Evaluation of Fatty Acid Oxidation in Cultured Bone Cells
Summary
The present protocol describes an assay to assess the capacity for fatty acid oxidation in cultures of primary bone cells or relevant cell lines.
Abstract
Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14C-labeled fatty acids to 14CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.
Introduction
The osteoblast, derived from progenitor cells present in the bone marrow and the periosteum, is responsible for synthesizing and secretion of the mineralized, collagen-rich matrix that composes bone tissue. To fulfill this energetically expensive endeavor and contribute to the lifelong maintenance of skeletal integrity, these specialized cells maintain an abundant rough endoplasmic reticulum essential for synthesizing extracellular matrix proteins1,2 and numerous high membrane-potential mitochondria to harvest the requisite chemical energy from fuel substrates3,4. The importance of this latter function is exemplified by the cessation of bone growth and the development of osteopenia5,6 associated with energy deficits as in caloric restriction. The identity of the osteoblast's preferred fuel source and the data on the metabolic requirements of the osteoblast during differentiation or in response to anabolic signaling are emerging7,8.
Long-chain fatty acids represent a rich supply of chemical energy present in serum and released from adipose tissue in response to reduced caloric intake or heightened energy expenditure. After traversing the cell membrane and ligating to Coenzyme A to increase solubility, fatty acyl-CoAs are shuttled into the mitochondrial matrix by the dual carnitine palmitoyltransferase enzymes on the outer and inner mitochondrial membranes and carnitine-acylcarnitine translocase7. Within the mitochondrial matrix, the β-oxidation machinery chain shortens acyl-CoA in a 4-step process that generates acetyl-CoA that enters the TCA cycle (tricarboxylic acid cycle) and reducing equivalents. Catabolism of palmitate (C16), the most common fatty acids in animals, via this pathway yields ~131 ATP, substantially more than the ~38 ATP generated by glucose oxidation7.
Tracing studies using radiolabeled lipids indicated that bone takes up a significant fraction of circulating lipids9,10, while genetic ablation of enzymes critical to lipid catabolism leads to a reduction in osteoblast activity and bone loss9,11. The protocol presented here uses a 14C-labeled fatty acid to evaluate the capacity of cultured osteoblasts to fully metabolize fatty acids to CO2 or acid-soluble metabolites, which represent intermediate steps in the oxidation process. While the use of radioactivity is required, the method is straightforward, requires limited investment, and is adaptable for the benefit of other radiolabeled metabolites and cell types.
Protocol
Representative Results
Discussion
The procedure described above allows for the direct assessment of fatty acid oxidation as the measured outputs are 14CO2 collected on the NaOH soaked filter paper and acid-soluble metabolites collected after the acidification of the culture with perchloric acid. Commercially available assay kits that use fluorescently labeled lipid molecules or lipid analogs can measure lipid uptake, but they do not determine catabolism for energy generation. Other measures of catabolism, such as 13C-meta…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by a Merit Review Award from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development (BX003724) and a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK099134).
Materials
| [1-14C]-Oleic acid | Perkin Elmer | NEC317050UC | |
| 15 x 30 mm rubber sleeve stoppers | VWR | 89097-542 | |
| 1 mL syringe | BD precision | 309628 | |
| 25 G needle (25 G x 1 1/2 in) | BD precision | 305127 | |
| Ascorbic Acid | Sigma Aldrich | A4403 | |
| BCA Protein Assay Kit | Thermo Fisher | 23225 | Other kits are also suitable |
| Beckman Scintillation Counter, or equivalent | Beckman Coulter | LS6000SC | |
| Beta-glycerol phosphate | Sigma Aldrich | G6626 | |
| Bovine Serum Albumin | Sigma Aldrich | 126609 | |
| Carnitine | Sigma Aldrich | C0283 | |
| Cellular lysis buffer | The protocol is amenable to typical lysis buffers (i.e. RIPA) | ||
| Dissecting forceps | Available from multiple sources | ||
| DNA quantification Kit | Available from multiple sources | ||
| Dulbecco’s Phosphate-Buffered Saline | Corning | 20-030-CV | |
| Fetal Bovine Serum | Available from multiple sources | ||
| Microcentrifuge tubes, 1.5 mL | Available from multiple sources | ||
| Minimum Essensial Medium, Alpha modification | Corning | 10-022-CV | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Perchloric Acid | Sigma Aldrich | 50439 | |
| Polypropylene center wells | VWR | 72760-048 | |
| Sodium hydroxide | Sigma Aldrich | S5881 | |
| T-25 canted neck tissue culture flask | Corning | 430639 | |
| Tissue Culture Incubator | |||
| Trypsin (0.25%)-EDTA | Gibco | 25200056 | |
| Ultima Gold (Scintillation solution) | PerkinElmer | 6013329 | |
| Whatman Chromatography paper | Sigma Aldrich | WHA3030917 |
References
- Cameron, D. A. The fine structure of osteoblasts in the metaphysis of the tibia of the young rat. Journal of Biophysical and Biochemical Cytology. 9, 583-595 (1961).
- Dudley, H. R., Spiro, D. The fine structure of bone cells. Journal of Biophysical and Biochemical Cytology. 11 (3), 627-649 (1961).
- Shapiro, I., Haselgrove, J. i. n., Hall, B. K. Ch. 5, Bone Metabolism and Mineralization. Bone Vol. 4. , 99-140 (1991).
- Komarova, S. V., Ataullakhanov, F. I., Globus, R. K. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts. American Journal of Physiology: Cell Physiology. 279 (4), 1220-1229 (2000).
- Bolton, J. G., Patel, S., Lacey, J. H., White, S. A prospective study of changes in bone turnover and bone density associated with regaining weight in women with anorexia nervosa. Osteoporosis International. 16 (12), 1955-1962 (2005).
- Nussbaum, M., Baird, D., Sonnenblick, M., Cowan, K., Shenker, I. R. Short stature in anorexia nervosa patients. Journal of Adolescent Health Care. 6 (6), 453-455 (1985).
- Alekos, N. S., Moorer, M. C., Riddle, R. C. Dual effects of lipid metabolism on osteoblast function. Frontiers in Endocrinology. 11, 578194 (2020).
- Karner, C. M., Long, F. Glucose metabolism in bone. Bone. 115, 2-7 (2018).
- Kim, S. P., et al. Fatty acid oxidation by the osteoblast is required for normal bone acquisition in a sex- and diet-dependent manner. JCI Insight. 2 (16), 92704 (2017).
- Niemeier, A., et al. Uptake of postprandial lipoproteins into bone in vivo: impact on osteoblast function. Bone. 43 (2), 230-237 (2008).
- Helderman, R. C., et al. Loss of function of lysosomal acid lipase (LAL) profoundly impacts osteoblastogenesis and increases fracture risk in humans. Bone. 148, 115946 (2021).
- Jonason, J. H., O’Keefe, R. J. Isolation and culture of neonatal mouse calvarial osteoblasts. Methods in Molecular Biology. 1130, 295-305 (2014).
- Haynie, K. R., Vandanmagsar, B., Wicks, S. E., Zhang, J., Mynatt, R. L. Inhibition of carnitine palymitoyltransferase1b induces cardiac hypertrophy and mortality in mice. Diabetes, Obesity & Metabolism. 16 (8), 757-760 (2014).
- Long, C. P., Antoniewicz, M. R. High-resolution (13)C metabolic flux analysis. Nature Protocols. 14 (13), 2856-2877 (2019).
- Divakaruni, A. S., et al. Etomoxir inhibits macrophage polarization by disrupting CoA homeostasis. Cell Metabolism. 28 (3), 490-503 (2018).
- Raud, B., et al. Etomoxir actions on regulatory and memory T cells are independent of cpt1a-mediated fatty acid oxidation. Cell Metabolism. 28 (3), 504-515 (2018).
- Frey, J. L., et al. Wnt-Lrp5 signaling regulates fatty acid metabolism in the osteoblast. Molecular and Cellular Biology. 35 (11), 1979-1991 (2015).
- Frey, J. L., Kim, S. P., Li, Z., Wolfgang, M. J., Riddle, R. C. beta-catenin directs long-chain fatty acid catabolism in the osteoblasts of male mice. Endocrinology. 159 (1), 272-284 (2018).
- Li, Z., et al. Glucose transporter-4 facilitates insulin-stimulated glucose uptake in osteoblasts. Endocrinology. 157 (11), 4094-4103 (2016).
- Lee, J., et al. Loss of hepatic mitochondrial long-chain fatty acid oxidation confers resistance to diet-induced obesity and glucose intolerance. Cell Reports. 20 (3), 655-667 (2017).
- Kim, S. P., et al. Sclerostin influences body composition by regulating catabolic and anabolic metabolism in adipocytes. Proceedings of the National Academy of Sciences of the United States of America. 114 (52), 11238-11247 (2017).
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