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. 2019 Dec 17;116(51):25974-25981.
doi: 10.1073/pnas.1908137116. Epub 2019 Dec 2.

Targeting liver aldehyde dehydrogenase-2 prevents heavy but not moderate alcohol drinking

Adrien Guillot  1 , Tianyi Ren  1 , Tony Jourdan  2 , Robert J Pawlosky  3 , Elaine Han  1 , Seung-Jin Kim  1 , Li Zhang  4 , George F Koob  5 , Bin Gao  6
Affiliations

Targeting liver aldehyde dehydrogenase-2 prevents heavy but not moderate alcohol drinking

Adrien Guillot et al. Proc Natl Acad Sci U S A. .

Abstract

Aldehyde dehydrogenase 2 (ALDH2), a key enzyme for detoxification the ethanol metabolite acetaldehyde, is recognized as a promising therapeutic target to treat alcohol use disorders (AUDs). Disulfiram, a potent ALDH2 inhibitor, is an approved drug for the treatment of AUD but has clinical limitations due to its side effects. This study aims to elucidate the relative contribution of different organs in acetaldehyde clearance through ALDH2 by using global- (Aldh2-/-) and tissue-specific Aldh2-deficient mice, and to examine whether liver-specific ALDH2 inhibition can prevent alcohol-seeking behavior. Aldh2-/- mice showed markedly higher acetaldehyde concentrations than wild-type (WT) mice after acute ethanol gavage. Acetaldehyde levels in hepatocyte-specific Aldh2 knockout (Aldh2Hep-/-) mice were significantly higher than those in WT mice post gavage, but did not reach the levels observed in Aldh2-/- mice. Energy expenditure and motility were dramatically dampened in Aldh2-/- mice, but moderately decreased in Aldh2Hep-/- mice compared to controls. In the 2-bottle paradigm and the drinking-in-the-dark model, Aldh2-/- mice drank negligible volumes from ethanol-containing bottles, whereas Aldh2Hep-/- mice showed reduced alcohol preference at high but not low alcohol concentrations. Glial cell- or neuron-specific Aldh2 deficiency did not affect voluntary alcohol consumption. Finally, specific liver Aldh2 knockdown via injection of shAldh2 markedly decreased alcohol preference. In conclusion, although the liver is the major organ responsible for acetaldehyde metabolism, a cumulative effect of ALDH2 from other organs likely also contributes to systemic acetaldehyde clearance. Liver-targeted ALDH2 inhibition can decrease heavy drinking without affecting moderate drinking, providing molecular basis for hepatic ALDH2 targeting/editing for the treatment of AUD.

Keywords: acetaldehyde; alcohol metabolism; alcohol use disorder; neuron; shAldh2.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Mouse ALDH2 protein expression and enzyme activity are widespread in the organism. To evaluate ALDH2 expression distribution in the organism, major organs from naïve C57BL/6N mice were collected. (A) ALDH2 protein expression was evaluated by Western blot, and beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as loading controls. (B) ALDH2 enzymatic activity was measured on fresh tissue homogenates and is represented as the relative mean ± SEM. Relative ALDH2 activity was compared to liver (n = 3 to 6 per organ). Liver samples from Aldh2−/− mice were used as blank negative controls. BAT, brown adipose tissue; WAT, white adipose tissue; ND, not detectable.
Fig. 2.
Fig. 2.
Organ-specific ALDH2 deficiency reveals a potent but not exclusive role of the liver in acetaldehyde clearance. (A) Tissue-specific ALDH2 protein deletion was confirmed at the protein level by Western blot analysis. Beta-actin and GAPDH were used as loading controls. (B) Relative liver ALDH2 enzymatic activity was measured in Aldh2−/−, Aldh2Hep−/−, and their WT control mice. Liver samples from Aldh2−/− mice were used as blank negative controls. Mice were given a single oral ethanol gavage (5 g/kg), then (C) ethanol and (D) acetaldehyde serum concentrations were measured by GC-MS at the indicated time points. ND, not detectable. Data are represented as mean ± SEM (n = 3 to 12). *P < 0.05, **P < 0.01, ***P < 0.005, unpaired Student’s t test (in B) and 1-way ANOVA (in C and D).
Fig. 3.
Fig. 3.
Global and hepatocyte-specific Aldh2 deficiency leads to severe and moderate inhibition, respectively, of metabolic rates. WT control or liver or global Aldh2-deficient mice received a single dose of 5 g/kg ethanol gavage and were placed in metabolic cages for indirect and noninvasive measurement of metabolic rates. The following parameters were evaluated: (A) ambulatory movements, (B) total energy expenditure, (C) carbohydrate oxidation, (D) fat oxidation, (E) respiratory quotient, and (F) heat production. In each panel, the red asterisk and line indicate the time at which oral gavage was performed. Yellow and blue bars represent the 12-h periods when the lights were on or off, respectively, in the animal holding room. Data are represented as mean ± SEM (n = 3 to 4 mice per group).
Fig. 4.
Fig. 4.
Hepatocyte-specific Aldh2 deficiency decreases excessive, but not light-to-moderate, alcohol drinking preference and binge-like drinking behavior in mice. (A–C) (Left) Mice were subjected to a 2-bottle choice paradigm, and consumption of regular drinking water and ethanol-containing water were measured daily. (A) Alcohol preference is represented as the percentage drank from the alcohol-containing bottle relative to cumulated water and alcohol drinking volumes. (B) Volumes drank from the alcohol-containing bottle per day per gram body weight. (C) Absolute alcohol intake per day per gram body weight. (Right) For all measurements in A−C, the relative area under the curve. (D) A DID was used to study binge-like drinking behavior of Aldh2f/f, Aldh2Hep−/−, B6N, and Aldh2−/− mice. Data are represented as mean ± SEM (n = 5 to 7 mice per group). *P < 0.05, ***P < 0.005, 1-way ANOVA.
Fig. 5.
Fig. 5.
Forebrain neuron or glial cell targeted Aldh2 deficiency does not affect alcohol drinking preference. Forebrain neuron- (Aldh2Camk2a−/−) and glial cell-specific (Aldh2Gfap−/−) Aldh2-deficient mice were subjected to the 2-bottle paradigm. (A and D) Alcohol preference and (B, C, E, and F) alcohol intake were evaluated in Aldh2Camk2a−/− and Aldh2Gfap−/− mice, respectively. Data are represented as mean ± SEM (n = 5 mice per group), unpaired Student’s t test (in C and F).
Fig. 6.
Fig. 6.
Hepatocyte-specific knockdown of Aldh2 decreases excessive alcohol preference in mice. (A) Naïve mice were injected with scrambled shRNA or shAldh2 adenovirus prior to being subjected to the 2-bottle paradigm. Briefly, mice were injected with adenovirus for 7 d and subsequently presented with 2 bottles, one containing regular drinking water, and the other one containing 3% or 6% ethanol in drinking water. Ethanol preference as well as alcohol intake were calculated. (B and C) (Lower) Mice were subjected to the 2-bottle paradigm with increasing ethanol concentration up to (B) 9% or (C) 18%, before being injected with scramble shRNA or shAldh2 adenovirus. Briefly, mice were given free choice between water or ethanol-containing water at the indicated concentrations (for 4 d each). Adenoviruses were then injected when ethanol concentration reached (B) 9% or (C) 18% in their drinking water. Mice were continuously kept on the 2-bottle choice test. (Upper) For each experiment, the experimental outline indicating the percentage of alcohol (vol/vol) in the drinking water; 1 w, 1 wk. Data are represented as mean ± SEM (n = 5 to 8 mice per group). *P < 0.05, ***P < 0.005, unpaired Student’s t test.
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