Microbially-derived indole-3-acetate alleviates diet induced steatosis and inflammation in mice

Reviewer #1 (Public Review):

The study by Ding et al demonstrated that microbial metabolite I3A reduced western diet induced steatosis and inflammation mice. They showed that I3A mediates its anti-inflammatory activities through AMP-activated protein kinase (AMPK)-dependent manner in macrophages. Translationally, they proposed that I3A could be a potential therapeutic molecule in preventing the progression of steatosis to NASH.

Major strengths
• Authors clearly demonstrated that the Western Diet (WD)-induced steatosis and I3A treatment reduced steatosis and inflammation in pre-clinical models. Data clearly supports these statements.
• I3A treatment rescued WD-altered bile acids as well partially rescued the metabolome, proteome in the liver.
• I3A treatment reduced the levels of enzymes in fatty acid transport, de novo lipogenesis and β-oxidation
• I3A mediates its anti-inflammatory activities through AMP-activated protein kinase (AMPK)-dependent manner in macrophages.

Minor Weakness
Although data strongly support the notion that I3A reduced WD-induced steatosis and I3A treatment reduced steatosis and inflammation, the following concerns need to be addressed.
• Authors suggested that I3A anti-inflammatory activities do not require AhR by using AhR-inhibitor in RAW cell lines. In the literature, studies do show that RAW cells do respond to AhR ligands such as TCDD and FICZ.
• AhR-dependency needs to be confirmed by bone marrow derived macrophages isolated from AhR+/+ and AhR-/- or siRNA/ShRNA knockdown experiments.
• Utilization of known AhR ligands as controls will strengthen the interpretation of the conclusions.

Reviewer #2 (Public Review):

This article examines the ability of dietary supplementation with indole-3-actetate (I3A) to attenuate western diet-induced fatty liver disease. The experiments are appropriately described, and convincing data are provided that I3A can attenuates fat accumulation in the liver. Several possible mechanisms of action were explored and one likely mechanism, an alteration in AMPK signaling pathway was observed, and is likely involved in the observed phenotype. However, I3A has already been shown to yield similar data in a high fat diet mouse model system (PMID: 31484323), although the I3A was administered through IP injection, not in the drinking water. In both studies the effects seen may well be due to activation of PPAR-alpha. Another study (PMID: 19469536) gave acetic acid in the drinking water and obtained data similar to this manuscript, supporting that the effect seen in this study may not be specific to I3A. These references should be included and discussed. Overall, the data and experimental approach taken support the stated conclusions.

Reviewer #3 (Public Review):

Ding et al. address the experimental question of whether the microbially derived I3A can exert pro-metabolic effects in an experimental model of diet induced obesity/hepatic steatosis. This was based on previous findings by the authors that high fat diet alters levels of I3A, and that I3A can exert anti-steatotic and anti-inflammatory effects in vitro. The data are robust and the authors provide a plethora of omics-based platforms including proteomics and metabolomics under a variety of treatment paradigms. By performing these studies in vivo in mouse liver tissue, these atlases of proteomic and metabolomic datasets would be of interest to the field of metabolism for future analysis. However, there are several weaknesses identified within this manuscript. Primarily, weaknesses in the interpretation and organization of presented data overshadow the robust data presented and make it difficult for the reader to draw any new biological conclusions. Specifically, this manuscript in its current form is primarily of descriptive nature and does not distill any of the complex datasets presented into digestable conclusions that shed new insight into regulation of hepatic metabolism and inflammation by I3A. In essence, this manuscript in its current form is an in vivo extension to the author's previous in vitro assessment of I3A on liver function. Finally, there is a flaw in the model presented (Supplemental Fig. 9) with regards to the authors linking the anti-inflammatory effects of I3A with the metabolic effects. In fact, the authors present data (Fig. 1&2) that show the opposite of this interpretation in which inflammation is uncoupled from the metabolic effects of I3A in the low dose treatment group. While the authors achieved their main goal of addressing the metabolic effects of I3A in vivo, the organization and interpretation of the data presented in its current form is likely to result in a modest impact on the field.