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Coordinated action of a gut–liver pathway drives alcohol detoxification and consumption

Nature Metabolism volume 6, pages 1380–1396 (2024)Cite this article

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Abstract

Alcohol use disorder (AUD) affects millions of people worldwide, causing extensive morbidity and mortality with limited pharmacological treatments. The liver is considered as the principal site for the detoxification of ethanol metabolite, acetaldehyde (AcH), by aldehyde dehydrogenase 2 (ALDH2) and as a target for AUD treatment, however, our recent data indicate that the liver only plays a partial role in clearing systemic AcH. Here we show that a liver–gut axis, rather than liver alone, synergistically drives systemic AcH clearance and voluntary alcohol drinking. Mechanistically, we find that after ethanol intake, a substantial proportion of AcH generated in the liver is excreted via the bile into the gastrointestinal tract where AcH is further metabolized by gut ALDH2. Modulating bile flow significantly affects serum AcH level and drinking behaviour. Thus, combined targeting of liver and gut ALDH2, and manipulation of bile flow and secretion are potential therapeutic strategies to treat AUD.

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Fig. 1: Liver and gut synergistically promote circulating AcH clearance via ALDH2.
Fig. 2: Bile flow is an important pathway for AcH clearance from liver into intestinal lumen.
Fig. 3: Gut microbiota play a minor role in gut luminal AcH clearance.
Fig. 4: Gut AcH is metabolized by gut ALDH2 with a small portion absorbed back and metabolized by the liver ALDH2.
Fig. 5: Bile flow controls systemic and liver AcH detoxification.
Fig. 6: Manipulating intrahepatic bile flow affects drinking behaviour.
Fig. 7: Gut and liver ALDH2 synergistically control blood AcH clearance and drinking behaviour.
Fig. 8: The schematic for a liver–gut loop controlling alcohol detoxification and drinking behaviour.

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No custom code was used in this paper.

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Acknowledgements

We thank D. Lovinger (NIAAA, National Institutes of Health) for critical comments and suggestions during the study. We also thank the National Institute of Allergy and Infectious Diseases Gnotobiotic Animal Facility for providing GF mice and V. Ling (British Columbia Cancer) for providing Bsep−/− mice. This work was supported by the intramural programme of NIAAA, National Institutes of Health (B.G., G.K., P.P.). H.Z. is the recipient of a Research Career Scientist Award from the Department of Veterans Affairs (grant no. IK6BX004477). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Yaojie Fu, Bryan Mackowiak.

Authors and Affiliations

  1. Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Yaojie Fu, Bryan Mackowiak, Yu-Hong Lin, Luca Maccioni, Taylor Lehner, Hongna Pan, Yukun Guan, Hongkun Lu, Cheng Chen, Dechun Feng & Bin Gao

  2. Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Luca Maccioni, Grzegorz Godlewski, Cheng Chen & George Kunos

  3. Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Shoupeng Wei & Li Zhang

  4. Laboratory of Cardiovascular Physiology and Tissue Injury, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Janos Paloczi & Pal Pacher

  5. Department of Microbiology and Immunology, Virginia Commonwealth University and Richmond Veterans Affairs Medical Center, Richmond, VA, USA

    Huiping Zhou

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Contributions

Y.F. and B.M. were responsible for the conception, experimental design, performing experiments and writing the paper. Y.L. conducted the GC–MS experiments. L.M., T.L., H.P., Y.G., G.G., H.L., C.C., S.W., D.F. and J.P. performed the animal experiments and staining. H.Z., P.P., L.Z. and G.K. carried out the conception, data analysis, and reviewing and editing of the paper. B.G. was responsible for conceptualization, resources and supervision, and writing, reviewing and editing of the paper.

Corresponding author

Correspondence to Bin Gao.

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Nature Metabolism thanks Daria Mochly-Rosen, Claudia Fuchs-Steiner and the other, anonymous, reviewer(s) 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 Confirmation of ALDH2 deletion in various strains of single organ Aldh2 KO mice.

(a) To identify which organ(s) in addition to the liver controls blood AcH clearance, several lines of tissue-specific KO mice were generated and listed in the table. (b) Measurement of AcH levels in serum from Aldh2f/f mice (n = 6), Aldh2E2a−/− mice (n = 5), and global Aldh2 KO mice (n = 4) 3 h post ethanol oral gavage (5 g/Kg). (c) Western blot analysis or immunofluorescent staining was performed to confirm the deletion of ALDH2 in tissues (IF staining: upper left panel: smooth muscle tissue; upper right panel: liver tissue; lower left panel: blood vessels of heart tissue were shown, scale bar: 50 μm) from various organ-specific Aldh2 KO mice listed in Table (a), representative of two independent experiments. Values represent means ± SEM. Two-way ANOVA and two-sided Student’s t-test was performed for the comparison between indicated two groups. ns: No significance.

Source data

Extended Data Fig. 2 Drinking behavior in various strains of single organ Aldh2 KO mice.

(a–d). 2-bottle choice (2-BC) experiments were performed in endothelial cell (Male: n = 8, n = 10; Female: n = 8, n = 10) in Aldh2Tie2−/−, (Male: n = 9, n = 7; Female: n = 6, n = 5) in Aldh2Tek2−/−, *P = 0.0377, *P = 0.0357), smooth muscle (n = 8, n = 9), and skeletal muscle (n = 8, n = 10) (*P = 0.0321, *P = 0.0345) specific Aldh2 KO mice. Sex of mice were indicated, ‘M’ means ‘male’ and ‘F’ means ‘female’. Values represent means ± SEM. *p < 0.05. Two-way ANOVA and two-sided Student’s t-test was used for the comparison between indicated two groups.

Source data

Extended Data Fig. 3 Expression of ALDH2 protein in the gut, and liver injury in gut-specific Aldh2 KO mice post ethanol feeding.

(a) Western blot analysis was performed to determine the ALDH2 protein expression in liver and different segments of intestine tissues, including duodenum, mid small intestine, and terminal ileum (n = 4 in each group) (**p = 0.0021, **p = 0.0078). (b) Representative immunofluorescence images showing expression of ALDH2 in liver and duodenum tissues from WT, Aldh2Hep−/−, Aldh2villin−/−, and Aldh2Hep−/−Villin−/− mice. Scale bar: 100 μm. (c) Schematic of mouse model of chronic ethanol feeding plus acute binge (the NIAAA model) and its pair-fed control model (Created with Biorender.com). (d) Serum ALT (IU/L) (liver injury marker) measurements of chronic-plus-binge ethanol feeding mice (shown as ‘EtOH diet’) and pair-fed control mice (shown as ‘Paired diet’) of WT, Aldh2Hep−/−, Aldh2villin−/−, Aldh2Hep−/−Villin−/− mice (n = 4 in each paired fed group; n = 13, n = 12, n = 8, n = 7 in each EtOH-fed group), the results were from one experiment. Values represent means ± SEM. **p < 0.01. Two-sided Student’s t-test and one-way ANOVA were used for the comparison between two groups. ns: No significance.

Source data

Extended Data Fig. 4 Measurement of liver injury in liver and/or gut Aldh2 KO post chronic-plus-binge ethanol feeding.

(a) Representative images of hematoxylin-eosin (H&E) staining and IHC staining with anti-IBA1 (macrophage marker), anti-myeloperoxidase (MPO; neutrophil marker; positive cells were indicated with black arrows), Sirius red (fibrosis), and anti-OPN (bile duct marker) of liver sections from WT, Aldh2Hep−/−, Aldh2villin−/−, and Aldh2Hep−/−Villin−/− mice of chronic-plus-binge ethanol feeding model (n = 5 in each group). Scale bar:200μm. (b) Quantification of IHC staining shown in panel (a) (count of positive cell or fold change of positive area/200×field) (n = 5 in each group) (**p = 0.0082, **p = 0.0059, **p = 0.0022, **p = 0.0016). Values represent means ± SEM. *p < 0.05; **p < 0.01. Two-sided Student’s t-test and one-way ANOVA were used for the comparison between indicated two groups.

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Extended Data Fig. 5 Bile AcH levels are much higher than serum AcH after ethanol administration and effects of glutathione depletion on acetaldehyde disposition.

(a) Measurement of EtOH and AcH in serum and bile samples from C57BL/6N mice (n = 5) by GC-MS 1 h and 3 h post i.p injection of ethanol (4 g/Kg). Box plot with whiskers (min to max), line at median were shown in (a). Two-sided paired Student’s t-test was performed, **p = 0.0028, **p = 0.0015. (b) Bile volume/body weight ratios (shown as Bile/BW ratio) of C57BL/6N mice received PBS gavage or ethanol (shown as EtOH gavage; 5 g/Kg) were determined 3 h, 6 h, and 9 h post oral gavage (n = 4 per group at each timepoint). (c) Schematic of potential AcH metabolite downstream of glutathione (GSH) and cysteinylglycine (CysGly). (d) Study timeline–male C57BL/6N mice (n = 7/group) were administered buthionine sulfoximine (BSO, 4 mmol/kg) or vehicle (control) 2.5 h before 5 g/kg EtOH gavage and mice were sacrificed 1 h later. (e, f) Measurement of EtOH and AcH in serum, liver (n = 7, n = 7), and bile samples from C57BL/6N mice (n = 4) by GC-MS (**p = 0.0098). (g–i). Study timeline of male C57BL/6N mice (n = 7/group) were administered BSO was shown in (g), and measurement of EtOH and AcH in serum, liver, and bile samples (n = 7/group) were shown in (h) and (i). (d) and (g) were created with Biorender.com. Values represent means ± SEM. Significance was evaluated via two-sided unpaired Student’s t-test (**p < 0.01). ns: No significance.

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Extended Data Fig. 6 No differences on ALDH2 protein levels between WT and GF mice.

(a) Western blot analysis of ALDH2 protein in liver tissues from WT and GF mice (n = 6). (b) Bile volumes (Left panel) and the fold change of bile volume/body weight ratio (shown as Bile/BW ratio) from WT and GF mice post ethanol gavage (5 g/Kg) were determined (n = 7) (***p < 0.0001). Values represent means ± SEM. ***p < 0.001. Two-sided Student’s t-test was used for the comparison between indicated two groups.

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Extended Data Fig. 7 Manipulation of intrahepatic bile flow does not affect EtOH transportation.

(a) EtOH levels in liver tissue, serum, bile samples from BDL mice (n = 5) and sham mice (n = 5) 3 h post ethanol (5 g/kg) gavage. (b) EtOH levels in liver, serum, bile samples from Mdr2 KO mice (Mdr2−/−) (n = 5) and control WT mice (n = 5) 3 h post ethanol (5 g/kg) gavage. (c) EtOH levels in liver, serum, bile samples from Bsep KO mice (Bsep−/−) (n = 6) and control WT mice (n = 7) 3 h post ethanol (5 g/kg) gavage. (d) EtOH levels in liver tissue, serum, and bile samples from C57BL/6N mice fed with control diet (n = 5) and Ursodeoxycholic acid (UDCA) diet (n = 5) 3 h post ethanol gavage (5 g/Kg). (e–h) EtOH levels in liver, serum, and bile samples from C57BL/6N mice pre-treated with vehicle, or Cyclo-1 (n = 3, n = 4), Novobiocin (n = 7, n = 4), Quinidine (n = 5, n = 5) and Rifampicin (n = 5, n = 5), respectively, were measured 3 h post ethanol gavage (5 g/Kg) (n = 3-6 each group) (*p = 0.0159). Values represent means ± SEM. Two-sided Student’s t-test was used for the comparison between indicated two groups. *p < 0.05.

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Extended Data Fig. 8 Expression and enzymatic activity of liver ALDH2 and DID in mice with various treatment or gene deletion.

(a, b) Liver tissues were obtained from bile duct ligated (BDL) (n = 6), Mdr2−/− (n = 6, **p = 0.0082), Bsep−/− (n = 5) or UDCA treated mice (n = 6), and their corresponding control mice. Western blot analyses were performed to determine ALDH2 expression. (c) Comparison of ALDH2 enzymatic activity in fresh liver homogenates was performed in the mice mentioned above (n = 6, n = 6, n = 5, n = 6). (d, e) DID assay in female C57BL/6N mice fed with chow diet (n = 10) or UDCA diet (n = 9, *p = 0.0464, *p = 0.0301), in female Mdr2 KO mice (Mdr2−/−) (n = 7, *p = 0.0256, **p = 0.0015) and their littermate control mice (WT) (n = 8), and in male Bsep KO mice (Bsep−/−) (n = 7, *p = 0.0117, *p = 0.0111) and their littermate control mice (WT) (n = 7). Values represent means ± SEM. Two-sided Student’s t-test was used for the comparison between indicated two groups. *p < 0.05, **p < 0.01.

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Extended Data Fig. 9 ALDH2 of liver-gut loop controls AcH clearance but does not affect EtOH concentration.

(a) AcH levels of cerebellar cortex, portal blood, bile and duodenal luminal content from four groups of mice were determined (female groups: n = 8, n = 8, n = 8, n = 7) (*p = 0.0228,***p < 0.0001,***p < 0.0001,**p = 0.0023,*p = 0.0194,***p = 0.0004,*p = 0.0207, ***p < 0.0001,***p < 0.0001,**p = 0.0043,***p = 0.0009,***p < 0.0001). (b) EtOH in cerebellar cortex, portal blood, bile and duodenal luminal content from four groups of mice were determined (male groups: n = 9, n = 8, n = 9, n = 11). (c) AcH levels from male WT and double KO mice treated with 2 g/kg EtOH gavage (*p = 0.0260, **p = 0.0087, *p = 0.0189, *p = 0.0260) (The scheme was created with Biorender.com). (d) The correlation of serum and cerebellar AcH levels 3 h post ethanol gavage (5 g/Kg) (n = 9, n = 9, n = 7, n = 11). (e) Schematic of different brain regions collected for AcH measurement post EtOH gavage (Created with Biorender.com). (f) AcH levels of prefrontal cortex (PFC) (n = 7, n = 7), hippocampus (n = 6, n = 7), thalamus (TH) (n = 6, n = 6), and hypothalamus (HTH) (n = 7, n = 7) from WT (Aldh2f/f) and Aldh2Hep−/−Villin−/− mice in (e) were determined 3 h post EtOH gavage (5 g/Kg) (**p = 0.0053, ***p = 0.0004). *p < 0.05, **p < 0.01, ***p < 0.001. Values represent means ± SEM. Two-sided student’s t-test and one-way ANOVA were used for the comparison between two groups in panels a and b; Two-sided student’s t-test was used for panels c and f. Two-tailed simple linear regression was used to determine the correlation.

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Extended Data Fig. 10 Liver and gut epithelium Aldh2 double knockout leads to significant inhibition of metabolic phenotypes after alcohol intake.

The following parameters of WT mice (Aldh2f/f), Aldh2Hep−/−, Aldh2Villin−/−, and Aldh2Hep−/−Villin−/− mice (n = 4/each group) were evaluated by using metabolic chambers after ethanol gavage (5 g/Kg): (a) Respiratory quotient (**p = 0.0077). (b) Carbohydrate oxidation (*p = 0.0133). (c) Cumulative food intake. (d) Cumulative water intake. (e) Total energy expenditure. (f) Fat oxidation. (g) Oxygen consumption. (h) Ambulatory movements. Values represent means ± SEM. A two-way ANOVA was performed for the comparisons among multiple groups, followed by two-sided Student’s t-test between WT mice and Aldh2Hep−/−Villin−/− mice in (a) and (b) at the timepoint of 48 h. No adjustments were made for multiple comparisons. *p < 0.05, **p < 0.01.

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Supplementary information

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Fu, Y., Mackowiak, B., Lin, YH. et al. Coordinated action of a gut–liver pathway drives alcohol detoxification and consumption. Nat Metab 6, 1380–1396 (2024). https://doi.org/10.1038/s42255-024-01063-2

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