Supplemental figure S1
Spectra for the isotopomers of glutamate used in tcaCALC
analysis and the spectra for glutamine C2.
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Supplemental methods
Cell lines and reagents
The cell lines used include the TGR-1 subclone of the Rat-1 fibroblast cell line (myc+/+)
and myc-/- and myc-/-MycER derivatives of TGR-1 as described (O'Connell et al, 2003).
[U-13C] labeled glucose was purchased from Cambridge Isotopes (Andover, MA),
methotrexate and 6-diazo-5-oxo-L-norleucine from Sigma.
Immunoblot analysis
Extracts were prepared as described (Slawson et al., 2005) and after SDS-PAGE, blotted
and probed with the CTD 110.6 antibody (Covance, Berkeley), Anti-O-GlcNAc
transferase (DM-17, Sigma) and tubulin (Oncogene, Biosciences ) .
Cell culture
Cells were plated in 10% serum and grown for 48 hours until confluent, media was
replaced with 0.25% bovine calf serum (BCS;InVitrogen) and cells grown a further 48
hours. Cells were then split and re-plated in 10% BCS for 12 hours. After washing with
PBS, cells were incubated in HEPES buffered saline (HBSS;114 mM NaCl/4.7 mmol
KCl/1.2 mM KH2PO4/1.16 mM MgSO4/20 mM Hepes/2.5 mM CaCl2/25.5 mM NaHCO3,
pH 7.2) in the presence of 10% dialyzed fetal calf serum (Hyclone) and 20 mM [U-13C]
glucose (Cambridge Isotopes) for 4 hours. Cells were washed, harvested by
trypsinization, washed, pelleted by centrifugation, and flash frozen in liquid nitrogen.
Cell extracts for NMR were prepared using chloroform methanol as described (McGrath
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et al, 2006). Extracts from equivalent numbers of cells were lyophilized and deutriated
water (Sigma) added prior to analysis by NMR. Cell growth assays were performed by
HO33342 staining as described previously (Morrish et al., 2008).
NMR
13
C NMR spectra were acquired at 500 MHz and 20 0C on a Varian Unity-500 NMR
spectrometer equipped with a 5 mm broadband “switchable” probe (Varian, Palo Alto,
CA). The acquisition parameters were as follows: radio frequency pulse angle of 450,
spectral width of 27.6 kHz, acquisition time of 1.185 s and 3 s repetition period between
consecutive scans. Proton decoupling was achieved using a Waltz decoupling pulse
sequence. The signal-to-noise ratio was enhanced by a 5 Hz exponential multiplication
function. TMPSA (1 mM) was used as an internal reference. Appropriate correction
factors for nuclear Overhauser enhancement (NOE) and signal saturation were
determined. Spectra were processed with one left shift, 2 levels of zero fill prior to
Fourier transformation. 13C NMR multiplets were deconvoluted using NUTS (Acorn
NMR Freemont, CA). Concentrations of 13C labeled metabolites were calculated by
comparison to the relative integral of the TMPSA standard (1mM), following subtraction
of natural abundance integral values. To confirm spectral assignments heteronuclear
single quantum correlation (HSQC Varian pulse program , with TD (F2)= 1024 (512
complex pairs), TD(F1)= 512 (256 complex pairs), SW (1H)= 11 ppm, SW (13C)=180
ppm, GARP decoupling NS=128, AQ=0.78 s, D1=1.2 s, States-TPPI (phase = 1,2)
hypercomplex phase cycling, and heteronuclear multiple bond correlation (HMBC Varian
pulse program optimized for long range couplings, TD(F2)= 2048 (1024 complex pairs),
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TD(F1)=800 (400 complex pairs), NS=128, D1= 1.2s, SW (1H)=11ppm,
SW(13C)=210ppm, AQ=0.155 s, GARP decoupling experiments were performed to
correlated 1H and 13C resonances. These spectra were acquired on a 600 MHz
spectrometer interfaced with a 14.1 T superconducting magnet using a Varian Cold
ProbeTM.
LC/MS/MS
Glutamic acid was analyzed using mass spectrometry in the form of butyl ester (Chace et
al., 1993). For improved signal to noise ratio and to remove tryptophane interference a
short reverse phase LCMS method was used. Agilent 1100 HPLC with Zorbax SB-1 C18
column (2.1x100mm, 80A pores and 3.6um particles) was coupled to EsquireLC ion trap
mass spectrometer operated in positive ionization mode. Water with 5% acetonitrile, 1%
acetic acid and acetonitrile with 1% acetic acid were used as solvents A and B
respectively. Gradient from 10% B to 64% B in 9 minutes was followed by 5min at
100%B and column was equilibrated during 12 minutes at a flow rate of 200 uL/min.
Glutamic acid retention time was 8.5 minutes.
Quantitative isotopic incorporation was analyzed by deconvolution of the
experimental isotope pattern using linear combination of contributions of unlabeled and
all theoretically possible
13
C labeled glutamate molecules. The percent value of
individual contributions was iteratively optimized to match the experimental isotope
pattern using Microsoft Excel.
Metabolic analysis
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Metabolic substrate provision to the TCA from different pathways was estimated from
the relative areas of the multiplets of glutamate C2, C3 and C4 in the 13C NMR spectra
using tcaCALC (Malloy et al., 1990). The 13C NMR data was fitted to a standard model
of catabolic mammalian cell metabolism. The model parameters that were considered
were: (1) the fraction of acetyl-CoA derived from the oxidation of [U-13C] pyruvate via
pyruvate dehydrogenase; (2) the fractional contribution of [U-13C] pyruvate to
oxaloacetate via pyruvate carboxylase; (3) the fraction of acetyl-CoA derived from
unlabeled acyl sources; (4) relative pyruvate carboxylase and pyruvate dehydrogenase
flux. All flux parameters are relative to that of citrate synthase, arbitrarily set at 1.0. The
standard error associated with each parameter estimate was calculated by Monte Carlo
simulation. Fittings were performed systematically until the experimental and calculated
13
C multiplet data showed the best fit.
Statistical analysis
Data are expressed as means +/- S.D. values for the indicated number of experiments.
Statistical comparisons were made using student T test and a p-value <0.05 was
considered statistically significant.
References
Chace, D.H., Millington, D.S., Terada, N., Kahler, S.G., Roe, C.R. and Hofman L.F.
(1993) Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and
tyrosine in neonatal blood spots by tandem mass spectrometry. Clin Chem. 39, 66-71.
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McGrath, B.M., Greenshaw, A.J., McKay, R., Slupsky, C.M., and Silverstone, P.H.
(2006). Lithium alters regional rat brain myo-inositol at 2 and 4 weeks: an ex-vivo
magnetic resonance spectroscopy study at 18.8 T. Neuroreport 17, 1323-1326.
Malloy, C.R., Sherry, A.D., and Jeffrey, F.M. (1990). Analysis of tricarboxylic acid cycle
of the heart using 13C isotope isomers. Am J Physiol 259: H987-95.
Morrish, F.M., Neretti, N. Sedivy, J.M. and Hockenbery, D. M. (2008) The oncogene cMyc coordinates regulation of metabolic networks to enable rapid cell cycle entry. Cell
Cycle 7: 1054-1066.
O'Connell, B.C., Cheung, A.F., Simkevich, C.P., Tam, W., Ren, X., Mateyak, M.K., and
Sedivy, J.M. (2003). A large scale genetic analysis of c-Myc-regulated gene expression
patterns. J Biol Chem 278, 12563-73.
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