Impairment of rat fetal beta-cell development by maternal exposure to dexamethasone during different time-windows.
ABSTRACT Glucocorticoids (GCs) take part in the direct control of cell lineage during the late phase of pancreas development when endocrine and exocrine cell differentiation occurs. However, other tissues such as the vasculature exert a critical role before that phase. This study aims to investigate the consequences of overexposure to exogenous glucocorticoids during different time-windows of gestation for the development of the fetal endocrine pancreas.
Pregnant Wistar rats received dexamethasone acetate in their drinking water (1 µg/ml) during the last week or throughout gestation. Fetuses and their pancreases were analyzed at day 15 and 21 of gestation. Morphometrical analysis was performed on pancreatic sections after immunohistochemistry techniques and insulin secretion was evaluated on fetal islets collected in vitro.
Dexamethasone given the last week or throughout gestation reduced the beta-cell mass in 21-day-old fetuses by respectively 18% or 62%. This was accompanied by a defect in insulin secretion. The alpha-cell mass was reduced similarly. Neither islet vascularization nor beta-cell proliferation was affected when dexamethasone was administered during the last week, which was however the case when given throughout gestation. When given from the beginning of gestation, dexamethasone reduced the number of cells expressing the early marker of endocrine lineage neurogenin-3 when analyzed at 15 days of fetal age.
GCs reduce the beta- and alpha-cell mass by different mechanisms according to the stage of development during which the treatment was applied. In fetuses exposed to glucocorticoids the last week of gestation only, beta-cell mass is reduced due to impairment of beta-cell commitment, whereas in fetuses exposed throughout gestation, islet vascularization and lower beta-cell proliferation are involved as well, amplifying the reduction of the endocrine mass.
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ABSTRACT: Epidemiological studies related poor maternal nutrition and subsequent growth retardation in the progeny to the development of diabetes later in life. Low protein diet during gestation altered the beta-cell development of the rat progeny, by decreasing beta-cells proliferation, and increasing their sensitivity to nitric oxide and cytokines in the foetus. This disturbed maternal environment had lasting consequences since the higher beta-cell vulnerability was maintained at adulthood. AIM: The aim was to determine whether early malnutrition influences the vulnerability and the regeneration capacity of beta-cells after streptozotocin (STZ) damage at adulthood. METHODS: Gestating rats were fed either a control or a low protein diet until weaning. Adult female offspring received weekly during five weeks, injections of Freund's adjuvant followed 24h later by STZ. Half of the cohort was killed at d34 whereas the other half was maintained until d48 to analyse the regeneration capacity of the beta-cells. RESULTS: Although control and low protein rats had equivalent pancreatic insulin content and beta-cell volume density at d34, hyperglycaemia appeared earlier and was more dramatic in low protein than in control rats. STZ treatment increased beta-cell proliferation similarly in both groups. At d48, apoptotic rate was higher in the low protein group. Regeneration appeared in control but not in the low protein rats, where beta-cell aggregates/surface area and Reg1-positive area were decreased compared to control. CONCLUSION: Maternal malnutrition programs a more vulnerable endocrine pancreas in the progeny which is unable to regenerate after injury therefore predisposing it to develop glucose intolerance and diabetes later in life. This article is protected by copyright. All rights reserved.Acta Physiologica 05/2013; · 4.38 Impact Factor
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ABSTRACT: Experimental and epidemiologic data have confirmed that undernutrition or overnutrition during critical periods of life can result in metabolic dysfunction, leading to the development of obesity, hypertension, and type 2 diabetes, later in life. These studies have contributed to the concept of the developmental origins of health and disease (DOHaD), which involves metabolic programming patterns. Beyond the earlier phases of development, puberty can be an additional period of plasticity, during which any insult can lead to changes in metabolism. Impaired brain development, associated with imbalanced autonomous nervous system activity due to metabolic programming, is pivotal to the creation of pathophysiology. Excess glucocorticoid exposure, due to hypothalamic-pituitary-adrenal axis deregulation, is also involved in malprogramming in early life. Additionally, the pancreatic islets appear to play a decisive role in the setup and maintenance of these metabolic dysfunctions as key targets of metabolic programming, and epigenetic mechanisms may underlie these changes. Moreover, studies have indicated the possibility that deprogramming renders the islets able to recover their functioning after malprogramming. In this review, we discuss the key roles of the pancreatic islets as targets of malprogramming; however, we also discuss their roles as important targets for the treatment and prevention of metabolic diseases.Nutrition 10/2013; · 2.86 Impact Factor
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ABSTRACT: Peroxisome proliferator-activated receptor gamma-coactivator-1α (PGC-1α) has recently been implicated as a crucial factor in the glucocorticoid-suppressed expansion and transdifferentiation of porcine neonatal pancreatic cell clusters (NPCCs). However, the molecular mechanism has not been clarified. We investigated whether the suppression of PGC-1α expression protects against β-cell dysfunction induced by dexamethasone (Dx) treatment in vitro and in vivo and determined the mechanism of action of PGC-1α in porcine NPCCs. The reduction in Pdx-1 gene expression caused by either Dx treatment or PGC-1α overexpression was normalized by siPGC-1α. Nuclear FOXO1 and cytoplasmic Pdx-1 were detected after Dx treatment. However, FOXO1 was observed in the cytoplasm, and Pdx-1 was observed in the nucleus after siPGC-1α. Suppression of PGC-1α by siPGC-1α improved the Dx-induced repression of insulin secretion and insulin content. In vivo studies showed that the glucose level in the Ad-siPGC-1α-infected group was significantly lower than that in the Dx-treated group. Insulin expression in the graft tissue disappeared in the Dx-injected group. However, the siPGC-1α- and Dx-treated group showed increased insulin expression and an increase in graft mass, β-cell mass, and β-cell % in the graft. Conversely, the Dx-induced ductal cystic area was markedly reduced in the siPGC-1α- and Dx-treated group. Our results suggest that the transdifferentiation of porcine NPCCs into β cells is influenced by the duration of the Dx treatment, which might result from the suppression of key pancreatic transcription factors. PGC-1α is an attractive target for modulating the deleterious effects of glucocorticoids on pancreatic stem cells.Transplantation 01/2014; · 3.78 Impact Factor
Impairment of Rat Fetal Beta-Cell Development by
Maternal Exposure to Dexamethasone during Different
Olivier Dumortier, Nicolas Theys, Marie-The ´re `se Ahn, Claude Remacle, Brigitte Reusens*
Laboratoire de Biologie Cellulaire, Universite ´ catholique de Louvain, Institut des Sciences de la Vie, Louvain-la-Neuve, Belgium
Aim: Glucocorticoids (GCs) take part in the direct control of cell lineage during the late phase of pancreas development
when endocrine and exocrine cell differentiation occurs. However, other tissues such as the vasculature exert a critical role
before that phase. This study aims to investigate the consequences of overexposure to exogenous glucocorticoids during
different time-windows of gestation for the development of the fetal endocrine pancreas.
Methods: Pregnant Wistar rats received dexamethasone acetate in their drinking water (1 mg/ml) during the last week or
throughout gestation. Fetuses and their pancreases were analyzed at day 15 and 21 of gestation. Morphometrical analysis
was performed on pancreatic sections after immunohistochemistry techniques and insulin secretion was evaluated on fetal
islets collected in vitro.
Results: Dexamethasone given the last week or throughout gestation reduced the beta-cell mass in 21-day-old fetuses by
respectively 18% or 62%. This was accompanied by a defect in insulin secretion. The alpha-cell mass was reduced similarly.
Neither islet vascularization nor beta-cell proliferation was affected when dexamethasone was administered during the last
week, which was however the case when given throughout gestation. When given from the beginning of gestation,
dexamethasone reduced the number of cells expressing the early marker of endocrine lineage neurogenin-3 when analyzed
at 15 days of fetal age.
Conclusions: GCs reduce the beta- and alpha-cell mass by different mechanisms according to the stage of development
during which the treatment was applied. In fetuses exposed to glucocorticoids the last week of gestation only, beta-cell
mass is reduced due to impairment of beta-cell commitment, whereas in fetuses exposed throughout gestation, islet
vascularization and lower beta-cell proliferation are involved as well, amplifying the reduction of the endocrine mass.
Citation: Dumortier O, Theys N, Ahn M-T, Remacle C, Reusens B (2011) Impairment of Rat Fetal Beta-Cell Development by Maternal Exposure to Dexamethasone
during Different Time-Windows. PLoS ONE 6(10): e25576. doi:10.1371/journal.pone.0025576
Editor: Damien Keating, Flinders University, Australia
Received April 28, 2011; Accepted September 7, 2011; Published October 3, 2011
Copyright: ? 2011 Dumortier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the European Commission (NUTRIX/QLK1-200-00083, 5th FP and EARNEST/FOOD-CT-2005-007036, 6th FP), the Parthenon
Trust (London, UK), and the Belgian Fonds National de la Recherche Scientifique. O. Dumortier was a recipient of a Fonds pour la Recherche dans l’Industrie et
l’Agriculture fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Epidemiological studies performed in distinct populations
throughout the world have clearly reported that individuals who
had a low birth weight or were thin at birth feature a higher
prevalence of glucose intolerance and type 2 diabetes (T2D)
during adult life [1,2,3,4]. Indeed various nutritional changes
during embryonic development have the potential of altering gene
expression and modifying T2D susceptibility. Thus, a key question
arises concerning the nature of the components of the intrauterine
metabolic environment which initiate the deleterious cascade.
Evidence supports elevated levels of maternal (and thus fetal)
glucocorticoids may be a key unifying mechanism linking maternal
malnutrition to increased diabetes risk in adult life [5,6,7].
Maternal use of glucocorticoids during pregnancy in human and
experimental animal may cause low birth weight and intrauterine
growth retardation (IUGR) and later may lead to chronic diseases
such as hypertension and type 2 diabetes [8,9,10,11]. In the
pathogenesis of T2D, insulin resistance coupled to beta cell failure
leads to chronic hyperglycemia defining diabetes. In growth-
retarded animals or humans, functional disruption in multiple
tissues including muscle, adipose tissue, liver, and pancreas is
observed in adults. In such low birth weight offspring, one may
wonder whether the beta cell alteration observed later in life has
been already acquired in utero or is a consequence of an adaption to
the disturbed insulin sensitivity of target tissues.
Shen et al (2003) have investigated the direct effect of a
glucocorticoid excess during the last week of gestation in rat on the
development of the endocrine pancreas in the progeny. Like
others, they found that glucocorticoids treatment led to IUGR and
showed that at 3 weeks of age, the IUGR progeny featured a
normal distribution of beta and alpha cell within islets, but
reported a lower level of insulin expression in beta cells compared
to normal pancreas . However Shen et al did not investigate
PLoS ONE | www.plosone.org1October 2011 | Volume 6 | Issue 10 | e25576
the beta cell mass and function immediately after the treatment
i.e.at the end of gestation or at birth.
In order to understand the mechanisms by which glucocorti-
coids may affect the pancreas development, Shen et al 2003
investigated also the role of glucocorticoids on pancreatic buds
from mouse embryo in vitro. They used pancreatic buds from
E11.5 mouse embryo and treated them with dexamethasone.
They showed that the expression of Pancreatic and duodenal
specific transcription factor (Pdx-1), a homeodomain protein that
is expressed in the entire pancreatic anlagen at early stages, that
declines during later embryonic stages in most of the pancreas but
remains only in beta cells later in life, was not affected in an early
stage of development but rather suppressed in a later phase,
leading to less differentiated beta cells after one week of culture
One year later, Gesina et al. (2004) further analyzed the role of
glucocorticoids in the development of the endocrine pancreas.
When pancreatic bud from normal E15.5 mouse embryo were
cultured in presence of glucocorticoids, the number of endocrine
precursor cells was not affected but fewer differentiated beta-cells
and more differentiated acinar cells were reported . They
concluded that glucocorticoids favor exocrine pancreas develop-
ment at the detriment of the endocrine tissue. Using transgenic
mouse lacking glucocorticoid receptor (GR) they showed that the
selective inactivation of the GR gene in insulin-expressing beta-
cells in mice had no consequences on beta- or alpha-cell mass,
whereas the absence of GR in the expression domain of Pdx-1 led
to a twofold increased beta-cell mass, with increased islet numbers
and size. In addition, they showed that GR does not appear
necessary for early phases but its accurate dosage is required to
unable beta cell mass expansion in later stages . Since GR is
very weakly expressed in the rodent pancreas before E14 , the
organ should be rather insensitive to a direct action of
glucocorticoids during the first two weeks of gestation. However,
during this earlier period the proper development of the endocrine
pancreas requires the specific influence of tissues like vasculature
Therefore, the aim of our study was to investigate the beta cell
mass expansion and function at the end of gestation when
glucocorticoids were given from the beginning of gestation and to
compare the effects when given only during the last week. We
demonstrate that in addition to compromising the beta and alpha
cell differentiation, glucocorticoids given from the beginning of
gestation inhibit the beta-cell proliferation as well as function and
reduce the islet vascularization which presumably may contribute
to amplify the reduction of the endocrine mass.
Materials and Methods
All procedures were performed with the approval of the Animal
Ethics Committee of the Universite ´ catholique de Louvain (Permit
number LA 1220028).
Animals and prenatal treatments
Nulliparous 200 to 250 g female Wistar rats (Janvier, Le Genest
Saint Isle, France) were mated with males overnight and
copulation was verified next morning by inspection of vaginal
smears. Midnight was arbitrarily considered as time of mating at
day 0 of gestation. The pregnant females were individually housed
at 25uC with 14 h light: 10 h dark cycle and had free access to
water and food (20% protein wt/wt; Hope Farm, Woerden, the
Netherlands). Dexamethasone acetate (1 mg/ml, dexamethasone
21-acetate; Sigma-Aldrich, St Louis, MO, USA) was given in the
drinking water of the mother as previously described . This
doses is comparable to that given IP by Shen et al, 2003 in
pregnant rats  and is of the same range as that given to
pregnant women at risk of preterm delivery . Three groups of
animals were created. The DEX group received dexamethasone
acetate in the drinking water (1 mg/mL) throughout gestation. The
DEXL group received dexamethasone during the last week of
gestation whereas the C group received normal drinking water. A
minimum of 3 litters per group and per age and 3 fetuses per
mother were analyzed in each experiment.
Blood and tissue sampling and analysis
At day 20 of gestation, 24 hours before sacrifice, the dams were
injected intraperitoneally with BrdU (50 mg/kg; Sigma-Aldrich).
At day 21 or at day 15, the mothers were sacrificed and fetuses
were rapidly removed. The fetal blood samples were collected at
day 21 from the axillary vein, when the feto-maternal circulation
was maintained. Fetuses were weighed and their pancreas, liver,
placenta, adrenal glands and brain were removed and weighed.
Pancreases from 3 fetuses per litter were used for immunohisto-
chemistry, 3 others were used for the determination of islet
vascularization and 3 others for the analysis of pancreatic insulin
For measurement of glucose concentration, 50 mL of blood was
added to 500 mL HC104 (0.33 N) to precipitate proteins. After
centrifugation, supernatants were kept at 220uC until analysis.
Blood glucose was measured by the glucose oxidase method using
glucose Trinder’s reagent (Sigma, Saint-Louis, MO, USA and
Stanbio Laboratory, Boerne, TX, USA). Plasma was prepared and
kept at 220uC for the determination of insulin. Plasma insulin
levels were assessed using the ultrasensitive rat insulin ELISA and
the pancreatic insulin content using the high range rat insulin
ELISA (Mercodia, Uppsala, Sweden).
Fixation and tissue processing for immunohistochemistry
Pancreases from 15 and 21 day-old fetuses were fixed in 3.7%
formalin solution, dehydrated, and embedded in paraffin. Tissue
sections (7 mm) were collected on poly-L-lysine-coated glass slides.
The slides were left at 37uC overnight and stored at 4uC until
processed for immunohistochemistry.
Tissue sections were submitted to a 10-min microwave
treatment in a citrate buffer (Antigen Retrieval Citra Solution;
Biogenex, Alphelys, Plaisir, France), permeabilized for 20 min
with 0.1% Triton X-100 in Tris-buffered saline, and incubated
30 min with a blocking buffer solution (0.1% Tween 20/3% BSA
in Tris-buffered saline) before a 4uC overnight incubation with the
primary antibodies. Secondary antibodies were incubated for 1 h
at room temperature. Primary antibodies were rabbit anti-PDX1
(1/1000) and rabbit anti- Neurogenin 3 (Ngn3) (NEUROG3) (1/
2000) (generous gifts from Dr R. Scharfmann), rabbit anti-
Pancreas specific transcription factor (PTF1A) (1/2000) (10),
mouse anti-insulin (1/1000; Sigma), anti-glucagon (1/1000; Novo
Nordisk, Bogsvaerd, Denmark ) and mouse anti-BrdU (1/200;
Amersham Pharmacia Biotech Europe, Saclay, France), Second-
ary antibodies were anti-mouse AP Conjugate (1/200; Promega,
Madisson, WI, USA), biotin conjugated anti-rabbit (1/200;
Jackson Immuno Research Laboratories, West Grove, PA,
USA), peroxidase-conjugated anti-rabbit (1/200; Promega). Bio-
tin-coupled antibodies were revealed using peroxidase-conjugated
streptavidin (Amersham Pharmacia Biotech). Peroxidase was
detected with diaminobenzidine (Dako, Carpinteria, CA) and
Maternal Glucocorticoid and Fetal Pancreas
PLoS ONE | www.plosone.org2October 2011 | Volume 6 | Issue 10 | e25576
alkaline phosphatase with the Vector blue alkaline phosphatase
substrate kit III (Vector Laboratories, Inc., Burlingam, USA).
Assessment of cell death by apoptosis was examined on tissue
section using the TUNEL method with an in situ cell death
detection kit (Roche, Indianapolis, IN, USA). Total nuclei were
stained in blue with DAPI. In addition to staining for apoptosis,
insulin detection was performed as described above.
In F15 fetuses, cells expressing NEUROG3 were counted on 3
pancreatic sections per animal, a total of six animals from 3
different mothers being analyzed per group. Pancreatic area was
determined by computer-assisted measurements on the adjacent
section stained with toluidine blue using an Axioskop2 Mot Plus
Zeiss microscope coupled with the Zeiss KS 400 3.0 software (Carl
Zeiss GmbH, Jena, Germany).
On pancreatic sections of 21 day-old fetuses, cells co-expressing
PDX-1 and insulin as well as cells positive for BrdU and insulin
were counted. Insulin and glucagon positive area was morpho-
metrically measured on 3 sections per animal, a total of nine
animals being analyzed per group. The beta-cell and alpha-cell
fractions (%) were measured as the ratio of the insulin or glucagon
positive cell area to the total tissue area on the entire section. The
beta-cell and the alpha-cell mass was obtained by multiplying the
beta-cell or the alpha-cell fraction by the weight of the pancreas, as
previously described .
The profile diameter of the islets was calculated using the Zeiss
KS 400 3.0 software. The diameter of islets was calculated
assuming that the islets are spheroid structures. The Zeiss KS 400
3.0 software was calibrated to measure such as an islet a cluster of
minimum 3 insulin positives cells.
Tissue preparation and epon-embedding for
Pancreases of 21-day old fetuses were placed in ice-cold fixative
(2.5%v/v)glutaraldhehydein0.1 Mphosphatebuffer(pH 7.2)during
2 hours, rinsed and post-fixed in 1% osmium tetroxide in phosphate
buffer for 1 hour. The pieces were washed in phosphate buffer,
ethanol-dehydrated, and epon-embedded as described previously .
Vascularization was measured on semi-thin sections allowing
visualizing the lumen of blood vessel (see results). Three semi-thin
pancreas, and stained with toluidine blue. The area of the each islet
presents on the 3 sections and of the area of the different blood
vessels in each islet were measured, using NIH-Image 1.56 software
in a Reichert Polyvar microscope (Wien, Austria). Volume density
of blood vessels in the islet was calculated as follow: Total area of
blood vessel/islet reported to the islet area. To estimate the number
density of islet blood vessels, the number of capillaries was counted
in each islet and reported to the islet area. Nine fetuses from 3
different litters were used in each group (C, DEXL, and DEX).
Culture of fetal islets
Fetal neoformed islets were obtained as previously described
 .Briefly, pancreases of 21 day-old fetuses were removed
aseptically. All the preparation, including the pancreatic tissue
digestion, was carried out with RPMI 1640 medium containing
11.1 mM glucose (Gibco, Grand Island, NY, U.S.A.). The medium
was supplemented with 10% (v/v) heat-inactivated fetal bovine
serum (Gibco) and antibiotics (penicillin 200 U/ml, streptomycin
0.2 mg/ml, Gibco). The pancreases were minced and digested with
collagenase (Sigma-Aldrich, specific activity 381 U/ml, 1.6 mg/ml
per 12 pancreases) at 37uC. The digestion was stopped by adding
ice-cold medium. After washing, tissue samples were suspended in
10 ml medium and gently stirred at room temperature for 60 min.
The digested pancreases were pelleted by centrifugation, and re-
suspended ina ratioof onepancreas perml ofmedium.Finally2 ml
of this suspension were poured into 35 mm Petri dishes (Falcon
3001; Falcon Plastics, Los Angeles, CA, USA). The culture dishes
were incubated for 7 days at 37uC, in a humidified atmosphere of
5% CO2in air. The culture medium was changed daily after the
second day. During the culture, exocrine cells disappear rapidly,
whereas fibroblasts and endocrine cells proliferate. The endocrine
cells are first arranged in monolayers but progressively reorganize in
islets essentially composed of beta-cells (90–95% of beta-cells) that
aggregated progressively on the layer of non-endocrine cells and
whichgraduallyacquire the capacitytosecreteinsulin inresponseto
Insulin secretion assay
All these experiments were performed using a Krebs-Ringer
solution containing (mmol/L): NaCl, 120;KCl,5;CaCl2, 2;MgCl2,
1; NaHCO3 which was supplemented with 5 g/L bovine serum
albumin (BSA) (Fraction V, Calbiochem-Behring, San Diego, CA,
USA). This solution was gassed with 95% O2/5% CO2 to maintain
a pH of 7.4. Glucose was added into the incubation medium without
correction for osmolarity. Batches of 10 free-floating fetal neoformed
islets were picked up and incubated at 37uC in 1 mL of Krebs-
Ringermediumcontainingglucoseat2.5 mmol/Lor16.7 mmol/L.
After 120 min, the incubation medium was removed and placed in a
watch glass to verify that no islet had been taken. Then, the medium
was frozen until the insulin assay was performed. To determine
insulin content, islets were collected under microscopic observation
andhomogenizedbysonification(30 s,40 W)in0N5 mlacid-ethanol
[0.15 mol/L HCl in 75% (v/v) ethanol in water] to extract insulin.
To eliminate variations due to differences in individual batches of
islets, insulin secretion during incubation was expressed as a
percentage of the islet insulin content at the start of the incubation,
which is referred to as fractional insulin release. The latter was
obtained by adding the content measured at the end to the amount
of released insulin. Islets size was determined by measuring the
diameters of islets collected in vitro.
All results were expressed as means 6 SEM. The statistical
significance of variations was evaluated with Prism software
(GraphPad software INC., San Diego, CA, USA). Cell number,
cell proliferation, beta-cell fraction and mass, blood vessel number
and density were tested by a one way ANOVA followed by
Newman-Keuls. P values,0.05 were considered significant.
Maternal body weight gain and food intake during
Dexamethasone impaired the normal body weight gain in
pregnant animals in relation to the duration of the treatment
(Figure 1). At day 21, the maternal weight was respectively
reduced by 18.5% in the DEXL and by 25% in DEX groups.
When the entire gestation period was taken into account, no
significant difference in food intake between groups was apparent,
but a small reduction was noted during 2–3 days after the first
administration of dexamethasone (data not shown).
Maternal Glucocorticoid and Fetal Pancreas
PLoS ONE | www.plosone.org3October 2011 | Volume 6 | Issue 10 | e25576
Effect of dexamethasone on body and organ weight in
Litter size was similar in the three groups (range: 10–12 fetuses).
Compared to Controls, fetal body weight was reduced by 18%
when dexamethasone was administered during the last week of
gestation and by 30% when given throughout gestation (Table 1).
The weight of the pancreas, liver, adrenal gland, and placenta was
reduced according to the time window of dexamethasone
treatment (Table 1). The relative brain weight was significantly
increased in dexamethasone treated fetuses suggesting that brain
development was spared in these animals.
Dexamethasone and fetal pancreas development
No disturbance of the intra-islet organization of the different
endocrine cell populations was observed in DEXL and DEX
groups at the end of gestation. Indeed, beta-cells were located in
the core of islets and were surrounded by glucagon and
somatostatin cells (not shown). Morphometrical analysis revealed
however that the beta- and the-alpha mass was significantly
reduced in the DEX and DEXL groups (Table 2), and the
inhibitory effect of dexamethasone was significantly more marked
when given from the first day of gestation than during the last
week only. Dexamethasone given from the first day of gestation
significantly increased the number of small islets and reduced the
number of large islets compared to control (Figure 2).
Effects of dexamethasone on beta-cell proliferation,
apoptosis and pancreatic transcription factors
Proliferation, apoptosis and neogenesis are cellular mechanisms
potentially involved in the modulation of the beta-cell mass. BrdU
incorporation into DNA was used as an index of cell proliferation
(Figure 3A). The percentage of beta-cells positive for BrdU was
similar in Controls and in fetuses overexposed to GCs during the
last week of gestation (Figure 3B), but when dexamethasone was
administered throughout gestation, the beta-cell proliferation was
reduced by 20% (Figure 3B).
Figure 1. Maternal body weight during pregnancy. Dexamethasone was administered to the mother during the last week of gestation (DEXL)
or throughout gestation (DEX). Values are means 6 SEM, n=4, **P,0.01, *P,0.05 vs Controls (C).
Table 1. Body (g) and organ weight (mg) of 21-day-old fetuses from control mother and mother treated with dexamethasone.
Fetus (n=45) 5.4760.06 4.4960.06*** 3.7860.07***$$$
Pancreas (n=35) 31.460.7427.160.62** 22.960.85***$$
Relative pancreatic weight, mg/g body wt 5.7260.086.0660.126.0460.16
Liver (n=12) 316611 21168*** 143613***$$$
Relative liver weight, mg/g body wt 57.261.8 50.261.7$$$ 37.862.8***
Brain (n=12) 181641796317766
Relative brain weight, mg/g body wt 32.560.641.561*** 47.161.2***$$
Relative adrenal weight, mg/g body wt 0.5560.020.2960.01***0.3360.02***
Relative placenta weight, mg/g body wt1156310263**93625***
Values are means 6 SEM,
**P,0.01, vs C ;
$$P,0.01, vs DEXL.
Maternal Glucocorticoid and Fetal Pancreas
PLoS ONE | www.plosone.org4October 2011 | Volume 6 | Issue 10 | e25576
Apoptosis was revealed by DNA fragmentation assessed by
molecular cytochemistry by counting the TUNEL positive nuclei
in insulin-positive cells on the last day of gestation. In every
groups, the beta-cell apoptotic rate was very low (range: 0.1%–
0.2%, data not shown) suggesting that apoptosis was not
implicated in the impairment of islet development by dexameth-
asone at this age.
The number of cells immunoreactive for NEUROG3, a marker
expressed in endocrine precursor cells, was measured on
pancreatic sections at day E15 of gestation in DEX animals to
assess the rate of cell commitment to the endocrine lineage
(Figure 4A). Clearly, fetuses overexposed to GCs featured a 74%
reduction in the number of NEUROG3-positive nuclei in the
pancreatic epithelium (Figure 4B). To examine the differentiated
status of the committed beta-cells in islets, the co-expression of
insulin and PDX1 was also analyzed on pancreatic sections from
21-day old fetuses in the 3 groups. In all groups (C, DEX, DEXL),
every beta-cells co-expressed insulin and PDX1 at this late fetal
age which indicates a similar stage of maturation (data not shown). A
normal immunostaining of PTF1A, the main transcription factor
involved in the development of exocrine cells, was detected in all
groups of fetuses at the same intensity (data not shown).
Fetal islet vascularization
To study the consequences of GCs overexposure on fetal islet
vascularization, the volume and number density of islet blood
vessels was measured by morphometrical analysis on semi-thin
sections in pancreas of 21 day-old rat fetuses (Figure 5A–5B). Both
volume and number density were markedly decreased respectively
by about 30 and 40% in islets of DEX group compared to
Controls (Figure 5C–5D), whereas the administration of dexa-
methasone during only the last week of gestation did not affect the
fetal islet vascularization.
Effect of dexamethasone on plasma glucose, plasma
insulin levels, and pancreatic insulin content
Dexamethasone treatment did not alter either the maternal or
fetal plasma glucose level. It significantly reduced the fetal plasma
insulin level (Table 3), which was decreased by 40% in the DEXL
group and by 60% in the DEX group. DEX and DEXL fetuses
had higher pancreatic insulin content (Table 3).
Insulin secretion by fetal islets in vitro
Batches of 10 fetal islets ‘‘neoformed’’ (see M&M) were
incubated in Krebs-Ringer solution containing 2.5 mmol/L or
16.7 mmol/L of glucose. DEX islets collected after 7 days of
culture were smaller and contained less insulin than Controls
(Figure 6A–B). If the insulin content of neoformed islets was
adjusted for their size, the insulin content per ‘‘islet volume’’ was
increased in DEX group (Figure 6C) however they secreted less
Figure 2. Islet size distribution in the 21-day-old fetuses. Effect
of dexamethasone on size-frequency distribution of the 21-day-old rat
pancreatic islets. Values are means 6 SEM, n=9, * P,0.05, ** P,0.01, vs
Table 2. Beta- and Alpha-cell mass of 21-day-old fetuses from
control mother and mother treated with dexamethasone.
Beta-cell mass (mg) 1.7560.2 1.3860.07*0.7560.1**$$
Relative beta-cell mass
mg/g body wt
Alpha-cell mass (mg)0.5160.080.2860.05**0.1860.02***
Relative alpha-cell mass
mg/g body wt
Values are means 6 SEM, n=9,
***P,0.001 vs C;
$$P,0.01, vs DEXL.
Figure 3. Effect of dexamethasone on BrdU incorporation in
beta-cells of 21-day-old fetuses. (A) Beta-cells in S phase (blue)
were counted on pancreatic section in colocalisation with insulin
(brown). Beta-cells that are proliferating are indicated with arrowheads.
(B) Percentage of proliferating beta-cells over total beta-cells. A total of
4500 to 6000 nuclei were counted in each group. Values are means 6
SEM, n=9 **P,0.01 vs Controls. Scale bar=50 mm.
Maternal Glucocorticoid and Fetal Pancreas
PLoS ONE | www.plosone.org5October 2011 | Volume 6 | Issue 10 | e25576
insulin in response to glucose stimulation. Indeed, although at low
glucose concentration, both control and DEX islets released about
5% of their insulin content after 2 hours incubation, when control
islets were stimulated with 16.7 mmol/L glucose, they released
22% of their insulin content, whereas DEX islets released only
12% which was not statistically different from the basal release
(2.5 mmol/L) (Figure 6D).
This paper clearly shows that excessive glucocorticoid levels
during gestation have dramatic consequences for the growth of the
fetus and especially for the establishment of alpha and beta cell
mass in the pancreas. When dexamethasone was given only during
the last week of gestation, alpha and beta-cell mass was reduced
due to impairment of cell commitment probably through a direct
effect of dexamethasone. The novelty of our study is that when
dexamethasone treatment was extended to the whole gestation
period, fetal islet vascularization and beta-cell proliferation were
additionally altered. This further contributes to amplify the
reduction of the beta-cell mass resulting from the direct effect of
dexamethasone later during fetal development. Taking together,
our data suggest that the sensitive window of the endocrine
pancreas to high GCs level is less narrow than previously suggested
[13,14]. In addition, we showed that beta cell function was altered
after intrauterine exposure to high level of GCs.
Pancreas develops from the anterior midgut region of the
endoderm epithelium through evaginations induced by adjacent
mesodermal structures such as the notochord and the dorsal aorta
[23,24,25]. The expression of Sonic hedgehog (shh) is suppressed
whereas that one of Pdx1 is stimulated. This occurs between E10.5
to E13.5 in mouse . Later, the exo-endocrine specification is
controlled by the Notch/Hes signaling system, which when active
leads, in the exocrine and ductal progenitors and to the
suppression of the pro-endocrine factor neurogenin-3, a transcrip-
tion factor transiently expressed in pancreas, which controls the
commitment of multipotent pancreatic endodermal progenitors to
the endocrine fate [27,28]. After the exo-endocrine specification,
the endocrine cell differentiation is based on the successive
expression of transcription factors which are different for each
endocrine cell (for review see [23,25,29]. In addition to its early
role in the pancreas development, Pdx1 reappears later and
intervenes in beta-cell differentiation.
In the mouse pancreas, the GR is only detected from E12 
and in rat, from E15 . This suggests that the potential critical
period in the pancreas for direct effect GCs susceptibility should be
beyond E12 in mice and E15 in rat. However, the precise stage at
which glucocorticoids act during pancreas development is still
controversial. GCs have been shown to act during exo-endocrine
specification favouring the exocrine differentiation at the expense of
endocrine cells . Indeed, when glucocorticoid was added in vitro
to the culture medium, an increased expression of genes associated
with exocrine development was observed while markers of
endocrine differentiation were reduced . On the other hand,
Gesina and colleagues (2006) have shown that when GR null/null
mice were studied at E15.5 they featured to be indistinguishable
from wild-type regarding pancreatic size, tissue structure, beta cell
fraction, Ngn3 and Pdx1 expression, as well as the production of
transcription factors involved in exocrine differentiation . These
data suggest that the GCs control of beta cell development occurs
Figure 4. Effect of dexamethasone on NEUROG3 Expression in pancreas of 15-day-old fetuses. (A) Detection of NEUROG3 positive cells
in the duct network at day 15 (brown; arrowheads in insets), n=6, Scale bar=25 mm. (B) The number of positive nuclei was evaluated per pancreas
surface area (100 mm2). Values are means 6 SEM, **P,0.01 vs Controls. Scale bar=20 mm.
Maternal Glucocorticoid and Fetal Pancreas
PLoS ONE | www.plosone.org6 October 2011 | Volume 6 | Issue 10 | e25576
after the Ngn3 production, thus after exo-endocrine specification.
This relatively late effect of GCs on endocrine differentiation would
suggest that the positive regulation of GCs on exocrine differenti-
ation is secondary rather than part of an exo-endocrine switch
mechanism. More recently, Valtat et al (2010) have shown that, in
mice, the impact of maternal undernutrition on the impairment of
beta cell expansion was mediated by the presence of glucocorticoid
receptor (GR) on beta cell progenitors. The progeny of undernour-
ished dams displayed a high level of GCs accompanied by a
decrease number of precursor cells expressing Ngn3 and Pdx1
expression leading, to a reduction of beta- and alpha-cell mass. On
the other hand, the expression of genes encoding for exocrine
marker was increased in the pancreas of undernourished fetuses.
When GCs-GR signaling was disrupted in underfed fetuses through
Knocking-out GR in pancreatic precursor cells, the alteration
observedinthe wild type underfedwerenotobservedinthe KO.
These data indicate that GR signaling is important between
progenitors and insulin-positive cells, but as mentioned by Valtat
the precise stage at which this occurs is not yet clear.
Here we showed that at day 15, the pancreatic ducts from the
DEX group expressed less Ngn3 positive cells. In consequence, the
beta and alpha cell mass was lower in these animals on the last day
of gestation, consistently with the hypothesis of GCs intervention
Figure 5. Effect of dexamethasone on islet vascularization of 21 day-old fetuses. (A and B) Semi-thin section (1 mm) of a pancreatic islet.
Scale bar=20 mm. (A) Inset: magnification of a blood vessel containing an erythrocyte (*). The arrow shows an endothelial cell. (B) The islet is
surrounded by exocrine tissue and 6 blood vessels are highlighted in red in this islet. (C) Volumic and (D) numerical density of islet blood vessels as
measured on semi-thin sections. Values are means 6 SEM, n=9,* P,0.05, vs Controls.
Table 3. Effect of dexamethasone on pancreatic insulin
content (PIC, ng/mg), plasma glucose (mg/dL) and insulin
PIC (n=12)14361 17869*17167*
Fetal insulin (n=20)4.4860.6 2.6660.2** 1.7560.15***
Fetal glucose (n=20)54.861.9 55.462.6 48.662.9
Maternal glucose (n=4)87.161.0 91.263.1 8165.1
Values are means 6 SEM,
*P,0.05, vs C.
Maternal Glucocorticoid and Fetal Pancreas
PLoS ONE | www.plosone.org7 October 2011 | Volume 6 | Issue 10 | e25576
in modulating the exocrine/endocrine balance. At day E21, the
pancreas of offspring from mothers having received dexametha-
sone throughout gestation featured a normal organization. Indeed,
the exocrine tissue exhibited a normal expression of PTF1A and
the islets were composed of a core of beta-cells co-expressing
PDX1 and insulin, surrounded by alpha- and delta-cells. When
dexamethasone was given during the last week only, the pancreas
of 21 day-old offspring displayed a normal organization but alpha
and beta cell mass was less reduced compared to DEX pancreas.
This suggests that the direct intervention of GCs on endocrine
specification does not account for the complete reduction of
endocrine mass observed in fetuses treated during the entire
Proliferation is obviously involved in the modulation of the beta-
cell mass. GCs have already been reported to reduce proliferation
in various cell types [31,32,33,34]. Despite extensive research
effort, there is no consensus on the mechanisms by which GCs
may reduce cell proliferation. In the present publication, we
showed that at day 21 of pregnancy, beta cell proliferation was
reduced when dexamethasone was given during the entire
gestation period which led to less large islets and more small islets
in this group. This observation is consistent with the recent finding
of Valtat et al (2010). Indeed, when GR was lacking in pancreatic
precursors cells, the proliferative index was increased in pancreatic
beta cell . In our experiment, when dexamethasone was given
during the last week only, the proliferative index of beta cell was
unchanged and the islet size distribution was similar to that of the
controls. This suggests that the sensitive window of beta cell
proliferation to GCs is placed before the onset of GR expression in
Other tissues could contribute to the indirect effect of GCs on
beta cell proliferation. GRs are expressed in the placenta and the
liver earlier than in the pancreas, being already detected in these
organs at day E9.5 in mice . Both tissues are major producers
of growth factors and are susceptible to GCs during development
[35,36,37]. In our study, the growth of these organs was strongly
affected by GCs over-exposure. Maternal glucocorticoid treatment
appeared to reduce the placental transfer of glucose  and
leptin , two master regulators of fetal growth. In another study,
dexamethasone-induced IUGR was associated with dysregulated
expression of IGF-II and prolactin in the junctional zone of the
placenta . It has been previously shown that dexamethasone
was unable to reduce the proliferative activity of 6 day-old rat islets
in vitro, except when the latter were stimulated by prolactin .
This supports the idea that anti-proliferative activity of GCs on
beta-cell was mediated through indirect effect.
Islet vascular alteration may also be implicated in reduced beta-
cell proliferation in DEX group. The influence of high GCs level
on endothelial cells has already been reported in other tissues.
Germinal matrix is a highly cellular and highly vascularized region
in the brain from which cells migrate out during brain
development. GC administration to pregnant rabbit reduced
endothelial proliferation of this region in the developing brain of
the progeny . The same observation was made in germinal
matrix of premature babies exposed to GC . Then, it is
possible that GC may have reduced the beta cell mass through an
inhibitory effect on endothelial cell knowing the importance of the
endothelium for islet development, highlighted during the last
decade. Recently, signals from the endothelium that promote beta-
cell proliferation were identified [16,17,42,43,44]. Here we
showed that islet blood vessels were reduced in DEX group. In
parallel the beta-cell proliferation was lower. This was not the case
in DEXL group in which the first administration of the treatment
took place at day F15, day at which islet vascularization can
Figure 6. Effect of dexamethasone given to the mother throughout gestation on fetal neoformed islets. (A) Dexamethasone reduced
the islet insulin content. Values are the means of 9 observations pooled from three independent cultures (n=3). Bars represent SEM,* P,0.05 vs C. (B)
Dexamethasone also reduced the size of islets after the culture ,* P,0.05 vs C. (C) The ratio islets insulin content per islet size was increased by
dexamethasone treatment. Values are means of 100 observations pooled from 3 independent cultures. Bars represent SEM,* P,0.05. (D) Insulin
secretion capacity of fetal islets. Islets were incubated in Krebs-Ringer medium containing glucose at 2.5 or 16.7 mmol/L. Values are the means of 9
observations pooled from three independent cultures (n=3). SEM, ** P,0.01.
Maternal Glucocorticoid and Fetal Pancreas
PLoS ONE | www.plosone.org8 October 2011 | Volume 6 | Issue 10 | e25576
already be observed. Dexamethasone was probably administered
too late to significantly influence the early mechanisms involved in
islet angiogenesis. In this group, the beta-cell proliferation was also
unaffected despite the beta-cell mass was lower. Interestingly, in
ZDF rat, a model of type 2 diabetes, the beta-cell mass and
function were impaired, together with an alteration of the islet
vascular integrity . It was proposed that such vascular
alteration played a role in the beta-cell deficiency. In addition,
in a recent study examining the process of beta-cell loss in IUGR
rat offspring after birth, a reduced vascularity was observed in islet
before the deterioration of beta cell mass . These strong
associations between islet vascularization and beta-cell prolifera-
tion reinforce the concept of a developmental association between
endothelial cells and beta-cells.
To characterize the impact of dexamethasone on fetal beta cell
function, we evaluated the insulin secretion in cultured neoformed
islets from foetus of dexamethasone-treated mother. We showed
that islet from DEX foetuses secreted less insulin in response to
glucose compared to controls. These data are consistent with
previous observations showing decreased glucose stimulated
insulin secretion following glucocorticoid treatment in vitro .
The precise mechanisms mediating the inhibitory intervention of
GCs on insulin secretion is still elusive [47,48,49,50]. The
programming of beta cell function was expected since a lasting
alteration of the insulin secretion in adult offspring after
dexamethasone treatment during early life has been recorded
. Indeed, prenatal exposure to dexamethasone produces
fasting hyperglycaemia and/or glucose intolerance later in life.
In conclusion, the developing pancreas is sensitive to an excess
of glucocorticoids in vivo which reduces the beta- and alpha-cell
mass by different mechanisms according the stage of development
during which they were applied. GCs may act directly on pancreas
during the last week of gestation which corresponds to active
endocrine neogenesis but when present from the beginning of the
pancreas development, it reduces islet vascularization and
pancreatic endocrine cell proliferation, which further compromises
the beta and the alpha cell mass. So, our work shows new
alterations of the endocrine pancreas development and proposes
possible pathways to be investigated such as the contribution of the
IGFs and islet vascularization. Because glucocorticoids are
overproduced in stress condition that may be present throughout
gestation and because they may program glucose intolerance in
the progeny, this study provides further insights into the
pathogenesis of common metabolic disorders.
We thank R. Scharfmann (INSERM. E363) for providing PDX1 and
NEUROG3 antibodies. We thank also B. Bre ´ant and B. Blondeau for their
fruitful discussion and the generous gift of rabbit anti Ptf1a .
Conceived and designed the experiments: OD CR BR. Performed the
experiments: OD NT M-TA. Analyzed the data: OD NT CR BR.
Contributed reagents/materials/analysis tools: M-TA. Wrote the paper:
OD CR BR.
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