FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2120553786> ?p ?o ?g. }

• W2120553786 endingPage "385" @default.
• W2120553786 startingPage "372" @default.
• W2120553786 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 [1–13C]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. 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 [1–13C]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. HIF is necessary and sufficient to induce reductive carboxylation in RCC cell lines HIF promotes the reductive carboxylation flux by reducing citrate levels HIF-expressing tumors display reductive carboxylation in vivo HIF renders RCC cells sensitive to glutaminase inhibition in vitro and in vivo Cancer cells undergo fundamental changes in their metabolism to support rapid growth, adapt to limited nutrient resources, and compete for these supplies with surrounding normal cells. One of the metabolic hallmarks of cancer is the activation of glycolysis and lactate production even in the presence of adequate oxygen. This is termed the Warburg effect, and efforts in cancer biology have revealed some of the molecular mechanisms responsible for this phenotype (Cairns et al., 2011Cairns R.A. Harris I.S. Mak T.W. Regulation of cancer cell metabolism.Nat. Rev. Cancer. 2011; 11: 85-95Crossref PubMed Scopus (3564) Google Scholar). More recently, 13C isotopic studies have elucidated the complementary switch of glutamine metabolism that supports efficient carbon utilization for anabolism and growth (DeBerardinis and Cheng, 2010DeBerardinis R.J. Cheng T. Q’s next: the diverse functions of glutamine in metabolism, cell biology and cancer.Oncogene. 2010; 29: 313-324Crossref PubMed Scopus (896) Google Scholar). Acetyl-CoA is a central biosynthetic precursor for lipid synthesis, being generated from glucose-derived citrate in well-oxygenated cells (Hatzivassiliou et al., 2005Hatzivassiliou G. Zhao F. Bauer D.E. Andreadis C. Shaw A.N. Dhanak D. Hingorani S.R. Tuveson D.A. Thompson C.B. ATP citrate lyase inhibition can suppress tumor cell growth.Cancer Cell. 2005; 8: 311-321Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar). Warburg-like cells, and those exposed to hypoxia, divert glucose to lactate, raising the question of how the tricarboxylic acid (TCA) cycle is supplied with acetyl-CoA to support lipogenesis. We and others demonstrated, using 13C isotopic tracers, that cells under hypoxic conditions or defective mitochondria primarily utilize glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) or 2 (IDH2) (Filipp et al., 2012Filipp F.V. Scott D.A. Ronai Z.A. Osterman A.L. Smith J.W. Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is required for lipogenesis in hypoxic melanoma cells.Pigment Cell Melanoma Res. 2012; 25: 375-383Crossref PubMed Scopus (136) Google Scholar; Metallo et al., 2012Metallo C.M. Gameiro P.A. Bell E.L. Mattaini K.R. Yang J. Hiller K. Jewell C.M. Johnson Z.R. Irvine D.J. Guarente L. et al.Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia.Nature. 2012; 481: 380-384Crossref Scopus (1232) Google Scholar; Mullen et al., 2012Mullen A.R. Wheaton W.W. Jin E.S. Chen P.H. Sullivan L.B. Cheng T. Yang Y. Linehan W.M. Chandel N.S. DeBerardinis R.J. Reductive carboxylation supports growth in tumour cells with defective mitochondria.Nature. 2012; 481: 385-388Crossref Scopus (898) Google Scholar; Wise et al., 2011Wise D.R. Ward P.S. Shay J.E. Cross J.R. Gruber J.J. Sachdeva U.M. Platt J.M. DeMatteo R.G. Simon M.C. Thompson C.B. Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of α-ketoglutarate to citrate to support cell growth and viability.Proc. Natl. Acad. Sci. USA. 2011; 108: 19611-19616Crossref PubMed Scopus (723) Google Scholar). The transcription factors hypoxia inducible factors 1α and 2α (HIF-1α, HIF-2α) have been established as master regulators of the hypoxic program and tumor phenotype (Gordan and Simon, 2007Gordan J.D. Simon M.C. Hypoxia-inducible factors: central regulators of the tumor phenotype.Curr. Opin. Genet. Dev. 2007; 17: 71-77Crossref PubMed Scopus (376) Google Scholar; Semenza, 2010Semenza G.L. HIF-1: upstream and downstream of cancer metabolism.Curr. Opin. Genet. Dev. 2010; 20: 51-56Crossref PubMed Scopus (989) Google Scholar). In addition to tumor-associated hypoxia, HIF can be directly activated by cancer-associated mutations. The von Hippel-Lindau (VHL) tumor suppressor is inactivated in the majority of sporadic clear-cell renal carcinomas (RCC), with VHL-deficient RCC cells exhibiting constitutive HIF-1α and/or HIF-2α activity irrespective of oxygen availability (Kim and Kaelin, 2003Kim W. Kaelin Jr., W.G. The von Hippel-Lindau tumor suppressor protein: new insights into oxygen sensing and cancer.Curr. Opin. Genet. Dev. 2003; 13: 55-60Crossref PubMed Scopus (165) Google Scholar). Previously, we showed that VHL-deficient cells also relied on RC for lipid synthesis even under normoxia. Moreover, metabolic profiling of two isogenic clones that differ in pVHL expression (WT8 and PRC3) suggested that reintroduction of wild-type VHL can restore glucose utilization for lipogenesis (Metallo et al., 2012Metallo C.M. Gameiro P.A. Bell E.L. Mattaini K.R. Yang J. Hiller K. Jewell C.M. Johnson Z.R. Irvine D.J. Guarente L. et al.Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia.Nature. 2012; 481: 380-384Crossref Scopus (1232) Google Scholar). The VHL tumor suppressor protein (pVHL) has been reported to have several functions other than the well-studied targeting of HIF. Specifically, it has been reported that pVHL regulates the large subunit of RNA polymerase (Pol) II (Mikhaylova et al., 2008Mikhaylova O. Ignacak M.L. Barankiewicz T.J. Harbaugh S.V. Yi Y. Maxwell P.H. Schneider M. Van Geyte K. Carmeliet P. Revelo M.P. et al.The von Hippel-Lindau tumor suppressor protein and Egl-9-Type proline hydroxylases regulate the large subunit of RNA polymerase II in response to oxidative stress.Mol. Cell. Biol. 2008; 28: 2701-2717Crossref PubMed Scopus (96) Google Scholar), p53 (Roe et al., 2006Roe J.S. Kim H. Lee S.M. Kim S.T. Cho E.J. Youn H.D. p53 stabilization and transactivation by a von Hippel-Lindau protein.Mol. Cell. 2006; 22: 395-405Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), and the Wnt signaling regulator Jade-1. VHL has also been implicated in regulation of NF-κB signaling, tubulin polymerization, cilia biogenesis, and proper assembly of extracellular fibronectin (Chitalia et al., 2008Chitalia V.C. Foy R.L. Bachschmid M.M. Zeng L. Panchenko M.V. Zhou M.I. Bharti A. Seldin D.C. Lecker S.H. Dominguez I. Cohen H.T. Jade-1 inhibits Wnt signalling by ubiquitylating beta-catenin and mediates Wnt pathway inhibition by pVHL.Nat. Cell Biol. 2008; 10: 1208-1216Crossref PubMed Scopus (142) Google Scholar; Kim and Kaelin, 2003Kim W. Kaelin Jr., W.G. The von Hippel-Lindau tumor suppressor protein: new insights into oxygen sensing and cancer.Curr. Opin. Genet. Dev. 2003; 13: 55-60Crossref PubMed Scopus (165) Google Scholar; Ohh et al., 1998Ohh M. Yauch R.L. Lonergan K.M. Whaley J.M. Stemmer-Rachamimov A.O. Louis D.N. Gavin B.J. Kley N. Kaelin Jr., W.G. Iliopoulos O. The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix.Mol. Cell. 1998; 1: 959-968Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar; Thoma et al., 2007Thoma C.R. Frew I.J. Hoerner C.R. Montani M. Moch H. Krek W. pVHL and GSK3beta are components of a primary cilium-maintenance signalling network.Nat. Cell Biol. 2007; 9: 588-595Crossref PubMed Scopus (189) Google Scholar; Yang et al., 2007Yang H. Minamishima Y.A. Yan Q. Schlisio S. Ebert B.L. Zhang X. Zhang L. Kim W.Y. Olumi A.F. Kaelin Jr., W.G. pVHL acts as an adaptor to promote the inhibitory phosphorylation of the NF-kappaB agonist Card9 by CK2.Mol. Cell. 2007; 28: 15-27Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Hypoxia inactivates the α-ketoglutarate-dependent HIF prolyl hydroxylases, leading to stabilization of HIF. In addition to this well-established function, oxygen tension regulates a larger family of α-ketoglutarate-dependent cellular oxygenases, leading to posttranslational modification of several substrates, among which are chromatin modifiers (Melvin and Rocha, 2012Melvin A. Rocha S. Chromatin as an oxygen sensor and active player in the hypoxia response.Cell. Signal. 2012; 24: 35-43Crossref PubMed Scopus (97) Google Scholar). It is therefore conceivable that the effect of hypoxia on RC that was reported previously may be mediated by signaling mechanisms independent of the disruption of the pVHL-HIF interaction. Here we (1) demonstrate that HIF is necessary and sufficient for RC, (2) provide insights into the molecular mechanisms that link HIF to RC, (3) detected RC activity in vivo in human VHL-deficient RCC cells growing as tumors in nude mice, (4) provide evidence that the reductive phenotype of VHL-deficient cells renders them sensitive to glutamine restriction in vitro, and (5) show that inhibition of glutaminase suppresses growth of VHL-deficient cells in nude mice. These observations lay the ground for metabolism-based therapeutic strategies for targeting HIF-driven tumors (such as RCC) and possibly the hypoxic compartment of solid tumors in general. To test whether the inhibition of RC by pVHL requires its ability to bind and inactivate the alpha regulatory subunit of HIF, we tested a panel of VHL germline mutations that are linked to different clinical phenotypes of the VHL disease and differ in their affinity to bind HIF. Missense germline type 2A VHL mutations confer a low risk for RCC to their carrier individuals and retain an attenuated HIF binding and regulatory activity. In contrast, type 2B mutations, which are defective in HIF binding and regulation, confer a high risk for RCC. On the other hand, type 2C VHL germline mutations are associated with an increased risk of pheochromocytomas, but not RCC, and they retain the ability to bind and inactivate HIF in a manner similar to wild-type protein, an observation that suggests that type 2C mutations inactivate HIF-independent function(s) of pVHL (Li et al., 2007Li L. Zhang L. Zhang X. Yan Q. Minamishima Y.A. Olumi A.F. Mao M. Bartz S. Kaelin Jr., W.G. Hypoxia-inducible factor linked to differential kidney cancer risk seen with type 2A and type 2B VHL mutations.Mol. Cell. Biol. 2007; 27: 5381-5392Crossref PubMed Scopus (83) Google Scholar). We infected VHL-deficient human UMRC2 renal carcinoma cells with retroviruses and generated polyclonal cell populations expressing wild-type pVHL or type 2A (Y112H), type 2B (Y98N, Y112N), and type 2C (L188V) pVHL mutants. First, we confirmed that expression of HIF-1α/HIF-2α and their downstream target GLUT1 were downregulated in cells expressing wild-type pVHL or type 2C mutant protein (Figure 1A). The effect of type 2A pVHL mutations appeared intermediate between wild-type and type 2B mutations regarding GLUT1 (Figure 1A, GLUT1 lane). To profile the metabolic phenotype of the engineered cell lines, we labeled the cells with either [U13-C6]glucose or [1-13C1]glutamine and determined the incorporation of each tracer in TCA cycle metabolites using gas chromatography-mass spectrometry (GC-MS) (Figure 1B). Reintroduction of wild-type pVHL or type 2C pVHL mutant suppressed the contribution of reductive carboxylation from glutamine (Figure 1C), as determined by the degree of labeled carbon (M1 enrichment) of the indicated metabolites (see Figure 1B, green circles). Conversely, type 2B mutants (Y112N/Y98N, which are defective in HIF inactivation) exhibited active RC to a level comparable to VHL-deficient control cells. We observed a concurrent regulation in glucose metabolism in the different VHL mutants. Reintroduction of wild-type or type 2C pVHL mutant, which can meditate HIF-α destruction, stimulated glucose oxidation via pyruvate dehydrogenase (PDH), as determined by the degree of 13C-labeled TCA cycle metabolites (M2 enrichment) (Figures 1D and 1E). In contrast, reintroduction of an HIF nonbinding Type 2B pVHL mutant failed to stimulate glucose oxidation, resembling the phenotype observed in VHL-deficient cells (Figures 1D and 1E). Additional evidence for the overall glucose utilization was obtained from the enrichment of M3 isotopomers using [U13-C6]glucose (Figure S1A), which shows a lower contribution of glucose-derived carbons to the TCA cycle in VHL-deficient RCC cells (via pyruvate carboxylase and/or continued TCA cycling). To test the effect of HIF activation on the overall glutamine incorporation in the TCA cycle, we labeled an isogenic pair of VHL-deficient and VHL-reconstituted UMRC2 cells with [U-13C5]glutamine, which generates M4 fumarate, M4 malate, M4 aspartate, and M4 citrate isotopomers through glutamine oxidation. As seen in Figure S1B, VHL-deficient/VHL-positive UMRC2 cells exhibit similar enrichment of M4 fumarate, M4 malate, and M4 asparate (but not citrate) showing that VHL-deficient cells upregulate reductive carboxylation without compromising oxidative metabolism from glutamine. Next, we tested whether HIF inactivation by pVHL is necessary to regulate the reductive utilization of glutamine for lipogenesis. To this end, we traced the relative incorporation of [U-13C6]glucose or [5-13C1]glutamine into palmitate. Labeled carbon derived from [5-13C1]glutamine can be incorporated into fatty acids exclusively through RC, and the labeled carbon cannot be transferred to palmitate through the oxidative TCA cycle (Figure 1B, red carbons). Tracer incorporation from [5-13C1]glutamine occurs in the one carbon (C1) of acetyl-CoA, which results in labeling of palmitate at M1, M2, M3, M4, M5, M6, M7, and M8 mass isotopomers. In contrast, lipogenic acetyl-CoA molecules originating from [U-13C6]glucose are fully labeled, and the labeled palmitate is represented by M2, M4, M6, M8, M10, M12, M14, and M16 mass isotopomers. VHL-deficient control cells and cells expressing pVHL type 2B mutants exhibited high palmitate labeling from the [5-13C1]glutamine; conversely, reintroduction of wild-type or type 2C pVHL mutant (L188V) resulted in high labeling from [U-13C6]glucose (Figures 2A and 2B , box inserts highlight the heavier mass isotopomers). Next, to determine the specific contribution from glucose oxidation or glutamine reduction to lipogenic acetyl-CoA, we performed isotopomer spectral analysis (ISA) of palmitate labeling patterns. ISA indicates that wild-type pVHL or pVHL L188V mutant-reconstituted UMRC2 cells relied mainly on glucose oxidation to produce lipogenic acetyl-CoA, while UMRC2 cells reconstituted with a pVHL mutant defective in HIF inactivation (Y112N or Y98N) primarily employed RC. Upon disruption of the pVHL-HIF interaction, glutamine becomes the preferred substrate for lipogenesis, supplying 70%–80% of the lipogenic acetyl-CoA (Figure 2C). This is not a cell-line-specific phenomenon, but it applies to VHL-deficient human RCC cells in general; the same changes are observed in 786-O cells reconstituted with wild-type pVHL or mutant pVHL or infected with vector only as control (Figure S2). Type 2A pVHL mutants (Y112H, which retain partial HIF binding) confer an intermediate reductive phenotype between wild-type VHL (which inactivates HIF) and type 2B pVHL mutants (which are totally defective in HIF regulation) as seen in Figures 1 and 2. Taken together, these data demonstrate that the ability of pVHL to regulate reductive carboxylation and lipogenesis from glutamine tracks genetically with its ability to bind and degrade HIF, at least in RCC cells. To test the hypothesis that HIF-2α is sufficient to promote RC from glutamine, we expressed a pVHL-insensitive HIF-2α mutant (HIF-2α P405A/P531A, marked as HIF-2α P-A) in VHL-reconstituted 786-O cells (Figure 3). HIF-2α P-A is constitutively expressed in this polyclonal cell population, despite the reintroduction of wild-type VHL, reflecting a pseudohypoxia condition (Figure 3A). We confirmed that this mutant is transcriptionally active by assaying for the expression of its targets genes GLUT1, LDHA, HK1, EGLN, HIG2, and VEGF (Figures 3B and S3A). As shown in Figure 3C, reintroduction of wild-type VHL into 786-O cells suppressed RC, whereas the expression of the constitutively active HIF-2α mutant was sufficient to stimulate this reaction, restoring the M1 enrichment of TCA cycle metabolites observed in VHL-deficient 786-O cells. Expression of HIF-2α P-A also led to a concomitant decrease in glucose oxidation, corroborating the metabolic alterations observed in glutamine metabolism (Figures 3D and 3E). Additional evidence of the HIF2α-regulation on the reductive phenotype was obtained with [U-13C5]glutamine, which generates M5 citrate, M3 fumarate, M3 malate, and M3 aspartate through RC (Figure 3F). Next, we tested whether VHL-independent HIF-2α expression was sufficient not only to stimulate RC in the TCA cycle but also to switch the substrate preference for lipogenesis from glucose to glutamine. Expression of HIF-2α P-A in 786-O cells phenocopied the loss-of-VHL with regards to glutamine reduction for lipogenesis (Figure 3G), suggesting that HIF-2α can induce the glutamine-to-lipid pathway in RCC cells per se. Although reintroduction of wild-type VHL restored glucose oxidation in UMRC2 and UMRC3 cells (Figures S3B–S3I), HIF-2α P-A expression did not measurably affect the contribution of each substrate to the TCA cycle or lipid synthesis in these RCC cells (data not shown). UMRC2 and UMRC3 cells endogenously express both HIF-1α and HIF-2α, whereas 786-O cells exclusively express HIF-2α. There is compelling evidence suggesting, at least in RCC cells, that HIF-α isoforms have overlapping—but also distinct—functions and their roles in regulating bioenergetic processes remain an area of active investigation. Overall, HIF-1α has an antiproliferative effect, and its expression in vitro leads to rapid death of RCC cells while HIF-2α promotes tumor growth (Keith et al., 2011Keith B. Johnson R.S. Simon M.C. HIF1α and HIF2α: sibling rivalry in hypoxic tumour growth and progression.Nat. Rev. Cancer. 2011; 12: 9-22Crossref PubMed Scopus (306) Google Scholar; Raval et al., 2005Raval R.R. Lau K.W. Tran M.G. Sowter H.M. Mandriota S.J. Li J.L. Pugh C.W. Maxwell P.H. Harris A.L. Ratcliffe P.J. Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma.Mol. Cell. Biol. 2005; 25: 5675-5686Crossref PubMed Scopus (757) Google Scholar). Because of this, we were not able to stably express the HIF-1α P-A mutant in cells that endogenously express HIF-2α only. To get insights into the role of HIF-α paralogs in promoting RC, we used mouse neonatal epithelial kidney (NEK) cells and selectively induced the expression of mouse HIF-1α or HIF-2α P-A in normoxia. Expressing HIF-1α P-A activated RC, consistent with our observation in cancer cell lines. In this model, HIF-2α P-A did not affect the contribution of this reaction to any of the TCA cycle metabolites, at least in the condition studied (Figure S3J). Thus, it is possible that the induction of RC by HIF-1α or HIF-2α is species- or cell-type-specific. Alternatively, there may be a redundant role of the paralogs, and/or one may adapt the control of the metabolic program in the absence of the other paralog. To determine absolute fluxes in RCC cells, we employed 13C metabolic flux analysis (MFA) as previously described (Metallo et al., 2012Metallo C.M. Gameiro P.A. Bell E.L. Mattaini K.R. Yang J. Hiller K. Jewell C.M. Johnson Z.R. Irvine D.J. Guarente L. et al.Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia.Nature. 2012; 481: 380-384Crossref Scopus (1232) Google Scholar). Herein, we performed MFA using a combined model of [U-13C6]glucose and [1-13C1]glutamine tracer data sets from the 786-O derived isogenic clones PRC3 (VHL−/−)/WT8 (VHL+) cells, which show a robust metabolic regulation by reintroduction of pVHL. To this end, we first determined specific glucose/glutamine consumption and lactate/glutamate secretion rates. As expected, PRC3 exhibited increased glucose consumption and lactate production when compared to WT8 counterparts (Figure 4A). While PRC3 exhibited both higher glutamine consumption and glutamate production rates than WT8 (Figure 4A), the net carbon influx was higher in PRC3 cells (Figure 4B). Importantly, the fitted data show that the flux of citrate to α-ketoglutarate was negative in PRC3 cells (Figure 4C). This indicates that the net (forward plus reverse) flux of isocitrate dehydrogenase and aconitase (IDH + ACO) is toward citrate production. The exchange flux was also higher in PRC3 than WT8 cells, whereas the PDH flux was lower in PRC3 cells. In agreement with the tracer data, these MFA results strongly suggest that the reverse IDH + ACO fluxes surpass the forward flux in VHL-deficient cells. The estimated ATP citrate lyase (ACLY) flux was also lower in PRC3 than in WT8 cells. Furthermore, the malate dehydrogenase (MDH) flux was negative, reflecting a net conversion of oxaloacetate into malate in VHL-deficient cells (Figure 4C). This indicates an increased flux through the reductive pathway downstream of IDH, ACO, and ACLY. Additionally, some TCA cycle flux estimates downstream of α-ketoglutarate were not significantly different between PRC and WT8 (Table S1). This shows that VHL-deficient cells maintain glutamine oxidation while upregulating reductive carboxylation (Figure S1B). This finding is in agreement with the higher glutamine uptake observed in VHL-deficient cells. Table S1 shows the metabolic network and complete MFA results. Similar MFA results were obtained in VHL-deficient vector versus VHL-reconstituted UMRC2 cells (data not shown). Together, the MFA data show that HIF expression reverses the net IDH flux, perhaps to compensate for a deficient citrate production due to inhibition of PDH. In support of the existing literature, our MFA data showed that HIF inhibits the absolute pyruvate dehydrogenase (PDH) flux (Kim et al., 2006Kim J.W. Tchernyshyov I. Semenza G.L. Dang C.V. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia.Cell Metab. 2006; 3: 177-185Abstract Full Text Full Text PDF PubMed Scopus (2593) Google Scholar; Papandreou et al., 2006Papandreou I. Cairns R.A. Fontana L. Lim A.L. Denko N.C. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption.Cell Metab. 2006; 3: 187-197Abstract Full Text Full Text PDF PubMed Scopus (1614) Google Scholar). We observed that citrate levels were depleted in VHL-deficient UMRC2 cells (Figure 5A), with the citrate-to-α-ketoglutarate ratio being equally decreased in the VHL-deficient and HIF-2α P-A-expressing RCC cell lines (Figures 5B and S4A). We hypothesized that low citrate levels are key in promoting RC through mass action. To test if the mechanism by which HIF promotes RC lies in its ability to reduce citrate levels, we restored the intracellular pools of citrate, hoping to suppress RC in the TCA cycle. First, we knocked down the HIF target PDK-1 (Figure 5C) and observed a decreased contribution of RC to the TCA cycle in VHL-deficient PRC3 cells (Figure 5D). Similarly, knocking down the ATP citrate lyase enzyme (Figure 5E) inhibited RC in PRC3 cells (relative contribution of RC in Figure 5F, total contribution of RC in Figure S4B). Next, we labeled VHL-deficient and VHL-reconstituted UMRC2 cells with [1-13C1]glutamine and supplemented the medium with 1 mM and 4 mM acetate. Addition of acetate decreased the relative (Figure 5G) and total contribution (Figure 5H) of RC in VHL-deficient cells, with minimal effects observed in the VHL-reconstituted counterparts. The M1 enrichment informs on RC for the formation of TCA cycle metabolites. The absence of a major effect of acetate on the minimal RC of VHL-reconstituted cells is consistent with the observation that addition of acetate to these cells did not increase the intracellular citrate levels significantly (Figures 5I and S4C). In contrast, addition of acetate to VHL-deficient cells led to a significant increase in their citrate levels (Figures 5I and S4C) (to a level similar to the one observed in VHL-positive cells) as well as significant inhibition of RC. To pinpoint the metabolic route of the acetate-to-citrate flux, we labeled the control and ACLY knockdown PRC3 cells with [U-13C2]acetate and observed an increased percentage of M2 citrate under ACLY knockdown conditions (Figure S4D), suggesting that the acetate rescue of citrate levels in VHL-deficient cells probably occurs through condensation with oxaloacetate via citrate synthase rather than through the reversibility of ACLY. We confirmed that acetate can serve as a lipogenic source in the pair of PRC3/WT8 cells (Figure S4E). We also cultured PRC3/WT8 cells with [5-13C1]glutamine in the presence of naturally labeled acetate. In line with the previous observations, the contribution of glutamine reduction to lipogenic acetyl-CoA was decreased under the presence of acetate (Figure S4F). Next, we directly supplemented the medium with citrate and rinsed the cells thoroughly with PBS before extraction to ensure measurement of the intracellular metabolite. Addition of 8 mM citrate decreased the M1 enrichment of citrate in VHL-deficient UMRC2 cells to half but, in contrast to acetate, did have a moderate effect on the minimal RC observed in VHL-reconstituted cells (Figure 5J). Interestingly the level of M1 fumarate, M1 aspartate, and M1 malate were unaffected. One could expect the addition of nonlabeled citrate to decrease, by definition, the percent enrichment of M1 citrate, regardless of its putative effect on RC. To control for this effect of exogenous citrate on the total formation of RC-derived citrate, we labeled the cells with [1-13C1]glutamine and [U-13C6]citrate and subtracted the total M6 citrate ions (which are exogenous and not metabolically generated by the cell) from the total citrate ions and calculated the total contribution of RC using the formula % M1 × (total ions−% M6 × total ions). Likewise, exogenous citrate significantly decreased the total contribution of RC in the VHL-deficient UMRC2 cells but also had a moderate effect in the RC observed in VHL-reconstituted cells (Figure 5K). This differential effect between acetate and citrate on RC is consistent with and supports our hypothesis that endogenous citrate levels regulate RC by mass action. Addition of citrate in the medium, in contrast to acetate, led to an increase in the citrate-to-α-ketoglutarate ratio (Figure 5L) and absolute citrate levels (Figure S4H) not only in VHL-deficient but also VHL-reconstituted cells. The ability of exogenous citrate, but not acetate, to also affect RC in VHL-reconstituted cells may be explained by compartmentalization differences or by allosteric inhibition of citrate synthase (Lehninger, 2005Lehninger A.L. The Citric Acid Cycle.in: Nelson D.L. Cox M.M. Lehninger Principles of Biochemistry. 4th edition. W.H. Freeman and Company, New York2005: 621-622Google Scholar); that is, the ability of acetate to raise the intracellular levels of citrate may be limited in (VHL-reconstituted) cells that exhibit high endogenous levels of citrate. Whatever the mechanism, the results imply that increasing the pools of intracellular citrate has a direct biochemical effect in cells with regards to their reliance on RC. Finally, we assayed the transcript and protein levels of enzymes involved in the reductive utilization of glutamine and did not observe significant differences between VHL-deficient and VHL-reconstituted UMRC2 cells (Figures S4I and S4J), suggesting that HIF does not promote RC by direct transactivation of these enzymes. The IDH1/IDH2 equilibrium is defined as follows:[α−ketoglutarate][NADPH][CO2][Isocitrate][NADP+]=K(IDH) Therefore, we sought to investigate whether HIF could affect the driving force of the IDH reaction by also enhancing NADPH" @default.
• W2120553786 created "2016-06-24" @default.
• W2120553786 creator A5009763658 @default.
• W2120553786 creator A5013547888 @default.
• W2120553786 creator A5022366237 @default.
• W2120553786 creator A5023526233 @default.
• W2120553786 creator A5024094716 @default.
• W2120553786 creator A5029244263 @default.
• W2120553786 creator A5038992904 @default.
• W2120553786 creator A5054893992 @default.
• W2120553786 creator A5082240684 @default.
• W2120553786 creator A5082836651 @default.
• W2120553786 creator A5089981020 @default.
• W2120553786 creator A5090901321 @default.
• W2120553786 date "2013-03-01" @default.
• W2120553786 modified "2023-10-17" @default.
• W2120553786 title "In Vivo HIF-Mediated Reductive Carboxylation Is Regulated by Citrate Levels and Sensitizes VHL-Deficient Cells to Glutamine Deprivation" @default.
• W2120553786 cites W1525523952 @default.
• W2120553786 cites W1571407612 @default.
• W2120553786 cites W1738677317 @default.
• W2120553786 cites W1811132283 @default.
• W2120553786 cites W1969734439 @default.
• W2120553786 cites W1979521719 @default.
• W2120553786 cites W1990607537 @default.
• W2120553786 cites W1994303821 @default.
• W2120553786 cites W1998351143 @default.
• W2120553786 cites W1998664010 @default.
• W2120553786 cites W2010356452 @default.
• W2120553786 cites W2016253024 @default.
• W2120553786 cites W2027705428 @default.
• W2120553786 cites W2028897202 @default.
• W2120553786 cites W2031485253 @default.
• W2120553786 cites W2033958147 @default.
• W2120553786 cites W2034427729 @default.
• W2120553786 cites W2035983112 @default.
• W2120553786 cites W2042702043 @default.
• W2120553786 cites W2046108263 @default.
• W2120553786 cites W2050397881 @default.
• W2120553786 cites W2056174640 @default.
• W2120553786 cites W2060592426 @default.
• W2120553786 cites W2064921878 @default.
• W2120553786 cites W2072885613 @default.
• W2120553786 cites W2075394689 @default.
• W2120553786 cites W2079121632 @default.
• W2120553786 cites W2082584138 @default.
• W2120553786 cites W2086546138 @default.
• W2120553786 cites W2087408461 @default.
• W2120553786 cites W2087714970 @default.
• W2120553786 cites W2088972647 @default.
• W2120553786 cites W2089346177 @default.
• W2120553786 cites W2092152920 @default.
• W2120553786 cites W2098972920 @default.
• W2120553786 cites W2101404359 @default.
• W2120553786 cites W2116516144 @default.
• W2120553786 cites W2125435853 @default.
• W2120553786 cites W2131078129 @default.
• W2120553786 cites W2134960373 @default.
• W2120553786 cites W2145675430 @default.
• W2120553786 cites W2149343603 @default.
• W2120553786 cites W2151980465 @default.
• W2120553786 cites W2164816766 @default.
• W2120553786 cites W2414026282 @default.
• W2120553786 doi "https://doi.org/10.1016/j.cmet.2013.02.002" @default.
• W2120553786 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4003458" @default.
• W2120553786 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23473032" @default.
• W2120553786 hasPublicationYear "2013" @default.
• W2120553786 type Work @default.
• W2120553786 sameAs 2120553786 @default.
• W2120553786 citedByCount "262" @default.
• W2120553786 countsByYear W21205537862013 @default.
• W2120553786 countsByYear W21205537862014 @default.
• W2120553786 countsByYear W21205537862015 @default.
• W2120553786 countsByYear W21205537862016 @default.
• W2120553786 countsByYear W21205537862017 @default.
• W2120553786 countsByYear W21205537862018 @default.
• W2120553786 countsByYear W21205537862019 @default.
• W2120553786 countsByYear W21205537862020 @default.
• W2120553786 countsByYear W21205537862021 @default.
• W2120553786 countsByYear W21205537862022 @default.
• W2120553786 countsByYear W21205537862023 @default.
• W2120553786 crossrefType "journal-article" @default.
• W2120553786 hasAuthorship W2120553786A5009763658 @default.
• W2120553786 hasAuthorship W2120553786A5013547888 @default.
• W2120553786 hasAuthorship W2120553786A5022366237 @default.
• W2120553786 hasAuthorship W2120553786A5023526233 @default.
• W2120553786 hasAuthorship W2120553786A5024094716 @default.
• W2120553786 hasAuthorship W2120553786A5029244263 @default.
• W2120553786 hasAuthorship W2120553786A5038992904 @default.
• W2120553786 hasAuthorship W2120553786A5054893992 @default.
• W2120553786 hasAuthorship W2120553786A5082240684 @default.
• W2120553786 hasAuthorship W2120553786A5082836651 @default.
• W2120553786 hasAuthorship W2120553786A5089981020 @default.
• W2120553786 hasAuthorship W2120553786A5090901321 @default.
• W2120553786 hasBestOaLocation W21205537861 @default.
• W2120553786 hasConcept C161790260 @default.
• W2120553786 hasConcept C185592680 @default.
• W2120553786 hasConcept C207001950 @default.
• W2120553786 hasConcept C24428623 @default.

• next