The Journal of Nutrition Nutrition and Disease Hepatoprotective Effects of Indole, a Gut Microbial Metabolite, in Leptin-Deficient Obese Mice Christelle Knudsen,1,2 Audrey M Neyrinck,1 Quentin Leyrolle,1 Pamela Baldin,3 Sophie Leclercq,1,4 Julie Rodriguez,1 Martin Beaumont,2 Patrice D Cani,1,5 Laure B Bindels,1 Nicolas Lanthier,6,7 and Nathalie M Delzenne1 1Metabolism and Nutrition Research Group, Louvain Drug Research Institute, UCLouvain, Université catholique de Louvain, Brussels, Downloaded from https://academic.oup.com/jn/article/151/6/1507/6166153 by guest on 27 September 2021 Belgium; 2GenPhySE, Université de Toulouse, INRAE, ENVT, 31320, Castanet Tolosan, France; 3Service d’Anatomie Pathologique Cliniques Universitaires Saint-Luc, Brussels, Belgium; 4Institute of Neuroscience, UCLouvain, Université catholique de Louvain, Brussels, Belgium; 5WELBIO–Walloon Excellence in Life Sciences and BIOtechnology, UCLouvain, Université catholique de Louvain, Brussels, Belgium; 6Service d’Hépato-gastroentérologie, Cliniques universitaires Saint-Luc, Brussels, Belgium; and 7Laboratory of Gastroenterology and Hepatology, Institut de Recherche Expérimentale et Clinique, UCLouvain, Université catholique de Louvain, Brussels, Belgium ABSTRACT Background: The gut microbiota plays a role in the occurrence of nonalcoholic fatty liver disease (NAFLD), notably through the production of bioactive metabolites. Indole, a bacterial metabolite of tryptophan, has been proposed as a pivotal metabolite modulating inflammation, metabolism, and behavior. Objectives: The aim of our study was to mimic an upregulation of intestinal bacterial indole production and to evaluate its potential effect in vivo in 2 models of NAFLD. Methods: Eight-week-old leptin-deficient male ob/ob compared with control ob/+ mice (experiment 1), and 4–5-wk- old C57BL/6JRj male mice fed a low-fat (LF, 10 kJ%) compared with a high-fat (HF, 60 kJ%) diet (experiment 2), were given plain water or water supplemented with a physiological dose of indole (0.5 mM, n ≥6/group) for 3 wk and 3 d, respectively. The effect of the treatments on the liver, intestine, adipose tissue, brain, and behavior was assessed. Results: Indole reduced hepatic expression of genes involved in inflammation [C-C motif chemokine ligand 2(Ccl2), C-X-C motif chemokine ligand 2 (Cxcl2); 3.3- compared with 5.0-fold, and 2.4- compared with 3.3-fold of control ob/+ mice, respectively, P < 0.05], and in macrophage activation [Cd68, integrin subunit α X(Itgax); 2.1- compared with 2.5-fold, and 5.0- compared with 6.4-fold of control ob/+ mice, respectively, P < 0.01] as well as markers of hepatic damage (alaninine aminotransferase; −32%, P < 0.001) regardless of genotype in experiment 1. Indole had no effect on hepatic inflammation in mice fed the LF or HF diet in experiment 2. Indole did not change hepatic lipid content, anxiety- like behavior, or inflammation in the ileum, adipose tissue, and brain in experiment 1. Conclusions: Our results support the efficacy of indole to reduce hepatic damage and associated inflammatory response and macrophage activation in ob/ob mice. These modifications appear to be attributable to direct effects of indole on the liver, rather than through effects on the adipose tissue or intestinal barrier. J Nutr 2021;151:1507–1516. Keywords: gut-liver axis, microbiota, ob/ob mice, steatosis, tryptophan Introduction metabolites are derived from dietary carbohydrates such as SCFAs, from dietary lipids such as PUFA-derived metabolites Concurrently with the increase in obesity and metabolic syn- or bile acids, or from dietary proteins and amino acids such as dromes, nonalcoholic fatty liver disease (NAFLD) is becoming indoles or phenols (3, 4). Due to its close anatomical proximity the most important cause of chronic liver disease, with a global with the digestive tract, the liver can easily be reached by prevalence of 24%. However, no pharmacological therapy is metabolites transiting from the gut lumen through the portal currently available (1). Diet plays an important role in the vein. Amino acid–derived metabolites such as phenylacetic acid development of NAFLD, but growing evidence suggests that (5), imidazole propionate (6), and 3-(4-hydroxyphenyl)lactate the gut microbiota also has its part to play in the occurrence (7) are in the spotlight and have been identified as potential and evolution of this disease, notably through the production inducers of steatosis and hepatic inflammation whereas others, of specific bioactive metabolites (2, 3). Most known bacterial C The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Manuscript received August 28, 2020. Initial review completed October 30, 2020. Revision accepted January 28, 2021. First published online March 10, 2021; doi: https://doi.org/10.1093/jn/nxab032. 1507 such as indolic compounds, seem to preserve liver integrity inflammation and steatosis in diet-induced obese mice(22). A (8–11). large spectrum of Gram-negative and Gram-positive bacteria Indole is a product of l-tryptophan deamination catalyzed produce indole at variable levels (23). However, no data are by the bacterial enzyme tryptophanase. It is the main bacterial currently available on the direct and indirect (mediated by the metabolite derived from tryptophan in the gut (12, 13), and intestine and adipose tissue) impact of an upregulation of the it can be detected in healthy human and mouse feces at bacterial indole production on hepatic health and metabolism. concentrations close to millimolar (12–15). In gnotobiotic The aim of our study was thus to mimic an upregulation of rats, and at high doses [500 mg/kg body weight (BW)] intestinal bacterial indole production (chronic treatment with in conventional rats, indole has been shown to modulate a low dose) and evaluate its impact on the liver, intestine, behavior, including anxiety-like and depression-like behaviors adipose tissue, and brain and on behavior. The model of genetic (16), and response to chronic mild stress (17). Conversely, leptin deficiency was chosen because previous data support the at the intestinal level, indole decreases mucosal inflammation decrease in proinflammatory cytokine expression in ob/ob liver in vivo (at 20 mg/kg BW) and in vitro (at 1 mM) (18, slices upon incubation with indole (8). The impact of indole 19) and enhances epithelial barrier function (at 20 mg/kg was also evaluated in a short-term dietary challenge model BW) (20). Indole (1 mM) can also modulate intestinal cell with a high-fat diet, that we previously established (24), to metabolism in vitro resulting in a change in glucagon-like determine the potential preventive properties of an upregulation Downloaded from https://academic.oup.com/jn/article/151/6/1507/6166153 by guest on 27 September 2021 peptide-1 secretion (21). Due to the close anatomical proximity of bacterial indole production on the onset of hepatic between the intestine and the liver, the immunomodulatory disorders. effects of indole on gut barrier and intestinal immune response and inflammation18 ( , 20) could potentially impact the liver. Recent studies conducted by our laboratory have shown Methods direct effects of indole on the liver with a reduction in inflammatory markers ex vivo and in vivo after an acute LPS In vivo experiments challenge (8). This mechanistic study demonstrated that the All mice were purchased from Janvier Labs and housed in a specific anti-inflammatory effects of indole were partly dependent on opportunistic pathogen–free environment. Housing conditions were as specified by the Belgian law of May 29, 2013 on the protection of Kupffer cells (KCs) and associated with a downregulation of κ laboratory animals (Agreement LA 1,230,314). Animals were placed in key proteins of the NF- B pathway. In this model, microarray individually ventilated cages with bedding (Rehofix Corncob bedding analyses highlighted modulations of the expression of genes MK1500; Technilab-BMI) in groups of 3. Hygrometry and temperature regulating specific metabolic pathways, including cholesterol were monitored, and a 12- h daylight cycle was applied. Housing metabolism. Thus, indole appears as a potential therapeutic was enriched with a plastic tunnel for the mice to hide and a soft tool for NAFLD treatment. In accordance with this hypothesis, paper tissue. Bedding and tissues were changed weekly. Mice had free a recent article reported that the concentration of circulating access to water and food [AIN-93M (25), D10012Mi; Research Diets] indole was inversely associated with the BMI and with the for 10–15 d before experiment. The experiments performed in this degree of liver lipid accumulation in humans, and that a high study were approved by the local Ethics Committee under the number dose of indole (50 mg/kg) given over 3 wk decreased hepatic 2017/UCL/MD/005. Experiment 1. CK is a beneficiary of the AgreenSkills+ fellowship program, which has received Eighteen B6.V-Lep ob/+ JRj and 18 B6.V-Lep ob/ob JRj 8-wk-old funding from the EU’s Seventh Framework Programme under grant agreement male mice were randomly allocated (9 mice/group; Figure 1A) to N◦FP7-609398 (AgreenSkills+ contract). PDC is a senior research associate at either receive water with no supplementation (ob/+ Candob/ob C FRS-FNRS (Fonds de la Recherche Scientifique) and recipient of grants from mice) or water supplemented with indole (ob/+ Iandob/ob I mice) FNRS (WELBIO-CR-2019C-02R, “The Excellence of Science: EOS 30770923”) (Sigma; dosage as specified below). Standard diet [AIN93M, 9%kJ and the Funds Baillet Latour (Grant for Medical Research 2015). NMD is a fat (25), D10012Mi; Research Diets] and water was provided ad recipient of grants from SPW-EER (convention 1610365, ERA-HDHL cofunded libitum.