A Drosophila model of combined D-2- and L-2-hydroxyglutaric aciduria reveals a mechanism linking mitochondrial citrate export with oncometabolite accumulation

Abstract

The enantiomers of 2-hydroxyglutarate (2HG) are potent regulators of metabolism, chromatin modifications and cell fate decisions. Although these compounds are associated with tumor metabolism and commonly referred to as oncometabolites, both D- and L-2HG are also synthesized by healthy cells and likely serve endogenous functions. The metabolic mechanisms that control 2HG metabolism in vivo are poorly understood. One clue towards how cells regulate 2HG levels has emerged from an inborn error of metabolism known as combined D- and L-2HG aciduria (D-/L-2HGA), which results in elevated D- and L-2HG accumulation. Because this disorder is caused by mutations in the mitochondrial citrate transporter (CIC), citrate must somehow govern 2HG metabolism in healthy cells. The mechanism linking citrate and 2HG, however, remains unknown. Here, we use the fruit fly Drosophila melanogaster to elucidate a metabolic link between citrate transport and L-2HG accumulation. Our study reveals that the Drosophila gene scheggia (sea), which encodes the fly CIC homolog, dampens glycolytic flux and restricts L-2HG accumulation. Moreover, we find that sea mutants accumulate excess L-2HG owing to elevated lactate production, which inhibits L-2HG degradation by interfering with L-2HG dehydrogenase activity. This unexpected result demonstrates that citrate indirectly regulates L-2HG stability and reveals a feedback mechanism that coordinates L-2HG metabolism with glycolysis and the tricarboxylic acid cycle. Finally, our study also suggests a potential strategy for preventing L-2HG accumulation in human patients with CIC deficiency.This article has an associated First Person interview with the first author of the paper.

Keywords: 2-Hydroxyglutarate; L2HGDH; Oncometabolite; SLC25A1; Scheggia.

Fig. 1.

sea mutant larvae accumulate excess L-2HG. (A) L- and D-2HG in larvae were detected separately using a chiral derivatization method coupled with GC-MS. (B) Relative abundance of L-2HG and D-2HG in sea mutant (sea^Δ24/Df) and control (sea^Prec/Df) larvae. (C) The PCA score plots of GC-MS spectra show that the metabolic profile of sea^Δ24/Df mutants is significantly different from that of the sea^Prec/Df and w¹¹¹⁸/Df controls. (D) Targeted GC-MS analysis reveals that sea^Δ24/Df mutants display significant changes in pyruvate (pyr), lactate (lac), 2-hydroxyglutarate (2HG), citrate (cit), fumarate (fum) and malate (mal). 2-oxoglutarate (2OG) and succinate (suc) were not significantly altered in the mutant. (E,F) Ubiquitous expression of a UAS-sea transgene restores sea mRNA levels (E) in sea mutant larvae and rescues the metabolic phenotypes (F). For all panels, data are shown as mean±s.e.m., n=5 samples containing 15 mid-L3 larvae collected from independent mating bottles. *P<0.05, **P<0.01. Data were analyzed using a two-tailed Student's t-test with Bonferroni correction for multiple comparisons. Data are representative of at least two independent experiments.

Fig. 2.

sea mutants exhibit elevated levels of glycolytic flux. (A) The relative metabolic flux rates from ¹³C₆-glucose into pyruvate (pyr), lactate (lac), 2HG and citrate (cit). n=4. (B) Relative Pfk mRNA levels in sea^Δ24/Df mutant larvae that ubiquitously express a UAS-Pfk-RNAi transgene. n=3 samples containing 15 mid-L3 larvae collected from independent mating bottles. (C) Pfk-RNAi reduces pyruvate, lactate, and 2HG levels in sea mutant larvae. n=6 samples containing 15 mid-L3 larvae collected from independent mating bottles. All data are shown as mean±s.e.m. *P<0.05, **P<0.01. ***P<0.001. Data were analyzed using a two-tailed Student's t-test with Bonferroni correction for multiple comparisons. Data are representative of at least two independent experiments.

Fig. 3.

Drosophila CIC activity regulates glycolysis at a post-transcriptional level. (A) A schematic diagram illustrating the Drosophila glycolytic pathway. The number in parentheses represents the change in gene expression observed in sea mutants. (B) A comparison of Pfk, dLdh and dL2HGDH mRNA levels in the sea^Δ24/Df mutants and sea^Prec/Df controls. n=3 samples containing 15 mid-L3 larvae collected from independent mating bottles. (C) sea^Δ24/Df mutant larvae fed a semi-defined diet supplemented with 10 mM citrate for 24 h accumulated excess citrate (cit) and displayed significant decreases in pyruvate (pyr), lactate (lac) and 2HG. n=6 samples containing 15 mid-L3 larvae collected from independent mating bottles. All data are shown as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001. Data were analyzed using a two-tailed Student's t-test. Data are representative of at least two independent experiments.

Fig. 4.

sea mutants accumulate excess L-2HG as a result of decreased degradation. (A) GC-MS analysis of sea^Δ24/Df mutants and sea^Prec/Df controls reveals that lactate and 2HG levels are highly correlated in individual larval samples. (B) The relative abundance of lactate and 2HG in sea^Δ24/Df mutant larvae that ubiquitously express a UAS-dLdh-RNAi (dLdh-RNAi) transgene. (C) The relative abundance of pyruvate (pyr), lactate (lac), 2HG and citrate (cit) in dL2HGDH^12/14 single mutants compared with dL2HGDH^12/14; sea^Δ24/Df double mutants. Note that 2HG levels are similar in both strains. Data are shown as mean±s.e.m., n=6 samples containing 15 mid-L3 larvae collected from independent mating bottles. *P<0.05, **P<0.01, ***P<0.001. Data were analyzed using a two-tailed Student's t-test with Bonferroni correction for multiple comparisons. Data are representative of at least two independent experiments.

Fig. 5.

Schematic of how the CIC influences L-2HG accumulation. In wild-type larvae, CIC exports citrate from the mitochondria into the cytosol, which dampens glycolytic flux and restricts the amount of lactate produced by glycolysis. In contrast, mutations in sea result in decreased CIC activity, decreased cytosolic citrate levels and increased glycolytic flux. As a result, sea mutants synthesize excess lactate, which interferes with L2HGDH activity and promotes L-2HG accumulation. CIC, mitochondrial citrate carrier; LDH, lactate dehydrogenase; L-2HG, L-2-hydroxyglutarate; TCA, tricarboxylic acid; 2OG, 2-oxoglutarate.