Intracellular metabolic gradients dictate dependence on exogenous pyruvate

Abstract

During developmental transitions, cells frequently remodel metabolic networks, including changing reliance on metabolites such as glucose and glutamine to fuel intracellular metabolic pathways. Here we used embryonic stem (ES) cells as a model system to understand how changes in intracellular metabolic networks that characterize cell state transitions affect reliance on exogenous nutrients. We find that ES cells in the naive ground state of pluripotency increase uptake and reliance on exogenous pyruvate through the monocarboxylate transporter MCT1. Naive ES cells, but not their more committed counterparts, rely on exogenous pyruvate even when other sources of pyruvate (glucose, lactate) are abundant. Pyruvate dependence in naive ES cells is a consequence of their elevated mitochondrial pyruvate consumption at the expense of cytosolic NAD⁺ regeneration. Indeed, across a range of cell types, increased mitochondrial pyruvate consumption is sufficient to drive demand for extracellular pyruvate. Accordingly, restoring cytosolic NAD⁺ regeneration allows naive ES cells to tolerate pyruvate depletion in diverse nutrient microenvironments. Together, these data demonstrate that intracellular metabolic gradients dictate uptake and reliance on exogenous pyruvate and highlight mitochondrial pyruvate metabolism as a metabolic vulnerability of naive ES cells.

Extended Data Fig. 1. Metabolite measurement in ESC conditioned media

a, Principal component analysis of nutrients present in conditioned media from S/L- and S/L+2i-cultured ESCs or media alone. Ellipses represent 95% confidence intervals within each sample group. Analysis is representative of 80 detected metabolites by LC-MS. b, Pyruvate consumption measured over 24 h in S/L- and S/L+2i-cultured ESCs normalized to area under the growth curve. c,d, Schematics showing experimental setup for ESC fate transitions. ESCs were converted to the naive ground state of pluripotency by the addition of inhibitions against MEK and GSK3β (2i) (c) and were induced to exit naive pluripotency by either removal of 2i and LIF with or without the addition of FGF2, Activin A, and KSR (d). For b, data are mean +/− s.d., n = 6 independent replicates, and significance was assessed using unpaired two-tailed Student’s t-test.

Extended Data Fig. 2. MCT1 levels are not limiting for pyruvate consumption by ESCs.

a, Expression of monocarboxylate transporters in S/L+2i-cultured ESCs. Slc16a7 and Slc16a8 were not detected. Data are from ref. b, Pyruvate consumption in S/L- or S/L+2i-cultured ESCs with MCT4 inhibition (VB124, 10 μM) added during 24 h of nutrient uptake measurement. c, Immunoblot of mouse ESCs deficient for MCT1 with addition of either cDNA encoding HA-tagged MCT1 or vector control. d, Pyruvate consumption in S/L+2i-cultured ESCs shown in panel c. e,f, RNA-seq (e) or proteomic (f) analysis of Slc16a1/MCT1 and Slc16a3/MCT4 during conversion from S/L to 2i/L medium. g,h, RNA-seq (g) or proteomic (h) analysis of Slc16a1/MCT1 and Slc16a3/MCT4 during conversion from 2i/L to S/L medium. Data in e-h are from ref. Data are mean +/− s.d., n=2 (a), 6 (b,d), or 3 (f,h) independent replicates. For b, significance was assessed by two-way ANOVA with Sidak’s multiple comparisons post-test relative to vehicle-treated cells. Significance was assessed for d using unpaired two-tailed Student’s t-test relative to each respective MCT1-deficient cell line. For f,h, significance was assessed by one-way ANOVA with Sidak’s multiple comparisons post-test relative to S/L (f) or 2i/LIF (h).

Extended Data Fig. 3. Mitochondrial pyruvate demand sets pyruvate uptake.

a, Bubble plot of gene set enrichment analysis for gene essentiality correlation with Slc16a1. GO:BP, Gene Ontology Biological Process; KEGG, Kyoto Encyclopedia of Genes and Genomes. Genes correlated with Slc16a1 are highly enriched for mitochondrial, TCA cycle, and oxidative phosphorylation gene processes. b,c, Schematic showing tracing of [U-¹³C]pyruvate (b) or [^1–13C]pyruvate (c) into the TCA cycle. d, Fraction of m+1 labeled citrate (left), malate (middle) or aspartate (right) from [^1–13C]pyruvate in S/L- or S/L+2i- cultured ESCs. e, Representative oxygen consumption rate profiles for experiments shown in Fig. 3c,d. f, Population doublings of S/L+2i-cultured ESCs treated with either 1 mM pyruvate, phenformin (50µM), or phenformin and 1 mM pyruvate for 48 h. Cells were additionally treated with either AZD3965 (MCT1i, 100 nM), UK5099 (MPCi, 10µM), or vehicle control. g-i, Pyruvate consumption in MEFs (g), C2C12 myoblasts (h), and H170 cells (i) treated with either mitochondrial pyruvate carrier inhibition (MPCi, UK5099, 10µM) or dichloroacetate (DCA, 5 mM) added during 24 h of nutrient uptake measurement. Data are mean +/− s.d., n = 3 (d, f) or n = 6 (g-i) independent replicates. For gene set enrichment analysis in a, p-values and false discovery rate were calculated using clusterProfiler v4.4.4 with Bonferroni correction. Significance was assessed by unpaired two-tailed Student’s t-test relative to S/L (d), by two-way ANOVA with Sidak’s multiple comparisons post-test relative to phenformin (f), or by one-way ANOVA with Sidak’s multiple comparisons post-test relative to vehicle (g-i).

Extended Data Fig. 4. Effect of manipulating mitochondrial pyruvate capture on cellular pyruvate uptake and retention

a, Schematic depicting regulation of mitochondrial pyruvate metabolism through the pyruvate dehydrogenase complex (PHDC). PDK, pyruvate dehydrogenase kinase. b, Immunoblot demonstrating increased levels of PDK1 and p-PDH in S/L relative to S/L+2i-cultured ESCs. c, Pdk1–4 transcript levels in ESCs following withdrawal of 2i/LIF. Data are from ref. d, Immunoblot of clonal mouse ESCs in which CRISPR/Cas9-mediated editing was used to target either a non-genic region of chromosome 8 (Ctrl) or Pdk1 (PDK-1 and PDK-2). e-f, Fractional enrichment of citrate from [U-¹³C]glucose (e) or pyruvate uptake (f) in ESC lines shown in panel d cultured as indicated. For f, pyruvate uptake is shown for cells cultured with either 1 mM (left) and 0.2 mM (right) extracellular pyruvate. g-j, Fractional enrichment of citrate from [U-¹³C]glucose (g,h) or pyruvate release (i,j) in either adipocyte (g,h) or myotube (i,j) differentiation. Data are mean +/− s.d., n = 2 (c), n = 3 (e, g, i), or n = 6 (f, h, j) independent replicates. Significance was assessed by two-way ANOVA with Sidak’s multiple comparisons post-test relative to control cells (e,f), or by unpaired two-tailed Student’s t-test relative to MEFs (g,h) or blasts (i,j).

Extended Data Fig. 5. Pyruvate facilitates NAD⁺ regeneration in naive ESCs.

a, Colony formation assay using S/L-cultured ESCs seeded at clonal density into medium containing 0.2 mM pyruvate and either MCT1i (AZD3965, 100 nM) or vehicle control. MCT1i was added 48 h before seeding and maintained for the duration of the assay. Following alkaline phosphatase staining, colonies were scored as naive, mixed, or differentiated. b-c, Pyruvate consumption in either MCT1-deficient ESC lines versus control (b) or MCT1-deficient ESC lines with addition of either cDNA encoding HA-tagged MCT1 or vector control (c) in S/L+2i media containing 0.2 mM pyruvate. d, Immunoblot of clonal mouse ESCs in which CRISPR/Cas9-mediated editing was used to target either a non-genic region of chromosome 8 (Ctrl) or Pdha1 (PDHA-1 and PDHA-2). For all subsequent experiments, the marked control cell line (*) was used. e-g, Fraction of m+2 labeled citrate from [U-¹³C]glucose (e), pyruvate/lactate ratio (f), or fraction of labeled serine from [U-¹³C]glucose (g) in S/L+2i-cultured control or PDHA1-deficient ESCs. Data are mean +/− s.d., n = 6 (a-c) or 3 (e-g) independent replicates. Significance was calculated in b,e-g by one-way ANOVA with Sidak’s multiple comparisons post-test relative to control cells or in a,c, by using unpaired two-tailed Student’s t-test relative to vehicle (a) or respective MCT1-deficient cell line (c).

Extended Data Fig. 6. Pyruvate supports naive ESCs by enabling cytosolic NAD⁺ regeneration.

a, Labeling of lactate from [U-¹³C]pyruvate in S/L- or S/L+2i-cultured ESCs. b, Simplified schematic depicting cytosolic NAD⁺ regeneration and de novo serine synthesis from glucose. LDH, lactate dehydrogenase. c,d, Fraction of serine labeled from [U-¹³C]glucose in ESCs cultured in S/L+2i with either inhibition of LDH (GSK2837808A, 40 μM) and indicated pyruvate concentration (c) or in control or MCT1-deficient ESCs with 1 mM pyruvate (d). e,f, Naive colony formation in S/L+2i-cultured ESCs (e) or percentage of Nanog^high cells in S/L-cultured ESCs (f) cultured with 1 mM pyruvate and treated with either LDHi (GSK2837808A, 40 μM) or vehicle. For e, LDHi was added 48 h before seeding and was maintained for the duration of the assay. g, Quantification of colony formation of S/L+2i-cultured ESCs in medium containing 1 or 0 mM pyruvate and 1 mM alpha-ketobutyrate (⍺KB) or vehicle control. h,i, Immunoblot showing Flag-tagged LbNOX expression in S/L+2i-cultured ESCs (h) and the pyruvate/lactate ratio (i) in these cells. Data are mean +/− s.d., n = 3 independent replicates, except for e,g (n = 6). Significance was assessed in c,g, by two-way ANOVA with Sidak’s multiple comparisons post-test relative to vehicle treatment. For d, signficance was assessed by one-way ANOVA with Sidak’s multiple comparisons post-test relative to control cells. In e,f,i, significance was assessed using unpaired two-tailed Student’s t-test relative to vehicle or vector control.

Extended Data Fig. 7. ESC identity determines pyruvate uptake and demand for NAD⁺ regeneration.

a, Principal component analysis of normalized gene counts in control or MCT1-deficient ESCs cultured in S/L or S/L+2i media. b, Heatmap depicting relative expression of the genes that are the top 500 most differentially expressed genes when comparing control and MCT1-deficient ESCs cultured in S/L+2i-media. Data are from the RNA-sequencing experiment shown in a. c, Relative expression of naive pluripotency and post-implantation associated genes in the samples shown in a. Values shown are log₂ fold change relative to row mean. d, Expression of pluripotency and trilineage differentiation genes in embryoid bodies generated from either control or MCT1-deficient ESCs. e, Differential gene expression of S/L-cultured LbNOX-expressing ESCs relative to vector control. Genes with adjusted p-value < 0.5 are shown in red. f, Relative expression of naive pluripotency and post-implantation associated genes in LbNOX-expressing ESCs or vector control. Values shown are log₂ fold change relative to row mean. j, Pyruvate uptake in LbNOX-expressing or control ESCs cultured in either S/L or S/L+2i. Data are mean +/− s.d., n = 3 (d) or 6 (g) independent replicates. For all transcriptional profiling experiments, n = 2 or 3 independent replicates. In e, p-values were calculated by DESeq2 using the Wald test. Significance was assessed in g by unpaired two-tailed Student’s t-test relative to vector control.

Extended Data Fig. 8. Lactate is a substrate for the TCA cycle in naive ESCs.

a, Quantification of naive colony formation by S/L+2i-cultured ESCs cultured in indicated concentrations of pyruvate and lactate. b, Schematic showing tracing of [U-¹³C]lactate into the TCA cycle. c-e, Fractional enrichment of citrate from [U-¹³C]pyruvate in S/L- or S/L+2i-cultured ESCs (c), control or MCT1-deficient S/L+2i-cultured ESCs (d), or S/L+2i-cultured ESCs with or without l0 mM lactate. Data are mean +/− s.d., n = 6 (a) or n = 3 (c-e) independent replicates. For a, significance was assessed by one-way ANOVA with Sidak’s multiple comparisons post-test relative to cells without pyruvate or lactate. For c-e, significance of overall fraction labeled was assessed by unpaired two-tailed Student’s t-test and individual isotopologue significance for citrate was assessed by two-way ANOVA with Sidak’s multiple comparisons post-test relative to S/L (c), control cells (d) or vehicle (e).

Figure 1.. Pyruvate is avidly consumed by naive ESCs.

a, Differences in nutrient consumption between S/L- and S/L+2i-cultured ESCs over 24 h. Values were normalized to starting amount of each metabolite present and protein concentration to determine the molar amount of each metabolite consumed per mg protein. b, Pyruvate consumption measured over 24 h in S/L- and S/L+2i-cultured ESCs. c, Separation of Nanog^high and Nanog^low populations by FACS. Total populations of Nanog-GFP ESCs cultured in S/L are shown in gray. The top 10% and bottom 10% populations (highlighted in green) were sorted and plated for nutrient uptake measurement (left). Nanog-GFP distribution at the time of media harvest (72 h after initial sorting) is shown on the right. d, Quantification of pyruvate uptake in Nanog^high, Nanog^low, and total Nanog-GFP populations shown in c (right). e, Pyruvate consumption during conversion to the naive, ground state of pluripotency upon addition of inhibitors against MEK and GSK3β (2i). f,g, Pyruvate consumption in ESCs cultured in 2i/LIF and subjected to 2i/LIF withdrawal (f) or EpiLC induction (g). Pyruvate consumption was measured over the final 24 h of indicated cell state transitions. For b,d,e,f,g, data are mean +/− s.d., n = 6 independent replicates. In b,f,g, significance was assessed in comparison to S/L (b) or 2i/LIF (f,g) using unpaired two-tailed Student’s t-test. For d and e, significance was assessed relative to total Nanog-GFP population (d) or S/L (e) using one-way ANOVA with Sidak’s multiple comparisons post-test.

Figure 2.. MCT1 is required for pyruvate consumption in ESCs.

a, Schematic depicting monocarboxylate transport by the Slc16 family of transporters. b, Pyruvate consumption in S/L- or S/L+2i-cultured ESCs with MCT1 inhibition (AZD3965, 100 nM) added during 24 h of nutrient uptake measurement. c, Immunoblot of clonal mouse ESCs in which CRISPR/Cas9-mediated editing was used to target either a non-genic region of chromosome 8 (Ctrl) or Slc16a1 (MCT1–1 and MCT1–2). d, Pyruvate consumption in ESC lines shown in panel c cultured as indicated. e, Immunoblot of ESCs expressing either HA-tagged Slc16a1 cDNA (encoding MCT1) or vector control. f, Representative immunofluorescence images of S/L-cultured ESCs expressing HA-tagged MCT1 (red) or vector control. DAPI staining is shown in blue. g, Pyruvate consumption of cells shown in panels e,f, cultured in either S/L or S/L+2i. Data are mean +/− s.d., n = 6 independent replicates. Significance was assessed for b,d, by two-way ANOVA with Sidak’s multiple comparisons post-test relative to control or vehicle-treated cells.

Figure 3.. Mitochondrial pyruvate demand sets pyruvate uptake.

a, Pearson correlation between Slc16a1 and all genes using dependency scores from CRISPR/Cas9 screens provided in DepMap 23Q4 release. Bsg, encoding the obligate chaperone for Slc16a1, is shown in blue; genes from the KEGG oxidative phosphorylation gene set are shown in red. b, Fractional enrichment of citrate (left), malate (middle) and aspartate (right) from [U-¹³C]pyruvate in S/L- or S/L+2i-cultured ESCs. Isotopologues are colored as indicated. c,d, Oxygen consumption rate of ESCs cultured in S/L and S/L+2i (c) or control and MCT1-deficient ESCs cultured in S/L+2i (d) with the addition of indicated substrates. Values shown are the first measurement following FCCP injection. e, Schematic depicting pharmacological manipulation of mitochondrial pyruvate metabolism. MPC, mitochondrial pyruvate carrier; PHDC, pyruvate dehydrogenase complex; DCA, dichloroacetate. f,g, Pyruvate consumption in S/L- or S/L+2i-cultured ESCs treated with either MPCi (f, UK5099, 10µM) or DCA (g, 5 mM) for 24 h. h, Intracellular pyruvate concentrations in ESCs cultured in S/L- or S/L+2i media containing pyruvate concentrations as indicated for 24 h. i, Pyruvate consumption in ESCs cultured in S/L or S/L+2i with the indicated concentrations of pyruvate for 24 h. j, Intracellular pyruvate concentrations of S/L- or S/L+2i-cultured ESCs treated with either MPCi (UK5099, 10µM) or DCA (5 mM) for 24 h. Data are mean +/− s.d., n = 3 (b) or n = 5 or 6 (c,d,f-j) independent replicates. In b, significance of overall fraction labeled was assessed by unpaired two-tailed Student’s t-test relative to S/L, and individual isotopologue significance for citrate (left) was assessed by two-way ANOVA with Sidak’s multiple comparisons post-test relative to S/L. For all other panels, significance was assessed by two-way ANOVA with Sidak’s multiple comparisons post-test relative to S/L, vehicle, or control cells (c,d,f,h-j), except for g where significance was assessed by unpaired two-tailed Student’s t-test relative to vehicle.

Figure 4.. Loss of pyruvate uptake impairs naive ESC growth.

a,b, Nanog-GFP distribution of ESCs cultured in S/L medium containing 1 or 0 mM pyruvate for 48 h. Percentage of Nanog^high cells is quantified in b. c, Colony formation assay using S/L-cultured ESCs seeded at clonal density into medium containing indicated pyruvate concentrations. Following alkaline phosphatase staining, colonies were scored as naive, mixed, or differentiated. d,e, Colony formation assay of S/L+2i-cultured ESCs seeded at clonal density into medium containing indicated pyruvate concentrations. Representative wells are shown in d and quantified in e. f-h, Quantification of colony formation assay of control and MCT1-deficient S/L+2i ESCs (f), MCT1-deficient versus MCT1-cDNA expressing S/L+2i ESCs (g), or control versus MCT1-deficient ESCs cultured with S/L+2i at varying pyruvate concentrations (h). Data are mean +/− s.d., n = 6 independent replicates, except for b (n = 4). For b,g, significance was assessed using unpaired two-tailed Student’s t-test relative to 1 mM pyruvate (b) or to each respective MCT1-deficient cell line (g). Significance was assessed in c,e,f, by one-way ANOVA with Sidak’s multiple comparisons post-test relative to 1 mM pyruvate (c,e) or control cells (f). For h, significance was assessed by two-way ANOVA with Sidak’s multiple comparisons post-test relative to 1 mM pyruvate.

Figure 5.. Pyruvate supports ESCs by enabling NAD⁺ regeneration

a, Pyruvate consumption in control or PDHA1-deficient ESC lines cultured in either S/L or S/L+2i. b, Quantification of colony formation assay of control or PDHA1-deficient ESC lines cultured in S/L+2i. c, Simplified schematic of pyruvate metabolism. LDH, lactate dehydrogenase. d, Pyruvate/lactate ratio in ESCs cultured in either S/L or S/L+2i medium. e, Quantification of colony formation assay of control or PDHA1-deficient ESC lines cultured in S/L+2i with indicated pyruvate concentrations. f, Schematic showing methods of NAD⁺ regeneration by electron acceptors or LbNOX. g,h, Quantification of colony formation of S/L+2i-cultured ESCs expressing LbNOX or vector control in medium containing 1 or 0 mM pyruvate (g) or in medium containing 1 mM pyruvate with or without MCT1i (h, AZD3965, 100nM). For h, MCT1i was added 48 h before seeding and maintained for the entire assay. Data are mean +/− s.d., n = 6 independent replicates, except for d (n = 3). Significance was assessed in a,e,g,h, by two-way ANOVA with Sidak’s multiple comparisons post-test relative to control cells. For b, significance was assessed by one-way ANOVA with Sidak’s multiple comparisons post-test relative to control cells, and for d significance was assessed using unpaired two-tailed Student’s t-test relative to S/L.

Figure 6.. Pyruvate consumption enables proliferation in uterine-like media.

a, Heatmap displaying relative abundance of metabolites in uterine fluid, plasma, and commercial stem cell media. Data from Harris et al. b,c,d, Growth curve of either control or MCT1-deficient ESCs (b) or unedited ESCs (c,d) in S/L+2i medium containing indicated concentrations of glucose, pyruvate, and lactate. e,f, Lactate (e) or pyruvate (f) consumption by S/L+2i-cultured ESCs in medium containing indicated concentrations of glucose, pyruvate, and lactate. g, Quantification of naive colony formation by either LbNOX-expressing or control ESCs cultured in S/L+2i medium with or without 10 mM lactate. h, Growth curve of either LbNOX-expressing or control ESCs cultured in S/L+2i medium with indicated concentrations of glucose, pyruvate, and lactate with or without MCT1i (AZD3965, 100nM). Data are mean +/− s.d., n = 3 (b,c,d,h) or n = 6 (e,f,g) independent replicates. Significance was assessed by two-way ANOVA with Sidak’s multiple comparisons post-test relative to control cells (b,h), to 0 mM lactate (c), to 0 mM pyruvate (d), or to each respective cell line and pyruvate condition without lactate (g). For e,f, significance was assessed using unpaired two-tailed Student’s t-test relative to 0 mM lactate.