Abstract
The role of hepatic insulin resistance (HIR) in the development of fatty liver, diabetes and cardiovascular diseases is well known, yet the molecular basis of HIR remains unclear, limiting targeted therapeutic strategies. Here we show that insulin signalling-inactivated phosphorylated GSK-3β (p-GSK-3β) is revitalized via reactive oxygen species-mediated sulfenylation, leading to glycogenesis termination and gluconeogenesis initiation, two hallmarks of HIR. Mechanistically, sulfenylated or ‘oxidatively activated’ p-GSK-3β regains the enzymatic activity to phosphorylate liver glycogen synthase, thereby blocking glucose storage. This activated p-GSK-3β can further phosphorylate insulin-suppressed Forkhead box O1, thus liberating its transcriptional activity to promote the expression of gluconeogenic enzymes. Notably, this dual-pathway mechanism is conserved in clinically relevant human liver samples and organoids. These findings elucidate the molecular mechanism by which HIR is formed and provide potential strategies against HIR by targeting sulfenylated or ‘oxidatively activated’ p-GSK-3β.
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Data availability
All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Information. All data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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No custom code was used in this study.
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Acknowledgements
This work was supported by National Key Research and Development Program of China (2022YFA1206000 to B.H.), National Natural Science Foundation of China (82388201 to B.H., 82401014 to J.C., 82171759 to J.G. and 32394003 to J.M.) and CAMS Innovation Fund for Medical Sciences (2024-I2M-ZD-006 to B.H., 2021-I2M-1-021 to B.H., 2025-I2M-XHXX-063 to J.C. and 2025-I2M-KJ-008 to J.L.). The authors acknowledge the use of BioRender for creating schematic diagrams.
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B.H. conceived the project. J.C., Z.L., K.C., Y.J., N.Z., Y.Z., D.W. and C.Z. and performed the experiments. J.C., Y.J., Z.L., K.C., Z.W., L. Zhou, J.Y., T.L. and L. Zhang developed the methodology. Z.L., X.S., J.G. and T.H. contributed to clinical simple collection and analysis. J.C., K.T., J.M., H.Z. and J.L. performed data analysis. B.H. and J.C. wrote the paper.
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Extended data
Extended Data Fig. 1 HIR occurrence relies on increased ROS levels.
a. The average daily food intake of C57BL/6 J mice was determined over a 3-day period (n = 5/group). b. The body weight of C57BL/6 J mice was measured after 3 days on their respective diets (n = 5/group). c. Serum insulin levels of C57BL/6 J mice were measured by ELISA following a 6-hour fast (n = 5/group). d. Hepatic H2O2 levels of C57BL/6 J mice were quantified using a fluorescence microplate assay (n = 5/ group). e. HGP was assessed by hyperinsulinemic–euglycemic clamps (n = 5/ group). f. M + 6-glucose enrichment in hepatic glycogen was analysed by LC–MS/MS (n = 5/ group). g. Serum M + 3-glucose enrichment in C57BL/6 J mice, measured by LC–MS/MS (n = 5/ group). h. Hepatocyte ROS levels were measured by flow cytometry (n = 3/ group). i. Hepatocyte glycogen M + 6-glucose enrichment was analysed by LC–MS/MS (n = 3/ group). j. Hepatocyte glucose output was measured using a colorimetric assay (n = 3/ group). k-o. C57BL/6 J mice were fed a ND or a HFD for 3 days (n = 5/ group). Blood and liver tissue were obtained. Basal glucose level was measured using a strip-based glucometer following an overnight fast (k). FFA in liver (l) and in blood (m) was analysed by the colorimetric assay. TG in liver (n) and in blood (o) was analysed by the colorimetric assay. p-r. C57BL/6 J mice fed with a ND or HFD were treated daily with 50 mg/kg ABT (i.g.) or 20 mg/kg Etomoxir (i.p.) for 3 consecutive days. The H2O2 levels in the livers were detected using fluorescence microplate (p). GIR (q) and HGP (r) under clamp conditions were detected by LC–MS/MS (n = 5/ group). s. C57BL/6 J mice were fed a ND or a HFD for 3 days (n = 5/ group). And liver tissue was obtained. And the hepatic levels of IL-1β, IL-6, and TNF were measured by ELISA. Data are presented as mean ± S.E.M. Individual data points represent biological replicates. Individual data points represent biological replicates. p values were calculated using one-way ANOVA (d-j and p-r) or unpaired two-tailed Student’s t-test (a-c, k-o, and s).
Extended Data Fig. 2 Increased ROS revitalizes p-GSK-3β to phosphorylate Gys.
a. Mice treated with SP600125(7.5 mg/kg), SB202190(5 mg/kg), U0126(10 mg/kg), or vehicle were fed with HFD or ND for 3 days (i.p.). The expression of p-GYS2, and GYS2 in livers of the mice treated with insulin (0.75 U/kg body weight) for 10 min was analysed by immunoblotting (n = 3/ group). b. Hepatocytes were transfected with Gsk-3β siRNA for 24 h. Gsk-3β expression was determined by real-time PCR. c. Hepatocytes were transfected with Gsk-3α siRNA for 24 h. Gsk-3α expression was determined by real-time PCR. d. Six-week-old male C57BL/6 J mice were fed either a ND or a HFD for 3 days. Levels of newly synthesized proteins labelled with puromycin were analysed by immunoblotting (n = 3/ group). e. Hepatocytes were pretreated with 1 mM GEE for 2 h prior to stimulation with 500 μM PA for 3 h followed by a 30 min incubation with 100 nM insulin. Dimedone, p-GSK-3β and p-GYS2 detected by immunofluorescence staining. P-GSK-3β, green. p-GYS2, red. Dimedone, magenta. (Scale bars = 20 mm) Data are presented as mean ± S.E.M. p values were calculated using one-way ANOVA (b and c).
Extended Data Fig. 3 Purification and identification of GSK-3β.
a. Purified murine p-GSK-3β protein was detected by silver staining. b. Hepatocytes with Gsk-3β overexpression were treated with 500 μM PA for 3 h followed by 30 min incubation with 100 nM insulin and 5 mM dimedone. Sulfenic acid modification of p-GSK-3β with dimedone was determined by LC–MS/MS. c. Separation of the three GSK-3β phosphoisotypes on phos-tag SDS-PAGE. Gsk-3β plasmid was expressed in hepatocytes and subjected to phos-tag SDS-PAGE, followed by immunoblotting with anti-GSK-3β, anti-phospho-Ser9 (pS9) and anti-phospho-Tyr216 (pY216) antibodies, as indicated. The phosphorylation states of the three bands of GSK-3β on phos-tag SDS-PAGE are indicated on the right side of the blot; the double arrowheads indicate GSK-3β that is phosphorylated at both Ser9 and Tyr216, the arrowhead indicates GSK-3β that is phosphorylated at Tyr216, and the bar indicates non-phosphorylated GSK-3β. d. The RMSDs of backbone Cα atoms with respect to the first frame during MD simulation for the wild-type (black) and complex with a Cys178 sulfenic acid (red). e, f. The variations of the distance between SER9@P and residues ARG180@CZ (e) and LYS94@NZ (f) during MD simulation for the wild-type complex (black) and complex with Cys178 sulfenic acid (red). SER9@P, the main chain phosphorus atom of Ser9. ARG180@CZ, the side chain carbon atom of Arg180. LYS94@NZ, the side chain amide nitrogen of Lys94.
Extended Data Fig. 4 FoxO1 was phosphorylated at Thr464 for gluconeogenesis.
a-b. Six-week-old male C57BL/6 J mice were fed either a ND or a HFD for 3 days (n = 5/ group). Expression of Pnpla2 (ATGL, adipose triglyceride lipase; a), and β-oxidation-related genes (b) were measured by real-time PCR. c. FoxO1 phosphorylation sites in hepatocytes overexpressing Foxo1 were detected by mass spectrometry following insulin treatment. d. FoxO1 phosphorylation sites were detected by mass spectrometry in hepatocytes overexpressing Foxo1 following insulin stimulation. e, f. Recombinant murine FoxO1 was incubated with p-GSK-3β in the presence of 200 μM ATP and 100 μM H2O2 for 6 h. Phosphorylation sites of FoxO1 were determined by mass spectrometry (e). Purified murine Foxo1 protein was detected by coomassie brilliant blue staining (f). g, h. Hepatocytes were transfected with Foxo1 siRNA in the presence of exogenous RNAi-resistant Foxo1 cDNA. After transfection with indicated Foxo1 and Gsk-3β plasmids for 45 h, hepatocytes were treated with 500 μM PA for 3 h followed by a 30 min incubation with 100 nM insulin. The expression of cytoplasmic or nuclear FoxO1 was analysed by immunoblotting. i, FoxO1T464 is a conserved site across difference species. Alignment of FoxO1 sequences from the indicated species was performed using Jalview software, based on the Clustal sequence alignment algorithm, with the Thr464 residue boxed in red. The colour scheme used for the alignment is default in Clustal X. Conserved amino acids are highlighted according to the Clustal X colour scheme57: hydrophobic (that is A, F, I, L, M, V, and W; in blue), positively charged (that is K and R; in red), negatively charged (that is D and E; in purple), polar (that is N, Q, S, and T; in green), aromatic (that is H and Y; in turquoise), glycine (G; in orange) and proline (P; in yellow). j, FOXO1T467 is a conserved site in other Homo sapiens FOXO family members. The Thr467 residue in FOXO1 and the corresponding residue in other FOXO members were boxed in red. Data are presented as mean ± S.E.M. p values were calculated using unpaired two-tailed Student’s t-test (a and b).
Extended Data Fig. 5 HIR can be reversed by targeting sulfenylated p-GSK-3β.
a. Hepatic GSK-3β expression in mice with AAV8-mediated Gsk-3β knockdown, assessed by immunoblotting. b, c. Mice treated with 4 mg/kg AR-A014418 (i.p.) were fed with HFD for 5 days. Then insulin tolerance tests (0.5 U/kg, b) and pyruvate tolerance tests (2 g/kg, c) of the mice were detected (n = 5/group). d. Hepatic GYS2 expression was altered in mice subjected to AAV8-mediated Gys2-shRNA knockdown, as shown by immunoblotting. e-f. Hepatic sulfenylation of p-GSK-3β and GSK-3β were analysed by immunoblotting in mice fed a ND or HFD for 8 (e) or 12 weeks (f). g. Immunofluorescence staining of consecutive liver sections from control and T2D mice(HFD, 24 weeks) following insulin stimulation: glutamine synthetase (CV zone, magenta), ROS (red), and p-GSK-3β (green). Scale bar= 200 μm. h. GIR was measured in wild-type (WT) and C178A mutant mice fed a ND or HFD (n = 5/group). i. p-GSK-3β was analysed by immunoblotting in ‘Young’ and ‘Aged’ mice. j. Hepatic sulfenylation of total GSK-3β and p-GSK-3β were analysed by immunoblotting in young and aged mice. k. Hepatic expression levels of p-GYS2, GYS2, pFoxO1 (T464), and FoxO1 were analysed by Western blot in ‘Young’ and ‘Aged’ mice following insulin treatment. l. The expression of nuclear FoxO1 in the livers of ‘Young’ and ‘Aged’ mice was analysed by immunoblotting. m. Hepatic sulfenic acid modification of p-GSK-3β was analysed by immunoblotting in ‘Aged’ mice. n. Nuclear FoxO1 expression in liver tissue was assessed by immunoblotting in ‘Aged’ mice. o. Hepatic levels of p-FoxO1 (T464), FoxO1, p-GYS2, and GYS2 were analysed by immunoblotting following insulin stimulation in ‘Aged’ mice. p. Nuclear FoxO1 expression in liver tissue was analysed by immunoblotting in ‘Aged’ mice. q. Hepatic expression of p-FoxO1 (T464), FoxO1, p-GYS2, and GYS2 was analysed by immunoblotting in ‘Aged’ mice following insulin stimulation. Data are presented as mean ± S.E.M. p values were calculated using one-way ANOVA(h) or unpaired two-tailed Student’s t-test (b and c).
Extended Data Fig. 6 ROS activates p-GSK-3β via sulfenylation to induce insulin resistance in human hepatocyte organoids.
a–d. Surgical liver tissues with or without NAFLD were collected (n = 15/group, a). The H2O2 (b) and glycogen (c) levels were measured using a colorimetric assay. The expression of PCK1 in human liver tissues was determined using real-time PCR (d). e. Immunoblot analysis of dimedone-labelled p-GSK-3β sulfenylation in human liver. f. The expression of p-GYS2 and GYS2 was analysed using immunoblotting. g. The expression of nuclear FoxO1 in the human liver tissue was analysed using immunoblotting. h. Human liver tissue was treated with 5 mM dimedone for 30 min. Sulfenic acid modification of dimedone-labelled GSK-3β was analysed by immunoblotting (upper). In addition, the expression of p-AKT, AKT, p-GSK-3β, and GSK-3β in liver tissues was analysed by immunoblotting (lower panel). i, GSK-3β sequence alignment among human, mouse, macaca mulatta and danio-rerio. The alignment was performed by using ClustalW2 algorithm and ESPript 3.0. Identical residues were highlighted by red background58. The black box marks the cysteine site for p-GSK-3β sulfenylation. Deoxynucleotide residue numbers were indicated on the top of the sequence. j. Immunoblot analysis of dimedone-labelled p-GSK-3β sulfenylation of purified human p-GSK-3β. k. Mass spectrometric analysis of dimedone-labelled p-GSK-3β sulfenylation of purified human p-GSK-3β. l. Colorimetric analysis of glucose release from human hepatocyte organoids. m. p-GYS2 and GYS2 expression in human hepatocyte organoids was analysed by digital immunoblotting. n. The FoxO1 location of organoids was observed by confocal microscope. Scale bar, 20 μm. o. The expression of p-GYS2 and GYS2 in organoids was analysed by digital immunoblotting. p. The FoxO1 location of organoids was observed by confocal microscope. Scale bar, 20 μm. Data are presented as mean ± S.E.M. p values were calculated using one-way ANOVA (l) or unpaired two-tailed Student’s t-test (b-d). Panel a created in BioRender; Chen, J. https://biorender.com/fdrz3xd (2026).
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Chen, J., Liu, Z., Gu, J. et al. Revitalizing p-GSK-3β via cysteine sulfenylation promotes hepatic insulin resistance by differentially regulating glycogenesis and gluconeogenesis. Nat Metab (2026). https://doi.org/10.1038/s42255-026-01507-x
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DOI: https://doi.org/10.1038/s42255-026-01507-x
