK1(-/-) mice had significantly reduced trabecular bone mass when analyzed by microCT (p<0.01). Histomorphometric analyses demonstrated a 31% reduction in the number of osteoblasts (p=0.0003) and a 23% reduction in osteoid surface (p=0.0005). The number of osteoclasts was no different in control and knock out animals. Consistent with the in vivo findings in osteoblasts, the number of osteoblast colony forming units in LIMK1(-/-) bone marrow was reduced by nearly 50%. Further, osteoblasts isolated from LIMK1(-/-) mice showed significantly reduced rates of mineralization in vitro. Osteoclasts from LIMK1(-/-) mice evidenced more rapid cytoskeletal remodeling in response to treatment with CSF1. In keeping with this latter finding, basal levels of phospho-cofilin were reduced in LIMK1(-/-) osteoclasts. LIMK1(-/-) osteoclasts also resorbed dentine slices to a greater extent in vitro and were more active in a pit assay. These data support the hypothesis that LIMK1 is required for normal osteoblast differentiation. In addition, its absence leads to increased cytoskeletal remodeling and bone resorption in osteoclasts.

Dilatational band formation in bone.

Toughening in hierarchically structured materials like bone arises from the arrangement of constituent material elements and their interactions. Unlike microcracking, which entails micrometer-level separation, there is no known evidence of fracture at the level of bone's nanostructure. Here, we show that the initiation of fracture occurs in bone at the nanometer scale by dilatational bands. Through fatigue and indentation tests and laser confocal, scanning electron, and atomic force microscopies on human and bovine bone specimens, we established that dilatational bands of the order of 100 nm form as ellipsoidal voids in between fused mineral aggregates and two adjacent proteins, osteocalcin (OC) and osteopontin (OPN). Laser microdissection and ELISA of bone microdamage support our claim that OC and OPN colocalize with dilatational bands. Fracture tests on bones from OC and/or OPN knockout mice (OC(-/-), OPN(-/-), OC-OPN(-/-;-/-)) confirm that these two proteins regulate dilatational band formation and bone matrix toughness. On the basis of these observations, we propose molecular deformation and fracture mechanics models, illustrating the role of OC and OPN in dilatational band formation, and predict that the nanometer scale of tissue organization, associated with dilatational bands, affects fracture at higher scales and determines fracture toughness of bone.

Osteoblasts mediate the adverse effects of glucocorticoids on fuel metabolism.

Long-term glucocorticoid treatment is associated with numerous adverse outcomes, including weight gain, insulin resistance, and diabetes; however, the pathogenesis of these side effects remains obscure. Glucocorticoids also suppress osteoblast function, including osteocalcin synthesis. Osteocalcin is an osteoblast-specific peptide that is reported to be involved in normal murine fuel metabolism. We now demonstrate that osteoblasts play a pivotal role in the pathogenesis of glucocorticoid-induced dysmetabolism. Osteoblast-targeted disruption of glucocorticoid signaling significantly attenuated the suppression of osteocalcin synthesis and prevented the development of insulin resistance, glucose intolerance, and abnormal weight gain in corticosterone-treated mice. Nearly identical effects were observed in glucocorticoid-treated animals following heterotopic (hepatic) expression of both carboxylated and uncarboxylated osteocalcin through gene therapy, which additionally led to a reduction in hepatic lipid deposition and improved phosphorylation of the insulin receptor. These data suggest that the effects of exogenous high-dose glucocorticoids on insulin target tissues and systemic energy metabolism are mediated, at least in part, through the skeleton.

Vitamin K-dependent carboxylation of osteocalcin: friend or foe?

Osteocalcin originates from osteoblastic synthesis and is deposited into bone or released into circulation, where it correlates with histological measures of bone formation. The presence of 3 vitamin K-dependent γ carboxyglutamic acid residues is critical for osteocalcin's structure, which appears to regulate the maturation of bone mineral. In humans, the percentage of the circulating osteocalcin that is not γ-carboxylated (percent ucOC) is used as a biomarker of vitamin K status. In contrast, when ucOC is not corrected for total osteocalcin, the interpretation of this measure is confounded by osteoblastic activity, independent of vitamin K. Observational studies using percent ucOC have led to the conclusion that vitamin K insufficiency leads to age-related bone loss. However, clinical trials do not provide overall support for the suggestion that vitamin K supplementation of the general population will reduce bone loss or fracture risk. More recently, results from in vitro and in vivo studies using animal models indicate that ucOC is an active hormone with a positive role in glucose metabolism. By inference, vitamin K, which decreases ucOC, would have a detrimental effect. However, in humans this hypothesis is not supported by the limited data available, nor is it supported by what has been established regarding osteocalcin chemistry. In summary, the specific function of osteocalcin in bone and glucose metabolism has yet to be elucidated.