Figure 1.
13C labeling patterns of glutamate derived from [2-13C]glucose.
Plain arrows represent flux through the indicated enzymatic process. Dotted arrows represent flux through the indicated multistep metabolic pathway. Circles represent the carbon backbone of the molecule. Filled black circles indicate the location of the 13C label upstream of PC and PDH. Filled green and purple circles indicate the location of the 13C label after metabolism through PC or PDH, respectively, and the first turn of the TCA cycle. Half-filled green and purple circles indicate the locations of the 13C label after metabolism through the second and subsequent turns of the TCA cycle on a population level (i.e.: may or may not be on the same molecule). The boxes represent the glutamate/glutamine molecules detected in the MR spectra. Adapted from Brekke et al. [27]. PC = pyruvate carboxylase, PDH = pyruvate dehydrogenase, TCA = tricarboxylic acid, α-KG = α-ketoglutarate.
Figure 2.
Representative 13C MR spectra of cell extracts post-incubation with [2-13C]glucose.
MR spectroscopy was performed on the aqueous phase of NHA IDH1 wild-type (top) and IDH1 mutant (bottom) cell extracts following 18 hours of incubation with medium containing 1 g/L [2-13C]glucose. [13C]glutamate peaks relevant for calculating pyruvate carboxylase and pyruvate dehydrogenase fractional fluxes, namely [1-13C], [2-13C], [3-13C], and [5-13C]glutamate, are highlighted in green (flux via pyruvate carboxylase) and purple (flux via pyruvate dehydrogenase). This data combined with 4 other spectra served to generate the results presented in Figure 3 and Table 1. Glu = glutamate, Gln = glutamine, 2-HG = 2-Hydroxyglutarate, IDH = isocitrate dehydrogenase, PC = pyruvate carboxylase, PDH = pyruvate dehydrogenase.
Figure 3.
[2-13C]glucose-derived fractional flux to glutamate and glutamine.
Fractional flux to glutamate (A&B) and to glutamine (C&D) via pyruvate carboxylase (in green) and (pyruvate dehydrogenase (in purple) for NHA IDHwt (solid) and IDHmut (striped) cells (data presented are averages of 3 repeats per cell line). Error bars represent standard deviations. Asterisks represent statistical significance (*: p<0.05). PC = pyruvate carboxylase, PDH = pyruvate dehydrogenase, IDHwt = isocitrate dehydrogenase wild-type, IDHmut = isocitrate dehydrogenase mutant.
Table 1.
13C labeling of glutamate and glutamine (fmol/cell and nmol/mg protein) from [2-13C]glucose, and PC, PDH and backflux fractional fluxes in IDHwt and IDHmut immortalized normal human astrocytes.
Figure 4.
Pyruvate carboxylase and pyruvate dehydrogenase enzyme activities, expression and protein levels.
(A) Enzymatic activities (n = 3), transcript levels (n = 9), and protein levels (n = 3) for pyruvate carboxylase (green) and pyruvate dehydrogenase (purple) in NHA IDHmut cells expressed as a percentage of their IDHwt counterparts. The horizontal dotted lines represents the baseline (i.e.: 100% of IDHwt). Error bars represent standard deviation. Asterisks represent statistical significance (*: p<0.05, **: p<0.01). (B) Representative Western blot bands used to quantify pyruvate carboxylase protein levels of NHA IDHwt and IDHmut cells. (C) Representative Western blot bands used to quantify pyruvate dehydrogenase protein levels of NHA IDHwt and IDHmut cells. Pyruvate carboxylase and pyruvate dehydrogenase quantification were normalized to glyceraldehyde-3-phosphate dehydrogenase and tubulin respectively. PC = pyruvate carboxylase, PDH = pyruvate dehydrogenase, IDHwt = isocitrate dehydrogenase wild-type, IDHmut = isocitrate dehydrogenase mutant, GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
Table 2.
Inhibitory pyruvate dehydrogenase phosphorylation and pyruvate dehydrogenase kinases and phosphatases transcript expression.
Figure 5.
Comparison of normalized expression scores (z-scores) for pyruvate carboxylase across de novo glioblastoma (A) and low grade glioma (B).
Boxes denote mean z-score and whiskers denote 2.5 to 97.5% for tumors in the specified group; IDH1/IDH2 mutant n = 8 and n = 174 and IDH wild-type n = 146 and n = 44 in (A) and (B), respectively. Data obtained from The Cancer Genome Atlas Data Portal (http://cancergenome.nih.gov.) A negative z-score denotes expression value below the glioblastoma population mean. Asterisks represent statistical significance (* p<0.01, ** p<0.0001). abbreviations.