Discussion

Microbiome GLs produced by Bacteroidota accumulate in host tissues11,14 and undergo cellular hydrolysis, though L1256 degrades slowly enough to promote retention.39 We demonstrate these lipids circulate in portal blood and intestinal lymph and that their chronic administration confers hepatoprotection against diet-induced MASH. Moreover, dietary fermentable fiber supplementation restores microbiome GL production during HFD feeding, establishing a novel mechanistic link between dietary fiber, gut microbial metabolism, and liver health.

Importantly, we establish that microbiome GLs upregulate hepatic PGC-1α (Ppargc1a) mRNA expression and increase mtDNA content. This represents a potential mechanism underlying the hepatoprotective effects observed with L654 and L1256 in the current study and with L567 and L654 in previous studies.14 PGC-1α is a key transcriptional coactivator involved in promoting PPARα-dependent fatty acid oxidation and PPARα-independent mitochondrial biogenesis.40 It is known to play a key role in preventing MASLD, and its dysfunction is associated with liver disease pathogenesis.40 Whether the regulation of PGC-1α expression and mitochondrial enhancement was linked to TLR signaling or some other mechanism is unclear. Activation of TLR2 signaling has been linked to Ppargc1a mRNA expression in the AML12 hepatocyte cell line33 and mouse liver.34

We also observed that bacterial GL extracts enriched in L1256, but not L654, enhanced mtDNA content and function in HepG2 cells. Increases in cell respiration are unlikely to be due to the GLs providing fatty acid substrate for oxidation, as cells were washed and replaced with fetal bovine serum-free assay buffer prior to respiration testing. Currently, it is unclear whether the differences observed between L654 and L1256 are related to potency in their TLR2 activation/regulation or result from unknown interactions with other receptors. Non-TLR effects of other glycine aminolipids have been previously reported, including activation of PPARα41 and G2 accumulation protein/ G protein-coupled receptor 132.13 Future studies should elucidate the upstream signaling mediating these effects and their direct impact on lipid metabolism across hepatic cell populations.

To date, most research on microbiome-derived GLs has centered on their immunomodulatory effects. Acute, high-dose exposure to these bacterial GLs can activate pro-inflammatory responses via TLR2/TLR6 heterodimers, which may contribute to pathogenesis in certain disease contexts like periodontal disease.42 However, a chronic, low-dose exposure, which is more reflective of physiological gut microbiome exposure, appears to induce tolerance and reduce inflammatory responses.16,17 In the present study, classical inflammatory biomarkers in the liver showed only minor changes at the mRNA level; however, the L1256-enriched extract significantly increased hepatic Trem2 mRNA expression. This up-regulation links our findings to emerging evidence implicating TREM2 in metabolic liver disease.43 TREM2 is present on myeloid cells and can act as a sensor for endogenous and exogenous lipid ligands.44 Prior work from Mihori et al17,45 showed that a Bacteroidota-targeting antibiotic regimen in mice downregulated Trem2 expression in splenic monocytes and TREM2 surface expression in peritoneal macrophages, whereas chronic L654 injections restored these measures. Our findings showing selective TREM2 upregulation by L1256 in liver, not L654, suggests a compartment-specific regulation by these lipids. Recent studies have highlighted the protective effects of liver TREM2 related to inhibition of inflammation, promotion of efferocytosis, and improvements in mitochondrial energy metabolism.46, 47, 48 Our observed reduction in MASLD histopathological features, including portal inflammation and hepatocyte ballooning, is consistent with the protective role of TREM2 in liver diseases.

The dramatic responsiveness of microbiome GLs to dietary fiber manipulation establishes these molecules as new targets for dietary interventions. This fiber-dependence likely reflects the polysaccharide utilization capabilities of Bacteroidota producers. We chose pea fiber and citrus pectin for this investigation and observed an increase in fecal Bacteroidota relative abundance. Further investigation is warranted to determine how other dietary fiber types regulate microbiome GL levels in feces and tissues, as well as to evaluate their broader translational potential and specific effects on liver disease pathology.

Although we revealed diet dependence and hepatic effects of microbiome GLs, limitations remain. While detected in human liver, we did not evaluate whether levels differ in MASLD patients. Additionally, to isolate direct hepatic effects via portal drainage, we utilized intraperitoneal injections.49 Because this bypasses potential gastrointestinal interactions, future studies should specifically investigate these local effects. Moving forward, dietary fiber may serve as an ideal intervention to stimulate in situ gut production of GLs during future bioactivity experiments.