Mechanism of Terpinen‐4‐ol in Suppressing β‐Cell Dedifferentiation: Restoring Mitophagy and Alleviating Mitochondrial Calcium Overload via Parkin‐Mediated MCU Degradation

ABSTRACT Background and Objective Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterised by insulin resistance and β‐cell impairment. Dedifferentiation of pancreatic β‐cells contributes to the progression of T2DM, influencing insulin secretion through mechanisms such as mitochondrial dysfunction, Ca 2+ overload and impaired autophagy. This study investigated the effects of terpinen‐4‐ol (T4O) on β‐cell dedifferentiation and mitochondrial autophagy, with an emphasis on the role of the Parkin/MCU signalling pathway. Methods A T2DM mouse model was generated using a high‐fat diet (HFD, 60% fat) and STZ (50 mg/kg) injection, and β‐cell dedifferentiation was induced by high glucose (HG) treatment in MIN6 cells. T4O was administered, and its effects on mitochondrial autophagy and β‐cell dedifferentiation were evaluated. The mitochondrial autophagy agonist NMN and the MCU inhibitor RU360 were used to assess the interplay between mitophagy and Ca 2+ signalling. Parkin was overexpressed in vitro and in vivo to determine its contribution to mitochondrial autophagy and Ca 2+ regulation. The investigation of the mechanism of the Parkin/MCU signalling pathway involved co‐immunoprecipitation (Co‐IP), mass spectrometry (MS) and protein synthesis inhibition assays. Results T4O improved blood glucose levels and insulin resistance in diabetic mice. In vitro and in vivo, T4O reduced the expression of dedifferentiation markers (such as OCT4, MafA and Pdx1 and Ngn3), increased the expression of mitochondrial autophagy‐related proteins (PINK1, Parkin, Beclin and LC3‐II/I) and modulated the expression of MCU and Drp1 (downregulated) as well as MFN2 (upregulated). T4O also alleviated HG‐induced mitochondrial damage, including ultrastructural abnormalities, decreased membrane potential, elevated reactive oxygen species and Ca 2+ overload. In addition, preincubation of MIN6 cells with T4O or the mitochondrial autophagy agonist NMN can reduce HG‐induced mitochondrial Ca 2+ overload, promote mitochondrial autophagy, reduce MCU protein levels and inhibit pancreatic β‐cell dedifferentiation. Similar results were observed when Parkin was overexpressed both in vivo and in vitro. Moreover, the opposite results were obtained in Parkin‐knockdown MIN6 cells. Mechanistically, T4O facilitated Parkin‐mediated MCU ubiquitination degradation by interacting with MCU at lysine residue K320; notably, mutation of K320 to arginine (K320R) abolished T4O‐induced MCU ubiquitination and reversed the protective effects against β‐cell dedifferentiation. These findings indicate that T4O increases Parkin expression, increases MCU ubiquitination and reduces mitochondrial Ca 2+ accumulation, thereby protecting against T2DM‐induced β‐cell dedifferentiation. Conclusion T4O enhances Parkin expression and targets the K320 site of MCU to promote Parkin‐mediated, ubiquitination‐mediated degradation of MCU, thereby alleviating mitochondrial calcium overload and mitophagy and consequently inhibiting the transdifferentiation of pancreatic islet β‐cells in diabetes. These data support T4O as a potential therapeutic candidate for T2DM by targeting mitochondrial Ca 2+ signalling and autophagy.