Stable-isotope tracing, metabolite profiling, and flux analysis

For stable-isotope tracing experiments, cells were plated into 6-well plates at 0.25-0.5x10⁶ cells/well and cultured for 24 hours in DMEM supplemented with 1mM labeled alanine and 10% dialyzed FBS (Gibco). For MPC inhibition tracing experiments, cells were plated at 0.25-0.5x10⁶ cells/well and washed once with PBS before addition of 1mM ¹³C₃-alanine or L-alanine and either vehicle (DMSO) or UK-5099 for 24 hours. Conditioned media was collected and briefly centrifuged at 1,000xg for 10 minutes to remove cell debris prior to extraction. For alanine exchange experiments, PSCs were plated at 0.1x10⁶ cells/well into 6-well plates and cultured with 1mM ¹³C₃-alanine and 10% dialyzed FBS. Conditioned media and intracellular metabolites were collected every 24 hours from independent plates per timepoint.

For metabolite extraction, cells were washed with cold 0.9% NaCl followed by addition of 500 μl methanol (−20°C) and 200 μl HPLC grade water (4°C). Cells were scraped and transferred into vials containing 500 μl chloroform (4°C) and vortexed at 4°C for 10-15 minutes. Aqueous and inorganic layers were separated by cold centrifugation for 15 minutes. 300 μl of the aqueous layer containing polar metabolites was transferred to sample vials (Agilent) and evaporated by SpeedVac (Savant Thermo) prior to derivatization and GC-MS analysis. Fatty acid labeling from ¹³C₃-alanine was measured by transferring 400 μl of the inorganic layer to a glass vial and evaporating under nitrogen flow in a needle evaporator prior to transesterification and GC-MS analysis. To measure proteogenic amino acids, the insoluble interphase containing proteins was washed three times with HPLC-grade acetone, to remove free metabolites, and allowed to dry overnight with gentle nitrogen flow. The resulting pellet was hydrolyzed in 2N HCl at 95°C for two hours with occasional vortexing and dried overnight under nitrogen flow prior to derivatization and GC-MS analysis. For amino acid and metabolite profiling, approximately 0.5-1x10⁶ cells were extracted using methanol:water:chloroform containing 2.5 nmol of uniformly (¹³C_x,¹⁵N_x)-labeled amino acids including L-alanine, L-lysine, L-hisitidine, L-arginine, L-tyrosine, L-phenylalanine, L-methionine, L-glutamic acid, L-aspartic acid, L-leucine, L-isoleucine, L-valine, L-threonine, L-proline, L-serine, and glycine; 4 nmol of ¹³C₅-labeled L-glutamine; 0.5 nmol ¹³C₃-labeled pyruvate; 5 nmol ¹³C₃-labeled lactate; 1.25 nmol ¹³C₆,¹⁵N₂-labeled cystine, and 1μg of norvaline. For media extraction, 5 μl of initial or conditioned media was extracted in 250 μl of 80% methanol solution containing the above isotope-labeled internal standards. The abundance of each metabolite was quantified by the following equation:

Where X_standard is the molar amount for each added standard (e.g. 2.5 nmol for alanine, 5 nmol for lactate) and %M0_X is the relative abundance of unlabeled (M+0) species ‘X’ corrected for natural isotope abundance. Multiple surrogate wells were pooled and counted to normalize metabolite abundances by cell number.

For cDNA withdrawal experiments, cells were plated into 6-well plates at 0.1-0.25x10⁶ cells/well in DMEM containing doxycycline (0.5 μg/ml). For time course cDNA withdrawal, cells were washed twice with PBS to remove residual doxycycline and cultured in DMEM with or without doxycycline to initiate time course. Metabolites were extracted at indicated times and surrogate wells were counted for normalization. Kinetic changes in relative amino acid levels with acute SLC38A2 cDNA withdrawal were calculated by subtracting a baseline value (chronic SLC38A2 KO; set to “0%”) from the control (+dox) and experimental conditions (acute KO) followed by normalization to positive control level (+dox; set to “100%”).

To quantify extracellular uptake and secretion fluxes, the molar change in extracellular metabolites was normalized to the cell density over the time course of the experiment using the following equation:

Where AA_t and AA_o are the final and initial molar amounts of each amino acid, respectively, quantified using labeled internal standards, X_o is the initial cell density, k is the exponential growth rate (hr⁻¹), and t is the media conditioning time. To quantify alanine exchange flux, the percent dilution of exogenous ¹³C₃-alanine was multiplied by the molar quantity of alanine in the media over the conditioning period normalized to cell density over time.