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 proteins^1,2 and numerous high membrane-potential mitochondria to harvest the requisite chemical energy from fuel substrates^3,

Protocol

This protocol uses the conversion of [1-¹⁴C]-oleic acid to ¹⁴CO₂ as an indicator of fatty acid oxidation capacity. The local Radiation Safety office approved the protocol for using radioactive materials before initiating the experiments. All radiation procedures were performed behind a plexiglass shield using appropriate personal protective equipment. Local Animal Care and Use Committee approved the protocol prior to using primary cells.

1. Fatty acid oxidation in cultured osteoblasts

1. Sub-culture the primary calvarial osteoblasts¹² or an osteoblastic ....

Results

Enzymatic activity of both carnitine palmitoyltransferase-1 (Cpt1) and carnitine palmitoyltransferase-2 (Cpt2) are required for mitochondrial long-chain fatty acid oxidation. Cpt1 is the rate-limiting enzyme in the metabolic pathway, but three isoforms (Cpt-1a, Cpt-1b, and Cpt-1c) are encoded in mammalian genomes, and the genetic disruption of one isoform can lead to compensation upregulation of another isoform¹³. By contrast, Cpt2 is encoded by a single gene. In the experiment illustrated in

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
[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