Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts
Summary
This protocol presents a radiolabeled amino acid uptake assay, which is useful for evaluating amino acid consumption either in primary cells or in isolated bones.
Abstract
Bone development and homeostasis is dependent upon the differentiation and activity of bone forming osteoblasts. Osteoblast differentiation is sequentially characterized by proliferation followed by protein synthesis and ultimately bone matrix secretion. Proliferation and protein synthesis require a constant supply of amino acids. Despite this, very little is known about amino acid consumption in osteoblasts. Here we describe a very sensitive protocol that is designed to measure amino acid consumption using radiolabeled amino acids. This method is optimized to quantify changes in amino acid uptake that are associated with osteoblast proliferation or differentiation, drug or growth factor treatments, or various genetic manipulations. Importantly, this method can be used interchangeably to quantify amino acid consumption in cultured cell lines or primary cells in vitro or in isolated bone shafts ex vivo. Finally, our method can be easily adapted to measure the transport of any of the amino acids as well as glucose and other radiolabeled nutrients.
Introduction
Amino acids are organic compounds that contain an amino (-NH2) and carboxyl (-COOH) functional groups with a variable side chain that is specific to each amino acid. In general, amino acids are well known as the basic constituent of protein. More recently, novel uses, and functions of amino acids have been elucidated. For example, individual amino acids can be metabolized to generate intermediate metabolites that contribute to bioenergetics, function as enzymatic cofactors, regulate reactive oxygen species or are used to synthesize other amino acids1,2,3,4,5,6,7,8,9,10. Many studies demonstrate that amino acid metabolism is critical for cell pluripotency, proliferation, and differentiation in various contexts3,6,11,12,13,14,15,16,17.
Osteoblasts are secretory cells that produce and secrete the Collagen Type 1 rich extracellular bone matrix. To sustain high rates of protein synthesis during bone formation, osteoblasts demand a constant supply of amino acids. To meet this demand, osteoblasts must actively acquire amino acids. Consistent with this, recent studies reveal the importance of amino acid uptake and metabolism in osteoblast activity and bone formation15,16,17,18,19,20.
Osteoblasts acquire cellular amino acids from three major sources: extracellular milieu, intracellular protein degradation and de novo amino acid biosynthesis. This protocol will focus on the evaluation of amino acid uptake from extracellular environment. The most common methods to measure amino acid uptake rely on either radiolabeled (e.g., 3H or 14C) or heavy isotope labeled (e.g., 13C) amino acids. Heavy isotopomer assays can analyze amino acid uptake and metabolism more thoroughly and safely but are more time consuming taking multiple days to complete as it takes a day to prepare and derivatize samples and multiple days to analyze on the mass spectrometer depending on the number of samples21,22. By comparison, radiolabeled amino acid uptake assays are not informative about downstream metabolism but are cheap and relatively fast, being able to be completed within 2-3 h from the start of the experiment23,24. Here, we describe an easily modifiable basic protocol designed to evaluate radiolabeled amino acid uptake in cultured primary cells or cell lines in vitro or individual bone shafts ex vivo. The application of these two protocols can be extended to other radiolabeled amino acids and other bone associated cell types and tissues.
Protocol
Representative Results
Discussion
The protocol described herein provides a fast and sensitive approach to evaluate amino acid uptake in response to various experimental permutations either in vitro or ex vivo. When compared to commercially available kits (e.g., Glutamine and Glutamate Determination Kit), this method is much more sensitive, quicker, and less labor intensive16,17,25. In our protocol, we evaluate uptake in the Krebs Ringers HEPES …
Disclosures
The authors have nothing to disclose.
Acknowledgements
The Karner lab is supported by National Institute of Health R01 grants (AR076325 and AR071967) to C.M.K.
Materials
| 0.25% trypsin | Gibco | 25200 | |
| 12-well plate | Corning | 3513 | |
| 1mL syringe | BD precision | 309628 | |
| 30G Needle | BD precision | 305106 | |
| Arginine Monohydrochloride L-[2,3,4-3H]-, 1mCi | PerkinElmer | NET1123001MC | |
| Beckman LS6500 scintillation counter | |||
| Calcium chloride | Sigma | C1016 | |
| choline chloride | Sigma | C7077 | |
| D-(+)-Glucose solution | Sigma | G8769 | |
| Dissection Tool | Forceps, scissors, scapels | ||
| DPBS | Gibco | 14190 | |
| Ethylenediaminetetraacetic acid | Sigma | E9884 | |
| HEPES(1M) | Gibco | 15630 | |
| L-[3,4-3H(N)]-Glutamine | PerkinElmer | NET551250UC | |
| Liquid scintilation vials | Sigma | Z190535 | |
| lithium chloride solution, 8M | Sigma | L7026 | |
| Magnesium chloride | Sigma | M8266 | |
| MEMα | Gibco | 12561 | |
| Microcentrifuge tube, 15mL | Biotix | 89511-256 | |
| NP-40 | Sigma | 492016 | |
| Potassium chloride | Sigma | P3911 | |
| Sodium bicarbonate | Sigma | S6014 | |
| sodium chloride | Sigma | S9888 | |
| Sodium Deoxycholate | Sigma | D6750 | |
| Sodium dodecyl sulfate | Sigma | 436143 | |
| Sonicator | Sonic&Materials | VCX130 | |
| Tris Base | Sigma | 648311 | |
| Ultima Gold (Scintillation solution) | PerkinElmer | 6013329 | |
| α-(Methylamino)isobutyric acid | Sigma | M2383 |
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