Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is ...

University of Texas Southwestern Medical Center

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   and , 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 β-catenin (CTNNB1)-dependent pathway to rapidly induce  expression. Conversely,  expression is regulated by the transcription factor ATF4 downstream of the mTORC1 pathway. Targeting either  or  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.

Biphasic regulation of glutamine consumption by WNT during osteoblast differentiation.

Osteoarthritis is a debilitating disease characterized by cartilage degradation and altered cartilage mechanical properties. Furthermore, it is well established that obesity is a primary risk factor for osteoarthritis. The purpose of this study was to investigate the influence of obesity on the mechanical properties of murine knee cartilage. Two-month old wild type mice were fed either a normal diet or a high fat diet for 16 weeks. Atomic force microscopy-based nanoindentation was used to quantify the effective indentation modulus of medial femoral condyle cartilage. Osteoarthritis progression was graded using the OARSI system. Additionally, collagen organization was evaluated with picrosirius red staining imaged using polarized light microscopy. Significant differences between diet groups were assessed using t tests with p < 0.05. Following 16 weeks of a high fat diet, no significant differences in OARSI scoring were detected. However, we detected a significant difference in the effective indentation modulus between diet groups. The reduction in cartilage stiffness is likely the result of disrupted collagen organization in the superficial zone, as indicated by altered birefringence on polarized light microscopy. Collectively, these results suggest obesity is associated with changes in knee cartilage mechanical properties, which may be an early indicator of disease progression.

Obesity alters the collagen organization and mechanical properties of murine cartilage.

Osteoblast differentiation is sequentially characterized by high rates of proliferation followed by increased protein and matrix synthesis, processes that require substantial amino acid acquisition and production. How osteoblasts obtain or maintain intracellular amino acid production is poorly understood. Here, we identify SLC1A5 as a critical amino acid transporter during bone development. Using a genetic and metabolomic approach, we show SLC1A5 acts cell autonomously to regulate protein synthesis and osteoblast differentiation. SLC1A5 provides both glutamine and asparagine which are essential for osteoblast differentiation. Mechanistically, glutamine and to a lesser extent asparagine support amino acid biosynthesis. Thus, osteoblasts depend on  to provide glutamine and asparagine, which are subsequently used to produce non-essential amino acids and support osteoblast differentiation and bone development.

SLC1A5 provides glutamine and asparagine necessary for bone development in mice.

Osteoblasts are responsible for bone matrix production during bone development and homeostasis. Much is known about the transcriptional regulation and signaling pathways governing osteoblast differentiation. However, less is known about how osteoblasts obtain or utilize nutrients to fulfill the energetic demands associated with osteoblast differentiation and bone matrix synthesis. The goal of this review is to highlight and discuss what is known about the role and regulation of bioenergetic metabolism in osteoblasts with a focus on more recent studies.

Bioenergetic Metabolism In Osteoblast Differentiation.

Enchondromas and chondrosarcomas are common cartilage neoplasms that are either benign or malignant, respectively. The majority of these tumors harbor mutations in either IDH1 or IDH2. Glutamine metabolism has been implicated as a critical regulator of tumors with IDH mutations. Using genetic and pharmacological approaches, we demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1 or IDH2 mutations. Glutamine affected cell differentiation and viability in these tumors differently through different downstream metabolites. During murine enchondroma-like lesion development, glutamine-derived α-ketoglutarate promoted hypertrophic chondrocyte differentiation and regulated chondrocyte proliferation. Deletion of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. In contrast, pharmacological inhibition of glutaminase in chondrosarcoma xenografts reduced overall tumor burden partially because glutamine-derived non-essential amino acids played an important role in preventing cell apoptosis. This study demonstrates that glutamine metabolism plays different roles in tumor initiation and cancer maintenance. Supplementation of α-ketoglutarate and inhibiting GLS may provide a therapeutic approach to suppress enchondroma and chondrosarcoma tumor growth, respectively. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Distinct Roles of Glutamine Metabolism in Benign and Malignant Cartilage Tumors With IDH Mutations.

Cellular differentiation is associated with the acquisition of a unique protein signature which is essential to attain the ultimate cellular function and activity of the differentiated cell. This is predicted to result in unique biosynthetic demands that arise during differentiation. Using a bioinformatic approach, we discovered osteoblast differentiation is associated with increased demand for the amino acid proline. When compared to other differentiated cells, osteoblast-associated proteins including RUNX2, OSX, OCN and COL1A1 are significantly enriched in proline. Using a genetic and metabolomic approach, we demonstrate that the neutral amino acid transporter SLC38A2 acts cell autonomously to provide proline to facilitate the efficient synthesis of proline-rich osteoblast proteins. Genetic ablation of SLC38A2 in osteoblasts limits both osteoblast differentiation and bone formation in mice. Mechanistically, proline is primarily incorporated into nascent protein with little metabolism observed. Collectively, these data highlight a requirement for proline in fulfilling the unique biosynthetic requirements that arise during osteoblast differentiation and bone formation.

Leyao Shen

Department of Internal Medicine

University of Texas Southwestern Medical Center

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Leyao Shen

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Internal Medicine

University of Texas Southwestern Medical Center

Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Department of Internal Medicine