Ampk-regulated glycerol excretion maintains metabolic crosstalk between reductive and energetic stress

ABSTRACT Glucose metabolism has been studied extensively, but the role of glucose-derived excretory glycerol remains unclear. Here we show that hypoxia induces NADH accumulation to promote

glycerol excretion and this pathway consumes NADH continuously, thus attenuating its accumulation and reductive stress. Aldolase B accounts for glycerol biosynthesis by forming a complex

with glycerol 3-phosphate dehydrogenases GPD1 and GPD1L. Blocking GPD1, GPD1L or glycerol 3-phosphate phosphatase exacerbates reductive stress and suppresses cell proliferation under hypoxia

and tumour growth in vivo. Overexpression of these enzymes increases glycerol excretion but still reduces cell viability under hypoxia and tumour proliferation due to energy stress. AMPK

inactivates aldolase B to mitigate glycerol synthesis that dissipates ATP, alleviating NADH accumulation-induced energy crisis. Therefore, glycerol biosynthesis/excretion regulates the

trade-off between reductive stress and energy stress. Moreover, this mode of regulation seems to be prevalent in reductive stress-driven transformations, enhancing our understanding of the

metabolic complexity and guiding tumour treatment. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

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Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS THE ALTERNATIVE ACTIVITY OF NUCLEAR PHGDH CONTRIBUTES TO TUMOUR GROWTH UNDER NUTRIENT STRESS Article 18 October 2021 TARGETING

ALDOLASE A IN HEPATOCELLULAR CARCINOMA LEADS TO IMBALANCED GLYCOLYSIS AND ENERGY STRESS DUE TO UNCONTROLLED FBP ACCUMULATION Article Open access 20 January 2025 GFAT1-LINKED TAB1

GLUTAMYLATION SUSTAINS P38 MAPK ACTIVATION AND PROMOTES LUNG CANCER CELL SURVIVAL UNDER GLUCOSE STARVATION Article Open access 09 August 2022 DATA AVAILABILITY MS data have been deposited in

ProteomeXchange with the primary accession code PXD056351 (http://proteomecentral.proteomexchange.org)50,51. The human cancer data were derived from the TCGA Research Network at

http://cancergenome.nih.gov/. The dataset derived from this resource that supports the findings of this study is available in Supplementary Table 1. All other data supporting the findings of

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integrated proteome resource. _Nucleic Acids Res._ 47, D1211–D1217 (2019). PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank X. Liu (Metabolomics Facility at Tsinghua

University Branch of China National Center for Protein Sciences, China) for technical help. This work was supported by the National Natural Science Foundation of China (82325038 and 82030093

to B.L.) and China Postdoctoral Science Foundation (GZB20230454 to R.Y.) AUTHOR INFORMATION Author notes * These authors contributed equally: Xuewei Zhai, Ronghui Yang. AUTHORS AND

AFFILIATIONS * Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing, China Xuewei Zhai, Ronghui Yang, Zihao Guo, Pengjiao Hou, Xuexue Li, Ziwen Lu, 

Luxin Qiao & Binghui Li * Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Capital Medical University, Beijing, China Qiaoyun Chu, Yanxia Fu, Jing Niu 

& Binghui Li * Department of Cancer Cell Biology and National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China Changsen Bai 

& Binghui Li Authors * Xuewei Zhai View author publications You can also search for this author inPubMed Google Scholar * Ronghui Yang View author publications You can also search for

this author inPubMed Google Scholar * Qiaoyun Chu View author publications You can also search for this author inPubMed Google Scholar * Zihao Guo View author publications You can also

search for this author inPubMed Google Scholar * Pengjiao Hou View author publications You can also search for this author inPubMed Google Scholar * Xuexue Li View author publications You

can also search for this author inPubMed Google Scholar * Changsen Bai View author publications You can also search for this author inPubMed Google Scholar * Ziwen Lu View author

publications You can also search for this author inPubMed Google Scholar * Luxin Qiao View author publications You can also search for this author inPubMed Google Scholar * Yanxia Fu View

author publications You can also search for this author inPubMed Google Scholar * Jing Niu View author publications You can also search for this author inPubMed Google Scholar * Binghui Li

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS B.L. conceived the study and designed experiments; X.Z. and R.Y. performed experiments;

Q.C. prepared some constructs and cell lines; Z.G., P.H., X.L., C.B. and Z.L. collected and analysed data; L.Q., Y.F. and J.N. provided conceptual advice and gave technical support; B.L.

wrote the manuscript; X.Z. and R.Y. edited the manuscript. CORRESPONDING AUTHOR Correspondence to Binghui Li. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Cell Biology_ thanks Lluis Fajas, Constantinos Koumenis and the other, anonymous, reviewer(s) for their contribution to the peer review

of this work. Peer reviewer reports are available. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and

institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 HYPOXIA PROMOTES GLYCEROL 3-PHOSPHATE BIOSYNTHESIS. A, The relative abundance of metabolites of glycolysis in HeLa cells under

normoxia, hypoxia, or antimycin A (AntA, 2 μM) treatment for 8 h. B, The relative abundance of glycerol 3-phosphate and its precursor DHAP in HeLa cells under normoxia, hypoxia, or AntA (2 

μM) treatment for 8 h. C, Isotopomer tracing analysis of glycerol 3-phosphate biosynthesis in HeLa cells cultured with 13C6-glucose (25 mM) or 13C5-glutamine (2 mM) for 8 h. D,E, Using

N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) to modify the hydroxyl groups of glycerol and glycerol 3-phosphate and detect them by gas chromatography-mass spectrometry (GC-MS). F,

Isotopomer tracing analysis of excreted glycerol in HeLa cells cultured with 13C6-glucose (25 mM) under normoxia or hypoxia for 8 h. G,H, The levels of excreted glycerol determined by GC-MS

or a liquid sample glycerol assay kit in HeLa cells cultured under normoxia and hypoxia for 24 h. Values are shown as mean ± SD, n = 3 biologically independent samples, two-tailed Student’s

t-tests. Source data EXTENDED DATA FIG. 2 HYPOXIA PROMOTES GLUCOSE UPTAKE AND LACTATE EXCRETION. A, Glucose uptake of several cell types including tumor cells and endothelial cells cultured

under normoxia and hypoxia for 24 h. B, Lactate excretion of several cell types including tumor cells and endothelial cells cultured under normoxia and hypoxia for 24 h. C-H, The glycerol

excretion, glucose uptake, and lactate excretion in HeLa and A549 cells cultured under hypoxia as indicated. I, Western blot analysis of PGP, GPD1, GPD1L, GPD2, ALDOA, ALDOB, TPI1, and

β-Actin expression in different cells as indicated. J, Immunoblot verification of GPD2 knockout in HeLa and A549 cells. K, Effects of GPD2 knockout on glycerol excretion of HeLa cells

cultured under normoxia and hypoxia for 24 h. Values are shown as mean ± SD, n = 3 biologically independent samples (a-h, k), two-tailed Student’s t-tests. In i, data are verified in two

replicates with similar results. Source data EXTENDED DATA FIG. 3 EFFECTS OF GLYCEROL EXCRETION ON CELL VIABILITY UNDER HYPOXIA. A, Effects of GPD1 and/or GPD1L knockout on colony formation

in HeLa cells cultured for 10 days. B, Effects of PGP knockout and PGP re-expression on colony formation in HeLa cells cultured for 10 days. C, Effects of PGP knockout and PGP re-expression

on glycerol excretion of A549 cells cultured under normoxia and hypoxia for 24 h. D, Effects of PGP knockout and PGP re-expression on cell viability of A549 cells cultured under normoxia and

hypoxia for 48 h. E, Effects of PGP knockout and PGP re-expression on colony formation of A549 cells cultured for 10 days. F, Effects of PGP knockout and PGP re-expression on glycerol

excretion of HepG2 cells cultured under normoxia and hypoxia for 24 h. G, Effects of PGP knockout and PGP re-expression on cell viability of HepG2 cells cultured under normoxia and hypoxia

for 48 h. H, Effects of PGP knockout and PGP re-expression on colony formation of HepG2 cells cultured for 10 days. I, Analysis of the mRNA levels of aquaporins (AQPs) based on the data from

Cancer Cell Line Encyclopedia (CCLE). J, Effects of AQP3 knockout on glycerol excretion and cell viability of HeLa and A549 cells cultured under normoxia and hypoxia for 24 h (glycerol

excretion) or 72 h (cell proliferation). Values are shown as mean ± SD, n = 3 biologically independent samples, two-tailed Student’s t-tests. Source data EXTENDED DATA FIG. 4 EFFECTS OF NADH

ACCUMULATION ON GLUCOSE UPTAKE, LACTATE EXCRETION, AND GLYCEROL EXCRETION. A, Immunoblot verification of CHOP and ATF4 activation in GPD1/GPD1L DKO, PGP KO and PGP re-expression HeLa cells

cultured under normoxia or hypoxia for 24 h. B, Effects of different concentrations of αKB on glucose uptake and lactate excretion of HeLa, A549, and HepG2 cells under hypoxia for 24 h. C,

Effects of different concentrations of LDHi on glucose uptake and lactate excretion of HeLa, A549, and HepG2 cells under normoxia for 24 h. D, Glucose uptake and lactate excretion of HeLa,

A549 and HepG2 cells cultured as indicated for 24 h. E, Effects of different concentrations of SC-26196 on the ratios of NADH/NAD+, glycerol excretion, glucose uptake, and lactate excretion

of HeLa, A549, and HepG2 cells cultured under normoxia for 8 h (NADH/NAD+ ratios) or 24 h (glycerol excretion, glucose uptake, and lactate excretion). F, Effects of αKB (2 mM) on cell

viability of HeLa PGP KO or GPD1/GPD1L DKO cells cultured under hypoxia for 48 h. Values are shown as mean ± SD, n = 3 biologically independent samples (b-f), two-tailed Student’s t-tests.

In a, data are verified in three replicates with similar results. Source data EXTENDED DATA FIG. 5 EFFECTS OF GLUCOSE UPTAKE ON GLYCEROL EXCRETION. A, Western blots for the expression of

PKM2, LDHA, GAPDH, and β-Actin in HeLa and A549 cells treated with normoxia, hypoxia, or AntA (2 μM) for 24 h. B, Effects of HIF2α knockdown on glycerol excretion of HeLa and A549 cells

cultured under hypoxia for 24 h. C, Immunoblot verification of GLUT1 knockdown in HeLa and A549 cells. D-F, Effects of GLUT1 knockdown on glucose uptake, lactate excretion, and glycerol

excretion of HeLa and A549 cells cultured under normoxia and hypoxia for 24 h. G, Immunoblot verification of GLUT1 over-expression in HeLa and A549 cells. H-J, Effects of GLUT1

over-expression on glucose uptake, lactate excretion, and glycerol excretion of HeLa and A549 cells cultured under normoxia, hypoxia, or AntA (2 μM) treatment for 24 h. Values are shown as

mean ± SD, n = 3 biologically independent samples (b, d-f, h-j), two-tailed Student’s t-tests. In a, data are verified in two replicates with similar results. Source data EXTENDED DATA FIG.

6 EFFECTS OF PGP, GPD1, OR GPD1L OVER-EXPRESSION ON GLUCOSE UPTAKE, LACTATE EXCRETION, GLYCEROL EXCRETION, AND CELL GROWTH. A, Effects of PGP, GPD1, or GPD1L over-expression on glucose

uptake and lactate excretion of HeLa and A549 cells cultured under normoxia and hypoxia for 24 h. B, Immunoblot verification of LKB1 expression in HeLa, A549, SK-Hep-1, and HCC-LM3 cells. C,

Immunoblot verification of over-expression of PGP, GPD1L, or GPD1 in SK-Hep-1 and HCC-LM3 cells. D, Effects of PGP, GPD1, or GPD1L over-expression on glycerol excretion of SK-Hep-1 and

HCC-LM3 cells cultured under normoxia and hypoxia for 24 h. E, Effects of PGP, GPD1, or GPD1L over-expression on cell viability of SK-Hep-1 and HCC-LM3 cells cultured with low nutrient

medium (10% fetal bovine serum medium containing 2 mM of glucose, without glutamine and pyruvate) under normoxia and hypoxia for 8 h. F, Effects of PGP, GPD1, or GPD1L over-expression on

cell viability of SK-Hep-1 and HCC-LM3 cells cultured in normal or nutrient-deprived media. Nutrient-deprived media contained 10% fetal bovine serum but without glucose, glutamine, and

pyruvate. G, Effects of PGP, GPD1L, or GPD1 over-expression on AMPK activation in SK-Hep-1 and HCC-LM3 cells cultured in nutrient-deprived medium. H, Tumor formation ability in nude mice of

SK-Hep-1 cells with PGP, GPD1, or GPD1L over-expression. Values are shown as mean ± SD, n = 3 biologically independent samples (a,d,e,f) or n = 5 biologically independent mice (h),

two-tailed Student’s t-tests. In g, data are verified in two replicates with similar results. Source data EXTENDED DATA FIG. 7 EFFECTS OF ALDOLASES AND AMPK ON GLUCOSE UPTAKE, LACTATE

EXCRETION, AND GLYCEROL EXCRETION. A, Effects of ALDOA over-expression on glycerol excretion, glucose uptake, and lactate excretion of HeLa and A549 cells cultured under normoxia and hypoxia

for 24 h. B, Effects of ALDOC over-expression on glycerol excretion, glucose uptake, and lactate excretion of HeLa and A549 cells cultured under normoxia and hypoxia for 24 h. C, Effects of

A769662 treatment on glycerol excretion, glucose uptake, and lactate excretion of A549, SK-Hep-1, and HCC-LM3 cells cultured under normoxia and hypoxia for 12 h. D, Effects of Compound C on

AMPK pathway in HeLa cells. E, Effects of Compound C on glycerol excretion, glucose uptake, and lactate excretion of HeLa and A549 cells cultured under normoxia and hypoxia for 12 h. F,

Effects of AMPKα knockdown and AMPKα1 re-expression on glycerol excretion, glucose uptake, and lactate excretion of A549 cells cultured under normoxia and hypoxia for 24 h. G.

Immunoprecipitation (IP) analysis of the interaction between endogenous ALDOB, GPD1 and GPD1L in HepG2 cells with anti-ADLOB antibody. Rabbit IgG was used as a negative control. WCL,

whole-cell lysate. pALDOB T245, AMPK, pAMPK T172, ACC1 and pACC1 S79 were also blotted. Values are shown as mean ± SD, n = 3 biologically independent samples (a-c, e-f), two-tailed Student’s

t-tests. In d,g, data are verified in two replicates with similar results. Source data EXTENDED DATA FIG. 8 INACTIVATION OF ADLOB BY AMPK-MEDIATED PHOSPHORYLATION. A, HEK293T cells were

transfected with vector, Flag-GPD1, or Flag-GPD1L plasmids, and then cultured under normoxia or hypoxia for 8 h. IP assays were performed using anti-FLAG affinity M2 beads followed by

immunoblotting for the phosphorylation of GPD1 or GPD1L. B, Mass spectrometry was used to detect the phosphorylation site of ALDOB in HeLa cells after treatment with A769662 for 8 h. C,

Conservation of the phosphorylation site in ALDOB among different species. Amino acid residues around Thr245 are shown. D, The activity of GST-ALDOB-WT, T245A, and T245D mutants purified

from an _E. coli_ expression system incubated with or without active AMPK as indicated. Values are shown as mean ± SD, data are verified in two replicates with similar results (a) and n = 3

biologically independent samples (d). Source data EXTENDED DATA FIG. 9 THE TRADE-OFF REGULATION BETWEEN REDUCTIVE STRESS AND ENERGY STRESS. A, Effects of αKB and _Lb_NOX on AMPK activation

of A549 cells cultured under respiratory chain inhibition or hypoxia for 24 h. αKB, 2 mM; Dox, 0.1 μg/mL. B,C, Effects of αKB and _Lb_NOX on cellular NADH/NAD+ ratio (b) and ATP/AMP ratio

(c) of A549 cells cultured under respiratory chain inhibition or hypoxia for 24 h. αKB, 2 mM; Dox, 0.1 μg/mL. D, Effects of Torin-1 (0.1 μM) and Rapamycin (1 μM) on AMPK activation of A549

cells cultured under respiratory chain inhibition or hypoxia for 24 h. E,F, Effects of Torin-1 (0.1 μM) and Rapamycin (1 μM) on cellular NADH/NAD+ ratio (e) and ATP/AMP ratio (f) of A549

cells cultured under respiratory chain inhibition or hypoxia for 24 h. G, Effects of Compound C (2 μM) on cell death of A549 cells cultured in nutrient-deprived medium under respiratory

chain inhibition or hypoxia for 12 h. H, Immunoblot verification of AMPKα knockout and AMPKα re-expression in HeLa cells. I, The effect of metformin (100 mg/kg/day, i.g.) or Compound C (20

mg/kg/day, i.p.) treatment alone or in combination on tumor formation ability in nude mice of HeLa cells. Metformin (Metf, 4 mM) and Ant A (2 μM) were used. Values are shown as mean ± SD, n

= 3 biologically independent samples (b,c,e,f,g) or n = 6 biologically independent mice (i), two way ANOVA (i), and two-tailed Student’s t-tests for others. In a,d, data are verified in two

replicates with similar results. Source data EXTENDED DATA FIG. 10 THE WORKING MODEL FOR TRADE-OFF BETWEEN REDUCTIVE STRESS AND ENERGY STRESS. A, Schematic diagram illustrating hypoxic

regulation of glycerol biosynthesis. Hypoxia promotes glycerol biosynthesis and excretion by inducing NADH accumulation and glucose uptake. Glycerol biosynthesis consumes NADH to reduce

reductive stress, but this process is accompanied by ATP consumption and thus may potentially provoke an energy crisis. Cells have evolved a negative feedback loop to suppress glycerol

synthesis through AMPK-mediated phosphorylation of ALDOB. B, The trade-off regulation between reductive stress and energy stress. Reductive stress induced by hypoxia or ETC inhibition

primarily contributes to energy stress and AMPK activation. To alleviate reductive stress, some metabolic pathways are promoted to consume NADH, along with ATP. In turn, the activated AMPK

negatively regulates these metabolic reactions to prevent catastrophic energy depletion. SUPPLEMENTARY INFORMATION REPORTING SUMMARY PEER REVIEW FILE SUPPLEMENTARY TABLE 1 Survival _P_-value

analysis based on TCGA datasets. SOURCE DATA SOURCE DATA FIG. 1 Unprocessed western blots. SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 2 Unprocessed western blots. SOURCE

DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 3 Unprocessed western blots. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Unprocessed western blots. SOURCE DATA FIG.

4 Statistical source data. SOURCE DATA FIG. 5 Unprocessed western blots. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA FIG. 6 Unprocessed western blots. SOURCE DATA FIG. 6

Statistical source data. SOURCE DATA FIG. 7 Unprocessed western blots. SOURCE DATA FIG. 7 Statistical source data. SOURCE DATA FIG. 8 Unprocessed western blots. SOURCE DATA FIG. 8

Statistical source data. SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 2 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 2 Unprocessed

western blots. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data.

SOURCE DATA EXTENDED DATA FIG. 4 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 5 Unprocessed western blots. SOURCE DATA

EXTENDED DATA FIG. 6 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 6 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 7 Statistical source data. SOURCE DATA EXTENDED DATA

FIG. 7 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 8 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 8 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 9

Statistical source data. SOURCE DATA EXTENDED DATA FIG. 9 Unprocessed western blots. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive

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excretion maintains metabolic crosstalk between reductive and energetic stress. _Nat Cell Biol_ 27, 141–153 (2025). https://doi.org/10.1038/s41556-024-01549-x Download citation * Received:

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