Phenylacetylglutamine restrains epithelial ferroptosis in intestinal ischemia-reperfusion by coupling lipid droplet-mitochondria remodeling to metabolic execution.
Abstract
Background
Intestinal ischemia-reperfusion injury (IIR) involves epithelial barrier failure, oxidative stress, and ferroptosis-associated lipid damage, yet how lipid droplet-mitochondria coupling influences ferroptotic susceptibility remains incompletely understood.
Methods
Untargeted plasma metabolomics was combined with murine IIR and Caco-2 oxygen-glucose deprivation/reoxygenation models. Epithelial injury, lipid peroxidation, mitochondrial function, lipid droplet-mitochondria association, and fluorescent fatty-acid analogue localization were assessed. Ferrostatin-1 (Fer-1), etomoxir, PLIN5 silencing, and the ADRB2 antagonist ICI-118551 were used for mechanistic interrogation, together with molecular docking, molecular dynamics simulations, and cellular thermal shift assays.
Results
Untargeted plasma metabolomics identified phenylacetylglutamine (PAGln) as an IIR-responsive circulating metabolite. Exogenous PAGln pretreatment attenuated epithelial injury, lipid peroxidation, and ferroptosis-associated alterations. PAGln restored perilipin 5 (PLIN5)-associated lipid droplet-mitochondria remodeling, increased the relative colocalization of a fluorescent fatty-acid analogue with mitochondria, and improved mitochondrial metabolic activity. Etomoxir markedly attenuated mitochondrial metabolic recovery, lipid peroxide control, and epithelial protection while largely preserving PAGln-induced organelle remodeling, whereas PLIN5 silencing disrupted both spatial remodeling and its downstream metabolic consequences. Upstream, PAGln engaged β2-adrenergic receptor (ADRB2)-dependent cyclic adenosine monophosphate/protein kinase A signaling. ADRB2 blockade attenuated PLIN5 restoration and organelle remodeling in vitro and weakened mitochondrial metabolic recovery and epithelial protection in both cell and animal models. Computational analyses and cellular thermal shift assays further supported a functional PAGln-ADRB2 relationship.
Conclusion
PAGln restrains ferroptosis-associated epithelial injury through an ADRB2-PLIN5-regulated organelle program in which lipid droplet-mitochondria spatial remodeling must be coupled to downstream mitochondrial fatty-acid utilization. These findings functionally distinguish spatial remodeling from metabolic execution and identify organelle-level fatty-acid handling as a regulatory layer of ferroptotic susceptibility in IIR.