Cell culture and drug treatments
Unless otherwise noted, cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, 4.5 g/L D-glucose) (Life Technologies) containing 10% Fetal Bovine Serum (FBS) (Life Technologies) and 1% penicillin/streptomycin (Life Technologies) at 37 °C with 5% CO2. In each drug experiment, either oxamate or GSK-2837808A (3-[[3-[(Cyclopropylamino) sulfonyl]-7-(2,4-dimethoxy-5-pyrimidinyl)-4-quinolinyl] amino]-5-(3,5-difluorophenoxy) benzoic acid, TOCRIS) was added into the assay buffer. To account for effects of DMSO, DMSO was added to the assay buffer in all experiments (including oxamate conditions and vehicle conditions). The final concentration of DMSO was 1%, unless otherwise stated. Three biological replicates were used for each condition tested.
Lactate production assay
Approximately 7 × 105 HeLa cells were seeded in a 12-well plate and allowed to attach overnight. Cells were then washed and supplemented with FBS-free, low-glucose media (1 g/L D-glucose) and treated with oxamate, GSK-2837808A, or DMSO alone (vehicle). After 6 h, the culture media were collected and extracted as described previously and detailed below . Samples were analyzed by liquid chromatography/mass spectrometry (LC/MS) in negative ion mode with a triple quadrupole mass spectrometer (6460, Agilent Technologies). Samples were separated with a Luna Aminopropyl column (3 μm, 150 mm × 1.0 mm I.D., Phenomenex) coupled to an Agilent 1260 LC system. A flow rate of 50 μL/min was used. The mobile phases and linear gradient were A = 95% water, 5% acetonitrile (ACN), 20 mM ammonium hydroxide (NH4OH), 20 mM ammonium acetate (NH4Ac); B = 100% ACN; 85% B from 0 to 3 min, 85% to 50% B from 3 to 7 min, 50% to 5% B from 7 to 11 min, and 5% B from 11 to 13 min.
Purification of mitochondria
Mitochondria were purified as described previously . Briefly, cells were harvested, pelleted, and re-suspended in cold mitochondrial isolation media (MIM) (300 mM sucrose, 10 mM HEPES, 0.2 mM EDTA, and 1 mg/mL bovine serum albumin (BSA), pH 7.4) and then homogenized with a glass-Teflon potter. Next, samples were centrifuged at 700×g (4 °C) for 7 min to separate mitochondria from the remaining cellular material. The supernatant was decanted after centrifugation and set aside. The remaining pellets were homogenized again in MIM to recover more mitochondria. The supernatant was then pooled with the supernatant from above and centrifuged at 10,000×g (4 °C) for 10 min to obtain mitochondrial pellets. Mitochondrial pellets were washed and quantified by performing a Bradford assay, unless otherwise noted.
LDH activity assay
LDH activity was assessed in a 96-well plate. First, mitochondria were purified from ~ 6 × 107 HeLa cells as above. Mitochondrial pellets were then lysed with 1% triton X-100/50 mM Tris (pH 7.4). The mitochondrial lysates were treated with oxamate, GSK-2837808A, or DMSO alone (vehicle). The 1% triton X-100/50 mM Tris solution was used as a negative control (blank). A standard mixture was prepared containing phenazine methosulphate (360 μg/mL), p-iodonitrotetrazolium violet (1.3 mg/mL), and NAD+ (340 μg/mL). A 50 μL aliquot of the standard mixture, 200 mM Tris (pH 8), and 50 mM lactate were added to each well before adding 50 μL of sample (38 μg of mitochondrial protein/well). The final concentration of DMSO was 0.4% in all three conditions. The kinetic assay was run at 490 nm with a Cytation 5 microplate reader (BioTek), and LDH activity was determined by the maximum slope.
Labeling whole cells with U-13C lactate
HeLa cells were grown to ~ 35% confluency in 100-mm culture dishes. The culture media were then changed to fresh low-glucose (5 mM) media supplemented with 3 mM uniformly 13C-labeled lactate (U-13C lactate, Cambridge Isotope Laboratories). Cells were treated with 50 mM oxamate, 75 µM GSK-2837808A, or DMSO alone (vehicle) for 24 h. The final concentration of DMSO was 0.3% in all three conditions. After 24 h, cells were washed with phosphate-buffered saline (PBS) and HPLC-grade water, quenched with 1 mL cold HPLC-grade methanol, scraped from the plate, and pelleted. Pellets were dried on a SpeedVac (Thermo Fisher Scientific) and subsequently lyophilized (Labconco). Dried samples were weighed and extracted by using the protocol described below . Experiments were performed with n = 3 cultures per sample group.
Oxygen consumption rate
Respiration of intact mitochondria was measured with an XFp analyzer (Seahorse Bioscience) or a high-resolution OROBOROS Oxygraph-2k respirometer (Oroboros Instruments). For the Seahorse experiments, after purifying mitochondria from ~ 2 × 107 HeLa cells, approximately 8 μg of mitochondrial pellets were re-suspended in cold mitochondria assay solution (MAS, 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, and 0.2% (w/v) fatty acid-free BSA, pH 7.2) with 10 mM lactate and 5 mM malate. Before measuring respiration, mitochondria were brought to room temperature. ADP (4 mM) was added to induce respiration. The oxygen consumption rate (OCR) of HeLa mitochondria was monitored under three different conditions: oxamate, GSK-2837808A, or DMSO alone (vehicle). To remove background contributions, the OCR value before the addition of ADP was subtracted from the OCR value after the addition of ADP (Fig. 3b). For assessing mitochondrial function, no drugs or DMSO were added. Sample sizes were used that produced OCR numbers within the recommended range of the vendor. For the Oroboros experiments, mitochondrial respiration media were used. Approximately the same number of HeLa cell mitochondria (300 μg mitochondrial protein) was added to each chamber followed by metabolic substrates and inhibitors.
Labeling whole cells with 2-2H lactate prior to mitochondrial purification
Cells were cultured in a T-150 flask until reaching 90% confluency. Cells were then transferred to glucose-free media for 4 h. After 4 h, cells were supplemented with 10 mM 2-2H lactate for 45 min prior to being washed, harvested, and pelleted. For mitochondrial purification, the cell pellets were re-suspended in 500 μL of cold MIM with 100 mM oxamate. The isolated mitochondrial pellets were lyophilized and subsequently treated with a methanol/acetonitrile/water (2:2:1) solution prior to being reconstituted in 40 μL acetonitrile/water (1:1) per milligram dry weight. LC/MS analysis was performed as described below.
Labeling purified mitochondria with U-13C lactate
Approximately 2 × 108 HeLa cells were harvested at 90% confluence. Mitochondria were purified as above. Purified mitochondria were split into wells (170 μg of mitochondrial protein/well) and incubated in 1 mL MAS buffer with 5 mM malate and 5 mM lactate. Samples were treated with oxamate, GSK-2837808A, or DMSO alone (vehicle) for 10 min. The final concentration of DMSO was 0.3% in all three conditions. After 10 min, 10 mM U-13C lactate was added to the MAS buffer for 20 min before harvesting. Mitochondrial pellets were washed, collected, and snap frozen in liquid nitrogen prior to extraction.
Metabolite extraction and LC/MS analysis
Cell pellets or purified mitochondria were extracted and analyzed by LC/MS as described before [6, 10]. Cell pellets were treated with a methanol/acetonitrile/water (2:2:1) solution and reconstituted in 40 μL acetonitrile/water (1:1) per milligram dry weight. Mitochondrial pellets were treated with a methanol/acetonitrile/water (2:2:1) solution and reconstituted in 50 μL acetonitrile/water (1:1) per 170 μg of mitochondrial protein, unless otherwise noted. Samples were separated with a Luna Aminopropyl column (3 μm, 150 mm × 1.0 mm I.D., Phenomenex) coupled to a Dionex UltiMate® 3000 RSLCnano LC system. MS detection was performed on a Thermo Q Exactive Plus mass spectrometer (Thermo Fischer Scientific) in negative ion mode at 140,000 resolving power. The column was used in hydrophilic interaction mode with a flow rate of 50 μL/min. The following mobile phases and linear gradient were used: A = 95% water, 5% ACN, 20 mM NH4OH, 20 mM NH4Ac; B = 95% ACN, 5% water; 100% B from 0 to 3 min, 100% B to 0% B from 3 to 40 min, and 0% B from 40 to 45 min.
Data are reported as means ± SD. Dataset comparisons were performed with a Student’s unpaired, two-tailed t test.