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Mitochondrial fatty acid oxidation is facilitated by the combined activities of carnitine palmitoyltransferase 1 (Cpt1) and Cpt2, which generate and utilize acylcarnitines, respectively. We compare the response of mice with liver-specific deficiencies in the liver-enriched Cpt1a or the ubiquitously expressed Cpt2 and discover that they display unique metabolic, physiological, and molecular phenotypes. The loss of Cpt1a or Cpt2 results in the induction of the muscle-enriched isoenzyme Cpt1b in hepatocytes in a Pparα-dependent manner. However, hepatic Cpt1b does not contribute substantively to hepatic fatty acid oxidation when Cpt1a is absent. Liver-specific double knockout of Cpt1a and Cpt1b or Cpt2 eliminates the mitochondrial oxidation of non-esterified fatty acids. However, Cpt1a/Cpt1b double knockout mice retain fatty acid oxidation by utilizing extracellular long-chain acylcarnitines that are dependent on Cpt2. These data demonstrate the non-cell-autonomous intercellular metabolism of fatty acids in hepatocytes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Etomoxir has been used for decades as a popular small molecule inhibitor of carnitine palmitoyltransferase I, Cpt1, to block mitochondrial fatty acid β-oxidation. To test the specificity of etomoxir, we generated click chemistry-enabled reagents to label etomoxir binding proteins in situ . Etomoxir bound to Cpt1, but also bound to a large array of diverse proteins that metabolize and transport fatty acids in the cytoplasm, peroxisome, and mitochondria. Many of the most abundant proteins identified in primary hepatocytes were peroxisomal proteins. The loss of Pex5, required for the import of peroxisomal matrix proteins, eliminated many of these etomoxir-labeled proteins. By utilizing the promiscuous, covalent, and fatty acid mimetic properties of etomoxir, etomoxir targets of fatty acid ω-oxidation were revealed following the loss of Pex5. These data demonstrate that etomoxir is not specific for Cpt1 and is not appropriate as a tool to distinguish the biological effects of fatty acid oxidation., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Growth deficiency is a characteristic feature of both Kabuki syndrome 1 (KS1) and Kabuki syndrome 2 (KS2), Mendelian disorders of the epigenetic machinery with similar phenotypes but distinct genetic etiologies. We previously described skeletal growth deficiency in a mouse model of KS1 and further established that a Kmt2d-/- chondrocyte model of KS1 exhibits precocious differentiation. Here we characterized growth deficiency in a mouse model of KS2, Kdm6atm1d/+. We show that Kdm6atm1d/+ mice have decreased femur and tibia length compared to controls and exhibit abnormalities in cortical and trabecular bone structure. Kdm6atm1d/+ growth plates are also shorter, due to decreases in hypertrophic chondrocyte size and hypertrophic zone height. Given these disturbances in the growth plate, we generated Kdm6a-/- chondrogenic cell lines. Similar to our prior in vitro model of KS1, we found that Kdm6a-/- cells undergo premature, enhanced differentiation towards chondrocytes compared to Kdm6a+/+ controls. RNA-seq showed that Kdm6a-/- cells have a distinct transcriptomic profile that indicates dysregulation of cartilage development. Finally, we performed RNA-seq simultaneously on Kmt2d-/-, Kdm6a-/-, and control lines at Days 7 and 14 of differentiation. This revealed surprising resemblance in gene expression between Kmt2d-/- and Kdm6a-/- at both time points and indicates that the similarity in phenotype between KS1 and KS2 also exists at the transcriptional level., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests:HTB is a consultant for Mahzi therapeutics. The authors declare no other conflicts of interest relevant to this manuscript., (Copyright: © 2024 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Genome-wide association studies (GWAS) have identified a large number of candidate genes believed to affect longitudinal bone growth and bone mass. One of these candidate genes, TMEM263 , encodes a poorly characterized plasma membrane protein. Single nucleotide polymorphisms in TMEM263 are associated with bone mineral density in humans and mutations are associated with dwarfism in chicken and severe skeletal dysplasia in at least one human fetus. Whether this genotype-phenotype relationship is causal, however, remains unclear. Here, we determine whether and how TMEM263 is required for postnatal growth. Deletion of the Tmem263 gene in mice causes severe postnatal growth failure, proportional dwarfism, and impaired skeletal acquisition. Mice lacking Tmem263 show no differences in body weight within the first 2 weeks of postnatal life. However, by P21 there is a dramatic growth deficit due to a disrupted growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, which is critical for longitudinal bone growth. Tmem263 -null mice have low circulating IGF-1 levels and pronounced reductions in bone mass and growth plate length. The low serum IGF-1 in Tmem263 -null mice is associated with reduced hepatic GH receptor (GHR) expression and GH-induced JAK2/STAT5 signaling. A deficit in GH signaling dramatically alters GH-regulated genes and feminizes the liver transcriptome of Tmem263-null male mice, with their expression profile resembling wild-type female, hypophysectomized male, and Stat5b-null male mice. Collectively, our data validates the causal role for Tmem263 in regulating postnatal growth and raises the possibility that rare mutations or variants of TMEM263 may potentially cause GH insensitivity and impair linear growth., Competing Interests: DS, JG, MS, RR, GW No competing interests declared, (© 2023, Sarver et al.)

Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters, indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-Seq comparing osteoblasts differentiated from Ezh2R684C/+, and Ezh2+/+ BM-mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases KDM6A and KDM6B substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.

Genome-wide association studies (GWAS) have identified a large number of candidate genes believed to affect longitudinal bone growth and bone mass. One of these candidate genes, TMEM263 , encodes a poorly characterized plasma membrane protein. Single nucleotide polymorphisms in TMEM263 are associated with bone mineral density in humans and mutations are associated with dwarfism in chicken and severe skeletal dysplasia in at least one human fetus. Whether this genotype-phenotype relationship is causal, however, remains unclear. Here, we determine whether and how TMEM263 is required for postnatal growth. Deletion of the Tmem263 gene in mice causes severe postnatal growth failure, proportional dwarfism, and impaired skeletal acquisition. Mice lacking Tmem263 show no differences in body weight within the first two weeks of postnatal life. However, by P21 there is a dramatic growth deficit due to a disrupted GH/IGF-1 axis, which is critical for longitudinal bone growth. Tmem263 -null mice have low circulating IGF-1 levels and pronounced reductions in bone mass and growth plate length. The low serum IGF-1 in Tmem263 -null mice is associated with reduced hepatic GH receptor (GHR) expression and GH-induced JAK2/STAT5 signaling. A deficit in GH signaling dramatically alters GH-regulated genes and feminizes the liver transcriptome of Tmem263-null male mice, with their expression profile resembling a wild-type female, hypophysectomized male, and Stat5b-null male mice. Collectively, our data validates the causal role for Tmem263 in regulating postnatal growth and raises the possibility that rare mutations or variants of TMEM263 may potentially cause GH insensitivity and impair linear growth., Competing Interests: COMPETING INTERESTS We declare that none of the authors has a conflict of interest.

Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2 , which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2 R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2 R684C/ + mice had abnormal bone parameters indicative of skeletal overgrowth, and Ezh2 R684C/ + osteoblasts showed increased osteogenic activity. RNA-seq comparing osteoblasts differentiated from Ezh2 R684C/ + and Ezh2 +/+ bone marrow mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases Kdm6a/6b substantially reversed the excessive osteogenesis in Ezh2 R684C/ + cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state, and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.

The nuclear receptor peroxisome proliferator activated receptor-γ (PPARγ) is a key contributor to metabolic function via its adipogenic and insulin-sensitizing functions, but it has negative effects on skeletal homeostasis. Here, we questioned whether the skeletal and metabolic actions of PPARγ are linked. Ablating Pparg expression in osteoblasts and osteocytes produced a high bone mass phenotype, secondary to increased osteoblast activity, and a reduction in subcutaneous fat mass because of reduced fatty acid synthesis and increased fat oxidation. The skeletal and metabolic phenotypes in Pparg mutants proceed from the regulation of sclerostin production by PPARγ. Mutants exhibited reductions in skeletal Sost expression and serum sclerostin levels while increasing production normalized both phenotypes. Importantly, disrupting the production of sclerostin synergized with the insulin-sensitizing actions of a PPARγ agonist while preventing bone loss. These data suggest that modulating sclerostin action may prevent bone loss associated with anti-diabetic therapies and augment their metabolic actions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

The energetic costs of bone formation require osteoblasts to coordinate their activities with tissues, like adipose, that can supply energy-dense macronutrients. In the case of intermittent parathyroid hormone (PTH) treatment, a strategy used to reduce fracture risk, bone formation is preceded by a change in systemic lipid homeostasis. To investigate the requirement for fatty acid oxidation by osteoblasts during PTH-induced bone formation, we subjected mice with osteoblast-specific deficiency of mitochondrial long-chain β-oxidation as well as mice with adipocyte-specific deficiency for the PTH receptor or adipose triglyceride lipase to an anabolic treatment regimen. PTH increased the release of fatty acids from adipocytes and β-oxidation by osteoblasts, while the genetic mouse models were resistant to the hormone's anabolic effect. Collectively, these data suggest that PTH's anabolic actions require coordinated signaling between bone and adipose, wherein a lipolytic response liberates fatty acids that are oxidized by osteoblasts to fuel bone formation.

Sclerostin, the product of the SOST gene has primarily been studied for its profound impact on bone mass. By interacting with LRP5 and LRP6, the glycoprotein suppresses the propagation of Wnt signals to β-catenin and thereby suppresses new bone formation. In this review, we discuss emerging data which suggest that sclerostin also acts outside the skeleton to influence metabolism. In humans, serum sclerostin levels are associated with body mass index and indices of metabolic function. Likewise, genetic mouse models of Sost gene deficiency indicate sclerostin influences adipocyte development and insulin signaling. These data raise the possibility that sclerostin neutralization may be effective at treating two epidemic conditions: osteoporosis and obesity., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Citrate is a critical metabolic substrate and key regulator of energy metabolism in mammalian cells. It has been known for decades that the skeleton contains most (>85%) of the body's citrate, but the question of why and how this metabolite should be partitioned in bone has received singularly little attention. Here, we show that osteoblasts use a specialized metabolic pathway to regulate uptake, endogenous production, and the deposition of citrate into bone. Osteoblasts express high levels of the membranous Na + -dependent citrate transporter solute carrier family 13 member 5 ( Slc13a5 ) gene. Inhibition or genetic disruption of Slc13a5 reduced osteogenic citrate uptake and disrupted mineral nodule formation. Bones from mice lacking Slc13a5 globally, or selectively in osteoblasts, showed equivalent reductions in cortical thickness, with similarly compromised mechanical strength. Surprisingly, citrate content in mineral from Slc13a5 -/- osteoblasts was increased fourfold relative to controls, suggesting the engagement of compensatory mechanisms to augment endogenous citrate production. Indeed, through the coordinated functioning of the apical membrane citrate transporter SLC13A5 and a mitochondrial zinc transporter protein (ZIP1; encoded by Slc39a1 ), a mediator of citrate efflux from the tricarboxylic acid cycle, SLC13A5 mediates citrate entry from blood and its activity exerts homeostatic control of cytoplasmic citrate. Intriguingly, Slc13a5 -deficient mice also exhibited defective tooth enamel and dentin formation, a clinical feature, which we show is recapitulated in primary teeth from children with SLC13A5 mutations. Together, our results reveal the components of an osteoblast metabolic pathway, which affects bone strength by regulating citrate deposition into mineral hydroxyapatite.

Gene mutations causing loss of dystrophin result in the severe muscle disease known as Duchenne muscular dystrophy (DMD). Despite efforts at genetic repair, DMD therapy remains largely palliative. Loss of dystrophin destabilizes the sarcolemmal membrane, inducing mechanosensitive cation channels to increase calcium entry and promote cell damage and, eventually, muscle dysfunction. One putative channel is transient receptor potential canonical 6 (TRPC6); we have shown that TRPC6 contributed to abnormal force and calcium stress-responses in cardiomyocytes from mice lacking dystrophin that were haplodeficient for utrophin (mdx/utrn+/- [HET] mice). Here, we show in both the HET mouse and the far more severe homozygous mdx/utrn-/- mouse that TRPC6 gene deletion or its selective pharmacologic inhibition (by BI 749327) prolonged survival 2- to 3-fold, improving skeletal and cardiac muscle and bone defects. Gene pathways reduced by BI 749327 treatment most prominently regulated fat metabolism and TGF-β1 signaling. These results support the testing of TRPC6 inhibitors in human trials for other diseases as a novel DMD therapy.

Skeletal remodeling is an energy demanding process that is linked to nutrient availability and the levels of metabolic hormones. While recent studies have examined the metabolic requirements of bone formation by osteoblasts, much less is known about the energetic requirements of bone resorption by osteoclasts. The abundance of mitochondria in mature osteoclasts suggests that the production of an acidified micro-environment conducive to the ionization of hydroxyapatite, secretion of matrix-degrading enzymes, and motility during resorption requires significant energetic capacity. To investigate the contribution of mitochondrial long chain fatty acid β-oxidation to osteoclast development, we disrupted the expression of carnitine palmitoyltransferase-2 (Cpt2) in myeloid-lineage cells. Fatty acid oxidation increases dramatically in bone marrow cultures stimulated with RANKL and M-CSF and microCT analysis revealed that the genetic inhibition of long chain fatty acid oxidation in osteoclasts significantly increases trabecular bone volume in female mice secondary to reduced osteoclast numbers. In line with these data, osteoclast precursors isolated from Cpt2 mutants exhibit reduced capacity to form large-multinucleated osteoclasts, which was not rescued by exogenous glucose or pyruvate, and signs of an energetic stress response. Together, our data demonstrate that mitochondrial long chain fatty acid oxidation by the osteoclast is required for normal bone resorption as its inhibition produces an intrinsic defect in osteoclast formation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kushwaha, Alekos, Kim, Li, Wolfgang and Riddle.)

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14 C-labeled fatty acids to 14 CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.

Despite advances in imaging, image-based vascular systems biology has remained challenging because blood vessel data are often available only from a single modality or at a given spatial scale, and cross-modality data are difficult to integrate. Therefore, there is an exigent need for a multimodality pipeline that enables ex vivo vascular imaging with magnetic resonance imaging, computed tomography and optical microscopy of the same sample, while permitting imaging with complementary contrast mechanisms from the whole-organ to endothelial cell spatial scales. To achieve this, we developed 'VascuViz'-an easy-to-use method for simultaneous three-dimensional imaging and visualization of the vascular microenvironment using magnetic resonance imaging, computed tomography and optical microscopy in the same intact, unsectioned tissue. The VascuViz workflow permits multimodal imaging with a single labeling step using commercial reagents and is compatible with diverse tissue types and protocols. VascuViz's interdisciplinary utility in conjunction with new data visualization approaches opens up new vistas in image-based vascular systems biology., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Low oxygen (O 2 ) diffusion into large tissue engineered scaffolds hinders the therapeutic efficacy of transplanted cells. To overcome this, we previously studied hollow, hyperbarically-loaded microtanks (μtanks) to serve as O 2 reservoirs. To adapt these for bone regeneration, we fabricated biodegradable μtanks from polyvinyl alcohol and poly (lactic-co-glycolic acid) and embedded them to form 3D-printed, porous poly-ε-caprolactone (PCL)-μtank scaffolds. PCL-μtank scaffolds were loaded with pure O 2 at 300-500 psi. When placed at atmospheric pressures, the scaffolds released O 2 over a period of up to 8 h. We confirmed the inhibitory effects of hypoxia on the osteogenic differentiation of human adipose-derived stem cells (hASCs and we validated that μtank-mediated transient hyperoxia had no toxic impacts on hASCs, possibly due to upregulation of endogenous antioxidant regulator genes. We assessed bone regeneration in vivo by implanting O 2 -loaded, hASC-seeded, PCL-μtank scaffolds into murine calvarial defects (4 mm diameters × 0.6 mm height) and subcutaneously (4 mm diameter × 8 mm height). In both cases we observed increased deposition of extracellular matrix in the O 2 delivery group along with greater osteopontin coverages and higher mineral deposition. This study provides evidence that even short-term O 2 delivery from PCL-μtank scaffolds may enhance hASC-mediated bone tissue regeneration., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Wnt signaling antagonist, sclerostin, is a potent suppressor of bone acquisition that also mediates endocrine communication between bone and adipose. As a result, Sost -/- mice exhibit dramatic increases in bone formation but marked decreases in visceral and subcutaneous adipose that are secondary to alterations in lipid synthesis and utilization. While interrogating the mechanism by which sclerostin influences adipocyte metabolism, we observed paradoxical increases in the adipogenic potential and numbers of CD45 - :Sca1 + :PDGFRα + adipoprogenitors in the stromal vascular compartment of fat pads isolated from male Sost -/- mice. Lineage tracing studies indicated that sclerostin deficiency blocks the differentiation of PDGFRα + adipoprogenitors to mature adipocytes in association with increased Wnt/β-catenin signaling. Importantly, osteoblast/osteocyte-specific Sost gene deletion mirrors the accumulation of PDGFRα + adipoprogenitors, reduction in fat mass, and improved glucose metabolism evident in Sost -/- mice. These data indicate that bone-derived sclerostin regulates multiple facets of adipocyte physiology ranging from progenitor cell commitment to anabolic metabolism., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Maternal stressors during the prenatal and perinatal periods are associated with increased susceptibility for and severity of chronic disease phenotypes in adult offspring. In this study, we used a rat model of maternal high-fat diet (HFD) exposure during pregnancy and lactation to investigate the impact on skeletal homeostasis in offspring. In the distal femur, young male and female offspring (up to 3 weeks of age) from dams fed a HFD exhibited marked increases in trabecular bone volume relative to offspring from dams fed a chow diet, but this was followed by sustained bone loss. By 15 weeks of age, male offspring of HFD fed dams exhibited a 33% reduction in trabecular bone volume fraction that histomorphometric analyses revealed was due to a nearly threefold increase in the abundance of bone-resorbing osteoclasts, while there were no differences between female control and HFD offspring by 15 weeks of age. The osteoblastic differentiation of male offspring-derived bone marrow stromal cells was not affected by maternal diet. However, osteoclastic precursors isolated from the male offspring of HFD fed dams exhibited enhanced differentiation in vitro, forming larger osteoclasts with higher expression of the fusion marker DC-STAMP. This effect appears to be mediated by a cell autonomous increase in the sensitivity of precursors to RANKL. Taken together, these results suggest that maternal stressors like HFD exposure have persistent consequences for the skeletal health of offspring that may ultimately lead to a predisposition for osteopenia/osteoporosis.

Background: Radium-223 dichloride ([ 223 Ra]RaCl2) is the first approved alpha particle-emitting therapy and is indicated for treatment of bone metastatic castrate resistant prostate cancer. Approximately half of the dose is absorbed into the gastrointestinal (GI) tract within minutes of administration, limiting disease-site uptake and contributing to toxicity. Here, we investigate the role of enteric ion channels and their modulation for improved therapeutic efficacy and reduced side effects. Methods: Utilizing primary human duodenal organoids (enteroids) as in vitro models of the functional GI epithelium, we found that Amiloride (ENaC blocker) and NS-1619 (K+ channel activator) presented significant effects in 223 Ra membranal transport. The radioactive drug distribution was evaluated for lead combinations in vivo, and in osteosarcoma and prostate cancer models. Results: Amiloride shifted 223 Ra uptake in vivo from the gut, to nearly double the uptake at sites of bone remodeling. Bone tumor growth inhibition with the combination as measured by bioluminescent and X-ray imaging was significantly greater than single agents alone, and the combination resulted in no weight loss. Conclusion: This combination of approved agents may be readily implemented as a clinical approach to improve outcomes of bone metastatic cancer patients with the benefit of ameliorated tolerability., (Copyright © 2021 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

The activation of the Wnt/β-catenin signaling pathway is critical for skeletal development but surprisingly little is known about the requirements for the specific frizzled (Fzd) receptors that recognize Wnt ligands. To define the contributions of individual Fzd proteins to osteoblast function, we profiled the expression of all 10 mammalian receptors during calvarial osteoblast differentiation. Expression of Fzd4 was highly upregulated during in vitro differentiation and therefore targeted for further study. Mice lacking Fzd4 in mature osteoblasts had normal cortical bone structure but reduced cortical tissue mineral density and also exhibited an impairment in the femoral trabecular bone acquisition that was secondary to a defect in the mineralization process. Consistent with this observation, matrix mineralization, markers of osteoblastic differentiation, and the ability of Wnt3a to stimulate the accumulation of β-catenin were reduced in cultures of calvarial osteoblasts deficient for Fzd4. Interestingly, Fzd4-deficient osteoblasts exhibited an increase in the expression of Fzd8 both in vitro and in vivo, which suggests that the two receptors may exhibit overlapping functions. Indeed, ablating a single Fzd8 allele in osteoblast-specific Fzd4 mutants produced a more severe effect on bone acquisition. Taken together, our data indicate that Fzd4 is required for normal bone development and mineralization despite compensation from Fzd8., (© 2020 Wiley Periodicals, Inc.)

The skeleton is a dynamic and metabolically active organ with the capacity to influence whole body metabolism. This newly recognized function has propagated interest in the connection between bone health and metabolic dysfunction. Osteoblasts, the specialized mesenchymal cells responsible for the production of bone matrix and mineralization, rely on multiple fuel sources. The utilization of glucose by osteoblasts has long been a focus of research, however, lipids and their derivatives, are increasingly recognized as a vital energy source. Osteoblasts possess the necessary receptors and catabolic enzymes for internalization and utilization of circulating lipids. Disruption of these processes can impair osteoblast function, resulting in skeletal deficits while simultaneously altering whole body lipid homeostasis. This article provides an overview of the metabolism of postprandial and stored lipids and the osteoblast's ability to acquire and utilize these molecules. We focus on the requirement for fatty acid oxidation and the pathways regulating this function as well as the negative impact of dyslipidemia on the osteoblast and skeletal health. These findings provide key insights into the nuances of lipid metabolism in influencing skeletal homeostasis which are critical to appreciate the extent of the osteoblast's role in metabolic homeostasis., (Copyright © 2020 Alekos, Moorer and Riddle.)

Objective: Dose optimization and pharmacokinetic evaluation of α-particle emitting radium-223 dichloride ( 223 RaCl 2 ) by planar γ-camera or single photon emission computed tomography (SPECT) imaging are hampered by the low photon abundance and injected activities. In this study, we demonstrate SPECT of 223 Ra using phantoms and small animal in vivo models. Methods: Line phantoms and mice bearing 223 Ra were imaged using a dedicated small animal SPECT by detecting the low-energy photon emissions from 223 Ra. Localization of the therapeutic agent was verified by whole-body and whole-limb autoradiography and its radiobiological effect confirmed by immunofluorescence. Results: A state-of-the-art commercial small animal SPECT system equipped with a highly sensitive collimator enables collection of sufficient counts for three-dimensional reconstruction at reasonable administered activities and acquisition times. Line sources of 223 Ra in both air and in a water scattering phantom gave a line spread function with a full-width-at-half-maximum of 1.45 mm. Early and late-phase imaging of the pharmacokinetics of the radiopharmaceutical were captured. Uptake at sites of active bone remodeling was correlated with DNA damage from the α particle emissions. Conclusions: This work demonstrates the capability to noninvasively define the distribution of 223 RaCl 2 , a recently approved α-particle-emitting radionuclide. This approach allows quantitative assessment of 223 Ra distribution and may assist radiation-dose optimization strategies to improve therapeutic response and ultimately to enable personalized treatment planning.

C1q/TNF-related protein 3 (CTRP3) is a cytokine known to regulate a variety of metabolic processes. Though previously undescribed in the context of bone regeneration, high throughput gene expression experiments in mice identified CTRP3 as one of the most highly upregulated genes in fracture callus tissue. Hypothesizing a positive regulatory role for CTRP3 in bone regeneration, we phenotyped skeletal development and fracture healing in CTRP3 knockout (KO) and CTRP3 overexpressing transgenic (TG) mice relative to wild-type (WT) control animals. CTRP3 KO mice experienced delayed endochondral fracture healing, resulting in abnormal mineral distribution, the presence of periosteal marrow compartments, and a nonunion-like state. Decreased osteoclast number was also observed in CTRP3 KO mice, whereas CTRP3 TG mice underwent accelerated callus remodeling. Gene expression profiling revealed a broad impact on osteoblast/osteoclast lineage commitment and metabolism, including arrested progression toward mature skeletal lineages in the KO group. A single systemic injection of CTRP3 protein at the time of fracture was insufficient to phenocopy the chronic TG healing response in WT mice. By associating CTRP3 levels with fracture healing progression, these data identify a novel protein family with potential therapeutic and diagnostic value. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:00-19966, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Osteoblasts are specialized mesenchymal cells that synthesize bone matrix and coordinate the mineralization of the skeleton. These cells work in harmony with osteoclasts, which resorb bone, in a continuous cycle that occurs throughout life. The unique function of osteoblasts requires substantial amounts of energy production, particularly during states of new bone formation and remodelling. Over the last 15 years, studies have shown that osteoblasts secrete endocrine factors that integrate the metabolic requirements of bone formation with global energy balance through the regulation of insulin production, feeding behaviour and adipose tissue metabolism. In this article, we summarize the current understanding of three osteoblast-derived metabolic hormones (osteocalcin, lipocalin and sclerostin) and the clinical evidence that suggests the relevance of these pathways in humans, while also discussing the necessity of specific energy substrates (glucose, fatty acids and amino acids) to fuel bone formation and promote osteoblast differentiation.

Kabuki syndrome 1 (KS1) is a Mendelian disorder of the epigenetic machinery caused by mutations in the gene encoding KMT2D, which methylates lysine 4 on histone H3 (H3K4). KS1 is characterized by intellectual disability, postnatal growth retardation, and distinct craniofacial dysmorphisms. A mouse model (Kmt2d+/βGeo) exhibits features of the human disorder and has provided insight into other phenotypes; however, the mechanistic basis of skeletal abnormalities and growth retardation remains elusive. Using high-resolution micro-CT, we show that Kmt2d+/βGeo mice have shortened long bones and ventral bowing of skulls. In vivo expansion of growth plates within skulls and long bones suggests disrupted endochondral ossification as a common disease mechanism. Stable chondrocyte cell lines harboring inactivating mutations in Kmt2d exhibit precocious differentiation, further supporting this mechanism. A known inducer of chondrogenesis, SOX9, and its targets show markedly increased expression in Kmt2d-/- chondrocytes. By transcriptome profiling, we identify Shox2 as a putative KMT2D target. We propose that decreased KMT2D-mediated H3K4me3 at Shox2 releases Sox9 inhibition and thereby leads to enhanced chondrogenesis, providing a potentially novel and plausible explanation for precocious chondrocyte differentiation. Our findings provide insight into the pathogenesis of growth retardation in KS1 and suggest therapeutic approaches for this and related disorders.

The fat mass and obesity-associated gene ( FTO ) encodes an m6A RNA demethylase that controls mRNA processing and has been linked to both obesity and bone mineral density in humans by genome-wide association studies. To examine the role of FTO in bone, we characterized the phenotype of mice lacking Fto globally ( Fto KO ) or selectively in osteoblasts ( Fto Oc KO ). Both mouse models developed age-related reductions in bone volume in both the trabecular and cortical compartments. RNA profiling in osteoblasts following acute disruption of Fto revealed changes in transcripts of Hspa1a and other genes in the DNA repair pathway containing consensus m6A motifs required for demethylation by Fto Fto KO osteoblasts were more susceptible to genotoxic agents (UV and H 2 O 2 ) and exhibited increased rates of apoptosis. Importantly, forced expression of Hspa1a or inhibition of NF-κB signaling normalized the DNA damage and apoptotic rates in Fto KO osteoblasts. Furthermore, increased metabolic stress induced in mice by feeding a high-fat diet induced greater DNA damage in osteoblast of Fto Oc KO mice compared to controls. These data suggest that FTO functions intrinsically in osteoblasts through Hspa1a-NF-κB signaling to enhance the stability of mRNA of proteins that function to protect cells from genotoxic damage., Competing Interests: The authors declare no conflict of interest.

Tumor progression to metastasis is not cancer cell autonomous, but rather involves the interplay of multiple cell types within the tumor microenvironment. Here we identify asporin (ASPN) as a novel, secreted mesenchymal stromal cell (MSC) factor in the tumor microenvironment that regulates metastatic development. MSCs expressed high levels of ASPN, which decreased following lineage differentiation. ASPN loss impaired MSC self-renewal and promoted terminal cell differentiation. Mechanistically, secreted ASPN bound to BMP-4 and restricted BMP-4-induced MSC differentiation prior to lineage commitment. ASPN expression was distinctly conserved between MSC and cancer-associated fibroblasts (CAF). ASPN expression in the tumor microenvironment broadly impacted multiple cell types. Prostate tumor allografts in ASPN-null mice had a reduced number of tumor-associated MSCs, fewer cancer stem cells, decreased tumor vasculature, and an increased percentage of infiltrating CD8 + T cells. ASPN-null mice also demonstrated a significant reduction in lung metastases compared with wild-type mice. These data establish a role for ASPN as a critical MSC factor that extensively affects the tumor microenvironment and induces metastatic progression. SIGNIFICANCE: These findings show that asporin regulates key properties of mesenchymal stromal cells, including self-renewal and multipotency, and asporin expression by reactive stromal cells alters the tumor microenvironment and promotes metastatic progression., (©2019 American Association for Cancer Research.)

Adipose-derived stem cells (ASCs) are a promising cell source for regenerating critical-sized craniofacial bone defects, but their clinical use is limited due to the supraphysiological levels of bone morphogenetic protein-2 required to induce bone formation in large grafts. It has been recently reported that platelet-derived growth factor-BB (PDGF) directly enhances the osteogenesis of ASCs when applied at physiological concentrations. In this study, a biomimetic delivery system that tethers PDGF to decellularized bone matrix (DCB) is developed to enhance osteogenic signaling in bone grafts by colocalizing PDGF-extracellular matrix cues. Heparin is conjugated to DCB particles (HC-DCB) to promote sustained binding of PDGF via electrostatic interactions. HC-DCB particles bind to PDGF with >99% efficiency and release significantly less PDGF over 21 days compared to nonconjugated DCB particles (1.1% vs 22.8%). HC-DCB-PDGF signaling in polycaprolactone (PCL)-fibrin grafts promotes >40 µg Ca 2+ µg -1 DNA deposition by ASCs during in vitro osteogenic culture compared to grafts without HC-DCB or PDGF. Furthermore, more bone formation is observed in grafts with HC-DCB-PDGF at 12 weeks following implantation of grafts into murine critical-sized calvarial defects. Collectively, these results demonstrate that HC-DCB enhances the osteogenic signaling of PDGF to ASCs and may be applied to promote ASC-mediated bone regeneration in critical-sized defects., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Sclerostin exerts profound local control over bone acquisition and also mediates endocrine communication between fat and bone. In bone, sclerostin's anti-osteoanabolic activity is enhanced by low-density lipoprotein receptor-related protein 4 (Lrp4), which facilitates its interaction with the Lrp5 and Lrp6 Wnt co-receptors. To determine whether Lrp4 similarly affects sclerostin's endocrine function, we examined body composition as well as glucose and fatty acid metabolism in mice rendered deficient of Lrp4 in the adipocyte (AdΔLrp4) or the osteoblast (ObΔLrp4). AdΔLrp4 mice exhibit a reduction in adipocyte hypertrophy and improved glucose and lipid homeostasis, marked by increased glucose and insulin tolerance and reduced serum fatty acids, and mirror the effect of sclerostin deficiency on whole-body metabolism. Indeed, epistasis studies place adipocyte-expressed Lrp4 and sclerostin in the same genetic cascade that regulates adipocyte function. Intriguingly, ObΔLrp4 mice, which exhibit dramatic increases in serum sclerostin, accumulate body fat and develop impairments in glucose tolerance and insulin sensitivity despite development of a high bone mass phenotype. These data indicate that expression of Lrp4 by both the adipocyte and osteoblast is required for normal sclerostin endocrine function and that the impact of sclerostin deficiency on adipocyte physiology is distinct from the effect on osteoblast function.

The emergence of bone as an endocrine organ able to influence whole body metabolism, together with comorbid epidemics of obesity, diabetes, and osteoporosis, have prompted a renewed interest in the intermediary metabolism of the osteoblast. To date, most studies have focused on the utilization of glucose by this specialized cell, but the oxidation of fatty acids results in a larger energy yield. Osteoblasts express the requisite receptors and catabolic enzymes to take up and then metabolize fatty acids, which appears to be required during later stages of differentiation when the osteoblast is dedicated to matrix production and mineralization. In this article, we provide an overview of fatty acid β-oxidation and highlight studies demonstrating that the skeleton plays a significant role in the clearance of circulating lipoproteins and non-esterified fatty acids. Additionally, we review the requirement for long-chain fatty acid metabolism during post-natal bone development and the effects of anabolic stimuli on fatty acid utilization by osteoblasts. These recent findings may help to explain the skeletal manifestations of human diseases associated with impaired lipid metabolism while also providing additional insights into the metabolic requirements of skeletal homeostasis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Wnt/β-catenin signaling plays a critical role in the achievement of peak bone mass, affecting the commitment of mesenchymal progenitors to the osteoblast lineage and the anabolic capacity of osteoblasts depositing bone matrix. Recent studies suggest that this evolutionarily-conserved, developmental pathway exerts its anabolic effects in part by coordinating osteoblast activity with intermediary metabolism. These findings are compatible with the cloning of the gene encoding the low-density lipoprotein related receptor-5 (LRP5) Wnt co-receptor from a diabetes-susceptibility locus and the now well-established linkage between Wnt signaling and metabolism. In this article, we provide an overview of the role of Wnt signaling in whole-body metabolism and review the literature regarding the impact of Wnt signaling on the osteoblast's utilization of three different energy sources: fatty acids, glucose, and glutamine. Special attention is devoted to the net effect of nutrient utilization and the mode of regulation by Wnt signaling. Mechanistic studies indicate that the utilization of each substrate is governed by a unique mechanism of control with β-catenin-dependent signaling regulating fatty acid β-oxidation, while glucose and glutamine utilization are β-catenin-independent and downstream of mammalian target of rapamycin complex 2 (mTORC2) and mammalian target of rapamycin complex 1 (mTORC1) activation, respectively. The emergence of these data has provided a new context for the mechanisms by which Wnt signaling influences bone development., Competing Interests: No potential conflict of interest relevant to this article was reported., (Copyright © 2018 Korean Endocrine Society.)

Wnt-initiated signaling through a frizzled receptor and the low-density lipoprotein-related receptor-5 coreceptor instructs key anabolic events during skeletal development, homeostasis, and repair. Recent studies indicate that Wnt signaling also regulates the intermediary metabolism of osteoblastic cells, inducing glucose consumption in osteoprogenitors and fatty acid utilization in mature osteoblasts. In this study, we examined the role of the canonical Wnt-signaling target, β-catenin, in the control of osteoblast metabolism. In vitro, Wnt ligands and agonists that stimulated β-catenin activation in osteoblasts enhanced fatty acid catabolism, whereas genetic ablation of β-catenin dramatically reduced oleate oxidation concomitant with reduced osteoblast maturation and increased glycolytic metabolism. Temporal ablation of β-catenin expression in osteoblasts in vivo produced the expected low-bone-mass phenotype and also led to an increase in white adipose tissue mass, dyslipidemia, and impaired insulin sensitivity. Because the expression levels of enzymatic mediators of fatty acid β-oxidation are reduced in the skeleton of β-catenin mutants, these results further confirm the role of the osteoblast in lipid metabolism and indicate that the influence of Wnt signaling on fatty acid utilization proceeds via its canonical signaling pathway., (Copyright © 2018 Endocrine Society.)

Sclerostin has traditionally been thought of as a local inhibitor of bone acquisition that antagonizes the profound osteoanabolic capacity of activated Wnt/β-catenin signaling, but serum sclerostin levels in humans exhibit a correlation with impairments in several metabolic parameters. These data, together with the increased production of sclerostin in mouse models of type 2 diabetes, suggest an endocrine function. To determine whether sclerostin contributes to the coordination of whole-body metabolism, we examined body composition, glucose homeostasis, and fatty acid metabolism in Sost -/- mice as well as mice that overproduce sclerostin as a result of adeno-associated virus expression from the liver. Here, we show that in addition to dramatic increases in bone volume, Sost -/- mice exhibit a reduction in adipose tissue accumulation in association with increased insulin sensitivity. Sclerostin overproduction results in the opposite metabolic phenotype due to adipocyte hypertrophy. Additionally, Sost -/- mice and those administered a sclerostin-neutralizing antibody are resistant to obesogenic diet-induced disturbances in metabolism. This effect appears to be the result of sclerostin's effects on Wnt signaling and metabolism in white adipose tissue. Since adipocytes do not produce sclerostin, these findings suggest an unexplored endocrine function for sclerostin that facilitates communication between the skeleton and adipose tissue., Competing Interests: The authors declare no conflict of interest.

Wnt signaling through the low-density lipoprotein-related receptor 5 (Lrp5) coreceptor regulates osteoblast maturation, matrix mineralization, and intermediary metabolism. In the mature osteoblast, signals downstream of Lrp5 are required for normal long-chain fatty acid β-oxidation. Mice rendered deficient for this coreceptor in osteoblasts and osteocytes accumulate body fat with elevated serum lipid levels but retain normal insulin sensitivity. In the present study, we challenged Lrp5-mutant mice with a high-fat diet (HFD) to determine whether they were more susceptible to diet-induced disturbances in glucose homeostasis. The HFD-fed Lrp5 mutant mice maintained a low bone mass phenotype with an increase in adipose tissue mass and hypertriglyceridemia and hypercholesterolemia. Examination of glucose metabolism revealed that Lrp5 deficiency in the osteoblast also resulted in hyperglycemia and hyperinsulinemia, with reductions in glucose tolerance, insulin sensitivity, and serum undercarboxylated osteocalcin. The results from in vivo genetic epistasis and in vitro studies suggest that this phenotype proceeds via the accumulation of diacylglycerol species and impaired insulin signaling in Lrp5-deficient osteoblasts. In turn, glucose uptake and osteocalcin production are diminished in mutant osteoblasts. Taken together, these data identify a link between Wnt-Lrp5 signaling and insulin signaling in the osteoblast that has the potential to influence energy balance and compound the detrimental effects of a HFD on whole-body metabolism., (Copyright © 2017 Endocrine Society.)

Postnatal bone formation is influenced by nutritional status and compromised by disturbances in metabolism. The oxidation of dietary lipids represents a critical source of ATP for many cells but has been poorly studied in the skeleton, where the prevailing view is that glucose is the primary energy source. Here, we examined fatty acid uptake by bone and probed the requirement for fatty acid catabolism during bone formation by specifically disrupting the expression of carnitine palmitoyltransferase 2 (Cpt2), an obligate enzyme in fatty acid oxidation, in osteoblasts and osteocytes. Radiotracer studies demonstrated that the skeleton accumulates a significant fraction of postprandial fatty acids, which was equal to or in excess of that acquired by skeletal muscle or adipose tissue. Female, but not male, Cpt2 mutant mice exhibited significant impairments in postnatal bone acquisition, potentially due to an inability of osteoblasts to modify fuel selection. Intriguingly, suppression of fatty acid utilization by osteoblasts and osteocytes also resulted in the development of dyslipidemia and diet-dependent modifications in body composition. Taken together, these studies demonstrate a requirement for fatty acid oxidation during bone accrual and suggest a role for the skeleton in lipid homeostasis.

The liver has a large capacity for mitochondrial fatty acid β-oxidation, which is critical for systemic metabolic adaptations such as gluconeogenesis and ketogenesis. To understand the role of hepatic fatty acid oxidation in response to a chronic high-fat diet (HFD), we generated mice with a liver-specific deficiency of mitochondrial long-chain fatty acid β-oxidation (Cpt2 L-/- mice). Paradoxically, Cpt2 L-/- mice were resistant to HFD-induced obesity and glucose intolerance with an absence of liver damage, although they exhibited serum dyslipidemia, hepatic oxidative stress, and systemic carnitine deficiency. Feeding an HFD induced hepatokines in mice, with a loss of hepatic fatty acid oxidation that enhanced systemic energy expenditure and suppressed adiposity. Additionally, the suppression in hepatic gluconeogenesis was sufficient to improve HFD-induced glucose intolerance. These data show that inhibiting hepatic fatty acid oxidation results in a systemic hormetic response that protects mice from HFD-induced obesity and glucose intolerance., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Biallelic mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a disease characterized by calcification in the skin, eyes, and blood vessels. The function of ATP-binding cassette C6 (ABCC6) and the pathogenesis of PXE remain unclear. We used mouse models and patient fibroblasts to demonstrate genetic interaction and shared biochemical and cellular mechanisms underlying ectopic calcification in PXE and related disorders caused by defined perturbations in extracellular adenosine 5'-triphosphate catabolism. Under osteogenic culture conditions, ABCC6 mutant cells calcified, suggesting a provoked cell-autonomous defect. Using a conditional Abcc6 knockout mouse model, we excluded the prevailing pathogenic hypothesis that singularly invokes failure of hepatic secretion of an endocrine inhibitor of calcification. Instead, deficiency of Abcc6 in both local and distant cells was necessary to achieve the early onset and penetrant ectopic calcification observed upon constitutive gene targeting. ABCC6 mutant cells additionally had increased expression and activity of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme that degrades pyrophosphate, a major inhibitor of calcification. A selective and orally bioavailable TNAP inhibitor prevented calcification in ABCC6 mutant cells in vitro and attenuated both the development and progression of calcification in Abcc6 -/- mice in vivo, without the deleterious effects on bone associated with other proposed treatment strategies., (Copyright © 2017, American Association for the Advancement of Science.)

Sensory nerves emanating from the dorsal root extensively innervate the surfaces of mammalian bone, a privileged location for the regulation of biomechanical signaling. Here, we show that NGF-TrkA signaling in skeletal sensory nerves is an early response to mechanical loading of bone and is required to achieve maximal load-induced bone formation. First, the elimination of TrkA signaling in mice harboring mutant TrkA F592A alleles was found to greatly attenuate load-induced bone formation induced by axial forelimb compression. Next, both in vivo mechanical loading and in vitro mechanical stretch were shown to induce the profound up-regulation of NGF in osteoblasts within 1 h of loading. Furthermore, inhibition of TrkA signaling following axial forelimb compression was observed to reduce measures of Wnt/β-catenin activity in osteocytes in the loaded bone. Finally, the administration of exogenous NGF to wild-type mice was found to significantly increase load-induced bone formation and Wnt/β-catenin activity in osteocytes. In summary, these findings demonstrate that communication between osteoblasts and sensory nerves through NGF-TrkA signaling is essential for load-induced bone formation in mice., Competing Interests: The authors declare no conflict of interest.

The rising incidence of metabolic diseases worldwide has prompted renewed interest in the study of intermediary metabolism and cellular bioenergetics. The application of modern biochemical methods for quantitating fuel substrate metabolism with advanced mouse genetic approaches has greatly increased understanding of the mechanisms that integrate energy metabolism in the whole organism. Examination of the intermediary metabolism of skeletal cells has been sparked by a series of unanticipated observations in genetically modified mice that suggest the existence of novel endocrine pathways through which bone cells communicate their energy status to other centers of metabolic control. The recognition of this expanded role of the skeleton has in turn led to new lines of inquiry directed at defining the fuel requirements and bioenergetic properties of bone cells. This article provides a comprehensive review of historical and contemporary studies on the metabolic properties of bone cells and the mechanisms that control energy substrate utilization and bioenergetics. Special attention is devoted to identifying gaps in our current understanding of this new area of skeletal biology that will require additional research to better define the physiological significance of skeletal cell bioenergetics in human health and disease., (Copyright © 2017 the American Physiological Society.)

Recent studies have identified the osteoblast as an insulin responsive cell that participates in global energy homeostasis. Here, we show that glucose transporter-4 (Glut4) is required for insulin-dependent uptake and oxidation of glucose in mature osteoblasts. In primary cultures of mouse osteoblasts, insulin increased uptake and oxidation of 14 C-glucose in a dose-dependent fashion but did not significantly affect uptake or oxidation of 14 C-oleate. In vitro, undifferentiated osteoblasts expressed 3 high-affinity Gluts: Glut1, Glut4, and Glut3. However, although levels of Glut1 and Glut3 remained constant during the course of osteoblast differentiation, Glut4 expression increased by 5-fold in association with enhanced insulin-stimulated glucose uptake. Glut4 ablation in osteoblasts in vitro eliminated insulin-stimulated glucose uptake, reduced proliferation and diminished measures of osteoblast maturation. In vivo, Glut4 expression was observed in osteoblasts, osteocytes, and chondrocytes at a level approaching that observed in adjacent skeletal muscle. To determine the importance of Glut4 in bone in vivo, we generated mice lacking Glut4 in osteoblasts and osteocytes (ΔGlut4). ΔGlut4 mice exhibited normal bone architecture but exhibited an increase in peripheral fat in association with hyperinsulinemia, β-cell islet hypertrophy, and reduced insulin sensitivity. Surprisingly, the expression of insulin target genes in liver, muscle, and adipose from ΔGlut4 mice were unchanged or increased, indicating that alterations in glucose homeostasis were the result of reduced clearance by bone. These findings suggest that Glut4 mediates insulin-stimulated glucose uptake by mature osteoblasts/osteocytes and that the magnitude of glucose use by bone cells is sufficient to impact global glucose disposal in the mouse.

Bone formation and remodeling occurs throughout life and requires the sustained activity of osteoblasts and osteoclasts, particularly during periods of rapid bone growth. Despite increasing evidence linking bone cell activity to global energy homeostasis, little is known about the relative energy requirements or substrate utilization of bone cells. In these studies, we measured the uptake and distribution of glucose in the skeleton in vivo using positron-emitting (18)F-fluorodeoxyglucose ([(18)F]-FDG) and non-invasive, high-resolution positron emission tomography/computed tomography (PET/CT) imaging and ex vivo autoradiography. Assessment of [(18)F]-FDG uptake demonstrated that relative to other tissues bone accumulated a significant fraction of the total dose of the glucose analog. Skeletal accumulation was greatest in young mice undergoing the rapid bone formation that characterizes early development. PET/CT imaging revealed that [(18)F]-FDG uptake was greatest in the epiphyseal and metaphyseal regions of long bones, which accords with the increased osteoblast numbers and activity at this skeletal site. Insulin administration significantly increased skeletal accumulation of [(18)F]-FDG, while uptake was reduced in mice lacking the insulin receptor specifically in osteoblasts or fed a high-fat diet. Our results indicated that the skeleton is a site of significant glucose uptake and that its consumption by bone cells is subject to regulation by insulin and disturbances in whole-body metabolism.

Osteocalcin is among the most abundant proteins in bone and is produced exclusively by osteoblasts. Initially believed to be an inhibitor of bone mineralization, recent studies suggest a broader role for osteocalcin that extends to the regulation of whole body metabolism, reproduction, and cognition. Circulating undercarboxylated osteocalcin, which is regulated by insulin, acts in a feed-forward loop to increase β-cell proliferation as well as insulin production and secretion, while skeletal muscle and adipose tissue respond to osteocalcin by increasing their sensitivity to insulin. Osteocalcin also acts in the brain to increase neurotransmitter production and in the testes to stimulate testosterone production. At least one putative receptor for osteocalcin, Gprc6a, is expressed by adipose, skeletal muscle, and the Leydig cells of the testes and appears to mediate osteocalcin's effects in these tissues. In this review, we summarize these new discoveries, which suggest that the ability of osteocalcin to function both locally in bone and as a hormone depends on a novel post-translational mechanism that alters osteocalcin's affinity for the bone matrix and bioavailability. This article is part of a Special Issue entitled Bone and diabetes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Bone-metastatic, castration-resistant prostate cancer (bmCRPC) represents a lethal stage of the most common noncutaneous cancer in men. The recent introduction of Radium-223 dichloride, a bone-seeking alpha particle (α)-emitting radiopharmaceutical, demonstrates statistically significant survival benefit and palliative effect for bmCRPC patients. Clinical results have established safety and efficacy, yet questions remain regarding pharmacodynamics and dosing for optimized patient benefit., Methods: We elucidated the biodistribution of (223)Ra as well as interaction with the bone and tumor compartments in skeletally mature mice (C57Bl/6 and CD-1, n = 3-6) and metastasis models (LNCaP and PC3, n = 4). Differences in uptake were evaluated by µCT and histological investigation. Novel techniques were leveraged on whole-mount undecalcified cryosections to determine microdistribution of Radium-223. All statistical tests were two-sided., Results: (223)Ra uptake in the bones (>30% injected activity per gram) at 24 hours was also accompanied by non-negligible remnant activity in the kidney (2.33% ± 0.36%), intestines (5.73% ± 2.04%), and spleen (10.5% ± 5.9%) Skeletal accumulation across strains did not correspond with bone volume or surface area but instead to local blood vessel density (P = .04). Microdistribution analysis by autoradiography and α camera revealed targeting of the ossifying surfaces adjacent to the epiphyseal growth plate. In models of PCa metastasis, radioactivity does not localize directly within tumors but instead at the apposite bone surface. Osteoblastic and lytic lesions display similar intensity, which is comparable with uptake at sites of normal bone remodeling., Conclusions: Profiling the macro- and microdistribution of (223)Ra in healthy and diseased models has important implications to guide precision application of this emerging α-therapy approach for bmCRPC and other bone metastastic diseases., (© The Author 2015. Published by Oxford University Press.)

The skeleton, populated by large numbers of osteoblasts and long-lived osteocytes, requires a constant supply of energy-rich molecules to fuel the synthesis, deposition and mineralization of bone matrix during bone modelling and remodelling. When these energetic demands are not met, bone acquisition is suppressed. Recent findings suggest that key developmental signals emanating from Wnt low-density lipoprotein-related receptor 5 and hypoxia-inducible factor pathways impact osteoblast bioenergetics to accommodate the energy requirements for bone cells to fulfil their function. In vivo studies in several mutant mouse strains have confirmed a link between bone cells and global metabolism, ultimately leading to the identification of hormonal interactions between the skeleton and other tissues. The hormones insulin and leptin affect postnatal bone acquisition, whilst osteocalcin produced by the osteoblast in turn stimulates insulin secretion by the pancreas. These observations have prompted additional questions regarding the nature of the mechanisms of fuel sensing and processing in the osteoblast and their contribution to overall energy utilization and homeostasis. Answers to such questions should advance our understanding of metabolic diseases and may ultimately improve management of affected patients. In this review, we highlight recent studies in this field and offer a perspective on the evolutionary implications of bone as a metabolic endocrine organ., (© 2015 The Association for the Publication of the Journal of Internal Medicine.)

The Wnt coreceptors Lrp5 and Lrp6 are essential for normal postnatal bone accrual and osteoblast function. In this study, we identify a previously unrecognized skeletal function unique to Lrp5 that enables osteoblasts to oxidize fatty acids. Mice lacking the Lrp5 coreceptor specifically in osteoblasts and osteocytes exhibit the expected reductions in postnatal bone mass but also exhibit an increase in body fat with corresponding reductions in energy expenditure. Conversely, mice expressing a high bone mass mutant Lrp5 allele are leaner with reduced plasma triglyceride and free fatty acid levels. In this context, Wnt-initiated signals downstream of Lrp5, but not the closely related Lrp6 coreceptor, regulate the activation of β-catenin and thereby induce the expression of key enzymes required for fatty acid β-oxidation. These results suggest that Wnt-Lrp5 signaling regulates basic cellular activities beyond those associated with fate specification and differentiation in bone and that the skeleton influences global energy homeostasis via mechanisms independent of osteocalcin and glucose metabolism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)