Am J Physiol Endocrinol Metab 306: E109 –E120, 2014.
First published November 12, 2013; doi:10.1152/ajpendo.00364.2013.
Long-term disruption of maternal glucose homeostasis induced by prenatal
glucocorticoid treatment correlates with miR-29 upregulation
Patrícia R. Gomes,1 Maria F. Graciano,1 Lucas C. Pantaleão,1 André L. Rennó,2 Sandra C. Rodrigues,1
Licio A. Velloso,3 Márcia Q. Latorraca,4 Angelo R. Carpinelli,1 Gabriel F. Anhê,2 and Silvana Bordin1
1
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil;
Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil; 3Department of
Internal Medicine, Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil; and 4Department of Food
and Nutrition, Faculty of Nutrition, Federal University of Mato Grosso, Cuiabá, Brazil
2
Submitted 1 July 2013; accepted in final form 13 November 2013
Gomes PR, Graciano MF, Pantaleão LC, Rennó AL, Rodrigues
SC, Velloso LA, Latorraca MQ, Carpinelli AR, Anhê GF, Bordin
S. Long-term disruption of maternal glucose homeostasis induced by
prenatal glucocorticoid treatment correlates with miR-29 upregulation. Am J Physiol Endocrinol Metab 306: E109 –E120, 2014. First
published November 12, 2013; doi:10.1152/ajpendo.00364.2013.—
Excess of glucocorticoids (GCs) during pregnancy is strongly associated with the programming of glucose intolerance in the offspring.
However, the impact of high GC levels on maternal metabolism is not
clearly documented. This study aimed to test the hypothesis that
mothers exposed to elevated levels of GCs might also display longterm disturbances in glucose homeostasis. Dexamethasone (DEX) was
administered noninvasively to the mothers via drinking water between
the 14th and the 19th days of pregnancy. Mothers were subjected to
glucose and insulin tolerance tests at 1, 2, 3, 6, and 12 mo postweaning. Pregnant rats not treated with DEX and age-matched virgin rats
were used as controls. Pancreatic islets were isolated at the 20th day
of pregnancy and 12 mo postweaning in order to evaluate glucosestimulated insulin secretion. The expression of the miR-29 family was
also studied due to its responsiveness to GCs and its well-documented
role in the regulation of pancreatic ␤-cell function. Rats treated with
DEX during pregnancy presented long-term glucose intolerance and
impaired insulin secretion. These changes correlated with 1) increased
expression of miR-29 and its regulator p53, 2) reduced expression of
syntaxin-1a, a direct target of miR-29, and 3) altered expression of
genes related to cellular senescence. Our data demonstrate that the use
of DEX during pregnancy results in deleterious outcomes to the
maternal metabolism, hallmarked by reduced insulin secretion and
glucose intolerance. This maternal metabolic programming might be
a consequence of time-sustained upregulation of miR-29s in maternal
pancreatic islets.
pregnancy; dexamethasone; insulin secretion; miR-29; cellular senescence
are essential for proper fetal
development (35) as well as for maternal homeostatic resetting
after delivery (3, 32, 46). On the other hand, fetal exposure to
excessive GCs leads to intrauterine growth restriction (IUGR),
which increases the risk of chronic metabolic diseases later in
life, such as obesity and type 2 diabetes, then reflecting the
noxious action of GC excess on metabolic programming of the
offspring. Glucose intolerance and impaired insulin secretion
are usually found in both rodents and humans that had IUGR
induced by prenatal GC excess (39, 47, 62).
GLUCOCORTICOID (GC) HORMONES
Address for reprint requests and other correspondence: S. Bordin, Dept. of
Physiology and Biophysics, Institute of Biomedical Sciences, Univ. of São
Paulo, Prof Lineu Prestes Ave. #1524, São Paulo, SP, Brazil 05508-000
(e-mail: [email protected]).
http://www.ajpendo.org
Metabolic programming of the IUGR phenotype is strongly
associated with an adverse fetal environment such as maternal
nutritional deficiency, restricted placental blood supply, stress,
and synthetic GC therapy (57, 9, 62). It is noteworthy that all
of these states share excessive maternal GC levels as a common feature (63). Several studies have shown that epigenetic
mechanisms such as DNA methylation and histone modifications are underlying mechanisms for the development of
IUGR-linked metabolic diseases (reviewed in Ref. 67). The
involvement of microRNAs (miRNAs) on IUGR outcomes is
still elusive (6, 20, 66).
miRNAs are endogenous single-stranded small noncoding
RNA molecules that control gene expression by binding to the
3=-untranslated region (UTR), thereby repressing translation or
decreasing messenger RNA stability. Deregulation of miRNA
expression in several tissues has been related to the pathogenesis
of type 2 diabetes. Due to its key role in the control of glucose
homeostasis, miRNA expression in pancreatic islets has drawn
special attention (17). Altered expression of miR-375 (7, 14, 15,
28, 29, 34, 54), miR-7 (71), miR-30d (73), miR-29 (4, 5, 55, 60),
and miR-338 –3p (25), among others, has been demonstrated to
correlate with pancreatic ␤-cell failure.
Numerous studies have been focusing on the metabolic
programming induced by gestational GC excess, with special
attention to the mechanisms leading to the late-onset development of metabolic syndrome in offspring subjected to IUGR.
However, the long-term impact of gestational GC excess on
maternal metabolism is not well documented. We therefore
examined whether GC excess during pregnancy would alter
maternal energy metabolism and pancreatic islet function later
in life. We also sought correlative changes on miRNAs and
their putative targets, particularly those that were demonstrated
to play a key role on pancreatic ␤-cell function.
MATERIALS AND METHODS
Experimental design and animal treatment. Nulliparous Wistar rats
at 8 wk of age were housed in a temperature-controlled room (22 ⫾
2°C) with the lights on from 7:00 AM to 7:00 PM; standard chow and
water were available ad libitum. Rats were allowed to acclimate for 2
wk in our animal facility prior to being randomized into four groups.
After habituation, two groups of female rats were housed with male
rats for 5 days (two female with one male per cage). The concomitant
presence of spermatozoa and estrous cells in a vaginal lavage indicated
day 0 of gestation. Pregnant rats were housed separately until weaning
but remained in visual, olfactory, and auditory contact with other animals
at all times. Daily food intake was assessed at the 13th and at the 20th
days of pregnancy by calculating the variation in chow mass within a
24-h interval (measurements started at the moments of lights on); the
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DEXAMETHASONE-INDUCED LONG-TERM MATERNAL GLUCOSE INTOLERANCE
same protocol was performed in age-matched virgin rats. Animals were
allowed to deliver undisturbed. On postnatal day 21, offspring were
weaned. The groups of age-matched virgin rats were housed in the same
environment. Treatment consisted of administration of 0.2 mg·kg⫺1·
day⫺1 water-soluble dexamethasone (Sigma-Aldrich, St. Louis, MO)
diluted in the drinking water from the 14th to the 19th day of pregnancy
or for 6 days for virgin rats. Random glucose levels were monitored
during the treatment every morning. Body weights were measured during
the treatment at weaning and at 1, 2, 3, 6, and 12 mo after weaning. All
studies were performed according to the guidelines of the Brazilian
College for Animal Experimentation (COBEA) and approved by the
Ethics Committee on Animal Use at the Institute of Biomedical Sciences,
University of São Paulo, Brazil.
Intraperitoneal glucose tolerance test. Rats were fasted for 12 h
prior to glucose injection (2 g/kg ip of a 20% solution of D-glucose).
Blood samples were collected from the tail at 0, 10, 15, 30, 60, and
120 min for the measurement of blood glucose. The area under the
curve (AUC) of blood glucose vs. time was calculated above each
individual baseline (basal glycemia) to estimate glucose tolerance.
Intraperitoneal insulin tolerance test. Fasted rats (12 h) were subjected to insulin injection (2 IU/kg ip). Blood samples were collected
from the tail at 0, 5, 10, 15, 20, 25, and 30 min for the measurement of
blood glucose. The constant rate for glucose disappearance (KITT) was
calculated from the slope of the least squares analysis of the blood
glucose concentrations during the linear phase of decay.
Pancreatic islet isolation and insulin secretion. Rats were narcotized in a CO2 chamber and euthanized by decapitation. Trunk blood
was collected to quantify circulating insulin levels, and islets were
isolated after perfusion and digestion of the exocrine pancreas with
collagenase solution (10). After isolation, groups of five islets were
preincubated at 37°C for 30 min in 0.5 ml of Krebs-Henseleit buffer
saturated with a mixture of O2 (95%) and CO2 (5%) and containing
0.2% BSA and 5.6 mM glucose. The medium was replaced with a
fresh buffer with 0.2% BSA in the presence of 5.6, 8.3, 11.1, or 16.7
mM glucose and incubated at 37°C for 1 h. At the end of the
experiment, the medium was collected and insulin measured by
radioimmunoassay using rat insulin as the standard.
RNA extraction and qPCR. Total RNA was extracted from a pool
of ⬃200 freshly isolated islets using TRIzol reagent, as previously
described (10). Extracted RNA was eluted in RNase-free water,
treated with Turbo DNA-free (Ambion, Austin, TX), and quantified
by spectrophotometry.
The analysis of miRNA expression was adapted from a previously
described method (64). For this, 250 ng of total RNA was polyadenylated using E. coli poly(A) polymerase (New England Biolabs,
Hitchin, Hertfordshire, UK) according to the manufacturer’s instructions at 37°C for 10 min. Then, poly(A) polymerse was heat inactivated (65°C, 5 min) and annealed to the adaptor [5=-GGCCACGCGTCGACTAGTAC(T)12-3=] by sequential incubations at 60°C (5
min) and then at 25°C (2 min). Annealed samples were chilled and
used for standard reverse transcription (Improm-II; Promega, Madison, WI) before setting up of qPCR reactions.
For mRNA expression analysis, 1 ␮g of total RNA was reversetranscribed using Improm-II reverse transcriptase (Promega) and
random primers, according to the manufacturer’s instructions. cDNAs
were used for qPCR as follows.
Real-time amplifications were performed using Kapa SYBR Fast
DNA polymerase (Kapa Biosystems, Boston, MA) and following
standard procedures (32). The primer sequences for miRNA amplification were as follows: antisense (universal): 5=-GGCCACGCGTCGACTAGTAC-3=; miR-29a sense: 5=-TAGCACCATCTGAAATCGGTTA-3=; miR-29b sense: 5=-TAGCACCATTTGAAATCAGTGTT3=; miR-29c 5=-TAGCACCATTTGAAATCGGTTA-3=; U1 snRNA
sense: 5=-CCAGGGCGAGGCTTATCCATTGC-3=; 18S rRNA sense:
5=-GACTCAACACGGGAAACCTCACC-3=. All reactions were performed using 60°C for the annealing step of amplification.
For mRNA expression analysis, primer sequences, accession number, TM, and product lengths were as follows: p53 (NM_030989)
sense: 5=-CGTTGCTCTGATGGTGACGG-3= and antisense: 5=AGCGTGATGATGGTAAGGATGG-3= (57°C; 230 bp); Bbc3 (NM_
173837) sense: 5=-CGGCGGAGACAAGAAGAGCAAC-3= and antisense: 5=-GGCACCTAGTTGGGCTCCATTTC-3= (58°C, 128 bp);
Ercc6 (NM_001107296) sense: 5=-CATCATAGATGGGTCCAGTCCG-3= and antisense: 5=-TGGGAACACCAGATGTTGCC-3= (56°C,
128 bp); Gadd45a (L32591) sense: 5=-TCAACATCCTGCGGGTCAGC-3= and antisense: 5=-TGTGGGTTCGTCACCAGCAC-3= (58°C,
133 bp); Nfkb1 (NM_001276711) sense: 5=-CTCAAGAACAGCAAGGCAGCAC-3= and antisense: 5=-AGAGGTGTCGTCCCATCGTAGG-3= (59°C, 266 bp); Rpl37a (X14069) sense: 5=-CAAGAAGGTCGGGATCGTCG-3= and antisense: 5=-ACCAGGCAAGTCTCAGGAGGTG-3= (57°C, 290 bp).
Values of miRNA and mRNA expression were normalized using
the geometric mean calculated from the internal control genes
(snRNA U1 and 18S rRNA for miRNA; 18S rRNA and Rpl37a for
mRNA). Fold changes were calculated by the 2⫺⌬⌬CT method.
Protein extraction and immunoblotting. Pools of ⬃350 freshly
isolated islets were processed for Western blotting as described
elsewhere (10). The primary antibodies were anti-p53 (BD Pharmigen, cat. no. 554157), anti-syntaxin-1a (Stx-1a; Abcam, cat. no.
ab78539, Cambridge, UK), and anti-␣-tubulin (Invitrogen, cat. no.
32–2500, Carlsbad, CA). A secondary antibody conjugated with
horseradish peroxidase (Bio-Rad, Hercules, CA) was used, followed
by the chemiluminescent detection of the bands on X-ray-sensitive
films. Optical densitometry was performed using the Scion Image
analysis software (Scion, Frederick, MD).
Immunohistochemistry. Rat pancreata were carefully excised,
cleared from lymph nodes and fat, and separated into a duodenal part
(“head”) and a splenic part (“tail”). After being weighed, head and tail
parts were fixed in Bouin’s solution for 12 h, dehydrated in ethanol
and embedded in paraffin. Sequential sections (5 ␮m) of the tail parts
were obtained with the use of a rotary microtome and placed on
individual silanized slides. Next, sections were deparaffinized in
xylene and rehydrated. Endogenous peroxidase activity was quenched
by incubating the slides with 3% H2O2 for 10 min. Sections were
washed with 0.1 M phosphate buffer (pH 7.4), blocked with 5%
nonfat dried milk (diluted in phosphate buffer containing 0.05%
Tween 20), and incubated with guinea pig anti-insulin antibody
(1:150) (Dako, cat. no. A0564, Glostrup, Denmark) at 4°C overnight.
After a washing, the sections were incubated with biotinilated antiguinea pig IgG at room temperature for 1 h. The streptavidin-biotin
complexes were detected with diaminobenzidine (DAB) solution
(0.1% DAB and 0.02% H2O2 in PBS). Finally, the sections were
rapidly stained with Harris’ hematoxylin and mounted for microscopic observation (LEICA DM 5000 B). Images were acquires using
a CCD camera (LEICA CTR 5000) and the software LEICA Q Win
Plus v. 3.2.0. Randomly 12–15 fields from tail parts of each animal
were analyzed. Pancreatic islet mass (mg per pancreas) was calculated
by multiplying relative pancreatic islet area (percentage of pancreatic
islet in each sliced pancreas area) by pancreas weight. The number of
pancreatic ␤-cell relative to number of islet cells was calculated by
dividing the number of insulin-positive cells by the number of cells in
the islet of a sequential section.
Statistical analysis. The results are presented as means ⫾ SE.
Comparisons were performed using one-way ANOVA followed by
Tukey-Kramer post hoc testing (INStat GraphPad Software, San
Diego, CA). P values of ⬍0.05 indicate a significant difference.
RESULTS
Changes in body weight, random and fasting glycemia,
insulinemia, and glucose tolerance induced by DEX treatment.
Six-day treatment with DEX led to a weight loss in virgin rats
and avoided the weight gain found in pregnant rats between the
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DEXAMETHASONE-INDUCED LONG-TERM MATERNAL GLUCOSE INTOLERANCE
Fig. 1. Changes in body weight, random
glycemia, and glucose tolerance during peripartum of pregnant rats treated with dexamethasone (DEX). Maternal body weight
was measured daily between 14th and 19th
days of pregnancy and 1, 7, 14, and 21 days
after delivery. Body weight of age-paired
virgin rats was also measured (A). Random
glucose levels were daily assessed starting 1
day before treatment (13th day of pregnancy) up to the 6th day (19th day of pregnancy) (B). Glucose tolerance tests were
performed on the day before end of DEX
treatment. Timeline changes in glucose levels were plotted vs. time (C) and the AUC of
each animal was calculated above each individual baseline (D). Results are shown as
means ⫾ SE. #P ⬍ 0.05 vs. pregnant group;
*P ⬍ 0.05 vs. virgin group (n ⫽ 8 –11).
14th and 19th days of gestation. Untreated pregnant rats
showed no changes in body weight throughout the 21 days of
lactation. On the other hand, rats that received DEX during
pregnancy showed a marked weight gain between the 1st and
the 14th day of lactation. At the 14th postpartum day, the body
weight of rats that received DEX during pregnancy was similar
to that of untreated mothers (Fig. 1A).
Aside from changes in maternal body weight during the last
week of pregnancy, pups from DEX-treated mothers had a
significantly low birth weight (4.4 ⫾ 0.4 vs. 7.2 ⫾ 0.2 g in
nontreated group, P ⬍ 0.0001).
A significant but transient increase in random glucose levels
was seen in virgin rats treated with DEX between the 2nd and
the 5th days of treatment. Glucose levels were lower in preg-
nant rats irrespective of the treatment (Fig. 1B). At the end of
treatment, only virgin rats that received DEX showed glucose
intolerance, as observed by timeline changes in glucose levels
after a glucose load and the AUC values (Fig. 1, C and D,
respectively).
Daily food intake of pregnant rats was higher than that of
virgin rats prior to the beginning of treatment with DEX (day
13 of pregnancy). One day after the end of treatment, DEX
induced a reduction in food intake of virgin but not in pregnant
rats. Insulin levels were similar between virgin and pregnant
rats before the beginning of DEX treatment. One day after the
end of treatment, insulin levels of virgin⫹DEX were higher
than in virgin rats. Pregnant⫹DEX rats also exhibited higher
insulin levels than untreated pregnant rats. As expected, insulin
Table 1. Food intake, insulinemia, and fasting glycemia in virgin and pregnant rats prior to and after DEX treatment
Food intake, g/day
Before treatment
After treatment
Insulinemia, ng/ml
Before treatment
After treatment
Fasting glycemia, mg/dl
After treatment
Virgin
Virgin⫹DEX
Pregnant
19.30 ⫾ 0.66 (10)
19.06 ⫾ 0.13 (7)
19.35 ⫾ 0.30 (10)
13.53 ⫾ 0.47*(5)
26.11 ⫾ 1.06*# (5)
25.37 ⫾ 1.18*# (5)
1.00 ⫾ 0.14 (4)
0.95 ⫾ 0.13 (6)
0.79 ⫾ 0.16 (4)
3.02 ⫾ 0.31* (9)
0.68 ⫾ 0.23 (4)
2.62 ⫾ 0.46* (7)
117.90 ⫾ 4.40 (7)
141.30 ⫾ 9.72* (8)
97.00 ⫾ 3.49*# (7)
Pregnant⫹DEX
26.97 ⫾ 0.80*# (6)
25.58 ⫾ 0.96*# (6)
0.56 ⫾ 0.21 (4)
6.50 ⫾ 1.42*#& (11)
98.75 ⫾ 5.92*# (8)
Values are means ⫾ SE (n). DEX, dexamethasone; before treatment, 13th day of pregnancy; after treatment, 20th day of pregnancy; virgin and virgin⫹DEX,
age-matched control rats. *P ⬍ 0.05 vs. virgin; #P ⬍ 0.05 vs. virgin⫹DEX; &P ⬍ 0.05 vs. pregnant.
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levels of pregnant rats at day 20th of pregnancy were higher
than in age-matched virgin rats. Also, at the end of treatment,
fasting glucose levels were higher in virgin⫹DEX trhan in
virgin rats. Fasting glucose levels were similar in pregnant and
pregnant⫹DEX rats, and both were lower than in virgin rats
(Table 1).
Late onset of glucose intolerance induced by DEX treatment
at the end of pregnancy was associated with impaired glucosestimulated insulin secretion (GSIS) but not with insulin resistance.
A glucose tolerance test was performed at 1, 2, 3, 6, and 12 mo
after weaning and in age-matched virgin rats previously treated or
not with DEX. At 1 and 2 mo after weaning, glucose tolerance
was similar among all groups (Fig. 2, A and B). At 3, 6, and 12
mo after weaning, glucose intolerance was found in rats treated
with DEX during pregnancy. These changes can be seen in
timeline changes of glycemia and in the AUC (Fig. 2, C–E). In
parallel, an increase in body weight was observed 3, 6, and 12
mo after weaning in mothers that were treated with DEX
during gestation (Fig. 2F).
To determine the mechanisms underlying the long-term
appearance of glucose intolerance in rats treated with DEX
during pregnancy, we next assessed whole body insulin sensitivity. An insulin tolerance test was performed at 1, 2, 3, 6, and
12 mo after weaning and in age-matched virgin rats previously
treated or not with DEX (Fig. 3). Although no differences were
found in the KITT, the rapid increase in glucose levels after
Fig. 2. Long-term changes of glucose tolerance in rats treated with DEX during pregnancy. Pregnant DEX-treated (p⫹D, solid
line and closed squares) and pregnant untreated (p, solid line and open squares) were
allowed to breastfeed and subjected to glucose tolerance tests at the 1st, 2nd, 3rd, 6th,
and 12th months after weaning. Virgin
DEX-treated (v⫹D, dotted line and closed
circles) and virgin untreated (v, dotted line
and closed circles) age-matched rats were
subjected to the same tests (A, B, C, D and E,
respectively). Timeline changes in glucose
levels vs. time and the AUC are shown in each
panel. Body weight was measured on the day
of glucose tolerance tests (F). Results are
shown as means ⫾ SE. #P ⬍ 0.05 vs. virgin,
virgin⫹DEX, and pregnant groups; $P ⬍
0.05 vs. virgin or pregnant untreated rats
(n ⫽ 4 –10).
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DEXAMETHASONE-INDUCED LONG-TERM MATERNAL GLUCOSE INTOLERANCE
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Fig. 3. Long-term insulin sensitivity in rats
treated with DEX during pregnancy. Pregnant DEX-treated (p⫹D, solid line and
closed squares) and pregnant untreated (p,
solid line and open squares) rats were allowed to breastfeed and subjected to insulin
tolerance tests at the end of the 1st, 2nd, 3rd,
6th, and 12th months after weaning. Virgin
DEX-treated (v⫹D, dotted line and closed
circles) and virgin untreated (v, dotted line
and open circles) age-matched rats were subjected to the same tests (A, B, C, D and E,
respectively). Timeline changes in glucose
levels vs. time and the KITT are shown in
each panel. Results are shown as means ⫾
SE. *P ⬍ 0.05 vs. virgin, pregnant, and
pregnant⫹DEX groups (n ⫽ 4 –10).
insulin injections (which was probably due to a stress response)
was transitorily suppressed in virgin⫹DEX rats. In this way,
glucose levels obtained 5 min after insulin injections were
lower in virgin⫹DEX rats (vs. the other groups) specifically 3
and 6 mo after the end of the treatment.
Increased glucose responsiveness is a hallmark of the adaptive changes of pancreatic islets to pregnancy (3, 52). To
explore both early- and late-onset effects of GC excess in
␤-cell function, we performed GSIS in islets isolated from rats
at the end of pregnancy and 1 yr after. One day after the end
treatment, 16.7 mM GSIS by islets from pregnant⫹DEX rats
was lower than that from untreated pregnant rats. As expected,
islets from untreated pregnant rats were more responsive to
stimulatory glucose concentrations than age-matched virgin
rats. DEX treatment of virgin rats did not change insulin
secretion by pancreatic islets (Fig. 4A).
Twelve months after weaning, insulin secretion stimulated
by 16.7 mM glucose was similar between rats that underwent
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Fig. 4. Long-term changes in insulin secretion by pancreatic islets isolated
from rats treated with DEX during pregnancy. Pregnant and virgin rats,
DEX-treated or untreated, were euthanized at the end of the 20th day of
pregnancy and subjective pregnancy, respectively. Pancreatic islets were
isolated and assayed for cumulative insulin secretion using 5.6, 8.3, 11.1, and
16.7 mM glucose (A). Another set of treated and untreated rats was euthanized
1 yr after weaning or 1 yr after subjective weaning (for pregnant and
age-matched virgin rats, respectively) (B). Results are shown as means ⫾ SE.
*P ⬍ 0.05 vs. corresponding basal insulin secretion (5.6 mM glucose); &P ⬍
0.05 vs. virgin and virgin-DEX (at 11.1 and 16.7 mM glucose); #P ⬍ 0.05 vs.
pregnant (at 16.7 mM glucose); $P ⬎ 0.05 vs. virgin, virgin-DEX, and
pregnant (at 11.1 and 16.7 mM glucose); (n ⫽ 4 – 6).
pregnancy without treatment and age-matched virgin rats.
Importantly, insulin secretion stimulated by both 11.1 and 16.7
mM glucose was found to be reduced 12 mo after weaning in
rats that were treated with DEX during pregnancy (Fig. 4B).
Long-term changes in mir-29 expression in islets of rats
treated with DEX during pregnancy correlates with impaired
insulin secretion. In a search for the mechanism responsible for
the long-term impairment of insulin secretion we screened
changes in islets’ miRNA profile induced by DEX treatment. The
contents of miR-29a and miR-29c were reduced in islets of virgin
rats 1 day after the end of DEX treatment (Fig. 5, A and G).
However, ⬃8 and 12 mo after the end of DEX treatment,
miR-29 expression showed different patterns. In DEX-treated
virgin rats, miR-29a returned to values similar to that of control
virgin rats (Fig. 5, B and C), whereas miR-29c significantly
increased (Fig. 5, H and I) and miR-29b showed a transient
increase specifically 8 mo after treatment (Fig. 5, E and F). On
the other hand, when DEX treatment was performed during
pregnancy, we observed an increase in miR-29s in islets 1 day
after the end of the treatment. In that case, the modulation was
characterized by a sustained response, as miR-29s remained
elevated at 8 and 12 mo after DEX treatment (Fig. 5, A–I).
Long-term changes in expression of putative targets of
mir-29 in islets of rats treated with DEX during pregnancy
correlates with impaired insulin secretion. As higher body
weigh gain and glucose intolerance appeared at the 3rd month
after weaning in pregnant⫹DEX rats, and increased miR-29
expressions were found in all periods studied, we performed
the next experimental protocols at an intermediate time point (8
mo after weaning).
The transcriptional factor p53 has already been described to
be involved in pancreatic ␤-cell viability (41). Importantly, p53
was also described as an indirect target that is upregulated by
miR-29 (27). The content of p53 was increased at the mRNA
and protein levels in islets of pregnant rats treated with DEX 1
day after the end of the treatment (Fig. 6, A and C, respectively). The levels of p53 mRNA remained elevated 8 mo after
weaning in islets from DEX-pregnant rats (Fig. 6B). The
content of p53 was increased later in life after DEX treatment
independently of whether it occurred during pregnancy or not
(Fig. 6D).
We next assessed the expression of Bbc3 [the gene that
encodes p53 upregulated modulator of apoptosis (Puma)], a
direct target of p53 transcriptional activity (45). The level of
the Bbc3 mRNA was increased in pancreatic islets of rats
treated with DEX during pregnancy. This upregulation was
detected 1 day after the end of DEX treatment and 8 mo after
weaning (Fig. 6, E and F, respectively).
Stx-1a is a t-SNARE protein that plays a crucial role in GSIS
(44, 50). Stx-1a mRNA is directly targeted by miR-29a,
thereby influencing insulin secretion (5). Accordingly to an
increase in miR-29a, treatment with DEX during pregnancy
resulted in reduced Stx-1a protein levels in maternal pancreatic
islets 8 mo after weaning (Fig. 6H).
DEX treatment during pregnancy did not change islet
morphology. It is well established that during normal pregnancy ␤-cell mass increases, peaking at the end of the second
gestational period, which corresponds in the rat to the 13th day
of pregnancy (31, 52). To verify whether the late-onset metabolic disturbances found in DEX-treated dams are associated
with morphological changes, we performed an immunohistochemical analysis of pancreatic islets at 8 mo after treatment.
The pancreatic islet architecture and insulin-positive ␤-cells
were relatively conserved in all groups (Fig. 7A). ␤-Cells were
always typically detected within the islet core, and islet size
and number were unchanged 8 mo after weaning. Histomorphometric measurements showed that the mean islets’ perimeters were less than 500 ␮m in the four groups [ranging from
414 ⫾ 24 ␮m in pregnant⫹DEX to 488 ⫾ 48 ␮m in
virgin⫹DEX; not significant (NS)]. Pancreas mass relative to
body weight was also similar among all groups (ranging from
0.36 ⫾ 0.015% in pregnant to 0.48 ⫾ 0.06% in pregnant⫹
DEX; NS). Also, no significant changes were observed in
average islet area, islet mass, or the percentage of ␤-cells per
islet (Fig. 7, B–D, respectively).
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DEXAMETHASONE-INDUCED LONG-TERM MATERNAL GLUCOSE INTOLERANCE
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Fig. 5. Changes in miR-29 expression in pancreatic islets
isolated from rats treated with DEX during pregnancy.
Pregnant rats, DEX-treated or untreated, were euthanized
at the end of the 20th day of pregnancy and 8 and 12 mo
after weaning. Age-matched virgin rats were used as
controls. Expression of miR-29a (A, B, and C), miR-29b
(D, E, and F), and miR-29c (G, H, and I) was analyzed by
qPCR. Results are shown as means ⫾ SE. *P ⬍ 0.05 vs.
virgin, pregnant, and pregnant⫹DEX; #P ⬍ 0.05 vs.
virgin, virgin⫹DEX, and pregnant (n ⫽ 5–11).
Expression of cellular senescence-related genes exhibits
long-term modulation in pancreatic islets of rats treated with
DEX during pregnancy. As miR-29s have been previously
related to cellular senescence (38), and the decrease in GSIS is
a feature of aged ␤-cells (58), we have searched for putative
mechanisms that could strengthen the correlation between
miR-29s and ␤-cell phenotype. The expression of Nfkb1, the
gene that encodes the 50-kDa subunit of the NF-␬B (nuclear
factor ␬B DNA binding subunit), is upregulated in islets from
untreated pregnant rats at gestational day 20. DEX treatment
abrogated the Nfkb1 overexpression induced by pregnancy
(Fig. 8A). One year after DEX treatment, the expression of
Nfkb1 in islets from DEX-treated dams was significantly higher
than from the other three groups (Fig. 8B).
Gadd45a (growth arrest and DNA damage protein 45a) and
Ercc6 (excision repair cross-complementing rodent repair deficiency, complementation group 6) are two genes that have
been associated with p53 signaling and cellular senescence (12,
18). One year after treatment, islets from DEX-treated dams
showed an increase in Gadd45a and a decrease in Ercc6
expression (vs. the other three groups; Fig. 8C). At that time
point, a significant increase in Ercc6 expression was found in
islets isolated from untreated pregnant rats (Fig. 8C).
DISCUSSION
Numerous studies dealing with GC excess in pregnancy and
metabolic programming have focused on late-onset metabolic
syndrome in IUGR pups (39, 47, 57), but the impact of prenatal
GC excess on maternal physiology has been neglected. Since
mothers and fetuses are exposed to a similar gestational environment, we hypothesized that metabolic programming of
maternal energy metabolism could also take place later in life.
Here we demonstrate that, as already described in their offspring, mothers treated with DEX during the last third of
pregnancy also develop long-term glucose intolerance associated with impaired GSIS. In addition, we show that miR-29
upregulation is an epigenetic process that is maintained until
the late onset of glucose intolerance, thus being a putative
mechanism for long-term pancreatic ␤-cell failure.
DEX treatment via drinking water reduced the body weight
of virgin rats and abrogated maternal body weight gain during
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DEXAMETHASONE-INDUCED LONG-TERM MATERNAL GLUCOSE INTOLERANCE
Fig. 6. Changes in p53, Bbc3, and Stx-1a expression in pancreatic islets
isolated from rats treated with DEX during pregnancy. Pregnant and virgin
rats, DEX-treated or untreated, were euthanized at the end of the 20th day of
pregnancy and subjective pregnancy and 8 mo after weaning or subjective
weaning. Pancreatic islets were isolated and processed for qPCR analysis of
p53 (A and B) and Bbc3 (E and F) and Western blot detection of p53 (C and
D) and Stx-1a (G and H) protein. Results are shown as means ⫾ SE. *P ⬍ 0.05
vs. virgin and pregnant; #P ⬍ 0.05 vs. virgin, virgin⫹DEX, and pregnant
(n ⫽ 4 –9).
the last third of pregnancy, which attests to the effectiveness of
our experimental protocol. As our protocol of treatment with
DEX did not reduce food intake in pregnant rats, it is plausible
to hypothesize that exogenous DEX administrated during the
last week of pregnancy might avoid body weight gain by
increasing energy expenditure. Also, glucose intolerance and
random hyperglycemia observed in nonpregnant rats immediately after a chronic DEX treatment are phenotypic traits of
DEX therapy (51). Finally, a significant reduction in pups’
weight was observed, which is a hallmark of fetal metabolic
programming due to maternal GC excess (40, 49).
Glucose homeostasis resetting of virgin⫹DEX rats later in
life was not unexpected. It has been previously demonstrated
that glucose intolerance and pancreatic ␤-cell dysfunction
induced by DEX in male rats are reversed after discontinuation
of the treatment (56). Importantly, either the interaction between DEX with some component of the gestational milieu or
the changes in body weight gain induced by DEX in pregnant
rats are possible causes for the long-term impacts of DEX
treatment during pregnancy on pancreatic islet physiology. In
accord with this hypothesis, a 65% food restriction during the
last week of pregnancy in rats reduced body weight and caused
maternal glucose intolerance (2).
Although we have shown that insulin secretion by pancreatic
islets isolated at end of pregnancy from pregnant⫹DEX rats is
reduced compared with pregnant rats, circulating insulin levels
of pregnant⫹DEX dams were higher than in untreated dams.
Similarly, others have described that DEX treatment of pregnant
rats leads to hyperinsulinemia at the end of pregnancy with
parallel reduction of GSIS by isolated pancreatic islets (23).
A plausible explanation for these in vivo and in vitro
differences might rely on extrapancreatic adaptations induced
by DEX during pregnancy that leads to increased in vivo
insulinemia. It was already demonstrated that women treated
with DEX during pregnancy display increased insulin levels
due to increased autonomic inputs to the pancreatic islets (1).
As we interpret it, this autonomic modulation is probably lost
in experiments with isolated pancreatic islets.
Interestingly, the appearance of glucose intolerance and
higher weight gain in pregnant⫹DEX rats was a delayed and a
time-sustained phenomenon. Epigenetic processes are primary
candidates for mechanisms that can stably modulate gene
expression involved in metabolic programming. In the search
for the underlying mechanism governing the late onset of
glucose intolerance in pregnant-DEX rats, we hypothesized
that alterations in miRNA expression could be involved. Our
hypothesis was based on 1) previous work showing that synthetic GC can regulate miRNA expression in several cell types
(65), 2) the pivotal role of miRNAs in pancreatic ␤-cell
function (17, 26), and 3) our previous screening of DEX effects
on miRNA expression in pancreatic islets (unpublished data).
We chose the miR-29 family for further analysis due to its
putative relevance to pancreatic ␤-cell function and maintenance (4, 5, 55, 60). Also, in our previous screening all
members of miR-29 family were regulated by DEX.
The miR-29 family has been widely studied because of its
deregulation in several processes such as tumorogenesis (72),
organ/tissue fibrosis (22, 59), senescence (38, 69), and regulation of extracellular matrix (24). The plethora of miR-29
actions is due to a common characteristic of miRNAs: each
miRNA can regulate hundreds of genes and each gene can be
regulated by multiple miRNAs (30). This feature results in a
complex combinatorial posttranscriptional regulation of gene
expression and is reflected in a cell type- and context-specific
response.
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DEXAMETHASONE-INDUCED LONG-TERM MATERNAL GLUCOSE INTOLERANCE
A
virgin
E117
virgin+DEX
negative control
D
cell / islet cells (%)
cell mass (mg)
C
Fig. 7. Long-term pancreatic islet morphology of
rats treated with DEX during pregnancy. DEXtreated and untreated pregnant rats were allowed
to breastfeed, and pancreata were removed 8 mo
postweaning. DEX-treated and untreated age-matched
virgin rats had their pancreata removed at end of the
8th month after subjective weaning. Tissues were
processed for immunohistochemistry detection of insulin (A). Average islet area (B), islet mass (C), and
percentage of ␤-cells per islet cells (D) were also
shown as means ⫾ SE (n ⫽ 3).
#
B
Islet mass (mg)
pregnant+DEX
Average islet area (mm2)
pregnant
In pancreatic islets and insulinoma cells, miR-29 overexpression led to impairment in GSIS and ␤-cell apoptosis (4,
60). These reports showed that both proinflammatory cytokines
and high glucose upregulated miR-29 expression, leading to
pancreatic ␤-cell dysfunction. The long-term effects observed
in DEX⫹pregnant rats are in agreement with these previous
studies. The lack of significant ␤-cell dysfunction immediately
after DEX treatment could be due to a weak direct effect of the
synthetic GC on mothers’ physiology, as seen in random
glycemia and glucose tolerance tests. Of note, GCs inhibit
inflammation by abrogating the activity of NF-␬B, a central
signaling molecule of proinflammatory cytokines (42), which
could be counteracting an inflammatory dependent miR-29
network by the time of pregnancy.
In the nonpregnant state, DEX induced a short-term downregulation of miR-29a/c in pancreatic islets. DEX-induced
miR-29a repression has already been described (70). On the
other hand, miR-29c has been suggested as a signature miRNA
in the diabetic environment (36). In kidney cells, knockdown
of miR-29c prevented high-glucose-induced cell apoptosis,
whereas forced expression of miR-29c induced cell death (36).
Although not definitely evidenced by our study, we believe that
the permanent differential modulation of the miR-29 family
might influence the cellular senescence process of pancreatic
␤-cells. We consider this hypothesis because the miR-29 family was previously reported to mediate the senescence process
in different cell types (38).
It has been suggested that the stress-activated p53 and
NF-␬B signaling pathways, acting in an antagonistic fashion,
are key players in the regulation of cellular senescence (61).
However, the tumor suppressor p53 is an important trigger of
cellular senescence (11), and NF-␬B signaling is involved in
the induction of the senescence-associated secretory phenotype, a condition where senescent cells secrete proinflammatory factors (61). Indeed, it has been recently reported that both
p53 and NF-␬B can operate synergistically (33). Since elevated
activation of NF-␬B is related to a poor cellular redox state and
the inflammatory process, it is possible that NF-␬B could be
accelerating the oxidative stress and/or an inflammatory response leading to ␤-cell dysfunction.
Gadd45a was the first well-defined p53 downstream gene
that behaves like a DNA damage-inducible gene (12). In
mammary tumor, Gadd45a activates the stress-induced JNK
and p38 kinases, which participate in increased apoptosis and
Ras-induced cellular senescence (68). The paralleled upregulation of Gadd45a and p53 in islets from rats exposed to GC
excess during pregnancy reinforces the putative increase in p53
activity in DEX-treated mothers.
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Fig. 8. Changes in Nfkb1, Gadd45a, and Ercc6 expression in pancreatic islets
isolated from rats treated with DEX during pregnancy. Pregnant and virgin
rats, DEX-treated or untreated, were euthanized at the end of the 20th day of
pregnancy and subjective pregnancy and 12 mo after weaning or subjective
weaning. Pancreatic islets were isolated and processed for qPCR analysis of
Nfkb1 (A and B) and Gadd45a and Ercc6 (C). Results are shown as means ⫾
SE. *P ⬍ 0.05 vs. virgin, virgin⫹DEX, and pregnant; #P ⬍ 0.05 vs. virgin,
virgin⫹DEX, and pregnant⫹DEX (n ⫽ 4 – 8).
As mentioned above, p53 integrates different physiological
signals. In response to different forms of cellular stress, p53
protein stabilizes and triggers multiple specific events to prevent damaged cells from undergoing tumor transformation.
These events can lead to cellular senescence and apoptosis due
to a permanent cell cycle arrest. It has been shown that the
absence of the DNA-binding protein CSB (Cockayne syndrome protein) can stabilize p53 (18), which gives rise to
pronounced cell fragility due the activation of p53 target genes
related to the apoptotic commitment. CSB, an essential DNAbinding protein involved in the transcription-coupled DNA
repair pathway is encoded by Ercc6, a gene that shows an
inverse correlation to p53 expression in islets from DEXtreated mothers. Thus, it is possible that the downregulation of
CBS and the upregulation of p53, as well as Gadd45a, could
mediate premature ␤-cell senescence, as suggested in other cell
types (reviewed in Ref. 19).
Importantly, aging of pancreatic islets displays two classic
features, reduced secretory capacity combined with increased
mass (58). As our data further demonstrate, reduced insulin
secretion later in life by islets from pregnant⫹DEX rats is
paralleled by a tendency in increase of pancreatic islet mass.
A mechanism that could explain the sustained miR-29 expression also involves the expression and activity of p53 (69).
Many signal transduction pathways converge on p53 and result
in differential regulation of its downstream targets. Because of
its functional diversity and its importance to cell fate decisions,
p53 levels and activity are tightly regulated through positive
and negative feedback loops (27). Recent studies have
shown that the cross talk between the p53 network and
miRNAs involves the regulation of miRNA transcription/
maturation by p53 as well as the role of miRNAs on the
abundance and activity of p53 (69). Among them, it has
been shown that miR-29 regulates the regulators of p53 to
indirectly activate it (27).
Our present data do not allow us to determine whether p53
is upstream to the long-lasting miR-29 expression or if elevated
miR-29 levels is maintaining increased p53 levels. However,
Bbc3, the gene that encodes Puma, and Gadd45a mRNAs are
directly induced by p53 (45, 12). Thus, the higher p53 content
seen in DEX-treated mothers suggests increased activity of this
transcription factor. We believe that positive feedback could be
involved in the sustained p53/miR29 upregulation.
Increased miR-29 (60) as well as Puma levels (21, 41), had
been associated with pancreatic ␤-cell apoptosis. Here, we did
not further focused on pancreatic ␤-cell death, especially
because we did not find any relevant change in pancreatic gross
morphology. Instead, we focused on the exocytotic machinery,
which is a well-recognized target of miRNAs (16, 37, 53).
Stx-1a is a t-SNAREs involved in insulin exocytosis. Both in
rodents and humans, decreased expression of Stx-1a impairs
GSIS (44, 50). Moreover, using a luciferase-based reporter
gene assay, a recent study has demonstrated that miR-29a
directly targets the Stx-1a gene in the 3=-UTR (5). In agreement with this study, we found that a decrease in Stx-1a protein
levels paralleled GSIS impairment in pregnant⫹DEX rats later
in life. Furthermore, our results show that increased miR-29
expression is associated with Stx-1a gene downregulation and
decreased GSIS in an in vivo context.
Due to its intrinsic complexity, we cannot presently explain
how the combination of pregnancy and GC therapy results in a
long-lasting upregulation of miR-29s. However, given the
late-onset nature of the impairment of pancreatic ␤-cell function in DEX-treated mothers, we believe that determining the
changes in the gestational internal milieu responsible for this
maternal programming requires further investigation. We can
speculate that several factors are candidates for the disruption
of maternal environment. For example, secretion of prolactin,
the main regulator of pancreatic ␤-cell adjustment to pregnancy (8), is reduced by GCs (13). A fall in maternal plasma
concentration of estradiol, a steroid known to increase insulin
biosynthesis and to enhance GSIS (43), was also observed after
antenatal DEX therapy (48).
All together, our data describe a long-term impairment of
maternal pancreatic islet function caused by the use of DEX
during pregnancy. DEX-induced metabolic programming accounts for weigh gain and glucose intolerance later in life. The
present study also describes correlative data pointing to a
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00364.2013 • www.ajpendo.org
DEXAMETHASONE-INDUCED LONG-TERM MATERNAL GLUCOSE INTOLERANCE
putative mechanism underlying the late onset of inaccurate
energy metabolism regulation, which involves an aberrant
expression of miR-29s and p53 and a downregulation of Stx-1a
in pancreatic islets.
ACKNOWLEDGMENTS
We thank Marlene S. Rocha (University of Sao Paulo) for technical
assistance.
GRANTS
This study was supported by the Research Foundation of the State of São
Paulo (FAPESP) and National Council of Research (CNPq).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: P.R.G., M.F.G., L.A.V., M.Q.L., A.R.C., G.F.A., and
S.B. conception and design of research; P.R.G., M.F.G., L.C.P., A.L.R., and
S.C.R. performed experiments; P.R.G., L.A.V., M.Q.L., A.R.C., G.F.A., and S.B.
analyzed data; P.R.G. and S.B. interpreted results of experiments; P.R.G. and S.B.
prepared figures; P.R.G., G.F.A., and S.B. drafted manuscript; P.R.G., M.F.G.,
L.C.P., A.L.R., S.C.R., L.A.V., M.Q.L., A.R.C., G.F.A., and S.B. approved final
version of manuscript; L.A.V., M.Q.L., A.R.C., and S.B. edited and revised
manuscript.
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