Am J Physiol Endocrinol Metab 295: E262–E268, 2008.
First published June 10, 2008; doi:10.1152/ajpendo.90208.2008.
TRANSLATIONAL PHYSIOLOGY
Transient prenatal androgen exposure produces metabolic syndrome
in adult female rats
Marek Demissie,1,3* Milos Lazic,1* Eileen M. Foecking,2 Fraser Aird,1 Andrea Dunaif,1*
and Jon E. Levine2*
1
Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University, Chicago; 2Department
of Neurobiology and Physiology, Northwestern University, Evanston, Illinios; and 3Department of Endocrinology,
Diabetology, and Isotope Therapy, Wroclaw Medical School, Wroclaw, Poland
Demissie M, Lazic M, Foecking EM, Aird F, Dunaif A,
Levine JE. Transient prenatal androgen exposure produces metabolic syndrome in adult female rats. Am J Physiol Endocrinol
Metab 295: E262–E268, 2008. First published June 10, 2008;
doi:10.1152/ajpendo.90208.2008.—Androgen exposure during intrauterine life in nonhuman primates and in sheep results in a
phenocopy of the reproductive and metabolic features of polycystic
ovary syndrome (PCOS). Such exposure also results in reproductive features of PCOS in rodents. We investigated whether transient prenatal androgen treatment produced metabolic abnormalities in adult female rats and the mechanisms of these changes.
Pregnant dams received free testosterone or vehicle injections
during late gestation, and their female offspring were fed regular or
high-fat diet (HFD). At 60 days of age, prenatally androgenized
(PA) rats exhibited significantly increased body weight; parametrial and subcutaneous fat; serum insulin, cholesterol and triglyceride levels; and hepatic triglyceride content (all P ⬍ 0.0125).
There were no significant differences in insulin sensitivity by
intraperitoneal insulin tolerance test or insulin signaling in liver or
skeletal muscle. HFD had similar effects to PA on body weight and
composition as well as on circulating triglyceride levels. HFD
further increased hepatic triglyceride content to a similar extent in
both PA and control rats. In PA rats, HFD did not further increase
circulating insulin, triglyceride, or cholesterol levels. In control
rats, HFD increased insulin levels, but to a lesser extent than PA
alone (⬃2.5- vs. ⬃12-fold, respectively). We conclude that transient prenatal androgen exposure produces features of the metabolic syndrome in adult female rats. Dyslipidemia and hepatic
steatosis appear to be mediated by PA-induced increases in adiposity, whereas hyperinsulinemia appears to be a direct result
of PA.
prenatal androgen exposure; metabolic syndrome; polycystic ovary
syndrome; hepatic steatosis
(PCOS) is among the most common disorders of premenopausal women, affecting ⬃7% of
this population (35). Hyperandrogenemia is the cardinal reproductive feature of the syndrome (11, 28), whereas insulin
resistance is the central metabolic phenotype (12). Abnormalities associated with insulin resistance are common in PCOS,
such as obesity, dyslipidemia, and an increased risk for Type 2
POLYCYSTIC OVARY SYNDROME
* These authors contributed equally to this work.
Address for reprint requests and other correspondence: A. Dunaif,
Division of Endocrinology, Metabolism and Molecular Medicine, Feinberg
School of Medicine, Northwestern Univ., Chicago, IL 60611 (e-mail:
[email protected]).
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diabetes mellitus and, possibly, cardiovascular disease (38).
Hepatic steatosis, another finding associated with insulin resistance (22), is also increased in frequency in PCOS (20).
The mechanism for the association between these reproductive and metabolic abnormalities has been the subject of
intense investigation since this phenomenon was first recognized in 1980 (8). There is extensive evidence that insulin
functions as a cogonadotropin to stimulate ovarian androgen
production (6). Furthermore, because lowering androgen levels
results in, at best, modest improvements in insulin sensitivity
(33), it has been widely accepted that insulin resistance causes
hyperandrogenemia rather than vice versa (12). However, recent studies have challenged this view. First, androgen exposure during intrauterine life in nonhuman primates and in sheep
produces many features of not only the reproductive but also of
the metabolic phenotype of PCOS (1, 18). Human studies
suggest that androgens play an important role in the abnormalities of feedback regulation of gonadotropin secretion (13) and
that they contribute to certain metabolic defects characteristic
of the syndrome (21). Consistent with an essential role of
androgens in the development of PCOS, girls at high risk for
the disorder may have early androgen excess (23, 31). Thus the
developmental role of androgen excess in the etiology of PCOS
has received renewed attention (1).
It is clear that androgens can act on the brain to induce major
sex differences in neural structure and function (9). We have
reported defeminization of the gonadotropin-releasing hormone (GnRH) neurosecretory system with accelerated basal
GnRH pulse generator activity, similar to women with PCOS,
in prenatally androgenized female rats (17). There has also
been growing interest in their role in metabolic programming
(43). Both transient neonatal and continuous pre- to postpubertal exposure resulted in insulin resistance and alterations in
body composition in adult female rats (3, 29, 34). Accordingly,
we sought to determine whether fetal exposure to androgen
excess in female rats would also program a PCOS-like metabolic phenotype and to examine the mechanisms of such
changes.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0193-1849/08 $8.00 Copyright © 2008 the American Physiological Society
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Submitted 2 February 2008; accepted in final form 5 June 2008
METABOLIC SYNDROME IN PRENATALLY ANDROGENIZED RATS
MATERIALS AND METHODS
Animal treatments and diet. All animal procedures were approved
by the Animal Care and Use Committee at Northwestern University
(Evanston, IL). Rats were housed at 23–24°C on a 10:14-h light-dark
cycle.
Time-pregnant female Sprague-Dawley rats were obtained from
Charles River (Portage, WI) at day 14 of gestation and treated from
embryonic day (E) 16 to E19 with daily subcutaneous (sc) injections
of 5 mg of free testosterone (T-1500; Sigma, St. Louis, MO) dissolved
in 500 ␮l sesame oil (S3547; Sigma)/benzyl benzoate (B6630; Sigma)
(prenatally androgenized: PA n ⫽ 8) or of 500 ␮l sesame oil vehicle
as a control (C, n ⫽ 12). This hormonal paradigm mimics the fetal
testosterone surge that is observed in male rats (17). In preliminary
experiments, we found no significant difference in either the femaleto-male offspring ratio, number of pups per litter, or birth weights
between PA and C animals (unpublished observations). Pups were
culled from control litters to equalize group sizes. All litters were
weaned, and females were separated from males at 21 days of age and
fed regular chow diet (RD) (3.30 kcal/g: kcal from fat 15%, carbohydrate 62%, and protein 23%; 7912 Harlan-Teklad, Madison, WI)
and water ad libitum. Body weights were monitored weekly from the
day of weaning to tissue harvest at age 60 days.
Study protocols. At 35 days of age, female rats were either kept on
RD (n ⫽ 37) or switched to the high-fat diet (HFD; n ⫽ 19) (5.24
kcal/g: kcal from fat 60%, carbohydrate 20%, and protein 20%,
D12492 Test Diet; Purina Mills, Richmond, IN). There were 20 C RD,
17 PA RD, 11 C HFD, and 8 PA HFD female rats. Adult female rats
(aged 60 – 63 days) on their respective diets were fasted overnight,
anesthetized with CO2, weighed, and decapitated. To examine
insulin signaling, one-half of the animals in all experimental
groups were injected intraperitoneally with 5 U of regular human
insulin (Novo Nordisk, Princeton, NJ) in 100 ␮l 0.9% sodium
chloride (Abraxis, Schaumburg, IL), and half with 100 ␮l 0.9%
sodium chloride 10 min before death. Trunk blood was collected and
centrifuged, and serum was stored at ⫺80°C. Glucose, insulin, cholesterol, and triglyceride (TG) levels were determined in samples from
saline-treated animals only (10 C RD, 8 PA RD, 6 C HFD, 4 PA
HFD). Liver, adipose tissue depots (parametrial and sc), and soleus
muscles were excised, weighed, frozen, and stored at ⫺80°C for
further analyses. Tissues from both saline- and insulin-treated rats
were used for analyses of body composition and hepatic TG content
(15 C RD, 13 PA RD, 11 C HFD, 8 PA HFD), since administration of
insulin would not acutely alter these end points (Fig. 1).
Dynamic studies. Dynamic studies of glucose homeostasis were
performed in separate groups of animals at age 60 days after a 6-h fast
with jugular catheters implanted 1 day before testing. Intraperitoneal
(ip) glucose tolerance test (IPGTT) was performed in 5 C RD and 6
PA RD rats. A baseline blood sample was followed by intraperitoneal
injection of 2 g/kg body wt dextrose and blood sampling at 2, 5, 10,
15, 30, 60, 90, and 120 min. Intraperitoneal insulin tolerance test
(IPITT) was performed in a separate group of 4 C RD and 6 PA RD
rats. A baseline blood sample was obtained followed by ip injection of
insulin (5 U/rat) with blood sampling at 15, 30, 60, 90 and 120 min.
Hormone assays and hepatic TG content. Whole blood glucose
levels were measured using a Prestige Smart System Glucose Monitor
(Home Diagnostics, Ft. Lauderdale, FL). Insulin levels were measured
by a rat insulin ELISA kit (Crystal Chem, Downers Grove, IL).
Circulating TG and cholesterol levels and hepatic TG content were
assessed by spectrophotometric assay (Infinity Triglyceride Reagent
Kit; Thermo Electron, Pittsburgh, PA). Hepatic TG content was
Fig. 2. A: body weight change with age in control (C; ‚)
and prenatally androgenized (PA; Œ) rats on regular chow
diet (RD). B: body weight change with age in C (䊐) and
PA (■) rats on a high-fat diet (HFD). Parametrial (C) and
sc (D) fat pad weights in C (open bars) and PA (filled
bars) rats on RD and HFD. Values are means ⫾ SE; C RD
n ⫽ 20, PA RD n ⫽ 17, C HFD n ⫽ 11, and PA HFD n ⫽
8. **P ⬍ 0.01 vs C RD (A). *P ⬍ 0.0125 and **P ⬍
0.005 vs C RD (C and D).
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Fig. 1. Study schema with the number of animals used in each experiment.
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METABOLIC SYNDROME IN PRENATALLY ANDROGENIZED RATS
analyzed as described previously (10). Phosphorylation was expressed
as the ratio of phosphorylated to total protein.
Statistical analysis. Repeated-measures two-way ANOVA with
time and treatment as factors was applied to body weight, IPITT, and
IPGTT with a Bonferroni’s post hoc test to determine which groups
differed significantly. For all other analyses, comparisons of interest,
1) C RD vs PA RD, 2) C RD vs C HFD, 3) C HFD vs PA HFD, and
4) PA RD vs PA HFD, were made separately using the appropriate
parametric (Student’s t-test) or nonparametric (Mann-Whitney) tests
according to the sample size (n ⱕ 6, nonparametric test used) and/or
the normality of the data. A Bonferroni correction for multiple testing
was used to adjust the threshold for statistical significance to P ⬍
0.0125 for these four comparisons. The data were transformed when
necessary to achieve homogeneity of variance. Data are reported as
means ⫾ SE. All analyses were performed using GraphPad Software
(GraphPad Software, San Diego, CA).
Fig. 3. Serum insulin (A) and glucose (B) levels in C (open bars) and PA (filled
bars) rats on RD and HFD. Values are means ⫾ SE; C RD n ⫽ 9, PA RD n ⫽
8, C HFD n ⫽ 6, and PA HFD n ⫽ 4 (samples from saline-treated rats only).
*P ⬍ 0.0125 and **P ⬍ 0.005 vs C RD. †P ⬍ 0.0125 vs C HFD.
expressed as percent of protein content as described previously (41).
Frozen livers were stained with Oil red-O for lipids (19).
Western blotting. Liver and soleus muscle lysates (100 ␮g protein)
were resolved using SDS-PAGE on 7.5% gels and incubated with
specific antibodies to insulin receptor substrate (IRS)-1 (Upstate
Biotechnology, Lake Placid, NY), IRS-2 (gift of Dr. M. White; Joslin
Diabetes Center, Boston, MA), protein kinase B (Akt), phospho-Akt
(Ser473), extracellular signal-regulated kinase (ERK)1/2, phosphoERK1/2 (Thr202/Tyr204) (Cell Signaling, Beverly, MA), insulin receptor ␤-subunit (IR-␤; BD Transduction Laboratories, San Jose, CA),
and appropriate secondary antibodies (goat anti-rabbit and -mouse
horseradish peroxidase conjugates; Cell Signaling), with PA and C
samples run together on the same gels. Bands were visualized and
Baseline features. PA treatment did not result in changes in
body weight at weaning (Fig. 2). Beginning at 42 days, body
weight began to increase in PA compared with C rats (Fig. 2).
PA rats had significantly increased body weight compared with
C rats at 60 days (P ⬍ 0.01, Fig. 2). The parametrial and
subcutaneous fat depots were increased by 30% at 60 days in
PA compared with C rats (P ⬍ 0.0125, Fig. 2). HFD produced
similar changes in C rats in body and fat pad weights to those
observed in PA rats with RD and did not further change these
parameters in PA rats (Fig. 2). PA rats exhibited ⬃12-fold
higher fasting insulin levels compared with C rats at 60 days
(P ⬍ 0.005, Fig. 3). However, fasting glucose levels at 60 days
did not differ in PA compared with C rats (Fig. 3). HFD
produced a significant ⬃2.5-fold increase in fasting insulin
levels in C rats (C RD vs C HFD, P ⬍ 0.0125). In PA rats,
HFD did not further increase circulating insulin levels (Fig. 3).
There was an increase in both fasting TG and cholesterol
levels in PA compared with C rats (P ⬍ 0.0125, Fig. 4). HFD
increased TG levels in C rats and did not further influence them
in PA rats (Fig. 4). Cholesterol levels in C and PA rats on HFD
did not differ from those in C RD rats. Hepatic TG content was
Fig. 4. Serum triglyceride (TG; A) and total cholesterol (B)
levels in C (open bars) and PA (filled bars) rats on RD and
HFD. C: hepatic TG content/␮g liver protein in C (open
bars) and PA (filled bars) rats. D: oil red-O stain for lipids
in the liver (top: C RD, PA RD; bottom: C HFD, PA HFD).
Values are means ⫾ SE; C RD n ⫽ 10, PA RD n ⫽ 8, C
HFD n ⫽ 6, PA and HFD n ⫽ 4 for serum TG and
cholesterol (samples from saline-treated rats only); C RD
n ⫽ 15, PA RD n ⫽ 13, C HFD n ⫽ 11, and PA HFD n ⫽
8 for hepatic TG content (samples from saline and insulintreated rats). *P ⬍ 0.0125, **P ⬍ 0.005, and ***P ⬍
0.001 vs C RD. ‡‡‡P ⬍ 0.001 vs PA RD.
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RESULTS
METABOLIC SYNDROME IN PRENATALLY ANDROGENIZED RATS
increased in PA compared with C rats (P ⬍ 0.0125), and the
increased lipid content was also visualized with oil red-O stain
(Fig. 4). HFD produced greater increases in hepatic TG content
in both PA and C rats than those seen with PA alone (Fig. 4).
Dynamic testing. Following IPGTT, there were no differences in glucose or insulin responses between PA and C groups
(Fig. 5). There were no differences in insulin sensitivity assessed by IPITT in PA and C rats (Fig. 5).
Insulin signaling. There were no significant changes in
IRS-1 or IRS-2 protein abundance in PA compared with C rats
in liver or skeletal muscle with either diet (Fig. 6). There were
no differences in the abundance of IR-␤ in the livers of PA
compared with C rats on either diet (data not shown). There
were also no differences in insulin-stimulated activation of Akt
(Fig. 7) or of ERK1/2 (not shown) in PA compared with C rat
skeletal muscle or liver with either diet.
DISCUSSION
These studies reveal that transient prenatal exposure to
androgens during late gestation programs adult female rat
AJP-Endocrinol Metab • VOL
offspring for increased: 1) body weight and visceral adiposity,
2) serum insulin, TG, and cholesterol levels and 3) hepatic TG
content, all features of the metabolic syndrome in humans (30).
However, there was no evidence for defects in insulin action in
PA compared with control rats. High-fat feeding in control rats
had similar effects to those of PA on body weight and composition, and circulating TG levels. High-fat feeding did not
exacerbate these changes in PA rats, whereas it substantially
increased hepatic TG content in both PA and control rats. In
contrast, HFD produced smaller but significant increases in
insulin levels in C rats than PA alone, despite similar body
weights, but did not further exacerbate this change in PA rats.
It is known that transient prenatal androgen exposure can
produce metabolic abnormalities in the adult sheep and rhesus
monkeys (1, 18). In sheep, PA treatment results in offspring
with reduced birth weight, insulin resistance (37), and higher
blood pressure (25). In both male and female rhesus monkeys,
such treatment results in impaired insulin secretion and reduced insulin sensitivity (2, 7), whereas it increases visceral
obesity only in females (14). The metabolic effects of prenatal
androgen exposure have not been investigated in rodents.
However, both transient neonatal and continuous pre- to postpubertal androgen treatment results in increased visceral fat
depots and insulin resistance in adult female rats (3, 29, 34).
Neonatal testosterone treatment also produces increased serum
cholesterol and TG levels (3). Our study demonstrates that
prenatal testosterone treatment can produce similar metabolic
changes in adult female offspring. Furthermore, PA also resulted in hepatic steatosis, a novel finding that has not been
reported previously with either pre- or postnatal androgen
treatment in any species (1, 18, 43).
The contribution of body weight and composition to the
metabolic phenotype resulting from exposure to testosterone at
critical developmental periods has not been investigated in
prior studies (1, 3, 18, 29). Our findings suggest that increased
adiposity mediates most of the observed metabolic changes
since they were reproduced in control animals by HFD, which
resulted in similar increases in weight and visceral adiposity. Thus one of the major mechanisms for PA-mediated
metabolic changes may be through increased food intake
and/or decreased energy expenditure, resulting in greater
adiposity (32, 40).
The only prominent metabolic change specific to PA was the
increase in circulating insulin levels, despite similar increases
in body weight in control rats during HFD. These findings
suggest that prenatal testosterone has an impact on glucose
homeostasis that is independent of increased adiposity. Because glucose levels did not fall despite increased circulating
insulin levels, these findings suggest the presence of insulin
resistance (4), despite our failure to detect significant decreases
in insulin sensitivity or signaling. Indeed, it is quite plausible
that modest decreases in insulin action escaped detection because the IPITT is less sensitive than the glucose clamp
technique for assessing this parameter (5). The observation that
PA had a more substantial effect on circulating insulin levels
than HFD may be due to androgen-mediated decreases in
insulin clearance, such as those that have been reported with
postnatal testosterone treatment (27). However, we detected no
decreases in IR-␤ abundance in livers of PA females, regardless of the diet, suggesting that there were no alterations in
receptor-mediated insulin clearance due to PA treatment. It is
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Fig. 5. Glucose (A) and insulin (B) levels during intraperitoneal glucose
tolerance test (IPGTT); %basal glucose during intraperitoneal insulin tolerance
test (IPITT; C) in C (‚) and PA (Œ) rats on RD . Values are means ⫾ SE. For
IPITT, C RD n ⫽ 5 and PA RD n ⫽ 6. For IPGTT, C RD n ⫽ 4 and PA
RD n ⫽ 6.
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METABOLIC SYNDROME IN PRENATALLY ANDROGENIZED RATS
also possible that PA caused increases in pancreatic ␤-cell
mass similar to those observed in other models for metabolic
programming (36). These possibilities will be directly assessed
in future studies.
Studies of postnatal androgen exposure suggest that androgen acting directly through activation of the androgen receptor
(3, 29) or by aromatization to estrogen (3) can result in
metabolic abnormalities in rodents. The timing of androgen
Fig. 7. Insulin signaling [represented as a phospho-protein
kinase B (Akt)-to-Akt ratio] in RD and HFD rats after 10
min ⫾ insulin (5 U) in liver (A and C, respectively) and
skeletal muscle (B and D, respectively) from C (open bars)
and PA (black bars) rats. Representative gel images for
phospho-proteins are shown above each graph. Values are
means ⫾ SE; C RD n ⫽ 10 saline, n ⫽ 10 insulin; PA RD
n ⫽ 8 saline, n ⫽ 9 insulin; C HFD n ⫽ 6 saline, n ⫽ 5
insulin; and PA HFD n ⫽ 4 saline, n ⫽ 4 insulin.
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Fig. 6. Insulin receptor substrate (IRS)-1 and IRS-2 in the
liver (A and C) and the skeletal muscle (B and D) from C
(open bars) and PA (black bars) rats on RD and HFD.
Representative gel images are shown above each graph.
Values are means ⫾ SE; C RD n ⫽ 10, PA RD n ⫽ 8, C
HFD n ⫽ 6, and PA HFD n ⫽ 4 (samples from salinetreated rats only).
METABOLIC SYNDROME IN PRENATALLY ANDROGENIZED RATS
ACKNOWLEDGMENTS
We thank Jorie Aardema for performing certain hormone assays.
GRANTS
This work was supported by National Institute of Child Health and Human
Development Grants P50 HD-044405, U54 HD-041859, and R01 HD-020677.
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exposure may also be critical. Testosterone treatment earlier
during gestation results in defects in insulin secretion, whereas
treatment later in gestation results in decreased insulin sensitivity in adult female rhesus monkeys (2). In female rats, both
transient neonatal (3) and continuous pre- to postpubertal (29)
dihydrotestosterone treatments result in insulin resistance,
whereas only the pre- to postpubertal treatment results in
increased total body and fat weight. Moreover, other prenatal
insults, such as maternal caloric restriction, nutrient excess, or
stress, result in metabolic abnormalities in adult offspring, such
as increased circulating lipid levels, hepatic steatosis, and
insulin resistance (15, 16). These observations suggest that
prenatal androgen treatment could cause other changes in the
maternal-fetal environment that contribute to the phenotype.
In conclusion, hyperandrogenemia is the cardinal reproductive feature of PCOS (11, 28), and its development may be one
of the earliest harbingers of the disorder (24). We have recently
mapped a susceptibility variant for hyperandrogenemia in
PCOS to an intron of the fibrillin-3 gene (42), consistent with
the hypothesis that hyperandrogenemia is a fundamental defect
in this disorder. Increased endogenous androgen levels are also
associated with metabolic syndrome in epidemiological studies
in both pre- and postmenopausal women (26, 39). Our study
suggests that androgen excess at critical prenatal developmental periods could play a role in the pathogenesis of PCOS,
obesity, and metabolic syndrome.
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