Triple GLP-1R/GIP-R/GCGR activation synergistically suppresses macrophage-driven oxidative stress and activation in human epicardial adipose tissue from patients with a history of atrial fibrillation

Background Epicardial adipose tissue (EAT) contributes to atrial remodeling through macrophage-driven inflammation, oxidative stress, and fibrosis. Glucagon-like peptide-1 receptor (GLP-1R) agonists provide cardiometabolic benefits, and combined incretin strategies including GLP-1R, glucose-dependent insulinotropic polypeptide receptor (GIP-R), and glucagon receptor (GCGR) agonism are emerging. However, their relevance in human EAT in atrial fibrillation (AF) is unknown. Purpose To determine the expression, localization, and functional impact of GLP-1R/GIP-R/GCGR activation in human EAT from patients with a history of AF. Methods EAT was obtained from cardiac surgery patients with AF history and matched sinus rhythm (SR) controls (n=20/group; age: 74.5 (61.8–78.0) vs 73.5 (61.5–76.3) years; BMI: 24.8 (23.0–27.6) vs 26.3 (23.2–27.3) kg/m² [median (Q1–Q3)]; male sex: 90% vs 85%; diabetes: 10% vs 0%). Gene expression was assessed by RT-qPCR and protein levels by Western blot. Oxidative stress was quantified by DHE fluorescence. Immunofluorescence was used for receptor localization. EAT-conditioned medium (CM) was generated by 24 h incubation in serum-free medium; cytokines/adipokines were quantified by ELISA. CM was applied to healthy donor PBMCs (30% v/v, 24 h). A GLP-1 receptor agonist (GLP-1RA), GIP, and glucagon were tested individually or combined (10 nM each). Adenylyl cyclase and PKA signaling were investigated using SQ22536 and PKI(14–22). Synergy was assessed by Bliss independence. Results EAT from patients with a history of AF exhibited increased levels of IL-1β, IL-6, TNF-α, MCP-1, CD86, NOX2, remodeling markers (TGF-β, MMP-2) and ROS formation compared with those of SR patients. In AF-EAT, GLP-1R, GIP-R, and GCGR expression were elevated and positively correlated to CD86 (r²=0.7464, 0.3053, 0.6634, respectively; all P<0.0001), with strong colocalization in CD86⁺ macrophages. Elevated levels of ROS in AF-EAT were unaffected by a single incretin agonist. However, combined GLP-1RA/GIP/glucagon mitigated ROS formation in a synergistic manner (69.4% inhibition vs expected additive 29.2%). CM from AF-EAT showed increased IL-1β, IL-6, TNF-α, MCP-1, leptin, sICAM-1, sVCAM-1 and reduced adiponectin levels, and induced PBMC activation evidenced by elevated ROS, calcium signal and glucose uptake. The triple combination reduced PBMC activation, an effect abolished by SQ22536 or PKI. Conclusions EAT from patients with a history of AF exhibits incretin receptor enrichment in resident pro-inflammatory macrophages and drives inflammatory, oxidative, and remodeling responses. Combined GLP-1RA/GIP/glucagon synergistically suppresses EAT oxidative stress and EAT-driven immune activation via cAMP/PKA signaling, supporting triple incretin receptor agonism as a strategy to target EAT-mediated atrial remodeling.