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A D-2-hydroxyglutarate dehydrogenase mutant reveals a critical role for ketone body metabolism in Caenorhabditis elegans development [1]

['Olga Ponomarova', 'Department Of Systems Biology', 'University Of Massachusetts Chan Medical School', 'Worcester', 'Massachusetts', 'United States Of America', 'Hefei Zhang', 'Xuhang Li', 'Shivani Nanda', 'Thomas B. Leland']

Date: 2023-04

In humans, mutations in D-2-hydroxyglutarate (D-2HG) dehydrogenase (D2HGDH) result in D-2HG accumulation, delayed development, seizures, and ataxia. While the mechanisms of 2HG-associated diseases have been studied extensively, the endogenous metabolism of D-2HG remains unclear in any organism. Here, we find that, in Caenorhabditis elegans, D-2HG is produced in the propionate shunt, which is transcriptionally activated when flux through the canonical, vitamin B12-dependent propionate breakdown pathway is perturbed. Loss of the D2HGDH ortholog, dhgd-1, results in embryonic lethality, mitochondrial defects, and the up-regulation of ketone body metabolism genes. Viability can be rescued by RNAi of hphd-1, which encodes the enzyme that produces D-2HG or by supplementing either vitamin B12 or the ketone bodies 3-hydroxybutyrate (3HB) and acetoacetate (AA). Altogether, our findings support a model in which C. elegans relies on ketone bodies for energy when vitamin B12 levels are low and in which a loss of dhgd-1 causes lethality by limiting ketone body production.

Funding: This work was supported by grants GM122502 and DK068429 from the National Institutes of Health to A.J.M.W. and DK115690 to A.J.M.W. and F.S., and by a grant from the Li Weibo Institute for Rare Disease at University of Massachusetts Chan Medical School to A.J.M.W. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we use a loss-of-function mutation in C. elegans D-2-hydroxyglutarate dehydrogenase (D2HGDH), which converts D-2HG to αKG, to study endogenous D-2HG metabolism. We identified the C. elegans gene F54D5.12 as a one-to-one ortholog of human D2HGDH and named this gene dhgd-1 for D-2-hydroxyglutarate dehydrogenase. We find that D-2HG is produced by the propionate shunt, in the step in which HPHD-1 oxidizes 3-hydroxypropionate (3HP) to malonic semialdehyde (MSA). dhgd-1 deletion mutants are embryonic lethal and have mitochondrial defects. Surprisingly, however, while mitochondrial defects can be explained by 3HP accumulation [ 30 ], lethality is neither simply caused by accumulation of 3HP nor D-2HG. Therefore, loss of viability and mitochondrial defects are caused by distinct mechanisms and can be uncoupled. We find that dhgd-1 mutant animals up-regulate ketone body metabolism genes, suggesting that ketone bodies are limiting in these animals. Indeed, our metabolomic analysis shows that the breakdown of the ketogenic amino acids leucine and lysine is impaired in these mutants. Moreover, the ketone bodies 3HB and AA can partially rescue dhgd-1 mutant lethality. Altogether, our findings support a model in which ketone bodies are important for C. elegans viability.

The nematode Caenorhabditis elegans has been a powerful model organism for decades, and research on this “simple” animal has yielded great insights into development, aging, and other processes. Recently, C. elegans has also become a major model to study metabolism. Of its 20,000 or so protein-coding genes, more than 2,000 are predicted to encode metabolic enzymes. Of these, 1,314 have been annotated to specific metabolic reactions and incorporated into a genome-scale metabolic network model that can be used with flux balance analysis (FBA) to computationally model the animal’s metabolism [ 22 , 23 ]. Like in humans, vitamin B12 plays an important metabolic role in C. elegans propionate breakdown [ 24 – 27 ]. Vitamin B12 is exclusively synthesized by bacteria and, perhaps, some archaea, and is therefore mostly acquired by diet. On bacterial diets low in vitamin B12, such as the standard laboratory diet of Escherichia coli OP50, C. elegans transcriptionally activates 5 genes that comprise an alternative propionate breakdown pathway or propionate shunt [ 27 – 29 ]. This shunt is activated only when high levels of propionate persist and detoxifies this SCFA to acetyl-CoA in the mitochondria [ 28 ].

Ketone bodies provide another important energy source under conditions where glucose is limiting, such as in diabetic patients, or on low carbohydrate, or “keto” diets. Ketone bodies are produced in the liver and are essential for energy metabolism in peripheral tissues such as muscle and the brain. There are 3 ketone bodies, 2 of which, acetoacetate (AA) and 3-hydroxybutyrate (3HB), can serve as energy sources, with 3HB being the most prevalent. Ketone bodies are produced from the breakdown of fatty acids and amino acids. Two amino acids, lysine and leucine, are exclusively ketogenic: degradation of leucine yields acetyl-CoA and acetoacetate, while breakdown of lysine yields 3HB. Furthermore, acetyl-CoA produced by leucine (and fatty acids) is an important precursor for ketone body production.

In eukaryotes, the degradation of the branch chain amino acids (BCAAs) valine and isoleucine yields propionyl-CoA, which can be converted to the short chain fatty acid (SCFA) propionate. In addition, propionate is produced by the gut microbiota during the breakdown of dietary fiber [ 17 ]. Together with the other major SCFAs, acetate and butyrate, propionate forms an important source of energy for muscle, colon, and liver [ 18 , 19 ]. Thus, propionate serves an important metabolic and physiological function. However, propionate is toxic when it accumulates to high levels in the blood, which occurs in patients with propionic acidemia that carry mutations in either of the 2 propionyl-CoA carboxylase subunits [ 20 ]. These enzymes function together in the canonical, vitamin B12-dependent propionyl-CoA breakdown pathway that leads to the production of succinyl-CoA, which can anaplerotically enter the TCA cycle to produce energy [ 21 ].

The metabolite 2HG occurs as 2 enantiomers, L-2HG and D-2-hydroxyglutarate (D-2HG), each of which is oxidized by a specific dehydrogenase. Mutations in these dehydrogenases result in the inborn errors of human metabolism, L- and D-2-hydroxyglutaric aciduria, respectively [ 1 , 2 ]. These diseases cause the accumulation of 2HG in bodily fluids, delayed development, neurological and muscle dysfunction, and early death [ 3 ]. Both enantiomers are oncometabolites, but they are produced differently and have distinct effects on metabolism and physiology. L-2HG accumulates during hypoxia and is produced by malate and lactate dehydrogenases [ 4 , 5 ]. Both L-2HG and D-2HG accumulate in humans with mutated mitochondrial citrate transporter [ 6 ], and an underlying mechanism of this disorder was proposed by studies in model organism Drosophila [ 7 ]. D-2HG drives oncogenic transformation in cells with neomorphic mutations in the isocitrate dehydrogenases IDH1 and IDH2 [ 8 , 9 ]. D-2HG inhibits multiple enzymes, including alpha-ketoglutarate (αKG)-dependent dioxygenases [ 10 , 11 ], BCAT transaminases [ 12 ], αKG dehydrogenase [ 13 ], and ATP synthase [ 14 ]. D-2HG can be produced by several enzymes, including the hydroxyacid-oxoacid transhydrogenase ADHFE1 [ 15 ], the phosphoglycerate dehydrogenase PHGDH [ 16 ], and wild-type IDH1 and IDH2 [ 5 ]. However, it remains unclear if D-2HG production bears any functional significance or is due to promiscuous enzyme activity.

Results

hphd-1 RNAi and vitamin B12 supplementation rescue embryonic lethality of Δdhgd-1 mutants High levels of 3HP cause mitochondrial defects in Δdhgd-1 mutants [30]. We therefore wondered whether these defects cause embryonic lethality. To test this, we performed RNAi knock-down of hphd-1, which we predicted to reduce 2HG but not 3HP levels (Fig 3A). Indeed, RNAi of hphd-1 reduced 2HG to levels that are similar to wild-type animals but did not affect 3HP levels (Fig 3B). Remarkably, RNAi of hphd-1 almost fully rescued lethality of Δdhgd-1 mutants (Fig 3C). This result shows that embryonic lethality can be uncoupled from mitochondrial defects, which are not rescued by hphd-1 perturbation [30]. Both dhgd-1 mutation and hphd-1 RNAi block HPHD-1 function [30]. However, hphd-1 RNAi rescues embryonic lethality in Δdhgd-1 mutants and is not lethal in wild-type animals. Therefore, we conclude that lack of flux through the propionate shunt does not explain embryonic lethality in Δdhgd-1 mutant animals. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Rescue of lethality in Δdhgd-1 mutants by vitamin B12 supplementation and hpdh-1 RNAi. (A) Schematic of predicted HPHD-1 and DHGD-1 contributions to 2HG (blue) and 3HP (orange) accumulation. HPHD-1 is a main source of 2HG, and therefore, its knockdown prevents 2HG accumulation in case of DHGD-1 dysfunction. 3HP is expected to accumulate if either DHGD-1 or HPHD-1 is perturbed because these reactions are coupled to facilitate 3HP oxidation. (B) 2HG and 3HP abundance in Δdhgd-1 mutants upon RNAi of hphd-1. Boxplot midline represents median of 4 independent biological replicates (dots). (C) hphd-1 RNAi rescues lethality in Δdhgd-1 mutants. The RNAi-compatible E. coli OP50 strain [34] was used because conventional RNAi-compatible E. coli HT115 rescued embryonic lethality of Δdhgd-1 animals. Each dot represents an independent biological replicate and bars indicate means. (D) Vitamin B12 rescues lethality in Δdhgd-1 mutants. Each dot represents an independent biological replicate and bars indicate means. (E) 3HP and 2HG abundance in Δdhgd-1 mutants supplemented with vitamin B12. Boxplot midline represents median of independent biological replicates (dots). All panels: The means of 3 or more groups were compared with ANOVA, followed by unpaired t test (*p < 0.05, **p < 0.01, ***p < 0.001). The data underlying Fig 3B–3E can be found in S1 Data. WT, wild type. https://doi.org/10.1371/journal.pbio.3002057.g003 Flux through the propionate shunt is transcriptionally repressed by vitamin B12, which enables flux through the canonical propionate degradation pathway [26–28]. We found that supplementation of vitamin B12 rescued both mitochondrial defects and embryonic lethality in Δdhgd-1 mutants (Figs 3D and S2A). Surprisingly, however, while 3HP levels went down in Δdhgd-1 mutants upon supplementation of vitamin B12, 2HG levels decreased very little (Fig 3E). This observation suggests that there is still some flux through the propionate shunt pathway in Δdhgd-1 mutant animals supplemented with vitamin B12. More importantly, this result indicates that high levels of 2HG are not sufficient to elicit embryonic lethality in these mutants. Importantly, levels of 3HP and 2HG were strongly correlated even in vitamin B12-supplemented Δdhgd-1 animals, indicating that their metabolism is still coupled (S5 Fig). This coordination is even more evident on a diet of RNAi competent E. coli OP50 (xu363) [34] where vitamin B12 reduces the levels of both 3HP and 2HG by more than 2 folds, in adults and embryos (S3F and S3I Fig). Therefore, we hypothesized that vitamin B12 supplementation rescued embryonic lethality in Δdhgd-1 mutants not by lowering 2HG accumulation but by compensating for its detrimental effects, for example, its inhibition of the activity of metabolic enzymes involved in the breakdown of leucine and/or lysine.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002057

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