This work was supported by the National Institute of Health (NIH) and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) Grants AR072123 (to ERR), AR076325 and AR071967 (to CMK); and National Institute on Aging (NIA) Grant AG069795 (to ERR).

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The authors have nothing to disclose.

Bone is a dynamic tissue that undergoes continuous remodeling, involving bone-resorbing osteoclasts and bone-forming osteoblasts. Importantly, these processes are coupled with other cells residing in the marrow space such as hematopoietic cells and bone marrow adipocytes. A disconnect in bone turnover results in skeletal fragility and a subsequent increase in fractures1. Recent studies have demonstrated the importance of metabolic substrates and energetic metabolism to regulate both bone formation and bone resorption2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17. Targeting metabolic pathways is used to treat various cancers and could be a provocative tool that could be applied to combat various conditions that lead to increased fracture incidence18. Due to&#160;this, it is important to have a complete understanding of cellular metabolism in all bone cells. Unfortunately, studying bone cells in their native environment is challenging due to their localization within the mineralized tissue. Compared to unmineralized tissues, the requisite steps required to process bone makes molecular and mechanistic studies of their metabolic status difficult. As such, this collection aims to highlight established techniques used to monitor metabolic pathways in cells isolated from their skeletal niche.
&#34;Osteoenergetics&#34; describes the metabolic processes and bioenergetic potential of osteoblasts and remains an active area of scientific interest and inquiry. Preceding the cells&#8217; ability to make and utilize ATP, however, energetic substrates must first be acquired. Three articles within this collection highlight methods used to monitor specific substrate fates within bone cells. First, Shen and Karner19 describe techniques to evaluate amino acid consumption in cultured cells in vitro or isolated bones ex vivo. In the in vitro system, cultured ST2 cells are incubated with L-[3,4-3H]-glutamine for a predetermined time, and glutamine uptake is quantified using a scintillation counter. Similarly, in the ex vivo system, bones harvested from neonatal mice are incubated with radio-labeled amino acids, and amino acid uptake is estimated using a scintillation counter. These methods allow for a rapid and sensitive evaluation of amino acid uptake in bone cells or in isolated bones. Importantly, these methods are easily modifiable to evaluate different amino acids or other energetic substrates and to test various scientific questions.
In addition to amino acids, glucose and long chain fatty acids (LCFA) represent a dense supply of cellular energy or ATP. In this capacity, LCFA enter the mitochondria via carnitine palmitoyl transferase (CPTI/II), where they are readily oxidized via &#946;-oxidation, and can subsequently&#160;enter the tricarboxylic acid (TCA) or oxidative phosphorylation cycle to yield ATP. This series of chemical reactions ultimately results in carbon being released as gaseous carbon dioxide (CO2), which is difficult to quantify. Song et al. and Kim et al. expertly describe two related methods to measure the metabolites generated in the oxidation process20,21. Both techniques use a 14C-labeled carbon substrate and filter paper as a &#34;trapping&#34; tool to quantify the released 14CO2. However, each method is equally important as they describe unique culture vessels and data normalization techniques. Additionally, Song et al.20 describe how to calculate substrate oxidation, while Kim et al.21 include an additional method to measure acid-soluble metabolites.
These tracing studies allow the investigator to determine the metabolic fate of various substrates. As a complement, Jayapalan et al.22 use metabolic flux analyses to monitor metabolic processes, including glycolysis and oxidative phosphorylation via mitochondrial respiration. This equipment fundamentally measures oxygen and pH in live cell cultures in real time. Therefore, by culturing primary bone marrow stromal cells (BMSCs) through osteoblast differentiation, a variety of experiments can be conducted while monitoring the metabolic pathways23. This technique can be performed with four different injections, which allows researchers to adapt the experiments to specific scientific questions while also being able to follow standardized protocols, including ATP rate, fuel/substrate utilization, mitochondrial respiration, and glycolytic rate assays. Finally, since many of these experiments are mechanistic in nature, it is imperative to maintain a homogenous cell population without contamination by other cell types. The in vitro cell culture systems used to monitor bone cell metabolic processes have many inherent limitations. Guo and Wu24 offer an excellent overview of the limitations introduced during standard BMSC cultures and contribute a detailed method to enrich for stromal cell populations and minimize hematopoietic contamination using hypoxia.
In summary, this collection demonstrates techniques to monitor substrate utilization and bioenergetic capacity in a variety of skeletal models both in vitro and ex vivo. Most of these methods are performed using cell culture systems and, therefore, present with limitations due to the artificial environment. While it is true that primary cells allow for a more physiological system, these cells are often cultured in high oxygen in media containing excess concentrations of metabolic substrates and, sometimes, even with contaminating cell populations. Therefore, it is critical that these mechanistic experiments be supported with additional in vivo data. Nonetheless, this exciting area of research has already proven beneficial. In this regard, continuing to characterize &#34;normal&#34; versus pathological metabolic processes is a promising scientific niche. Furthermore, studying osteoenergetics and metabolism to understand how, why, and when bone cells utilize various substrates and the implications these have on overall bone health opens a wide area for therapeutic targets. To this end, first-generation anti-resorptive bisphosphonates (i.e., etidronate and clodronate) used to treat osteoporosis have been found to impact osteoclast energetics. These widely prescribed drugs represent a perfect example of how metabolic pathways can be exploited to impact overall bone health and improve a patient&#8217;s quality of life. In addition to antiresorptive treatments, there are two FDA-approved anabolic therapies, Teriparatide and Romosozumab, which activate the PTH and WNT pathways, respectively. Both PTH and WNT stimulate glutamine uptake and metabolism in osteoblasts6,11,25,26. Highlighting the therapeutic implications of this, inhibiting glutamine metabolism activity either pharmacologically or genetically reduces bone formation in response to either WNT or anabolic PTH treatments6,11. Therefore, continued investigation is warranted to study metabolism and bioenergetics in bone cells. The techniques described in this collection provide a foundation to address various scientific questions related to osteoenergetics and metabolism in bone cells.

methods for studying osteoenergetics and metabolism

ARTICLES DISCUSSED:
Shen, L., Karner, C. M. Evaluation of amino acid consumption in cultured bone cells and isolated bone shafts. Journal of Visualized Experiments. (182), e62995 (2022).
Jayapalan, S., Nandy, A., Rendina-Ruedy, E. Using real-time cell metabolic flux ananlyzer to monitor osteoblast bioenergetics. Journal of Visualized Experiments. (181), e63142 (2022).
Song, C., Valeri, A., Long, F. Assessing energy substrate oxidation in vitro with 14CO2 trapping. Journal of Visualized Experiments. (181), e63568 (2022).
Kim, S. P., Li, Z., Riddle, R. C. Evaluation of fatty acid oxidation in cultured bone cells. Journal of Visualized Experiments. (182), e63638 (2022).
Guo, W., Wu, C. Utilizing low oxygen tension to reduce hemopoietic cells in murine bone marrow stromal cell cultures. Journal of Visualized Experiments. Epub ahead of print (2022).

Read this Scientific Article Protocol about Methods For Studying Osteoenergetics And Metabolism at JoVE.com

Methods For Studying Osteoenergetics And Metabolism

ARTICLES DISCUSSED:
Shen, L., Karner, C. M. Evaluation of amino acid consumption in cultured bone cells and isolated bone shafts. Journal of Visualized ...

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656 visualizações.  Vanderbilt University Medical Center. Methods For Studying Osteoenergetics And Metabolism