ABSTRACT
AIMS/HYPOTHESIS: Pro-inflammatory cytokines induce death of beta cells and hamper engraftment of transplanted islet mass. Our aim was to reveal novel genes involved in this process, as a platform for innovative therapeutic approaches. METHODS: Small interfering RNA (siRNA) high-throughput screening (HTS) of primary human islets was employed to identify novel genes involved in cytokine-induced beta cell apoptosis. Dispersed human islets from nine human donors, treated with a combination of TNF-α, IL-1ß and IFN-γ were transfected with â¼730 different siRNAs. Caspase-3/7 activity was measured, results were analysed and potential anti- and pro-apoptotic genes were identified. RESULTS: Dispersed human pancreatic islets appeared to be suitable targets for performance of siRNA HTS. Using this methodology we found a number of potential pro- and anti-apoptotic target hits that have not been previously associated with pancreatic beta cell death. One such hit was the de-ubiquitinating enzyme otubain 2 (OTUB2). OTUB2 knockdown increased caspase-3/7 activity in MIN6 cells and primary human islets and inhibited insulin secretion and increased nuclear factor-κB (NF-κB) activity both under basal conditions and following cytokine treatment. CONCLUSIONS: Use of dispersed human islets provides a new platform for functional HTS in a highly physiological system. Employing this technique enabled the identification of OTUB2 as a novel promoter of viability and insulin secretion in human beta cells. OTUB2 acts through the inhibition of NF-κB signalling, which is deleterious to beta cell survival. siRNA screens of human islets may therefore identify new targets, such as OTUB2, for therapeutic intervention in type 1 diabetes and islet transplantation.
Subject(s)
Insulin-Secreting Cells/cytology , Islets of Langerhans/cytology , RNA, Small Interfering/metabolism , Thiolester Hydrolases/metabolism , Animals , Caspase 3/metabolism , Caspase 7/metabolism , Cell Survival , Flow Cytometry , HEK293 Cells , Humans , Insulin-Secreting Cells/pathology , Interferon-gamma/metabolism , Interleukin-1beta/metabolism , Islets of Langerhans Transplantation , Mice , NF-kappa B/metabolism , Promoter Regions, Genetic , Signal Transduction , Tumor Necrosis Factor-alpha/metabolismABSTRACT
AIMS/HYPOTHESIS: Pro-inflammatory cytokines induce death of pancreatic beta cells, leading to the development of type 1 diabetes. We sought to identify novel players and the underlying mechanisms involved in this process. METHODS: A high-throughput screen of 3,850 mouse small interfering RNAs (siRNAs) was performed in cytokine-treated MIN6 beta cells. Cells were transfected with the different siRNAs and then treated with a combination of TNFα, IL-1ß and IFNγ. Cellular apoptosis (caspase-3/7 activity), and changes in cellular reducing power and cell morphology were monitored. The resulting data were analysed and the corresponding z scores calculated. RESULTS: Several gene families were identified as promoting cytokine-induced beta cell apoptosis, the most prominent being those encoding ubiquitin ligases and serine/threonine kinases. Conversely, deubiquitinating enzymes appeared to reduce apoptosis, while protein phosphatases were mainly associated with lowering cellular reducing power. The screen suggested with high confidence the involvement of several novel genes in cytokine-induced beta cell death, including Camkk2, Epn3, Foxp3 and Tm7sf3, which encodes an orphan seven transmembrane receptor. siRNAs to Tm7sf3 promoted cytokine-induced death of MIN6 cells and human pancreatic islets, and abrogated insulin secretion in these cells. These findings implicate transmembrane 7 superfamily member 3 as a potential new player in the inhibition of cytokine-induced death and in the promotion of insulin secretion from pancreatic beta cells. CONCLUSIONS/INTERPRETATION: The signalling pathways and novel genes that we identified in this screen and that mediate beta cell death offer new possible targets for therapeutic intervention in diabetes and its adverse complications.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , Cytokines/metabolism , Insulin-Secreting Cells/metabolism , Membrane Glycoproteins/metabolism , RNA, Small Interfering/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Caspases, Effector/metabolism , Cell Line , Female , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/cytology , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Male , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Oxidation-Reduction , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Signal Transduction , Tissue Culture Techniques , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Heterologous binding of rat brain hexokinase to wild type, porinless, and recombinant yeast mitochondria expressing human porin was assessed, partially characterized, and compared to that in the homologous system (rat liver mitochondria). With porin-containing yeast mitochondria it is shown that (i) a significant, saturable association occurs; (ii) its extent and apparent affinity, correlated with the origin of porin, are enhanced in the presence of dextran; (iii) the binding requires Mg ions and apparently follows a complex cooperative mechanism. This heterologous association does not seem to differ fundamentally from that in the homologous system and represents a good basis for molecular studies in yeast. With porinless yeast mitochondria, binding occurs at much lower affinity, but to many more sites per mitochondrion. The results indicating a major but not exclusive role for porin in the binding are discussed in terms of (i) the mode and mechanism of binding, and (ii) the suitability of the rat hexokinase-yeast mitochondria couple for the study of heterogeneous catalysis in reconstituted cellular model systems.
