Abstract
Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, and is crucial for efficient glycolysis–oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyses reversible O-GlcNAcylation, a post-translational modification influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of the regulatory domain of HK1, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances the mitochondrial association with HK1, orchestrating glycolytic and mitochondrial ATP production. Mutation in HK1’s O-GlcNAcylation site reduces ATP generation in multiple cell types, specifically affecting metabolic efficiency in neurons. This study reveals a previously unappreciated pathway that links neuronal metabolism and mitochondrial function through OGT and the formation of the glycolytic metabolon, providing potential strategies for tackling metabolic and neurological disorders.
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Data availability
Source data are provided with this paper.
Code availability
Agilent Seahorse XF96e Metabolic Flux Analyzer data normalization is performed with the custom-written macro ‘FluxNormalyzer’, available at https://doi.org/10.5281/zenodo.11111133 (ref. 105). The electrical field stimulation code is available at https://doi.org/10.5281/zenodo.11462693 (ref. 106). HK1 structure simulation data are available at https://doi.org/10.5281/zenodo.11462797 (ref. 107). Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
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Acknowledgements
We gratefully acknowledge the invaluable contributions of the Pekkurnaz laboratory members, as well as the generous sharing of key instrument resources by E. Hui. We also extend our appreciation to the technical team members of the University of California San Diego Biomolecular/Proteomics Mass Spectrometry Facility and Nikon Imaging Center for their expert assistance, and Arizona State University Research Computing for providing High Performance Computing resources. This project was made possible by the support of a grant from the National Institutes of Health (NIH) to G.P. (R35GM128823), NIH (2T32GM007240) to S.B.Y., NIH (5T32GM133351) to A.A.A., NIH to M.H. (R01NS094219), University of California San Diego TRELS fellowship to A.Z., NIH (5T32NS007220) to V.L., NIH (5T32NS007220-40) to Z.W., NIH (5T32EB009380-15) to N.M.C, the San Diego IRACDA Scholars Program (K12GM068524) to R.S., URS Ledell Family Research Scholarship for Science and Engineering to A.Z., NSF Graduate Research Fellowship (2020298734) to J.W.V and NSF (MCB-1942763) to A.S. We also acknowledge the Gordon and Betty Moore Foundation (7555.04) and the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (2020-222005) for their contributions to this project.
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G.P. and H.W. designed the experiments, with technical contributions from H.W., R.S., M.L.M., A.Z., S.B.Y., S.Y., Z.W., N.M.C., V.L., M.J., Q.G., S.W. and A.A.A. J.W.V. and A.S. contributed structural data and performed molecular simulations, and C. elegans experiments were carried out by Y.W. and M.H. T.M. contributed lentiviral particle production. M.G. performed mass spectrometry experiments and analysis, and E.G. provided expert technical assistance for microscopy. H.W. and G.P. wrote the manuscript with input from all co-authors.
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Nature Metabolism thanks Matthew Merrins, Matthew Pratt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.
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Extended data
Extended Data Fig. 1 The mitochondrial localization of Hexokinase 1 depends on glucose metabolism.
a, Experimental scheme detailing the sequence of plating, transfection, imaging, and alteration of extracellular glucose levels in cultured rat hippocampal neurons. b, Evaluation of HK1-shRNA knock-down efficiency in Neuro-2a cells. HK1-shRNA and shRNA resistant eGFP tagged HK1 (HK1-GFP) were expressed for 48–72 hrs, and whole cell lysate (Input) were probed with anti-HK1 and anti-Tubulin (loading control) antibodies. Asterisk indicates endogenous HK1. c, Quantification of endogenous HK1 (left) and eGFP-tagged HK1 (WT-HK1-GFP) (right) expression levels as shown in (b). All values are shown as mean ± SEM. n = 4 (two-tailed Mann-Whitney U test). d, Experimental scheme detailing the sequence of plating, transfection, and imaging conditions for the 0 mM glucose experiments in cultured rat hippocampal neurons. e, Axonal localization of HK1 in cultured rat hippocampal neurons transfected with HK1-shRNA, shRNA-resistant eGFP-tagged HK1 (pseudo-color, fire), and Mito-DsRed (gray). Representative axonal images were captured at 5 mM glucose, following a 2-hour exposure to 0 mM glucose, and at 5 mM glucose after 2 hours exposure to 0 mM glucose (1 mM lactate and pyruvate), as depicted in (d). Scale bar represents 5 µm. f, The mitochondrial (Mito) and cytoplasmic (Cyto) HK1 intensity ratios were quantified along axons. Data are presented as a violin plot with individual data points and associated p-value. n = 94–117 mitochondria, 9-10 axons from three biological replica (one-way ANOVA with post hoc Tukey’s multiple comparison test). g, Axonal localization of HK1 in cultured rat hippocampal neurons transfected with HK1-shRNA, shRNA-resistant eGFP-tagged HK1 (pseudo-color, fire), and Mito-DsRed (gray). Representative images of axons were captured after 2 hours in 2 mM glucose, and subsequently following 2 hours in 5 mM glucose after initial exposure to 2 mM glucose. The switch from 2 mM to 5 mM glucose was performed in the presence of either vehicle or OSMI-4 treatments. Scale bar represents 5 µm. h, Quantification of mitochondrial (Mito) and cytoplasmic (Cyto) HK1 intensity ratios along axons. Data are presented as a violin plot with individual data points and associated p-value. n = 65–79 mitochondria, 10, 11 axons (unpaired two-tailed t-test). i, Blood glucose measurements from the Fasted and Re-fed mice used for comparing subcellular localization of Hexokinase 1 as shown in Fig. 1. n = 3 mice for each condition, three biological replicas. All values are shown as mean ± SEM (Mann-Whitney U test). j, HK1 distribution pattern in the CA3 region of the hippocampus in Ad-lib, and after 6 hours fasting (6hrs Fasted) states. Scale bar represents 10 µm. k, Co-localization analysis to measure the percent intensity of HK1 on mitochondria for each condition. Data are presented as violin plots with individual data points and associated p-values. n = 9 hippocampal CA3 regions, 9 mice from three biological replica (unpaired two-tailed t-test).
Extended Data Fig. 2 O-GlcNAcylation promotes mitochondrial enrichment of Hexokinase 1 in various cell types.
a, The experimental timeline illustrating the sequence of HEK293T cell plating, OGT transfection, administration of vehicle or Thiamet-G, and mitochondrial isolation. b, Analysis of mitochondrial size in cultured rat hippocampal neurons co-transfected with HK1-shRNA and eGFP-tagged HK1 to achieve endogenous HK1 levels. O-GlcNAcylation level was upregulated by ectopic OGT expression and Thiamet-G treatment, and downregulated by OGA expression and OSMI-4 treatment. Data are presented as a violin plot with individual data points and associated p-values. n = 83–120 mitochondria, 11–13 neurons, three biological replicas (one-way ANOVA with post hoc Kruskal-Wallis multiple comparison test). c, Experimental timeline outlining the sequence of plating, transfection, Thiamet-G and OSMI-4 treatments, and imaging of cultured rat hippocampal neurons in 5 mM glucose for experiments illustrated in Fig. 2d. d, Western blot analysis of whole cell lysate (Input), isolated mitochondrial and cytoplasmic fractions from HEK293T. The samples were probed with antibodies against HK1, ATP5B (mitochondrial marker), and Tubulin (cytoplasmic marker) with or without ectopic OGT expression and Thiamet-G or vehicle treatments. e, Quantification of HK1 levels in mitochondrial (left), cytoplasmic (middle), and whole cell lysate (right) under indicated different conditions. n = 3 (all values are shown as mean ± SEM, one-tailed Mann-Whitney U test). f, A schematic illustration of Native and Tissue-specific Fluorescence (NATF) method used for endogenous labeling of HK1 in C. elegans DA9 neuron.
Extended Data Fig. 3 Elucidating the O-GlcNAc modification of Hexokinase 1 and 2.
a, Illustration of CRISPR-based approach to add eGFP tag at the C-terminal of HK1 in HEK293T cells. The strategy is based on transcript-202 (NM_000188.2) and was implemented by BioCytogen. b, Western blot analysis of the whole cell lysate (Input), used for the generation of mitochondrial and cytoplasmic fractions as shown in Fig. 3, from HEK293T cells. The whole cell lysate (Input) from CRISPR edited HEK293T cells was probed with antibodies against O-GlcNAc (RL2), GFP (HK1), OGT and tubulin (loading control), with or without OGT overexpression and Thiamet-G treatments. c, Schematic demonstrating the sequence of mitochondrial isolation and O-GlcNAc immunoprecipitation (IP) using the anti-O-GlcNAc antibody RL2 from cultured rat hippocampal neurons. d, Western blot analysis of mitochondrial fraction (Input) and O-GlcNAc IP using antibody against HK1 and ATP5B (mitochondrial marker). e, f, Western blot analysis of whole cell lysate from cortical neuron cultures. The lysate was probed with antibodies against O-GlcNAc (RL2), OGT, OGA, and tubulin (as a loading control), following treatments with Thiamet-G or OSMI-4. (f) Quantification of O-GlcNAcylation levels, normalized to tubulin. All values are presented as mean ± SEM. n = 3 independent experiments (one-tailed Mann-Whitney U test). g, eGFP tagged Hexokinase 2 (HK2) was expressed in HEK293T cells. GFP antibody was used to immunoprecipitate (IP) HK2, with or without OGT overexpression and Thiamet-G treatments. The IPs were probed with anti-GlcNAc (RL2) and anti-GFP antibodies. Whole cell lysates (Input) were probed with anti-GFP and anti-tubulin antibodies. Rabbit IgG serves as an IP control. h, Quantification of HK2 O-GlcNAcylation levels. All values are shown as mean ± SEM, unpaired one-tailed t-test. n = 3. i-k, Quantification of the expression levels of HK1-GFP (i), myc-OGA (1–400) (j) and nGFP-HA-OGA (544–706) (k) in COS-7 cells, as shown in Fig. 3d. Data are presented as a violin plot with individual data points and associated p-value. n = 42 cells, three independent experiments (Unpaired two-tailed t-test, and one-way ANOVA with post hoc Tukey’s multiple comparison test).
Extended Data Fig. 4 Identification and functional analysis of Hexokinase 1 T259 O-GlcNAcylation Site.
a, Tandem mass spectra showing O-GlcNAc on peptides derived from human Hexokinase 1. Data were acquired using HCD fragmentation and prominent y and b-type ions are labeled. Blue arrow indicates the O-GlcNAc modified threonine (T). Bottom figure demonstrating the survey scan and prominent y/b-type ions. b–e, Quantitative analysis of HK1 co-localization to measure the percentage of mitochondrial HK1 intensity in HEK293T cells, cultured in 5 mM glucose containing media. (b and d) Representative images of HEK293T cells expressing WT-HK1-GFP or T259A-HK1-GFP (green) and Mito-DsRed (magenta) with or without OGT overexpression and Thiamet-G treatments. Scale bars represent 10 μm (c and e) WT and T259A HK1 intensity on mitochondria, percentage of total WT and T259A HK1 on mitochondria and the Pearson’s correlation coefficient (R value) for each condition. Data are presented as violin plots with individual data points and associated p-values. n = 9 cells, three biological replicas (two-tailed Mann-Whitney U test). f, Representative images of hippocampal neurons expressing HK1-shRNA, WT-HK1-GFP, and the O-GlcNAc mutant T259A HK1-GFP (T259-HK1-GFP) are shown in green, along with Mito-DsRed in magenta. These images were stained with an HK1 antibody (in cyan) to visualize the total HK1 expression. Scale bar represents 5 µm. g, Quantification of HK1 expression levels was performed retrospectively for all experiments by analyzing the anti-HK1 staining to ensure consistent endogenous HK1 levels throughout all experiments. All values are shown as mean ± SEM (one-way ANOVA with post hoc Tukey’s multiple comparison test). h, Quantification of the size of the mitochondria along the axons as depicted in Fig. 4g. n = 81–86 mitochondria from 10–13 axons from three biological replica (one-way ANOVA with post hoc Kruskal-Wallis multiple comparison test).
Extended Data Fig. 5 O-GlcNAcylation modifies Hexokinase 1 activity and contributes to the formation of mitochondrial glycolytic metabolon.
a, b, Quantification of WT and T259A-HK1 expression levels in HEK293T cells, corresponding to the Fig. 5b. Whole cell lysates (Input) were probed with anti-HK1 and anti-tubulin (loading control) antibodies. All values are shown as mean ± SEM. n = 4 (one-way ANOVA with post hoc Tukey’s multiple comparison test). c–e, Glucose-6-phosphate (G6P) levels were measured in HEK293T cells (maintaining endogenous HK1 levels) following OGT overexpression and treatment with either Thiamet-G or vehicle. G6P levels in untreated cells were set as 1, and fold changes in response to Thiamet-G treatment and OGT overexpression were calculated (mean ± SEM, one-tailed Mann-Whitney U test). (d and e) Endogenous HK1 levels were quantified from whole cell lysates (Input) using anti-HK1 and anti-tubulin (loading control) antibodies (mean ± SEM, Kruskal-Wallis test). f, Mitochondrial oxygen consumption rates (left) and extracellular acidification rates (right) were measured in HEK293T cells expressing control vector, eGFP-tagged WT, or T259A HK1, following treatment with either vehicle (DMSO) or overnight Thiamet-G treatment. The subsequent injections of Oligomycin (Oligo, 2 μM), FCCP (2 μM), and a combination of rotenone (Rot, 0.5 μM) and antimycin A (AA, 0.5 μM) were used to calculate ATP production rates. All values are presented as the mean ± SEM. g–i, HK1 levels in HEK293T cells used for metabolic measurements were quantified using western blot analysis of whole cell lysates, probed with antibodies against HK1 and tubulin (serving as a loading control). The asterisk indicates the presence of endogenous HK1. All values are shown as mean ± SEM. n = 3 (Mann-Whitney U test and and post hoc Kruskal-Wallis multiple comparison test). j, k, Western blot analysis of mitochondrial (Mito) and cytoplasmic (Cyto) fractions from HEK293T (j) and Cortical neurons (k) using antibodies against ATP5B (mitochondrial marker), Actin (cytosolic marker), Golgin 97 (Golgi Marker), KDEL and CKAP4 (endoplasmic reticulum marker), PEX19 (peroxisome marker), LAMP2 (lysosome marker) and Lamin A (nuclear marker). Input indicates whole cell lysate from Cortical neurons. Total loading amount per lane is indicated as percentage for each fraction. l, m, Analysis of glycolytic enzymes in mitochondrial and cytoplasmic fractions from rat cortical neurons. Mitochondrial (left) and cytoplasmic fractions (right) from rat cortical neurons, treated overnight with vehicle or Thiamet-G to upregulate O-GlcNAcylation, were analyzed for all glycolytic enzymes using the following antibodies: HK1, Glucose-6-phosphate isomerase (GPI), Phosphofructokinase muscle isoform (PFK M), Aldolase A (Aldo A), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Phosphoglycerate kinase (PGK), Phosphoglycerate mutase 1 (PGAM1), Neuron-specific enolase (NSE), Triosephosphate isomerase (TPI), Pyruvate kinase (PKM), ATP5B (mitochondrial loading control), and Tubulin (cytoplasmic loading control) (l). Quantified enzyme levels under baseline conditions (m) were normalized to 1 (dashed line), and fold changes in response to Thiamet-G treatment were calculated. All values are shown as mean ± SEM. n = 3-4 biological replica. n, o, Axonal segments of hippocampal neurons cultured in 5 mM glucose, co-transfected with Mito-DsRed (gray), and human PKM2 tagged with mEGFP (pseudocolor, fire). O-GlcNAcylation level was upregulated by Thiamet-G treatment, and downregulated by OSMI-4 treatment. Scale bar represents 5 µm. (o) Quantification of mitochondrial (Mito) and cytoplasmic (Cyto) PKM2-mEGFP intensity ratios along axons. Data are presented as a violin plot with individual data points and associated p-values. n = 93–110 mitochondria from 9–12 axons, three biological replicas (one-way ANOVA with post hoc Tukey’s multiple comparison test).
Extended Data Fig. 6 Hexokinase 1 O-GlcNAcylation is required for glycosome formation.
a-d, Analysis of glycolytic enzymes in mitochondrial and cytoplasmic fractions from HEK293T cells expressing WT-HK1-GFP or T259-HK1-GFP. Whole cell lysates, as well as mitochondrial and cytoplasmic fractions from HEK293T cells expressing WT (a and b) and T259A HK1 (c and d) with or without ectopic OGT expression and overnight vehicle (DMSO) or Thiamet-G treatment, were analyzed to quantify the glycolytic enzyme levels. The following antibodies were used: HK1, Aldolase A (Aldo A), Phosphoglycerate kinase (PGK), Pyruvate kinase 2 (PKM2), ATP5B (mitochondrial loading control), and Tubulin (cytoplasmic loading control). Quantified enzyme levels under baseline conditions were normalized to 1, and fold changes in response to OGT overexpression and Thiamet-G treatment were calculated. All values are shown as mean ± SEM. n = 3 biological replica (one-tailed Mann-Whitney U test).
Extended Data Fig. 7 Hexokinase 1 O-GlcNAcylation and neuronal functional measurements.
a, Representative images of neuronal soma expressing shRNA-resistant BFP tagged WT or T259A-HK1 (blue) with HK1-shRNA, mCherry cell filler (magenta), and vGLUT1-pH (green). b-c, Retrospective quantification of WT-HK1 and T259A-HK1-BFP, and vGLUT1-pH (c) in rat hippocampal neurons used for imaging experiments depicted in Fig. 7. Scale bar represents 5 µm. All values are presented as mean ± SEM. n = 12–13 neurons from three biological replica (unpaired two-tailed t-test). d-g, The experimental design outlines the timeline for the plating, transfection, Tetrodotoxin (TTX) treatment, and imaging of cultured rat hippocampal neurons. (e) Hippocampal neurons were transfected with shRNA-resistant BFP-tagged WT or T259A-HK1-BFP, HK1-shRNA, and vGLUT1-pH. Following transfection, 1 µM TTX was added to neuronal culture. Two hours before imaging, TTX was washed-off as shown in (d). Neurons were electrically stimulated with 100 APs 10 Hz. Images showing vGLUT1-pH (pseudo-color, fire) and the cell filler mCherry (gray) before and after stimulation with WT-HK1 or T259A-HK1-BFP expressing neurons. Neutralization of vGLUT1-pH vesicles with NH4Cl reveals total axonal vesicle pool. (f) Average traces of vGLUT1-pH with 100 APs 10 Hz stimulation in WT-HK1 (black) or T259A-HK1 (orange) expressing neurons, previously TTX treated. ∆F values were normalized to maximal ∆F obtained from NH4Cl treatment. Error bars represent SEM. n = 8-9 neurons and 20–55 presynaptic boutons from four biological replicas. (g) Baseline and maximal (after electrical stimulation) vGLUT1-pH ∆F/F values. All values are shown as mean ± SEM (unpaired two-tailed t-test).
Extended Data Fig. 8 Impact of Hexokinase 1 O-GlcNAcylation on Presynaptic Calcium Dynamics.
a, Hippocampal neurons were transfected with shRNA-resistant BFP-tagged WT or T259A-HK1-BFP, HK1-shRNA, and GCaMP6s. The displayed images illustrate the GCaMP6s signal (pseudo-colored, fire) and the cell filler mCherry (magenta) prior to and following stimulation with 100 APs at 10 Hz. in neurons expressing either WT-HK1 or T259A-HK1-BFP. The peak of the Ca2+ response elicited by KCl defines the maximum GCaMP6s intensity. The scale bar represents 5 µm. b, The average trace of GCaMP6s during the 100 APs at 10 Hz stimulation in neurons expressing either WT-HK1 or T259A-HK1. ∆F values were normalized to the maximal ∆F observed during KCl treatment. All values are presented as mean ± SEM. n = 132 ROIs, 12 neurons from three biological replica. c, Maximum GCaMP6s ∆F/Fmax values. Data are presented as a violin plot with individual data points and associated p-value (unpaired two-tailed t-test). d-f, Retrospective quantification of WT-HK1 and T259A-HK1-BFP (blue), GCaMP6s, and mCherry cell filler (magenta) in rat hippocampal neurons used for imaging experiments depicted in (d-f). Scale bar represents 10 µm. All values are presented as mean ± SEM. n = 12-13 neurons from three biological replica (unpaired two-tailed t-test). g, Representative raster plots of demonstrating the firing patterns of rat cortical neurons (cultured on microelectrode array plates) across 64 electrodes at different time points, following transduction with lentiviral particles containing shRNA-resistant GFP-tagged WT or T259A-HK1, and HK1-shRNA. Each black line indicates a detected spike (action potentials), while blue lines represent a single-channel burst, defined as a sequence of at least five spikes with an inter-spike interval not exceeding 100 milliseconds. Each magenta line indicates coordinated bursts across the electrodes, known as network bursts. h, Mean firing rate (Hz) and network bursts were calculated from MEA recordings of neurons expressing WT or T259A-HK1. Data are shown as mean values ± SEM with associated p-values (unpaired two-tailed t-test), n = 5-6 MEA recordings across conditions, from two independent primary neuron preparation.
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Wang, H., Vant, J.W., Zhang, A. et al. Organization of a functional glycolytic metabolon on mitochondria for metabolic efficiency. Nat Metab 6, 1712–1735 (2024). https://doi.org/10.1038/s42255-024-01121-9
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DOI: https://doi.org/10.1038/s42255-024-01121-9
