Sexual dimorphism of osteoclast reliance on mitochondrial oxidation of energy substrates in the mouse.

Osteoclasts specialize in bone resorption and are critical for bone remodeling. Previous studies have shown that osteoclasts possess abundant mitochondria and derive most energy through oxidative phosphorylation (OXPHOS). However, the energy substrates fueling OXPHOS in osteoclasts remain to be fully defined. Here, we showed that osteoclast differentiation was coupled with increased oxidation of glucose, glutamine, and oleate. Transcriptomic analyses with RNA sequencing revealed marked upregulation of genes participating in OXPHOS and mitochondrial fatty acid oxidation, during osteoclast differentiation. Increased mitochondrial oxidation of long-chain fatty acids was required for osteoclast differentiation in vitro. However, blocking fatty acid oxidation in vivo, by deletion of carnitine palmitoyltransferase 1a (Cpt1a) in osteoclast progenitors, impaired osteoclast formation only in the female mice. The Cpt1a-deficient females were further protected from osteoclast activation by a high-fat diet. The males, on the contrary, exhibited normal bone resorption despite Cpt1a deletion, regardless of the dietary fat content. Moreover, concurrent deletion of mitochondrial pyruvate carrier 1 and Cpt1a, blocking mitochondrial oxidation of both glucose and fatty acids in the osteoclast lineage, failed to impede bone resorption in the males. The study therefore uncovers a female-specific dependence on mitochondrial oxidation of fatty acids and glucose in osteoclasts in vivo.

Genetic activation of glycolysis in osteoblasts preserves bone mass in type I diabetes.

Type I diabetes (T1D) impairs bone accrual in patients, but the mechanism is unclear. Here in a murine monogenic model for T1D, we demonstrate that diabetes suppresses bone formation resulting in a rapid loss of both cortical and trabecular bone. Single-cell RNA sequencing uncovers metabolic dysregulation in bone marrow osteogenic cells of diabetic mice. In vivo stable isotope tracing reveals impaired glycolysis in diabetic bone that is highly responsive to insulin stimulation. Remarkably, deletion of the insulin receptor reduces cortical but not trabecular bone. Increasing glucose uptake by overexpressing Glut1 in osteoblasts exacerbates bone defects in T1D mice. Conversely, activation of glycolysis by Pfkfb3 overexpression preserves both trabecular and cortical bone mass in the face of diabetes. The study identifies defective glucose metabolism in osteoblasts as a pathogenic mechanism for osteopenia in T1D, and furthermore implicates boosting osteoblast glycolysis as a potential bone anabolic therapy.

Osteoblast-intrinsic defect in glucose metabolism impairs bone formation in type II diabetic male mice.

Skeletal fragility is associated with type 2 diabetes mellitus (T2D), but the underlying mechanism is not well understood. Here, in a mouse model for youth-onset T2D, we show that both trabecular and cortical bone mass is reduced due to diminished osteoblast activity. Stable isotope tracing in vivo with 13C-glucose demonstrates that both glycolysis and glucose fueling of the TCA cycle are impaired in diabetic bones. Similarly, Seahorse assays show suppression of both glycolysis and oxidative phosphorylation by diabetes in bone marrow mesenchymal cells as a whole, whereas single-cell RNA sequencing reveals distinct modes of metabolic dysregulation among the subpopulations. Metformin not only promotes glycolysis and osteoblast differentiation in vitro, but also improves bone mass in diabetic mice. Finally, osteoblast-specific overexpression of either Hif1a, a general inducer of glycolysis, or Pfkfb3 which stimulates a specific step in glycolysis, averts bone loss in T2D mice. The study identifies osteoblast-intrinsic defects in glucose metabolism as an underlying cause of diabetic osteopenia, which may be targeted therapeutically.

Osteoblast-intrinsic defect in glucose metabolism impairs bone formation in type II diabetic mice.

Skeletal fragility is associated with type 2 diabetes mellitus (T2D), but the underlying mechanism is not well understood. Here, in a mouse model for youth-onset T2D, we show that both trabecular and cortical bone mass are reduced due to diminished osteoblast activity. Stable isotope tracing in vivo with 13 C-glucose demonstrates that both glycolysis and glucose fueling of the TCA cycle are impaired in diabetic bones. Similarly, Seahorse assays show suppression of both glycolysis and oxidative phosphorylation by diabetes in bone marrow mesenchymal cells as a whole, whereas single-cell RNA sequencing reveals distinct modes of metabolic dysregulation among the subpopulations. Metformin not only promotes glycolysis and osteoblast differentiation in vitro, but also improves bone mass in diabetic mice. Finally, targeted overexpression of Hif1a or Pfkfb3 in osteoblasts of T2D mice averts bone loss. The study identifies osteoblast-intrinsic defects in glucose metabolism as an underlying cause of diabetic osteopenia, which may be targeted therapeutically.

Glucose metabolism in skeletal cells.

The mammalian skeleton is integral to whole body physiology with a multitude of functions beyond mechanical support and locomotion, including support of hematopoiesis, mineral homeostasis and potentially other endocrine roles. Formation of the skeleton begins in the embryo and mostly from a cartilage template that is ultimately replaced by bone through endochondrial ossification. Skeletal development and maturation continue after birth in most species and last into the second decade of postnatal life in humans. In the mature skeleton, articular cartilage lining the synovial joint surfaces is vital for bodily movement and damages to the cartilage are a hallmark of osteoarthritis. The mature bone tissue undergoes continuous remodeling initiated with bone resorption by osteoclasts and completed with bone formation from osteoblasts. In a healthy state, the exquisite balance between bone resorption and formation is responsible for maintaining a stable bone mass and structural integrity, while meeting the physiological needs for minerals via controlled release from bone. Disruption of the balance in favor of bone resorption is the root cause for osteoporosis. Whereas osteoclasts pump molar quantities of hydrochloric acid to dissolve the bone minerals in a process requiring ATP hydrolysis, osteoblasts build bone mass by synthesizing and secreting copious amounts of bone matrix proteins. Thus, both osteoclasts and osteoblasts engage in energy-intensive activities to fulfill their physiological functions, but the bioenergetics of those and other skeletal cell types are not well understood. Nonetheless, the past ten years have witnessed a resurgence of interest in studies of skeletal cell metabolism, resulting in an unprecedented understanding of energy substrate utilization and its role in cell fate and activity regulation. The present review attempts to synthesize the current findings of glucose metabolism in chondrocytes, osteoblasts and osteoclasts. Advances with the other relevant cell types including skeletal stem cells and marrow adipocytes will not be discussed here as they have been extensively reviewed recently by others (van Gastel and Carmeliet, 2021). Elucidation of the bioenergetic mechanisms in the skeletal cells is likely to open new avenues for developing additional safe and effective bone therapies.

Glycemic Control and Bone in Diabetes.

PURPOSE OF REVIEW: This review summarizes recent developments on the effects of glycemic control and diabetes on bone health. We discuss the foundational cellular mechanisms through which diabetes and impaired glucose control impact bone biology, and how these processes contribute to bone fragility in diabetes. RECENT FINDINGS: Glucose is important for osteoblast differentiation and energy consumption of mature osteoblasts. The role of insulin is less clear, but insulin receptor deletion in mouse osteoblasts reduces bone formation. Epidemiologically, type 1 (T1D) and type 2 diabetes (T2D) associate with increased fracture risk, which is greater among people with T1D. Accumulation of cortical bone micro-pores, micro-vascular complications, and AGEs likely contribute to diabetes-related bone fragility. The effects of youth-onset T2D on peak bone mass attainment and subsequent skeletal fragility are of particular concern. Further research is needed to understand the effects of hyperglycemia on skeletal health through the lifecycle, including the related factors of inflammation and microvascular damage.

Impaired glucose metabolism underlies articular cartilage degeneration in osteoarthritis.

Osteoarthritis (OA) is the leading joint disease characterized by cartilage destruction and loss of mobility. Accumulating evidence indicates that the incidence and severity of OA increases with diabetes, implicating systemic glucose metabolism in joint health. However, a definitive link between cellular metabolism in articular cartilage and OA pathogenesis is not yet established. Here, we report that in mice surgically induced to develop knee OA through destabilization of medial meniscus (DMM), expression of the main glucose transporter Glut1 is notably reduced in joint cartilage. Inducible deletion of Glut1 specifically in the Prg4-expressing articular cartilage accelerates cartilage loss in DMM-induced OA. Conversely, forced expression of Glut1 protects against cartilage destruction following DMM. Moreover, in mice with type I diabetes, both Glut1 expression and the rate of glycolysis are diminished in the articular cartilage, and the diabetic mice exhibit more severe cartilage destruction than their nondiabetic counterparts following DMM. The results provide proof of concept that boosting glucose metabolism in articular chondrocytes may ameliorate cartilage degeneration in OA.

Rational Design of Bisphosphonate Lipid-like Materials for mRNA Delivery to the Bone Microenvironment.

The development of lipid nanoparticle (LNP) formulations for targeting the bone microenvironment holds significant potential for nucleic acid therapeutic applications including bone regeneration, cancer, and hematopoietic stem cell therapies. However, therapeutic delivery to bone remains a significant challenge due to several biological barriers, such as low blood flow in bone, blood-bone marrow barriers, and low affinity between drugs and bone minerals, which leads to unfavorable therapeutic dosages in the bone microenvironment. Here, we construct a series of bisphosphonate (BP) lipid-like materials possessing a high affinity for bone minerals, as a means to overcome biological barriers to deliver mRNA therapeutics efficiently to the bone microenvironment in vivo. Following in vitro screening of BP lipid-like materials formulated into LNPs, we identified a lead BP-LNP formulation, 490BP-C14, with enhanced mRNA expression and localization in the bone microenvironment of mice in vivo compared to 490-C14 LNPs in the absence of BPs. Moreover, BP-LNPs enhanced mRNA delivery and secretion of therapeutic bone morphogenetic protein-2 from the bone microenvironment upon intravenous administration. These results demonstrate the potential of BP-LNPs for delivery to the bone microenvironment, which could potentially be utilized for a range of mRNA therapeutic applications including regenerative medicine, protein replacement, and gene editing therapies.

Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping.

Mitochondria host the machinery for the tricarboxylic acid (TCA) cycle and electron transport chain (ETC), which generate adenosine triphosphate (ATP) to maintain energy homeostasis. Glucose, fatty acids, and amino acids are the major energy substrates fueling mitochondrial respiration in most somatic cells. Evidence shows that different cell types may have a distinct preference for certain substrates. However, substrate utilization by various cells in the skeleton has not been studied in detail. Moreover, as cellular metabolism is attuned to physiological and pathophysiological changes, direct assessments of substrate dependence in skeletal cells may provide important insights into the pathogenesis of bone diseases. The following protocol is based on the principle of carbon dioxide release from substrate molecules following oxidative phosphorylation. By using substrates containing radioactively labeled carbon atoms (14C), the method provides a sensitive and easy-to-use assay for the rate of substrate oxidation in cell culture. A case study with primary calvarial preosteoblasts versus bone marrow-derived macrophages (BMMs) demonstrates different utilization of the main substrates between the two cell types.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

Teriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

WNT7B overexpression rescues bone loss caused by glucocorticoids in mice.

Glucocorticoids, widely prescribed for anti-inflammatory and immunosuppressive purposes, are the most common secondary cause for osteoporosis and related fractures. Current anti-resorptive and anabolic therapies are insufficient for treating glucocorticoid-induced osteoporosis due to contraindications or concerns of side effects. Glucocorticoids have been shown to disrupt Wnt signaling in osteoblast-lineage cells, but the efficacy for Wnt proteins to restore bone mass after glucocorticoid therapy has not been examined. Here by using two mouse genetic models wherein WNT7B expression is temporally activated by either tamoxifen or doxycycline in osteoblast-lineage cells, we show that WNT7B recovers bone mass following glucocorticoid-induced bone loss, thanks to increased osteoblast number and function. However, WNT7B overexpression in bone either before or after glucocorticoid treatments does not ameliorate the abnormal accumulation of body fat. The study demonstrates a potent bone anabolic function for WNT7B in countering glucocorticoid-induced bone loss.

The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair.

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1-lineage cells. Normally, meniscal tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh agonist right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.

Functional interaction between Wnt and Bmp signaling in periosteal bone growth.

Wnt and Bmp proteins are well known to regulate bone development and homeostasis. Although both signals are extensively studied, their potential interaction in vivo is less well understood. Previous studies have shown that deletion of Bmpr1a, a type I receptor for Bmp signaling, results in excessive trabecular bone formation while diminishing periosteal bone growth. Moreover, forced-expression of the Wnt antagonist Sost suppresses the overgrowth of trabecular bone caused by Bmpr1a deletion, thus implicating hyperactive Wnt signaling in the excessive trabecular bone formation. However, it remains uncertain whether Wnt and Bmp signaling interacts in regulating the periosteal bone growth. Here we show that multiple Wnt genes are markedly suppressed in the cortical bone without Bmpr1a. Importantly, overexpression of Wnt7b fully rescues periosteal bone growth in the Bmpr1a-deficient mice. Thus, pharmacological activation of Wnt signaling can restore normal bone size without intact Bmp signaling.

Biphasic regulation of glutamine consumption by WNT during osteoblast differentiation.

Osteoblasts are the principal bone-forming cells. As such, osteoblasts have enhanced demand for amino acids to sustain high rates of matrix synthesis associated with bone formation. The precise systems utilized by osteoblasts to meet these synthetic demands are not well understood. WNT signaling is known to rapidly stimulate glutamine uptake during osteoblast differentiation. Using a cell biology approach, we identified two amino acid transporters, γ(+)-LAT1 and ASCT2 (encoded by Slc7a7 and Slc1a5, respectively), as the primary transporters of glutamine in response to WNT. ASCT2 mediates the majority of glutamine uptake, whereas γ(+)-LAT1 mediates the rapid increase in glutamine uptake in response to WNT. Mechanistically, WNT signals through the canonical ß-catenin (CTNNB1)-dependent pathway to rapidly induce Slc7a7 expression. Conversely, Slc1a5 expression is regulated by the transcription factor ATF4 downstream of the mTORC1 pathway. Targeting either Slc1a5 or Slc7a7 using shRNA reduced WNT-induced glutamine uptake and prevented osteoblast differentiation. Collectively, these data highlight the critical nature of glutamine transport for WNT-induced osteoblast differentiation.This article has an associated First Person interview with the joint first authors of the paper.

Malic Enzyme Couples Mitochondria with Aerobic Glycolysis in Osteoblasts.

The metabolic program of osteoblasts, the chief bone-making cells, remains incompletely understood. Here in murine calvarial cells, we establish that osteoblast differentiation under aerobic conditions is coupled with a marked increase in glucose consumption and lactate production but reduced oxygen consumption. As a result, aerobic glycolysis accounts for approximately 80% of the ATP production in mature osteoblasts. In vivo tracing with 13C-labeled glucose in the mouse shows that glucose in bone is readily metabolized to lactate but not organic acids in the TCA cycle. Glucose tracing in osteoblast cultures reveals that pyruvate is carboxylated to form malate integral to the malate-aspartate shuttle. RNA sequencing (RNA-seq) identifies Me2, encoding the mitochondrial NAD-dependent isoform of malic enzyme, as being specifically upregulated during osteoblast differentiation. Knockdown of Me2 markedly reduces the glycolytic flux and impairs osteoblast proliferation and differentiation. Thus, the mitochondrial malic enzyme functionally couples the mitochondria with aerobic glycolysis in osteoblasts.

Both aerobic glycolysis and mitochondrial respiration are required for osteoclast differentiation.

Excessive bone resorption over bone formation is the root cause for bone loss leading to osteoporotic fractures. Development of new antiresorptive therapies calls for a holistic understanding of osteoclast differentiation and function. Although much has been learned about the molecular regulation of osteoclast biology, little is known about the metabolic requirement and bioenergetics during osteoclastogenesis. Here, we report that glucose metabolism through oxidative phosphorylation (OXPHOS) is the predominant bioenergetic pathway to support osteoclast differentiation. Meanwhile, increased lactate production from glucose, known as aerobic glycolysis when oxygen is abundant, is also critical for osteoclastogenesis. Genetic deletion of Glut1 in osteoclast progenitors reduces aerobic glycolysis without compromising OXPHOS, but nonetheless diminishes osteoclast differentiation in vitro. Glut1 deficiency in the progenitors leads to osteopetrosis due to fewer osteoclasts specifically in the female mice. Thus, Glut1-mediated glucose metabolism through both lactate production and OXPHOS is necessary for normal osteoclastogenesis.

The Amino Acid Sensor Eif2ak4/GCN2 Is Required for Proliferation of Osteoblast Progenitors in Mice.

Skeletal stem/progenitor cells (SSPC) are critical regulators of bone homeostasis by providing a continuous supply of osteoblasts throughout life. In response to inductive signals, SSPC proliferate before osteoblast differentiation. Proliferation requires the duplication of all cellular components before cell division. This imposes a unique biosynthetic requirement for amino acids that can be used for biomass production. Thus, the ability to sense and respond to amino acid availability is likely a major determinant for proliferation. Using a cellular and genetic approach, we demonstrate the amino acid sensor GCN2 is required to support the robust proliferative capacity of SSPC during bone homeostasis. GCN2 ablation results in decreased postnatal bone mass due primarily to reduced osteoblast numbers. Decreased osteoblast numbers is likely attributed to reduced SSPC proliferation as loss of GCN2 specifically affected proliferation in cultured bone marrow stromal cells (BMSCs) without impacting osteoblast differentiation in vitro. Mechanistically, GCN2 regulates proliferation by increasing amino acid uptake downstream of the transcriptional effector ATF4. Collectively, these data suggest amino acid sensing through the GCN2/ATF4 pathway is indispensable for robust SSPC proliferation necessary for bone homeostasis. © 2020 American Society for Bone and Mineral Research.

Single cell transcriptomics identifies a unique adipose lineage cell population that regulates bone marrow environment.

Bone marrow mesenchymal lineage cells are a heterogeneous cell population involved in bone homeostasis and diseases such as osteoporosis. While it is long postulated that they originate from mesenchymal stem cells, the true identity of progenitors and their in vivo bifurcated differentiation routes into osteoblasts and adipocytes remain poorly understood. Here, by employing large scale single cell transcriptome analysis, we computationally defined mesenchymal progenitors at different stages and delineated their bi-lineage differentiation paths in young, adult and aging mice. One identified subpopulation is a unique cell type that expresses adipocyte markers but contains no lipid droplets. As non-proliferative precursors for adipocytes, they exist abundantly as pericytes and stromal cells that form a ubiquitous 3D network inside the marrow cavity. Functionally they play critical roles in maintaining marrow vasculature and suppressing bone formation. Therefore, we name them marrow adipogenic lineage precursors (MALPs) and conclude that they are a newly identified component of marrow adipose tissue.

Diet-Induced Metabolic Dysregulation in Female Mice Causes Osteopenia in Adult Offspring.

Bone mass and quality in humans are controlled by numerous genetic and environmental factors that are not fully understood. Increasing evidence has indicated that maternal metabolic dysregulation impairs multiple physiological processes in the adult offspring, but a similar effect on bone health is yet to be established. Here, we have analyzed the bones of first-generation offspring from murine dams that present metabolic syndrome due to a high-fat and high-sugar (HF/HS) diet. Micro-CT analyses show that the long bones of HF/HS offspring possess lower cortical bone mass and weaker mechanical strength than normal, even though the trabecular bone is not affected. Histomorphometry and serum biochemistry indicate that both bone formation and resorption are diminished in the HF/HS offspring. In vitro, both osteoblast and osteoclast progenitors from the HF/HS offspring are deficient in differentiation, likely due to impairment of mitochondrial respiration. The study, therefore, identifies maternal metabolic health as an important environmental factor influencing bone volume and strength.