Glucagon-like peptide-1 (GLP-1) and dual GLP-1/glucose-dependent insulinotropic polypeptide (GIP) receptor agonists show high efficacy in treating obesity and metabolic dysfunction-associated liver diseases, conditions that collectively represent one of the most challenging One Health crises of our time. However, their hepatic molecular mechanisms remain poorly understood. This study defined targeted hepatic proteomic signatures associated semaglutide (GLP-1 agonist) and tirzepatide (dual GLP-1/GIP) in diet-induced obese (DIO) male C57BL/6J mice using an liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted-proteomics approach and validated using western blotting. After 18 weeks of high-fat diet (HFD), obese mice received once-daily subcutaneous semaglutide (10 nmol/kg), tirzepatide (3 nmol/kg), or vehicle for 4 weeks. Tirzepatide produced greater systemic efficacy (24.6% body weight and 30.3% fat mass reduction, preserved lean mass, improved glucose homeostasis), yet both drugs yielded overlapping targeted hepatic proteomic signatures despite different receptor pharmacology. Both drugs significantly downregulated CD36 and CPT1A fatty acid importers for β-oxidation without altering lipogenic enzymes (FASN, ACC1, ACLY, ACSS2) or glucose-handling proteins (PDH, GLUT2). These findings suggest a substrate limitation over enhanced β-oxidation or broad lipogenesis-suppression, suggesting GLP-1 receptor activation is sufficient for the core liver response and GIP co-agonism mainly augments extrahepatic effects. From a One Health proteomics perspective, CD36/CPT1A anchored targeted proteomic signature provides a mechanistically informed benchmark for evaluating novel GLP-1 and non-GLP-1 investigational therapeutics.
