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.

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.

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.

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.

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.

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.

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.

Age group and sex do not influence responses of vitamin K biomarkers to changes in dietary vitamin K.

Inadequate vitamin K intake has been associated with abnormal soft tissue calcification. Older adults may have insufficient intakes of vitamin K and respond less to vitamin K supplementation compared with younger adults. However, little is known about the determinants that influence the response to vitamin K supplementation. Our primary objective was to assess dietary and nondietary determinants of vitamin K status in healthy younger and older adults. In a nonrandomized, nonmasked study, 21 younger (18-40 y) and 21 older (55-80 y) men and women consumed a baseline diet (200 µg phylloquinone/d) for 5 d, a phylloquinone-restricted diet (10 µg phylloquinone/d) for 28 d, and a phylloquinone-supplemented diet (500 µg phylloquinone/d) for 28 d. Changes in vitamin K status markers in response to vitamin K depletion and repletion were studied and the influences of BMI, body fat, and circulating TG were assessed by including them as covariates in the model. Despite baseline differences in measures of vitamin K status, plasma phylloquinone tended to increase (P = 0.07) and the percentage of uncarboxylated osteocalcin and uncarboxylated prothrombin both improved with phylloquinone supplementation (P < 0.007), regardless of age group or sex. Only the excretion of urinary menadione, a vitamin K metabolite, was greater among younger adults in response to depletion than in older adults (P = 0.012), regardless of sex. Adiposity measures and circulating TG did not predict response of any measures. In conclusion, poor vitamin K status can be similarly improved with vitamin K supplementation, regardless of age group or sex.

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).

Circulating uncarboxylated matrix gla protein is associated with vitamin K nutritional status, but not coronary artery calcium, in older adults.

Matrix Gla protein (MGP) is a calcification inhibitor in vascular tissue that must be carboxylated by vitamin K to function. Evidence suggests circulating uncarboxylated MGP (ucMGP) is elevated in persons with disease characterized by vascular calcification. The primary purpose of this study was to determine cross-sectional and longitudinal associations between plasma ucMGP, vitamin K status, and coronary artery calcium (CAC) in older adults without coronary heart disease. Genetic determinants of ucMGP were also explored. Cross-sectional associations among baseline plasma ucMGP, vitamin K status biomarkers [plasma phylloquinone, uncarboxylated prothrombin (PIVKA-II), serum uncarboxylated osteocalcin (%ucOC)], CAC, and plausible genetic polymorphisms were examined in 438 community-dwelling adults (60-80 y, 59% women). The effect of phylloquinone supplementation (500 µg/d) for 3 y on plasma ucMGP was determined among 374 participants. At baseline, plasma phylloquinone was lower and %ucOC and PIVKA-II were greater across higher plasma ucMGP quartiles (all P < 0.001, age-adjusted). Major allele homozygotes for MGP rs1800801 and rs4236 had higher plasma ucMGP than heterozygotes or minor allele homozygotes. (P ≤ 0.004). The decrease in plasma ucMGP was greater in the 190 participants who received phylloquinone (mean ± SD) (-345 ± 251 pmol/L) than in the 184 who did not (-40 ± 196 pmol/L) (P < 0.0001). CAC did not differ according to ucMGP quartile (P = 0.35, age-adjusted). In the phylloquinone-supplemented group, the 3-y change in ucMGP was not associated with the 3-y change in CAC [unstandard ß (SE) = -0.02 (0.02); P = 0.44]. Plasma ucMGP was associated with vitamin K status biomarkers and was reduced following phylloquinone supplementation, suggesting it may be a useful marker of vitamin K status in vascular tissue. Plasma ucMGP did not reflect CAC in healthy older adults.

Expression of an estrogen receptor agonist in differentiating osteoblast cultures.

Osteoblasts respond in direct and indirect ways to estrogens, and age-dependent changes in hormone levels and bone health can be limited by focused hormone replacement therapy. In this study, we report the release and isolation of an estrogen receptor agonist from osteoblast cultures. This entity reprises many aspects of estradiol activity in isolated osteoblasts, but differs from authentic estradiol by several biochemical and physical criteria. At levels that occur in conditioned medium from differentiating osteoblast cultures, the agonist directly drives gene expression through estrogen-sensitive response elements, activates the obligate osteoblast transcription factor Runx2, and potently enhances Smad-dependent gene expression in response to TGF-beta, but exhibits relatively lesser suppressive effects on gene expression through C/EBP and AP-1-binding protein transcription factors. Estrogen receptor agonist activity is resistant to heating at 100 degrees C and separable from the bulk of the remaining alcohol- and hexane-soluble molecules by C18 chromatography. MS and molecular fragmentation analyses predict a M(r) of 415.2 to 437.2. Therefore, in addition to earlier studies showing that osteoblasts readily respond to and metabolize various sex steroid-like substrates, we find that they also generate a potent estrogen receptor agonist during differentiation in vitro. Changes in the availability of a molecule like this within bone may relate to differences in skeletal integrity with aging or metabolic disease.

Adulthood obesity is positively associated with adipose tissue concentrations of vitamin K and inversely associated with circulating indicators of vitamin K status in men and women.

Increased adiposity is associated with increased storage of several fat-soluble nutrients. However, the extent to which vitamin K is stored in fat and the association between vitamin K status and adiposity are unknown. Our objectives in this study were to determine whether vitamin K is stored in human adipose tissue and the association between vitamin K status and body fat in older men and women. In study A, the vitamin K concentration of subcutaneous and visceral adipose tissue was quantified in samples taken from 16 gastric bypass patients [13 women, 3 men, age 40 +/- 10 y (mean +/- SD)] using HPLC. In study B, cross-sectional associations between percent body fat (%BF) and circulating measures of vitamin K status were examined in 260 women and 183 men [age = 68 +/- 5 y]. The phylloquinone (K(1)) concentrations in subcutaneous and visceral adipose tissue were 148.2 +/- 71.8 and 175 +/- 112 nmol/kg, respectively, which is higher than the reported concentrations of other organs known to store vitamin K. There was an inverse association between %BF and plasma K(1) in women (P-trend < 0.001). Higher %BF was associated with greater circulating concentrations of uncarboxylated prothrombin, indicative of lower hepatic utilization of vitamin K in both men (P-trend = 0.02) and women (P-trend = 0.002) but not with the percentage of undercarboxylated osteocalcin. Adipose tissue contained high concentrations of vitamin K, and increased adiposity was associated with poorer vitamin K status in older adults. Additional studies are needed to further explore the relationships among body fat, storage of vitamin K in adipose tissue, and implications for vitamin K status and function.

Effect of vitamin K supplementation on bone loss in elderly men and women.

CONTEXT: Vitamin K has been implicated in bone health, primarily in observational studies. However, little is known about the role of phylloquinone supplementation on prevention of bone loss in men and women. OBJECTIVE: The objective of this study was to determine the effect of 3-yr phylloquinone supplementation on change in bone mineral density (BMD) of the femoral neck bone in older men and women who were calcium and vitamin D replete. DESIGN, PARTICIPANTS, AND INTERVENTION: In this 3-yr, double-blind, controlled trial, 452 men and women (60-80 yr) were randomized equally to receive a multivitamin that contained either 500 mug/d or no phylloquinone plus a daily calcium (600 mg elemental calcium) and vitamin D (400 IU) supplement. MAIN OUTCOME MEASURES: Measurements of the femoral neck, spine (L2-L4), and total-body BMD, bone turnover, and vitamins K and D status were measured every 6-12 months. Intent-to-treat analysis was used to compare change in measures in 401 participants who completed the trial. RESULTS: There were no differences in changes in BMD measurements at any of the anatomical sites measured between the two groups. The group that received the phylloquinone supplement had significantly higher phylloquinone and significantly lower percent undercarboxylated osteocalcin concentrations compared with the group that did not receive phylloquinone. No other biochemical measures differed between the two groups. CONCLUSIONS: Phylloquinone supplementation in a dose attainable in the diet does not confer any additional benefit for bone health at the spine or hip when taken with recommended amounts of calcium and vitamin D.

The presence of both an energy deficiency and estrogen deficiency exacerbate alterations of bone metabolism in exercising women.

BACKGROUND: Bone loss in amenorrheic athletes has been attributed to energy deficiency-related suppression of bone formation, but not increased resorption despite hypoestrogenism. OBJECTIVE: To assess the independent and combined effects of energy deficiency and estrogen deficiency on bone turnover markers in exercising women. DESIGN: PINP, osteocalcin, U-CTX-I, TT3, leptin, and ghrelin were measured repeatedly, and bone mineral density (BMD) was measured once in 44 exercising women. Resting energy expenditure (REE) was used to determine energy status (deficient or replete) and was corroborated with measures of metabolic hormones. Daily levels of urinary estrone and pregnanediol glucuronides (E1G, PdG), were assessed to determine menstrual and estrogen status. Volunteers were then retrospectively categorized into 4 groups: 1) Energy Replete+Estrogen Replete (EnR+E2R), (n=22), 2) Energy Replete+Estrogen Deficient (EnR+E2D), (n=7), 3) Energy Deficient+Estrogen Replete (EnD+E2R), (n=7), and 4) Energy Deficient+Estrogen Deficient (EnD+E2D), (n=8). RESULTS: The groups were similar (p>0.05) with respect to age (24.05+/-1.75 yrs), weight (57.7+/-2.2 kg), and BMI (21.05+/-0.7 kg/m2). By design, REE/FFM (p=0.028) and REE:pREE (p<0.001) were lower in the EnD vs. EnR group, and the E2D group had a lower REE:pREE (p=0.005) compared to the E2R group. The EnD+E2D group had suppressed PINP (p=0.034), and elevated U-CTX-I (p=0.052) and ghrelin (p=0.028) levels compared to the other groups. These same women also had convincing evidence of energy conservation, including TT3 levels that were 29% lower (p=0.057) and ghrelin levels that were 44% higher (p=0.028) than that observed in the other groups. Energy deficiency was associated with suppressed osteocalcin, and TT3 (p<0.05), whereas estrogen deficiency was associated with decreased E1G (p<0.02), and lower L2-L4 BMD (p=0.033). Leptin was significant in predicting markers of bone formation, but not markers of bone resorption. CONCLUSIONS: When the energy status of exercising women was adequate (replete), there were no apparent perturbations of bone formation or resorption, regardless of estrogen status. Estrogen deficiency in exercising women, in the presence of an energy deficiency, was associated with bone loss and involved suppressed bone formation and increased bone resorption. These findings underscore the importance of avoiding energy deficiency, which is associated with hypoestrogenism, to avoid bone health problems.

Efficacy of high dose phylloquinone in correcting vitamin K deficiency in cystic fibrosis.

BACKGROUND: Subclinical deficiencies of vitamin K are universally present in unsupplemented cystic fibrosis (CF) patients. The dose required to prevent deficiencies cannot be estimated from the existing literature. The aim of this study is determine if a supplemental dose of 1 mg/day or 5 mg/day vitamin K1 per day would normalize vitamin K status in a population of children with cystic fibrosis. METHODS: Fourteen pancreatic insufficient CF children, between the ages of 8 to 18 years old, were randomized to receive either 1 mg/day or 5 mg/day vitamin K1 per day, for one month. Fasting blood tests were done at baseline and after one month of the intervention. The degree of undercarboxylation of osteocalcin (%Glu-OC), and serum vitamin K1, were evaluated by descriptive statistics and nonparametric Wilcoxon matched-pair test and Mann-Whitney U test. RESULTS: Of the 50% of subjects who were below the optimal serum vitamin K1 at baseline, all rose into the normal range with supplementation. Supplementation also significantly reduced the overall %Glu-OC from a median of 46.8 to 29.1% (p<0.0003). CONCLUSION: Our results suggest that both 1 mg and 5 mg of vitamin K1, given over a one-month period in pancreatic insufficient pediatric cystic fibrosis patients improve vitamin K status.

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.

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.

Repetitive exposure to TGF-beta suppresses TGF-beta type I receptor expression by differentiated osteoblasts.

Transforming growth factor-beta (TGF-beta) has potent, cell phenotype restricted effects. In bone, it controls multiple activities by osteoblasts through three predominant receptors. Of these, the relative amounts of TGF-beta receptor I (TbetaRI) vary directly with TGF-beta sensitivity. The rat TbetaRI gene promoter includes cis-acting elements for transcription factor Runx2. Here we show conservation and selective partitioning of TbetaRI and retention of TGF-beta activity with osteoblast differentiation, Runx2 binding to the TbetaRI gene promoter on osteoblast chromatin, and decreased promoter activity by Runx2 binding site mutation. Furthermore, in contrast to the stimulatory effects induced by single or limited exposure to TGF-beta, we found that osteoblasts became resistant to TGF-beta after high dose and repetitive treatment. TbetaRI protein, mRNA, and gene promoter activity all decreased after three daily TGF-beta treatments, in parallel with a reduction in Runx2 protein and Runx dependent gene expression. In this way, sustained TGF-beta exposure can limit its own effectiveness by suppressing the expression of its primary signaling receptor. This tightly controlled system may constitute a feedback loop to protect against TGF-beta excess, and impose important limitations that are required for the progression of events during skeletal growth, remodeling and repair.