Gut Microbiota and Serum Metabolic Signatures of High-Fat-Induced Bone Loss in Mice

BackgroundAccumulating evidence indicates that high-fat diet (HFD) is a controllable risk factor for osteoporosis, but the underlying mechanism remains to be elucidated. As a primary biological barrier for nutrient entry into the human body, the composition and function of gut microbiota (GM) can be altered rapidly by HFD, which may trigger abnormal bone metabolism. In the current study, we analyzed the signatures of GM and serum metabolomics in HFD-induced bone loss and explored the potential correlations of GM and serum metabolites on HFD-related bone loss.MethodsWe conducted a mouse model with HFD-induced bone loss through a 12-week diet intervention. Micro-CT, Osmium-μCT, and histological analyses were used to observe bone microstructure and bone marrow adipose tissue. Quantitative Real-Time PCR was applied to analyze gene expression related to osteogenesis, adipogenesis, and osteoclastogenesis. Enzyme-linked immunosorbent assay was used to measure the biochemical markers of bone turnover. 16s rDNA sequencing was employed to analyze the abundance of GM, and UHPLC-MS/MS was used to identify serum metabolites. Correlation analysis was performed to explore the relationships among bone phenotypes, GM, and the metabolome.ResultsHFD induced bone loss accompanied by bone marrow adipose tissue expansion and bone formation inhibition. In the HFD group, the relative abundance of Firmicutes was increased significantly, while Bacteroidetes, Actinobacteria, Epsilonbacteraeota, and Patescibacteria were decreased compared with the ND group. Association analysis showed that thirty-two bacterial genera were significantly related to bone volume per tissue volume (BV/TV). One hundred and forty-five serum metabolites were identified as differential metabolites associated with HFD intervention, which were significantly enriched in five pathways, such as purine metabolism, regulation of lipolysis in adipocyte and cGMP-PKG signaling pathway. Sixty-four diffiential metabolites were matched to the MS2 spectra; and ten of them were positively correlated with BV/TV and five were negatively correlated with BV/TV.ConclusionsThese findings indicated that the alternations of GM and serum metabolites were related to HFD-induced bone loss, which might provide new insights into explain the occurrence and development of HFD-related osteoporosis. The regulatory effects of GM and metabolites associated with HFD on bone homeostasis required further exploration.

Osteal Macrophages and Megakaryocytes Increase Adipogenesis

Bone marrow adipose tissue (MAT) increases with aging and contributes to low bone density and skeletal fractures. However, the cells and factors within the bone marrow (BM) that regulate adipogenesis remain poorly understood. In the current study, we examined the role of osteal macrophages (OMs) and megakaryocytes (MKs) on the regulation of adipogenesis. We cultured murine osteoblasts/osteoblast progenitors (OBs from hereon) derived from neonatal calvarial cells (CCs, a combination of OBs and OMs) or OBs isolated by fluorescence activated cell sorting (FACS) in the presence or absence of fetal liver derived murine MK. The cells underwent induced adipogenesis for 5-7 days by supplementation of media with insulin, indomethacin, and dexamethasone, and then the number of adipocytes was quantified. We found that co-culturing MKs and OMs with OBs results in up to a 7.8-fold and 11.7-fold increase in adipocytes, respectively. We also elucidated that thrombopoietin (TPO), the major growth factor for MKs, inhibits adipogenesis in both OBs and CCs by approximately 60%. Similarly, we found that CCs and OBs derived from mice deficient in the TPO receptor, Mpl, had approximately 30% more adipocytes than their wild-type (WT) counterparts. Finally, in vitro findings were corroborated in vivo through quantification of MKs and adipocytes in mice in which MK number was elevated or reduced. Mice with significantly higher numbers of BM-residing MKs also had significantly higher numbers of BM-residing adipocytes. Because there is typically an inverse relationship between adipogenesis and osteogenesis, understanding ways to inhibit adipogenesis could lead to an increase in OB number and bone formation, which in turn could lead to new treatments for bone loss diseases such as osteoporosis.

Genetically Determined Obesity and Adipose Distribution Impact Human Blood Trait Variation across Cell Lineages

Introduction Genome wide association studies (GWAS) have catalogued thousands of loci that influence blood traits, but genetic mechanisms and impacted cell types are often unknown. While some regulatory factors are blood cell-autonomous, other extrinsic regulatory factors respond to systemic physiology. Epidemiologic studies have correlated generalized obesity (increased body mass index, BMI) and high cholesterol with anemia, but cell types and mechanisms that mediate these effects are incompletely understood, and these studies do not provide evidence for causality. Thus, we wanted to use genetic effects of BMI and cholesterol on hemoglobin level (HGB) and other blood traits to infer causal relationships, identify relevant cell types, and propose genetic mechanisms. Methods Mendelian randomization (MR), akin to a "genetic randomized controlled trial", uses variants linked to an exposure trait to estimate causal effects of that exposure on a given outcome. 1 Random variant allele allocation at meiosis enables this approach to address confounding and reverse causality that can otherwise preclude causal inference from epidemiologic and cohort studies. Further, effects of multiple factors can be parsed using multivariable MR and causal mediation analyses to help explain genetic associations. 2 Our study used gender- and age-adjusted GWAS summary statistics from European individuals. Results We hypothesized that increased genetically determined BMI would decrease HGB. Using a MR framework, we found that a 5 kg/m 2 (1 standard deviation unit) increase in BMI decreased hemoglobin by 0.06±0.01 g/dL (p=1x10 -5, Fig). Increased BMI also decreased erythrocyte count, and unexpectedly also decreased platelet and white blood cell counts (all with p<5x10 -3). These data showed that obesity-related mechanisms extended beyond the erythroid lineage, perhaps impacting multipotent hematopoietic progenitor cells (HPCs). Similar to BMI, a 1 SD unit increase in total cholesterol decreased HGB (0.10±0.03 g/dL, p=2x10 -3, Fig). However, genetic effects of cholesterol were restricted to erythroid traits (HGB and hematocrit). Multivariable and mediation analyses confirmed that effects of BMI and cholesterol functioned through distinct genetic mechanisms. We speculated that multilineage effects from BMI could reflect a genetic predisposition to accumulate bone marrow adipose tissue, which can impact HPCs and hematopoiesis. 3 A tendency for 'central' adiposity increases one's waist-hip ratio (WHR), and increases cardiovascular disease risk concordant with BMI. Unexpectedly, we found that increased WHR, in contrast to BMI, increased HGB (0.08±0.02 g/dL, p=9x10 -6) as well as erythrocyte, platelet, and white blood cell counts (all with p<4x10 -3, Fig). In multivariable experiments, the effects of WHR were exacerbated after accounting for BMI at the individual or population level. Thus, obesity impacts blood traits through genetically determined adipose distribution. Conclusions Our results confirm that BMI and cholesterol negatively impact HGB at a genetic level, consistent with clinical observations. The unexpected multilineage effects of genetically determined BMI most likely reflects a tendency to accumulate bone marrow adipose tissue, which in turn impacts HPCs and downstream blood cell production. Our findings suggest that adjustment for BMI and adiposity traits may be considered in blood trait GWAS analyses and illuminate opportunities to functionally dissect related genes and molecular pathways. References 1. Hemani, G. et al. Elife 7, (2018). PMID: 29846171. 2. Sanderson, E. et al. Int. J. Epidemiol. 48, 713-727 (2019). PMID: 30535378. 3. Wang, H. et al. Front. Endocrinol.. 9, 694 (2018). PMID: 30546345. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

I Can Get Fat Where? The Other Types of Fat

This chapter considers the more obscure non-white types of adipose tissue present in the human body. The first and better-known type discussed is brown fat, which contributes to the regulation of body temperature as it burns (excess) calories to generate heat. The second section explores the biology of bone marrow fat, whose enigmatic behaviour in the context of starvation and obesity does little to help define its role. After considering the relationship between bone marrow adipose tissue, bone strength, and overall metabolic health, this chapter concludes by briefly reviewing other, lesser-known types of (white) fat (e.g., epicardial fat) and their potential contribution to human biology.

Guidelines for Biobanking of Bone Marrow Adipose Tissue and Related Cell Types: Report of the Biobanking Working Group of the International Bone Marrow Adiposity Society

Over the last two decades, increased interest of scientists to study bone marrow adiposity (BMA) in relation to bone and adipose tissue physiology has expanded the number of publications using different sources of bone marrow adipose tissue (BMAT). However, each source of BMAT has its limitations in the number of downstream analyses for which it can be used. Based on this increased scientific demand, the International Bone Marrow Adiposity Society (BMAS) established a Biobanking Working Group to identify the challenges of biobanking for human BMA-related samples and to develop guidelines to advance establishment of biobanks for BMA research. BMA is a young, growing field with increased interest among many diverse scientific communities. These bring new perspectives and important biological questions on how to improve and build an international community with biobank databases that can be used and shared all over the world. However, to create internationally accessible biobanks, several practical and legislative issues must be addressed to create a general ethical protocol used in all institutes, to allow for exchange of biological material internationally. In this position paper, the BMAS Biobanking Working Group describes similarities and differences of patient information (PIF) and consent forms from different institutes and addresses a possibility to create uniform documents for BMA biobanking purposes. Further, based on discussion among Working Group members, we report an overview of the current isolation protocols for human bone marrow adipocytes (BMAds) and bone marrow stromal cells (BMSCs, formerly mesenchymal), highlighting the specific points crucial for effective isolation. Although we remain far from a unified BMAd isolation protocol and PIF, we have summarized all of these important aspects, which are needed to build a BMA biobank. In conclusion, we believe that harmonizing isolation protocols and PIF globally will help to build international collaborations and improve the quality and interpretation of BMA research outcomes.

Characterization of Osteogenesis and Chondrogenesis of Human Decellularized Allogeneic Bone with Mesenchymal Stem Cells Derived from Bone Marrow, Adipose Tissue, and Wharton’s Jelly

Allogeneic bone grafts are a promising material for bone implantation due to reduced operative trauma, reduced blood loss, and no donor-site morbidity. Although human decellularized allogeneic bone (hDCB) can be used to fill bone defects, the research of revitalizing hDCB blocks with human mesenchymal stem cells (hMSCs) for osteochondral regeneration is missing. The hMSCs derived from bone marrow, adipose tissue, and Wharton’s jelly (BMMSCs, ADMSCs, and UMSCs, respectively) are potential candidates for bone regeneration. This study characterized the potential of hDCB as a scaffold for osteogenesis and chondrogenesis of BMMSCs, ADMSCs, and UMSCs. The pore sizes and mechanical strength of hDCB were characterized. Cell survival and adhesion of hMSCs were investigated using MTT assay and F-actin staining. Alizarin Red S and Safranin O staining were conducted to demonstrate calcium deposition and proteoglycan production of hMSCs after osteogenic and chondrogenic differentiation, respectively. A RT-qPCR was performed to analyze the expression levels of osteogenic and chondrogenic markers in hMSCs. Results indicated that BMMSCs and ADMSCs exhibited higher osteogenic potential than UMSCs. Furthermore, ADMSCs and UMSCs had higher chondrogenic potential than BMMSCs. This study demonstrated that chondrogenic ADMSCs- or UMSCs-seeded hDCB might be potential osteochondral constructs for osteochondral regeneration.