Bacteroidota-derived Glycine Lipids Regulate Skeletal Muscle Inflammation and Mitochondrial Quality Control

Introduction: The Western diet (WD) is characterized by high levels of sugar and cholesterol, which contribute to the development of metabolic diseases in part by disrupting gut microbiome composition and altering production of microbial metabolites that influence host metabolism and inflammation. Among these metabolites, glycine lipids from Bacteroidota, including glycine lipids 654 (L654) and 1256 (L1256), are known as Toll-like receptor 2 (TLR2) activators. Despite such pro-inflammatory potential, these lipids have recently gained attention for their therapeutic properties, as both L654 and L1256 were shown to restore vascular function in WD-fed Ldlr-/- mice. However, their actions differ at the level of skeletal muscle, as only L1256 enhances mitochondrial respiration. The mechanism underlying this selective enhancement remains unclear, but may relate to differences in lipid structural stability, tissue distribution, and the degree to which each lipid engages TLR2-mediated cytoprotective signaling, including activation of nuclear factor (erythroid-derived 2)–like 2 (NRF2) and subsequent regulation of mitophagy. Therefore, this study examines the effects of L654 and L1256 on skeletal muscle inflammatory signaling and mitochondrial regulation under WD-induced stress, with a focus on uncovering the mechanism through how L1256 uniquely improves mitochondrial respiration. Methods: Forty-five male Ldlr-/- mice (8 weeks old) were randomly assigned to four groups: a chow diet vehicle group (LFD, n=12), a high-fat diet vehicle group (HFD, n=10), an HFD group treated with L654 (n=11), and an HFD group treated with L1256 (n=12). All HFD groups were fed a high-fat diet for 8 weeks and received intraperitoneal injections of vehicle, L654, or L1256 three times per week at a dose of 1 μg per injection. At the end of the intervention, the gastrocnemius muscles were collected for molecular analyses. Accumulation of the administered glycine lipids (L654 and L1256) in skeletal muscle was quantified using liquid chromatography–mass spectrometry (LC-MS). Protein expression of inflammatory markers (TLR2, IL-6, TNF-α, 4-HNE), cytoprotective markers (NRF2), and mitophagy-related proteins (PINK1, Parkin, p62) was evaluated by Western blotting. A one-way ANOVA was performed to assess group differences, followed by LSD post hoc tests to identify pairwise comparisons. Results: Total bacterial glycine lipid levels in gastrocnemius muscle were reduced in the HFD and HFD+L654 groups compared with LFD and HFD+L1256, and among the administered lipids, only L1256 was detectable within skeletal muscle. Both glycine lipids, L654 and L1256, increased muscle TLR2 protein expression and concurrently lowered IL-6 protein levels compared to LFD and HFD, while TNF-α and 4-HNE remained unchanged. In parallel, NRF2 and p62 expression were significantly elevated in both lipid-treated groups, indicating activation of cytoprotective and autophagy-related signaling. Regarding mitophagy, Parkin expression was reduced in HFD but restored only by L1256, whereas PINK1 levels remained unchanged across groups. Conclusion: Although only L1256 accumulated in skeletal muscle, both L654 and L1256 influenced TLR2-related signaling and exhibited cytoprotective effects, consistent with our previous finding that both lipids improved vascular function in WD-fed Ldlr-/- mice. However, only L1256 enhanced mitophagy, suggesting that its greater structural stability may confer a unique therapeutic advantage in restoring WD-disrupted mitochondrial function in skeletal muscle. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.