Lipid-induced S-palmitoylation of Insulin-Responsive Aminopeptidase (IRAP) drives the onset of insulin resistance in the heart.

Excessive accumulation of long-chain fatty acids (LCFAs) in cardiomyocytes leads to cardiac lipid-induced insulin resistance (CLIR), impairing insulin signaling and glucose uptake. This metabolic disruption marks a prediabetic state in which cardiomyocytes rely predominantly on lipids for energy provision, leading to lipotoxicity and reduced contractile function over time. Palmitate, the most common dietary LCFA, also serves as a substrate for protein S-palmitoylation, a reversible lipid-based post-translational modification (PTM) that governs protein localization and trafficking through distinct palmitoyl acyltransferase (PATs) isoforms, also denominated as DHHC. In this study, we demonstrate that pharmacological inhibition of protein S-palmitoylation with 2-bromopalmitate (2BP) prevents contractile dysfunction and CLIR in palmitate-overloaded cardiomyocytes. Analysis of the cardiomyocyte palmitoylome disclosed, among other proteins, the insulin-regulated aminopeptidase (IRAP). IRAP translocates together with glucose transporter GLUT4 to the plasma membrane in response to insulin. LCFA-induced hyper-S-palmitoylation of IRAP, mediated by DHHC5, disrupts glucose uptake. Knockdown of DHHC5 or transduction with S-palmitoylation-deficient IRAP rescues both cardiomyocyte glucose uptake and contractile function. These findings identify protein S-palmitoylation, particularly IRAP hyper-S-palmitoylation, as a novel driver of CLIR and associated myocardial dysfunction.