Diabetes Volume 65, October 2016 3015 Kiyoto Nishi,1 Yuichi Sato,2 Mikiko Ohno,1 Yoshinori Hiraoka,1,3 Sayaka Saijo,1 Jiro Sakamoto,1 Po-Min Chen,1 Yusuke Morita,1 Shintaro Matsuda,1 Kanako Iwasaki,2 Kazu Sugizaki,2 Norio Harada,2 Yoshiko Mukumoto,4 Hiroshi Kiyonari,4,5 Kenichiro Furuyama,6 Yoshiya Kawaguchi,6,7 Shinji Uemoto,6 Toru Kita,8 Nobuya Inagaki,2 Takeshi Kimura,1 and Eiichiro Nishi1 Nardilysin Is Required for Maintaining Pancreatic b-Cell Function Diabetes 2016;65:3015–3027 | DOI: 10.2337/db16-0178 Type 2 diabetes (T2D) is associated with pancreatic factors play important roles in the pathogenesis of the b-cell dysfunction, manifested by reduced glucose- disease, b-cell dysfunction, mainly manifested by deteriorated stimulated insulin secretion (GSIS). Several transcription glucose-stimulated insulin secretion (GSIS), seems to be pre- factors enriched in b-cells, such as MafA, control b-cell dominant for the transition from simple obesity to T2D (2). function by organizing genes involved in GSIS. Here we The observation that impaired GSIS in islets from patients demonstrate that nardilysin (N-arginine dibasic conver- with T2D cannot be accounted for by reduced insulin content tase; Nrd1 and NRDc) critically regulates b-cell function also supports this concept (1). Several biological processes, ISLET STUDIES Nrd12/2 through MafA. mice showed glucose intoler- including endoplasmic reticulum stress, inflammation, and ance and severely decreased GSIS. Islets isolated from oxidative stress, are suggested to impair GSIS in T2D (3,4). Nrd12/2 mice exhibited reduced insulin content and The pancreas comprises an exocrine compartment, impaired GSIS in vitro. Moreover, b-cell-specificNRDc- b consisting of acinar and ductal cells, and an endocrine com- deficient (Nrd1del ) mice showed a diabetic phenotype partment, consisting of a, b, d, e, and pancreatic polypep- with markedly reduced GSIS. MafA was specifically downregulated in islets from Nrd1delb mice, whereas tide cells. These different types of endocrine cells express overexpression of NRDc upregulated MafA and insulin and secrete the hormones glucagon, insulin, somatostatin, expression in INS832/13 cells. Chromatin immunopre- ghrelin, and pancreatic polypeptide, respectively. Sequen- cipitation assay revealed that NRDc is associated with tial expression of pancreatic transcription factors deter- Islet-1 in the enhancer region of MafA, where NRDc mines cell fate and pancreatic development. Among them, controls the recruitment of Islet-1 and MafA transcrip- several transcriptional factors enriched in b-cells, such as tion. Our findings demonstrate that NRDc controls b-cell Pdx-1, Nkx6.1, and MafA, play essential roles in the main- function via regulation of the Islet-1–MafA pathway. tenance of b-cell identity and function. b-Cell-specific ablation of Pdx-1 results in hyperglycemia with fewer Type 2 diabetes (T2D) is a common metabolic disorder insulin-positive cells and more glucagon-positive cells (5,6), that afflicts more than 300 million people globally (1). It whereas conditional Nkx6.1 inactivation in adult b-cells is characterized by impaired insulin secretion from pan- causes reduced insulin production (7). In contrast to the creatic b-cells and insulin resistance of the target tissues, earlier expression of Pdx-1 and Nkx6.1 in endocrine pro- such as liver, adipose tissue, and muscle. Although both genitor cells, MafA is first produced at embryonic day 13.5 1Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto 7Department of Clinical Application, Center for iPS Cell Research and Application, University, Sakyo-ku, Kyoto, Japan Kyoto University, Sakyo-ku, Kyoto, Japan 2Department of Diabetes, Endocrinology and Nutrition, Graduate School of Med- 8Kobe City Medical Center General Hospital, Chuo-ku, Kobe, Japan icine, Kyoto University, Sakyo-ku, Kyoto, Japan Corresponding author: Eiichiro Nishi, [email protected]. 3Division of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Kobe Gakuin Received 5 February 2016 and accepted 15 June 2016. University, Chuo-ku, Kobe, Japan 4Genetic Engineering Team, RIKEN Center for Life Science Technologies, Chuo-ku, This article contains Supplementary Data online at http://diabetes Kobe, Japan .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0178/-/DC1. 5Animal Resource Development Unit, RIKEN Center for Life Science Technologies, © 2016 by the American Diabetes Association. Readers may use this article as Chuo-ku, Kobe, Japan long as the work is properly cited, the use is educational and not for profit, and the 6Department of Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku, work is not altered. More information is available at http://www.diabetesjournals Kyoto, Japan .org/content/license. 3016 Nardilysin in Pancreatic b-Cell Function Diabetes Volume 65, October 2016 fl fl and is observed only in insulin-positive mature b-cells. ICR background (.98%). Nrd1 ox/ ox mice (accession no. MafA binds to an insulin promoter, like Pdx-1, and regu- CDB1019K; http://www.clst.riken.jp/arg/mutant%20mice% lates b-cell-selective insulin transcription (8). Mafa-deficient 20list.html) were generated by gene targeting in TT2 mice consistently develop diabetes because of impaired (23) embryonic stem (ES) cells (http://www.clst.riken.jp/ GSIS without a defect of islet cell development (9). More- arg/Methods.html). The targeting vector was designed to over, b-cell-specific ablation of pan-endocrine transcription insert loxP sites upstream and downstream of exon 1 of factors such as NeuroD1 and Islet-1 demonstrated their Nrd1 (Supplementary Fig. 1A). Successful homologous critical roles in b-cell maturation and function (10,11). In recombination in TT2 ES cells was confirmed by PCR particular, postnatal ablation of Islet-1 in b-cells resulted in (Supplementary Fig. 1A) and Southern blotting. Result- impaired GSIS without significantly reducing b-cell mass ingmutantmiceweregenotypedwithtailDNAbyPCR (11). Collectively, these findings demonstrated that tran- (Supplementary Fig. 1B) and crossed with CAG-FLPe trans- scription factors involved in the determination of endocrine genic mice to remove the neomycin selection cassette sur- or b-cell identity also play essential roles in b-cell functions. rounded by Flp recombinase target sites, then backcrossed A regulatory network of b-cell-enriched transcriptional to the C57BL/6J background (.98%). b-Cell-specificNRDc- fl fl factors (e.g., Pdx-1, NeuroD1, NKx6.1, and MafA) critically deficient mice were generated by crossing Nrd1 ox/ ox mice controls GSIS (5–10). These transcriptional factors also con- with rat insulin II promoter-Cre transgenic (RIP-Cre) nect oxidative stress with impaired GSIS in diabetic mouse mice (24). Male mice were used unless otherwise indi- models and human T2D (4). Among them, several lines of cated.Allanimalexperimentswereperformedaccording evidence have suggested that the Islet-1–MafA pathway to procedures approved by the Institute of Laboratory critically controls GSIS in adults. First, ablation of these Animals, Kyoto University. Mice were maintained on a genes in b-cells results in impaired GSIS without a severe diet of standard rodent chow in environmentally con- defect in b-cell development, as described above (9,11). trolled rooms. Second, MafA is a direct target gene of Islet-1 (11,12). Third, MafA directly regulates several genes critical for Glucose and Insulin Tolerance Tests GSIS, such as Ins1, Ins2,andGlut2 (also known as Slc2a2). For glucose tolerance tests, mice were fasted for 16 h and In this study, we demonstrate a novel regulatory intraperitoneally injected with 2 g/kg body weight of mechanism of GSIS by nardilysin (N-arginine dibasic D-glucose. For insulin tolerance tests, mice were fasted for convertase; Nrd1 and NRDc) through the Islet-1–MafA 5 h and intraperitoneally injected with insulin (Humulin pathway. NRDc is a zinc peptidase of the peptidase M16 R; Eli Lilly). To determine fasting plasma glucagon con- domain (M16) family that selectively cleaves dibasic sites centrations, mice were fasted for 16 h. To determine pan- (13,14). We rediscovered NRDc as a specificreceptorfor creatic insulin content and the proinsulin-to-insulin ratio, heparin-binding epidermal growth factor–like growth fac- whole pancreata were homogenized in 4 mL of a 2% tor (HB-EGF) (15), and our subsequent studies showed HCl/75% ethanol solution. After neutralization, samples multiple functions of NRDc, which depend on its cellular were diluted 1:1,000 in PBS before measuring insulin and localization. In the extracellular space, NRDc enhances proinsulin. Glucose was measured using a glucometer. Insulin ectodomain shedding of HB-EGF and other membrane pro- (MS303; Morinaga Institute of Biological Science, Yokohama, teins, such as tumor necrosis factor-a, amyloid precursor pro- Japan) and proinsulin (AKMPI-111; Shibayagi, Shibukawa- tein, and neuregulin-1 (16–20). We also revealed that NRDc City,Gunma,Japan)weremeasuredbyELISA.Glucagon in the nucleus works as a transcriptional coregulator was measured by enzyme immunosorbant assay (YK090; through the modulation of NCoR/SMRT corepressor or Yanaihara Institute, Fujinomiya-City, Shizuoka, Japan). PGC-1a coactivator function (21,22). We show here that Cell Culture fi 2/2 NRDc-de cient (Nrd1 ) mice and pancreatic b-cell- MIN6 and INS832/13 cells were gifts from Dr. Miyazaki fi fi delb 2 2 speci c NRDc-de cient (Nrd1 ) mice show glucose in- and Dr. Newgard, respectively. Nrd1 / mouse embryonic tolerance and severely impaired GSIS. Islets isolated from fi 2 2 broblasts (MEFs) were prepared as described previously / delb 2 2 Nrd1 and Nrd1 mice also show lower insulin pro- (19). MIN6 cells and Nrd1 / MEFs were grown in 2 duction and GSIS than wild-type islets. Notably, NRDc and DMEM (4.5 g L 1 glucose) supplemented with 10% FBS Islet-1 colocalize in the MafA enhancer, where NRDc reg- and antibiotics. INS832/13 cells were cultured as previ- ulates the recruitment of Islet-1 and MafA transcription, ously described (25). GM6001 (26) was purchased from indicating the essential role of NRDc in GSIS through Islet- Calbiochem (CA). For overexpression or gene knockdown – 1 MafA regulation.