title: Equilibrative nucleoside transporter 3 depletion in β-cells impairs mitochondrial function and promotes apoptosis: Relationship to pigmented hypertrichotic dermatosis with insulin-dependent diabetes.
abstract: Loss of function recessive mutations in the SLC29A3 gene that encodes human equilibrative nucleoside transporter 3 (ENT3) 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 β-cells and identify the effects of its depletion on β-cell mitochondrial activity and apoptosis. RT-PCR amplification identified ENT3 expression in human and mouse islets and exocrine pancreas, and in MIN6 β-cells. Immunohistochemistry using human and mouse pancreas sections exhibited extensive ENT3 immunostaining of β-cells, which was confirmed by co-staining with an anti-insulin antibody. In addition, exposure of dispersed human islet cells and MIN6 β-cells to MitoTracker and an ENT3 antibody showed co-localisation of ENT3 to β-cell mitochondria. Consistent with this, Western blot analysis confirmed enhanced ENT3 immunoreactivity in β-cell mitochondria-enriched fractions. Furthermore, ENT3 depletion in β-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 β-cells. These observations demonstrate that ENT3 is predominantly expressed by islet β-cells where it co-localises with mitochondria. Depletion of ENT3 causes mitochondrial dysfunction which is associated with enhanced β-cell apoptosis. Thus, apoptotic loss of islet β-cells may contribute to the occurrence of autoantibody-negative insulin-dependent diabetes in individuals with non-functional ENT3 mutations.
authors.affiliation: Diabetes Research Group, Division of Diabetes and Nutritional Sciences, Faculty of Life and Medical Sciences, King's College London, London SE1 1UL, United Kingdom.
authors.forename: B
authors.initials: B
authors.lastname: Liu
authors.affiliation: Diabetes Research Group, Division of Diabetes and Nutritional Sciences, Faculty of Life and Medical Sciences, King's College London, London SE1 1UL, United Kingdom.
authors.forename: A
authors.initials: A
authors.lastname: Czajka
authors.affiliation: Diabetes Research Group, Division of Diabetes and Nutritional Sciences, Faculty of Life and Medical Sciences, King's College London, London SE1 1UL, United Kingdom.
authors.forename: A N
authors.initials: AN
authors.lastname: Malik
authors.affiliation: Institute of Child Health, London WC1N 1EH, United Kingdom.
authors.forename: K
authors.initials: K
authors.lastname: Hussain
authors.affiliation: Diabetes Research Group, Division of Diabetes and Nutritional Sciences, Faculty of Life and Medical Sciences, King's College London, London SE1 1UL, United Kingdom.
authors.forename: P M
authors.initials: PM
authors.lastname: Jones
authors.affiliation: Diabetes Research Group, Division of Diabetes and Nutritional Sciences, Faculty of Life and Medical Sciences, King's College London, London SE1 1UL, United Kingdom. Electronic address: shanta.persaud@kcl.ac.uk.
authors.forename: S J
authors.initials: SJ
authors.lastname: Persaud
keywords: Apoptosis; Diabetes; Equilibrative nucleoside transporter 3; Islets of Langerhans; Mitochondria; β-Cells