Arachidonic Acid Metabolism by Cyp4A Modulates Caveolae-Mediated Transcytotic LPS Transport in Mouse Jejunum

Abstract

Arachidonic acid (AA) is metabolized not only by cyclooxygenases and lipoxygenases, but also by cytochrome P-450 4A family (Cyp4A) which is an androgen-driven enzyme that produces 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is an endogenous ligand for GPR75, which is highly associated with pathogenesis of obesity. Since metabolic endotoxemia is implicated in the induction of obesity, and the gut lumen is the primary source of endotoxin (lipopolysaccharides [LPS]), we hypothesized that AA metabolism may affect LPS entry through the small intestine. Furthermore, we have found that increased LPS transport during long-chain fatty acid (LCFA) absorption is abolished in caveolin-1 (Cav1) knockout (KO) jejunum, suggesting the involvement of caveolae-mediated endocytosis. We thus examined the effects of AA on LPS transport in murine jejunum. FITC-LPS (10 μg/ml) or FD4 (0.1 mM) was applied to the mucosal (m) bath of Ussing chambered muscle-stripped jejunum from male C57Bl6 wild type (WT) and Cav1 KO mice with measurement of the serosal (s) appearance of FITC-LPS or FD4 and transepithelial electrical resistance (TER). We examined the effects of the luminal application (10 mM) of oleic acid (OA), AA, eicosapentaenoic acid (EPA), or docosahexaenoic (DHA) with taurocholic acid (0.1 mM) on these parameters with or without the lipid raft inhibitor methyl-β-cyclodextrin (MβCD, 0.1 mM) or Cyp4A inhibitor HET0016 (10 μM). In another study, male and female WT and Cav1 KO mice were fed a standard diet (SD), or a diet enriched in saturated LCFA fat and sugar (LCD) for 6 wks. Luminal addition of OA and AA, but not EPA nor DHA, increased LPS m-to-s movement in WT jejunal mucosa. AA also increased FD4 m-to-s movement with no effect on TER. AA-induced LPS and FD4 movements were inhibited by luminal application of MβCD or HET0016, suggesting the involvement of lipid rafts and Cyp4A activity. Luminal AA had no effect on LPS and FD4 m-to-s movement in KO jejunum. LPS and FD4 m-to-s movement were also increased by luminal application of 20-HETE (0.3 and 1 μM). Immunostaining revealed that GPR75 was expressed on the plasma membranes of jejunal villous but not crypt cells. LCD induced obesity in WT, not in KO, with increased blood glucose levels, perigonadal fat weight, and liver weight, with changes more evident in male WT than in female WT. LCD increased LPS m-to-s movement in WT male jejunum, not in KO jejunum, which was inhibited by luminal addition of MβCD or HET0016. These results suggest that luminal AA increases 20-HETE production by Cyp4A, then activates Cav1-mediated endocytosis which in turn enhances LPS transport in the small intestine, possibly via GPR75 activation. The AA-Cyp4A-20-HETE-GPR75 system may be a new therapeutic target for metabolic endotoxemia associated with diet-induced obesity, especially in males. VA Merit Review and DoD grants. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.