Molecular and Cellular Endocrinology 364 (2012) 36–45 Contents lists available at SciVerse ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce Reduced insulin secretion correlates with decreased expression of exocytotic genes in pancreatic islets from patients with type 2 diabetes Sofia A. Andersson a, Anders H. Olsson b, Jonathan L.S. Esguerra a, Emilia Heimann e, Claes Ladenvall c, Anna Edlund a, Albert Salehi d, Jalal Taneera c, Eva Degerman e, Leif Groop c, Charlotte Ling b, ⇑ Lena Eliasson a, a Islet Cell Exocytosis, Lund University Diabetes Centre, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden b Epigenetics and Diabetes, Lund University Diabetes Centre, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden c Diabetes and Endocrinology, Lund University Diabetes Centre, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden d Islet Cell Physiology, Lund University Diabetes Centre, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden e Department of Experimental Medical Sciences, Biomedical Center, Lund University, Lund, Sweden article info abstract Article history: Reduced insulin release has been linked to defect exocytosis in b-cells. However, whether expression of Received 14 December 2011 genes suggested to be involved in the exocytotic process (exocytotic genes) is altered in pancreatic islets Received in revised form 7 August 2012 from patients with type 2 diabetes (T2D), and correlate to insulin secretion, needs to be further investi- Accepted 13 August 2012 gated. Available online 23 August 2012 Analysing expression levels of 23 exocytotic genes using microarray revealed reduced expression of five genes in human T2D islets (v2 = 13.25; p < 0.001). Gene expression of STX1A, SYT4, SYT7, SYT11, Keywords: SYT13, SNAP25 and STXBP1 correlated negatively to in vivo measurements of HbA1c levels and positively Type 2 diabetes to glucose stimulated insulin secretion (GSIS) in vitro in human islets. STX1A, SYT4 and SYT11 protein lev- Islet Beta-cell els correspondingly decreased in human T2D islets. Moreover, silencing of SYT4 and SYT13 reduced GSIS Exocytosis in INS1-832/13 cells. Our data support that reduced expression of exocytotic genes contributes to SYT impaired insulin secretion, and suggest decreased expression of these genes as part of T2D pathogenesis. GWAS Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction dependent exocytosis and the release of insulin (Eliasson et al., 2008). During the priming process the SNARE complex assemble Type 2 diabetes (T2D) is characterized by increased blood glu- to prepare the granules for Ca2+-dependent fusion. In short, the cose levels, due to both reduced insulin secretion from the pancre- plasma membrane associated proteins syntaxin 1A (Stx1A) and atic b-cell, and impaired insulin action at the target cells. Several synaptosomal-protein of 25 kD (Snap25) interact with vesicle- studies have shown that impaired insulin secretion occurs already associated membrane protein (Vamp2), which gives rise to a ter- before the onset of T2D (Del Prato and Tiengo, 2001; Gerich, 2002; tiary SNARE complex that promotes fusion by pulling the vesicle Hosker et al., 1989) signifying that processes involved in insulin membrane in close contact with the plasma membrane upon secretion are crucial in the development of the disease. Ca2+ influx (Gerber and Sudhof, 2002). Insulin release is biphasic with a first rapid phase followed by a The importance of the SNARE complex for insulin secretion in b- second slower phase. Patients with T2D characteristically display cells has been stated in several studies (reviews e.g. (Eliasson et al., reduced or complete absence of the first phase (Del Prato and Tien- 2008; Leung et al., 2007; Wang and Thurmond, 2009)). Stx1A pro- go, 2001; Gerich; 2002; Hosker et al., 1989), which is suggestive of teins are arranged in clusters along the plasma membrane in close impaired priming and/or fusion of the insulin-containing granules association with the insulin granules and the number of clusters with the plasma membrane (Barg et al., 2002). Priming and fusion are reduced in the GK-rat (Ohara-Imaizumi et al., 2004). In accor- are processes involved in the cascade of events that precedes Ca2+- dance, the diabetic GK-rat has reduced levels of stx1A (Gaisano et al., 2002). Inhibition of Stx1 in mice upon addition of an anti- body reduces Ca2+-dependent insulin secretion (Martin et al., Abbreviations: T2D, type 2 diabetes; GSIS, glucose-stimulated insulin secretion; 1995) and decreases the exocytotic response by 90%, an effect that SNP, polymorphism; DGI, diabetes Genetic Initiative; BIS, basal insulin secretion. is strongly associated with inhibition of the Ca2+current (Vikman ⇑ Corresponding author. Address: Islet Cell Exocytosis, Lund University Diabetes Centre, CRC 91-11, Jan Waldenströms gata 35, 205 02 Malmö, Sweden. Tel.: +46 40 et al., 2006). 2+ 391153; fax: +46 40 391222. The Ca -sensing element in regulated exocytosis is proposed to E-mail address: [email protected] (L. Eliasson). be aided by some isoforms in the family of synaptotagmins (Syts; 0303-7207/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2012.08.009 S.A. Andersson et al. / Molecular and Cellular Endocrinology 364 (2012) 36–45 37 (Pang and Sudhof, 2010)), namely Syt1–3, 5–7 and 9–10. These iso- tween the Human Tissue Laboratory at Lund University Diabetes 2+ forms display Ca -dependent phospholipid binding to the C2A-do- Centre and the Nordic Network for Clinical Islet Transplantation main, whereas the other members do not. The family of headed by Prof. Olle Korsgren at Uppsala University, Sweden. The synaptotagmins is comprised of at least16 members out of which donors had given their consent before death, or consent was given Syt1–6 and 9–13 has previously been shown to be expressed in by her/his relatives upon admission to Intensive Care Unit (ICU) to brain tissue (Pang and Sudhof, 2010; Sudhof, 2002) where Syt5, donate organs and the protocols were approved by the local ethics Syt9 and Syt13 have also been detected in primary b-cells (Grise committees. Islets were prepared by collagenase digestion and et al., 2007; Iezzi et al., 2004; Parton et al., 2006) and Syt4 tran- density gradient purification. After isolation, islets were cul- script has been shown to be expressed in clonal b-cell lines as well tured1–7 days, free floating in CMRL 1066 culture medium (ICN as in islet cells (Gao et al., 2000; Gut et al., 2001). Syt7, 8, 14 and 15 Biomedicals, Costa Mesa, CA, USA) containing human serum and are mainly expressed in heart, kidney, and pancreas (Gauthier and 5.56 mM glucose and supplemented with 10 mmol/l HEPES, Wollheim, 2008). The effect of Syt7 has previously been studied in 2 mmol/l 1-glutamine, 50 lg/ml gentamicin, 0.25 lg/ml Fungizone Syt7 knockout mice, which display impaired glucose tolerance and (GIBCO, BRL, Gaithersburg, MD, USA), 20 lg/ml ciprofloxacin lowered basal- and glucose-induced insulin levels (Gustavsson (Bayer Healthcare, Leverkusen, Germany), and 10 mmol/l nicotin- et al., 2008). amide at 37 °C (5% CO2) prior to RNA and DNA preparation. The Apart from Stx1A, Snap25, Vamp2 and the Syts, successful b-cell preparation of islets can be contaminated by exocrine tissue and exocytosis involve several proteins such as Rim1, Rim2, Stxbp (or is often therefore not 100% pure. However, the islet purity was sim- Munc-18) and Unc-13 (or Munc-13) (Gandini et al., 2011; Kwan ilar for non-diabetic and T2D donors (68 ± 18% vs 58 ± 19%, et al., 2006; Yasuda et al., 2010; Zhang et al., 2000). Most of the p = 0.15) using dithizone staining. We further used gene expression published work on exocytotic proteins and insulin secretion has of endocrine (somatostatin and glucagon) and exocrine (pancreatic investigated the functional role of these proteins in rodents and lipase, a2A amylase, and chymotrypsin 2) markers to determine is- less has been performed in human islets or in genetic studies. let purity, and again there was no difference in purity between One previous study performed on islets from 8 donors demon- non-diabetic and T2D donors (72% vs 68%, p = 0.29) (Olsson et al., strated that expression of STX1A, SNAP25, VAMP2 and STXBP is 2011). It was controlled that days in culture was not affecting down-regulated in donors with T2D compared to non-diabetic the expression of the investigated genes. Insulin secretion mea- controls (Ostenson et al., 2006). Transcripts of STX7, SYT5 and surements were performed on hand-picked islets, which obviously SYT1 in islets from a donor with T2D and expression of Syt13 in increased the purity. Wistar rats have been demonstrated to be up-regulated by palmi- tate (Ghanaat-Pour and Sjoholm, 2009, 2011). 2.2. Animal model T2D is a complex disease most likely comprised of both envi- ronmental and genetic factors where in the case of heritability, ef- The Sprague Dawley rat was purchased from Charles River Lab- fects seem to be more pronounced on insulin secretion in the oratories (Germany) and kept under standardized conditions in the pancreatic b-cells than on insulin action in the target tissue (Lehto- animal house facilities. The experimental procedure has been ap- virta et al., 2000). Interestingly, many of the identified polymor- proved by the Committee of ethical animal research in Malmö phisms associated with T2D are related to b-cell function (Koeck and Lund (permission number: M249-11). Rat brain was used as et al., 2011; Lyssenko et al., 2007; Olsson et al., 2011; Rosengren control tissue in the western blot analysis. et al., 2010), e.g. two studies link SNPs in STX1A to T2D (Tsunoda et al., 2001; Romeo et al., 2008). Genome-Wide Association Studies 2.3. Glucose-stimulated insulin secretion in human islets (GWAS) offer the possibility to explore single nucleotide polymor- phisms (SNPs) in the genome that may be of importance in T2D Islets were hand-picked under a stereomicroscope at room tem- (Saxena et al., 2007).