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ESTROGEN α REGULATES FORMATION

DURING DEVELOPMENT AND FOLLOWING INJURY

Yixing Yuchi1,*, Ying Cai1,*, Bart Legein1,6, Sofie De Groef1, Gunter Leuckx1,

Violette Coppens1, Eva Van Overmeire2,3, Willem Staels1,4, Nico De Leu1,5, Geert Martens1, Jo A. Van Ginderachter2,3, Harry Heimberg1,# Mark Van de Casteele1,#

1Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium; 2Myeloid Cell Immunology Laboratory, VIB, 1040 Brussels, Belgium; 3Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, 1040 Brussels, Belgium; 4Department of Pediatrics, Division of Pediatric Endocrinology, Ghent University Hospital and Department of Pediatrics and Genetics, Ghent University, Ghent, Belgium; 5Department of Endocrinology, UZ Brussel, 1090 Brussels, Belgium 6Current address: Experimental Vascular Pathology, Department of Pathology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands

*: equal contribution

#: equal contribution

(Total characters count: 33,957) Running Title: Estrogen regulates beta cell formation

Key words: beta cell/ development/ estrogen/ regeneration

Correspondence to: Harry Heimberg Diabetes Research Center Vrije Universiteit Brussel Laarbeeklaan 103 B1090 Brussels, Belgium Tel: 32 2 4774477 Fax: 32 2 4774472 email: [email protected]

Diabetes Publish Ahead of Print, published online May 26, 2015 Diabetes Page 2 of 45

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ABSTRACT

Identifying pathways for beta cell generation is essential for cell therapy in diabetes.

Here we investigate the potential of 17βestradiol (E2) and (ER) signaling for stimulating beta cell generation during embryonic development and in severely injured adult pancreas. Estradiol concentration, ER activity and number of

ERα transcripts were enhanced in pancreas injured by partial duct ligation (PDL), along with nuclear localization of ERα in beta cells. PDLinduced proliferation of beta cells depended on aromatase activity. The activation of Neurogenin3 (Ngn3) expression and beta cell growth in PDL pancreas were impaired when ERα was

/ turned off chemically or genetically (ERα ), while in situ delivery of E2 promoted beta cell formation. In embryonic pancreas, beta cell replication, number of Ngn3+ progenitor cells and expression of key factors of the endocrine lineage

were decreased by ERα inactivation. The present study reveals that E2 and ERα signaling can drive beta cell replication and formation in mouse pancreas.

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INTRODUCTION

Decreased functional beta cell mass is the major cause for hyperglycemia in diabetes.

Restoration of the endogenous beta cell mass as a therapeutic strategy, however,

requires better understanding of signaling pathways that control beta cell growth and

differentiation. Embryonic beta cells are generated by a developmental program

executed through the timed action of a number of key transcription factors among

which Ngn3 is key for endocrine specification. Ngn3+ cells delaminate from

pancreatic , are mitotically quiescent, and give rise to endocrine cells. Ngn3

cells appear maximally competent for driving beta cell formation at E14.5. Formed

beta cells expand through selfreplication, already evident at E18.5 and continuing

into early postnatal life (1). Also in severely injured partial duct ligated (PDL)

pancreas of adult mice, Ngn3+ cells are generated near duct epithelium and can

differentiate to beta cells (2). Beta cells are vastly generated through replication in

PDL (3; 4), but some derive from acinar (5) and duct (6), apparently via a Ngn3+ stage

(2; 5) as in embryonic pancreas.

It is uncertain how the numbers of Ngn3+ endocrine progenitors and of replicating

beta cells are controlled in embryonic or mature pancreas. Identifying factors that

control these processes and manipulating them may be of therapeutic advantage. It is

known that 17βestradiol (E2) enhances beta cell survival and glycemic control in

various animal models (7; 8) by signaling through estrogen receptor ERα (8; 9) and/

or ERβ (10).

However, little is known on the importance of estrogen and ER signaling for beta cell

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Yuchi et al. proliferation and differentiation. So far, no in vivo effects on beta cell formation have been reported for the ER antagonist tamoxifen (TAM) while this compound is used to conditionally activate Cre recombinase activity (CreERT) in genetic tracing studies on embryonic and adult endocrine progenitor and beta cell . Here we investigated whether estrogen receptor signalling is important for beta cell replication and neogenesis during embryogenesis and in injured adult pancreas.

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RESEARCH DESIGN AND METHODS

Animals and tissues

All mouse experiments were performed according to the guidelines of our

institutional ‘‘Ethical Committee for Animal Experiments’’ and national guidelines

and regulations. Balbc mice were obtained from Janvier laboratories (Le Genest

Saint Isle, France). The mouse strains Ngn3CreERT; R26RYFP, and RIPCreERT;R26RYFP,

and Ngn3 knockaddon EYFP (Ngn3YFP) were previously described (3; 11). ERα/

mice are infertile and were obtained by crossing ERα+/ mice, as described (12).

All partial pancreatic duct ligation (PDL) surgery in the current study was performed

on 8week old male mice as described (2). Embryos and embryonic pancreas and

were dissected as previously described (13). Mouse embryos were

obtained after timed mating. Pregnant mice were killed by cervical dislocation, uteri

dissected in ice cold sterile DPBS and embryos removed from the deciduas. For

immunohistochemistry studies, embryos were fixed in 10% % neutralbuffered

formalin overnight at 4°C and embedded in paraffin. For RNA analysis, embryos

were kept on icecold DPBS and the gut was dissected to remove the pancreas buds.

Pancreas and duodenum were collected in RNA later (R0901, Sigma Aldrich,

Diegem, Belgium).

Chemicals

Tamoxifen (T5648, Sigma) was dissolved in corn oil (C8267, Sigma) at 20mg/ml,

4hydroxytamoxifen (579002, Calbiochem, Darmstadt, Germany) dissolved in pure

Diabetes Page 6 of 45

Yuchi et al. ethanol to 20mg/ml was diluted to 5mg/ml in corn oil (C8267, Sigma). 17estradiol

(E2785, Sigma) dissolved in pure ethanol to 0.4mg/ml was diluted to 0.4ng/ml or

2ng/ml in 0.9% NaCl for intrapancreas injection and Aromasin (Pfizer, Brussels,

Belgium) was dissolved to 12.5mg/ml in 0.3% hydroxypropylcellulose (HPC,

191884, Sigma) in PBS.

For PDL studies in adult mice, tamoxifen, 4hydroxytamoxifen, Aromasin and the corresponding vehicles (corn oil, corn oil and 0.3% HPC/PBS, respectively) were given subcutaneously (s.c.). E2 (20 or 100pg in 50µl) and vehicle (50µl saline) were injected in the ligated tail portion of PDL pancreas. Before PDL and sham surgery or intrapancreatic injection, mice were sedated by intraperitoneal injection of ketamine

(3.5mg/kg bodyweight, Ceva Sante Animale, Brussels, Belgium) plus xylazine

(0.5mg/kg bodyweight, Bayer, Diegem, Belgium).

Immunohistochemistry

Samples for immunohistochemistry were fixed in 10% neutralbuffered formalin overnight at 4°C and embedded in paraffin. Sections (45µm) were deparaffinized, washed in 0.2% Tween 20 (P5927, Sigma) in PBS, antigen retrieval was performed in a steamer, and the slides were blocked in 10% donkey serum (Jackson

ImmunoResearch Laboratories, Suffolk, UK). Primary antibodies for (1:3000, guinea pig, generated at the Diabetes Research Center, Brussels, Belgium), ERα

(1:200, rabbit, E1644, Spring Biosciences, Fremont, CA, USA), GFP (1:100, goat,

Abcam, Cambridge, UK), IdU (1:100, mouse, BD Biosciences, Erembodegem,

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Belgium) and Ki67 (1:1000, rabbit, Leica Microsystems, Diegem, Belgium), Ngn3

(1:2000, rabbit, Millipore, Tenecula, CA, USA), Ecadherin (1:50, mouse, BD

Biosciences), were incubated overnight at 4°C at the indicated dilutions. Secondary

antibodies for detection of guinea pig, rabbit, goat, or rat antibodies were labeled by

fluorescence (Cy3, Cy2 or Cy5). Nuclei were stained by Hoechst 33342 (4mg/ml,

Sigma).

RNA and DNA analysis

Total RNA was isolated from tissue (74104, RNeasy, Qiagen, Venlo, The

Netherlands). Only RNA with RNA Integrity Number R≥7 (2100 BioAnalyzer,

Agilent) was further analyzed. cDNA synthesis and RealTime quantitive PCR were

done as described (14) with TaqMan Universal PCR master mix on an ABI Prism

7700 Sequence Detector, and data were analyzed using the Sequence Detection

Systems Software, version 1.9.1 (all Applied Biosystems, Life Technologies, Gent,

Belgium). Mousespecific primers and probes were for Ngn3:

5’GTCGGGAGAACTAGGATGGC3’ (forward primer), 5’GGAGCAGTCCCTA

GGTATG3’ (reverse primer) and 5’CGGAGCCTCG GACCACGAA3’ (probe),

Tert (Mm.PT.53a.13753537), Lcn2 (Mm.PT.53a. 10167155), ptma (Mm.PT.53a.

30222141), IL-6 (Mm.PT.49a.10005566), (Mm.PT.53a33146682), C-Jun

(Mm.PT.49a.8204422.g), Pax4 (N011038.1.m m), Pax6 (Mm.PT.51.14285402), Pdx1

(Mm.PT.51.11487144), Esr2 (Mm.PT. 49a.17681375.g), all from Integrated DNA

® Technologies (IDT, Leuven, Belgium), and Esr1 (Mm00433149_m1, TaqMan Gene

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Expression Assay, Life Technologies). Quantitative PCR reactions were performed with cDNA corresponding to 20ng RNA as described (3) . Data were normalized to

Cyclophilin A (Mm02342429, IDT). Conventional PCR primers for ERα (Exon3) were P1: 5’ AGAATGGCCGAGAGAGACTG3’, P2: 5’ TTCTCTTAA

AGAAAGCCTTGCAG3’ (IDT). Primers (IDT) for genotyping ERα+/+ (wild type) and ERα/ mice and embryos were previously described (12).

Western blot analysis

Protein extracts were prepared as described (15). 50 µg of was separated by

SDSPAGE and transferred onto a nitrocellulose membrane. ERα was detected with a rabbit polyclonal antibody (1:200, MC20, Santa Cruz Biotechnology, Heidelberg,

Germany). βactin (sc1616, Santa Cruz) was detected for verification of protein loading.

Beta cell volume, insulin content, and estradiol concentration

The measurement of beta cell volume (mm3) in PDL or Sham tail pancreas was performed as described previously (2; 3). The measurement for each pancreas tail was based on 4µmsections equally spaced (116µm apart) and spanning the whole tail tissue, corresponding to the analysis of 3% of the total volume. Insulin content of pancreatic tissue was measured as previously described (16). Estradiol (E2) concentration in whole pancreas tail tissue was determined after tissue homogenization using a Retsch Mill MM400 mixer (R20.745.0001, Haan, Germany)

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with grinding metal beads (5mm, R22.455.0003, Retsch). Lysates were cleared by

centrifugation after which 100µl samples were etherextracted. Estradiol was then

assayed using a RIA kit (DIAsource immunoassays, LouvainlaNeuve, Belgium).

Image analysis

Images were obtained by fluorescence microscopy (Olympus, Aartselaar, Belgium,

BX61 with Hamamatsu C10600 ORKAR2 camera). Ki67+ or IdU+ beta cells

(insulin+), and Ngn3+ or YFP+ Ecadherin+ cells were quantified in a nonautomated

manner (inspecting and counting individual cells). For the quantification of IdU+ or

Ki67+ beta cells, 10 nonconsecutive sections of each PDL pancreas (head or tail)

were studied and at least 1,000 beta cells per sample were counted. For the

quantification of Ngn3+ cells, 10 nonconsecutive sections of each embryonic

pancreas were studied and at least 4,000 Ecadherin+ cells per sample were counted.

Images were analyzed using SmartCapture3 (version 3.0.8, Digital Scientific UK,

Cambridge, UK) and NIH ImageJ (U.S. National Institutes of Health, Bethesda, MD,

USA) software, Photoshop CS (version 1.3.1, Adobe, San Jose, CA, USA).

Statistics

Data were expressed as mean ± S.E.M (standard error of the mean) of at least 3

independent experiments. Groups were compared using unpaired twotailed student’s

ttest, 1way ANOVA, or 2way ANOVA with Bonferroni or Sidak posthoc tests;

differences were considered statistically significant if p<0.05.

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RESULTS

Tamoxifen inhibits beta cell proliferation in PDL pancreas

We investigated whether tamoxifen influenced the outcome of PDLinduced beta cell proliferation. First, transgenic Ngn3CreERT;R26RYFP male mice of mixed genetic background as well as inbred Balbc male mice underwent PDL surgery followed by subcutaneous (s.c.) injection of either TAM (4mg per injection, 20mg in total over a

14day period) (Figure 1A), vehicle or nothing and application of

5iodo2’deoxyuridine (IdU) via the drinking water. Whereas more beta cells were

IdU+ in the ductligated tail portion of PDL pancreas (from hereon termed “PDL tail”) as compared to the nonligated head portion (from hereon termed “PDL head”)(Figure

1B), beta cell proliferation was blunted in PDL tail of TAMtreated mice as compared to nontreated or vehicletreated mice (Figure 1B,C, Supplementary Figure S1). Thus,

TAM inhibited beta cell proliferation in PDL tail regardless of the genetic background or the presence of Cre recombinase. In another cohort of Balbc mice with PDL surgery but without nucleotide analogues, beta cell proliferation in PDL tail was assessed by immunofluorescence for proliferation marker Ki67. The percentage of

Ki67+ beta cells in PDL tail was significantly lower in TAM vs. vehicletreated mice

(Figure 1D) corroborating that beta cell proliferation was inhibited by TAM.

We then examined the effect of 4hydroxytamoxifen (4OHT), a selective estrogen receptor modulator and metabolite of TAM that binds to endogenous estrogen receptors and inactivates them (17). At only 1mg per injection (5mg in total), 4OHT and TAM were equally efficient in inducing YFP expression (>90%) in beta cells of

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adult RipCreERT;R26RYFP mice (Supplementary Figure S2) and beta cell proliferation in

PDL tail was decreased to the same extent by both compounds (Figure 1E). Also a

single s.c. injection of either 1 or 2mg TAM at day 5 post PDL (Figure 1F) resulted in

significantly lower beta cell proliferation in PDL pancreas at day 7 (Figure 1G). Since

TAM can act as an agonist or antagonist of estrogen signaling, depending on the cell

type, its effect on beta cell proliferation was also examined in the pancreas of

pregnant mice. A single intraperitoneal injection of 1.5 mg TAM at day G13.5 of

gestation caused a marked decrease of the beta cell proliferation at day G15.5, while

there was no significant effect of TAM on the basal beta cell proliferation in

nonpregnant females (Figure 1H). Collectively, the data suggest that adult beta cell

proliferation in PDL pancreas or during pregnancy is suppressed by antagonism of

estrogen signaling through ERs.

Tamoxifen inhibits Ngn3 and beta cell expansion in PDL

pancreas

Ngn3+ cells have been identified in the ductal lining of ligated pancreatic tail and are

capable of differentiating in vivo or ex vivo to functional beta cells (2). Activation of

Ngn3 gene expression and the presence of Ngn3+ cells are required for in vivo beta

cell expansion in PDL pancreas (2; 3). Since Ngn3 gene expression is regulated by

estrogen in developing neurons (18), we assessed whether Ngn3 transcriptional level

in PDL pancreas was influenced by modulation of ER signaling.

As expected, the amount of Ngn3 transcripts increased in PDL tail compared with

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PDL head in mice that received vehicle, but it was significantly reduced following administration of TAM (Figure 2A). In addition, the constitutive expression of Ngn3 associated with enteroendocrine progenitor cells (19) was also decreased in the duodenum of TAMtreated mice (Figure 2B). These data suggest that TAM decreases

Ngn3 gene expression and/or the number of Ngn3 expressing cells in PDL pancreas and intestine.

We next evaluated the impact of TAM administration on increased beta cell volume in PDL pancreas. The total beta cell volume was significantly increased in PDL vs.

Sham tails of pancreas from mice that did not receive TAM and this was not the case in TAMtreated mice (Figure 2C). There was also a trend towards a lower average beta cell volume in PDL tails of TAM treated mice as compared to PDL tails of vehicle treated mice (Figure 2C). The insulin content of PDL tail pancreas was doubled in vehicletreated mice, but significantly lowered following TAM treatment

(Figure 2D). These data indicate that TAM blunts the increase in PDLinduced beta cell volume and insulin content, be it to a limited extent.

ERααα contributes to beta cell proliferation and Ngn3 gene expression in PDL pancreas

Above results suggest that ERs are involved in beta cell replication and Ngn3 gene induction in PDL pancreas. When estrogen receptors were studied at mRNA level in

PDL tail vs. head, Esr1 (estrogen receptor α) expression was 5fold higher in PDL tail, whereas subtype Esr2 (estrogen receptor β) mRNA was 18fold lower in PDL tail

(Figure 3A). Since Esr2 mRNA was nearly undetectable in PDL tail tissue

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(Ct=39.0±0.97), ERα appears the prime estrogen receptor subtype in PDL pancreas.

Studies in murine mammary cells showed that ERα can promote proliferation in

response to E2 primarily when ERβ is lowly expressed (20). The ERα (Esr1)

transcript levels in beta cells isolated from sham tail or PDL tail pancreas (6) were

similar (Figure 3B). To investigate whether ERα activity contributes to the increase of

beta proliferation induced by PDL, we analysed ERα/ (null mutant of ERα) mice,

obtained by breeding of ERα+/ parents (12). The full disruption of ERα in the

knockout mice was evidenced by absence of ERα mRNA and polypeptide in the

uterus of female ERα/ mice and by its abnormal development (Supplementary Figure

S3) (21). PDL was performed on 8week old male ERα/ and ERα+/+ littermates and

pancreas was studied following immune detection of insulin and Ki67 at day 14 after

surgery. ERα gene inactivation diminished beta cell proliferation specifically in PDL

tail (Figure 3C). In addition, ERα/ mice had decreased Ngn3 mRNA in PDL tail

pancreas (Figure 3D) and duodenum (Figure 3E) as compared with ERα+/+

littermates. Thus, genetic inactivation of ERα and administration of TAM had the

same effect on beta cell proliferation and Ngn3 transcript level in PDL pancreas and

duodenum, suggesting that ERα activity contributed to a higher Ngn3 gene expression

and/or number of Ngn3+ cells, and increased beta cell proliferation in ductligated

pancreas.

Increased estrogen levels and ERααα activity in PDL tail pancreas

To investigate how ERα activity could be influenced by PDL, we assayed the level of

its natural E2 by RIA in pancreas tail of male Balbc mice 7 days after Sham or

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PDL surgery. While the level of E2 in Sham tail was generally below the detection limit of the assay (2ng/L), its concentration was 17±2 ng/L in PDL tail tissue

(±20pg/PDL tail) (Table 1). Furthermore, the expression of several estrogen regulated (2228) was increased in PDL tail (Figure 4A), suggesting increased ER activity.

The activity of ERα in beta cells of PDL pancreas was investigated on the basis of its subcellular localization. In uterus, ERα was predominantly located in the nucleus

(Supplementary Figure S3D), which is consistent with other studies (29). ERα protein remained predominantly cytosolic (9; 29) in beta cells of Sham tail (Figure 4B) or

PDL head (Figure 4C), but was nuclear in beta cells of PDL tail (Figure

4D). The nuclear ERα signal in beta cells of PDL tail pancreas was strongly diminished following TAM administration (Figure 4E) and ERα protein was absent

/ from pancreas of ERα mice (Figure 4F). Thus, PDL increased E2 in the ligated portion of pancreas, and stimulated a nuclear localization of ERα in beta cells, which was counteracted by TAM.

We next examined whether artificial increase of E2 could stimulate in situ beta cell proliferation and Ngn3 gene expression in PDL pancreas. E2 or vehicle was injected on the day of surgery (day 0) and on day 3, and PDL tail pancreas was studied on day

7. E2 further increased beta cell proliferation and Ngn3 mRNA level in PDL tail

(Figure 4GI). To investigate whether endogenous synthesis of E2 was required for increased beta cell proliferation in PDL we s.c.administered Aromasin (ARO), an inhibitor of aromatase, the ratelimiting enzyme for E2 synthesis (Figure 4J). ARO

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blunted activation of the beta cell cycle (Figure 4K). Thus, beta cell proliferation in

PDL was dependent on E2 synthesis, and increasing E2 within the environment of

PDL was sufficient for stimulating proliferation. In addition, both TAM

administration and ERα knockout interfered with nuclear localization of ERα protein

in beta cells and with beta cell proliferation in the PDL pancreas. These data indicate

that estrogen regulation of beta cell growth is mediated by ERα. Since macrophages

were suggested to promote beta cell proliferation during PDL (30), we analyzed the

presence of myeloid cells in untreated or TAM treated PDL pancreas by flow

cytometry (31). No shift in the myeloid cell composition and no difference in the

number of macrophages (Supplementary Figure S4A) was induced by tamoxifen

treatment of mice with PDL. Moreover, no difference was observed for other immune

cell types (Tcells, Bcells, dendritic cells) (data not shown). As expected, the level of

mRNA encoding various macrophage markers (Supplementary Figure S4B) and

proinflammatory cytokines (Supplementary Figure S4C) was increased in PDL tail as

compared to Sham tail pancreas, but treatment with tamoxifen did not significantly

change the expression levels in PDL (Supplementary Figure S4B,C). Thus, estrogen

has no effect on myeloid cell infiltration or inflammatory cytokines in PDL pancreas.

That the expression of the ERαresponsive gene IL6 was not significantly affected by

TAM, suggests that additional regulatory factors control IL6 expression in PDL.

ERααα regulates beta cell proliferation and Ngn3+ progenitors in embryonic

pancreas

We next examined whether ERα played a role in embryonic beta cell proliferation

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Yuchi et al. and development. Proliferation was evaluated at embryonic stage E18.5, when massive beta cell replication occurs (32). TAM efficiently reached the embryonic beta cells of RipCreERT;R26RYFP mice since 89±1.9% of E18.5 beta cells were YFP+ when

1.5mg TAM was administered at day E16.5 to the pregnant mother (Supplementary

Figure S5). Embryonic beta cell proliferation evaluated at E18.5 was markedly lowered by the TAM treatment (Figure 5A). This suggested that ERs might be involved in embryonic beta cell expansion.

The majority of endocrine progenitor cells are generated at E15.5 and transiently express Ngn3 (33). Since Ngn3 gene expression is regulated by estrogen in developing neurons (18) and was reduced by ERα inactivation in PDL pancreas

(Figure 2A, 3D), we examined the Ngn3 mRNA level in E15.5 pancreas of ERα+/+ and ERα/ embryos and found it significantly decreased in ERα+/+ embryos that received TAM and in untreated ERα/ embryos, as compared with ERα+/+ control embryos (Figure 5B). Transcription factors Pax6 and Pax4 that are crucial for differentiation of the alpha and beta cell lineages downstream (34) were also reduced by TAM or knockout of ERα (Figure 5B), suggesting that embryonic endocrine cell specification was affected. In contrast, Pdx1, which is expressed already prior to Ngn3 in pancreas formation, was not influenced (Figure 5B). Moreover, Ngn3 gene expression in endocrine cell progenitors of E15.5 duodenum was significantly lower in TAMtreated ERα+/+ embryos and in ERα/ embryos as compared to ERα+/+ control embryos (Figure 5C).

Ngn3+ progenitor cells were identified in E15.5 pancreas either by the presence of

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YFP reporter in Ngn3 addon YFP (Ngn3YFP) embryos (11) or by detection with an

NGN3specific antibody. The percentages of Ecadherin+ cells that were YFP+ or

NGN3+ in E15.5 pancreas were significantly reduced when TAM was administered to

the mother at E13.5 (Figure 5D), indicating reduced numbers of embryonic endocrine

progenitor cells. Furthermore, the percentage of Ecadherin+ cells that expressed

Ngn3 protein was lower in pancreas of E15.5 ERα/ vs. ERα+/+ embryos (Figure 5E).

Together, the data suggest that the generation of Ngn3+ cells and the subsequent beta

cell proliferation in developing pancreas are both regulated by ERα.

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DISCUSSION

The present study shows that signaling via ERα is involved in control of beta cell progenitor activation and beta cell proliferation in embryonic and injured adult pancreas. Both genetic loss and competitive inhibition of ERα signaling decreased the number of Ngn3+ endocrine pancreas progenitors in embryonic mice, suggesting a regulatory role for estrogen during pregnancy. Specification of endocrine progenitor cells is counteracted by Notch signaling (35). As ERα signaling decreases Notch transcriptional activity in breast cancer cells (36), it is possible that E2mediated regulation of Ngn3 gene expression and endocrine cell differentiation in developing pancreas occurs through Notch signaling. In addition, in vertebrate brain, aromatase, that catalyzes estrogen synthesis, is expressed by neural progenitor cells (37) and regulates neural progenitor cell proliferation (38). It has been speculated that E2 lowers Notch signaling during brain development and results in Ngn3dependent neural cell differentiation (18). Interestingly, certain variants of the NGN3 gene are associated with more severe hyperglycemia in men with diabetes but not women (39).

Molecular pathways other than ERα signaling are likely as Ngn3+ cells are present, albeit at lowered numbers, in pancreas of ERα/ embryonic mice. In line with these data, the pancreatic beta cell mass was found similar in ERα null mutant and wild type mice (9; 40), possibly by progenitor cell activation through Ngn3independent pathways (41). Since ER signaling was decreased in the embryos of mice treated with

TAM at a dose generally used for activation of CreERT recombinase in transgenic mice, while this also lowered the number of Ngn3+ cells, as well as the proliferation

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of beta cells, we question the accuracy of data on the ontogeny of embryonic beta

cells or Ngn3+ cells obtained with CreERTexpressing mice. Our data at least suggest a

temporary inhibition of the estrogenregulated endocrine cell formation by TAM. We

report 5075% decrease of beta cell proliferation and Ngn3 expression by TAM or

ERα/, whereas TAM treatment does not cause such major changes of the beta cell

volume and insulin content in PDL tail. To further address the impact of ERα on beta

cell expansion in PDL, it is necessary to study mice with conditional knockout of ERα

in beta cells. In addition, it is not certain that the effects described here account for

ERα activation in the pancreas since this study is based on global ERα knockout or

drug administration. It will therefore be informative to study mice with ERα knockout

in pancreatic cells and in particular in beta cells.

Circulating levels of prolactin, placental lactogen and estrogen are high during late

pregnancy (42), when an increase of beta cell mass compensates for the increased

needs of insulin in the mother (43). ER signaling affects the maternal beta cell

function under these conditions (9; 40; 44), and based on our current data, it also

contributes to the increase of beta cells in fetal pancreas by stimulating neogenesis

and proliferation. Further studies are needed to clarify the precise mechanism of

steroid and lactogenic hormones in the regulation of fetal beta cell formation.

In pancreas of adult mice, severely injured by PDL, Ngn3 gene expression is activated

in endocrine progenitorlike cells and beta cell proliferation is stimulated, resulting in

increased beta cell volume (2; 3). We here report that ER signaling is increased under

these conditions, regulates Ngn3 expression levels and beta cell proliferation, and thus

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Yuchi et al. affects the regenerative processes in PDL pancreas. Using TAMbased genetic tracing is unlikely to be the most adequate approach for evaluating the extent of adult beta cell formation from alternative cell sources in PDL pancreas, since TAM clearly interferes with ER signaling and beta cell formation. A washout procedure might diminish the suppressive effects of TAM on ER signaling, however, it is not excluded that transient exposures to TAM may have long lasting effects on beta cell differentiation.

Both isoforms of ER are present in beta cells and contribute to control of function, proliferation and survival (810; 29; 40; 45) although mechanistic details are lacking

(reviewed in (46)). PDL increased the number of transcripts encoding ERα while decreasing ERβ mRNA to a nearly undetectable level. Deletion of only ERα prevented PDLinduced proliferation of beta cells and although this does not exclude a role for ERβ, the latter was not able to compensate for the defect. Interestingly, the cell cycle is activated through ERα signaling when ERβ is lowly expressed as in mouse mammary cells (20) and human isletderived precursor cells (47). Also, in breast cancer with ERdependent growth, the abundance of ERα encoding transcripts increased while that of ERβ mRNA decreased (48; 49). This suggests that tissues with an increased growth tendency may require elevated ERα expression and an increased

ERα/ERβ ratio. Moreover, in the case of beta cells, several conditions now appear to stimulate growth via ERα, including PDL injury, embryonic pancreas, and pregnancy.

Compared to shamtreatment, the tail part of PDL pancreas shows increased estrogen concentration, proliferation and nuclear ERα localization in beta cells. The latter two

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processes were blunted in ERα/ mice as well as by aromatase inhibition and

enhanced by intrapancreas injection of E2 at a dose similar to its concentration in

PDL pancreas, suggesting a role for synthesis of E2, possibly by newly formed

adipocytes in situ (50). In addition, we showed that local administration of E2

potentiated Ngn3 gene expression in PDL pancreas. Since estrogen and activation of

ERα do not increase betacell proliferation in rodent models for diabetes (51) and in

human islets transplanted in diabetic mice (52) we cannot exclude that the estrogen

effects described in this study would be specific to the PDL model. Nevertheless, our

data suggest also involvement of estrogen in other models of beta cell proliferation,

namely in both the embryonic and mature pancreas during pregnancy.

Estrogen effects also depend on dose and route of administration (53). Cellspecific

delivery of E2 stimulates beneficial activities without unwanted side effects. Estrogen

conjugated to GLP1, which is delivered preferentially to pancreas, reduces adiposity

and improves hyperglycemia, whereas systemic delivery of estrogen potentially

increases its adverse effects such as breast cancer (54).

In summary, the new effects of estrogen signaling in developing and regenerating

pancreas disclosed by the current report, support ERα as a candidate target for control

of glucose homeostasis and beta cell formation in diabetes therapy.

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ACKNOWLEDGEMENTS

Special thanks are to Dr. Pierre Chambon and Dr. Andrée Krust (Université Louis

Pasteur, Illkirch, France) for providing the ERα mutant mice, and Ann Demarré,

Veerle Laurysens, Jan De Jonge, Erik Quartier, Ellen Anckaert and Gaby Schoonjans for technical assistance, Yves Heremans for critical revision of the manuscript.

Financial support was from the VUB Research Council (HH, MVDC), the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT) (HH,

VC), the Beta Cell Biology Consortium (HH), the Chinese Scholarship Council (YY), the Innovative Medicines Initiative Joint Undertaking under grant agreement n°

155005 (IMIDIA) composed of financial contribution from the European Union's

Seventh Framework Programme (FP7/20072013) and EFPIA companies' in kind contribution (HH), Stichting Diabetes Onderzoek Nederland (HH), the Fund for

Scientific Research Flanders (FWO) (HH) and the Interuniversity Attraction Pole networks (HH).

AUTHOR CONTRIBUTIONS AND CONFLICT OF INTEREST

YY, YC, HH, MVDC: study concept and design; YY, YC, BL, SDG, EVO, WS,

NDL, GL, VC, GM, JAVG, HH, MVDC: acquisition, analysis or interpretation of data.

HH is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. The authors declare no conflict of interest.

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2010;34:523530 48. Iwao K, Miyoshi Y, Egawa C, Ikeda N, Tsukamoto F, Noguchi S: Quantitative analysis of estrogen receptoralpha and beta messenger RNA expression in breast carcinoma by realtime polymerase chain reaction. Cancer 2000;89:17321738 49. Leygue E, Dotzlaw H, Watson PH, Murphy LC: Altered estrogen receptor alpha and beta messenger RNA expression during human breast tumorigenesis. Cancer research 1998;58:31973201 50. CaveltiWeder C, Shtessel M, Reuss JE, Jermendy A, Yamada T, Caballero F, BonnerWeir S, Weir GC: Pancreatic duct ligation after almost complete betacell loss: exocrine regeneration but no evidence of betacell regeneration. Endocrinology 2013;154:44934502 51. Tiano JP, DelghingaroAugusto V, Le May C, Liu S, Kaw MK, Khuder SS, Latour MG, Bhatt SA, Korach KS, Najjar SM, Prentki M, MauvaisJarvis F: Estrogen receptor activation reduces lipid synthesis in pancreatic islets and prevents beta cell failure in rodent models of . The Journal of clinical investigation 2011;121:33313342 52. Liu S, Kilic G, Meyers MS, Navarro G, Wang Y, Oberholzer J, MauvaisJarvis F: Oestrogens improve human pancreatic islet transplantation in a mouse model of insulin deficient diabetes. Diabetologia 2013;56:370381 53. Gonzalez C, Alonso A, Diaz F, Patterson AM: Dose and timedependent effects of 17betaoestradiol on insulin sensitivity in insulindependent tissues of rat: implications of IRS1. The Journal of endocrinology 2003;176:367379 54. Finan B, Yang B, Ottaway N, Stemmer K, Muller TD, Yi CX, Habegger K, Schriever SC, GarciaCaceres C, Kabra DG, Hembree J, Holland J, Raver C, Seeley RJ, Hans W, Irmler M, Beckers J, de Angelis MH, Tiano JP, MauvaisJarvis F, PerezTilve D, Pfluger P, Zhang L, Gelfanov V, DiMarchi RD, Tschop MH: Targeted estrogen delivery reverses the metabolic syndrome. Nature medicine 2012;18:18471856

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Table 1.

Table 1. Estradiol concentration in PDL or Sham tail tissue of pancreas, 7 days after surgery.

Sham tail PDL tail Sham tail PDL tail Sham tail PDL tail

(ng/L) (ng/L) (ng/g protein) (ng/g protein) (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

Note: detection limit E2 RIA assay 2.0 ng/L. * undetectable.

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FIGURE LEGENDS

Figure 1. Tamoxifen inhibits beta cell proliferation in PDL pancreas. (A) 8week old male Balbc mice underwent PDL surgery and received either no injection or 5 injections of tamoxifen (TAM, 4mg per injection) or vehicle. 5iodo2’deoxyuridine

(1mg/ml) was administered via the drinking water until the time of sacrifice. (B) The percentage of IdU labeled beta cells increased in PDL tail of mice without injection

(27±3% PDL tail vs. 7±1% PDL head, n=3, ***p<0.001) or in vehicletreated mice

(28±4% PDL tail vs. 7±2% PDL head, n=3), but not in TAMtreated mice (8±2%

PDL tail vs. 5±0.8% PDL head, TAM, n=3, ns: not significant). (C)

Immunofluorescent staining of insulin and IdU in PDL tail (see Fig.1B). (D) Mice were treated as in (A), without IdU administration. The percentage of Ki67+ beta cells in PDL tail was significantly lower in TAM as compared to vehicletreated mice

(0.8±0.01% in TAM vs. 3.0±0.2% in vehicle, n=3, ***p<0.001). (E) Mice were treated as in (A) with TAM or 4OHT administered at 1mg per injection (5 injections).

TAM and 4OHT reduced the percentage of IdU labeled beta cells in PDL tail as compared to vehicle (10%±0.5 in TAM, and 11±3% in 4OHT, vs. 28±4% in vehicle, n=3, *p<0.05). (F) Mice underwent PDL and received one injection of 1 or 2mg TAM or vehicle at day 5 post PDL. (G) The percentage of Ki67+ beta cells was determined at day 7 (2.6±0.3% for vehicle vs. 0.88±0.14% for 1mg TAM, and 0.84±0.25% for

2mg TAM, n=3; **p<0.01). (H) Pregnant mice at day G13.5 of gestation and nonpregnant female mice of the same age, received one IP injection of either vehicle or 1.5mg TAM, and beta cell proliferation was determined at day G15.5 (n=4,

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**p<0.01, ***p<0.001, ns: not significant). Magnification bars are 20m.

Figure 2. Tamoxifen inhibits Ngn3 gene expression and beta cell expansion in

PDL pancreas. (A, B) 8 weeks old male Balbc mice were treated as in Figure 1A,

without IdU administration. Ngn3 mRNA expression was studied in PDL head and

tail tissues and in duodenum. (A) PDL tail Ngn3 mRNA data were normalized to

CycloA mRNA and expressed relative to PDL head pancreas of mice without injection

(None) (n=9; ***p<0.001, *p<0.05). (B) Ngn3 mRNA in duodenum tissue. Data are

normalized to CycloA mRNA and expressed relative to duodenum tissue of mice

without injection (None). (n=39, ***p<0.001). (C, D) Sham or PDL surgery was

performed and TAM administered as before (Figure1A). (C) Total beta cell volume in

PDL tail was significantly increased as compared to Sham tail in mice without

injection (none, 0.46±0.1mm3 PDL tail vs. 0.18±0.03mm3 Sham tail, n=3, *p<0.05),

but not in mice that received TAM (0.35±0.05mm3 PDL tail vs. 0.17±0.03mm3 Sham

tail, n=3, ns: not significant). (D) Insulin content was doubled in PDL tail as

compared to Sham tail pancreas of vehicletreated mice (25.0±2.12µg PDL tail vs.

12.9±0.46µg Sham tail, n=3, ***p<0.001), but the insulin content of PDL tail was

significantly decreased by TAM (20.4±0.6µg with TAM vs. 25.0±2.1µg without

TAM, n=6, *p<0.05).

Figure 3. ERααα contributes to beta cell proliferation and Ngn3 gene expression in

PDL pancreas. (A) 8 weeks old male Balbc mice underwent PDL. Estrogen receptor

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Yuchi et al. mRNA expression was studied in the indicated tissues at day 14. Data were normalized to CycloA mRNA. PDL increased Esr1 mRNA expression (103±7x104

PDL tail vs. 20±10x104 PDL head, n=3, ***p<0.001), whereas Esr2 mRNA was decreased (1.1±0.5x104 PDL tail vs. 18±2x104 PDL head, n=3, *p<0.05). (B) Esr1 mRNA quantity did not differ in FACS sorted beta cells from Sham tail and PDL tail

(n=79, ns: not significant). (C) The percentage of Ki67+ beta cells in 14 days post surgery PDL tail was determined in ERα/ and wildtype littermates. ERα gene inactivation decreased beta cell proliferation in PDL tail (1.28±0.08% ERα/ vs.

2.54±0.07% WT, n=6, ***p<0.001), while basal proliferation in the unligated head part was not changed (0.76±0.05% WT vs. 1.09±0.17% ERα/, n=6, ns: not significant). (D, E) Ngn3 mRNA expression in 14 days post surgery PDL head and tail pancreas and duodenum was determined in ERα/ and wildtype littermates. (D)

Data normalized to CycloA mRNA and expressed relative to the Ngn3 mRNA level in

WT PDL head tissue. Ngn3 mRNA was increased in PDL tail as compared to PDL head of WT mice (n=6, ***p<0.001). Knockout of ERα 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 wild type duodenum. (n=56, *p<0.05).

Figure 4. Increased estrogen levels and ERααα activity in PDL tail pancreas. (A) 8 weekold male Balbc mice underwent PDL surgery. The expression of estrogenregulated genes was studied in the indicated tissues 7 days post PDL (n=4).

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Data are normalized to CycloA mRNA and expressed relative to the gene mRNA level

in PDL head tissue (=1). (B, C, D) 8 weekold male Balbc mice underwent Sham or

PDL surgery and ERα protein was studied in Sham tail and PDL head and tail at day

7 (n=3). ERα protein was predominantly cytosolic in beta cells of (B) Sham tail and

(C) PDL head. (D) ERα was nuclear in beta cells of PDL tail. (E) Nuclear

localization in PDL tail was prevented by administration of TAM (2mg) at day 5

(PDL T+TAM). (F) No ERα protein was detected in pancreas tissue from ERα/

mice. (G, H, I) E2 or vehicle was injected locally into PDL tail at the day of surgery

and repeated 3 days later. Mice were studied at day 7 post surgery and IdU (50mg/kg)

was i.p.injected 16hrs and 2hrs before sacrifice. (G) Immunofluorescent staining of

insulin and IdU in sections of PDL tail pancreas. (H) The percentage of IdU labeled

beta cells in PDL tail pancreas was increased by E2 (2.4 ± 0.4% for vehicle vs.

3.3±0.3% for 20pg E2 and 4.5±0.4% for 100pg E2, n=6, **p<0.01). (I) Ngn3 mRNA

expression was normalized to CycloA mRNA and expressed relative to the Ngn3

mRNA level in vehicletreated PDL tail tissue (n=6, *p<0.05, ***p<0.001). (J)

Balbc male mice underwent PDL surgery and aromasin was s.c.injected. (K)

Quantification of Ki67+ beta cells in PDL tail at day 7. Aromasin (ARO) decreased

beta cell proliferation in PDL tail (1.5±0.51% ARO vs. 3.1±0.16% vehicle, n=3,

**p<0.01). Magnification bars are 20m.

Figure 5. ERααα regulates beta cell proliferation and Ngn3+ progenitors in

embryonic pancreas. (A) Pregnant mice received i.p. vehicle or 1.5mg TAM at

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E16.5 , embryonic pancreas was dissected at E18.5 and the percentage of Ki67+ beta cells quantified (6.3±0.01% TAM vs. 12.3±0.01% vehicle, n=34, **p<0.01). (B, C)

Wild type and ERα/ embryos were collected at E15.5. For embryos treated with

TAM, 1.5mg TAM was given i.p. to pregnant mice at E13.5. mRNA expression of indicated genes was studied by RTqPCR. (B) mRNA data are normalized to CycloA mRNA and expressed relative to the mRNA level in wild type embryonic pancreas.

Ngn3, Pax6 and Pax4 mRNA level of embryonic pancreas was significantly decreased in wild type embryos that received TAM and in ERα/ embryos, compared to wild type control embryos (n=49, *p<0.05, **p<0.01, ***p<0.001). (C) Ngn3 mRNA level of embryonic duodenum was significantly lower in TAMtreated and in

ERα/ embryos than in wild type control embryos (n=49, **p<0.01, ***p<0.001). (D)

Pregnant Ngn3YFP mice received i.p. vehicle or 1.5mg TAM at E13.5 and embryonic pancreas was dissected at E15.5. The percentage of Ecadherin+ epithelial cells that was NGN3+ or YFP+ was determined by immunofluorescent staining

(YFP+Ecadherin+ cells: 9.0±1.02% vehicle vs. 4.5±0.84% TAM, NGN3+ Ecadherin+ cells: 8.4±1.00% Vehicle vs. 3.4±0.25% TAM, n=3, *p<0.05, **p<0.01). (E) Wild type and ERα/ embryos were collected from pregnant ERα+/ mice at E15.5. The percentage of Ecadherin+ cells that expressed Ngn3 was quantified in ERα/ and wild type embryonic pancreas (5.4±0.5% WT vs. 3.1±0.5% ERα/, n=3, *p<0.05).

Magnification bars are 50m.

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SUPPLEMENTARY FIGURE LEGENDS

Figure S1. Tamoxifen decreases beta cell proliferation in PDL pancreas in

Ngn3CreERT;R26RYFP mice. 8weeks old male Ngn3CreERT;R26RYFP mice underwent

PDL surgery and received no injection or 5 injections of tamoxifen (4mg per

injection). IdU (1mg/ml) was sequentially administered via the drinking water until

the time of sacrifice. The percentage of beta cells that were IdU+ in PDL tail was

determined at day 14 by immunofluorescent staining for insulin and IdU (21±2.8%

vehicle vs. 11±0.5% TAM, n=3, *p<0.05).

Figure S2. Study of the recombination efficacy in RipCreERT;R26RYFP mice.

8weeks old RipCreERT;R26RYFP mice underwent PDL and followed with injections of

TAM or 4OHT (1mg/injection) according to the scheme (Figure 1A). Mice were

sacrificed at day 14 and immunofluorescent staining was performed for insulin and

YFP on paraffin sections of PDL tail. TAM and 4OHT are equally efficient in

inducing recombination (96±0.9% TAM and 94±0.2% 4OHT, n=2, ns: not

significant). Magnification bars are 20m.

Figure S3. Quality control of ERααα-/- mice. (A) Morphology analysis of uterus from

ERα/ and wild-type female littermates. Abnormal development of uterus in ERα/

mice. (B) Western blot analysis of ERα protein in WT and ERα/ uterus. Lack of ERα

immunoreactivity in ERα/ uterus (Right panel), βACTIN immunoblotting used as

internal control. Molecular weights are indicated, 50g protein was loaded for each

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Yuchi et al. sample. (C) Detection of ERα mRNA transcripts by RTPCR (35 cycles) using primers specific to exon3. No ERα RNA in uterus of ERα/ (/) mice containing exon3 could be detected (left panel, lane1 and 2), but very strong expression of ERα

(Right panel, lane3) in wild type mice (+/+). RTPCR products were separated on agarose gel, RTPCR of β-actin used as an expression control. 300ng cDNA was loaded from each sample. (D) Immunofluorescent staining for ERα in sections of uterus from wild type female mice; ERα protein (red) and DNA (blue); magnification bar is 50m.

Figure S4. Tamoxifen treatment has no effect on inflammatory signals and myeloid cells in the PDL pancreas. 8week old male Balb/c mice underwent PDL surgery or sham surgery. Mice with PDL either received 3 injections of tamoxifen

(TAM, 4 mg per injection) or vehicle. At 3 days postsurgery, (A) singlecell suspensions were analyzed by flow cytometry. Cells were gated on CD11b+ and checked for expression of F4/80, Ly6C, MHCII (for Ly6Chi monocytes, immature

MΦ and MHCIIlo and MHCIIhi MΦ), Ly6G (for neutrophils) and SiglecF (for eosinophils). MΦ=macrophage. Data are expressed as % of total cells (mean ±

SEM, n= 34, **p<0.01, ***p<0.001, ns: not significant, by 2way ANOVA with

Sidak posttest). (B,C) Quantitative RTPCR on tissue was performed for (B) markers of inflammatory cells: CD45 (CD45 antigen), or of macrophages: F4/80 (F4/80 antigen), CD163 (scavenger receptor CD163), NOS2 (inducible nitric oxide synthase),

MRC1 (macrophage mannose receptor C1), and (C) indicated inflammatory cytokines.

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The mRNA levels in PDL tail are expressed relative to sham tail pancreas (fold

increase, mean ± SEM, n=34, **p<0.01, ns: not significant, by multiple ttest with

Sidak posttest). Probes for RTqPCR were from Integrated DNA Technologies (IDT,

Leuven, Belgium): CD163 (Mm.PT.56a.15897116), NOS2 (Mm.PT.56a.43705194),

MRC1 (Mm.PT.56a.42560062), IL6 (Mm.PT.53.10005566), IFNγ

(Mm.PT.53.13380517), TNFα (Mm.PT.56a.12575861), IL1β (Mm.PT.53a.16901608),

IL10 (Mm.PT.53.13531087), or from Applied Biosystems (Life Technologies, Gent,

Belgium): CD45 (Mm00448463_m1), F4/80 (Mm00802530_m1), TGFβ

(Mm00441727_g1).

Figure S5. Study of the recombination efficacy in embryonic pancreas of

RipCreERT;R26RYFP mice. Pregnant RipCreERT;R26RYFP mice received 1.5mg TAM or

vehicle i.p. at E16.5. Embryonic pancreas was dissected and analyzed by

immunofluorescent staining for insulin and YFP at day E18.5 to check the

recombination efficacy (89.0±1.90% beta cells were labeled by YFP in TAM injected

group vs. 1.6±0.89% in vehicle group, n=3). Magnification bar is 100m.

Figure 1 Diabetes Page 36 of 45 A B D

ns 4 40 None *** ns *** *** Vehicle PDL * TAM IHC 30 TAM 3

***** 20 2

day 0 IdU 14 cells (% of beta cells) + 10 1 cells (% of beta cells) +

IdU 0 0 PDL head PDL tail Vehicle TAM

C PDL tail Ki67 None PDL tail Vehicle PDL tail TAM PDL tail Vehicle TAM

INS IdU INS IdU INS IdU INS Ki67 INS Ki67

E 40 * * ns Vehicle TAM 4OHT 30

20 cells (% of beta cells) + 10

INS IdU DNA INS IdU DNA INS IdU DNA 0 Vehicle TAM 4OHT PDL tail IdU

F G H 8 *** ** 4 ** Vehicle Vehicle 6 ** TAM TAM 3 PDL * TAM IHC 4 2 ns * beta cells (%) +

day 0 5 7 cells (% of beta cells) + 2

1 Ki67 For Peer Review Only 0 0 Vehicle 1 mg 2 mg Non-pregnant pregnant PDL tail Ki67 Figure 2 Page 37 of 45 Diabetes

A B

* 1.5 50 None *** *** Vehicle 40 TAM 1.0 30

20 0.5 Ngn3 mRNA 10 Doudenum Ngn3 mRNA

0 0.0 PDL Head PDL Tail NoneVehicle TAM

C D ** Sham tail Vehicle 30 *** * ns 0.6 * PDL tail TAM ) 3 ns 20 0.4

10

0.2 Insulin content ( µ g) Beta cell volume (mm 0.0 0 Vehicle TAM Sham tail PDL tail FigureDiabetes 3 Page 38 of 45

A B C ns ns

) 120 *** Esr1 WT -4 3 *** *** Esr2 ERα-/- 100 2.0 ns 80 1.5 2 40 ns * 1.0 1 20 cells (% of beta cells)

0.5 + Esr1 mRNA in beta cells Esr mRNA/ CycloA (x 10

0 0.0 Ki67 0 PDL head PDL tail Sham tail PDL tail PDL head PDL tail

D E 1.5 *** WT * WT 15 ns *** ERα-/- ERα-/- 1.0 10

Ngn3 mRNA 0.5

Ngn3 mRNA 5

0 0.0 PDL head PDL tail Duodenum Page 39 of 45 FigureDiabetes 4

A 35 ** PDL head 30 PDL tail 25 20

10 ** Target mRNA *** 5 *** * * 0 Tert Lcn2 ptma IL6 E2F1 C-Jun B Sham T Sham T Sham T H ** 6 ns **

4 cells (% of beta cells) + 2 INS ER DNA ER DNA ER C PDL H PDL H PDL H 0 PDL tail IdU Vehicle 20pg E2 100pg E2

*** I 4 * INS ER DNA + ER DNA ER D PDL T PDL T PDL T 3

2

INS ER DNA ER DNA ER 1 E PDL T + TAM PDL T + TAM PDL T + TAM PDL tail Ngn3 mRNA 0

Vehicle 20pg E2 100pg E2

INS ER DNA ER DNA ER J F ER -/- ER -/- ER -/- day -4 -2 0 2 4 7 ARO PDL IHC

INS ER DNA ER DNA ER K 4 ** G 3

Vehicle 20pg E2 100pg E2 2 cells (% of beta cells) +

1

For Peer Review Only

PDL tail Ki67 0 INS IdU INS IdU INS IdU Vehicle ARO Figure 5 Diabetes Page 40 of 45 A Vehicle E18.5 TAM E18.5 15 **

10 cells (% of beta cells) + 5

E18.5 Ki67 0 INS Ki67 DNA INS Ki67 DNA Vehicle TAM

1.5 1.5 B WT C *** WT ** * ** ** WT + TAM WT+TAM *** ** *** -/- ER -/- ER 1.0 1.0

0.5 0.5 Ngn3 mRNA E15.5 Target mRNA

0.0 0.0 Pdx1 Ngn3 Pax6 Pax4 Duodenum D Vehicle Vehicle

Vehicle 15 cells) + TAM * ** 10 Ecad NGN3 DNA Ecad YFP DNA TAM TAM 5 cells (% of E-cadherin +

0

Marker YFP NGN3

Ecad NGN3 DNA Ecad YFP DNA E WT ER -/-

cells) 8 + WT

* -/- 6 ER

4

2 cells (% of E-cadherin

For Peer Review Only + 0 NGN3 Ecad NGN3 DNA Ecad NGN3 DNA NGN3 Page 41 of 45 SupplementaryDiabetes Figure S1

30 *

20 cells (% of beta cells)

+ 10

0

PDL tail IdU None TAM

Figure S1. Tamoxifen decreases beta cell proliferation in PDL pancreas in Ngn3CreERT;R26RYFP mice. 8-weeks old male Ngn3CreERT;R26RYFP mice underwent PDL surgery and received no injection or 5 injections of tamoxifen (4mg per injection). IdU (1mg/ml) was sequentially administered via the drinking water until the time of sacrifice. The percentage of beta cells were IdU+ in PDL tail was determined at day 14 by immunofluorescent staining for insulin and IdU (21±2.8% vehicle vs. 11±0.5% TAM, n=3, *p<0.05 ). SupplementaryDiabetes Figure S2 Page 42 of 45 ns 100

80

60

40

cells (% of beta cells) 20 +

YFP 0 TAM 4OHT

TAM 4OHT

INS DNA INS DNA

YFP DNA YFP DNA Figure S2. Study of the recombination efficacy in RipCreERT; R26RYFP mice. 8-weeks old RipCreERT;R26RYFP mice underwent PDL and followed with injections of TAM or 4OHT (1mg/injection) according to the scheme (Figure 1A). Mice were sacrificed at day 14 and immunofluorescent staining was performed for insulin and YFP on paraffin sections of PDL tail. TAM and 4OHT are equally efficient in inducing recombination (96±0.9% TAM and 94±0.2% 4OHT, n=2, ns: not significant). Magnification bars are 20μm. Page 43 of 45 SupplementaryDiabetes Figure S3 A D ER -/- Uterus WT Uterus WT Uterus

ER DNA ER

B Ab ER C

98 64 50 ER 36 -/----/-

22 -actin Ab -ACTIN

64 1 2 3 50 -/- -/- +/+

36

+/+ -/- Figure S3. Quality control of ER -/- mice. (A) Morphology analysis of uterus from ER -/- and wild-type female littermates. Abnormal development of uterus in ER -/- mice. (B) Western blot analysis of ER protein in WT and ER -/- uterus. Lack of ER immunoreactivity in ER -/- uterus (Right panel), -ACTIN loaded for each sample. (C) Detection of ER mRNA transcripts by RT-PCR (35 cycles) using primers specific to exon3. No ER RNA in uterus of ER -/- (-/-) mice containing exon3 could be detected (left panel, lane1 and 2), but very strong expression of ER (Right panel, lane3) in wild type mice (+/+). RT-PCR products were separated on agarose gel, RT-PCR of -actin used as an expression control. 300ng cDNA was loaded from each sample. (D) Immunofluorescent staining for ER in sections of uterus from wild type female For Peer Review Only mice; ER protein (red) and DNA (blue); SupplementaryDiabetes Figure S4 Page 44 of 45

ns 50 *** ham A PDL PDL +TAM 40

30 ** ns 20 % of total cells total of % * ns 10

0

lo M hi M mono CD11b+ hi Eosino Neutro MHC-II MHC-II Ly6C Immature M

B 150 ** ns ns 100 ** ** ns

** ns ns 50 ** mRNA in PDL tail PDL in mRNA (fold of Sham tail mRNA) tail Sham of (fold 0

ns ns C 60 ** **

40

** ns** ns **ns ** ns 20 mRNA in PDL tail PDL in mRNA (fold of Sham tail) Sham of (fold

0

Figure S4. Tamoxifen treatment has no effect on inflammatory signals and myeloid cells in the PDL pancreas. 8-week old male Balb/c mice underwent PDL surgery or sham surgery. Mice with PDL either received 3 injections of tamoxifen (TAM, 4 mg per injection) or vehicle. At 3 days post-surgery, (A) single-cell suspensions were analyzed by flow cytometry. Cells were gated on CD11b+ and checked for expression of F4/80, Ly6C, MHC-II (for Ly6Chi monocytes, immature M and MHC-IIlo and MHC-IIhi M ), Ly6G (for neutrophils) and SiglecF (for eosinophils). M =macrophage. Data are expressed as % of total cells (mean ± SEM, n= 3-4, **p<0.01, ***p<0.001, ns: not significant, by 2-way ANOVA with Sidak post-test). (B,C) Quantitative RT-PCR on tissue was performed for (B) markers of inflammatory cells: CD45 (CD45 antigen), or of macrophages: F4/80 (F4/80 antigen), CD163 (scavenger receptor CD163), NOS2 (inducible nitric oxide synthase), MRC1 (macrophage mannose receptor C1), and (C) indicated inflammatory cytokines. The mRNA levels in PDL tail are expressed relative to sham tail pancreas (fold increase, mean ± SEM, n=3-4, **p<0.01, ns: not significant, by multiple t-test with Sidak post-test). Probes for RT-qPCR were from Integrated For Peer Review Only DNA Technologies (IDT, Leuven, Belgium): CD163 (Mm.PT.56a.15897116), NOS2 (Mm.PT.56a.43705194), MRC1 (Mm.PT.56a.42560062), IL6 (Mm.PT.53.10005566), IFN (Mm.PT.53.13380517), TNF (Mm.PT.56a.12575861), IL1 (Mm.PT.53a.16901608), IL10 (Mm.PT.53.13531087), or from Applied Biosystems (Life Technologies, Gent, Belgium): CD45 (Mm00448463_m1), F4/80 (Mm00802530_m1), TGF (Mm00441727_g1). Page 45 of 45 SupplementaryDiabetes Figure S5

100

80

60

40 ce ll s (% o f beta cel ls) +

20

E18.5 YFP 0 TAM Vehicle

E18.5 E18.5

YFP DNA INS DNA

Figure S5. Study of the recombination efficacy in embryonic pancreas of RipCreERT;R26RYFP mice. Pregnant RipCreERT;R26RYFP mice received 1.5mg TAM or vehicle i.p. at E16.5. Embryonic pancreas was dissected and analyzed by immunofluorescent staining for insulin and YFP at day E18.5 to check the recombination efficacy (89.0±1.90% beta cells were labeled by YFP in TAM injected group vs. 1.6±0.89% in vehicle group, n=3). Magnification bar is100μm.