Altered Osteoblast Metabolism with Aging Results in Lipid Accumulation and Oxidative Stress Mediated Bone Loss.

Cellular aging is associated with dysfunction of numerous tissues affecting multiple organ systems. A striking example of this is related to age-related bone loss, or osteoporosis, increasing fracture incidence. Interestingly, the two compartments of bone, cortical and cancellous or trabecular, rely on different mechanisms for development and maintenance during 'normal' aging. At a cellular level, the aging process disturbs a multitude of intracellular pathways. In particular, alterations in cellular metabolic functions thereby impacting cellular bioenergetics have been implicated in multiple tissues. Therefore, this study aimed to characterize how metabolic processes were altered in bone forming osteoblasts in aged mice compared to young mice. Metabolic flux analyses demonstrated both stromal cells and mature, matrix secreting osteoblasts from aged mice exhibited mitochondrial dysfunction. This was also accompanied by a lack of adaptability or metabolic flexibility to utilize exogenous substrates compared to osteoblasts cultured from young mice. Additionally, lipid droplets accumulated in both early stromal cells and mature osteoblasts from aged mice, which was further depicted as increased lipid content within the bone cortex of aged mice. Global transcriptomic analysis of the bone further supported these metabolic data as enhanced oxidative stress genes were up-regulated in aged mice, while osteoblast-related genes were down-regulated when compared to the young mice. Collectively, these data suggest that aging results in altered osteoblast metabolic handling of both exogenous and endogenous substrates which could contribute to age-related osteoporosis.

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.

Influence of Cholesterol on the Regulation of Osteoblast Function.

Bone is a dynamic tissue composed of cells, an extracellular matrix, and mineralized portion. Osteoblasts are responsible for proper bone formation and remodeling, and function. These processes are endergonic and require cellular energy in the form of adenosine triphosphate (ATP), which is derived from various sources such as glucose, fatty acids, and amino acids. However, other lipids such as cholesterol have also been found to play a critical role in bone homeostasis and can also contribute to the overall bioenergetic capacity of osteoblasts. In addition, several epidemiological studies have found a link between elevated cholesterol, cardiovascular disease, an enhanced risk of osteoporosis, and increased bone metastasis in cancer patients. This review focuses on how cholesterol, its derivatives, and cholesterol-lowering medications (statins) regulate osteoblast function and bone formation. It also highlights the molecular mechanisms underlying the cholesterol-osteoblast crosstalk.

Mitochondrial ß-oxidation of adipose-derived fatty acids by osteoblasts fuels parathyroid hormone-induced bone formation.

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 ß-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 ß-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.

Endocrinal metabolic regulation on the skeletal system in post-menopausal women.

Osteoporosis is a common endocrinologic disorder characterized as a chronic bone loss condition. Sexual dimorphism is ubiquitous in the incidence of osteoporosis with post-menopausal women being acutely affected. Gonadal sex hormones including estrogen act as crucial regulators of bone mass; therefore, loss of such hormones leads to an imbalance in skeletal turnover leading to osteoporosis. Estrogen can influence both bone formation as well as resorption by reducing osteoblast activity and enhancing osteoclastogenesis. Additionally, estrogen is a potent regulator of systemic metabolism. Recent studies have provided clues that estrogenic effect on bone might also involve alterations in bone cell metabolism and bioenergetic potential. While direct effects of gonadal hormones ability to alter intracellular metabolism of bone cells has not been studied, there is precedence within the literature that this is occurring and contributing to post-menopausal bone loss. This review aims to serve as a perspective piece detailing the prospective role of gonadal hormones regulating bone cell metabolic potential.

Common Dietary Modifications in Preclinical Models to Study Skeletal Health.

Bone is a highly dynamic tissue that undergoes continuous remodeling by bone resorbing osteoclasts and bone forming osteoblasts, a process regulated in large part by osteocytes. Dysregulation of these coupled catabolic and anabolic processes as in the case of menopause, type 2 diabetes mellitus, anorexia nervosa, and chronic kidney disease is known to increase fracture risk. Recent advances in the field of bone cell metabolism and bioenergetics have revealed that maintenance of the skeleton places a high energy demand on these cells involved in bone remodeling. These new insights highlight the reason that bone tissue is the beneficiary of a substantial proportion of cardiac output and post-prandial chylomicron remnants and requires a rich supply of nutrients. Studies designed for the specific purpose of investigating the impact of dietary modifications on bone homeostasis or that alter diet composition and food intake to produce the model can be found throughout the literature; however, confounding dietary factors are often overlooked in some of the preclinical models. This review will examine some of the common pre-clinical models used to study skeletal biology and its pathologies and the subsequent impact of various dietary factors on these model systems. Furthermore, the review will include how inadvertent effects of some of these dietary components can influence bone cell function and study outcomes.

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Parathyroid hormone (PTH) regulation of metabolic homeostasis: An old dog teaches us new tricks.

BACKGROUND: Late in the nineteenth century, it was theorized that a circulating product produced by the parathyroid glands could negatively impact skeletal homeostasis. A century later, intermittent administration of that protein, namely parathyroid hormone (PTH), was approved by the FDA and EMA as the first anabolic agent to treat osteoporosis. Yet, several unanswered but important questions remain about the skeletal actions of PTH. SCOPE OF REVIEW: Current research efforts have focused on improving the efficacy of PTH treatment by designing structural analogs and identifying other targets (e.g., the PTH or the calcium sensing receptor). A unique but only recently described aspect of PTH action is its regulation of cellular bioenergetics and metabolism, namely in bone and adipose tissue but also in other tissues. The current review aims to provide a brief background on PTH's previously described actions on bone and highlights how PTH regulates osteoblast bioenergetics, contributing to greater bone formation. It will also shed light on how PTH could alter metabolic homeostasis through its actions in other cells and tissues, thereby impacting the skeleton in a cell non-autonomous manner. MAJOR CONCLUSIONS: PTH administration enhances bone formation by targeting the osteoblast through transcriptional changes in several pathways; the most prominent is via adenyl cyclase and PKA. PTH and its related protein, PTHrP, also induce glycolysis and fatty acid oxidation in bone cells and drive lipolysis and thermogenic programming in adipocytes; the latter may indirectly but positively influence skeletal metabolism. While much work remains, alterations in cellular metabolism may also provide a novel mechanism related to PTH's temporal actions. Thus, the bioenergetic impact of PTH can be considered another of the myriad anabolic effects of PTH on the skeleton. Just as importantly from a translational perspective, the non-skeletal metabolic effects may lead to a better understanding of whole-body homeostasis along with new and improved therapies to treat musculoskeletal conditions.

Untargeted metabolomics in primary murine bone marrow stromal cells reveals distinct profile throughout osteoblast differentiation.

INTRODUCTION: Skeletal homeostasis is an exquisitely regulated process most directly influenced by bone resorbing osteoclasts, bone forming osteoblasts, and the mechano-sensing osteocytes. These cells work together to constantly remodel bone as a mechanism to prevent from skeletal fragility. As such, when an individual experiences a disconnect in these tightly coupled processes, fracture incidence increases, such as during ageing, gonadal hormone deficiency, weightlessness, and diabetes. While therapeutic options have significantly aided in the treatment of low bone mineral density (BMD) or osteoporosis, limited options remain for anabolic or bone forming agents. Therefore, it is of interest to continue to understand how osteoblasts regulate their metabolism to support the energy expensive process of bone formation. OBJECTIVE: The current project sought to rigorously characterize the distinct metabolic processes and intracellular metabolite profiles in stromal cells throughout osteoblast differentiation using untargeted metabolomics. METHODS: Primary, murine bone marrow stromal cells (BMSCs) were characterized throughout osteoblast differentiation using standard staining protocols, Seahorse XFe metabolic flux analyses, and untargeted metabolomics. RESULTS: We demonstrate here that the metabolic footprint of stromal cells undergoing osteoblast differentiation are distinct, and while oxidative phosphorylation drives adenosine triphosphate (ATP) generation early in the differentiation process, mature osteoblasts depend on glycolysis. Importantly, the intracellular metabolite profile supports these findings while also suggesting additional pathways critical for proper osteoblast function. CONCLUSION: These data are the first of their kind to characterize these metabolites in conjunction with the bioenergetic profile in primary, murine stromal cells throughout osteoblast differentiation and provide provocative targets for future investigation.

Bone marrow adipocytes - Good, bad, or just different?

Skeletal remodeling is essential for proper maintenance of adult bone mass, and due to its heavy energetic demands this process is closely tied to whole body metabolic. Thus, bone formation by the osteoblast, bone resorption by the osteoclast, and mechano-sensing by the osteocyte, are highly coupled processes that are essential for bone turnover. When one experiences a disruption in these processes, over time increased skeletal fragility and fracture can result. In addition to these primary cells, secondary cells within the skeletal niche are suspected to directly coordinate bone health as well. The bone marrow compartment provides a unique microenvironment in which communication occurs between white blood cells, red blood cells, platelets, and immune cells, in addition to classic bone cells (osteoblasts, osteoclasts, and osteocytes) that can both directly and indirectly impact skeletal homeostasis. One such cell population that has attracted much attention and scientific inquiry in the past decade are bone marrow adipocytes (BMAdipo) which can be found interspersed throughout the marrow compartment, and collectively are often referred to as bone marrow adipose tissue (BMAT). Although our understanding of BMAT has advanced significantly in the past decade, many questions remain relative to their lineage and function. The current collection presents recent studies and the up-to-date understanding of bone marrow adipocytes, along with future clinical perspectives related to this unique depot.

Loss of function of lysosomal acid lipase (LAL) profoundly impacts osteoblastogenesis and increases fracture risk in humans.

Lysosomal acid lipase (LAL) is essential for cholesteryl ester (CE) and triacylglycerol (TAG) hydrolysis in the lysosome. Clinically, an autosomal recessive LIPA mutation causes LAL deficiency (LALD), previously described as Wolman Disease or Cholesteryl Ester Storage Disease (CESD). LAL-D is associated with ectopic lipid accumulation in the liver, small intestine, spleen, adrenal glands, and blood. Considering the importance of unesterified cholesterol and fatty acids in bone metabolism, we hypothesized that LAL is essential for bone formation, and ultimately, skeletal health. To investigate the role of LAL in skeletal homeostasis, we used LAL-deficient (-/-) mice, in vitro osteoblast cultures, and novel clinical data from LAL-D patients. Both male and female LAL-/- mice demonstarted lower trabecular and cortical bone parameters , which translated to reduced biomechanical properties. Further histological analyses revealed that LAL-/- mice had fewer osteoblasts, with no change in osteoclast or marrow adipocyte numbers. In studying the cell-autonomous role of LAL, we observed impaired differentiation of LAL-/- calvarial osteoblasts and in bone marrow stromal cells treated with the LAL inhibitor lalistat. Consistent with LAL's role in other tissues, lalistat resulted in profound lipid puncta accumulation and an altered intracellular lipid profile. Finally, we analyzed a large de-identified national insurance database (i.e. 2016/2017 Optum Clinformatics®) which revealed that adults (≥18 years) with CESD (n = 3076) had a higher odds ratio (OR = 1.21; 95% CI = 1.03-1.41) of all-cause fracture at any location compared to adults without CESD (n = 13.7 M) after adjusting for demographic variables and osteoporosis. These data demonstrate that alterations in LAL have significant clinical implications related to fracture risk and that LAL's modulation of lipid metabolism is a critical for osteoblast function.

A regulatory variant at 3q21.1 confers an increased pleiotropic risk for hyperglycemia and altered bone mineral density.

Skeletal and glycemic traits have shared etiology, but the underlying genetic factors remain largely unknown. To identify genetic loci that may have pleiotropic effects, we studied Genome-wide association studies (GWASs) for bone mineral density and glycemic traits and identified a bivariate risk locus at 3q21. Using sequence and epigenetic modeling, we prioritized an adenylate cyclase 5 (ADCY5) intronic causal variant, rs56371916. This SNP changes the binding affinity of SREBP1 and leads to differential ADCY5 gene expression, altering the chromatin landscape from poised to repressed. These alterations result in bone- and type 2 diabetes-relevant cell-autonomous changes in lipid metabolism in osteoblasts and adipocytes. We validated our findings by directly manipulating the regulator SREBP1, the target gene ADCY5, and the variant rs56371916, which together imply a novel link between fatty acid oxidation and osteoblast differentiation. Our work, by systematic functional dissection of pleiotropic GWAS loci, represents a framework to uncover biological mechanisms affecting pleiotropic traits.

Lipids in the Bone Marrow: An Evolving Perspective.

Because of heavy energy demands to maintain bone homeostasis, the skeletal system is closely tied to whole-body metabolism via neuronal and hormonal mediators. Glucose, amino acids, and fatty acids are the chief fuel sources for bone resident cells during its remodeling. Lipids, which can be mobilized from intracellular depots in the bone marrow, can be a potent source of fatty acids. Thus, while it has been suggested that adipocytes in the bone marrow act as "filler" and are detrimental to skeletal homeostasis, we propose that marrow lipids are, in fact, essential for proper bone functioning. As such, we examine the prevailing evidence regarding the storage, use, and export of lipids within the skeletal niche, including from both in vitro and in vivo model systems. We also highlight the numerous challenges that remain to fully appreciate the relationship of lipid turnover to skeletal homeostasis.

Metabolic programming determines the lineage-differentiation fate of murine bone marrow stromal progenitor cells.

Enhanced bone marrow adipogenesis and impaired osteoblastogenesis have been observed in obesity, suggesting that the metabolic microenvironment regulates bone marrow adipocyte and osteoblast progenitor differentiation fate. To determine the molecular mechanisms, we studied two immortalized murine cell lines of adipocyte or osteoblast progenitors (BMSCsadipo and BMSCsosteo, respectively) under basal and adipogenic culture conditions. At baseline, BMSCsadipo, and BMSCsosteo exhibit a distinct metabolic program evidenced by the presence of specific global gene expression, cellular bioenergetics, and metabolomic signatures that are dependent on insulin signaling and glycolysis in BMSCsosteo versus oxidative phosphorylation in BMSCsadipo. To test the flexibility of the metabolic program, we treated BMSCsadipo with parathyroid hormone, S961 (an inhibitor of insulin signaling) and oligomycin (an inhibitor of oxidative phosphorylation). The treatment induced significant changes in cellular bioenergetics that were associated with decreased adipocytic differentiation. Similarly, 12 weeks of a high-fat diet in mice led to the expansion of adipocyte progenitors, enhanced adipocyte differentiation and insulin signaling in cultured BMSCs. Our data demonstrate that BMSC progenitors possess a distinct metabolic program and are poised to respond to exogenous metabolic cues that regulate their differentiation fate.

Bone Marrow Adiposity: Basic and Clinical Implications.

The presence of adipocytes in mammalian bone marrow (BM) has been recognized histologically for decades, yet, until recently, these cells have received little attention from the research community. Advancements in mouse transgenics and imaging methods, particularly in the last 10 years, have permitted more detailed examinations of marrow adipocytes than ever before and yielded data that show these cells are critical regulators of the BM microenvironment and whole-body metabolism. Indeed, marrow adipocytes are anatomically and functionally separate from brown, beige, and classic white adipocytes. Thus, areas of BM space populated by adipocytes can be considered distinct fat depots and are collectively referred to as marrow adipose tissue (MAT) in this review. In the proceeding text, we focus on the developmental origin and physiologic functions of MAT. We also discuss the signals that cause the accumulation and loss of marrow adipocytes and the ability of these cells to regulate other cell lineages in the BM. Last, we consider roles for MAT in human physiology and disease.

Montmorency tart cherry protects against age-related bone loss in female C57BL/6 mice and demonstrates some anabolic effects.

PURPOSE: Age-related bone loss is a consequence of endocrine and immune changes that disrupt bone remodeling. Functional foods (e.g., tart cherries) with antioxidant, anti-inflammatory and prebiotic activity can potentially counter this age-related phenomenon. The aim of this study was to determine if Montmorency tart cherry protects against early age-related bone loss and the culpable alterations in bone metabolism. METHODS: Female, 5-month-old, C57BL/6 mice were assigned to baseline or treatment groups: AIN-93M diet supplemented with 0, 1, 5, or 10% tart cherry for 90 days. Bone mineral density (BMD) and trabecular and cortical bone microarchitecture were assessed. Treatment effects on bone metabolism and regulators of bone formation, resorption and mineralization were determined. RESULTS: Mice consuming the 5% and 10% doses had higher vertebral and tibial BMD (p < 0.05) compared to controls. The age-related decrease in trabecular bone volume (BV/TV) of the distal femur was prevented with these doses. Vertebral trabecular BV/TV and cortical bone thickness of the femur mid-diaphysis were greater (p < 0.05) in the groups receiving the 5% and 10% cherry than the control diet. Notably, these improvements were significantly greater than the baseline controls, consistent with an anabolic response. Although no differences in systemic biomarkers of bone formation or resorption were detected at 90 days, local increases in Phex and decreases in Ppar-γ suggest a bone environment that supports increased mineralization. CONCLUSIONS: These findings demonstrate that cherry supplementation (5% and 10%) improves BMD and some indices of trabecular and cortical bone microarchitecture; these effects are likely attributed to increased bone mineralization.

Progenitor recruitment and adipogenic lipolysis contribute to the anabolic actions of parathyroid hormone on the skeleton.

Intermittent administration of parathyroid hormone (PTH) stimulates bone formation in vivo and also suppresses the volume of bone marrow adipose tissue (BMAT). In contrast, a calorie-restricted (CR) diet causes bone loss and induces BMAT in both mice and humans. We used the CR model to test whether PTH would reduce BMAT in mice by both altering cell fate and inducing lipolysis of marrow adipocytes. Eight-week-old mice were placed on a control (Ctrl) diet or CR diet. At 12 wk, CR and Ctrl mice were injected daily with PTH (CR/PTH or Ctrl/PTH) or vehicle for 4 wk. Two other cohorts were CR and simultaneously injected (CR + PTH or CR + Veh) for 4 wk. CR mice had low bone mass and increased BMAT in the proximal tibias. PTH significantly increased bone mass in all cohorts despite calorie restrictions. Adipocyte density and size were markedly increased with restriction of calories. PTH reduced adipocyte numbers in CR + PTH mice, whereas adipocyte size was reduced in CR/PTH-treated mice. In contrast, osteoblast number was increased 3-8-fold with PTH treatment. In vitro, bone marrow stromal cells differentiated into adipocytes and, treated with PTH, exhibited increased production of glycerol and fatty acids. Moreover, in cocultures of bone marrow adipocyte and osteoblast progenitors, PTH stimulated the transfer of fatty acids to osteoblasts. In summary, PTH administration to CR mice increased bone mass by shifting lineage allocation toward osteogenesis and inducing lipolysis of mature marrow adipocytes. The effects of PTH on bone marrow adiposity could enhance its anabolic actions by providing both more cells and more fuel for osteoblasts during bone formation.-Maridas, D. E., Rendina-Ruedy, E., Helderman, R. C., DeMambro, V. E., Brooks, D., Guntur, A. R., Lanske, B., Bouxsein, M. L., Rosen, C. J. Progenitor recruitment and adipogenic lipolysis contribute to the anabolic actions of parathyroid hormone on the skeleton.

Dried Plum Polyphenolic Extract Combined with Vitamin K and Potassium Restores Trabecular and Cortical Bone in Osteopenic Model of Postmenopausal Bone Loss.

Dried plum has unique anabolic effects on bone, but the responsible bioactive components have remained unclear. This study investigated components of dried plum with potential osteoprotective activity utilizing aged, osteopenic Sprague Dawley rats fed diets supplemented with a crude polyphenol extract, potassium, vitamin K or their combination. Whole body and femoral bone mineral density were restored with the polyphenol and combination treatments to a similar extent as the dried fruit. The combination treatment reversed trabecular bone loss in the spine and cortical bone in the femur mid-diaphysis in a similar manner. Biomarkers of bone resorption were reduced by the polyphenol and combination treatments. The polyphenol extract accounted for most of the anabolic effect of dried plum on bone. This study is the first to show the bioactive components in dried plum responsible for restoring bone in vivo.