Integrated proteomic and phosphoproteomic profiling unravels an O‐ GlcNAc ‐dependent mechanism in DGAT1 inhibition‐mediated protection of β‐cells from lipotoxicity

Abstract Aims Obesity‐induced lipotoxicity is a key driver of pancreatic β‐cell dysfunction in Type 2 diabetes. Although Diacylglycerol O‐acyltransferase 1 (DGAT1) inhibition has been reported to ameliorate lipotoxic injury, the underlying mechanisms remain incompletely understood. This study leverages integrated proteomic and phosphoproteomic analyses to systematically explore the novel protective mechanisms of DGAT1 inhibition. Methods We employed Min6 β‐cells subjected to palmitic acid (PA)‐induced lipotoxicity, comparing control, PA‐treated, and PA co‐treated with two distinct DGAT1 inhibitor groups. We assessed lipid accumulation, ER stress, and inflammation. Integrated proteomic and phosphoproteomic analyses, combined with bioinformatic interrogation and experimental validation, were employed to delineate novel molecular mechanisms. Results DGAT1 inhibition effectively alleviated PA‐induced lipid deposition, ER stress, and inflammatory responses. Our multi‐omics data revealed that PA triggered ferroptosis, which was suppressed by DGAT1 inhibitors. Mechanistically, DGAT1 inhibition attenuated global O‐GlcNAcylation, leading to the destabilization of the pro‐ferroptotic protein ACSL4. This cascade resulted in reduced lipid peroxidation and restored glutathione levels, ultimately enhancing β‐cell survival. Conclusion Our study delineates the dynamic proteomic and phosphoproteomic landscape in Min6 cells under PA‐induced lipotoxicity and its remediation by a DGAT1 inhibitor. We delineate the remodelling of key signalling pathways, changes in critical gene expression, and dysregulation of transcription factor activity that collectively contribute to PA‐induced β‐cell injury. Furthermore, we reveal that DGAT1 inhibition attenuates ferroptosis through a novel mechanism involving DGAT1‐driven regulation of O‐GlcNAcylation. This study provides new insights into DGAT1 inhibition‐mediated protection against lipotoxicity and offers valuable multi‐omics data resources for the research community.