Fig. 5: TCA rewiring after hexokinase isoenzyme switch in Huh7 cells.

a Glycogen quantification. b Creatinine and creatinine-P quantification. c This bubble chart compares intracellular metabolomes of Huh7 and Huh7-GCK⁺/HK2⁻ cells. Metabolite pool sizes larger in Huh7 are indicated in blue, whereas the one larger in Huh7-GCK⁺/HK2⁻ are shown in red. The size of bubbles inversely scales with p values between 5.10⁻² and 1.10⁻¹⁷ of differential metabolomics responses. d Metabolic fluxes for overall glucose consumption and lactate secretion by Huh7 and Huh7-GCK⁺/HK2⁻ cells. Indicated values correspond to differences in glucose or lactate concentrations in extracellular culture medium before and after 24 h of culture. e Mass isotopomer distribution vector of pyruvate in cells cultured with [U-¹³C]-glucose. Presented data correspond to n = 24 (c, d) or n = 16 (e) acquired spectra from N = 6 and N = 4 independent specimens, respectively. f Pyruvate carboxylase (PC) activity determined in cell homogenates. g Western-blot analysis of PC expression in Huh7 and Huh7-GCK⁺/HK2⁻ cells. h Western-blot analysis of pyruvate dehydrogenase (PDH) E1-alpha subunit phosphorylation at Ser293. i RNA-seq quantification of pyruvate dehydrogenase kinase 2 (PDK2) and pyruvate dehydrogenase phosphatase 2 (PDP2) (BH adjusted p value<0.05 from transcriptomic data). j Western-blot analysis of ATP-citrate Lyase (ACLY) phosphorylation at Ser455. k Succinate quantification in cell homogenates. l Succinate dehydrogenase (SDH) activity determined in cell homogenates. m Oxygen consumption rate (OCR) in Huh7 and Huh7-GCK⁺/HK2⁻ cells was determined with a Seahorse analyzer before and after the addition of oligomycin (Complex V inhibitor), FCCP (uncoupling agent), rotenone (Complex I inhibitor) and antimycin A (Complex III inhibitor) (n = 5). n Non-mitochondrial, complex I-dependent and complex III-dependent maximal OCR were calculated from m. Except otherwise indicated, data correspond to means ± SEM of 3 independent experiments and p values were determined by Student’s t-test.