Evaluation of Amino Acid Consumption in Cultured Bone Cells and Isolated Bone Shafts

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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^{1,2,3,4,5,6,7,8,9,10}. Many studies demonstrate that amino acid metabolism is critical for cell pluripotency, proliferation, and differentiation in various contexts^{3,6,11,12,13,14,15,16,17}.

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 formation^{15,16,17,18,19,20}.

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 samples^21,22. 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 experiment^23,24. 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.

Protokół

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 normalizations in step 1.12. Incubate cells in a humidified cell culture incubator at 37 °C with 5% CO₂.
2. Culture the cells for 2-3 days until confluent.
3. On the day of the experiment, prepare the following solutions: 1x Phosphate Buffered Saline (PBS), pH 7.4 and Krebs Ringers HEPES (KRH) buffer, pH 8.0: (120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, NaHCO₃, 5 mM HEPES, 1 mM D-Glucose). Prewarm to 37 °C.
4. Aspirate the medium and wash the cells twice with 1 mL of 1x PBS, pH 7.4.
   NOTE: This protocol is also appropriate for rapidly dividing non-confluent cells. In this case, it is important to normalize the radioactivity to either absolute cell number or DNA content. In addition, consider increasing the size of the culture plate or flask to increase the overall cell number and cpm values. This is important to determine empirically on an individual basis.
5. Aspirate 1x PBS and wash the cells once with 1 mL KRH.
6. Make 4 µCi/mL L-[3,4-³H]-Glutamine working media by diluting 4 µL of [1 µCi µL^-1] L-[3,4-³H]-Glutamine stock per 1 mL of KRH.
   NOTE: Before using radioactive materials, please contact the Office of Radiation Safety at your home institution to obtain approvals. All the procedures related to radiation must be performed behind the plexiglass shield.
7. Incubate cells with 0.5 mL of KRH containing 4 µCi/mL L-[3,4-³H]-Glutamine working media for 5 min.
8. Collect the radioactive medium and dispense into the liquid waste container. Wash the cells three times briefly with ice-cold KRH to terminate the reaction. Collect and discard all the washes in the radioactive liquid waste container.
9. Add 1 mL of 1% SDS to each well and triturate 10x to lyse and homogenize the cells. Transfer the cell lysates to 1.5 mL tubes. Discard cell culture plates, serological pipettes, and pipette tips in the solid radioactive waste container.
10. Centrifuge at >10,000 x g for 10 min. Transfer the supernatants to scintillation vials containing 8 mL of the scintillation solution. Mix by shaking the scintillation vials vigorously. Discard tubes and pipette tips in solid radioactive waste container.
11. Read radioactivity in counts per minute (cpm) using a Scintillation counter. Discard scintillation vials in scintillation vial waste container.
12. Trypsinize, resuspend, and count the cells from the remaining non-radioactive plates of cells (see step 1.1) to estimate the number of cells in the lysed, radioactive cultures. Using a hemocytometer, count the number of cells per non-radioactive well for each experimental condition. Normalize the cpm from step 1.11 to the estimated cell number from the non-radioactive plates.
13. After completion of the experiment, decontaminate the cell culture hood, bench, and all instruments with a radioactivity decontaminant spray. Finally, perform wipe tests to ensure the working area is radiation-free.

2. Amino acid uptake in freshly isolated bone tissues (Protocol II)

1. Prewarm KRH to 37 °C.
2. Euthanize a 3-day-old mouse and remove the skin on the arms. Disarticulate the humeri from the shoulder using scissors and dissect out both humeri. Remove all extemporaneous tissues using a scalpel and forceps. Remove the epiphyses from the bone.
   NOTE: In addition to humerii, this protocol can be adapted to neonatal femora, tibiae and calvariae as well as humerii, femora and tibiae isolated from 2- and 4-month-old mice (unpublished data).
3. Flush out marrow from the bone and weigh the bone shafts to normalize in step 2.14.
4. Boil one humerus in 1x PBS at 100 °C for 10 min to decellularize the bone. The decellularized boiled bone is used as a negative control.
   NOTE: As a quality control, paraffin embed the boiled and non-boiled bones for histological stains to visually confirm that boiling was efficacious in decellularizing the bone.
5. Equilibrate both humeri in 1 mL of KRH for 30 min in the cell culture incubator at 37 °C.
6. Make a working solution of 4 µCi/mL L-[2,3,4-³H]-Arginine in KRH by diluting 4 µL of 1µCi/µL L-[2,3,4-³H]- Arginine stock per 1 mL KRH.
   NOTE: This protocol is appropriate to evaluate the uptake of whichever radiolabeled nutrient is chosen. L-[2,3,4-³H]-glutamine, L-[¹⁴C_U]-alanine, and L-[2,3-³H]-proline have been tested with similar results. Arginine data is shown to highlight the utility of this protocol.
   CAUTION: All the procedures using radiation must be performed behind the plexiglass shield while using appropriate personal protective equipment (PPE).
7. Incubate both the experimental and boiled control bone separately in KRH containing 4 µCi/mL L-[2,3,4-³H]-Arginine for up to 90 min at 37 °C.
   NOTE: It is important to empirically determine the incubation times for different conditions including the age of the mice, different skeletal elements and the types of radiolabeled amino acids by performing a time course. Saturation mostly occurs after approximately 3-4 h. Uptake should be evaluated at a timepoint in the linear rage of uptake.
8. Remove the radioactive medium and discard in the liquid radioactive waste container. Wash humeri three times using ice cold 1 mL of KRH to terminate the reaction. Discard all the washes in the liquid radioactive waste container.
9. Transfer each bone into a 1.5 mL tube. Add 500 µL of RIPA buffer [150 mM NaCl; 5 mM EDTA; 50 mM Tris (pH 8.0); 0.5% NP40 (v/v); 0.5% DOX (w/v); 0.1% SDS (w/v)]. Discard the culture plates, serological pipettes, and pipette tips in a radioactive solid waste container.
10. Homogenize the humeri by chopping 100 times with scissors in a RIPA buffer.
11. Sonicate (Amplitude: 35%, Pulse 1 s) the resulting homogenized bone for 10 s.
    NOTE: Sonication is also known to produce aerosols. Sonication steps can be performed in a biological safety cabinet. To reduce aerosol formation, it is important to not overfill the tubes. Sonication of small sample volumes can result in incomplete sonication or sample loss due to foaming. If foaming occurs, centrifuge the sample for 5 min to settle.
12. Clarify the lysate by centrifugation for 10 min at >10,000 x g at room temperature. Transfer 200 µL of the supernatant to scintillation vials containing 8 mL of the scintillation solution. Mix by shaking the scintillation vials vigorously. Discard all solid radioactive waste (e.g., used tubes, pipette tips, plates, etc.) in the solid radioactive waste container.
13. Read radioactivity (cpm) using the Scintillation counter. Discard used scintillation vials in the radioactive waste container designated for scintillation vials.
14. Subtract the cpm of boiled bone (basal value) from the cpm of experimental bone. Normalize the radioactivity with bone weight from step 2.3.
15. Spray the cell culture hood, instruments, and bench with radioactivity decontaminant. Perform wipe tests to confirm the working area is radiation-free.

Wyniki

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 L...

Dyskusje

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 ...

Materiały

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