A host enzyme reduces non-alcoholic fatty liver disease by inactivating intestinal lipopolysaccharide

Reviewer #1 (Public review):

Lu et. al. proposed here a direct role of LPS in inducing hepatic fat accumulation and that the metabolism of LPS therefore can mitigate fatty liver injury. With an Acyloxyacyl hydrolase whole-body KO mice, they demonstrated that Acyloxyacyl hydrolase deletion resulted in higher hepatic fat accumulation over 8 months of high glucose/high fructose diet. Previous literature has found that hepatocyte TLR4 (which is a main receptor for binding LPS) KO reduced fatty liver in the MAFLD model, and this paper complements this by showing that degradation/metabolism of LPS can also reduce fatty liver. This result proposed a very interesting mechanism and the translational implications of utilizing Acyloxyacyl hydrolase to decrease LPS exposure are intriguing.

The strengths of the present study include that they raised a very simplistic mechanism with LPS that is of interest in many diseases. The phenotype shown in the study is strong. The mechanism proposed by the findings is generally well supported.

There are also several shortcomings in the findings of this study. As AOAH is a whole-body KO, the source production of AOAH in MAFLD is unclear. Although the authors used published single-cell RNA-seq data and flow-isolated liver cells, physiologically LPS degradation could occur in the blood or the liver. The authors linked LPS to hepatocyte fatty acid oxidation via SREBP1. The mechanism is not explored in great depth. Is this signaling TLR4? In this model, LPS could activate macrophages and mediate the worsening of hepatocyte fatty liver injury via the paracrine effect instead of directly signaling to hepatocytes, thus it is not clear that this is a strictly hepatocyte LPS effect. It would also be very interesting to see if the administration of the AOAH enzyme orally could mitigate MAFLD injury. Overall, this work adds to the current understanding of the gut-liver axis and development of MAFLD and will be of interest to many readers.

Reviewer #2 (Public review):

The authors of this article investigated the impact of the host enzyme AOAH on the progression of MASLD in mice. To achieve this, they utilized whole-body Aoah-/- mice. The authors demonstrated that AOAH reduced LPS-induced lipid accumulation in the liver, probably by decreasing the expression and activation of SREBP1. In addition, AOAH reduced hepatic inflammation and minimized tissue damage.

However, this paper is descriptive without a clear mechanistic study. Another major limitation is the use of who-body KO mice so the cellular source of the enzyme remains undefined. Moreover, since LPS-mediated SREBP1 regulation or LPS-mediated MASLD progression is already documented, the role of AOAH in SREBP1-dependent lipid accumulation and MASLD progression is largely expected.

Specific comments:

(1) The overall human relevance of the current study remains unclear.

(2) Is AOAH secreted from macrophages or other immune cells? Are there any other functions of AOAH within the cells?

(3) Due to using whole-body KO mice, the role of AOAH in specific cell types was unclear in this study, which is one of the major limitations of this study. The authors should at least conduct in vitro experiments using a co-culture system of hepatocytes and Kupffer cells (or other immune cells) isolated from WT or Aoah-/- mice.

(4) It has been well-known that intestinal tight junction permeability is increased by LPS or inflammatory cytokines. However, in Figure 3E, intestinal permeability is comparable between the groups in both diet groups. The authors should discuss more about this result. In addition, intestinal junctional protein should be determined by Western blot and IHC (or IF) to further confirm this finding.

(5) In Figure 6, LPS i.g. Aoah-/- group is missing. This group should be included to better interpret the results.

(6) The term NAFLD has been suggested to be changed to MASLD as the novel nomenclature according to the guidelines of AASLD and EASL.