Gut microbiota-derived gamma-aminobutyric acid from metformin treatment reduces hepatic ischemia/reperfusion injury through inhibiting ferroptosis

Reviewer #1 (Public Review):

Many drugs have off-target effects on the gut microbiota but the downstream consequences for drug efficacy and side effect profiles remain unclear. Herein, Wang et al. use a mouse model of liver injury coupled to antibiotic and microbiota transplantation experiments. Their results suggest that metformin-induced shifts in gut microbial community structure and metabolite levels may contribute to drug efficacy. This study provides valuable mechanistic insights that could be dissected further in future studies, including efforts to identify which specific bacterial species, genes, and metabolites play a causal role in drug response. Importantly, although some pilot data from human subjects is shown, the clinical relevance of these findings for liver disease remain to be determined.

The major strength of this work is its scope, including detailed mouse phenotyping, inter-disciplinary methods, and numerous complementary experiments. The antibiotic depletion and FMT experiments provide support for a role of the gut microbiota in this mouse model.

A major limitation is the lack of studies narrowing down which microbes are responsible. Sequencing data is shown, but no follow-up studies are done with bacterial isolates or defined communities.

The link to GABA is also somewhat tenuous. While it does match the phenotypic data, there are no targeted experiments in which GABA producing microbial communities/strains are compared to a control community/strain. As such, it seems difficult to know how much of the effects in this model are due to GABA vs. other metabolites.

My major recommendation would be to revise the title, abstract, and discussion to provide more qualification and to consider alternative interpretations.

Some key controls are also missing, which could be addressed by repeat experiments in the mouse model. The antibiotic depletion experiment would be improved by testing the effect of antibiotics in the absence of metformin, to see if the effect is just driven by the model itself as opposed to an interaction between metformin and antibiotics. The FMT experiment lacks a control group and suffers from pseudoreplication: multiple donors from metformin treated and untreated mice could be used to colonize separate groups of recipient mice.

Reviewer #2 (Public Review):

The authors examine the use of metformin in the treatment of hepatic ischemia/reperfusion injury (HIRI) and suggest the mechanism of action is mediated in part by the gut microbiota and changes in hepatic ferroptosis. While the concept is intriguing, the experimental approaches are inadequate to support these conclusions.

The histological and imaging studies were considered a strength and reveal a significant impact of metformin post-HIRI.

Weaknesses largely stem from the experimental design. First, use of the iron chelator DFO would be strengthened using the ferroptosis inhibitor, liproxstatin. Second, the impact of metformin on the microbiota is profound resulting in changes in bile acid, lipid, and glucose homeostasis. Throughout the manuscript no comparisons are made with metformin alone which would better capture the metformin-specific effects. Lastly, the absence of proper controls including germ free mice, metformin treated mice, FMT treated mice, etc make it difficult to understand the outcomes and to properly reproduce the findings in other labs.

Overall, while the concept is interesting and has the potential to better understand the pleiotropic functions of metformin, the limitations with the experimental design and lack of key controls make it challenging to support the conclusions.

Reviewer #3 (Public Review):

The study presented in this paper explores the role of gut microbiota in the therapeutic effect of metformin on HIRI, as supported by fecal microbiota transplantation (FMT) experiments. Through high throughput sequencing and HPLC-MS/MS, the authors have successfully demonstrated that metformin administration leads to an increase in GABA-producing bacteria. Moreover, the study provides compelling evidence for the beneficial impact of GABA on HIRI.