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Deficient Pancreatic Islets Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH RNA-sequencing of WFS1-deficient pancreatic islets Marilin Ivask1, Alison Hugill2 & Sulev Koks~ 1 1 Department of Pathophysiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia 2 Mammalian Genetics Unit, Medical Research Council, Harwell, Oxfordshire, United Kingdom Keywords Abstract Insulin, RNA-sequencing, Trpm5, Wfs1. Wolfram syndrome, an autosomal recessive disorder characterized by juvenile- Correspondence onset diabetes mellitus and optic atrophy, is caused by mutations in the WFS1 Marilin Ivask, Department of gene. WFS1 encodes an endoplasmic reticulum resident transmembrane pro- Pathophysiology, Institute of Biomedicine and tein. The Wfs1-null mice exhibit progressive insulin deficiency and diabetes. Translational Medicine, University of Tartu, The aim of this study was to describe the insulin secretion and transcriptome 19 Ravila Street, 50411 Tartu, Estonia. of pancreatic islets in WFS1-deficient mice. WFS1-deficient (Wfs1KO) mice Tel: 372 737 4374 Fax: 372 7 374372 had considerably less pancreatic islets than heterozygous (Wfs1HZ) or wild- E-mail: [email protected] type (WT) mice. Wfs1KO pancreatic islets secreted less insulin after incuba- tion in 2 and 10 mmol/L glucose and with tolbutamide solution compared to Funding Information WT and Wfs1HZ islets, but not after stimulation with 20 mmol/L glucose. This study was supported by the European Differences in proinsulin amount were not statistically significant although Science Foundation grant SV/3452 within the there was a trend that Wfs1KO had an increased level of proinsulin. After framework of “Frontiers of Functional incubation in 2 mmol/L glucose solution the proinsulin/insulin ratio in Genomics”, by Estonian Science Foundation grant 7479, by institutional research funding Wfs1KO was significantly higher than that of WT and Wfs1HZ. RNA-seq IUT20-46 of the Estonian Ministry of from pancreatic islets found melastatin-related transient receptor potential Education and Research (IUT20-46) and by subfamily member 5 protein gene (Trpm5) to be downregulated in WFS1- The European Regional Development Fund deficient mice. Functional annotation of RNA sequencing results showed that together with the Archimedes Foundation. WFS1 deficiency influenced significantly the pathways related to tissue mor- phology, endocrine system development and function, molecular transport Received: 18 July 2015; Revised: 29 February network. 2016; Accepted: 3 March 2016 doi: 10.14814/phy2.12750 Physiol Rep, 4 (7), 2016, e12750 doi:10.14814/phy2.12750 b Introduction associated with impaired -cell function as in the case of type 2 diabetes (T2D) (Fonseca et al. 2005). Genetic stud- Wolfram syndrome (WS, OMIM 222300) is a rare auto- ies have also shown a link between the risk of developing somal recessive neurodegenerative disorder caused by T2D and WFS1 (Minton et al. 2002; Sandhu et al. 2007; mutations in the wolframin gene (WFS1). The main Florez et al. 2008; Franks et al. 2008; van Hoek et al. symptoms of the disease are juvenile-onset diabetes melli- 2008; Cheurfa et al. 2011). tus, progressive optic atrophy, diabetes insipidus and WFS1 is a 100 kDa glycoprotein composed of 890 deafness (Barrett and Bundey 1997; Inoue et al. 1998; amino acids and has nine transmembrane segments. Strom et al. 1998). Nonautoimmune degeneration of pan- Inside the cell WFS1 localizes in the ER (Takeda et al. creatic b-cells is common for WS patients (Karasik et al. 2001; Hofmann et al. 2003; Philbrook et al. 2005). Its 1989). In patients with WS high levels of endoplasmic location in the ER suggests that WFS1 could participate reticulum (ER) stress and pancreatic b-cell death may be in membrane trafficking, processing proteins and/or regu- ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of 2016 | Vol. 4 | Iss. 7 | e12750 the American Physiological Society and The Physiological Society. Page 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. RNA-Sequencing of WFS1-Deficient Pancreatic Islets M. Ivask et al. lation of the Ca2+ homeostasis in the ER (Takeda et al. Generation of Wfs1 mutant (Wfs1KO, Wfs1tm1Koks) mice 2001). WFS1 itself may be a Ca2+-channel in the ER or has been described elsewhere (Luuk et al. 2008; Koks et al. its regulator (Osman et al. 2003). WFS1 is expressed at 2009). Briefly, most of exon 8 was replaced with a LacZ cas- highest levels in brain, heart and pancreatic b-cells (Inoue sette resulting in the deletion of amino acids 360–890 in et al. 1998; Strom et al. 1998; Hofmann et al. 2003; Ishi- the WFS1 protein. The expressed truncated protein is a hara et al. 2004). fusion between WFS1 residues 1–359 (N-terminal part) Wfs1-mutant mice have been generated in several labo- and LacZ lacking at least seven of the nine transmembrane ratories (Ishihara et al. 2004; Riggs et al. 2005; Luuk et al. domains and the C-terminal portion of endogenous WFS1 2008; Koks et al. 2009). One model is a knock-out of the protein (Luuk et al. 2008; Koks et al. 2009). In the study 3 second exon in [(129Sv 9 B6) 9 B6] F2 background and genotypes of mice were used: wild-type (WT), heterozy- the resulting WFS1 protein lacks first 183 amino acids gotes for Wfs1 mutation (Wfs1HZ) and homozygotes for and NH2-terminus (Ishihara et al. 2004). Riggs et al. Wfs1 mutation (Wfs1KO). All studies were performed on (2005) generated a pancreatic islet specific conditional male F2 hybrids (129S6/SvEvTac 9 C57BL/6) and mice Wfs1 mouse model disrupting exon 8. The WFS1-defi- were 5–6-month-old at the time of the experiment. Mice cient mice (Wfs1tm1Koks) used in current study have also were housed in groups of 6–8at20Æ 2°C under 12-h/12- the exon 8 disrupted resulting in the deletion of amino h light/dark cycle with free access to food and water. acids 360–890 (Luuk et al. 2008; Koks et al. 2009). Mice with a loss of function of the Wfs1 gene exhibit Isolation of pancreatic islets b-cell loss, which may result from high levels of ER stress, cell cycle dysfunction, and apoptosis (Fonseca et al. 2005; Pancreatic islets were isolated as previously described Riggs et al. 2005; Takei et al. 2006; Yamada et al. 2006). (Shimomura et al. 2009) from six mice in each genotype. Insulin secretion from isolated islets of mice with a dis- Briefly, mice were executed by cervical dislocation and rupted Wfs1 gene has been shown to be impaired (Ishi- the islets isolated by collagenase Type XI digestion hara et al. 2004). However, there is no more detailed (Sigma-Aldrich, St. Louis, MO, USA, final concentration analysis of insulin secretion in Wfs1-mutant mice. 1 mg/mL). The inflated pancreas was dissected out and RNA-sequencing (RNA-seq) has become a great tool incubated in a 37°C water bath for 13 min. Only pancre- for transcriptomic studies without the need of previous ata inflated 100% were used. The tissue was washed twice knowledge of the targets, allowing identifying novel tran- with 0.2% BSA (Sigma) and HBSS (Sigma) solution. The scripts and gene expression changes due to some disease islets were handpicked under stereomicroscope in 0.2% (Mortazavi et al. 2008). There have been studies regarding BSA and HBSS solution. Finally, the islets were picked pancreatic islets’ transcriptome and differential gene into high glucose (4.5 g/L) DMEM/Ham’s F12 media expression caused by diabetes (Eizirik et al. 2012; Ku (PAA/GE Healthcare Life Sciences, Chicago, IL, USA), et al. 2012; Moran et al. 2012). However, the transcrip- containing 10% FBS (Gibco/Thermo Fisher Scientific Inc., tional changes in the islets induced by the deletion of Waltham, MA, USA), 100 U/mL penicillin and 100 lg/ WFS1 are not known. mL streptomycin (Gibco) and 20 mmol/L L-glutamine The aim of this study was to perform analysis of insu- (Invitrogen/Thermo Fisher Scientific Inc., Waltham, MA, lin secretion and proinsulin content in isolated pancreatic USA). Islets were incubated overnight at 37°C before islets of WFS1-deficient mice (Wfs1tm1Koks) (Luuk et al. insulin secretion assay. During the picking the number of 2008; Koks et al. 2009). The transcriptome of islets was islets was also counted manually. analyzed using RNA-seq to describe transcriptional changes in pancreatic islets associated with Wfs1 defi- Insulin secretion assay ciency and potentially related to the changes in insulin secretion. Islets were incubated for 1 h at 37°C and 5% CO2 in 0.2% BSA and Krebs-Ringer solution (KRBH, 140 mmol/L NaCl, Methods 0.5 mmol/L NaH2PO4, 2 mmol/L NaHCO3, 3.6 mmol/L KCl, 0.5 mmol/L MgSO4, 2.6 mmol/L CaCl2Á2H2O, 5 mmol/L HEPES, pH 7.4) containing 2 mmol/L glucose. Animals Then, islets were incubated for 1 h at 37°C and 5% CO2 in The animal experiments described in this study were per- selected assay solution (KRBH and 2 mmol/L, 10 mmol/L formed with permission from Estonian National Board of or 20 mmol/L glucose or 200 lmol/L tolbutamide and Animal Experiments (No. 71, April 8th, 2011) and in 2 mmol/L glucose). Tolbutamide (Sigma) was first dis- accordance with the European Communities Directive solved in 0.2% DMSO (Sigma) with final concentration of (86/609/EEC). 200 lmol/L. Each assay media group contained 5 islets and 2016 | Vol. 4 | Iss. 7 | e12750 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 2 the American Physiological Society and The Physiological Society. M. Ivask et al. RNA-Sequencing of WFS1-Deficient Pancreatic Islets was in duplicate per genotype.
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