Nardilysin Is Required for Maintaining Pancreatic Β-Cell Function

Home , Metalloendopeptidase, Nardilysin, Secretion assay

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, STUDIES ISLET 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. of Nrd1 or Mafa in MIN6 and INS832/13, cells were infected with lentiviral vectors expressing Nrd1, Mafa,or RESEARCH DESIGN AND METHODS BLOCK-iT miR RNAi (Life Technologies) targeting Nrd1 or Mafa, respectively. Animals 2 2 Nrd1 / mice (accession no. CDB0466K; http://www Islet Isolation and Insulin Secretion Assay .clst.riken.jp/arg/mutant%20mice%20list.html) were gen- Pancreatic islets were isolated, and insulin release from erated as described previously (19) and backcrossed to the the islets was measured as previously described (27). The diabetes.diabetesjournals.org Nishi and Associates 3017 amount of immunoreactive insulin was determined by radio- showed elevated blood glucose concentrations 60 and immunoassay (Aloka Accuflex g 7000; Hitachi, Tokyo, Japan). 120 min after glucose challenge (Fig. 1A). Importantly, Insulin secretion from INS832/13 cells (25) was deter- the serum insulin concentration 30 min after glucose mined as previously described, with some modifications injection was strikingly low in the mutant mice (Fig. (28). In brief, cells cultured on six-well plates were washed 1B). At the same time, the insulin tolerance test showed 2 2 with Krebs-Ringer bicarbonate HEPES buffer (KRBH; increased insulin sensitivity in Nrd1 / mice (Fig. 1C). 140 mmol/L NaCl, 3.6 mmol/L KCl, 0.5 mmol/L MgSO4, On the basis of these observations, the glucose metab- 2/2 0.5mmol/LNaH2PO4,1.5mmol/LCaCl2, 2 mmol/L olism of the Nrd1 mice is characterized by mild NaHCO3, and 10 mmol/L HEPES; pH 7.4) with 0.1% glucose intolerance and significantly reduced GSIS, ac- BSA and 2.8 mmol/L glucose, preincubated at 37°C for companied by insulin hypersensitivity. 2 2 2 h in KRBH with 2.8 mmol/L glucose, and then incubated Histologically, islets of Nrd1 / mice showed no obvious at 37°C for 2 h in KRBH with 2.8 and 15 mmol/L glucose. anomalies in their size and structure; there was a predom- Insulin was measured by ELISA (MS303; Morinaga Insti- inance of insulin-positive b-cells in the core, which were tute of Biological Science). surrounded by glucagon-positive a-cells (Fig. 1D). Quantita- fi fi Chromatin Immunoprecipitation (ChIP) and Re-ChIP tive analysis con rmed no signi cant difference in b-cell – – +/+ 2/2 Assay mass and a-cell to b-cell ratio between Nrd1 and Nrd1 2/2 Chromatin was prepared from MIN6 cells (29) as previ- mice (Fig. 1E and F). Thus, impaired GSIS in Nrd1 mice is ously described (21). ChIP assays were performed using not the result of a gross developmental defect in the pan- ChIP-IT Express Chromatin Immunoprecipitation Kits creas, but disturbed islet function was suspected. (Active Motif). Antibodies against NRDc (mouse mono- To evaluate islet function directly, we isolated islets +/+ 2/2 clonal antibody 2E6), PDX-1 (sc-146643;SantaCruz from Nrd1 and Nrd1 mice and performed in vitro 2/2 Biotechnology, Inc.), Islet-1 (ab20670; Abcam), Neuro GSIS assays. Consistent with the in vivo results, Nrd1 D1 (sc-10843; Santa Cruz Biotechnology, Inc.), or con- islets showed severely reduced GSIS (Fig. 1G) compared +/+ trol IgG (Santa Cruz Biotechnology, Inc.) were used. For with Nrd1 islets. Because KCl-induced insulin secretion 2/2 the re-ChIP assay, Re-ChIP-IT Express Magnetic Chroma- was preserved in Nrd1 islets (Fig. 1G), NRDc may tin Re-Immunoprecipitation Kits (Active Motif) were regulate GSIS through the processes before membrane fi used according to the manufacturer’s protocol. The pri- depolarization. Insulin content was modestly but signi - 2/2 mers used are listed in Supplementary Table 1. cantly reduced in Nrd1 islets (Fig. 1H). Since NRDc has endopeptidase activity, we examined pancreatic pro- Luciferase Reporter Assays insulin-to-insulin ratios but found no significant differ- 2 2 For the luciferase reporter assays, PicaGene Promoter ence between Nrd1+/+ and Nrd1 / islets (Fig. 1I). Vector 2 (PGV-P2; Toyo Ink, Tokyo, Japan) with or without the insertion of MafA R3 (28,118 to 27,750 relative to b-Cell-Specific NRDc-Deficient Mice Show a Diabetic the transcriptional start site of Mafa) was transiently trans- Phenotype With Markedly Reduced GSIS fected into MIN6 cells. Luciferase activity was quantified The analysis of isolated islets strongly suggested that 48 h after transfection using the Dual-Luciferase Reporter NRDc is primarily involved in GSIS. To confirm the b-cell- fl fl Assay Kit (Promega) according to the manufacturer’spro- specific role of NRDc, we generated Nrd1 ox/ ox mice by tocol. pRL-TK (Promega) was cotransfected with PGV-P2 to inserting two loxP sites around exon 1 of the mouse control for transfection efficiency. Nrd1 gene (Supplementary Fig. 1A and B)andcrossed Statistics them with RIP-Cre transgenic mice (24) to produce fi fi delb delb Results are expressed as mean 6 SEM. The Student t test b-cell-speci cNRDc-de cient (Nrd1 )mice.Nrd1 was used for comparisons between two groups. ANOVA mice were born at the expected Mendelian frequency and was used for comparisons among multiple groups, with a showed no overt phenotypes, including body weight (Sup- fi fi Tukey-Kramer post hoc test. plementary Fig. 1C). We con rmed the speci cdecreaseof the Nrd1 mRNA level in pancreatic islets, but not in other organs, including the hypothalamus, heart, white adipose RESULTS tissue, liver, and skeletal muscle (Supplementary Fig. 1D). Nrd12/2 Mice and Islets Exhibit Impaired GSIS Because glucose intolerance and decreased GSIS of RIP- 2 2 We previously reported that Nrd1 / mice show a lean Cre mice has been reported (30), we set two controls, fl fl phenotype, which has been attributed to increased energy Nrd1 ox/ ox mice and RIP-Cre (Nrd1wt/wt;RIP-Cre) mice, expenditure as a result of enhanced thermogenesis in for comparison with Nrd1delb mice. While the global knock- brown adipose tissue and hyperactivity (21). To address out mice showed normal fasting blood glucose (Fig. 1A), whether NRDc is involved in glucose metabolism, we per- Nrd1delb mice demonstrated markedly elevated fasting formed the glucose tolerance test and the insulin toler- blood glucose compared with both control mice (Fig. 2A). 2 2 ance test in Nrd1 / mice. Although the fasting blood In glucose tolerance tests, RIP-Cre mice showed glucose glucose and serum insulin concentrations remained the intolerance and reduced insulin secretion, as previously 2 2 same as in the wild-type (Nrd1+/+) mice, Nrd1 / mice reported (30), whereas Nrd1delb mice displayed more severe 3018 Nardilysin in Pancreatic b-Cell Function Diabetes Volume 65, October 2016

Figure 1—Nrd12/2 mice and islets exhibit impaired GSIS. A and B: Glucose tolerance test (i.p. injection of 2 mg/kg glucose) reveals mild glucose intolerance (A) and impaired insulin secretion (B) in 3-month-old Nrd12/2 mice (n =7–10 per genotype). C: Insulin tolerance test (i.p. injection of 1 unit/kg insulin) reveals increased insulin sensitivity in 3-month-old Nrd12/2 mice (n = 4 per genotype). D:Pancreatafrom 3-month-old Nrd1+/+ and Nrd12/2 mice were stained with anti-insulin (red) and antiglucagon (green) antibodies. E: b-Cell (insulin-positive cell) mass per body weight is not changed in Nrd12/2 mice (n = 4 per genotype). F: Proportion of a-cell (glucagon-positive cell) areas among a- and b-cell (insulin-positive cell) areas is not changed in Nrd12/2 mice (n = 4 per genotype). G: Static incubation of islets from 3- to 4-month-old Nrd1+/+ and Nrd12/2 mice with 2.8 mmol/L glucose, 25 mmol/L glucose, and 30 mmol/L KCl. Secreted insulin was normalized to total insulin in the islets (n = 20 per group). H: Insulin contents normalized to protein concentration in isolated islets are reduced in Nrd12/2 mice (n =20per genotype). I: Proinsulin-to-insulin ratio is not changed in Nrd12/2 mice (n =5–8 per genotype). *P < 0.05; ** P < 0.01. NS, not signiﬁcant.

glucose intolerance and more reduced insulin secretion com- though without a statistically significant difference, than fl fl pared with RIP-Cre and Nrd1 ox/ ox mice (Fig. 2B and C). RIP-Cre control mice (Fig. 2H). The ratio of large islets fl fl Insulin sensitivity was similar between Nrd1 ox/ ox,RIP-Cre, was significantly decreased, whereas that of small islets and Nrd1delb mice (Supplementary Fig. 1E and F). These was increased in Nrd1delb mice compared with either fl fl results indicated that the b-cell-specificNRDcdepletionis Nrd1 ox/ ox miceorRIP-Cremice(Fig.2I). sufficient to cause glucose intolerance and impaired GSIS. We also performed a lineage tracing study of insulin- 2 2 fl fl In sharp contrast to the normal appearance of Nrd1 / expressing cells in Nrd1delb mice. By crossing Nrd1 ox/ ox islets, islets in Nrd1delb mice showed an impaired struc- mice with RIP-Cre and R26ECFP mice (31), we generated fl fl ture (Fig. 2D). Glucagon-positive a-cells increased in Nrd1 ox/ ox;RIP-Cre;R26ECFP mice, in which the simulta- number, and their locations were not restricted to neous deletion of NRDc and expression of enhanced cyan the periphery of the islet but intermingled with b-cells fluorescent protein (ECFP) occurred specifically in b-cells, at the core. When quantified, the a-cell–to–b-cell ratio and compared them with control Nrd1wt/wt;RIP-Cre; was significantly increased in Nrd1delb islets (Fig. 2E). In R26ECFP mice (Supplementary Fig. 2A). As shown in agreement with these results, fasting plasma glucagon and Supplementary Fig. 2B and C, several ECFP-positive fl fl the mRNA level of islet Gcg in Nrd1delb mice were signif- cells lost insulin staining in Nrd1 ox/ ox;RIP-Cre;R26ECFP icantly higher than those of control mice (Fig. 2F and G). mice. Furthermore, a small subset of glucagon-positive cells fl fl b-Cell mass in Nrd1delb mice was significantly smaller was clearly labeled with ECFP in Nrd1 ox/ ox;RIP-Cre; fl fl than that in Nrd1 ox/ ox mice and tended to be smaller, R26ECFP islets (Supplementary Fig. 2D and E). When diabetes.diabetesjournals.org Nishi and Associates 3019

upregulated in Nrd1delb islets (Supplementary Fig. 3A–C). These results suggested that some b-cells may lose their identity and a subset of these b-cells may be converted to glucagon-positive a-like cells in Nrd1delb islets. MafA Is Specifically Downregulated in Islets Isolated From Nrd1delb Mice Consistent with the results of the in vivo glucose tolerance test, islets isolated from Nrd1delb mice showed markedly reduced GSIS. Compared with islets isolated fl fl from Nrd1 ox/ ox and RIP-Cre control mice, both low- (2.8 mmol/L) and high-glucose–stimulated (25 mmol/L) insulin secretion (Fig. 3A) was significantly reduced in Nrd1delb islets. Furthermore, the insulin content of the freshly isolated islets (Fig. 3B) was significantly lower in Nrd1delb mice. To obtain mechanistic insights that explain the reduced insulin content and impaired GSIS of the Nrd1delb islets, we examined the mRNA levels of genes involved in GSIS and found that Glut2, Pcx, Ins1, Ins2, and Pcsk1 are significantly decreased in Nrd1delb islets compared with those in RIP-Cre islets (Fig. 3C). Reduction of Glut2, Pcx, Ins1, and Ins2 levels in Nrd1delb islets was fl fl also confirmed when compared with Nrd1 ox/ ox islets. However, Pcsk1 was increased in Nrd1delb islets compared fl fl with that in Nrd1 ox/ ox islets, since it was markedly increased in RIP-Cre islets (Supplementary Fig. 4A). We next investigated the expression of transcription factors in b-cells that potentially regulate Glut2, Pcx, Ins1 and 2, and Pcsk1. Intriguingly, Mafa was specifically downregulated in islets from Nrd1delb mice, whereas the expression of Pdx1 and others, including NKx6.1, NeuroD1,and Islet-1,wasnotsignificantly changed (Fig. 3D and Sup- Figure 2—b-Cell-specific NRDc-deficient (Nrd1delb) mice show a plementary Fig. 4B). Histological findings confirmed the diabetic phenotype with markedly reduced GSIS. A: Fasting hyper- Nrd1delb n – B reduced protein expression of GLUT2, MafA, and insulin glycemia in 6-month-old mice ( =9 14 per genotype). delb and C: Glucose tolerance test (i.p. injection of 2 mg/kg glucose) in Nrd1 islets (Fig. 3E and F). These results suggested reveals glucose intolerance (B) and impaired insulin secretion (C) that NRDc in b-cells acts upstream of MafA. in 6-month-old Nrd1delb mice (n =4–8 per genotype). *P < 0.05 for Nrd1delb vs. Rip-Cre; #P < 0.05 for Nrd1delb vs. floxed; ##P < 0.01 NRDc Regulates Insulin Production Through MafA for Nrd1delb vs. floxed; †P < 0.05 for Rip-Cre vs. floxed. D: Our loss-of-function experiments using Nrd1delb mice/ Pancreata from 6-month-old Nrd1delb and control mice were stained with anti-insulin (red) and antiglucagon (green) antibodies. E:Propor- islets revealed that NRDc is essential for proper islet function of glucagon-positive areas among glucagon- and insulin-positive tion, especially for GSIS, via maintaining the expression of areas is increased in Nrd1delb mice (n =4pergenotype).F: Fasting Mafa and its downstream genes such as Ins1, Ins2, Glut2, delb hyperglucagonemia in 6-month-old Nrd1 mice (n = 5 or 6 per ge- and Pcsk1. At the same time, islet structure was impaired notype). G: Relative mRNA levels of glucagon in isolated islets from delb 6-month-old Nrd1delb and control mice, as measured by quantitative in Nrd1 mice. Considering a previous report describing RT-PCR (n = 4 or 5 per genotype). H: Quantification of b-cell (insulin- that an abrogated structure itself disturbs islet function positive cell) mass in 6-month-old Nrd1delb and control mice (n = (32), we were prompted to perform loss-of-function or delb 4 per genotype). I: Distribution of islet size in 6-month-old Nrd1 gain-of-function experiments in a b-cell line to confirm and control mice (n = 4 per genotype). *P < 0.05; **P < 0.01; ***P < 0.001, unless otherwise noted. the cell-autonomous role of NRDc. INS832/13 cells are an INS-1-derived rat b-cell line that is strongly responsive to glucose in terms of insulin secretion (25). Similar to Nrd1delb islets, Nrd1-knocked down INS832/13 (Fig. 4A) quantified, glucagon and ECFP double-positive cells were showed decreased insulin secretion (Fig. 4B), insulin con- observed in 3.28% (n = 853) of the total glucagon-positive tent (Fig. 4C), and mRNA levels of genes involved in in- fl fl cells in Nrd1 ox/ ox;RIP-Cre;R26ECFP islets but in 0.85% sulin secretion (Fig. 4D). On the other hand, INS832/13 (n = 233) in control islets. In addition, genes transiently cells, in which NRDc was overexpressed by two- to three- expressed in endocrine progenitors (e.g., Sox9, Ngn3,and fold (Fig. 4E), showed augmented insulin secretion in Mafb)andAldh1a3, recently reported as a marker of their response to glucose (Fig. 4F) and increased insulin b-cell dedifferentiation, were significantly or tended to be content at a steady state (Fig. 4G). While the expression 3020 Nardilysin in Pancreatic b-Cell Function Diabetes Volume 65, October 2016

Figure 3—Islets from Nrd1delb mice exhibit reduced GSIS with decreased MafA expression. A: Static incubation of islets from 6- to 8-month-old Nrd1delb and control mice with 2.8 and 25 mmol/L glucose. Secreted insulin was normalized to total insulin in the islets (n = 10 per group). B: Insulin contents normalized to protein concentration in isolated islets are reduced in Nrd1delb mice (n = 10 per group). C and D: Relative mRNA levels of genes involved in insulin secretion (C) and transcriptional factors (D) in islets isolated from 6-month-old Nrd1delb and RIP-cre mice, as measured by quantitative RT-PCR (n = 4 or 5 per genotype). E: Pancreata from 6-month-old Nrd1delb and control mice were stained with anti-GLUT2 (red) and anti-insulin (green) antibodies. F: Pancreata from 6-month-old Nrd1delb and control mice were stained with anti-MafA (red) and anti-insulin (green) antibodies. *P < 0.05; **P < 0.01; ***P < 0.001. NS, not signiﬁcant.

levels of genes involved in glycolysis and ATP production NRDc regulates insulin expression through MafA, we per- were not changed, the mRNA levels of Ins1 and Ins2 were formed gene knockdown of Mafa in NRDc-overexpressed signiﬁcantly upregulated by the overexpression of NRDc INS832/13 cells. Mafa knockdown did not affect NRDc in INS832/13 cells (Fig. 4H). Notably, MafA expression expression, whereas it cancelled the augmentation of was upregulated at the mRNA level (Fig. 4H) and the Mafa expression by NRDc overexpression (Fig. 5A). As protein level in the nucleus (Fig. 4E). To ascertain whether aresult,Mafa knockdown almost fully cancelled the diabetes.diabetesjournals.org Nishi and Associates 3021

Figure 4—NRDc cell-autonomously regulates insulin secretion and MafA expression. A: Western blot analysis of cytosolic and nuclear fractions of INS832/13 cells infected with microRNAs (miRs) targeted to Nrd1 or a control (Cont) miR. B: Static incubation of INS832/13 cells described in A with 2.8 and 15 mmol/L glucose. Secreted insulin was normalized to protein concentration (n =9pergroup).C: Insulin contents normalized to protein concentration in INS832/13 cells described in A (n =4pergroup).D: Relative mRNA levels of genes in INS832/13 cells described in A,asmeasuredbyquantitativeRT-PCR(n =4pergroup).E: Western blot analysis of cytosolic and nuclear fractions of control or NRDc-overexpressing INS832/13 cells using the indicated antibodies. F: Static incubation of control or NRDc-overexpressing INS 832/13 cells with 2.8 and 15 mmol/L glucose. Secreted insulin was normalized to protein concentration (n =9pergroup).G: Insulin contents normalized to protein concentration in control or NRDc-overexpressing INS832/13 cells (n =6pergroup).H: Relative mRNA levels of genes in control or NRDc- overexpressing INS832/13 cells, as measured by quantitative RT-PCR (n = 5 per group). KD, knockdown. *P < 0.05; **P < 0.01. NS, not signiﬁcant.

NRDc-mediated increase of Ins2 and partially cancelled the start site, which contain six highly conserved sequence do- NRDc-mediated increase of Ins1 gene expression (Fig. 5B). mains, called regions 1–6 (33,34). Among them, region 3 Thus, NRDc regulates insulin production, at least in part, (R3; 28,118/27,750 bp) has been reported to be essen- by controlling MafA expression. tial for the expression of MafA in b-cells both in vitro and NRDc Regulates the Recruitment of Islet-1 to the in vivo (33,34). Several b-cell-enriched transcriptional MafA Enhancer Region factors, such as Pdx1, NeuroD1, NKx2.2, Islet-1, HNF1a, Previous reports showed that MafA expression is regu- HNF1b, FoxA2, and NKx6.1, have their consensus binding lated by roughly 10 kg base pairs (bp) 59 of the transcription motifs in R3 (35). Our ChIP assay using antimouse NRDc 3022 Nardilysin in Pancreatic b-Cell Function Diabetes Volume 65, October 2016

Figure 5—NRDc regulates insulin production through MafA. A: Relative mRNA levels of Nrd1 and Mafa in control (Cont) or NRDc- overexpressing INS832/13 cells, in which Mafa is knocked down by microRNA (miR) targeted to Mafa (or a control miR) (n = 4 per group). B: Relative mRNA levels of Ins1 and Ins2 in INS832/13 cells described in A. Gene knockdown of Mafa reduces NRDc-mediated augmentation of Ins1 gene expression in INS832/13 cells. Relative ratios of Ins1 and Ins2 mRNA levels in NRDc-overexpressing cells to that in control cells are shown in the right panels (n = 4 per group). KD, knockdown. *P < 0.05; **P < 0.01; ***P < 0.001.

monoclonal antibody showed that NRDc is recruited to R3 recruitment of Islet-1 to R3, we performed ChIP analysis of the MafA promoter in MIN6 cells, a mouse b-cell line using MIN6 cells in which NRDc was knocked down (Fig. (Fig. 6A). Furthermore, luciferase reporter assay showed 6E and F). Consistent with results in Nrd1delb islets, gene that the gene knockdown of NRDc reduced the enhancer knockdown of NRDc resulted in the decrease of MafA at activity of R3 in MIN6 cells (Fig. 6B). Because NRDc has the protein and mRNA levels (Fig. 6E and F). Intriguingly, no canonical DNA-binding motifs, we hypothesized that gene knockdown of NRDc signiﬁcantly reduced the re- NRDc forms a complex with other transcription factors cruitment of Islet-1, but not Pdx-1 and NeuroD1, to R3 and exists on R3. To this end, we performed coprecipitation (Fig. 6G). Of note, gene knockdown of NRDc did not assays using cells overexpressing NRDc and Pdx1, NeuroD1, change the nuclear expression level of Islet-1 (Fig. 6E). NKx2.2,Islet-1,HNF1a,HNF1b,FoxA2,orNKx6.1,and Given the critical roles of Islet-1 in MafA regulation and found that Islet-1 forms a complex with NRDc (Fig. 6C b-cell function, our results suggest that NRDc regulates and data not shown). b-cell function, at least in part, through controlling the Because ChIP analysis indicated that both NRDc (Fig. recruitment of Islet-1 to R3 of the MafA promoter. 6A) and Islet-1 (Supplementary Fig. 5D) exist on R3 of the In addition to its transcriptional coregulator function MafA promoter, we performed sequential ChIP/re-ChIP in the nucleus, NRDc enhances ectodomain shedding of analysis to determine the in vivo colocalization of NRDc membrane proteins (e.g., HB-EGF, tumor necrosis factor-a) and Islet-1. As shown in Fig. 6D, ChIP/re-ChIP analysis through the activation of ADAMs (A disintegrin and with anti-NRDc antibody and anti-Islet-1 antibody dem- metalloproteinase) (16–20). By immunoblotting and onstrated that NRDc and Islet-1 are colocalized in R3 of the immunostaining in pancreatic b-cell lines (Figs. 4A and E MafA promoter. To determine whether NRDc affects the and 6E, and Supplementary Fig. 5A) and immunostaining diabetes.diabetesjournals.org Nishi and Associates 3023

Figure 6—NRDc regulates the recruitment of Islet-1 to the enhancer region of MafA. A: ChIP with anti-NRDc antibody followed by quantitative RT-PCR demonstrates that NRDc binds to the enhancer region of MafA in MIN6 cells. Results were normalized with input DNA (n =6). MafA R3: 28,118 to 27,750, relative to the transcriptional start site (TSS) of Mafa;nonconserved:21,678 to 21,560, relative to the TSS of Mafa. B: Luciferase activity was quantified in MIN6 cells 48 h after the transfection of PGV-P2 with the insertion of MafA R3. The luciferase activity was normalized by the activity of PGV-P2 without MafA R3 (n =4).C: NRDc forms a complex with Islet-1. NRDc was cotransfected with FLAG-tagged Pdx1 (P), NeuroD1 (N), Islet-1 (I), or a control vector (2)intoNRDc2/2 MEFs. Arrowheads indicate nonspecific bands. IP, immunoprecipitation. D: ChIP/re-ChIP analysis of MIN6 cells with anti-NRDc antibody and anti-Islet-1 antibody followed by quantitative RT-PCR (qRT-PCR) targeting MafA R3 (n =4).E: Western blot analysis of cytosolic and nuclear fractions of MIN6 cells infected with microRNAs (miRs) targeted to Nrd1 or a control (Cont) miR. F: Relative mRNA levels of genes in the MIN6 cells described in D,asmeasured by qRT-PCR (n =5).G: ChIP with antibodies to PDX-1, NeuroD, and Islet-1 followed by qRT-PCR demonstrates that Nrd1 knockdown in MIN6 cells decreases Islet-1 recruitment to MafA R3. The results were normalized to input DNA and are shown as the ratio to that in the control miR-treated cells (IP: aPDX-1, aNeuroD, or aIslet1) (n = 6). KD, knockdown. *P < 0.05; ***P < 0.001. NS, not significant.

in the islets of wild-type mice and humans (Supplementary ectodomain shedding (26). As shown in Supplementary Fig. 5B and C), we demonstrate the intra- and extranuclear Fig. 5E, GM6001 had no effect on the augmentation of localization of NRDc in pancreatic b-cells. Broad-spectrum Mafa expression by NRDc overexpression, suggesting that ADAM and matrix metalloproteinase inhibitor GM6001 NRDc in the extracellular space has little, if any, effect on blocks the activity of extracellular NRDc from enhancing the transcription of MafA. 3024 Nardilysin in Pancreatic b-Cell Function Diabetes Volume 65, October 2016

Acidic Domain of NRDc Is Responsible for Islet-1 widespread in the whole body, the analysis of islets isolated 2 2 Interaction and Insulin Production from Nrd1 / mice and b-cell-specific Nrd1-deficient mice One unique characteristic of NRDc among the M16 family clearly indicates that NRDc in b-cells is critical for insulin of metalloendopeptidases is a highly acidic domain next production and secretion. Accumulating evidence supports to the catalytic domain (Fig. 7A), which is required for its the notion that several b-cell-enriched transcription fac- binding to several proteins, including HB-EGF, but is dis- tors, such as Pdx-1, Nkx6.1, and MafA, play essential roles pensable for its metalloendopeptidase activity (36,37). We in the maintenance of b-cell function. Among them, MafA transfected the mutant form of NRDc lacking this acidic was solely downregulated in islets from Nrd1delb mice. Fur- 2/2 domain (dAcD) into Nrd1 MEF cells and tested whether thermore, the Mafa mRNA level was increased by the over- the mutant interacts with Islet-1. Coimmunoprecipitation expression of NRDc, whereas it was decreased by the gene assays revealed that the acidic domain is responsible for knockdown of NRDc in b-cell lines, indicating that NRDc is the interaction between NRDc and Islet-1. On the other a cell-autonomous regulator of MafA transcription. Impor- hand, the enzymatic activity of NRDc was dispensable for tantly, NRDc overexpression in INS832/13 cells was ac- its binding to Islet-1 (Fig. 7A and B). companied by increased mRNA levels and the secretion Islet-1 is a member of the LIM-homeodomain protein of insulin, which was partially inhibited by the simulta- family, which is characterized by two LIM domains arrayed neousgeneknockdownofMafa. These results demon- in tandem at the N terminus, a DNA-binding homeodo- strated that NRDc regulates insulin production and GSIS main, and a LIM homeobox protein 3-binding domain through the control of MafA expression. (LBD) at the C terminus (Fig. 7C). To examine which do- MafA is first produced at embryonic day 13.5 exclusively main of Islet-1 is required for the interaction with NRDc, in insulin-positive cells, which is unusually late compared we performed coprecipitation assays of NRDc and two de- with other islet-enriched transcription factors (38). More- letion mutant forms of Islet-1 (Fig. 7C). The mutant Islet-1 over, Mafa mRNA levels show an age-dependent increase delLIM lacking the N-terminal LIM domain (Islet-1 ) showed from neonates to adults, which is similar to the expression markedly reduced coprecipitation with NRDc, whereas the pattern of insulin and other putative target genes of MafA mutant without the COOH-terminal LBD domain (Islet- (39). This unique spatial and temporal expression pattern delC 1 ) showed results similar to the wild type of Islet-1 of MafA is regulated by its well-established 59 flanking en- (Fig. 7D). Collectively, these results indicated that the hancer region, called R3 (28,118/27,750 bp) (34,40). We acidic domain of NRDc and the LIM domain of Islet-1 show here that NRDc binds to the MafA enhancer R3. We are responsible for their interaction. also demonstrated that the enhancer activity of MafA R3 is To assess the functional significance of NRDc and Islet-1 reduced by the gene knockdown of NRDc. In addition, we interaction in pancreatic b-cells, we overexpressed the performed unbiased screening of b-cell-enriched transcrip- wild-type NRDc or the mutants of NRDc in INS832/13 tional factors for binding to NRDc and found that Islet-1 cells and examined NRDc-mediated augmentation of Mafa associates with NRDc. Notably, ChIP/re-ChIP assay clearly gene expression. Consistent with the results of coprecipi- demonstrated the association of NRDc and Islet-1 in the tation assays, mRNA levels of Mafa were augmented by MafA enhancer R3. Furthermore, gene knockdown of NRDc the wild type of NRDc, but not by dAcD (Fig. 7E). Fur- was accompanied by a decrease of Islet-1 recruitment to R3. thermore, induction of the Ins1 mRNA level by NRDc Given that MafA is a direct target of Islet-1, MafA reg- expression was significantly reduced by the coexpression ulation by NRDc is, at least in part, attributed to NRDc- of dAcD. On the other hand, Islet1-induced upregulation mediated Islet-1 recruitment to the MafA enhancer. of Mafa and Ins1 mRNA levels were decreased by the We show that the acidic domain of NRDc and the LIM delLIM expression of Islet-1 (Fig. 7F). These results suggest domain of Islet-1 are required not only for complex that the interaction of NRDc and Islet-1 is required for formation but also for the full enhancement of Mafa and the proper transcriptional regulation of Mafa and Ins1. Ins1 mRNA. Islet-1 is a member of the LIM-homeodomain Although the enzymatically inactive E.Amutantbound protein family, characterized by two tandemly repeated to Islet-1 as the wild type of NRDc did (Fig. 7B), transcrip- LIM domains fused to a conserved homeodomain. The tional induction of Mafa was not recapitulated by the E.A LIM domain consists of two tandemly repeated zinc fin- mutant (Fig. 7E). These results indicate that not only phys- gers, structurally similar to GATA-type zinc fingers (41). ical association between NRDc and Islet-1 but also enzymatic However, unlike GATA-type zinc fingers, the LIM domain activity of NRDc seem to be involved in Mafa, Ins1,andIns2 in mammalian LIM proteins does not seem to bind to regulation. DNA. Instead, the domain seems to be important for di- verse protein–protein interactions (42). Two transcription factors, LIM domain-binding protein 1 (43) and NeuroD DISCUSSION (44), were previously reported to bind Islet-1 via the LIM In this report, we present the first in vivo evidence that domain and activate the transcriptional activity of Islet-1. NRDc is essential for proper b-cell function, especially for Meanwhile, the deletion of the LIM domains from Islet-1 2 2 GSIS. Nrd1 / mice develop glucose intolerance and se- was reported to enhance insulin promoter–driven luciferase verely impaired GSIS. Although the expression of NRDc is activity in HEK-293 cells (44). As opposed to that result, diabetes.diabetesjournals.org Nishi and Associates 3025

Figure 7—The acidic domain of NRDc is responsible for its association with Islet-1 and insulin production. A: Schematics of the mutants of NRDc. AcD, acidic domain; dAcD, the mutant form of NRDc in which the acidic domain is deleted; E>A, enzymatically inactive mutant. B: The highly acidic domain of NRDc is required for complex formation between NRDc and Islet-1. Wild-type NRDc or indicated mutants described in A were cotransfected with Islet-1 into NRDc2/2 MEFs. C: Schematics of Islet-1 and deletion mutants. HD, homeo domain; LIM1, ﬁrst LIM domain; LIM2, second LIM domain. D: The LIM domain of Islet-1 is required for the complex formation between NRDc and Islet-1. FLAG-tagged wild-type Islet-1 or deletion mutants described in C were cotransfected with NRDc into NRDc2/2 MEFs. Arrowheads indicate nonspeciﬁcbands.E: Relative mRNA levels of Nrd1, Mafa, Ins1,andIns2 in INS832/13 cells infected with wild-type or mutated NRDc (n = 8 per group). Expression levels of the mock infected cells are normalized to 1. F: Relative mRNA levels of Nrd1, Mafa, Ins1,andIns2 in INS832/13 cells infected with wild-type or mutated Islet-1 (n = 5 per group). Expression levels of the mock infected cells are normalized to 1. Cont, control; IB, immunoblotting; IP, immunoprecipitation. *P < 0.05; **P < 0.01; ***P < 0.001.

the LIM domain–deleted mutant of Islet-1, overexpressed our results in b-cells are more likely to recapitulate the in INS832/13 cells, failed to elevate the mRNA levels of physiological regulation of insulin promoter activity. Some Mafa and Ins1. Although these findings are inconsistent, factors in b-cells, such as NeuroD and NRDc, may activate 3026 Nardilysin in Pancreatic b-Cell Function Diabetes Volume 65, October 2016 the transcription of insulin through interacting with the a-cell–to–b-cell ratio, and an increased ratio of small is- LIM domains. lets. Future studies using Glut2-rescued Nrd1delb mice 2 2 Although both Nrd1 / and Nrd1delb mice show glu- might clarify the precise role of Glut2 in the regulation cose intolerance and severely impaired GSIS, there are of b-cell function and proliferation by NRDc. some differences between these two mouse lines. First, NRDc in the nucleus has been identified as a histone- 2 2 fasting blood glucose was normal in Nrd1 / mice, whereas binding protein, specifically recognizing dimethyl-H3K4 it was elevated in Nrd1delb mice. Second, b-cell mass was (22). The roles of NRDc in transcriptional regulation have 2 2 unaltered in Nrd1 / mice but reduced in Nrd1delb mice. been illustrated by the interaction of NRDc with various 2 2 Third, the a-cell–to–b-cell ratio was unchanged in Nrd1 / nuclear proteins such as HDAC3 in the NCoR/SMRT com- mice but significantly increased in Nrd1delb mice. Nrd1delb plex and PGC-1a (21,22). We demonstrate here that Islet-1 mice also showed the dysregulated distribution patterning of is a novel binding partner of NRDc in b-cells. Considering glucagon-positive a-cells, a phenotype that is similar to that the context-dependent multiple binding partners and wide 2 2 of Mafa-deficient mice (9). Consistently, Nrd1delb mice range of phenotypes of Nrd1 / mice (19,21,53), NRDc showed hyperglucagonemia. Considering that insulin sensi- may function as a transcriptional coregulator in general. 2 2 tivity was enhanced in Nrd1 / mice, hyperglucagonemia Since NRDc has its own peptidase activity, which seems and unaltered insulin sensitivity in Nrd1delb mice may ac- to be involved in MafA regulation, further study using count for their severe glucose intolerance. Insulin hypersen- peptidase-dead mutant knockin mice will be required to 2 2 sitivity in Nrd1 / mice may arise from an increase in energy clarify the link between the biological role and the protein expenditure caused by enhanced thermogenesis in brown function of NRDc. adipose tissue and increased physical activity (21). Our on- going studies using organ-specific (e.g., liver, adipose tissue, and skeletal muscle) NRDc-deficient mice will further clarify Acknowledgments. The authors are grateful to H. Iwai, K. Shimada, and the role of NRDc in the regulation of insulin sensitivity. N. Nishimoto (Kyoto University) for technical assistance and to C.B. Newgard Recent reports have indicated that several transcrip- (Duke University), J. Miyazaki (Osaka University), and T. Masui and A. Sato (Kyoto tion factors, such as Pdx1 (6), Nkx6.1 (7), and FoxO1 University) for materials. (45), are critical for b-cells to maintain their differenti- Funding. This study was supported by KAKENHI Grants-in-Aid (26293068, ated states. Loss of FoxO1 in mature b-cells leads to the 26670139, 26116715, 15K19376, and 15K19513) and a research grant of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct) dedifferentiation of b-cells, characterized by upregulation from the Ministry of Education, Culture, Sports, Science and Technology of of Ngn3, an endocrine progenitor cell marker. A subset of Japan. It was also supported by a Health Labour Sciences Research Grant from dedifferentiated cells was shown to adopt an a-cell fate the Ministry of Health, Labour and Welfare of Japan (Comprehensive Research (45). Importantly, possible dedifferentiation and the on Lifestyle-Related Diseases including Cardiovascular Diseases and Diabetes conversion of b-cells to a-cells have also been reported Mellitus), the Takeda Science Foundation, the Mitsui Sumitomo Insurance Welfare in Mafa-deficient mice (46). Our results of lineage trac- Foundation, the Kao Research Council for the Study of Healthcare Science, the ing experiments demonstrated that a small portion of Uehara Memorial Foundation, and Otsuka Pharmaceutical Co., Ltd. Sponsored glucagon-positive cells in Nrd1delb islets originated from Research Program. b-cells. Moreover, endocrine progenitor cell markers such Duality of Interest. No potential conflicts of interest relevant to this article as Sox9, Ngn3, Mafb,andAldh1a3 (47) were upregulated in were reported. Author Contributions. K.N. and E.N. designed experiments and wrote Nrd1delb islets. Together, these results suggest that dedif- the manuscript. K.N., M.O., Y.H., S.S., J.S., P.-M.C., Y.Mo., and S.M. performed ferentiation of b-cells and redifferentiation to the glucagon- experiments and analyzed data. Y.S., K.I., K.S., N.H., and N.I. isolated islets and expressing a-like cells might occur in a subset of b-cells in performed the insulin secretion assay. Y.Mu. and H.K. generated Nrd1flox/flox an islet environment where NRDc-positive a-cells and mice. K.F., Y.K., and S.U. carried out the histological procedures and provided NRDc-negative b-cells are intermingled. As the b-to-a con- human samples. Y.K, N.I., T.Kit., and T.Kim. supervised the work. 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