Estrogens protect pancreatic beta-cells from apoptosis and prevent insulin-deficient diabetes mellitus in mice.
ABSTRACT In diabetes, the death of insulin-producing beta-cells by apoptosis leads to insulin deficiency. The lower prevalence of diabetes in females suggests that female sex steroids protect from beta-cell injury. Consistent with this hypothesis, 17beta-estradiol (estradiol) manifests antidiabetic actions in humans and rodents. In addition, estradiol has antiapoptotic actions in cells that are mediated by the estrogen receptor-a (ERalpha), raising the prospect that estradiol antidiabetic function may be due, in part, to a protection of beta-cell apoptosis via ERalpha. To address this question, we have used mice that were rendered estradiol-deficient or estradiol-resistant by targeted disruption of aromatase (ArKO) or ERalpha (alphaERKO) respectively. We show here that in both genders, ArKO(-/-) mice are vulnerable to beta-cell apoptosis and prone to insulin-deficient diabetes after exposure to acute oxidative stress with streptozotocin. In these mice, estradiol treatment rescues streptozotocin-induced beta-cell apoptosis, helps sustain insulin production, and prevents diabetes. In vitro, in mouse pancreatic islets and beta-cells exposed to oxidative stress, estradiol prevents apoptosis and protects insulin secretion. Estradiol protection is partially lost in beta-cells and islets treated with an ERalpha antagonist and in alphaERKO islets. Accordingly, alphaERKO mice are no longer protected by estradiol and display a gender nonspecific susceptibility to oxidative injury, precipitating beta-cell apoptosis and insulin-deficient diabetes. Finally, the predisposition to insulin deficiency can be mimicked in WT mice by pharmacological inhibition of ERalpha by using the antagonist tamoxifen. This study demonstrates that estradiol, acting, at least in part, through ERalpha, protects beta-cells from oxidative injury and prevents diabetes in mice of both genders.
- [Show abstract] [Hide abstract]
ABSTRACT: Background:We conducted a population-based cohort study to assess whether tamoxifen treatment is associated with an increased incidence of diabetes.Methods:Data obtained from the Taiwanese National Health Insurance Research Database were used for a population-based cohort study. The study cohort included 22 257 breast cancer patients diagnosed between 1 January 2000 and 31 December 2004. Among them, 15 210 cases received tamoxifen treatment and 7047 did not. Four subjects without breast cancer were frequency-matched by age and index year as the control group. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using multivariate Cox proportional hazards regression analysis.Results:Breast cancer patients exhibited a 14% higher rate of developing diabetes (adjusted HR=1.14, 95% CI=1.08-1.20) compared with non-breast cancer controls, but the significant difference was limited to tamoxifen users. In addition, tamoxifen users exhibited a significantly increased risk of diabetes compared with non-tamoxifen users among women diagnosed with breast cancer (adjusted HR=1.31, 95% CI=1.19-1.45). Stratification by age groups indicated that both younger and older women diagnosed with breast cancer exhibited a significantly higher risk of diabetes than the normal control subjects did, and tamoxifen users consistently exhibited a significantly higher diabetes risk than non-tamoxifen users or normal control subjects did, regardless of age. Both recent and remote uses of tamoxifen were associated with an increased likelihood of diabetes.Conclusions:The results of this population-based cohort study suggested that tamoxifen use in breast cancer patients might increase subsequent diabetes risk. The underlying mechanism remains unclear and further larger studies are mandatory to validate our findings.British Journal of Cancer advance online publication 16 September 2014; doi:10.1038/bjc.2014.488 www.bjcancer.com.British Journal of Cancer 09/2014; · 4.82 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Beta-cell dysfunction is an early event in the natural history of type 2 diabetes. However, its progression is variable and potentially influenced by several clinical factors. We report the baseline data of the BetaDecline study, an Italian prospective multicenter study on clinical predictors of beta-cell dysfunction in type 2 diabetes.PLoS ONE 10/2014; 9(10):e109702. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Defects in insulin secretion play a central role in the pathogenesis of type 2 diabetes, yet the mechanisms driving beta-cell dysfunction remain poorly understood, and therapies to preserve glucose-dependent insulin release are inadequate. We report a luminescent insulin secretion assay that enables large-scale investigations of beta-cell function, created by inserting Gaussia luciferase into the C-peptide portion of proinsulin. Beta-cell lines expressing this construct cosecrete luciferase and insulin in close correlation, under both standard conditions or when stressed by cytokines, fatty acids, or ER toxins. We adapted the reporter for high-throughput assays and performed a 1,600-compound pilot screen, which identified several classes of drugs inhibiting secretion, as well as glucose-potentiated secretagogues that were confirmed to have activity in primary human islets. Requiring 40-fold less time and expense than the traditional ELISA, this assay may accelerate the identification of pathways governing insulin secretion and compounds that safely augment beta-cell function in diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.Cell metabolism. 01/2015; 21(1):126-37.
Estrogens protect pancreatic ?-cells from apoptosis
and prevent insulin-deficient diabetes mellitus in mice
Cedric Le May*, Khoi Chu†, Min Hu*, Christina S. Ortega*, Evan R. Simpson‡, Kenneth S. Korach§, Ming-Jer Tsai†,
and Franck Mauvais-Jarvis*†¶
*Division of Diabetes, Endocrinology & Metabolism, Department of Medicine and†Department of Molecular & Cellular Biology, Baylor College of Medicine,
Houston, TX 77030;‡Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia; and§National Institute of Environmental Health Sciences,
National Institutes of Health, Research Triangle Park, NC 27709
Communicated by Jean D. Wilson, University of Texas Southwestern Medical Center, Dallas, TX, April 11, 2006 (received for review October 28, 2005)
In diabetes, the death of insulin-producing ?-cells by apoptosis
leads to insulin deficiency. The lower prevalence of diabetes in
females suggests that female sex steroids protect from ?-cell
injury. Consistent with this hypothesis, 17?-estradiol (estradiol)
manifests antidiabetic actions in humans and rodents. In addition,
estradiol has antiapoptotic actions in cells that are mediated by the
estrogen receptor-a (ER?), raising the prospect that estradiol an-
tidiabetic function may be due, in part, to a protection of ?-cell
apoptosis via ER?. To address this question, we have used mice
that were rendered estradiol-deficient or estradiol-resistant by
targeted disruption of aromatase (ArKO) or ER? (?ERKO) respec-
tively. We show here that in both genders, ArKO?/?mice are
vulnerable to ?-cell apoptosis and prone to insulin-deficient dia-
betes after exposure to acute oxidative stress with streptozotocin.
In these mice, estradiol treatment rescues streptozotocin-induced
?-cell apoptosis, helps sustain insulin production, and prevents
diabetes. In vitro, in mouse pancreatic islets and ?-cells exposed to
oxidative stress, estradiol prevents apoptosis and protects insulin
secretion. Estradiol protection is partially lost in ?-cells and islets
treated with an ER? antagonist and in ?ERKO islets. Accordingly,
?ERKO mice are no longer protected by estradiol and display a
gender nonspecific susceptibility to oxidative injury, precipitating
?-cell apoptosis and insulin-deficient diabetes. Finally, the predis-
position to insulin deficiency can be mimicked in WT mice by
pharmacological inhibition of ER? by using the antagonist tamox-
ifen. This study demonstrates that estradiol, acting, at least in part,
through ER?, protects ?-cells from oxidative injury and prevents
diabetes in mice of both genders.
estradiol ? oxidative stress
events, in type 1 and the late stages of type 2 diabetes, proin-
flammatory cytokines and?or chronic elevation of blood glucose
generate oxidative stress in pancreatic islets, which ultimately
provokes ?-cell death by apoptosis (1, 2). To date, little progress
has been made in understanding how to halt the loss of insulin-
Recent data from human and animal studies suggest that the
ovarian estrogen, 17?-estradiol (estradiol), protects insulin pro-
duction in diabetic states. First, the prevalence of diabetes is
lower in females (3). More specifically, this observed ‘‘female
protection’’ is more pronounced in diabetic syndromes with
severe insulin deficiency (4, 5). Second, in most rodent models
of diabetes, females are protected from ?-cell death and hyper-
glycemia, whereas, conversely, males develop overt insulin-
deficient diabetes (reviewed in ref. 6). Furthermore, estradiol,
used in pharmacological concentrations, protects human pan-
creatic islets from apoptosis induced by proinflammatory cyto-
kines in vitro (7). In both sexes, estradiol is biosynthesized by the
cytochrome P450 enzyme complex called aromatase (8) and acts
ER? and ER? (9). ER? is predominant in many biological
n diabetes, hyperglycemia arises from the loss of insulin
production from pancreatic ?-cells. Disregarding the causal
functions of estradiol (9). In neurons, notably, estradiol sup-
presses apoptosis in vivo. It is considered that this estrogenic
effect is primarily mediated through ER? (10). Together, this
information raises the prospect that estradiol antidiabetic func-
tion may be due, in part, to a protection of ?-cell apoptosis
through activation of ER?.
Estradiol Protects Pancreatic Islet Survival and Function in Vivo. To
study the protection of estradiol in vivo, we used estradiol-
deficient (?ERKO) aromatase-deficient (ArKO?/?) mice. We
provoked oxidative stress in ?-cells, using a single high-dose
injection of streptozotocin (STZ) (150 mg?kg of body weight),
which augments the generation of reactive oxygen species in
pancreatic islets (11). We determined the STZ dose used from
a dose–response study that defined the minimal amount causing
diabetes with a gender dimorphism (see Table 1, which is
published as supporting information on the PNAS web site). In
basal conditions, WT and ArKO?/?mice of both genders
displayed a normal islet architecture, with insulin-producing
?-cells in a central location, glucagon-producing ?-cells at the
periphery (Fig. 1 A and B), similar ?-cell numbers (Fig. 1 C and
D), and pancreatic insulin concentration (Fig. 1 E and F). After
exposure to STZ in WT mice, females were protected and
retained a normal islet architecture, ?-cell numbers, and pan-
creas insulin concentration (Fig. 1 A, C, and E); conversely,
males were vulnerable to STZ and exhibited a loss of ?-cells and
pancreas insulin concentration (Fig. 1 B, D, and F). We found
that exposure to STZ in ArKO?/?mice of both genders pro-
voked severe ?-cell destruction and a dramatic decrease in
pancreatic insulin concentration (Fig. 1 A–F). Furthermore, in
males, the fall in pancreas insulin concentration was more severe
in WT than in ArKO?/?mice (Fig. 1F). In WT and mutant mice
the destruction of ?-cells (Fig. 1 A–D) and helped retain
pancreas insulin concentration in ArKO?/?mice (Fig. 1 E
Estradiol Prevents ?-Cell Apoptosis in Vivo and Protects Mice from
estradiol protection from ?-cell death in vivo. ?-cell apoptosis
(TUNEL staining) and proliferation (KI67 staining) were as-
sessed on pancreas sections from male mice. In STZ-exposed
WT and ArKO?/?males, we observed a major increase in
apoptotic ?-cells (Fig. 2). Conversely, estradiol treatment sup-
pressed apoptosis in both groups, demonstrating the importance
Conflict of interest statement: No conflicts declared.
Abbreviations: ArKO, aromatase-deficient; ?ERKO, ?ER-deficient; ER, estrogen receptor;
¶To whom correspondence should be addressed at: Department of Medicine, Division of
TX 77030. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
June 13, 2006 ?
vol. 103 ?
of this hormone in preventing acute oxidative injury in vivo (Fig.
2). In contrast, estradiol treatment was not associated with
increased ?-cell proliferation (Fig. 2) or ?-cell hypertrophy (see
web site). The glucose analogue STZ requires the ?-cell glucose
transporter GLUT2 to allow for its apoptotic effect (12). Thus,
we studied the effect of estradiol on GLUT2 protein expression
in mouse islets, but we observed no effect, ruling out the
possibility that estradiol down-regulates GLUT2 expression,
thereby preventing STZ entry in ?-cells (see Fig. 8, which is
published as supporting information on the PNAS web site).
We next determined the consequences of estradiol deficiency
and treatment on whole-animal glucose homeostasis. Consistent
with the results shown previously, female WT mice were less
prone to STZ-induced insulin-deficient diabetes than WT males
(Fig. 3, compare A–C with D–F); however, ArKO?/?mice of
both genders showed a severe predisposition to insulin-deficient
diabetes compared with WT littermates (Fig. 3). Conversely,
estradiol treatment prevented the development of insulin-
deficient diabetes in WT and ArKO?/?mice regardless of their
sex (Fig. 3). In all experiments described above, estradiol treat-
ment was associated with physiological serum hormone concen-
trations (data not shown).
ER? Is Expressed In Pancreatic ?-Cells. The predominant biological
effects of estradiol are mediated through two members of the
nuclear receptor family, ER? and ER? (9). We focused on ER?
because no significant role of ER? has been demonstrated in
energy metabolism (13). Using qualitative and real-time quan-
titative RT-PCR, we confirmed that the full-length ER? mRNA
was expressed (see Fig. 9 A and B, which is published as
supporting information on the PNAS web site). The study of
protein expression by Western blotting demonstrated that the
staining for insulin (green) and glucagon (red) was performed in the indicated mice in basal conditions (vehicle) and after STZ injection (day 8). (C and D) ?-cell
(24) in basal conditions and after STZ challenge (day 8). Values are represented as scatter plot and mean ? SE in ng?mg of pancreas (n ? 4–19).*, P ? 0.05;**,
P ? 0.01.
Disruption of islet architecture and function in ArKO?/?mice exposed to STZ. (A and B) Representative pancreatic sections showing immunofluorescent
immunostaining from pancreas section after vehicle or STZ injection in male
mice.**, P ? 0.01.
Estradiol protects mice from STZ-induced ?-cell apoptosis in vivo.
Le May et al.
June 13, 2006 ?
vol. 103 ?
no. 24 ?
islets and MIN6 cells (Fig. 4A), thus confirming results obtained
by RT-PCR. A smaller 58-kDa isoform was expressed in mouse
islets but not in MIN6. The functionality of this isoform is
unknown. Neither islets nor ?-cells expressed the 46-kDa iso-
form, which is expressed in human breast cancer cells, MCF7
(Fig. 4A). We next studied ER? localization in mouse pancreatic
section by immunofluorescence using the same antibody. ER?
was present in ?-cells with an exclusive nuclear localization (Fig.
4B). As a control of antibody specificity, we confirmed that the
antibody detected ER? in the nuclei of mouse uterine cells. ER?
was expressed in both sexes, and its expression was present in
exocrine cells (see Fig. 10, which is published as supporting
information on the PNAS web site).
asked whether estradiol acts on ER? to suppress apoptosis in
mouse islets from WT and ER?-deficient mice (?ERKO). We
exposed islets to hydrogen peroxide (H2O2), which causes ?-cell
injury during exposure to cytokines or chronic hyperglycemia in
diabetic states (2). In WT islets, exposure to H2O2provoked a
4-fold increase in apoptotic cells (Fig. 5A) that was suppressed
by estradiol treatment. Conversely, treatment with the selective
ER? antagonist MPP (14) partially reversed estradiol protec-
tion, whereas exposure to the ER antagonist tamoxifen totally
reversed estradiol’s antiapoptotic property (Fig. 5A). Accord-
MPP, estradiol protection was partially abolished. In addition,
estradiol’s antiapoptotic property through ER? was also ob-
served in islets treated by STZ (data not shown) and in the ?-cell
line MIN6 treated by either STZ or H2O2(see Fig. 11 A and B,
which is published as supporting information on the PNAS web
site). Furthermore, consistent with its antiapoptotic properties,
in WT islets, after exposure to H2O2, estradiol prevented
alteration of insulin release, which was reversed by MPP and
tamoxifen (Fig. 5B). Accordingly, in ?ERKO?/?islets, estradiol
protection of insulin release was partially eliminated (Fig. 5B).
Elimination of ER? in Mice Predisposes to Insulin-Deficient Diabetes.
We determined the physiological relevance of the results pre-
sented above in ?ERKO mice. At basal levels, young female WT
and ?ERKO?/?mice showed similar insulin secretion in re-
sponse to glucose (Fig. 6A), pancreas insulin concentration, and
islet ?-cell numbers (Fig. 6 B and C). On the contrary, exposure
calculated by Kaplan–Meier estimation in female (A) (n ? 10–28) and male (D) (n ? 10–30) mice after STZ challenge (150 mg?kg of body weight). (B and E)
Random-fed blood glucose (day 8), in female (B) and male (E) mice. (C and F) The ratio of random-fed of insulin (pg?ml) and glucose (mg?dl) at day 8 was used
as an index of insulin deficiency in female (C) and male (F) mice.*, P ? 0.05;**, P ? 0.01.
www.pnas.org?cgi?doi?10.1073?pnas.0602956103Le May et al.
to STZ caused severe islet ?-cell destruction in ?ERKO?/?
compared with WT female mice (Fig. 6C). We further studied
the effect of ER? deletion on the occurrence of STZ-induced
diabetes in WT and mutant mice of both genders. In female
?ERKO?/?mice, exposure to STZ caused a rise in the incidence
of insulin-deficient diabetes compared with WT mice (Fig. 6D).
This phenotypic abnormality was dominant, because it was also
observed in ?ERKO?/?mice. In addition, treatment with the
antagonist tamoxifen did not protect female ?ERKO?/?mice
from STZ toxicity. Rather, as observed in islets, tamoxifen
reversed the protection of endogenous estradiol in WT female
mice, resulting in an increased incidence of insulin-deficient
diabetes (Fig. 6D). These results demonstrate that pharmaco-
Furthermore, in ?ERKO?/?mice, suppression of estradiol
Accordingly, in ovariectomized ?ERKO?/?mice, treatment by
estradiol partially reversed diabetes (Fig. 6D), suggesting that, in
the absence of ER?, as observed in islets, estradiol partially
protects ?-cells via an alternate signaling pathway. Finally, male
WT and ?ERKO?/?mice were exposed to a nondiabetogenic
single low dose of STZ (75 mg?kg of body weight; Table 1). Such
a low dose of STZ caused only a moderate increase in diabetes
incidence in WT mice, whereas it provoked an early and severe
insulin-deficient diabetes in ?ERKO?/?and ?ERKO?/?mice
This study establishes that estradiol, acting at least in part
by oxidative stress, thus maintaining insulin production and
preventing diabetes in mice of both genders. The analysis of
human and rodent models of aromatase or ER? deficiency has
challenged the traditional belief of gender-specificity in sex
steroid actions and has revealed critical functions of the estra-
diol–ER? axis in the prevention of obesity and insulin resistance
in mammals of both sexes (9, 13, 15–18).
The first finding of this study is the identification of circulating
estradiol as a protective hormone for ?-cell survival in mice.
Mice of both sexes develop a vulnerability to STZ-induced
insulin deficiency when estradiol production is suppressed. We
show that estradiol treatment, at physiological concentrations,
prevents ?-cell apoptosis in these mice.
The second observation is the authentication of ER? function
in ?-cell survival. After exposure to oxidative stress, elimination
of ER? impairs the ability of circulating estradiol to prevent
?-cell apoptosis, resulting in insulin-deficient diabetes. This
finding has implications for genetic predisposition to diabetes.
Young mice of both sexes lacking ER? show no detectable
alteration in insulin secretion or insulin sensitivity. They are
vulnerable, however, to oxidative injury in ?-cells. Moreover,
although only one copy of ER? is sufficient for reproduction
(19), haploinsufficiency for ER? in mice aggravates the ?-cell
vulnerability to STZ toxicity. This finding suggests that, in mice,
both ER? alleles are required to guard ?-cells from oxidative
stress. Thus, ER? gene variations may participate in the genetic
predisposition to human diabetes.
Another significance of this study is therapeutic. Estradiol is
an ER? agonist, and estrogen replacement therapy (ERT)
reduces the incidence of diabetes in postmenopausal women (20,
21). However, estradiol has undesirable side effects, such as
breast cancer, and ERT is no longer suitable. Rather, selective
ER modulators are compounds that act as ER agonists or
antagonists, depending on the tissue considered. Tamoxifen, for
example, acts as an ER? antagonist in breast and as an ER?
agonist in bone. Accordingly, tamoxifen is used in the treatment
of breast cancer while preventing osteoporosis. In our study,
tamoxifen reverses estradiol protection of ?-cell survival in vitro
and exacerbates the predisposition to insulin-deficient diabetes.
Together, these data suggest that tamoxifen acts as an ER?
antagonist in ?-cells and may precipitate insulin dependence in
diabetic women treated for breast cancer. This critical issue
The last observation of this study is that estradiol partially
prevents ?-cell apoptosis in the absence of ER?, suggesting the
existence of an alternate estradiol signaling pathway in ?-cells.
One possibility is that estradiol functions via ER?. Alternatively,
estradiol may activate nongenomic pathways via a membrane
receptor unrelated to ERs and mediating estradiol survival
Materials and Methods
Creation of Mutant Mice, Exogenous Substance Infusion, and Induc-
tion of Experimental Diabetes. ArKO?/?and ?ERKO?/?were
generated as described (19, 23). Estradiol (0.48 mg), 4-
hydroxytamoxifen (0.48 mg), and vehicle (cholesterol) were
administered by s.c. pellets (Innovative Research of America,
Sarasota, FL). One week after pellet insertion, diabetes was
in MCF7 cells, MIN6, and mouse islets. (B) Female pancreas section showing a
single islet with ER? nuclear staining in ?-cells (ER?). The insulin (red), nuclear
(blue) (DAPI), and triple staining (merge) are shown.
oxidative stress. (A) WT or ?ERKO mouse islets were incubated with E2 (10?8
M), MPP (10?7M), and TMX (10?7M) for 72 h, followed by H2O2(100 ?M) for
TUNEL?PI double labeling. Values represent five independent experiments.
(B) Insulin secretion in response to glucose was assessed in static incubations
from WT or ?ERKO?/?mouse islets cultured with E2 (10?8M), MPP (10?7M),
and TMX (10?7M) for 72 h, followed by H2O2(100 ?M) for the last hour. The
stimulation index represents the ratio of insulin released at 16.7 mM glucose
to insulin released at 2.8 mM glucose in four to seven replicate experiments).
*, P ? 0.05.
ER? is important for ?-cell survival and function after exposure to
Le May et al.
June 13, 2006 ?
vol. 103 ?
no. 24 ?
induced in 8-week-old mice by a single i.p. injection of either 75
or 150 mg?kg of body weight STZ prepared in 50 mM citrate
buffer (pH 4.5). Blood glucose was measured every 48 h after
STZ injection with a glucose monitor (OneTouch Ultra; Life-
scan, Mountain View, CA).
Islet Isolation. Mouse islets were isolated by collagenase diges-
tion. Briefly, the pancreas was injected through the pancreatic
duct with 3 ml of 2 mg?ml collagenase (Sigma) in Hanks’
buffered saline solution (HBSS), dissected out, incubated at
37°C for 12 min, and passed through a 400-mm wire mesh. The
digested pancreas was rinsed with HBSS, and islets were sepa-
rated by density gradient in Histopaque (Sigma). After several
washes with HBSS, islets were hand picked under a dissection
Islet Culture and Compound Stimulation. Islets were cultured in
phenol-red-free DMEM and 5 mM glucose containing 10%
charcoal-stripped FBS. Islets were incubated with estradiol
(10?8M; Steraloids, Newport, RI), Methyl–Piperidino–Pyrazole
weight) in female mice (n ? 8–10) was measured as described (24). (B) Pancreas insulin concentration was measured in female mice at basal levels or after 150
mg?kg STZ injection (day 8). Values are represented as scatter plot and mean ? SE (n ? 5–14). (C) ?-cell number per pancreas section at basal levels and after
STZ injection in female mice at day 8 (n ? 4). (D and E) (Left) Cumulative incidence of diabetes in mice after STZ injection. (Center) Random-fed blood glucose
in mice (day 8). (Right) Ratio of random-fed insulin (pg?ml) to glucose (mg?dl) in female and male mice after STZ injection (day 8).*, P ? 0.05;**, P ? 0.01.
?ERKO mice are predisposed to STZ-induced insulin-deficient diabetes. (A) Acute-phase insulin secretion after i.p. glucose injection (3 g?kg of body
www.pnas.org?cgi?doi?10.1073?pnas.0602956103Le May et al.
(MPP, 10?7M; Tocris Cookson, Ellisville, MO), and 4-
hydroxytamoxifen (10?7M; Sigma) for 72 h. After ER ligand
treatment, islets were exposed to H2O2(100 ?M; Sigma) for the
last 6 h before assessment of apoptosis. For additional informa-
tion, see Supporting Materials and Methods, which is published as
supporting information on the PNAS web site.
Insulin Secretion in Static Incubations. Insulin release from islets
was measured by static incubation. Batches of 10 islets were
cultured for 72 h in phenol-red-free DMEM. Islets were washed
twice for 30 min in KRB buffer containing 0.1% BSA and 2.8
mM glucose and estradiol or ER antagonists. Oxidative stress
was induced by addition of 100 ?M H2O2 during the second
wash. Islets were then incubated for 30 min in the presence of 2.8
or 16.7 mM glucose. Insulin was measured by using a sensitive
rat insulin radioimmunoassay (RIA) kit (Linco Research, St.
Pancreas and Islet Immunostaining. Tissues were fixed by cardiac
perfusion with 4% paraformaldehyde, dissected out, further
fixed overnight in 10% neutral formalin, and embedded in
paraffin. Sections were cut at 5 ?m. After dewaxing, slides were
washed, microwave antigen retrieval was performed, and the
slides were permeabilized with 0.2% Triton X-100 and blocked
in 10% serum. Primary antibodies were incubated overnight at
4°C with the following dilutions: guinea pig anti-insulin (1:1,000;
Linco), rabbit anti-glucagon (1:1,000; DiaSorin, Sallugia, Italy),
and rabbit anti-ER? (E1644, 1:200; Spring Biosciences, Fre-
mont, CA). Slides were then incubated with the appropriate
secondary antibodies: FITC-donkey anti-guinea pig, (1:400;
Jackson ImmunoResearch) and Cy3-donkey anti-rabbit (1:800;
Jackson ImmunoResearch). For ER?, tyramide signal amplifi-
cation (Molecular Probes) was performed. For TUNEL assay,
sections were incubated with terminal deoxytransferase enzyme
in the presence of Biotin-dUTP (Roche). TUNEL-positive cells
were revealed with Alexa Fluor 488-Streptavidin (Molecular
Measurement of Apoptosis and Proliferation in Vivo.Pancreatawere
fixed 12 h after STZ injection. Quantification of apoptosis and
proliferation were performed by TUNEL and KI67 staining,
respectively. Percent apoptosis or proliferation was calculated by
dividing the number of TUNEL- or KI67-positive ?-cells by the
total number of ?-cell stained nuclei. At least four mice per
condition and 50 islets per mouse were counted.
Calculation of ?-Cell Number per Pancreas Section. We counted the
?-cell number from merged pictures of pancreas sections im-
munostained with insulin and DAPI in all islets present in each
section. ?-Cell number per section was quantified by using five
sections per pancreas and five mice per condition.
Measurement of Apoptosis by Flow Cytometry. Apoptosis was mea-
sured by a DNA fragmentation assay (FlowTACS; R & D
Systems) according to the manufacturer’s instructions, and cells
were analyzed by flow cytometry by using a Beckman Coulter
EPICS XL-MCL flow cytometer. Double staining was per-
formed in duplicate by incubating FITC-labeled cells with
ptotic populations was used as a reference.
Statistical Analysis. Results are presented as mean ? SE unless
otherwise stated. All statistical analyses were performed by
using the unpaired Student t test. Cumulative incidence of
diabetes was determined by Kaplan–Meier estimates, and sta-
tistical analysis of differences was determined by log-rank test. A
value of P ? 0.05 was considered significant.
We thank Rachel E. Webb for editorial expertise. This work was
supported by National Institutes of Health Grant R21 DK069362-01 (to
F.M.-J.), and a Carolyn Weiss Law Award in Molecular Medicine (to
F.M.-J.). C.L.M. is the recipient of a Juvenile Diabetes Research
Foundation Postdoctoral Fellowship.
1. Mathis, D., Vence, L. & Benoist, C. (2001) Nature 414, 792–798.
2. Robertson, R. P., Harmon, J., Tran, P. O., Tanaka, Y. & Takahashi, H. (2003)
Diabetes 52, 581–587.
3. Wild, S., Roglic, G., Green, A., Sicree, R. & King, H. (2004) Diabetes Care 27,
4. Gale, E. A. & Gillespie, K. M. (2001) Diabetologia 44, 3–15.
5. Mauvais-Jarvis, F., Sobngwi, E., Porcher, R., Riveline, J. P., Kevorkian, J. P.,
Vaisse, C., Charpentier, G., Guillausseau, P. J., Vexiau, P. & Gautier, J. F.
(2004) Diabetes 53, 645–653.
6. Louet, J. F., Le May, C. & Mauvais-Jarvis, F. (2004) Curr. Atheroscler. Rep. 6,
7. Contreras, J. L., Smyth, C. A., Bilbao, G., Young, C. J., Thompson, J. A. &
Eckhoff, D. E. (2002) Transplantation 74, 1252–1259.
8. Simpson, E. R., Misso, M., Hewitt, K. N., Hill, R. A., Boon, W. C., Jones, M. E.,
Kovacic, A., Zhou, J. & Clyne, C. D. (2005) Endocr. Rev. 26, 322–330.
9. Couse, J. F. & Korach, K. S. (1999) Endocr. Rev. 20, 358–417.
10. Behl, C. (2002) Nat. Rev. Neurosci. 3, 433–442.
11. Friesen, N. T., Buchau, A. S., Schott-Ohly, P., Lgssiar, A. & Gleichmann, H.
(2004) Diabetologia 47, 676–685.
12. Schnedl, W. J., Ferber, S., Johnson, J. H. & Newgard, C. B. (1994) Diabetes 43,
13. Ohlsson, C., Hellberg, N., Parini, P., Vidal, O., Bohlooly, M., Rudling, M.,
Lindberg, M. K., Warner, M., Angelin, B. & Gustafsson, J. A. (2000) Biochem.
Biophys. Res. Commun. 278, 640–645.
14. Harrington, W. R., Sheng, S., Barnett, D. H., Petz, L. N., Katzenellenbogen,
J. A. & Katzenellenbogen, B. S. (2003) Mol. Cell. Endocrinol. 206, 13–22.
15. Morishima, A., Grumbach, M. M., Simpson, E. R., Fisher, C. & Qin, K. (1995)
J. Clin. Endocrinol. Metab. 80, 3689–3698.
T. C., Lubahn, D. B. & Korach, K. S. (1994) N. Engl. J. Med. 331, 1056–1061.
Proc. Natl. Acad. Sci. USA 97, 12729–12734.
18. Jones, M. E., Thorburn, A. W., Britt, K. L., Hewitt, K. N., Wreford, N. G.,
Proietto, J., Oz, O. K., Leury, B. J., Robertson, K. M., Yao, S. & Simpson, E. R.
(2000) Proc. Natl. Acad. Sci. USA 97, 12735–12740.
19. Lubahn, D. B., Moyer, J. S., Golding, T. S., Couse, J. F., Korach, K. S. &
Smithies, O. (1993) Proc. Natl. Acad. Sci. USA 90, 11162–11166.
20. Margolis, K. L., Bonds, D. E., Rodabough, R. J., Tinker, L., Phillips, L. S.,
Allen, C., Bassford, T., Burke, G., Torrens, J. & Howard, B. V. (2004)
Diabetologia 47, 1175–1187.
21. Kanaya, A. M., Herrington, D., Vittinghoff, E., Lin, F., Grady, D., Bittner, V.,
Cauley, J. A. & Barrett-Connor, E. (2003) Ann. Intern. Med. 138, 1–9.
22. Nadal, A., Rovira, J. M., Laribi, O., Leon-Quinto, T., Andreu, E., Ripoll, C.
& Soria, B. (1998) FASEB J. 12, 1341–1348.
23. Fisher, C. R., Graves, K. H., Parlow, A. F. & Simpson, E. R. (1998) Proc. Natl.
Acad. Sci. USA 95, 6965–6970.
24. Mauvais-Jarvis, F., Virkamaki, A., Michael, M. D., Winnay, J. N., Zisman, A.,
Kulkarni, R. N. & Kahn, C. R. (2000) Diabetes 49, 2126–2134.
Le May et al.
June 13, 2006 ?
vol. 103 ?
no. 24 ?