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From the Lewis-Sigler Institute for Integrative Genomics andthe Department of Chemistry and Molecular Biology, Princeton University, Princeton, New Jersey 08544,the Rutgers Cancer Institute of New Jersey, New Brunswick, New Jersey 08903, and
Acetyl-CoA is an important anabolic precursor for lipid biosynthesis. In the conventional view of mammalian metabolism, acetyl-CoA is primarily derived by the oxidation of glucose-derived pyruvate in mitochondria. Recent studies have employed isotope tracers to show that in cancer cells grown in hypoxia or with defective mitochondria, a major fraction of acetyl-CoA is produced via another route, reductive carboxylation of glutamine-derived α-ketoglutarate (catalyzed by reverse flux through isocitrate dehydrogenase, IDH). Here, we employ a quantitative flux model to show that in hypoxia and in cells with defective mitochondria, oxidative IDH flux persists and may exceed the reductive flux. Therefore, IDH flux may not be a net contributor to acetyl-CoA production, although we cannot rule out net reductive IDH flux in some compartments. Instead of producing large amounts of net acetyl-CoA reductively, the cells adapt by reducing their demand for acetyl-CoA by importing rather than synthesizing fatty acids. Thus, fatty acid labeling from glutamine in hypoxia can be explained by spreading of label without net reductive IDH flux.
Background: Cancer cells in hypoxia were claimed to rely on reductive isocitrate dehydrogenase (IDH) for lipogenesis based on increased isotopic labeling of fatty acids from glutamine.
plays a central role in cellular adaptation to hypoxia, up-regulating glucose intake, glycolytic enzymes, and lactate secretion. HIF further suppresses glycolytic flux entering the TCA cycle through pyruvate dehydrogenase by inducing transcription of pyruvate dehydrogenase kinase 1 (PDK1), which is an inhibitor of pyruvate dehydrogenase (
). In some tumors, however, mitochondrial function is impaired by mutated mitochondrial proteins. For example, somatic mutations in the TCA cycle genes fumarate hydratase and succinate dehydrogenase are tumorigenic (
). In both cases, the mutations lead to the activation of HIF, which causes a pseudohypoxic state, resulting in similar phenotypes to those of hypoxic cells even in the presence of oxygen.
An important intermediate of oxidative mitochondrial metabolism is acetyl-CoA. Cytosolic acetyl-CoA is the main precursor for de novo fatty acid biosynthesis. The canonical pathway for production of cytosolic acetyl-CoA begins with the oxidation of glucose-derived pyruvate in mitochondria (see Fig. 1). The resulting mitochondrial acetyl-CoA is consumed by citrate synthase to convert oxaloacetate into citrate. Citrate may then be either oxidized in TCA cycle or shuttled to cytoplasm, where its cleavage by ATP citrate lyase produces cytosolic acetyl-CoA (
Recent studies have employed isotope tracing to study how acetyl-CoA is produced in mammalian cell by feeding with 13C-labeled glutamine, glutamate, or succinate and measuring the resulting labeling of citrate and fatty acids (
). Experiments in liver and cardiac cells established that a fraction of citrate and fatty acid 2-carbon units originates from α-ketoglutarate through reductive carboxylation of isocitrate dehydrogenase (IDH) (
Although these isotope tracer experiments unambiguously demonstrate reverse IDH flux, they do not address the question of whether there is actually net flux in the reductive direction. In general, isotope labeling patterns reflect gross (i.e. total) flux in a given direction, which may be offset by yet greater flux in the opposite direction and not necessarily net conversion. This key principle was elucidated more than half a century ago to refute claims that liver synthesizes glucose from fatty acids based on the experimental observation that feeding cells with labeled fatty acids results in glucose labeling (
In this work, we employ a quantitative flux model to examine oxidative and reductive IDH flux in cancer cells grown in hypoxia and in cells with defective mitochondria. We analyze the lung cancer cell line A549 grown in hypoxia and the osteosarcoma cell line 143B-CYTB with a defective electron transport chain. In both cell types, reductive IDH flux was recently claimed to have a central role in lipogenesis (
). We show that the observed fatty acid labeling from glutamine does not necessarily imply net reductive IDH flux. Indeed, by placing analytical bounds on the oxidative and reductive IDH fluxes based on metabolite isotope labeling, we find evidence for oxidative net flux in pseudohypoxia and for modest or no net flux in either direction in hypoxia. Thus, reductive IDH flux is not a major net contributor to acetyl-CoA production. Instead, cells cope with limited oxidative acetyl-CoA production by reducing the biosynthetic utilization of acetyl-CoA for fatty acid synthesis (
Cell lines were grown in Dulbecco's modified Eagle's media (DMEM) without pyruvate (Cellgro), supplemented with 10% dialyzed fetal bovine serum (HyClone). Isogenic 143B human osteosarcoma cells that contained (143B-CYTB) or lacked (143B-WT) a loss-of-function mutation in mitochondrial complex III were grown in an incubator containing 5% CO2 and ambient oxygen at 37 °C. For hypoxia experiments, A549 cells were grown inside a hypoxic chamber (Coy Laboratory Products) containing 1% oxygen and 5% CO2 at 37 °C. For labeling experiments, medium was prepared from DMEM without glutamine (Cellgro), with the desired isotopic form of glutamine added to a final concentration 0.584 g/liter. Metabolite extractions were conducted at 70–80% confluency.
For all metabolomic and isotope tracer experiments, metabolism was quenched, and metabolites were extracted by quickly aspirating media and immediately adding −80 °C 80:20 methanol:water extraction solution. Samples were analyzed for water-soluble metabolites and saponified fatty acids by a stand-alone orbitrap mass spectrometer (Exactive) operating in negative ion mode and coupled to reversed-phase ion-pairing chromatography as described previously (
). In addition, confirmatory measurements of water-soluble metabolites were acquired with a TSQ Quantum Discovery triple-quadrupole mass spectrometer operating in negative ion, multiple-reaction monitoring mode coupled to reversed-phase ion-pairing chromatography as described (
) and normalized by packed cell volume. Acetate secretion rate was measured using the acetic acid (ACS manual format) test kit (Megazyme; catalogue number K-ACET) according to manufacturer's instructions. Oxygen consumption was measured by a Seahorse XF24 flux analyzer (Seahorse Bioscience, North Billerica, MA). To measure oxygen uptake in hypoxia, the Seahorse instrument was placed in the hypoxia chamber with 1% oxygen.
Several recent studies have employed isotopic tracers to investigate how acetyl-CoA is produced in cancer cell lines in hypoxia and with defective mitochondria (
). Their conclusion was that acetyl-CoA is primarily made through reductive carboxylation of glutamine-derived α-ketoglutarate, suggesting potential therapeutic targets along this pathway for inhibiting hypoxic tumor growth. These studies followed previous studies of IDH reductive carboxylation flux in normal liver and cardiac cells (
). Here, we followed up on these studies and employed a quantitative flux model to analyze IDH flux in the same cancer cell lines. Our analysis shows that although reductive IDH flux indeed occurs in hypoxia and with mitochondrial deficiency, oxidative IDH flux persists, with net flux much less than flux in either direction.
A limitation of our analysis (as well as prior related analyses) is that it does not account for subcellular compartmentalization of most metabolites. For acetyl-CoA, we do account for the possibility of distinct labeling patterns in the mitochondria versus cytoplasm, using fatty acid labeling to infer cytosolic acetyl-CoA labeling. For other metabolites, the LC-MS approach employed here measures the isotopic labeling of the overall cellular pool, which may represent a mixture of different labeling patterns in distinct compartments (depending on the relative concentration of the metabolite in the various compartments and compartment volumes). Thus, we cannot rule out net reductive IDH flux in at least some compartments, e.g. if cytosolic and mitochondrial IDH are working in opposite directions, perhaps as a means for shuttling high energy electrons from mitochondria to cytoplasm (
Nevertheless, a simple mechanistic explanation for the observed labeling patterns involves simultaneous oxidative and reductive IDH flux due to near equilibrium between the isocitrate oxidation and α-ketoglutarate reductive carboxylation. Such bidirectional flux can result in extensive citrate and lipid labeling via reductive carboxylation without reductive IDH being a net contributor to citrate or acetyl-CoA production. In such cases, net acetyl-CoA production may come from glucose or other carbon sources, with these influxes mixing with the larger TCA influx from glutamine via the reversible IDH reaction. The outcome is an apparent predominance of glutamine as the source of two-carbon units, despite net production coming from other sources.
More complete examination of fatty acid labeling patterns in hypoxia and pseudohypoxia reveals that glutamine labeling occurs in parallel with a rise in fatty acids that do not label from glucose or from glutamine. Such fatty acids are scavenged from media, and their assimilation into lipids decreases cellular requirements for 2-carbon unit production, thereby mitigating the need for either pyruvate dehydrogenase flux or reductive carboxylation.
We thank I. F. M. deCoo and Carlos Moraes for the 143B cells, as well as Ralph Deberardinis, Eyal Gottlieb, Michel Nofal, and Gregory Stephanopoulos for commenting on this paper.