In vivo HIF-mediated reductive carboxylation is regulated by citrate levels and sensitizes VHL-deficient cells to glutamine deprivation

Abstract

Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [(1-13)C]glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.

Figure 1. HIF Inactivation Is Necessary for Downregulation of Reductive Carboxylation by pVHL

(A) Expression of HIF-1 α, HIF-2α, and their target protein GLUT1 in UMRC2-derived cell lines, as indicated. (B) Carbon atom transition map: the fate of [1-¹³C₁] and [5-¹³C₁]glutamine used to trace reductive carboxylation in this work (carbon atoms are represented by circles). The [1-¹³C₁] (green circle) and [5-¹³C₁] (red circle) glutamine-derived isotopic labels are retained during the reductive TCA cycle (bold red pathway). Metabolites containing the acetyl-CoA carbon skeleton are highlighted by dashed circles. (C) Relative contribution of reductive carboxylation. (D and E) Relative contribution of glucose oxidation to the carbons of indicated metabolites (D) and citrate (E). Student’s t test compared VHL-reconstituted to vector-only or to VHL mutants (Y98N/Y112N). Error bars represent SEM. Pyr, pyruvate; Lac, lactate; AcCoA, acetyl-CoA, Cit, citrate; IsoCit, isocitrate; Akg, α-ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; OAA, oxaloacetate; Asp, aspartate; Glu, glutamate; PDH, pyruvate dehydrogenase; ME, malic enzyme; IDH, isocitrate dehydrogenase enzymes; ACO, aconitase enzymes; ACLY, ATP-citrate lyase; GLS, glutaminase. See also Figure S1.

Figure 2. HIF Inactivation Is Necessary for Downregulation of Reductive Lipogenesis by pVHL

(A and B) VHL-deficient UMRC2 cells were cultured for 3 days in the presence of either [U-¹³C₆]glucose or [5-¹³C₁]glutamine. Mass isotopomer distributions (MIDs) of biomass-extracted palmitate in parental cells reconstituted with empty vector control (vector), wild-type EE-VHL, or HA-VHL mutants and labeled with [5-¹³C₁]glutamine (A) or [U-¹³C₆]glucose (B). Note: M0 equals 70%–80%; scale shown up to 20%. Error bars represent SEM. (C) Specific contribution to lipogenic acetyl-CoA as estimated by the ISA method. Error bars represent 95% confidence intervals. See also Figure S2.

Figure 3. Expression of HIF-2α Is Sufficient to Induce Reductive Carboxylation and Lipogenesis from Glutamine in RCC Cells

(A) Schematic of stabilized HIF-2α expression under normoxia conditions. (B) Expression of pVHL, HIF-2α, and its target protein GLUT1 in 786-O-derived cells, as indicated. (C) Relative contribution of reductive carboxylation. (D and E) Relative contribution of glucose oxidation to the carbons of indicated metabolites (D) and citrate (E). (F) Evidence for induction of reductive carboxylation by HIF-2α P-A using [U-¹³C₅]glutamine. Student’s t test compared VHL-reconstituted or mutant HIF-2α-expressing cells to corresponding controls. Error bars represent SEM. (G) ISA results showing the specific contribution of glucose oxidation and glutamine reduction to lipogenic acetyl-CoA (error bars denote 95% confidence intervals). See also Figure S3.

Figure 4. Metabolic Flux Analysis of VHL-Deficient and VHL-Positive Cells

(A) Extracellular fluxes (Glc and Gln uptake, Glu and lactic acid secretion) in PRC3 (VHL^−/−) and WT8 (VHL⁺) isogenic cells. Error bars denote SEM. (B) Net influx of glutamine-derived carbons determined by the difference between glutamine uptake and glutamate secretion. Error bars denote SEM. (C) Flux estimates from combined [U-¹³C₆]glucose/[1-¹³C₁]glutamine MFA model. Student’s t test compared WT8 to PRC3 cells. GDH, glutamate dehydrogenase; AAT, aspartate aminotransferase; PDH, pyruvate dehydrogenase; CS, citrate synthase; IDH, isocitrate dehydrogenase; ACO, aconitase; MDH, malate dehydrogenase; ACLY, ATP-citrate lyase; Pyr transp, pyruvate transport between cytosol and mitochondria. Error bars denote 95% confidence intervals.

Figure 5. Regulation of HIF-Mediated Reductive Carboxylation by Citrate Levels

(A and B) Citrate ion counts (A) and citrate-to-α-ketoglutarate ratio (B) in RCC cells. Student’s t test compared VHL-reconstituted to VHL-deficient cells. (C) Validation of PDK-1 knockdown in VHL-deficient PRC3 cells. (D) Relative contribution of reductive carboxylation to the TCA cycle under PDK-1 knockdown conditions. Student’s t test compared PDK-1 knockdown to control cells. (E) Validation of ACLY knockdown in PRC3 cells. (F) Effect of ACLY knockdown on the relative contribution of reductive carboxylation. Student’s t test compared ACLY knockdown to control cells. (G and H) Effect of acetate and citrate on the activity of reductive carboxylation in VHL-deficient/VHL-reconstituted UMRC2 cells. Acetate suppresses the relative (G) and total (H) contribution of reductive carboxylation in VHL-deficient UMRC2 cells. (I) Rescue of citrate-to-α-ketoglutarate ratio by acetate addition. (J and K) Exogenous citrate suppresses the relative (J) and total (K) contribution of reductive carboxylation in VHL-deficient UMRC2 cells. (L) Rescue of citrate-to-α-ketoglutarate ratio by exogenous citrate in (K). ACLY, ATP-citrate lyase; PDK-1, pyruvate dehydrogenase kinase. Error bars denote SEM. See also Figure S4.

Figure 6. Evidence for Reductive Carboxylation Activity In Vivo

(A) Time course of ¹³C enrichment (%) of glutamine in tumor, normal kidney, and plasma extracts during infusion with [1-¹³C₁]glutamine. ¹³C enrichment at time zero was determined using control noninfused mice. (B) Percent M1 citrate in the different tissues as determined by GC-MS analysis. (C) High-resolution ¹³C NMR spectra showing ¹³C enrichment of carboxylic groups in tumor extracts of a [1-¹³C₁]glutamine-infused mouse when compared to a control mouse. (D) NMR analysis of ¹³C enrichment in tumor citrate during the time course of infusion determined by deconvolution of the carboxyl group signals. The area of the citrate signal was divided by tumor weight and normalized to the dioxane signal. Error bars represent SEM of n = 3.

Figure 7. VHL-Deficient Cells and Tumors Are Sensitive to Glutamine Deprivation

(A–E) Cell proliferation is normalized to the corresponding cell type grown in 1 mM glutamine-containing medium. Effect of treatment with glutaminase (GLS) inhibitor 968 in PRC3/WT8 (A) and UMRC2 cells (B). Rescue of GLS inhibition with dimethyl alpha-ketoglutarate (DM-Akg; 4 mM) or acetate (4 mM) in PRC3/WT8 clonal cells (C) and polyclonal 786-O cells (D). Effect of GLS inhibitor BPTES in UMRC2 cells (E). Student’s t test compares VHL-reconstituted cells to control cells in (A), (B), and (E) and DM-Akg or acetate-rescued cells to correspondent control cells treated with 968 only in (C) and (D) (asterisk in parenthesis indicates comparison between VHL-reconstituted to control cells). Error bars represent SEM. (F) GLS inhibitor BPTES suppresses growth of human UMRC3 RCC cells as xenografts in nu/nu mice. When the tumors reached 100mm³, injections with BPTES or vehicle control were carried out daily for 14 days (n = 12). BPTES treatment decreases tumor size and mass (see insert). Student’s t test compares control to BPTES-treated mice (F). Error bars represent SEM. (G) Diagram showing the regulation of reductive carboxylation by HIF.