Quantitative metabolic flux analysis reveals an unconventional pathway of fatty acid synthesis in cancer cells deficient for the mitochondrial citrate transport protein

Abstract

The mitochondrial citrate transport protein (CTP), encoded by SLC25A1, accommodates bidirectional trafficking of citrate between the mitochondria and cytosol, supporting lipid biosynthesis and redox homeostasis. Genetic CTP deficiency causes a fatal neurodevelopmental syndrome associated with the accumulation of L- and D-2-hydroxyglutaric acid, and elevated CTP expression is associated with poor prognosis in several types of cancer, emphasizing the importance of this transporter in multiple human pathologies. Here we describe the metabolic consequences of CTP deficiency in cancer cells. As expected from the phenotype of CTP-deficient humans, somatic CTP loss in cancer cells induces broad dysregulation of mitochondrial metabolism, resulting in accumulation of lactate and of the L- and D- enantiomers of 2-hydroxyglutarate (2HG) and depletion of TCA cycle intermediates. It also eliminates mitochondrial import of citrate from the cytosol. To quantify the impact of CTP deficiency on metabolic flux, cells were cultured with a set of 13C-glucose and 13C-glutamine tracers with resulting data integrated by metabolic flux analysis (MFA). CTP-deficient cells displayed a major restructuring of central carbon metabolism, including suppression of pyruvate dehydrogenase (PDH) and induction of glucose-dependent anaplerosis through pyruvate carboxylase (PC). We also observed an unusual lipogenic pathway in which carbon from glucose supplies mitochondrial production of alpha-ketoglutarate (AKG), which is then trafficked to the cytosol and used to supply reductive carboxylation by isocitrate dehydrogenase 1 (IDH1). The resulting citrate is cleaved to produce lipogenic acetyl-CoA, thereby completing a novel pathway of glucose-dependent reductive carboxylation. In CTP deficient cells, IDH1 inhibition suppresses lipogenesis from either glucose or glutamine, implicating IDH1 as a required component of fatty acid synthesis in states of CTP deficiency.

Functional Properties of the Mitochondrial Carrier System

“…Although the identity of the mitochondrial alanine carrier is currently unknown, it is functionally redundant with the MPC because of cytoplasmic and mitochondrial pyruvate-alanine interconversions. This principle is also illustrated by a recent report of an α-ketoglutarate bypass of the CIC in H460 non-small lung cancer cells [69]. Mitochondria support lipogenesis by importing pyruvate derived from glucose, and converting it to citrate.…”

“…The IDH2 reaction also reduces NADP + to NADPH, which is utilized to reduce glutathione and augment mitochondrial ROS defense (Figure 3C). Re-export of α-ketoglutarate may then be utilized for lipogenesis, as outlined earlier in this review [69]. This raises the possibility that citrate and α-ketoglutarate may be perpetually cycled to regenerate mitochondrial NADPH.…”

“…Citrate is then exported by the CIC and converted to acetyl-CoA by ATP-dependent citrate lyase as the starting molecule for acyl-CoA chain elongation. Yet, similar to the effects of MPC disruption on gluconeogenesis, disruption of the CIC impaired, but did not block, lipogenesis from glucose [69]. 13 C tracer metabolomic experiments showed that mitochondrial α-ketoglutarate, produced from citrate was exported to the cytoplasm, reductively carboxylated to isocitrate, isomerized to citrate, and utilized for lipogenesis [69].…”

“…Yet, similar to the effects of MPC disruption on gluconeogenesis, disruption of the CIC impaired, but did not block, lipogenesis from glucose [69]. 13 C tracer metabolomic experiments showed that mitochondrial α-ketoglutarate, produced from citrate was exported to the cytoplasm, reductively carboxylated to isocitrate, isomerized to citrate, and utilized for lipogenesis [69]. Thus, mitochondrial α-ketoglutarate transport capacity provides a level of functional redundancy for CIC activity.…”

“…Increased reductive carboxylation of α-ketoglutarate partially sustained lipogenesis, but numerous metabolite concentrations and patterns of flux were altered [69]. Critically, CIC knockout cells remained competent for the numerous biosynthetic processes required for the higher-order process of cell division.…”