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 acids^1,2,3,

Protocol

All mouse procedures described herein were approved by the Animal Studies Committees at the University of Texas Southwestern Medical Center at Dallas. The radiation protocol was approved by the Radiation Safety Advisory Committee at the University of Texas Southwestern Medical Center at Dallas.

1. Amino acid uptake in cells (Protocol I)

1. Plate 5 x 10⁴ ST2 cells in each well of a 12-well tissue culture plate. Plate cells in α-MEM containing 10% FBS, 100 U/mL penicillin and 0.1 µg/mL streptomycin (Pen/Strep). Plate extra wells of cells to quantify the cell number per condition for the normaliza....

Results

Amino acid transport is regulated by many membrane-bound amino acid transporters that have been categorized into distinct transport systems based on numerous characteristics, including substrate specificity, kinetics, as well as ion and pH dependence²⁵. For example, glutamine uptake can be mediated by the Na⁺-dependent transport systems A, ASC, γ+L and N or the Na⁺-independent System L. The Na⁺-dependent systems are distinguished by the ability to substitute Li

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 intensive^16,17,25. In our protocol, we evaluate uptake in the Krebs Ringers HEPES .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% trypsin	Gibco	25200
12-well plate	Corning	3513
1 mL 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