ORIGINAL ARTICLE Inhibition of Glucose-Stimulated Insulin Secretion by KCNJ15, a Newly Identified Susceptibility Gene for Type 2 Diabetes Koji Okamoto,1,2 Naoko Iwasaki,3,4,5 Kent Doi,2 Eisei Noiri,2 Yasuhiko Iwamoto,3 Yasuko Uchigata,3 Toshiro Fujita,2 and Katsushi Tokunaga1 Potassium inwardly rectifying channel, subfamily J, member 15 also called IRKK, KIR1.3, and KIR4.2 (8), as a suscepti- (KCNJ15) is a type 2 diabetes–associated risk gene, and Kcnj15 bility gene for type 2 diabetes (9) in subjects with a mean 2 overexpression suppresses insulin secretion in rat insulinoma BMI of ;23.0 kg/m (10). The synonymous single nucleo- (INS1) cells. The aim of the current study was to characterize tide polymorphism of KCNJ15 (C566T at rs3746876 in the role of Kcnj15 by knockdown of this gene in vitro and in vivo. exon 4) increases the risk of type 2 diabetes in Japanese, Human islet cells were used to determine the expression of and the risk allele (T) is associated with increased levels of KCNJ15. Expression of KCNJ15 mRNA in islets was higher in KCNJ15 mRNA in the peripheral blood of healthy volun- subjects with type 2 diabetes. In INS1 cells, Kcnj15 expression teers (9). Clinical observations reveal that subjects with was induced by high glucose–containing medium. Regulation of Kcnj15 by glucose and its effect on insulin secretion were ana- type 2 diabetes carrying the risk allele are more likely to lyzed in INS1 cells and in normal mice and diabetic mice by the require insulin, indicating that insulin secretion is low in inactivation of Kcnj15 using small interfering RNA. Knockdown this group. Moreover, Kcnj15 overexpression decreases of Kcnj15 increased the insulin secretion in vitro and in vivo. the insulin response to glucose in rat insulinoma (INS1) KCNJ15 and Ca2+-sensing receptor (CsR) interact in the kidney. cells (9). Binding of Kcnj15 with CsR was also detected in INS1 cells. In In the current study, we examined KCNJ15 expression conclusion, downregulation of Kcnj15 leads to increased insulin in human pancreatic islets from normal and type 2 diabetic secretion in vitro and in vivo. The mechanism to regulate insulin patients and investigated the effects of Kcnj15 on insulin se- secretion involves KCNJ15 and CsR. cretioninvitroandinvivo.Kcnj15 was upregulated by glucose and had a significant inhibitory effect on insulin secretion. We also confirmed that Kcnj15 interacts with Ca2+-sensing receptor (SCARA3, CsR) in INS1 cells, suggesting that ype 2 diabetes is a leading health problem in KCNJ15 is involved in regulating insulin secretion. the developed world, and the disease is becom- ing increasingly common in developing countries Tas well (1,2). Clinical studies indicate that type 2 RESEARCH DESIGN AND METHODS diabetes comprises heterogeneous phenotypes among Immunofluorescence staining of KCNJ15 protein in islets of Langerhans. various ethnic groups. The prevalence of type 2 diabetes in Immunofluorescence double staining for KCNJ15 and insulin was performed Japan has dramatically increased in the past 40 years, and using frozen sections of pancreas derived from humans (Human Tissues & the same trend was recently reported in other Asian Biofluids Bank, Asterand, Detroit, MI), C57BL/6 mice (10-week-old male), and countries, due primarily to genetic factors in combination Sprague-Dawley rats (10-week-old male). When possible, the human tissues with behavioral changes, such as high-calorie diets and obtained from Asterand are obtained with written informed consent and – institutional review board approval; see the following web site for details on sedentary lifestyles (3 6). Despite the rise in type 2 diabetes, Asterand’s policies regarding responsible research and informed consent (http:// Asian patients are still characterized as having lower body www.asterand.com/Asterand/about/ethics.htm). Human pancreatic tissue was mass indices and insulin levels compared with European, prepared from diabetic patients and nondiabetic control subjects during sur- Mexican American, or African American type 2 diabetic gery. Tissue sections (5-mm thick) were cut with a cryotome and mounted on patients (7). slides. The sections were then dried at room temperature and fixed with ace- fi tone for 10 min. The slides were washed three times with PBS and incubated We recently identi ed potassium inwardly rectifying overnight with anti-KCNJ15 goat antibody (Santa Cruz Biotechnology, Santa channel, subfamily J, member 15 (KCNJ15), a member of Cruz, CA) and then anti-insulin rabbit antibody (Santa Cruz Biotechnology) for the potassium inwardly rectifying channel (KIR) family, 1 h. After washing with PBS, the slides were incubated for 40 min with either Alexa-488– or Alexa-633–conjugated secondary antibody (Invitrogen, Carlsbad, CA). After washing with PBS, sections were visualized using a confocal laser From the 1Department of Human Genetics, Graduate School of Tokyo Univer- scanning microscope (LSM 510 Meta NLO Imaging System; Carl Zeiss, sity, Tokyo, Japan; the 2Department of Nephrology and Endocrinology, Grad- Oberkochen, Germany). uate School of Medicine, University of Tokyo, Tokyo, Japan; the 3Diabetes KCNJ15 mRNA expression in the islets of human subjects. Optimal Center, Tokyo Women’s Medical University, Tokyo, Japan; the 4Institute of cutting temperature compound (Sakura Finetechnical, Tokyo, Japan) was added Integrated Medical Science, Tokyo Women’s Medical University, Tokyo, to fresh-frozen human pancreatic tissues (Human Tissues & Biofluids Bank), and 5 ’ Japan; and the Institute of Medical Genetics, Tokyo Women s Medical Uni- the tissue was sectioned at a thickness of 10 mm. The sections were mounted on versity, Tokyo, Japan. membrane-coated slides, and the optimal cutting temperature compound was Corresponding authors: Katsushi Tokunaga, [email protected], and Naoko Iwasaki, [email protected]. removed by dipping the slides six times in ice-cold RNase-free water. The Received 5 September 2011 and accepted 27 February 2012. sections were then dehydrated in ice-cold 70% ethanol for 3 min. Laser micro- DOI: 10.2337/db11-1201 dissection and pressure catapulting were performed on the caps of microtubes Ó 2012 by the American Diabetes Association. Readers may use this article as filled with 20 mL lysis buffer (PALM MicroLaser System; Carl Zeiss). Total RNA long as the work is properly cited, the use is educational and not for profit, was extracted from microdissected cells from both nondiabetic control subjects and the work is not altered. See http://creativecommons.org/licenses/by and patients with type 2 diabetes. First-strand cDNA synthesis was performed -nc-nd/3.0/ for details. using a MessageBOOSTER cDNA synthesis kit (Epicentre Biotechnologies, See accompanying editorial, p. XXX. Madison, WI). Relative mRNA expression was compared as described below. diabetes.diabetesjournals.org DIABETES 1 Diabetes Publish Ahead of Print, published online May 7, 2012 Kcnj15 REGULATES INSULIN SECRETION TABLE 1 siRNA sequences Target gene Sequence Rat Kcnj15 siRNA Sense 59-GCAGGCGCUUAAUCUGGGCAUUGAA-39 Antisense 59-UUCAAUGCCCAGAUUAAGCGCCUGC-39 Rat CsR siRNA Sense 1 59-CAGGGAUGUACUGUGUGUCACUGAA-39 Antisense 1 59-UUCAGUGACACACAGUACAUCCCUG-39 Sense 2 59-GAGAUGGGCACUUGUUGGUUCUCCA-39 Antisense 2 59-UGGAGAACCAACAAGUGCCCAUCUC-39 Sense 3 59-CAGCUACAACUGGACAAUAUCUCUU-39 Antisense 3 59-AAGAGAUAUUGUCCAGUUGUAGCUG-39 Mouse Kcnj15 siRNA Sense 59-CCUGCUACAGCAGAGCAAUGUCUGA-39 Antisense 59-UCAGACAUUGCUCUGCUGUAGCAGG-39 Regulation of Kcnj15 expression by glucose in INS1 cells and HEK293 59-ATGTCAGAAGACAAGATCACCGGA-39, reverse: 59-CCCGAGCCACCCAC- cells. Changes in the expression of Kcnj15 mRNA in INS1 cells induced by CAAAGAAT-39;forratIns1&2,forward:59-TGCCCGGGCTTTTGTCAAAC-39,re- 5 and 25 mmol/L glucose were compared with changes in the expression of verse: 59-CTCCAGTGCCAAGGTCTGAA-39; and for human & rat & mouse B-actin, Kcnj11, Glut2,andinsulin genes. Changes in the expression of KCNJ15 mRNA in forward: 59-CGCACCACTGGCATTGTCAT-39,reverse:59-TTCTCCTTGATGT- HEK293 cells induced by 5, 25, 45, and 65 mmol/L glucose were also compared. CACGCAC-39. All were obtained from Invitrogen. Kcnj15 and actin protein levels Total RNA was isolated from cultured cells and tissues using TRIzol (Invitrogen). in cultured INS1 cell lysates were detected by Western blotting. INS1 cells were After DNase I treatment (Roche, Lewes, East Sussex, U.K.), cDNA was synthesized incubated for 48 h and harvested. using oligo (dT) primers and ImProm-II reverse transcriptase (Promega, Madison, Inactivation of Kcnj15 and CsR in INS1 cells by small interfering RNA. WI). Quantitative real-time PCR was performed using SYBR green (Applied Bio- To clarify the role of Kcnj15, we knocked down Kcnj15 in INS1 cells using systems, Foster City, CA). The relative expression of those genes was normalized small interfering (si)RNA. A control siRNA (Stealth RNAi Negative Control to b-actin. All samples were analyzed in duplicate. The primers are as follows: Med GC), predesigned siRNA against Kcnj15 (NM_001107285.1_stealth_1056), for human KCNJ15,forward:59-GGAATGTCCTCATGCCATCT-39, reverse: 59- and CsR (NM_001108870.1) were obtained from Invitrogen (Table 1). Cultured TTCTGCTTGGTGATGACTGC-39;forratKcnj15,forward:59-CCGTTCCATCACA- INS1 cells were transfected with either control siRNA, Kcnj15 siRNA, and/or GAGGAGT-39, reverse: 59-GCTTTTTGGGTCTTGCAATC-39; for mouse Kcnj15, CsR siRNA using Lipofectamine 2000 (Invitrogen). Cells were incubated for forward: 59-GTGCCAGCTCTCTGGAAAAC-39, reverse: 59-AGGGACTCTCGGAA- 72 h to