Equilibrative nucleoside transporter 3 depletion in β-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes

Loss of function recessive mutations in the SLC29A3 gene that encodes human equilibrative nucleoside transporter 3 (END) have been identified in patients with pigmented hypertrichotic dermatosis with insulin-dependent diabetes (PHID). ENT3 is a member of the equilibrative nucleoside transporter (ENT) family whose primary function is mediating transport of nucleosides and nucleobases. The aims of this study were to characterise ENT3 expression in islet beta-cells and identify the effects of its depletion on beta-cell mitochondrial activity and apoptosis. RT-PCR amplification identified ENT3 expression in human and mouse islets and exocrine pancreas, and in MIN6 beta-cells. Immunohistochemistty using human and mouse pancreas sections exhibited extensive ENT3 immunostaining of beta-cells, which was confirmed by co-staining with an anti-insulin antibody. In addition, exposure of dispersed human islet cells and MIN6 beta-cells to MitoTracker and an ENT3 antibody showed co-localisation of END to beta-cell mitochondria. Consistent with this, Western blot analysis confirmed enhanced ENT3 immunoreactivity in beta-cell mitochondria-enriched fractions. Furthermore, ENT3 depletion in beta-cells increased mitochondrial DNA content and promoted an energy crisis characterised by enhanced ATP-linked respiration and proton leak. Finally, inhibition of ENT3 activity by dypridamole and depletion of ENT3 by siRNA-induced knockdown resulted in increased caspase 3/7 activities in beta-cells. These observations demonstrate that END is predominantly expressed by islet beta-cells where it co-localises with mitochondria. Depletion of ENT3 causes mitochondrial dysfunction which is associated with enhanced beta-cell apoptosis. Thus, apoptotic loss of islet beta-cells may contribute to the occurrence of autoantibody-negative insulin-dependent diabetes in individuals with non-functional END mutations. (C) 2015 Elsevier B.V. All rights reserved.