Hyperuricemia Drives Pancreatic β‐cell Death Facilitated by DEPTOR uratylation

Abstract

Introduction Uric acid (UA) is the final metabolite of the human purine metabolism, also representing the main antioxidant in the blood. However, elevated serum uric acid (SUA) called hyperuricemia and causing gout has been linked to diabetes mellitus (DM). Loss of viable pancreatic β-cells is a hallmark of both development and progression of DM. We aimed to determine how elevated UA levels can induce a loss of cell viability through the AMPK-mTOR pathway, involving the uric acid transporter glucose transporter 9 (GLUT9). Material and methods We employed the human (1.1B4) and mouse (MIN6) pancreatic β-cell lines. Co-immunoprecipitation, siRNA knock-down, western blot analyses, autophagy, MTT and Caspase 3/7 assays were used to determine the effects of chronic hyperuricemia on molecular mechanisms of β-cell viability. Results Hyperuricemia reduced pancreatic β-cell viability by both reduced metabolic activity and increased autophagy/apoptosis, which could be reverted by the GLUT9 inhibitor benzbromarone as well as by GLUT9 knock-down. Phosphorylation of AMP-activated protein kinase (AMPK) was increased upon elevated UA conditions, whereas phosphorylation of mTOR complex-1 (mTORC1) activator Raptor was reduced. However, hyperuricemia increased expression of the mTORC1 negative regulator DEP domain-containing mTOR-interacting protein (DEPTOR). DEPTOR stabilization was mediated by changes in DEPTOR ubiquitination due to the down-regulation of the E3 ligase β-TrCP and upregulation of a newly identified DEPTOR-regulating ubiquitin-specific protease, USP3, in cells exposed to high UA conditions. In addition, we document for the first time that a protein is UA-modified (uratylated) in a cellular context. Interestingly, DEPTOR uratylation seems to contribute to the change of its ubiquitination. We observed this to be different between mouse and human β-cells, where in MIN6 cells only ubiquitin seems to be uratylated, whereas in 1.1B4 cells DEPTOR seems to be directly uratylated. This difference in uratylation of DEPTOR between mouse and human pancreatic β-cells possibly reflects a further evolved, more sophisticated post-transcriptional regulation of proteins based on the difference in UA handling between these species. Our observation also puts into question if mouse models are an accurate representation of the regulatory processes of basic physiological processes such as insulin secretion or β-cell viability in humans. Conclusion Hyperuricemic conditions resembling elevated SUA levels result in a decrease in pancreatic β-cell viability initiated by changes in DEPTOR ubiquitination/uratylation. This results in an increase of autophagy and ultimately apoptosis. Our findings thus suggest a causal basis for the connection between elevated SUA and diabetes mellitus.