3218 Diabetes Volume 64, September 2015

Yixing Yuchi,1 Ying Cai,1 Bart Legein,1 Sofie De Groef,1 Gunter Leuckx,1 Violette Coppens,1 Eva Van Overmeire,2,3 Willem Staels,1,4 Nico De Leu,1,5 Geert Martens,1 Jo A. Van Ginderachter,2,3 Harry Heimberg,1 and Mark Van de Casteele1

Estrogen a Regulates b-Cell Formation During Development and Following Injury

Diabetes 2015;64:3218–3228 | DOI: 10.2337/db14-1798

Identifying pathways for b-cell generation is essential among which Neurogenin3 (Ngn3) is key for endocrine for cell therapy in diabetes. We investigated the potential specification. Ngn3+ cells delaminate from pancreatic ep- b of 17 -estradiol (E2) and (ER) signaling ithelium, are mitotically quiescent, and give rise to endo- for stimulating b-cell generation during embryonic de- crine cells. Ngn3 cells appear maximally competent for velopment and in the severely injured adult pancreas. E2 driving b-cell formation at embryonic day (E) 14.5. concentration, ER activity, and number of ERa tran- Formed b-cells expand through self-replication, already scripts were enhanced in the pancreas injured by partial evident at E18.5, and continue into early postnatal life (1). duct ligation (PDL) along with nuclear localization of Also in adult mice with severely injured pancreas by partial a b b ER in -cells. PDL-induced proliferation of -cells duct ligation (PDL), Ngn3+ cells are generated near duct depended on aromatase activity. The activation of Neuro- and can differentiate into b-cells (2). b-Cells genin3 (Ngn3) expression and b-cell growth in PDL are vastly generated through replication in PDL (3,4), but pancreas were impaired when ERa was turned off chem- a2/2 some derive from acinar (5) and duct (6) cells, apparently ically or genetically (ER ), whereas in situ delivery of + ISLET STUDIES b through an Ngn3 stage (2,5) as in embryonic pancreas. E2 promoted -cell formation. In the embryonic pancreas, + b-cell replication, number of Ngn3+ progenitor cells, and How the numbers of Ngn3 endocrine progenitors and b expression of key factors of the endocrine replicating -cells are controlled in the embryonic or mature lineage were decreased by ERa inactivation. The current pancreas is uncertain. Identifying factors that control these processes and manipulating them may be of thera- study reveals that E2 and ERa signaling can drive b-cell replication and formation in mouse pancreas. peutic advantage. What is known is that 17b-estradiol (E2) enhances b-cell survival and glycemic control in var- ious animal models (7,8) by signaling through estrogen Decreased functional b-cell mass is the major cause for receptor (ER) a (8,9) and/or ERb (10). hyperglycemia in diabetes. Restoration of the endogenous However, little is known about the importance of b-cell mass as a therapeutic strategy, however, requires estrogen and ER signaling for b-cell proliferation and dif- a better understanding of signaling pathways that control ferentiation. So far, no in vivo effects on b-cell formation b-cell growth and differentiation. Embryonic b-cells are have been reported for the ER antagonist tamoxifen generated by a developmental program executed through (TAM), although this compound is used to conditionally the timed action of a number of key transcription factors activate Cre recombinase activity (CreERT) in genetic

1Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium This article contains Supplementary Data online at http://diabetes 2Myeloid Cell Immunology Laboratory, Vlaams Instituut voor Biotechnologie, Brus- .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1798/-/DC1. sels, Belgium Y.Y. and Y.C. contributed equally to this work. 3Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, H.H. and M.V.d.C. contributed equally to this work. Brussels, Belgium 4Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University B.L. is currently affiliated with Experimental Vascular Pathology, Department of Hospital, and Department of Pediatrics and Medical Genetics, Ghent University, Pathology, Cardiovascular Research Institute Maastricht, Maastricht University, Ghent, Belgium Maastricht, the Netherlands. 5Department of Endocrinology, Universitair Ziekenhuis Brussel, Brussels, Belgium © 2015 by the American Diabetes Association. Readers may use this article as Corresponding author: Harry Heimberg, [email protected]. long as the work is properly cited, the use is educational and not for profit, and the work is not altered. Received 3 December 2014 and accepted 16 May 2015. diabetes.diabetesjournals.org Yuchi and Associates 3219 tracing studies on embryonic and adult endocrine progenitor blocked in 10% donkey serum (Jackson ImmunoResearch and b-cell . We investigated whether ER signaling Inc.,Suffolk,U.K.).Primary antibodies for is important for b-cell replication and neogenesis during (1:3,000, guinea pig, generated at the Diabetes Research embryogenesis and in injured adult pancreas. Center, Vrije Universiteit Brussel, Brussels, Belgium), ERa (1:200, rabbit, E1644; Spring Bioscience, Fremont, CA), RESEARCH DESIGN AND METHODS green fluorescent (1:100, goat; Abcam, Cambridge, Animals and Tissues U.K.), 5-iodo-2’-deoxyuridine (IdU) (1:100, mouse; BD All mouse experiments were performed according to the Biosciences, Erembodegem, Belgium), Ki67 (1:1,000, rabbit; guidelines of our institutional ethics committee for animal Leica Microsystems, Diegem, Belgium), Ngn3 (1:2,000, rab- experiments and national guidelines and regulations. Balb-c bit; Millipore, Temecula, CA), and E-cadherin (1:50, mouse; mice were obtained from Janvier Labs (Le Genest-Saint-Isle, BD Biosciences) were incubated overnight at 4°C at the France). The mouse strains Ngn3CreERT;R26RYFP and indicated dilutions. Secondary antibodies for detection of RIPCreERT;R26RYFP and Ngn3 knock-add-on enhanced guinea pig, rabbit, goat, or rat antibodies were labeled by yellow fluorescent protein (YFP) (Ngn3YFP)werepreviously fluorescence (Cy3, Cy2, or Cy5). Nuclei were stained by 4 2 2 described (3,11). ERa / mice are infertile and were mg/mL Hoechst 33342 (Sigma). 2 obtained by crossing ERa+/ mice as previously described RNA and DNA Analysis (12). Total RNA was isolated from tissue (74104, RNeasy; All PDL pancreatic surgeries in the current study were performed in 8-week-old male mice as previously de- QIAGEN, Venlo, the Netherlands). Only RNA with RNA integrity number R $7 (2100 Bioanalyzer; Agilent) was scribed (2). Embryos and embryonic pancreas and duode- further analyzed. cDNA synthesis and real-time quantitative num were dissected as previously described (13). Mouse PCR were done as previously described (14) with TaqMan embryos were obtained after timed mating. Pregnant mice Universal PCR Master Mix on an ABI Prism 7700 Sequence were killed by cervical dislocation, uteri dissected in ice- Detector, and data were analyzed using Sequence Detection cold sterile Dulbecco’s PBS, and embryos removed from System version 1.9.1 software (all Applied Biosystems, Life the deciduas. For immunohistochemistry studies, em- fi fi Technologies, Ghent, Belgium). Mouse-speci cprimers bryos were xed in 10% neutral-buffered formalin over- 9 night at 4°C and embedded in paraffin. For RNA analysis, and probes were for Ngn3 (5 -GTCGGGAGAACTAGGA TGGC-39 [forward primer], 59-GGAGCAGTCCCTA GGTA embryos were kept on ice-cold Dulbecco’s PBS, and the TG-39 [reverse primer], and 59-CGGAGCCTCG GACC gut was dissected to remove the pancreas buds. Pancreas ACGAA-39 [probe]), Tert (Mm.PT.53a.13753537), Lcn2 and were collected in RNAlater (R0901; (Mm.PT.53a.10167155), ptma (Mm.PT.53a.30222141), IL-6 Sigma-Aldrich, Diegem, Belgium). (Mm.PT.49a.10005566), (Mm.PT.53a.33146682), C-Jun Chemicals (Mm.PT.49a.8204422.g), Pax4 (Mm01159043_g1), Pax6 TAM (T5648; Sigma) was dissolved in corn oil (C8267; (Mm.PT.51.14285402), Pdx1 (Mm.PT.51.11487144), Esr2 Sigma) at 20 mg/mL, and 4-hydroxytamoxifen (4OHT) (Mm.PT.49a.17681375.g) (all from Integrated DNA Technol- (579002; Calbiochem, Darmstadt, Germany) dissolved in ogies [IDT], Leuven, Belgium), and Esr1 (Mm00433149_m1, pure ethanol to 20 mg/mL was diluted to 5 mg/mL in corn TaqMan Assay; Life Technologies). Quan- oil (C8267; Sigma). E2 (E2785; Sigma) dissolved in pure titative PCR reactions were performed with cDNA corre- ethanolto0.4mg/mLwasdilutedto0.4ng/mLor2ng/mL sponding to 20 ng RNA as previously described (3). Data in 0.9% NaCl for intrapancreas injection, and aromasin (ARO) were normalized to cyclophilin A (CycloA) (Mm02342429, (Pfizer, Brussels, Belgium) was dissolved to 12.5 mg/mL in IDT). Conventional PCR primers for ERa (Exon3) were P1: 0.3% hydroxypropyl cellulose (191884; Sigma) in PBS. 59-AGAATGGCCGAGAGAGACTG-39 and P2: 59-TTCTCTT For PDL studies in adult mice, TAM, 4OHT, ARO, and AAAGAAAGCCTTGCAG-39 (IDT). Primers (IDT) for geno- 2 2 the corresponding vehicles (corn oil, corn oil, and 0.3% typing ERa+/+ (wild-type) and ERa / mice and embryos hydroxypropyl cellulose/PBS, respectively) were given sub- were previously described (12). cutaneously. E2 (20 or 100 pg in 50 mL) and vehicle (50 mL saline) were injected in the ligated tail portion of PDL pan- Western Blot Analysis creas. Before PDL and sham surgery or intrapancreatic in- Protein extracts were prepared as previously described jection, mice were sedatedbyketamine3.5mg/kgbody (15). Fifty micrograms of protein were separated by SDS- weight i.p. (Ceva Santé Animale, Brussels, Belgium) plus PAGE and transferred onto a nitrocellulose membrane. a xylazine 0.5 mg/kg body weight i.p. (Bayer, Diegem, Belgium). ER was detected with a rabbit polyclonal antibody (1:200, MC20; Santa Cruz Biotechnology, Heidelberg, Ger- Immunohistochemistry many). b-Actin (sc1616; Santa Cruz) was detected for fi Samples for immunohistochemistry were xed in 10% verification of protein loading. neutral buffered formalin overnight at 4°C and embedded in paraffin. Sections (4–5 mm) were deparaffinized and b-Cell Volume, Insulin Content, and E2 Concentration washed in 0.2% Tween 20 (P5927; Sigma) in PBS, antigen The measurement of b-cell volume (in cubic millimeters) retrieval was performed in a steamer, and slides were in PDL or sham tail pancreas was performed as described 3220 Estrogen Regulates b-Cell Formation Diabetes Volume 64, September 2015 previously (2,3). The measurement for each pancreas tail nucleotide analogs, b-cell proliferation in PDL tail was was based on 4-mm sections equally spaced (116 mm assessed by immunofluorescence for proliferation marker apart) and spanning the whole tail tissue, corresponding Ki67. The percentage of Ki67+ b-cells in PDL tail was to the analysis of 3% of the total volume. Insulin content significantly lower in TAM- versus vehicle-treated mice of pancreatic tissue was measured as previously described (Fig. 1D), corroborating that b-cell proliferation was (16). E2 concentration in whole pancreas tail tissue was inhibited by TAM. determined after tissue homogenization using a Retsch We then examined the effect of 4OHT, a selective ER Mixer Mill MM 400 (R20.745.0001; Retsch GmbH, modulator and metabolite of TAM that binds to endoge- Haan, Germany) using 5-mm grinding metal beads nous ERs and inactivates them (17). At only 1 mg per in- (R22.455.0003; Retsch). Lysates were cleared by centrifugation jection (5 mg total), 4OHT and TAM were equally efficient m . b after which 100- L samples were ether extracted. E2 was in inducing YFP expression ( 90%) in -cells of adult then assayed using a radioimmunoassay (RIA) kit (DIA- RipCreERT;R26RYFP mice (Supplementary Fig. 2), and b-cell source ImmunoAssays, Louvain-la-Neuve, Belgium). proliferation in PDL tail was decreased to the same extent Image Analysis by both compounds (Fig. 1E). Additionally, a single subcu- taneous injection of either 1 or 2 mg TAM at day 5 after PDL Images were obtained by fluorescence microscopy (Olym- (Fig. 1F)resultedinsignificantly lower b-cell proliferation in pus [Aartselaar, Belgium] BX61 with Hamamatsu C10600 ORCA-R2 camera). Ki67+ or IdU+ b-cells (insulin-positive) PDL pancreas at day 7 (Fig. 1G). Because TAM can act as an + + agonist or antagonist of estrogen signaling, depending on and Ngn3 or YFP E-cadherin–positive cells were quanti- the cell type, its effect on b-cell proliferation was also fied in a nonautomated manner (inspecting and counting + + examined in the pancreas of pregnant mice. A single in- individual cells). For the quantification of IdU or Ki67 traperitoneal injection of 1.5 mg TAM at gestation day b-cells, 10 nonconsecutive sections of each PDL pancreas (G) 13.5 caused a marked decrease of b-cell prolifera- (head or tail) were studied, and at least 1,000 b-cells per + tion at G15.5, whereas no significant effect of TAM on sample were counted. For the quantification of Ngn3 cells, the basal b-cell proliferation in nonpregnant females (Fig. 10 nonconsecutive sections of each embryonic pancreas b were studied, and at least 4,000 E-cadherin–positive cells 1H) was seen. Collectively, the data suggest that adult -cell proliferation in PDL pancreas or during pregnancy is sup- per sample were counted. Images were analyzed using pressed by antagonism of estrogen signaling through ERs. SmartCapture3 version 3.0.8 (Digital ScientificUK,Cambridge, b U.K.), ImageJ (National Institutes of Health, Bethesda, TAM Inhibits Ngn3 Gene Expression and -Cell Expansion in PDL Pancreas MD), and Photoshop CS version 1.3.1 (Adobe, San Jose, Ngn3+ cells have been identified in the ductal lining of ligated CA) software. pancreatic tail and are capable of differentiating in vivo or ex Statistics vivo to functional b-cells (2). Activation of Ngn3 gene expres- Data are expressed as mean 6 SEM of at least three in- sion and the presence of Ngn3+ cells are required for in vivo dependent experiments. Groups were compared using un- b-cell expansion in PDL pancreas (2,3). Because Ngn3 gene paired two-tailed Student t test, one-way ANOVA, or two-way expression is regulated by estrogen in developing neurons  ANOVA with Bonferroni or Sidák post hoc tests. Differences (18), we assessed whether Ngn3 transcriptional level in PDL were considered statistically significant if P , 0.05. pancreas was influenced by modulation of ER signaling. As expected, the amount of Ngn3 transcripts increased RESULTS in PDL tail compared with PDL head in mice that received TAM Inhibits b-Cell Proliferation in PDL Pancreas vehicle, but it was significantly reduced following admin- We investigated whether TAM influenced the outcome istration of TAM (Fig. 2A). In addition, the constitutive of PDL-induced b-cell proliferation. First, transgenic expression of Ngn3 associated with enteroendocrine pro- Ngn3CreERT;R26RYFP male mice of mixed genetic back- genitor cells (19) was also decreased in the duodenua of ground as well as inbred Balb-c male mice underwent TAM-treated mice (Fig. 2B). These data suggest that TAM PDL surgery followed by subcutaneous injection of either decreases Ngn3 gene expression and/or the number of TAM (4 mg per injection, 20 mg in total over a 14-day Ngn3-expressing cells in PDL pancreas and intestine. period) (Fig. 1A), vehicle, or nothing and application of We next evaluated the impact of TAM administration IdU through drinking water. Although more b-cells were on increased b-cell volume in PDL pancreas. The total IdU+ in the tail portion of PDL pancreas (from here on b-cell volume was significantly increased in PDL versus termed “PDL tail”) than in the nonligated head portion sham tails of pancreas from mice that did not receive (from here on termed “PDL head”) (Fig. 1B), b-cell pro- TAM, and this was not the case in TAM-treated mice (Fig. liferation was blunted in PDL tail of TAM-treated mice 2C). There was also a trend toward a lower average b-cell compared with nontreated or vehicle-treated mice (Fig. 1B volume in PDL tails of TAM-treated mice compared with and C and Supplementary Fig. 1). Thus, TAM inhibited PDL tails of vehicle-treated mice (Fig. 2C). The insulin con- b-cell proliferation in PDL tail regardless of the genetic tent of PDL tail was doubled in vehicle-treated mice but background or the presence of Cre recombinase. In an- significantly lowered following TAM treatment (Fig. 2D). other cohort of Balb-c mice with PDL surgery but without ThesedataindicatethatTAMbluntstheincreasein diabetes.diabetesjournals.org Yuchi and Associates 3221

Figure 1—TAM inhibits b-cell proliferation in PDL pancreas. A: Eight-week-old male Balb-c mice underwent PDL surgery and received either no injection or five injections of TAM 4 mg or vehicle. IdU 1 mg/mL was administered through drinking water until kill. B: The percentage of IdU-labeled b-cells increased in PDL tail of mice without injection (27 6 3% PDL tail vs. 7 6 1% PDL head, n =3)orin vehicle-treated mice (28 6 4% PDL tail vs. 7 6 2% PDL head, n = 3) but not in TAM-treated mice (8 6 2% PDL tail vs. 5 6 0.8% PDL head, n = 3). ***P < 0.001. C: Immunofluorescent staining of insulin and IdU in PDL tail (see B). D: Mice were treated as in A without IdU administration. The percentage of Ki67+ b-cells in PDL tail was significantly lower in TAM- vs. vehicle-treated mice (0.8 6 0.01% in TAM vs. 3.0 6 0.2% in vehicle, n = 3). ***P < 0.001. E: Mice were treated as in A with TAM or 4OHT administered at 1 mg/injection (five injections). TAM and 4OHT reduced the percentage of IdU-labeled b-cells in PDL tail compared with vehicle (10 6 0.5% in TAM and 11 6 3% in 4OHT vs. 28 6 4% in vehicle, n = 3). *P < 0.05. F: Mice underwent PDL and received one injection of 1 or 2 mg TAM or vehicle at day 5 after PDL. G: The percentage of Ki67+ b-cells was determined at day 7 (2.6 6 0.3% for vehicle vs. 0.88 6 0.14% for 1 mg TAM and 0.84 6 0.25% for 2 mg TAM, n = 3). **P < 0.01. H: Pregnant mice at G13.5 and nonpregnant mice of the same age received one intraperitoneal injection of either vehicle or 1.5 mg TAM, and b-cell proliferation was determined at G15.5 (n = 4). **P < 0.01, ***P < 0.001. Scale bars are 20 mm. IHC, immunohistochemistry; INS, insulin; ns, not significant.

PDL-induced b-cell volume and insulin content, albeit to pancreas. When ERs were studied at the mRNA level in a limited extent. PDL tail versus head, Esr1 (ERa) expression was fivefold higher in PDL tail, whereas subtype Esr2 (ERb) mRNA ERa Contributes to b-Cell Proliferation and Ngn3 Gene was 18-fold lower in PDL tail (Fig. 3A). Because Esr2 Expression in PDL Pancreas mRNA was nearly undetectable in PDL tail tissue (Ct = The aforementioned results suggest that ERs are involved 39.0 6 0.97), ERa appears to be the prime ER subtype in in b-cell replication and Ngn3 gene induction in PDL PDL pancreas. Studies in murine mammary cells showed 3222 Estrogen Regulates b-Cell Formation Diabetes Volume 64, September 2015

Figure 2—TAM inhibits Ngn3 gene expression and b-cell expansion in PDL pancreas. A and B: Eight-week-old male Balb-c mice were treated as in Fig. 1A without IdU administration. Ngn3 mRNA expression was studied in PDL head and tail and in duodenum. A: PDL tail Ngn3 mRNA data were normalized to CycloA mRNA and expressed relative to PDL head of mice without injection (none) (n = 9). *P < 0.05, ***P < 0.001. B: Ngn3 mRNA in duodenum tissue. Data are normalized to CycloA mRNA and expressed relative to duodenum tissue of mice without injection (none) (n =3–9). ***P < 0.001. C and D: Sham or PDL surgery was performed and TAM administered as in Fig. 1A. C: Total b-cell volume in PDL tail was significantly increased compared with sham tail in mice without injection (none) (0.46 6 0.1 mm3 PDL tail vs. 0.18 6 0.03 mm3 sham tail, n = 3) but not in mice that received TAM (0.35 6 0.05 mm3 PDL tail vs. 0.17 6 0.03 mm3 sham tail, n = 3). *P < 0.05. D: Insulin content was doubled in PDL tail compared with sham tail of vehicle-treated mice (25.0 6 2.12 mg PDL tail vs. 12.9 6 0.46 mg sham tail, n = 3), but the insulin content of PDL tail was significantly decreased by TAM (20.4 6 0.6 mg with TAM vs. 25.0 6 2.1 mg without TAM, n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

a a that ER can promote proliferation in response to E2 Increased Estrogen Levels and ER Activity in PDL Tail primarily when ERb is lowly expressed (20). The ERa To investigate how ERa activity could be influenced by (Esr1) transcript levels in b-cells isolated from sham PDL, we assayed the level of its natural E2 by RIA tail or PDL tail (6) were similar (Fig. 3B). To investigate in the pancreas tail of male Balb-c mice 7 days after sham whether ERa activity contributes to the increase of or PDL surgery. Although the level of E2 in sham tail was 2 2 b-proliferation induced by PDL, we analyzed ERa / generally below the detection limit of the assay (2 ng/L), 2 (null mutant of ERa) mice obtained by breeding of ERa+/ its concentration was 17 6 2 ng/L in PDL tail (620 pg/ parents (12). The full disruption of ERa in the knockout PDL tail) (Table 1). Furthermore, the expression of several mice was evidenced by the absence of ERa mRNA and poly- estrogen-regulated (22–28) was increased in PDL 2 2 peptide in the uterus of female ERa / mice and by its tail (Fig. 4A), suggesting increased ER activity. abnormal development (Supplementary Fig. 3) (21). The activity of ERa in b-cells of PDL pancreas was 2 2 PDL was performed in 8-week-old male ERa / and investigated on the basis of its subcellular localization. ERa+/+ littermates, and the pancreas was studied following In uterus, ERa was predominantly located in the nucleus immune detection of insulin and Ki67 at day 14 after (Supplementary Fig. 3D), which is consistent with other surgery. ERa gene inactivation diminished b-cell prolifer- studies (29). ERa protein remained predominantly cyto- 2 2 ation specifically in PDL tail (Fig. 3C). In addition, ERa / solic (9,29) in b-cells of sham tail (Fig. 4B)orPDL mice had decreased Ngn3 mRNA in PDL tail (Fig. 3D)and head (Fig. 4C) but was nuclear in b-cells of PDL tail (Fig. duodenum (Fig. 3E)comparedwithERa+/+ littermates. 4D). The nuclear ERa signal in b-cells of PDL tail was Thus, genetic inactivation of ERa and administration of strongly diminished following TAM administration (Fig. TAM had the same effect on b-cell proliferation and 4E), and ERa protein was absent from the pancreas of 2/2 Ngn3 transcript level in PDL pancreas and duodenum, ERa mice (Fig. 4F). Thus, PDL increased E2 in the suggesting that ERa activity contributed to a higher ligated portion of the pancreas and stimulated a nuclear Ngn3 gene expression and/or number of Ngn3+ cells localization of ERa in b-cells, which was counteracted by and increased b-cell proliferation in PDL pancreas. TAM. diabetes.diabetesjournals.org Yuchi and Associates 3223

Figure 3—ERa contributes to b-cell proliferation and Ngn3 gene expression in PDL pancreas. A: Eight-week-old male Balb-c mice underwent PDL. ER mRNA expression was studied in the indicated tissues at day 14. Data were normalized to CycloA mRNA. PDL increased Esr1 mRNA expression (103 6 7 3 1024 PDL tail vs. 20 6 10 3 1024 PDL head, n = 3), whereas Esr2 mRNA was decreased (1.1 6 0.5 3 1024 PDL tail vs. 18 6 2 3 1024 PDL head, n = 3). *P < 0.05, ***P < 0.001. B: Esr1 mRNA quantity did not differ in FACS- sorted b-cells from sham tail and PDL tail (n =7–9). C: The percentage of Ki67+ b-cells in 14 days’ postsurgery PDL tail was determined in ERa2/2 and wild-type (WT) littermates. ERa gene inactivation decreased b-cell proliferation in PDL tail (1.28 6 0.08% ERa2/2 vs. 2.54 6 0.07% WT, n = 6), whereas basal proliferation in the unligated head part was not changed (0.76 6 0.05% WT vs. 1.09 6 0.17% ERa2/2, n = 6). ***P < 0.001. D and E: Ngn3 mRNA expression in 14 days’ postsurgery PDL head and tail and duodenum was determined in ERa2/2 and WT littermates. D: Data were normalized to CycloA mRNA and expressed relative to the Ngn3 mRNA level in WT PDL head. Ngn3 mRNA was increased in PDL tail compared with PDL head of WT mice (n = 6). Knockout of ERa decreased Ngn3 mRNA expression in PDL tail (n = 5). ***P < 0.001. E: For duodenum, Ngn3 mRNA data were normalized to CycloA mRNA and expressed relative to Ngn3 mRNA level in WT duodenum (n =5–6). *P < 0.05. ns, not significant.

We next examined whether an artificial increase of E2 b-cell proliferation in PDL depended on E2 synthesis, could stimulate in situ b-cell proliferation and Ngn3 gene and increasing E2 within the environment of PDL was expression in PDL pancreas. E2 or vehicle was injected on sufficient for stimulating proliferation. In addition, both the day of surgery (day 0) and on day 3, and PDL tail was TAM administration and ERa knockout interfered with b a b studied on day 7. E2 further increased -cell proliferation nuclear localization of ER protein in -cells and with and Ngn3 mRNA level in PDL tail (Fig. 4G–I). To investigate b-cell proliferation in the PDL pancreas. These data in- b whether endogenous synthesis of E2 was required for dicate that estrogen regulation of -cell growth is medi- increased b-cell proliferation in PDL, we subcutane- ated by ERa. Because macrophages were suggested to ously administered ARO, an inhibitor of aromatase, the promote b-cell proliferation during PDL (30), we analyzed rate-limiting enzyme for E2 synthesis (Fig. 4J). ARO the presence of myeloid cells in untreated or TAM-treated blunted activation of the b-cell cycle (Fig. 4K). Thus, PDL pancreas by flow cytometry (31). No shift in the

Table 1—E2 concentration in PDL or sham tail tissue of pancreas 7 days after surgery Sham tail PDL tail PDL tail Sham tail (ng/L) PDL tail (ng/L) (ng/g protein) (ng/g protein) Sham tail (ng/mg tissue) (ng/mg tissue) 2.2 14.0 0.37 10.4 0.06 0.60 ,2.0* 14.0 ,0.34 10.4 ,0.06 0.60 ,2.0* 21.0 ,0.34 15.6 ,0.06 0.89 ,2.0* 17.4 ,0.34 12.9 ,0.06 0.74

Detection limit E2 RIA 2.0 ng/L. *Undetectable. 3224 Estrogen Regulates b-Cell Formation Diabetes Volume 64, September 2015

myeloid cell composition and no difference in the number of macrophages (Supplementary Fig. 4A) were induced by TAM treatment of mice with PDL. Moreover, no difference was observed for other immune cell types (T cells, B cells, dendritic cells) (data not shown). As expected, the level of mRNA encoding various macrophage markers (Supple- mentary Fig. 4B) and proinflammatory cytokines (Supple- mentary Fig. 4C) was increased in PDL tail compared with sham tail, but treatment with TAM did not significantly change the expression levels in PDL (Supplementary Fig. 4B and C). Thus, estrogen has no effect on myeloid cell infiltration or inflammatory cytokines in PDL pancreas. That the expression of the ERa-responsive gene IL6 was not significantly affected by TAM suggests that additional regulatory factors control IL6 expression in PDL.

ERa Regulates b-Cell Proliferation and Ngn3+ Progenitors in the Embryonic Pancreas We next examined whether ERa plays a role in embryonic b-cell proliferation and development. Proliferation was evaluated at embryonic stage E18.5 when massive b-cell replication occurs (32). TAM efficiently reached the em- bryonic b-cells of RipCreERT;R26RYFP mice because 89 6 1.9% of E18.5 b-cells were YFP+ when 1.5 mg TAM was administered at day E16.5 to the pregnant mother (Sup- plementary Fig. 5). Embryonic b-cell proliferation evaluated at E18.5 was markedly lowered by the TAM treatment (Fig. 5A), suggesting that ERs might be involved in embryonic b-cell expansion. The majority of endocrine progenitor cells are generated at E15.5 and transiently express Ngn3 (33). Because Ngn3 gene expression is regulated by estrogen in developing neurons (18) and was reduced by ERa inactivation in PDL pancreas (Figs. 2A and 3D), we examined the Ngn3 2 2 mRNA level in E15.5 pancreas of ERa+/+ and ERa / em- +/+ Figure 4—Increased estrogen levels and ERa activity in PDL tail pan- bryos and found it significantly decreased in ERa em- 2 2 creas. A: Eight-week-old male Balb-c mice underwent PDL surgery. The bryosthatreceivedTAMandinuntreatedERa / expression of estrogen-regulated genes was studied in the indicated embryos compared with ERa+/+ control embryos (Fig. tissues 7 days after PDL (n = 4). Data are normalized to CycloA mRNA and expressed relative to the gene mRNA level in PDL head 5B). Transcription factors Pax6 and Pax4, which are cru- (n =1).*P < 0.05, **P < 0.01, ***P < 0.001. B–D: Eight-week-old cial for differentiation of the a- and b-cell lineages down- male Balb-c mice underwent sham or PDL surgery, and ERa protein stream (34), were also reduced by TAM or knockout of was studied in sham tail and PDL head and tail at day 7 (n =3).ERa a b ER (Fig. 5B), suggesting that embryonic endocrine cell protein was predominantly cytosolic in -cells of sham tail (B)andPDL fi head (C). D:ERa was nuclear in b-cells of PDL tail. E: Nuclear localiza- speci cation was affected. In contrast, Pdx1, which is al- tion in PDL tail was prevented by administration of TAM 2 mg at day 5. ready expressed before Ngn3 in pancreas formation, was 2 2 F:NoERa protein was detected in pancreas tissue from ERa / mice. not influenced (Fig. 5B). Moreover, Ngn3 gene expres- – G I:E2 or vehicle was injected locally into PDL tail at the day of surgery sion in endocrine cell progenitors of E15.5 duodenum and repeated 3 days later. Mice were studied at day 7 postsurgery, and +/+ fl was significantly lower in TAM-treated ERa embryos IdU 50 mg/kg i.p. was injected 16 and 2 h before kill. G: Immuno uo- 2 2 rescent staining of insulin and IdU in sections of PDL tail. H:Theper- and in ERa / embryos compared with ERa+/+ control b 6 centage of IdU-labeled -cells in PDL tail was increased by E2 (2.4 embryos (Fig. 5C). 6 6 0.4% for vehicle vs. 3.3 0.3% for 20 pg E2 and 4.5 0.4% for 100 pg + fi < Ngn3 progenitor cells were identi ed in E15.5 pancreas E2, n =6).**P 0.01. I: Ngn3 mRNA expression was normalized YFP to CycloA mRNA and expressed relative to the Ngn3 mRNA level in either by the presence of YFP reporter in Ngn3 embryos vehicle-treated PDL tail (n =6).*P < 0.05, ***P < 0.001. J: Balb-c male (11) or by detection with an NGN3-specific antibody. The mice underwent PDL surgery, and ARO was injected subcutaneously. K: – + + percentages of E-cadherin positive cells that were YFP or Quantification of Ki67 b-cells in PDL tail at day 7. ARO decreased + fi b-cell proliferation in PDL tail (1.5 6 0.51% ARO vs. 3.1 6 0.16% NGN3 in E15.5 pancreas were signi cantly reduced when vehicle, n =3).**P < 0.01. Scale bars are 20 mm. H, head; IHC, immu- TAM was administered to the mother at E13.5 (Fig. 5D), nohistochemistry; INS, insulin; ns, not significant; T, tail. indicating reduced numbersofembryonicendocrine progenitor cells. Furthermore, the percentage of diabetes.diabetesjournals.org Yuchi and Associates 3225

Figure 5—ERa regulates b-cell proliferation and Ngn3+ progenitors in embryonic pancreas. A: Pregnant mice received intraperitoneally vehicle or 1.5 mg TAM at E16.5, and the embryonic pancreas was dissected at E18.5 and the percentage of Ki67+ b-cells quantified (6.3 6 0.01% TAM vs. 12.3 6 0.01% vehicle, n =3–4). **P < 0.01. B and C: Wild-type (WT) and ERa2/2 embryos were collected at E15.5. For embryos treated with TAM, pregnant mice were given TAM 1.5 mg i.p. at E13.5. mRNA expression of indicated genes was studied by real- time quantitative PCR. B: mRNA data are normalized to CycloA mRNA and expressed relative to the mRNA level in WT embryonic pancreas. Embryonic pancreas levels of Ngn3, Pax6, and Pax4 mRNA were significantly decreased in WT embryos that received TAM and in ERa2/2 embryos compared with WT control embryos (n =4–9). *P < 0.05, **P < 0.01, ***P < 0.001. C: Ngn3 mRNA level of embryonic duodenum was significantly lower in TAM-treated and in ERa2/2 embryos than in WT control embryos (n =4–9). **P < 0.01, ***P < 0.001. D: Pregnant Ngn3YFP mice received intraperitoneal vehicle or 1.5 mg TAM at E13.5, and the embryonic pancreas was dissected at E15.5. The percentage of E-cadherin (Ecad)-positive epithelial cells that was NGN3+ or YFP+ was determined by immunofluorescent staining (YFP+ Ecad+ cells: 9.0 6 1.02% vehicle vs. 4.5 6 0.84% TAM; NGN3+ Ecad+ cells: 8.4 6 1.00% vehicle vs. 3.4 6 0.25% TAM; n = 3). *P < 0.05, **P < 0.01. E: WT and ERa2/2 embryos were collected from pregnant ERa+/2 mice at E15.5. The percentage of Ecad+ cells expressing Ngn3 was quantified in ERa2/2 and WT embryonic pancreas (5.4 6 0.5% WT vs. 3.1 6 0.5% ERa2/2, n = 3). *P < 0.05. Scale bars are 50 mm. INS, insulin.

E-cadherin–positive cells that expressed Ngn3 protein was generation of Ngn3+ cells and the subsequent b-cell pro- 2 2 lower in the pancreas of E15.5 ERa / versus ERa+/+ liferation in developing pancreas are both regulated by embryos (Fig. 5E). Together, the data suggest that the ERa. 3226 Estrogen Regulates b-Cell Formation Diabetes Volume 64, September 2015

DISCUSSION studies are needed to clarify the precise mechanism of The current study shows that signaling through ERa is steroid and lactogenic hormones in the regulation of fetal b involved in the control of b-cell progenitor activation and -cell formation. b-cell proliferation in embryonic and injured adult pan- In the pancreas of adult mice severely injured by PDL, – creas. Both genetic loss and competitive inhibition of ERa Ngn3 gene expression is activated in endocrine progenitor b signaling decreased the number of Ngn3+ endocrine pan- like cells and -cell proliferation is stimulated, resulting in b creas progenitors in embryonic mice, suggesting a regula- increased -cell volume (2,3). We report that ER signaling tory role for estrogen during pregnancy. Specification of is increased under these conditions, regulates Ngn3 ex- b endocrine progenitor cells is counteracted by Notch sig- pression levels and -cell proliferation, and thus affects naling (35). Because ERa signaling decreases Notch tran- regenerative processes in PDL pancreas. Using TAM- scriptional activity in breast cancer cells (36), it is possible based genetic tracing is unlikely to be the most adequate approach for evaluating the extent of adult b-cell for- that E2-mediated regulation of Ngn3 gene expression and endocrine cell differentiation in developing pancreas mation from alternative cell sources in PDL pancreas occurs through Notch signaling. In addition, in the vertebrate because TAM clearly interferes with ER signaling and b brain, aromatase, which catalyzes estrogen synthesis, is -cell formation. A washout procedure might diminish expressed by neural progenitor cells (37) and regulates the suppressive effects of TAM on ER signaling, but it is neural progenitor cell proliferation (38). E has been spec- not excluded that transient exposures to TAM may have 2 long-lasting effects on b-cell differentiation. ulated to lower Notch signaling during brain development, Both isoforms of ER are present in b-cells and contrib- resulting in Ngn3-dependent neural cell differentiation ute to control of function, proliferation, and survival (18). Of note, certain variants of the NGN3 gene are (8–10,29,40,45), although mechanistic details are lacking associated with more severe hyperglycemia in men but [reviewed in Tiano and Mauvais-Jarvis (46)]. PDL in- not in women with diabetes (39). Molecular pathways creased the number of transcripts encoding ERa while other than ERa signaling are likely because Ngn3+ cells 2 2 decreasing ERb mRNA to a nearly undetectable level. De- arepresent,albeitinlowernumbers,inthepancreasofERa / letion of only ERa prevented PDL-induced proliferation embryonic mice. In line with these data, the pancreatic of b-cells, and although this does not exclude a role for b-cell mass has been found to be similar in ERa null ERb, the latter could not compensate for the defect. Of mutant and wild-type mice (9,40), possibly by progenitor note, the cell cycle is activated through ERa signaling cell activation through Ngn3-independent pathways (41). when ERb is lowly expressed as in mouse mammary cells Because ER signaling was decreased in the embryos of (20) and human islet–derived precursor cells (47). Addi- mice treated with TAM at a dose generally used for acti- ERT tionally, studies in breast cancer with ER-dependent vation of Cre recombinase in transgenic mice, while a + growth have shown that the abundance of ER -encoding this also lowered the number of Ngn3 cells as well as transcripts increases, whereas that of ERb mRNA the proliferation of b-cells, we question the accuracy of + decreases (48,49), suggesting that tissues with an increased data on the ontogeny of embryonic b-cells or Ngn3 cells a ERT growth tendency may require elevated ER expression and generally obtained with Cre -expressing mice. The cur- an increased ERa/ERb ratio. Moreover, in the case of rent data suggest at least a temporary inhibition of b-cells, several conditions now appear to stimulate growth estrogen-regulated endocrine cell formation by TAM. We through ERa, including PDL injury, embryonic pancreas, – b report a 50 75% decrease of -cell proliferation and Ngn3 and pregnancy. a2/2 expression by TAM or ER , whereas TAM treatment Compared with sham treatment, PDL tail shows b does not cause such major changes of the -cell volume increased estrogen concentration, proliferation, and nu- andinsulincontentinPDLtail.Tofurtheraddressthe clear ERa localization in b-cells. The latter two processes a b 2 2 impact of ER on -cell expansion in PDL, it is necessary were blunted in ERa / mice as well as by aromatase to study mice with conditional knockout of ERa in b-cells. inhibition and enhanced by intrapancreas injection of E2 In addition, whether the effects described here account for at a dose similar to its concentration in PDL pancreas, ERa activation in the pancreas is uncertain because this suggesting a role for synthesis of E2 possibly by newly study is based on global ERa knockout or drug administra- formed adipocytes in situ (50). In addition, we showed a tion. Therefore, a study in mice with ER knockout in that local administration of E2 potentiated Ngn3 gene pancreatic cells, particularly in b-cells, will be informative. expression in PDL pancreas. Because estrogen and activa- Circulating levels of prolactin, placental lactogen, and tion of ERa do not increase b-cell proliferation in rodent estrogen are high during late pregnancy (42) when an models for diabetes (51) and in human islets transplanted increase of b-cell mass compensates for the increased in diabetic mice (52), we cannot exclude that the estrogen needs of insulin in the mother (43). ER signaling affects effects described in the current study would be specificto the maternal b-cell function under these conditions the PDL model. Nevertheless, the data also suggest in- (9,40,44), and on the basis of the current data, it also volvement of estrogen in other models of b-cell prolifer- contributes to the increase of b-cells in the fetal pancreas ation, namely in both the embryonic and the mature by stimulating neogenesis and proliferation. Further pancreas during pregnancy. diabetes.diabetesjournals.org Yuchi and Associates 3227

Estrogen effects also depend on dose and route of 8. Le May C, Chu K, Hu M, et al. Estrogens protect pancreatic beta-cells from fi apoptosis and prevent insulin-deficient diabetes mellitus in mice. Proc Natl Acad administration (53). Cell-speci c delivery of E2 stimulates beneficial activities without unwanted side effects. Estro- Sci U S A 2006;103:9232–9237 gen conjugated to GLP-1, which is delivered preferentially 9. Alonso-Magdalena P, Ropero AB, Carrera MP, et al. Pancreatic insulin content regulation by the estrogen receptor ER alpha. PLoS One 2008;3:e2069 to the pancreas, reduces adiposity and improves hyper- 10. Alonso-Magdalena P, Ropero AB, García-Arévalo M, et al. Antidiabetic ac- glycemia, whereas systemic delivery of estrogen potentially tions of an estrogen receptor b selective agonist. Diabetes 2013;62:2015–2025 increases its adverse effects, such as in breast cancer (54). In 11. Mellitzer G, Martín M, Sidhoum-Jenny M, et al. Pancreatic islet progenitor summary, the new effects of estrogen signaling in develop- cells in neurogenin 3-yellow fluorescent protein knock-add-on mice. Mol En- ing and regenerating pancreas support ERa as a candidate docrinol 2004;18:2765–2776 target for control of glucose homeostasis and b-cell forma- 12. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M. Effect of tion in diabetes therapy. single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive . Development 2000;127: 4277–4291 Acknowledgments. The authors thank Pierre Chambon and Andrée Krust 13. Percival AC, Slack JM. Analysis of pancreatic development using a cell (Université Louis Pasteur, Illkirch, France) for providing the ERa mutant mice; Ann lineage label. Exp Cell Res 1999;247:123–132 Demarré, Veerle Laurysens, Jan De Jonge, Erik Quartier (Diabetes Research 14. Mellitzer G, Bonné S, Luco RF, et al. IA1 is NGN3-dependent and essential Center, Vrije Universiteit Brussel, Brussels, Belgium), Ellen Anckaert, and Gaby for differentiation of the endocrine pancreas. EMBO J 2006;25:1344–1352 Schoonjans (Department of Endocrinology, Universitair Ziekenhuis Brussel, Brus- 15. Heimberg H, Bouwens L, Heremans Y, Van De Casteele M, Lefebvre V, sels, Belgium) for technical assistance; and Yves Heremans (Diabetes Research Pipeleers D. Adult human pancreatic duct and islet cells exhibit similarities in Center, Vrije Universiteit Brussel, Brussels, Belgium) for critical revision of the expression and differences in phosphorylation and complex formation of the manuscript. homeodomain protein Ipf-1. Diabetes 2000;49:571–579 Funding. Financial support was from the Chinese Scholarship Council (Y.Y.), 16. Grouwels G, Cai Y, Hoebeke I, et al. Ectopic expression of E2F1 stimulates Institute for the Promotion of Innovation by Science and Technology in Flanders beta-cell proliferation and function. 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