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A genetically encoded bifunctional enzyme mitigates redox imbalance and lipotoxicity

Nature Metabolism volume 8, pages 350–370 (2026)Cite this article

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Abstract

Dihydroxyacetone phosphate (DHAP), glycerol-3-phosphate (Gro3P) and reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD⁺) are key metabolites of the Gro3P shuttle, which transfers reducing equivalents between the cytosol and mitochondria. Targeted activation of Gro3P biosynthesis has recently emerged as a promising strategy to alleviate reductive stress. However, because Gro3P constitutes the backbone of triglycerides, its accumulation can promote extensive lipogenesis. Here we show that a genetically encoded tool based on a di-domain glycerol-3-phosphate dehydrogenase from the alga Chlamydomonas reinhardtii (CrGPDH) effectively operates both the alternative Gro3P shunt, which regenerates NAD⁺ while converting DHAP to Gro3P, and the glycerol shunt, which converts Gro3P to glycerol and inorganic phosphate, across transformed and primary mammalian cell cultures as well as mouse liver. CrGPDH expression supported proliferation of cancer cells under respiratory chain inhibition or hypoxia, as well as patient-derived fibroblasts with mitochondrial dysfunction. Moreover, CrGPDH decreased triglyceride levels in kidney cancer cell lines and reversed ethanol-induced triglyceride accumulation in mouse liver. Thus, CrGPDH represents a promising xenotopic tool to alleviate redox imbalance and associated impaired lipogenesis in conditions ranging from primary mitochondrial diseases to steatosis.

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Fig. 1: Initial screening of di-domain GPDHs in mammalian cells.
Fig. 2: Biochemical properties of di-domain GPDH from C. reinhardtii.
Fig. 3: Metabolic features of HeLa cells expressing CrGPDH.
Fig. 4: CrGPDH expression allows partial alleviation of growth arrest of HeLa cells with inhibited ETC or under hypoxia.
Fig. 5: CrGPDH expression increases fatty acid β-oxidation in HeLa cells.
Fig. 6: CrGPDH alters lipid profiles of 786-O and Caki-1 ccRCC cell lines.
Fig. 7: CrGPDH expression allows partial alleviation of growth arrest of cellular models of mitochondrial disease.
Fig. 8: CrGPDH alters ethanol-induced hepatic lipid metabolism.

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Data availability

RNA-seq data presented in this work are available at the Gene Expression Omnibus database under accession number GEO: GSE312066. The H. sapiens GRCh38 reference genome was used for mapping RNA-seq reads. Source data are provided with this paper.

Code availability

No new code was generated during this study.

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Acknowledgements

We thank David Scott (SBP Discovery Cancer Metabolism Core, La Jolla, CA) for technical support. This work was supported by grants from the National Institutes of Health (R35GM142495 and R03CA286706 to V.C. and R01DK134675 to R.P.G.). R.P.G. was supported by a Career Award for Medical Scientist (Burroughs Welcome Fund). The Seahorse XFe96 analyser in the Saez laboratory (TSRI) was supported by 1S10OD16357. The Metabolomics Platform (RRID: SCR_022932) was supported by the University of Chicago Comprehensive Cancer Center Support Grant (P30 CA014599). This is manuscript 1080 from the Scintillon Institute.

Author information

Author notes

  1. Austin L. Zuckerman

    Present address: Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY, USA

  2. Sara Violante

    Present address: Gulbenkian Institute for Molecular Medicine (GIMM), Lisboa, Portugal

Authors and Affiliations

  1. Scintillon Institute, Laboratory of Redox Biology and Metabolism, San Diego, CA, USA

    Xingxiu Pan, Subrata Munan, Austin L. Zuckerman, Andrew Pon & Valentin Cracan

  2. Program in Mathematics and Science Education, University of California San Diego and San Diego State University, San Diego, CA, USA

    Austin L. Zuckerman

  3. Metabolomics Platform, Comprehensive Cancer Center, The University of Chicago, Chicago, IL, USA

    Niklas B. Thompson, Rasangi M. Perera & Hardik Shah

  4. Liver Center, Division of Gastroenterology, Massachusetts General Hospital, Boston, MA, USA

    Nirajan Shrestha & Russell P. Goodman

  5. Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Sara Violante & Justin R. Cross

  6. Department of Molecular and Cellular Biology, The Scripps Research Institute, La Jolla, CA, USA

    Valentin Cracan

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Contributions

X.P. performed all experiments with assistance from A.L.Z., S.M. and A.P. S.M. performed protein purification and enzyme kinetics experiments. H.S., N.B.T. and R.M.P. performed all the lipid profiling, metabolomic profiling and stable-isotope-tracing experiments. R.P.G. and N.S. performed mouse experiments. S.V. and J.R.C. performed LC–MS experiments to screen GPDHs. X.P., V.C. and A.L.Z. wrote the paper, with input from all the authors.

Corresponding author

Correspondence to Valentin Cracan.

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Competing interests

V.C., X.P. and A.L.Z. are listed as inventors on a patent application (no. 63/812,699: ‘Enzyme-based compositions and methods for treating mitochondrial dysfunction’) based on sequences and activities of proteins described in this paper. V.C. is listed as an inventor on a patent application on the therapeutic uses of LbNOX and TPNOX (US patent application US20190017034A1). The other authors declare no competing interests.

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Nature Metabolism thanks Melanie McReynolds, Yi Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Gimenez-Cassina and Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Effect of CrGPDH expression in HeLa cells on metabolic features, bioenergetics and GSH/GSSG ratio.

Lactate (a) and pyruvate (b) levels in media incubated for 24 hours (spent media) with HeLa cells expressing Luciferase or CrGPDH. Glucose consumption (c), lactate production (d) and glutamine consumption (e) by HeLa cells expressing CrGPDH. Oxygen consumption rate (OCR) (f) and extracellular acidification rate (ECAR) (g) of HeLa cells expressing CrGPDH before and after separate additions of 1 µM piericidin A (Pier) or 1 µM antimycin A (ANT) measured in pyruvate-free HEPES/DMEM+dFBS media containing 5 mM glucose. GSH (h), GSSG (i) and the GSH/GSSG ratios (j) in HeLa cells expressing CrGPDH. Two hundred μM menadione (vitamin K3) was used as a positive control in (h-j). Experiments in (a-e, h-j) were performed with pyruvate-free DMEM supplemented with dialysed FBS (DMEM+dFBS) containing 25 mM glucose. Values are mean ± s.d.; n = 5, 6 in (a), n = 6, 6 in (b), n = 11, 6 in (c), n = 11, 11 in (d), n = 12, 10 in (e), n = 45, 15, 30, 55, 25, 30 in (f), n = 50, 20, 30, 60, 30, 30 in (g), n = 3 in (h-j) biologically independent samples. The statistical significance indicated for (a-e) represents a two-tailed unpaired t test; for (f-g) represents a One-Way ANOVA followed by Šídák multiple comparison test; for (h-j) represents a Two-Way ANOVA followed by uncorrected Fisher’s least significant difference test.

Source data

Extended Data Fig. 2 SoNar and iNAP1 imaging of HeLa cells expressing CrGPDH.

(a) Widefield images of HeLa cells with lentivirus mediated LUC and CrGPDH expression under Dox control transiently expressing SoNar. Experiments were performed in the imaging medium (DMEM without pyruvate, fluorescent vitamins and phenol red with 5 mM glucose, 25 mM HEPES, pH 7.4 and 1% dialyzed FBS), or after addition of 1 μM antimycin A. Scale bars: 20 µm. (b) Quantification of the time course measurements of the fluorescence ratio (F400/488) for HeLa cells with lentivirus mediated LUC and CrGPDH expression under Dox control transiently expressing SoNar for conditions shown in (a). (c) Widefield images of HeLa cells with lentivirus mediated LUC and CrGPDH expression under Dox control transiently expressing iNAP1. Experiments were performed in the imaging medium (DMEM without pyruvate, fluorescent vitamins and phenol red with 5 mM glucose, 25 mM HEPES, pH 7.4 and 1% dialyzed FBS), or when cells were switched to the imaging medium without glucose but with 10 mM pyruvate. Scale bars: 20 µm. Quantification of the time course measurements of the fluorescence ratio (F400/488) for HeLa cells with lentivirus mediated LUC and CrGPDH expression under Dox control transiently expressing iNAP1 (d) or inactive variant which does not bind NADPH (iNAPC) (e) in the imaging medium (DMEM without pyruvate, fluorescent vitamins and phenol red with 5 mM glucose, 25 mM HEPES, pH 7.4 and 1% dialyzed FBS), or when cells were switched to the basal medium without glucose but with 10 mM pyruvate. Values are mean ± s.d.; n = 27, 30 in (b), n = 29, 27 in (d), n = 20, 20 in (e) biologically independent samples.

Source data

Extended Data Fig. 3 Metabolic flux features of CrGPDH expression HeLa cells.

(a) Schematic showing carbons flux from glucose and glutamate. Brown circles represent 13C-labelled carbons from glucose, purple circles represent 13C-labelled carbons from glutamine. Heatmaps of the glucose carbon flux into glycolysis (b), TCA cycle (c), pentose phosphate pathway (PPP) and purine (d), and pyrimidine (e) metabolites, as well as TCA cycle intermediates from glutamine carbon transitions (f) in HeLa cells expressing CrGPDH and LUC after 8hr-culture with 10 mM U-¹³C₆ D-Glucose or 2 mM ¹³C₅, 99% L-Glutamine. Note that unless specified, the values in (b-f) are the abundances of the most labelled isotopologue. (g) Fractional labeling of citrate M + 4 (oxidative) and M + 5 (reductive) from ¹³C₅ glutamine. G6P: glucose-6-phosphate; F6P: fructose-6-phosphate; F1,6BP: fructose-1,6-bisphosphate; DHAP: dihydroxyacetone phosphate; Gro3P, glycerol-3-phosphate; AKG, α-ketoglutarate; 6PG: 6-phosphogluconate; R5P: ribose-5-phosphate. In heatmaps in (b-f), each column represents a biologically independent sample. Values are mean ± s.d.; n = 5 in (g) biologically independent samples. The statistical significance indicated for (g) represents a two-way ANOVA followed by Fisher’s LSD test without correction.

Extended Data Fig. 4 Analysis of transcriptomic features of CrGPDH expression in HeLa cells.

(a) Volcano plots that represent the log2 fold change (x axis) and adjusted p value for significance (y axis) of CrGPDH vs LUC expressing HeLa cells. Genes significantly different in expression at false discovery rate (FDR) of 5% are indicated in red (upregulated genes, log2 fold change above 1) or blue (downregulated genes, log2 fold change below -1). Grey dots represent genes without significant changes. (b) Gene ontology (GO) terms analysis of significant genes that are differentially changed between CrGPDH and LUC. BP: biological process; CC, cellular component; MF: molecular function. The Cnet plots for the subset of genes that correlate with GO terms BP in (c) or MF in (d). Analysis shown in (a-d) is based on n = 4 biologically independent samples per group.

Extended Data Fig. 5 Metabolic features of CrGPDH expression in 786-O cells.

Glycerol (a), lactate (b), pyruvate (c) and the lactate/pyruvate ratio (d) measured by GC-MS in media which was incubated for 24 hours (spent media) with 786-O cells expressing Luciferase (LUC) or CrGPDH. Glucose consumption (e), lactate production (f) and glutamine consumption (g) measured by the YSI 2900 Biochemistry Analyzer in media which was incubated for 24 hours (spent media) with 786-O cells expressing Luciferase (LUC) or CrGPDH. Targeted metabolomics of 786-O cells expressing CrGPDH relative to LUC control (h). Significantly increased metabolites (p value cutoff < 0.05, fold change >1) are highlighted in red, and significantly decreased metabolites (p value cutoff < 0.05, fold change < -1) are highlighted in dark blue, while gray dots represent metabolites without significant changes. Experiments in (a-g) were performed in pyruvate-free RPMI+dFBS containing 5 mM glucose; in (h) were performed in pyruvate-free RPMI+dFBS containing 25 mM glucose. UDP-GlcA: UDP-glucuronic acid. Values are mean ± s.d.; n = 6, 6 in (a-g) biologically independent samples. The statistical significance indicated for (a-g) represents a two-tailed unpaired t test. NS, no significant difference.

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Extended Data Fig. 6 Diagram summarizing metabolic rewiring in clear cell renal cell carcinoma (ccRCC) cells expressing CrGPDH.

(a) In ccRCC cells, the Gro3P shuttle is functionally truncated, as cytosolic cGPDH is uncoupled from mitochondrial mGPDH, thereby supporting a robust Gro3P pool for TG synthesis57. (b) Expression of CrGPDH in these cells facilitates efficient Gro3P clearance, leading to suppression of TG synthesis. FFA: free fatty acid; TG: triglycerides; GL/FFA cycle: glycerolipid/free fatty acid cycle; GK: glycerol kinase.

Extended Data Fig. 7 Metabolic features of CrGPDH expression in IMR-90 primary human fibroblasts.

Lactate (a) and pyruvate (b) measured by GC-MS in media which was incubated for 24 hours (spent media) with IMR-90 cells expressing Luciferase (LUC) and CrGPDH. Glucose consumption (c), lactate production (d) and glutamine consumption (e) measured by the YSI 2900 Biochemistry Analyzer in media which was incubated for 24 hours (spent media) with IMR-90 cells expressing CrGPDH or Luciferase (LUC). Targeted metabolomics of IMR-90 cells expressing CrGPDH relative to LUC control (f). Significantly increased metabolites (p value cutoff < 0.05, fold change > 0.7) are highlighted in red, and significantly decreased metabolites (p value cutoff < 0.05, fold change < -0.7) are highlighted in dark blue, while gray dots represent metabolites without significant changes. Experiments were performed in pyruvate-free DMEM+dFBS supplemented with 5 mM glucose in (a-e) or 25 mM glucose in (f). Values are mean ± s.d.; n = 6, 6 in (a-e) biologically independent samples. The statistical significance indicated for (a-e) represents a two-tailed unpaired t test. NS, no significant difference.

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Extended Data Fig. 8 Hepatic metabolic features of CrGPDH expression in mice.

KEGG pathway enrichment analysis of altered metabolites in (a) CrGPDH versus GFP under water gavage, (b) ethanol versus water in GFP-expressing mice, and (c) CrGPDH versus GFP under ethanol gavage. X axis represents KEGG terms, Y axis represents log10 p value. Pathway analysis was performed in MetaboAnalyst 6.0 using metabolites with p < 0.05 in (a, b) and p < 0.2 in (c). (d-f) The schematics highlighting related metabolomic pathways and selected metabolites for indicated comparisons in (a-c). Heatmaps of the most impacted pyrimidines (g), beta-Alanine (h), arginine (i), TCA cycle (j) and glycerophospholipid (k) metabolites in indicated group. AKG, α-ketoglutarate; Gro3P, glycerol-3-phosphate; Gro3P-Cho, glycerol-3-phosphate Choline; P-Cho, Phosphocholine; PEA, Phosphoethanolamine; CDP-Etn, CDP-Ethanolamine; CDP-Cho, CDP-Choline. In heatmaps in (g-k), each column represents a biologically independent sample.

Extended Data Fig. 9 Hepatic lipid profiles of CrGPDH expression in mice.

Volcano plots of summed lipid classes. Comparisons are: (a) CrGPDH versus GFP under water gavage, (b) ethanol versus water in GFP-expressing mice, and (c) CrGPDH versus GFP under ethanol gavage. (d) Heatmaps of all detected lipids, clustered by each lipid class across the indicated groups. Significantly increased summed lipids (log2FC > 0.1, p < 0.05) are shown in red, decreased summed lipids (log2FC < -0.1, p < 0.05) in dark blue, and nonsignificant ones in gray. ChE: Cholesterol ester, CoQ: Coenzyme Q-like molecules, DG: Diglycerides, HexCer: Ceramide with hexose, LBPA: Glycerophosphoglycerols, LPC: Lysophosphatidylcholine, LPE: Lysophosphatidylethanolamine, LPI: Lysophosphatidylinositol, MePC: Methyl phosphatidylcholine, PA: Phosphatidic acid, PC: Phosphatidylcholine, PE: Phosphatidylethanolamine, PG: Phosphatidylglycerol, PI: Phosphatidylinositol, PS: Phosphatidylserine, SM: Sphingomyelin, TG: triglycerides. In heatmaps in (d), each column represents a biologically independent sample.

Extended Data Table 1 Steady-state kinetic parameters of CrGPDH
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Pan, X., Munan, S., Zuckerman, A.L. et al. A genetically encoded bifunctional enzyme mitigates redox imbalance and lipotoxicity. Nat Metab 8, 350–370 (2026). https://doi.org/10.1038/s42255-025-01450-3

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