PPARG in osteocytes controls cell bioenergetics and systemic energy metabolism independently of sclerostin levels in circulation.

Objective: The skeleton is one of the largest organs in the body, wherein metabolism is integrated with systemic energy metabolism. However, the bioenergetic programming of osteocytes, the most abundant bone cells coordinating bone metabolism, is not well defined. Here, using a mouse model with partial penetration of an osteocyte-specific PPARG deletion, we demonstrate that PPARG controls osteocyte bioenergetics and their contribution to systemic energy metabolism independently of circulating sclerostin levels. Methods: In vivo and in vitro models of osteocyte-specific PPARG deletion, i.e. Dmp 1 Cre Pparγ flfl male and female mice (γOT KO ) and MLO-Y4 osteocyte-like cells with either siRNA-silenced or CRISPR/Cas9-edited Pparγ . As applicable, the models were analyzed for levels of energy metabolism, glucose metabolism, and metabolic profile of extramedullary adipose tissue, as well as the osteocyte transcriptome, mitochondrial function, bioenergetics, insulin signaling, and oxidative stress. Results: Circulating sclerostin levels of γOT KO male and female mice were not different from control mice. Male γOT KO mice exhibited a high energy phenotype characterized by increased respiration, heat production, locomotion and food intake. This high energy phenotype in males did not correlate with "beiging" of peripheral adipose depots. However, both sexes showed a trend for reduced fat mass and apparent insulin resistance without changes in glucose tolerance, which correlated with decreased osteocytic responsiveness to insulin measured by AKT activation. The transcriptome of osteocytes isolated from γOT KO males suggested profound changes in cellular metabolism, fuel transport and usage, mitochondria dysfunction, insulin signaling and increased oxidative stress. In MLO-Y4 osteocytes, PPARG deficiency correlated with highly active mitochondria, increased ATP production, shifts in fuel utilization, and accumulation of reactive oxygen species (ROS). Conclusions: PPARG in male osteocytes acts as a molecular break on mitochondrial function, and protection against oxidative stress and ROS accumulation. It also regulates osteocyte insulin signaling and fuel usage to produce energy. These data provide insight into the connection between osteocyte bioenergetics and their sex-specific contribution to the balance of systemic energy metabolism. These findings support the concept that the skeleton controls systemic energy expenditure via osteocyte metabolism. Highlights: Osteocytes function as a body energostat via their bioenergeticsPPARG protein acts as a "molecular break" of osteocyte mitochondrial activityPPARG deficiency activates TCA cycle, oxidative stress and ROS accumulationPPARG controls osteocyte insulin signaling and fuel utilization.

Deletion of FNDC5/irisin modifies murine osteocyte function in a sex-specific manner.

Irisin, released from exercised muscle, has been shown to have beneficial effects on numerous tissues but its effects on bone are unclear. We found significant sex and genotype differences in bone from wildtype (WT) mice compared to mice lacking Fndc5 (knockout [KO]), with and without calcium deficiency. Despite their bone being indistinguishable from WT females, KO female mice were partially protected from osteocytic osteolysis and osteoclastic bone resorption when allowed to lactate or when placed on a low-calcium diet. Male KO mice have more but weaker bone compared to WT males, and when challenged with a low-calcium diet lost more bone than WT males. To begin to understand responsible molecular mechanisms, osteocyte transcriptomics was performed. Osteocytes from WT females had greater expression of genes associated with osteocytic osteolysis and osteoclastic bone resorption compared to WT males which had greater expression of genes associated with steroid and fatty acid metabolism. Few differences were observed between female KO and WT osteocytes, but with a low-calcium diet, the KO females had lower expression of genes responsible for osteocytic osteolysis and osteoclastic resorption than the WT females. Male KO osteocytes had lower expression of genes associated with steroid and fatty acid metabolism, but higher expression of genes associated with bone resorption compared to male WT. In conclusion, irisin plays a critical role in the development of the male but not the female skeleton and protects male but not female bone from calcium deficiency. We propose irisin ensures the survival of offspring by targeting the osteocyte to provide calcium in lactating females, a novel function for this myokine.

PTH and the Regulation of Mesenchymal Cells within the Bone Marrow Niche.

Parathyroid hormone (PTH) plays a pivotal role in maintaining calcium homeostasis, largely by modulating bone remodeling processes. Its effects on bone are notably dependent on the duration and frequency of exposure. Specifically, PTH can initiate both bone formation and resorption, with the outcome being influenced by the manner of PTH administration: continuous or intermittent. In continuous administration, PTH tends to promote bone resorption, possibly by regulating certain genes within bone cells. Conversely, intermittent exposure generally favors bone formation, possibly through transient gene activation. PTH's role extends to various aspects of bone cell activity. It directly influences skeletal stem cells, osteoblastic lineage cells, osteocytes, and T cells, playing a critical role in bone generation. Simultaneously, it indirectly affects osteoclast precursor cells and osteoclasts, and has a direct impact on T cells, contributing to its role in bone resorption. Despite these insights, the intricate mechanisms through which PTH acts within the bone marrow niche are not entirely understood. This article reviews the dual roles of PTH-catabolic and anabolic-on bone cells, highlighting the cellular and molecular pathways involved in these processes. The complex interplay of these factors in bone remodeling underscores the need for further investigation to fully comprehend PTH's multifaceted influence on bone health.

Deletion of FNDC5/Irisin modifies murine osteocyte function in a sex-specific manner.

Irisin, released from exercised muscle, has been shown to have beneficial effects on numerous tissues but its effects on bone are unclear. We found significant sex and genotype differences in bone from wildtype (WT) mice compared to mice lacking Fndc5 (KO), with and without calcium deficiency. Despite their bone being indistinguishable from WT females, KO female mice were partially protected from osteocytic osteolysis and osteoclastic bone resorption when allowed to lactate or when placed on a low-calcium diet. Male KO mice have more but weaker bone compared to WT males, and when challenged with a low-calcium diet lost more bone than WT males. To begin to understand responsible molecular mechanisms, osteocyte transcriptomics was performed. Osteocytes from WT females had greater expression of genes associated with osteocytic osteolysis and osteoclastic bone resorption compared to WT males which had greater expression of genes associated with steroid and fatty acid metabolism. Few differences were observed between female KO and WT osteocytes, but with a low calcium diet, the KO females had lower expression of genes responsible for osteocytic osteolysis and osteoclastic resorption than the WT females. Male KO osteocytes had lower expression of genes associated with steroid and fatty acid metabolism, but higher expression of genes associated with bone resorption compared to male WT. In conclusion, irisin plays a critical role in the development of the male but not the female skeleton and protects male but not female bone from calcium deficiency. We propose irisin ensures the survival of offspring by targeting the osteocyte to provide calcium in lactating females, a novel function for this myokine.

Lipolysis supports bone formation by providing osteoblasts with endogenous fatty acid substrates to maintain bioenergetic status.

Bone formation is a highly energy-demanding process that can be impacted by metabolic disorders. Glucose has been considered the principal substrate for osteoblasts, although fatty acids are also important for osteoblast function. Here, we report that osteoblasts can derive energy from endogenous fatty acids stored in lipid droplets via lipolysis and that this process is critical for bone formation. As such, we demonstrate that osteoblasts accumulate lipid droplets that are highly dynamic and provide the molecular mechanism by which they serve as a fuel source for energy generation during osteoblast maturation. Inhibiting cytoplasmic lipolysis leads to both an increase in lipid droplet size in osteoblasts and an impairment in osteoblast function. The fatty acids released by lipolysis from these lipid droplets become critical for cellular energy production as cellular energetics shifts towards oxidative phosphorylation during nutrient-depleted conditions. In vivo, conditional deletion of the ATGL-encoding gene Pnpla2 in osteoblast progenitor cells reduces cortical and trabecular bone parameters and alters skeletal lipid metabolism. Collectively, our data demonstrate that osteoblasts store fatty acids in the form of lipid droplets, which are released via lipolysis to support cellular bioenergetic status when nutrients are limited. Perturbations in this process result in impairment of bone formation, specifically reducing ATP production and overall osteoblast function.

Refining Evaluation of Bone Mass and Adipose Distribution in Dunnigan Syndrome.

Familial partial lipodystrophies (FPLD) are rare diseases characterized by selective loss of subcutaneous adipose tissue at different sites. This cross-sectional observational study aimed to estimate adipose tissue in the bone marrow (BMAT), intra (IMCL) and extra-myocyte lipids (EMCL), and define the bone phenotype in the context of FPLD2/Dunnigan syndrome (DS). The subjects comprised 23 controls (C) and 18 DS patients, matched by age, weight and height. Blood samples, dual-energy X-ray absorptiometry for bone mineral density (BMD) and trabecular bone score (TBS) and 1H-spectroscopy using magnetic resonance to estimate BMAT in the lumbar spine, IMCL, EMCL and osteoclastogenesis were assessed. The prevalence of diabetes mellitus was 78% in DS patients. Glucose, HbA1c, triglycerides, insulin and HOMA-IR levels were elevated in DS, whereas HDLc, 25(OH)D, PTH and osteocalcin levels were reduced. BMD was similar between groups at all sites, except 1/3 radius, which was lower in DS group. TBS was reduced in DS. DS presented increased osteoclastogenesis and elevated BMAT, with greater saturation levels and higher IMCL than the C group. HOMA-IR and EMCL were negatively associated with TBS; osteocalcin and EMCL were correlated negatively with BMD. This study contributes to refining the estimation of adipose tissue in DS by showing increased adiposity in the lumbar spine and muscle tissue. DXA detected lower TBS and BMD in the 1/3 radius, suggesting impairment in bone quality and that bone mass is mainly affected in the cortical bone.

Effects of PTH on osteoblast bioenergetics in response to glucose.

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

Nutrient regulation of bone marrow adipose tissue: skeletal implications of weight loss.

Adipose tissue is a dynamic component of the bone marrow, regulating skeletal remodelling and secreting paracrine and endocrine factors that can affect haematopoiesis, as well as potentially nourishing the bone marrow during periods of stress. Bone marrow adipose tissue is regulated by multiple factors, but particularly nutrient status. In this Review, we examine how bone marrow adipocytes originate, their function in normal and pathological states and how bone marrow adipose tissue modulates whole-body homoeostasis through actions on bone cells, haematopoietic stem cells and extra-medullary adipocytes during nutritional challenges. We focus on both rodent models and human studies to help understand the unique marrow adipocyte, its response to the external nutrient environment and its effects on the skeleton. We finish by addressing some critical questions that to date remain unanswered.

Single-Cell Transcriptomics of Bone Marrow Stromal Cells in Diversity Outbred Mice: A Model for Population-Level scRNA-Seq Studies.

Genome-wide association studies (GWASs) have advanced our understanding of the genetics of osteoporosis; however, the challenge has been converting associations to causal genes. Studies have utilized transcriptomics data to link disease-associated variants to genes, but few population transcriptomics data sets have been generated on bone at the single-cell level. To address this challenge, we profiled the transcriptomes of bone marrow-derived stromal cells (BMSCs) cultured under osteogenic conditions from five diversity outbred (DO) mice using single-cell RNA-seq (scRNA-seq). The goal of the study was to determine if BMSCs could serve as a model to generate cell type-specific transcriptomic profiles of mesenchymal lineage cells from large populations of mice to inform genetic studies. By enriching for mesenchymal lineage cells in vitro, coupled with pooling of multiple samples and downstream genotype deconvolution, we demonstrate the scalability of this model for population-level studies. We demonstrate that dissociation of BMSCs from a heavily mineralized matrix had little effect on viability or their transcriptomic signatures. Furthermore, we show that BMSCs cultured under osteogenic conditions are diverse and consist of cells with characteristics of mesenchymal progenitors, marrow adipogenic lineage precursors (MALPs), osteoblasts, osteocyte-like cells, and immune cells. Importantly, all cells were similar from a transcriptomic perspective to cells isolated in vivo. We employed scRNA-seq analytical tools to confirm the biological identity of profiled cell types. SCENIC was used to reconstruct gene regulatory networks (GRNs), and we observed that cell types show GRNs expected of osteogenic and pre-adipogenic lineage cells. Further, CELLECT analysis showed that osteoblasts, osteocyte-like cells, and MALPs captured a significant component of bone mineral density (BMD) heritability. Together, these data suggest that BMSCs cultured under osteogenic conditions coupled with scRNA-seq can be used as a scalable and biologically informative model to generate cell type-specific transcriptomic profiles of mesenchymal lineage cells in large populations. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The Multifaceted Roles of Bone Marrow Adipocytes in Bone and Hematopoietic Homeostasis.

Bone marrow adipose tissue (BMAT) makes up a significant portion of the marrow space, ranging from 50% to 70%, in healthy adults. It expands with aging, obesity, anorexia nervosa, and irradiation, which are conditions associated with skeletal complications or hematopoietic disorders. Therefore, BMAT has been viewed as a negative component of the bone marrow niche for decades, although the mechanisms and causative relationships have not been well-addressed. Of note, recent studies have revealed that BMAT is a multifaceted tissue that can serve as an energy reservoir to fuel osteoblasts and hematopoietic cells under stressful situations, and also acts as an endocrine/paracrine organ to suppress bone formation and support hematopoiesis at steady-state conditions. In this review, we summarize the uniqueness of BMAT, the complex findings of previous studies, and update our understanding of the physiological roles of BMAT in bone and hematopoietic metabolism based on a newly established bone marrow adipocyte-specific mouse model.

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

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

New Insights into Calorie Restriction Induced Bone Loss.

Caloric restriction (CR) is now a popular lifestyle choice due to its ability in experimental animals to improve lifespan, reduce body weight, and lessen oxidative stress. However, more and more emerging evidence suggests this treatment requires careful consideration because of its detrimental effects on the skeletal system. Experimental and clinical studies show that CR can suppress bone growth and raise the risk of fracture, but the specific mechanisms are poorly understood. Reduced mechanical loading has long been thought to be the primary cause of weight loss-induced bone loss from calorie restriction. Despite fat loss in peripheral depots with calorie restriction, bone marrow adipose tissue (BMAT) increases, and this may play a significant role in this pathological process. Here, we update recent advances in our understanding of the effects of CR on the skeleton, the possible pathogenic role of BMAT in CR-induced bone loss, and some strategies to mitigate any potential side effects on the skeletal system.

PTH regulates osteogenesis and suppresses adipogenesis through Zfp467 in a feed-forward, PTH1R-cyclic AMP-dependent manner.

Conditional deletion of the PTH1R in mesenchymal progenitors reduces osteoblast differentiation, enhances marrow adipogenesis, and increases zinc finger protein 467 (Zfp467) expression. In contrast, genetic loss of Zfp467 increased Pth1r expression and shifts mesenchymal progenitor cell fate toward osteogenesis and higher bone mass. PTH1R and ZFP467 could constitute a feedback loop that facilitates PTH-induced osteogenesis and that conditional deletion of Zfp467 in osteogenic precursors would lead to high bone mass in mice. Prrx1Cre; Zfp467fl/fl but not AdipoqCre; Zfp467fl/fl mice exhibit high bone mass and greater osteogenic differentiation similar to the Zfp467-/- mice. qPCR results revealed that PTH suppressed Zfp467 expression primarily via the cyclic AMP/PKA pathway. Not surprisingly, PKA activation inhibited the expression of Zfp467 and gene silencing of Pth1r caused an increase in Zfp467 mRNA transcription. Dual fluorescence reporter assays and confocal immunofluorescence demonstrated that genetic deletion of Zfp467 resulted in higher nuclear translocation of NFκB1 that binds to the P2 promoter of the Pth1r and increased its transcription. As expected, Zfp467-/- cells had enhanced production of cyclic AMP and increased glycolysis in response to exogenous PTH. Additionally, the osteogenic response to PTH was also enhanced in Zfp467-/- COBs, and the pro-osteogenic effect of Zfp467 deletion was blocked by gene silencing of Pth1r or a PKA inhibitor. In conclusion, our findings suggest that loss or PTH1R-mediated repression of Zfp467 results in a pathway that increases Pth1r transcription via NFκB1 and thus cellular responsiveness to PTH/PTHrP, ultimately leading to enhanced bone formation.