Introduction

Metabolic dysfunction–associated steatotic liver disease (MASLD) is the most common liver disease worldwide, present in over 30% of adults globally.1 Approximately 16% of people with MASLD will progress to metabolic dysfunction–associated steatohepatitis (MASH), which is characterized by steatosis, inflammation, and fibrosis.1 Current therapeutic options remain limited, highlighting an urgent need for novel treatment strategies. The role of diet in the pathogenesis and management of MASLD is well-established.2 However, the contributions of the gut microbiome and its metabolites in the development of MASLD have only recently begun to be widely investigated.

The gut microbiome influences host metabolism through the production of bioactive metabolites. Members of the Bacteroidota phylum, which comprise up to 60% of the human gut microbiome, are known for their ability to break down complex glycans and generate fermentation products for the host.3 In addition to their glycan metabolism, Bacteroidota also produce a unique spectrum of bioactive lipids, including sphingolipids4 and glycine lipids (GLs).5

Bacterial GLs were first identified in Flavobacterium species,6,7 further characterized from the oral pathogen Porphyromonas gingivalis,8 and later found to be broadly expressed in members of the Bacteroidota phylum.5,8, 9, 10, 11, 12, 13 Bacteroidota contain GLs in their cell membranes and shed them in outer membrane vesicles.12 The microbiome GL classes identified thus far include the glycine amino lipids, Lipid 342 (L342) and Lipid 567 (L567), named for their negative ion masses, as well as the serine-glycine lipodipeptides, Lipid 430 (L430), Lipid 654 (L654), and Lipid 1256 (L1256).5,8,9 These lipid classes were named for their dominant ion species but are found alongside minor species variants with different fatty acid substitutions. Of these, L654 and L1256 classes are the major forms detected in most biological samples.

We previously reported that feeding a high-fat diet (HFD) reduces microbiome GLs in mouse feces, serum, and liver tissues.14 Prior research has also shown that microbiome GLs activate Toll-like receptor 2 (TLR2) in vitro and in mice.5,8,9,15 Despite signaling through TLR2, we have previously found that chronic administration of microbiome GLs attenuates atherosclerosis,14 liver injury,14 and autoimmune disease16 in mouse models. While short-term exposure to GLs can induce cytokine secretion in monocytes5 and macrophages9 via TLR2, chronic exposure induces TLR2 tolerance16,17 and cross-tolerance to TLR4.14

Previously, chronic intraperitoneal administration of L654- and L567-enriched lipid extracts was shown to be hepatoprotective in mouse models of atherosclerosis.14 However, critical knowledge gaps persist regarding their therapeutic potential in liver disease models. Additionally, the mechanisms by which these compounds influence hepatic metabolism and the extent to which dietary interventions can modulate their production remain unexplored. The current study was designed to address these knowledge gaps.