Cytosolic aldose metabolism contributes to progression from cirrhosis to hepatocarcinogenesis

Abstract

Oxidative stress modulates carcinogenesis in the liver; however, direct evidence for metabolic control of oxidative stress during pathogenesis, particularly, of progression from cirrhosis to hepatocellular carcinoma (HCC), has been lacking. Deficiency of transaldolase (TAL), a rate-limiting enzyme of the non-oxidative branch of the pentose phosphate pathway (PPP), restricts growth and predisposes to cirrhosis and HCC in mice and humans. Here, we show that mitochondrial oxidative stress and progression from cirrhosis to HCC and acetaminophen-induced liver necrosis are critically dependent on NADPH depletion and polyol buildup by aldose reductase (AR), while this enzyme protects from carbon trapping in the PPP and growth restriction in TAL deficiency. Both TAL and AR are confined to the cytosol; however, their inactivation distorts mitochondrial redox homeostasis in opposite directions. The results suggest that AR acts as a rheostat of carbon recycling and NADPH output of the PPP with broad implications for disease progression from cirrhosis to HCC.

Extended Data Figure 1.. Impact of AR on carbon recycling and NADPH output by PPP.

A, Bar chart shows opposite effects of AR inactivation on concentrations of substrates sequestered in the non-oxidative branch of the PPP with respect to those depleted in the oxidative PPP and connected pathways. Data represent individual and mean values from five mice per genotype. Changes in metabolite concentration affected by TAL deficiency at two-tailed t-test p<0.05 are shown. B, Global impact of TAL deficiency on the metabolome is indicated by analysis of pathways arranged according to the scores from enrichment analysis (y axis: −log p) and from topology analysis using betweenness centrality to estimate node importance (x axis: impact: number of detected metabolites with significant p value, using two-tailed t-test via Metaboanalyst). TAL deficiency most prominently impacted the PPP, glycerophospholipid, and glycine/serine/threonine (G/S/T), valine/leucine/isoleucine (V/L/I), and alanine, aspartate, glutamate (A/D/E) amino acid metabolism. C, Global impact of AR deficiency on metabolic pathways, using two-tailed t-test via Metaboanalyst Pathway analysis module. D, Global impact of combined TAL and AR deficiency on metabolic pathways by comparison of DKO and WT mice, using two-tailed t-test via Metaboanalyst Pathway analysis module. E, Western blot detection recombinant AR (rAR, left panel) and assays of its enzyme activity (right panel). Data represent independent measurements for the following substrates: glyceraldehyde (GAD, n=28), ascorbate (n=2), E4P (n=3), S7P (n=4), OAA (n=7), PGA (n=8), erythrose (n=8), sedoheptulose (n=18). F, Schematic diagram of metabolic pathways affected by inactivation of the TAL-AR axis.

Extended Data Figure 2.. Segregation of metabolites by impact of AR inactivation in TAL deficiency.

A, Metabolites affected synergistically by inactivation of the TAL-AR axis. B, Metabolite changes corrected by inactivation of AR. Displayed metabolites exhibited significant fold changes in TAL deficiency at two-tailed t-test p < 0.05.

Extended Data Figure 3.. Western blot analysis of expression in genes implicated by RNAseq changes in livers of TAL-deficient mice at FDR p< 0.05.

A, Western blot analysis of protein levels corrected by inactivation of AR. *, two-tailed t-test p < 0.05. B, Western blot analysis of protein levels uncorrected by inactivation of AR. Representative blots and bar charts of cumulative analysis of five mice per genotype are shown for each gene. *, two-tailed t-test p < 0.05.

Extended Data Figure 4.. Effect of the TAL-AR axis on de novo GSH biosynthesis.

A, Measurement of GSH synthesis intermediates in liver of WT (n=4), TALKO (n=4), ARKO (n=5), and DKO mice (n=5). *, p < 0.05 relative to WT using two-tailed t test. Brackets indicate differences between mouse strains at p < 0.05. B, Enrichment of [M1-^13C]-PGA, [M2-^13C]-PGA, and [M5-^13C]-GSH in hepatocytes of WT (n=4), TALKO (n=4), ARKO (n=4), and DKO mice (n=4) labelled with [U-¹³C]-glutamine (DLM-1150–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. *, p < 0.05 relative to WT using one-tailed t-test. C, Schematic diagram of GSH biosynthesis involving substrates regulated by the TAL-AR axis.

Extended Data Figure 5.. Effect of aldose reductase inhibitors, zopolrestat and sorbinil, on serum replenishment-induced proliferation of HepG2 hepatoma and MCF7 breast carcinoma cells.

MCF7 or HepG2 cells seeded 96-well plates at 5,000 cells/well in complete Dulbecco’s minimal essential medium with 10% fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 25 µg/ml amphotericin B (GIBCO/ThermoFisher catalogue number 15240096) at 37 °C with 5% CO2. After 24 hours, sub-confluent cells were growth arrested in 0.1% FBS with or without sorbinil (Millipore/Sigma catalogue number S7701) or zopolrestat (Millipore/Sigma catalogue number Z4527). After 24 h, 10% serum was added to the medium and the cells were incubated for another 24 h. Cells were counted after trypan blue staining (upper and lower left panels and upper right panel) and viability was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay (lower right panel) . MTT powder was dissolved in PBS to a final concentration of 5 mg/ml, and 500 µl of MTT solution was added to cells and incubated for 1 h at 37^oC. Subsequently, 500 µl of isopropyl alcohol with 0.04 N HCl was added to dissolve the precipitate. Absorbance was read at the 570-nm wavelength subtracting background reading at the 650-nm wavelength. Data represent four experiments. Brackets reflect p values <0.05 as compared by 1-way ANOVA.

Extended Data Figure 6.. Effect of siRNA-mediated knockdown of IDH2 on metabolic flux through the TCA cycle in primary hepatocytes from age-matched female WT, TALKO, ARKO, and DKO mice.

A, Effect of IDH2 knockdown on enrichment of [M4-^13C]-citrate in hepatocytes labelled with [U-¹³C]-glutamine (DLM-1150–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. B, Effect of IDH2 knockdown on enrichment of [M1-^13C]-citrate, and [M2-^13C]-citrate in hepatocytes labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. Data represent mean ± SE of experiments using 4 WT, 4 TALKO, 4 ARKO, and 4 DKO mice. *, Brackets reflect p values 0.05 using unpaired two-tailed t tests for comparison of mice with different genotypes. Effect of IDH2 knockdown was evaluated with two-tailed paired t-tests; p values <0.05 are displayed.

Extended Data Figure 7.. IDH2 moderates carbon sequestration in the non-oxidative PPP in primary DKO hepatocytes.

A, Effect of IDH2 knockdown on enrichment of [M1-^13C]-S7P and [M2-^13C]-S7P in WT, TALKO, ARKO, and DKO hepatocytes labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. B, Effect of IDH2 knockdown on enrichment of [M5-^13C]-O8P and [M6-^13C]-O8P in WT, TALKO, ARKO, and DKO hepatocytes labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. C, Effect of IDH2 knockdown on enrichment of [M1-^13C]-R5P and [M2-^13C]-R5P and [M1-¹³C]-G6P and [M2-¹³C]-G6P in WT, TALKO, ARKO, and DKO hepatocytes labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. D, Effect of IDH2 knockdown on enrichment of [M2-^13C]-erythronic acid in WT, TALKO, ARKO, and DKO hepatocytes labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. Data represent mean ± SE of experiments using 4 WT, 3 TALKO, 4 ARKO, and 4 DKO mice. *, p < 0.05 relative to WT using unpaired two-tailed t tests. Brackets reflect p values 0.05 using 2-way ANOVA.

Figure 1.. Inactivation of AR blocks hepatocarcinogenesis and susceptibility to APAP in TAL deficiency.

A, Western blot analyses of TAL, AR, pJNK, and c-jun in livers from wild-type (WT or TAL^+/+AR^+/+), TAL-deficient (TALKO or TAL^−/−AR^+/+), AR-deficient (ARKO or TAL^+/+AR^−/−), and double-deficient (DKO, or TAL^−/−AR^−/−) mice. B, Measurement of NADPH and GSH by LC-MS/MS. Data represent mean ± SEM of measurements were carried out from liver extracts of 4 mice per each genotype. Using two-tailed t-test, differences at p < 0.05 relative to WT are indicated by *; differences at p < 0.05 between other mouse strains are indicated by brackets. C, Detection of fibrosis in Gömöri-trichrome-stained liver tissues from TALKO and DKO mice. Pro-fibrotic Ito cells or fat-storing hepatic stellate cells are indicated with arrows in areas of higher magnification. Stellate cells were expanded in livers of TALKO (4.75-fold; p=0.007) and DKO mice relative to WT controls (6-fold; p=0.042). D, Detection of tumor (T) in TALKO but not in WT, ARKO, and DKO livers. E, Microscopy of tumor (T) reveals hepatocellular carcinoma (HCC) in TALKO liver. F, Frequency of HCC in WT, TALKO, ARKO, and DKO mice aged to 78 weeks. The number of animals that developed HCC is indicated: n; the total number of animals per genotype is shown in parenthesis; p values reflect comparison with two-tailed Fisher’s exact test. G, Prevalence of anisonucleosis was increased in TALKO mice relative to WT controls; p value reflect comparison with two-tailed chi-square test. H, Survival of WT, TALKO, ARKO, and DKO mice following APAP exposure. Percent survival of seven or more mice per strain were compared to eight WT mice by Mantel-Cox log-rank test using GraphPad software.

Figure 2.. TAL-AR axis controls growth and body size through carbon trapping in the non-oxidative branch of the PPP.

A, Representative images of body size (left panel) and cumulative weight differences at 24 weeks of age among WT, TALKO, ARKO, and DKO female mice (right panel). Data represent mean ± SE of five mice per genotype. Using two-tailed t-test, differences at p < 0.05 relative to WT are indicated by *; differences at p < 0.05 between other mouse strains are indicated by brackets. B, Metabolome analysis by LC-MS/MS shows progressive trapping of 4–8 carbon sugars in the non-oxidative branch of the PPP by inactivation of the TAL-AR axis. Heat diagrams show genotype-dependent changes of the top 30 metabolites. The color bar on the right side of the heat diagram depicts two-fold changes into positive (red) or negative direction (green) relative to the mean of each compound. C, Discrimination of the metabolomes of TALKO and WT mice by PLS-DA (left panel) and volcano plot (right panel). D, Discrimination of the metabolomes of ARKO and WT mice by PLS-DA (left panel) and volcano plot (right panel). E, Discrimination of the liver metabolomes of DKO and WT mice by PLS-DA (left panel) and volcano plot (right panel). The log2FC fold change and -log10p cut-off values in volcano plots of panels C-E were 2-fold and p<0.05, respectively.

Figure 3.. Effect of TAL, AR, and dual TAL and AR deletion on NADPH production and metabolic flux through the PPP in primary hepatocytes.

A, Schematic diagram of stable isotope labeling and tracing of isotopologue distributions across the PPP, glycolysis, and the TCA cycle. Hepatocytes were labelled with [3-²H]-glucose to assess NADPH production by the PPP, [1,2-¹³C]-glucose to assess carbon flux via the PPP, and [U-¹³C]-glutamine to assess carbon flux through the TCA cycle. Red circles mark ¹³C label within glucose or glutamine. Metabolites in blue are depleted in TALKO hepatocytes. NADPH molecules in purple are connected to enzyme reactions of regulatory impact, which are catalyzed by AR and IDH2. B, Enrichment of [3-²H]-NADPH in hepatocytes labelled with [3-²H]-glucose (DLM-9294-PK, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. Hydride formation was also noted in NADP, R5P, S7P, and G6P. C, Enrichment of [M1-¹³C]-S7P and [M2-¹³C]-S7P in hepatocytes labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. D, Enrichment of [M1-¹³C]-S7P and [M2-¹³C]-S7P and [M1-¹³C]-G6P and [M2-¹³C]-G6P in hepatocytes labelled with [U-¹³C]-glutamine (DLM-1150–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. Percent enrichment of labelled metabolites was calculated between cells cultured with and without stable isotopes. Data represent mean ± SE of experiments using 4 WT, 4 TALKO, 5 ARKO, and 5 DKO mice. *, p<0.05 reflect comparison with unpaired two-tailed t tests.

Figure 4.. Impact of AR inactivation on changes in gene transcription imposed by TAL deficiency.

The signaling cascade triggered by TAL deficiency and modulated by AR inactivation was evaluated by sequencing of RNA from livers of disease-free, 14–16-week-old, age-matched female WT, TALKO, ARKO, and DKO mice, 5 mice from each genotype. Out of 24,606 RNA sequences identified, 300 were significantly affected by TAL deficiency at a false discovery rate (FDR) p value < 0.05 (Supplementary Table 1). A, Segregation of changes in gene expression by impact of AR inactivation in TAL deficiency. Abnormal expression of 70 genes was improved, while expression of 13 genes was further distorted by inactivation of AR. Blue and red arrows indicate decreases and increases of gene expression, which have been confirmed on the protein level by western blot analyses. B, Impact of gene expression changes on biological processes after Bonferroni correction for multiple comparisons. Statistical analysis was performed using Panther gene ontology software (http://www.pantherdb.org/pathway). Pathway changes represent the input 300 genes in TAL mice and 526 genes in DKO mice, which were changed at FDR p<0.05 relative to WT controls.

Figure 5.. AR inactivation moderates the formation of polyols.

A, Steady state levels of C5-polyols and sorbitol in liver of WT (n=7), TALKO (n=6), ARKO (n=7), and DKO mice (n=8). Data represent mean ± SEM. Statistical analyses for all figure panels were performed with two-tailed t-test.*, p < 0.05 relative to WT. B, Enrichment of [M2-¹³C]-sorbitol in hepatocytes labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. Data represent mean ± SE of experiments using 4 WT, 4 TALKO, 5 ARKO, and 5 DKO mice. Statistical analyses for all figure panels were performed with two-tailed t-test. Brackets indicate differences between mouse strains at p < 0.05. C, Accumulation of sorbitol, C5-polyols, sedoheptitol, and erythritol in the urine of age-matched WT, TALKO, ARKO, and DKO mice. Metabolite concentrations were measured by LC-MS/MS and normalized to the mean of WT mice set at 1.0. Data represent mean ± SE of experiments using 5 WT, 8 TALKO, 9 ARKO, and 3 DKO mice. Statistical analyses for all figure panels were performed with two-tailed t-test. Brackets indicate differences between mouse strains at p < 0.05. D, Biomarkers of increased oxidative stress in the urine of age-matched WT (n=7), TALKO (n=6), ARKO (n=7), and DKO mice (n=8). Lipid hydroperoxides 4-hydroperoxy-2-nonenal (4-HpNE), 4-oxo-2-nonenal (4-ONE), and 4-hydroxy-2-nonenal (4-HNE), oxidized DNA 8-oxoguanine (8-oxoG) and 8-oxo-2-deoxyguanosine (8-oxo-dGuo), ophthalmic acid, and homocysteine were measured by LC-MS/MS. Data represent mean ± SEM of concentration values normalized to the mean WT mice set at 1.0. Statistical analyses for all figure panels were performed with two-tailed t-test. Brackets indicate differences between mouse strains at p < 0.05. E, Metabolism of sorbitol by 1875 TALKO hepatoma cells. 75,000 cells were cultured for 24 hours in 6-well plates and labelled with 25 mM or 50 mM [M6-¹³C]-sorbitol for 30 min (Cat. No. CLM-8529-PK, Cambridge Isotope Laboratories Inc., Tewksbury, MA). Before harvesting, 0.0125 mM 5-thio-glucose, an internal standard, was added to each well. Metabolite concentrations were measured by LC-MS/MS and normalized to the internal standard. Values are expressed as fold changes relative to the mean of control cells set at 1.0. Data represent mean ± SEM of 5 experiments. F, Effect of sorbitol on mitochondrial ROS production by 1875 TALKO hepatoma cells. 75,000 cells were seeded for 24 hours in 6-well plates and further treated in the absence (control) or presence of 25 mM or 50 mM sorbitol for 4 hours. The mitochondrial transmembrane potential, ΔΨm (TMRM/MTG), mitochondrial ROS production (DHR/MTG), superoxide (HE/MTG), H₂O₂ (DCF/MTG), and GSH (MCB/MTG) were measured by flow cytometry. Data represent mean ± SEM of mean fluorescent intensity (MFI) values of 5 experiments. Statistical analyses were performed with two-tailed t-test. Brackets indicate differences between mouse strains at p < 0.05. G, Effect of sorbitol on JNK phosphorylation in 1875 TALKO hepatoma cells. 150,000 cells were seeded for 24 hours in 6-well plates and further treated in the absence (control) or presence of 25 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500 mM sorbitol for 30 minutes. pJNK levels (Cell Signaling Technology Cat. No. 4668) were assessed relative to β-actin (Millipore Cat. No. Mab1501R) using western blot. Upper panel, representative western blot. Lower panel, cumulative analysis of dose-dependent pJNK activation by sorbitol. Values are expressed as fold changes relative to the mean of control cells set at 1.0. Data represent mean ± SEM of 5 experiments. Statistical analysis was performed with two-tailed t-test. *, p < 0.05 relative to control. H, Effect of sorbitol on proliferation of 1875 TALKO hepatoma cells. 2,500 cells were seeded for 24 hours in 6-well plates. After washing the wells twice with PBS, fresh medium was added to each well with or without (control) sorbitol for 4, 8, or 12 more hours and cellular DNA content was measured using the CyQUANT Cell Proliferation Assay (ThermoFisher Cat. No. C7026). Data represent mean ± SEM of CyQUANT fluorescence values of 5 experiments. Statistical analysis was performed with two-tailed t-test. Brackets indicate differences relative to control at p < 0.05.

Figure 6.. AR inactivation limits mitochondrial ETC activity at complex I and ROI production without affecting the accumulation of mitochondria, ATP synthesis or glycolytic capacity in TAL-deficient hepatocytes.

A, Cumulative analyses of O₂ consumption rates (OCR) through ETC complexes I, II, and IV. During the assay of each ETC complex, 150 uM ADP and 150 uM Pi were added to attain state 3 respiration; when the ADP has been exhausted state 4 respiration was attained; after achieving a stable rate for state 4 respiration, 2uM mClCCP was added to measure uncoupled O₂ consumption, which is an indicator of maximal ETC capacity. In all experiment, O₂ consumption was normalized to that of the WT control strain, which was studied in parallel and set at 1.0 for each ETC complex. Mitochondria were isolated from WT (n=8), TALKO (n=8), ARKO (n=3), and 8 DKO mice (n=8). Data represent mean ± SEM. Using two-tailed t-test, differences at p < 0.05 relative to WT are indicated by *; differences at p < 0.05 between other mouse strains are indicated by brackets. B, Measurement of ROI production (by HE and DHR fluorescence) and ΔΨm (by JC-1, DiOC6, and TMRM fluorescence) relative to mitochondrial mass (by NAO and MTG fluorescence) using flow cytometry. Mitochondria were isolated from WT (n=8), TALKO (n=8), ARKO (n=3), and 8 DKO mice (n=8). Data represent mean ± SEM. Using two-tailed t-test, differences at p < 0.05 relative to WT are indicated by *; differences at p < 0.05 between other mouse strains are indicated by brackets. C, Measurement of mitochondrial mass (by MTG fluorescence) and GSH in isolated mitochondria (MCB fluorescence). Mitochondria were isolated from 7 WT, 4 TALKO, 8 ARKO, and 5 DKO mice. Data represent mean ± SEM. D, Measurement of mitochondrial mass (by MTG fluorescence) and GSH in isolated hepatocytes (by MCB fluorescence) from 6 WT, 6 TALKO, 6 ARKO, and 6 DKO mice. Data represent mean ± SEM. E, Measurement of mitochondrial respiration via OCR by isolated hepatocytes from WT (n=5), TALKO (n=5), ARKO (n=8), and DKO mice (n=5). Data represent mean ± SEM. Left panel shows representative tracings from WT, TALKO, ARKO, and DKO mice recorded in parallel. Right panel shows cumulative analysis of baseline and maximal respiration and mitochondrial ATP production. F, Assessment of glycolytic activity by extracellular acidification rates (ECAR). Left panel shows representative tracings from WT, TALKO, ARKO, and DKO mice recorded in parallel. Right panel shows cumulative analysis of baseline ECAR, glycolysis rate, and glycolytic capacity from WT (n=6), TALKO (n=6), ARKO (n=8), and DKO mice (n=6). Data represent mean ± SEM. G, Steady-state levels of glycolysis and TCA metabolites in liver extracts of age-matched female WT, TALKO, ARKO, and DKO mice, using ≥ 4 mice per strain. Data represent mean ± SEM. H, Assessment of ATP, ADP, AMP and adenylate energy charge (AEC) calculated according to the Atkinson formula (AEC  =  ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP])) in liver extracts of age-matched female WT (n=5), TALKO (n=4), ARKO (n=5), and DKO mice (n=5). Data represent mean ± SEM. I, Schematic diagram of TCA cycle and glycolytic substrates that shuttle between mitochondria and the cytosol and transport reducing power. NADPH can be regenerated from NADP by oxidation of glutamate, isocitrate, malate, and oxaloacetate by GDH, IDH2, ME, and AR. The TCA cycle may operate in the forward direction, which supports electron transport and ATP production and involves NADPH production by IDH2. In the reverse reaction, IDH2 depletes NADPH.

Figure 7.. Effect of siRNA-mediated knockdown of IDH2 on metabolic flux through the TCA cycle and the PPP in 1875 TALKO hepatoma cells.

The data represent five independent experiments. P values reflect statistical analyses performed with two-tailed t-test in all panels. A, Left panels, Western blot detection of IDH2, TAL, and tubulin loading control in TALKO and WT HCC cell lines (ATCC CRL-2717 and ATCC CRL-2712). IDH2 was detected with mouse monoclonal antibody (Invitrogen catalogue number MA5–17271); TAL was detected with rabbit antibody 169 , tubulin was detected with rabbit monoclonal antibody (Cell Signaling catalogue number CST 2128L). Right panel, NADPH/NADP ratio was determined in 1875 TALKO cells treated with scrambled (Con) or IDH2 siRNA. P value represents comparison with two-tailed paired t-test of four independent experiments. B, Effect of IDH2 knockdown on enrichment of [M1-^13C]-citrate, [M2-^13C]-citrate, [M3-^13C]-citrate, [M4-^13C]-citrate, and [M5-^13C]-citrate in 1875 TALKO hepatoma cells labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) or [U-¹³C]-glutamine (DLM-1150–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. C, Effect of IDH2 knockdown on enrichment of [M1-^13C]-S7P, [M2-^13C]-S7P, [M3-^13C]-S7P, [M4-^13C]-S7P, [M5-^13C]-S7P, and [M6-^13C]-S7P, in 1875 TALKO hepatoma cells labelled with [1, 2-¹³C]-glucose (CLM-504–0.5, Cambridge Isotope Laboratories; Cambridge, MA) for 24 hours. D, Effect of IDH2 knockdown on the accumulation of [M2-¹³C]-sedoheptitol and [M2-¹³C]-C5-polyols in [1, 2-¹³C]-glucose-labelled TALKO hepatoma cells.