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The hepatic clock synergizes with HIF-1α to regulate nucleotide availability during liver damage repair

Nature Metabolism volume 7, pages 148–165 (2025)Cite this article

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Abstract

Nucleotide availability is crucial for DNA replication and repair; however, the coordinating mechanisms in vivo remain unclear. Here, we show that the circadian clock in the liver controls the activity of the pentose phosphate pathway (PPP) to support de novo nucleotide biosynthesis for DNA synthesis demands. We demonstrate that disrupting the hepatic clock by genetic manipulation or mistimed feeding impairs PPP activity in male mice, leading to nucleotide imbalance. Such defects not only elicit DNA replication stress to limit liver regeneration after resection but also allow genotoxin-induced hepatocyte senescence and STING signalling-dependent inflammation. Mechanistically, the molecular clock activator BMAL1 synergizes with hypoxia-inducible factor-1α (HIF-1α) to regulate the transcription of the PPP rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD), which is enhanced during liver regeneration. Overexpressing G6PD restores the compromised regenerative capacity of the BMAL1- or HIF-1α-deficient liver. Moreover, boosting G6PD expression genetically or through preoperative intermittent fasting potently facilitates liver repair in normal mice. Hence, our findings highlight the physiological importance of the hepatic clock and suggest a promising pro-regenerative strategy.

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Fig. 1: Disturbance of the hepatic clock leads to S phase progression failure and impairs liver regeneration.
Fig. 2: Disturbance of the hepatic clock impairs the rhythmicity and activity of the PPP that is crucial for liver regeneration.
Fig. 3: Elevated oxidative stress in hepatic clock disruption partially contributes to regenerative defects.
Fig. 4: Nucleotide shortage induced by PPP dysfunction contributes to replication stress and growth defects in BMAL1-deficient cells.
Fig. 5: BMAL1 synergizes with HIF-1α to control the rhythmicity and abundance of G6PDX expression in the mouse liver.
Fig. 6: Boosting PPP activity facilitates liver regeneration.
Fig. 7: Nucleotide shortage impedes DNA repair in Bmal1-depleted cells, causing senescence and STING-dependent inflammation.

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Data availability

The time-series microarray data for the postoperative mouse livers25 have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE215216. All other supporting data are included in this paper. Source data are provided with this paper.

Code availability

No custom code was used in this study.

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Acknowledgements

We thank all members of the laboratory of the late P. Sassone-Corsi (University of California, Irvine) for insightful discussions and advice, as well as J. Tamanini (Shenzhen University and ET Editing) for editing the paper before submission. This work was supported by grants from the National Key Research and Development Program of China (2022YFC2303200, 2023YFA1801900 to M.Q.); the National Natural Science Foundation of China (82022025, 91949124 and 82271602 to M.Q.; 32071259 to Z.W.; 81972894 to C.S.); the Innovation and Entrepreneurship (Shuangchuang) Program of Jiangsu Province (2023); the Natural Science Foundation of Chongqing (CSTB2024NSCQ-MSX1152); the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (SKLNMZZ2024JS39); and the ‘Double First-Class’ University Project of China Pharmaceutical University (CPUQNJC22). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

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  1. These authors contributed equally: Linyuan Peng, Siliang Xiang.

Authors and Affiliations

  1. State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, China

    Linyuan Peng, Siliang Xiang, Tianzhi Wang, Mei Yang, Yajun Duan, Xiaoyu Ma, Su Li, Xin Zhang, Haiyang Hu, Chen Wang, Zhongyuan Wang & Minxian Qian

  2. Guangdong Key Laboratory of Genome Instability and Human Disease Prevention, Center for Anti-aging and Regenerative Medicine, School of Basic Medical Sciences, Shenzhen University Medical School, Shenzhen University, Shenzhen, China

    Siliang Xiang, Cong Yu, Jie Sun & Baohua Liu

  3. School of Life Sciences, Hainan University, Haikou, China

    Zuojun Liu

  4. State Key Laboratory of Natural Medicines, NMPA Key Laboratory for Research and Evaluation of Pharmaceutical Preparations and Excipients, Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing, China

    Chunmeng Sun

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Contributions

M.Q., Z.W. and B.L. conceived of and designed the research and wrote the paper. L.P. and S.X. performed most experiments, with help from T.W., M.Y., Y.D., X.M., S.L., C.Y. and X.Z., and analysed the data. H.H., Z.L. and J.S. provided technical assistance and/or suggestions for experiments. C.S. and C.W. reviewed and edited the paper.

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Correspondence to Baohua Liu, Zhongyuan Wang or Minxian Qian.

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Nature Metabolism thanks Jean-Michel Fustin, Meritxell Huch and Tara Teslaa for their contribution to the peer review of this work. Primary Handling Editor: Yanina-Yasmin Pesch, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Expression profiles of the central clock genes under different experimental conditions.

a, qRT-PCR showing expression of Cry1, Per2, and Dbp in C57BL/6 J mouse livers at the indicated time points after PH. b. Heatmap showing differential expression of genes related to the circadian clock and cell cycle during liver regeneration. Original data were derived from GSE215216 dataset. c-e. The expression patterns of the core clock genes in the livers of WT and LBKO mice (c), or WT mice fed AL or DRF (d,e), determined by qRT-PCR. For a,c-e, n = 3 mice per group. Data are mean ± s.e.m. P-values were calculated using two-way ANOVA with Sidak’s test (a). Rhythmicity analysis was performed using JTK_CYCLE as shown in Supplementary Table 1.

Source data

Extended Data Fig. 2 Circadian clock disruption limits hepatocyte proliferation after resection.

a,b, Representative IHC images (a) and quantification (b) of Ki67-positive staining cells in AL, DRF and LBKO mice with sham- or PH- operation. More than three fields for each of three independent samples were counted. Scale bar, 100 μm. c-g, Graphs showing the expression of Bmal1 (c), the liver-body weight ratio (d), the contents of ALT (e) and AST (f), and representative IHC images and quantification of Ki-67 staining (g) in the livers on day 4 post-PH. Male adult Bmal1f/f mice were infected with control AAV8 (AAV-NC) or AAV8-TbgCre (AAV-Cre) and then subjected to PH. Five fields for each of three independent samples were counted. Scale bar, 100 μm. h-j, Graphs showing the liver-body weight ratio (h) and the contents of ALT (i) and AST (j) in mice which fed with DRF for 10 days were then subjected to PH. The samples were collected on day 4 post-PH. The red line indicates the mean value of DRF-D4 group. k, qPCR analysis showing the pattern of Pcna mRNA expression in the livers of WT, LBKO and DRF mice at various ZTs. For c,k, n = 3 mice per group. For d-f,h-j, n = 5 mice per group. Data are mean ± s.e.m. P-values were calculated using one-way ANOVA with Dunnett’s test (b) and unpaired two-tailed Student’s t-test (c-j). For k, JTK_CYCLE was applied to determine rhythmicity. P < 0.05 was considered to be rhythmic.

Source data

Extended Data Fig. 3 Bmal1 depletion alters glucose metabolic flux, while G6pdx deficiency attenuates hepatocyte proliferation.

a-c, Examples of rhythmic metabolites generated from the PPP. 6-PG, 6-phosphogluconate (a); R5P, ribose-5-phosphate (b); S7P, sedoheptulose 7-phosphate (c). * P < 0.05, ** P < 0.01, *** P < 0.001. d,e, Mass isotopomer distribution of nucleotides (d) and lactate (e) derived from 13C-labeled glucose in primary hepatocytes from WT and LBKO mice. n = 4 independent biological samples. ND, not detected. f-h, Flow cytometric analysis (f) and quantification (g) of 2-NBDG intake, and measurement of lactate contents (h) in primary hepatocytes from WT and LBKO mice. n = 3 independent biological replicates. i,j, The mRNA expression of glycolytic genes Ldha and Pdk1 in hepatocytes from WT and LBKO mice with or without overexpressing G6pdx (i) and those from WT mice infected with AAV-shNC or AAV-shG6pdx (j). k,l, Representative IF images (k) and quantification (l) of staining with anti-Ki-67 and anti-G6pdx antibodies for the livers of AAV8-shNC or AAV8-shG6pdx infected mice on day 4 post-PH. Seven fluorescent fields for each of three samples were counted. Scale bar, 20 μm. m, qRT-PCR showing the mRNA levels of G6pdx in the livers of mice infected with AAV-shNC or AAV-shG6pdx for four weeks. For a-c,i,j,m, n = 3 mice per group. Data are mean ± s.e.m. P-values were calculated using two-way ANOVA with Sidak’s test (a-c,e,i), unpaired two-tailed Student’s t-test (g,h,l,m) and multiple unpaired two-tailed Student’s t-test (j).

Source data

Extended Data Fig. 4 Redox imbalance in hepatic clock-disturbed mice after PH.

a, The rhythmicity of NADPH content in the livers from WT and LBKO mice at various ZTs. n = 3 mice per group. b,c, Representative IF images (b) and quantification (c) of DHE staining of liver tissues of AL, DRF, and LBKO mice after sham or PH. Scale bar, 20 μm. d,e, Representative IHC images (d) and quantification (e) of 8-OhdG staining for AL, DRF and LBKO mice with sham- or PH- operation. Scale bar, 100 μm. More than three fields for each of three samples were counted. Data are mean ± s.e.m. P-values were calculated using one-way ANOVA with Dunnett’s test (c,e). For a, JTK_CYCLE was applied to determine rhythmicity. P < 0.05 was considered to be rhythmic.

Source data

Extended Data Fig. 5 Bmal1-regulating nucleotide pool is required for cell proliferation and survival.

a, Measurement of the nucleoside/nucleotide contents by LC-MS/MS in WT mice with or without shG6pdx via the AAV8 system. b, Examples of oscillating nucleosides/nucleotides from the metabolome of WT and Bmal1-null mouse livers. The processing data were included in Supplementary Table 3. Box plots extend from the 25th and 75th percentile, with a horizontal line representing the median value, and the whiskers indicate the min and max values. c, Measurement of the contents of nucleotides in the livers from WT and LBKO mice before and after PH by LC-MS/MS. d, Representative images of hepatic organoids cultured in Matrigel assessed by bright field microscopy, with quantification of the numbers of hep-organoids formed from 104 cells. Primary hepatocytes from LBKO mice were treated with or without a combination of NAC and NTPs during the 14-day organoid culture. n = 3 independent experiments. e, Immunoblotting showing the protein levels of Bmal1 and G6pdx in primary hepatocytes, which were isolated from Bmal1f/f mouse livers and then treated with AAV-Cre. f-h, Graphs showing representative images (f) of hepatic organoids cultured in Matrigel and quantification of the numbers of hep-organoids formed from 104 cells (g) and the mean diameter (h). Primary hepatocytes, as pretreated in e, were subjected to 3D culture with or without a combination of NAC and dNTPs (N + N) for 14 days. n = 3 independent experiments. Scale bar, 50 μm. For a,c, n = 3 or 4 mice per group. Data are mean ± s.e.m. P-values were calculated using multiple unpaired two-tailed Student’s t-test (a), one-way ANOVA with Dunnett’s test (c,g,h) and two-tailed unpaired t-test (d).

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Extended Data Fig. 6 Bmal1 depletion has a less pronounced effect on the expression of other PPP-related genes.

a, Diagram outlining the oxidative and non-oxidative branches of the PPP in glucose metabolism. b-f, qRT-PCR showing oscillations of the PPP-related genes in AL, DRF, and LBKO mouse livers at various ZTs, including Pgd (b), Tkt (c), Rpe (d), Taldo1 (e), and Rpia (f). The arrows indicate the ZT point at which the expression of the indicated gene peaked in the livers of AL and DRF groups (b,c). g, qRT-PCR showing the mRNA levels of the PPP-related genes in Hepa1-6 cells expressing shNC or shBmal1. h, Immunoblotting showing the protein levels of Bmal1 and G6pdx in the livers from Bmal1f/f mice with AAV-NC or AAV-TbgCre injection. i, qRT-PCR showing the mRNA levels of G6pdx in the indicated tissues from WT and Bmal1-null mice. j, Representative immunofluorescence images of WT and Bmal1-null mouse intestines stained with the indicated antibodies. Nuclei were stained with DAPI. Scale bar, 15 μm. k, Quantification of the intensity of Ki67-positive staining shown in j. Four fluorescent fields for each of three independent samples were counted. For b-f,g-i, n = 3 mice per group. Data are mean ± s.e.m. P-values were calculated using multiple unpaired two-tailed Student’s t-test (g,i) and unpaired two-tailed Student’s t-test (k). For b-f, JTK_CYCLE was applied to determine rhythmicity as shown in Supplementary Table 4. P < 0.05 was considered to be rhythmic.

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Extended Data Fig. 7 G6pdx expression is regulated by Bmal1 and Hif-1α, which is required for facilitating liver regeneration.

a, Schematic diagram depicting the promoter region of murine G6pdx gene. Expanded fragments show the DNA sequence of the E-box in the P1 region and the E’-box in the P2 region. b, Measurement of the relative luciferase activity in Hepa1-6 cells transfected with the indicated expression plasmids. c,d, qRT-PCR showing G6pdx expression in Hepa1-6 cells expressing shNC, shBmal1, or shClock (c) and immortalized WT and Clock mutant MEFs (d). Delta 19 (Δ19), deletion of exon 19 in the Clock gene. e, Measurement of the activity of P2-driven luciferase reporter in Hepa1-6 cells expressing shNC, shBmal1, or shHif-1α. f, Representative images of IF staining showing the G6pdx expression in the livers. WT and LBKO mice were infected with AAV8-GFP or AAV8-G6pdx virus for three weeks. Scale bar, 15 μm. g, Representative IHC images and quantification of Ki67-positive cells in the livers of postoperative mice treated as in f. The red line indicates the mean value of the AL group. Scale bar, 100 μm. h, Graphs showing the liver-body weight ratio and the contents of ALT and AST in DRF mice that were injected with AAV8-GFP or AAV8-Flag-G6pdx. The samples were collected on day 4 post-PH. i,j, Graphs showing Ki-67 staining (i) and the indicated protein levels (j) in mice treated as in Fig. 6b. Scale bar, 100 μm. k-m, Identification of G6pdx overexpression in WT mice after AAV8 injection, as measured by immunoblotting (k), qRT-PCR (l) and enzymatic activity assay (m). n, Measurement of the nucleoside/nucleotide contents in WT mice with or without G6pdx overexpression by LC-MS/MS. For g,i, Five fields for each of three samples were counted. For b-e, n = 3 independent biological samples. For h, n = 6 mice per group. For l-n, n = 3 mice per group. Data are mean ± s.e.m. P-values were calculated using two-way ANOVA with Tukey’s test (b) or Sidak’s test (c), unpaired two-tailed Student’s t-test (d,g,h,l,m), one-way ANOVA with Dunnett’s test (e,i) and multiple unpaired two-tailed Student’s t-test (n).

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Extended Data Fig. 8 Changes to the feeding schedule alter G6pdx expression and affect hepatocyte proliferation.

a-c, Immunoblotting showing the indicated protein levels in Hepa1-6 cells cultured in glucose-free medium (a), and in the livers from mice fasted for 2 days (b) or AAV-infected (c). d, Representative IHC images and quantification of Ki67-positive cells from mice with the indicated treatment. More than three fields for each of three independent samples were counted. Scale bar, 100 μm. e,f, The mRNA levels (e) and enzymatic activity (f) of G6pdx in the livers of AL- or 10-day IF- mice. g, qRT-PCR showing the expression of other PPP related genes in the livers of mice fed AL or IF. h, Quantification of the ratio of phosphorylated (p)-Bmal1 to total Bmal1 from the blots as shown in Fig. 6h. i, Immunoblotting showing the levels of the indicated proteins in chromatin fraction. j,k, Representative IHC images (j) and quantification (k) of Ki67-positive cells from mice with the indicated treatment. Five fields for each of three independent samples were counted. For e-h, n = 3 or 4 mice per group. Data are mean ± s.e.m. P-values were calculated using two-way ANOVA with Tukey’s test (d), unpaired two-tailed Student’s t-test (e,f,h) and multiple unpaired two-tailed Student’s t-test (g,k).

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Extended Data Fig. 9 Accumulated DNA damage by Bmal1 depletion triggers senescence and inflammation.

a, Clonogenic survival experiments showing the colony formation capacity of NIH3T3 cells expressing shNC or shBmal1 after treatment with the indicated doses of CPT with or without dNTPs. b, Representative images and quantification of IHC staining for the indicated markers of livers from 6-month-old WT and whole-body Bmal1 knockout (BKO) mice. More than three fields for each of three samples were counted. c, Representative IF images of primary MEFs at passage 5 stained with the anti-γH2ax antibody and quantification of the number of γH2ax foci in individual cells. n = 53 cells counted for each condition taken from three independent experiments. Scale bar, 15 μm. d, qRT-PCR showing the expression of Bmal1, G6pdx, and the senescent markers P21 and P16, in shNC or shBmal1 MEFs at passage 5. e, Representative IHC images in mouse liver sections stained with anti-F4/80 antibody and optical density-based quantification. WT and LBKO mice with AAV8-GFP or AAV8-G6pdx were subjected to paraquat (20 mg kg−1) treatment and euthanized after 5 days. More than three fields for each of three independent samples were counted. Scale bar, 100 μm. f, qRT-PCR showing the expression of the indicated SASP genes in shNC- or shBmal1-expressing primary MEFs at passage 5. g, Immunoblotting showing the indicated protein levels in the liver lysates of WT or LBKO mice treated with 20 mg kg−1 paraquat and then with 0 or 5 mg kg−1 SN-011 treatment for 2 days before sacrifice. h-j, qRT-PCR showing the expression of the indicated genes (h,j) and representative images of IHC staining with the anti-F4/80 antibody (i) of livers from mice as treated in g. Scale bar, 100 μm. For a,d,f, n = 3 independent biological samples. For h,j, n = 3 mice per group. Data are mean ± s.e.m. P-values were calculated using RM two-way ANOVA (a), unpaired two-tailed Student’s t-test (b), two-way ANOVA with Sidak’s test (d,f,h,j) and one-way ANOVA with Dunnett’s test (e).

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Extended Data Fig. 10 A working model.

The proposal model depicting the hepatic clock controls the oscillation of pentose phosphate pathway through cooperating with HIF-1α, that is crucial for liver recovery from damage. Glu, Glucose; senescence-associated secretory phenotype, SASP. Created by figdraw.com.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and unprocessed blots for Supplementary Fig. 6.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–5. Rhythmicity analysis, primers and metabolome dataset.

Supplementary Data

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Peng, L., Xiang, S., Wang, T. et al. The hepatic clock synergizes with HIF-1α to regulate nucleotide availability during liver damage repair. Nat Metab 7, 148–165 (2025). https://doi.org/10.1038/s42255-024-01184-8

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