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Supplmental Figure 1: Metabolic pathways of [U-13C]glucose
Label distribution (stars) during [U-13C]glucose metabolism through the Krebs cycle
after glycolytic formation of [3-13C]pyruvate, which is converted via PDH and PC to [213
C]acetyl-CoA and [3-13C]oxaloacetate, respectively. The fluxes through PDH and PC lead
to a different 13C-labelling pattern in the Krebs cycle intermediate citrate, and are finally
measured by the isotopomer pattern of glutamate in 13C-NMR spectra. The same isotopomer
pattern as in glutamate is seen in glutamine and the (-glutamyl)-residue of glutathione. PC:
pyruvate carboxylase; PDH: pyruvate dehydrogenase.
Supplemental Figure 2: De novo synthesis of GSH and Glu through PDH after
treatment with NAC compared to alternative GSH precursors
The fractional
13
C-enrichments and total amounts (nmol/g ww) of
13
C-labelled GSH
(glutathione) and Glu (glutamate) synthesized after conversion of [U-13C]glucose via PDH
(pyruvate dehydrogenase) ([4,5-13C]isotopomers) were calculated from 1H- and
13
C-NMR
spectra of mouse liver extracts. The mice were treated with 500 mg/kg [U-13C]glucose
intravenously and 300 mg/kg NAC or equimolar amounts of cysteine, methionine or
hypotaurine intraperitoneally for 45 minutes, and then the freeze-clamped livers were
extracted. Each experiment was performed on at least 4 animals. The data are expressed as
means ± SD. *p<0.05 versus saline-treated controls.
Supplemental Figure 3: Changes in metabolic parameters and serum ALT after
treatment with 3-NPA
Tissue concentrations of GSH (glutathione), HTau (hypotaurine) and Tau (taurine)
(µmol/g ww) were calculated from their resonances in 1H-NMR spectra of mouse liver
extracts. Concentrations of ATP (adenonsine triphosphate) were calculated from
31
P-NMR
spectra. The fractional 13C-ernichments in [4,5-13C]glutamate, reflecting glucose flux through
PDH (pyruvate dehydrogenase), were calculated from 1H- and
13
C-NMR spectra. Serum
alanine aminotransferase (ALT) was analyzed with a biochemical multianalyzer. Mice were
treated with 50 mg/kg 3-NPA (3-nitropropionic acid), sacrificed 5 h later, and then the freezeclamped livers were extracted. 300 mg/kg NAC was administered 45 min before 3-NPA
treatment. 500 mg/kg [U-13C]glucose was administered intravenously 45 min before sacrifice.
Cys: cysteine; Met: methionine; HTau: hypotaurine. Each experiment was performed on at
least 4 animals. The data are expressed as means ± SD. *p<0.05 versus mice treated with 3NPA alone.