Am J Physiol Regul Integr Comp Physiol 296: R1124–R1131, 2009.

First published January 21, 2009; doi:10.1152/ajpregu.90947.2008.

Acupuncture and exercise restore adipose tissue expression

of sympathetic markers and improve ovarian morphology in rats
with dihydrotestosterone-induced PCOS
Louise Mannerås,1 Stefan Cajander,2 Malin Lönn,3 and Elisabet Stener-Victorin1
1
Institute of Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, University of Gothenburg,
Gothenburg, Sweden; 2 Department of Pathology and Cytology, Sunderby County Hospital, Luleå, Sweden; and 3 Institute
of Medicine, Wallenberg Laboratory, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
Submitted 22 November 2008; accepted in final form 15 January 2009

Mannerås L, Cajander S, Lönn M, Stener-Victorin E. Acu-
puncture and exercise restore adipose tissue expression of sympa-
thetic markers and improve ovarian morphology in rats with
dihydrotestosterone-induced PCOS. Am J Physiol Regul Integr
Comp Physiol 296: R1124 –R1131, 2009. First published January
21, 2009; doi:10.1152/ajpregu.90947.2008.—Altered activity of the
sympathetic nervous system, which innervates adipose and ovarian
tissue, may play a role in polycystic ovary syndrome (PCOS). We
hypothesize that electro-acupuncture (EA) and physical exercise re-
duce sympathetic activity by stimulating ergoreceptors and somatic
afferent pathways in muscles. Here we investigated the effects of
low-frequency EA and physical exercise on mRNA expression of
sympathetic markers in adipose tissue and on ovarian morphology in
female rats that received dihydrotestosterone (DHT) continuously,
starting before puberty, to induce PCOS. At age 11 wk, rats with
DHT-induced PCOS were randomly divided into three groups: PCOS,
PCOS plus EA, and PCOS plus exercise. The latter two groups
received 2-Hz EA (evoking muscle twitches) three times/week or had
free access to a running wheel for 4 –5 wk. In mesenteric adipose
tissue, expression of ␤3-adrenergic receptor (ADRB3), nerve growth
factor (NGF), and neuropeptide Y (NPY) mRNA was higher in
untreated PCOS rats than in controls. Low-frequency EA and exercise
downregulated mRNA expression of NGF and NPY, and EA also
downregulated expression of ADRB3, compared with untreated rats
with DHT-induced PCOS. EA and exercise improved ovarian mor-
phology, as reflected in a higher proportion of healthy antral follicles
and a thinner theca interna cell layer than in untreated PCOS rats.
These findings support the theory that increased sympathetic activity
contributes to the development and maintenance of PCOS and that the
effects of EA and exercise may be mediated by modulation of
sympathetic outflow to the adipose tissue and ovaries.
sympathetic activity; ␤3-adrenergic receptor; androgen receptor;
nerve growth factor; neuropeptide Y
THE AUTONOMIC NERVOUS SYSTEM has been suggested to contrib-
ute to polycystic ovary syndrome (PCOS) (15, 19, 20, 61).
Features of PCOS and the related metabolic syndromes, such
as hyperandrogenemia, hyperinsulinemia, insulin resistance,
and abdominal obesity, are associated with disturbed activity
of the sympathetic nervous system (13, 33). Women with
PCOS have increased sympathetic and decreased parasympa-
thetic components of heart rate variability, an indirect measure
of cardiac autonomic control (61), and abnormal heart rate
recovery, another measure of autonomic function (17). Fur-
thermore, increased ovarian sympathetic nerve activity stimu-
lates androgen secretion in rats (19, 47), and PCOS is associ-
ated with an increase in ovarian catecholaminergic nerve fibers
(20, 48) and altered catecholamine metabolism (15, 49), sug-
gesting increased sympathetic nervous system activity. In-
creased sympathetic nerve activity in the ovaries might con-
tribute to PCOS by stimulating androgen secretion (19, 47).
Ovarian surgeries reduce the amount of androgen-producing
theca cells and the regulation of the hypothalamic-pituitary-
ovarian axis is changed, which increases ovulatory response in
women with PCOS (21). Additionally, ovarian surgeries may
improve ovulatory dysfunction via disruption of ovarian sym-
pathetic innervation.
Recently, we performed direct intra-neural recordings of
sympathetic nerve activity in PCOS patients (58). This
novel study showed that PCOS is associated with increased
sympathetic nervous system activity, which correlated with
the elevated testosterone levels that characterize this syn-
drome. Increased sympathetic outflow might explain the
increased prevalence of vascular disease in women with
PCOS and might contribute to its etiology.
Obesity and metabolic disturbances exacerbate many of the
typical symptoms in women with PCOS and increase the risk
of long-term health consequences. Adipose tissue participates
in the integrative physiology of whole body glucose and fat
metabolism and is innervated by the autonomic nervous sys-
tem, mainly the sympathetic nervous system, which modulates
its metabolic and endocrine functions (6). Alterations in sym-
pathetic activity in adipose tissue can affect the metabolic and
endocrine functions of other tissues by altering the secretion of
fatty acids and adipokines. Indeed, in obese women with
metabolic syndrome, circulatory concentrations and gene ex-
pression of nerve growth factor (NGF), a marker of sympa-
thetic activity, are increased in subcutaneous adipose tissue (7).
However, the expression of genes encoding sympathetic mark-
ers and the influence of therapy on those markers has not been
studied in PCOS.
Since PCOS appears to be associated with increased sym-
pathetic nerve activity, interventions thought to modulate sym-
pathetic nerve activity, such as physical exercise (43) and
low-frequency electro-acupuncture (EA) (39, 51), might be
beneficial. In uncontrolled studies of women with well-defined
PCOS and women with undefined ovulatory dysfunction, acu-

Address for reprint requests and other correspondence: E. Stener-Victorin,

Institute of Neuroscience and Physiology, Dept. of Physiology, Sahlgrenska The costs of publication of this article were defrayed in part by the payment
Academy, Univ. of Gothenburg, Box 434, SE-405 30 Göteborg, Sweden of page charges. The article must therefore be hereby marked “advertisement”
(e-mail: elisabet.stener-victorin@neuro.gu.se). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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 ACUPUNCTURE AND EXERCISE RESTORE SYMPATHETIC MARKERS IN PCOS
puncture exerted long-lasting beneficial effects on endocrine
parameters and anovulation without negative side effects (8,
40, 57). In overweight women with PCOS, exercise together
with other lifestyle modification decreases central fat and
increases insulin sensitivity and ovulatory function (23, 27).
However, those studies were not focused on parameters related
to the autonomic nervous system or the mechanisms underly-
ing the effects of EA.
In rats with polycystic ovaries induced with estradiol valer-
ate (EV), we found that low-frequency (2 Hz) EA (3, 37,
53–56) and physical exercise (36) restored the ovarian expres-
sion of markers of sympathetic nervous system activity, con-
sistent with the notion that EA and exercise inhibit sympathetic
hyperactivity. In rats with PCOS induced with dihydrotestos-
terone (DHT), we showed that low-frequency EA and physical
exercise each reduced insulin resistance and the expression of
adipose tissue genes associated with insulin resistance, obesity,
and inflammation (35). EA did so without affecting adiposity
or adipose tissue cellularity.
Hyperandrogenism is the central feature of PCOS and may
be a key factor in the excessive sympathetic tone associated
with the syndrome. Our DHT-induced rat PCOS model de-
velop obesity and display ovarian dysfunction, both of which
are associated with high sympathetic activity. Therefore, we
aimed to investigate the mRNA expression of the ␤3-adrener-
gic receptor (Adrb3), Ngf, NGF receptor (Ngfr), and neuropep-
tide Y (Npy), all markers of sympathetic nerve activity, and of the
androgen receptor (Ar) in adipose tissue in our rat model of
DHT-induced PCOS (34). We also assessed the effects of
low-frequency EA and physical exercise on the expression of
those genes and on ovarian morphology.
MATERIALS AND METHODS
Animals
Six Wistar dams, each with 8 –9 female pups, were purchased from
Charles River (Frankfurt, Germany). Pups were raised with a lactating
dam until 21 days of age and were then housed four to five per cage
under controlled conditions (21–22°C, 55– 65% humidity, 12:12-h
light-dark cycle). Rats were fed commercial chow containing 18.7%
protein, 4.7% fat, 63% carbohydrates, vitamins, minerals (B&K Uni-
versal, Sollentuna, Sweden), and tap water ad libitum. Animals were
cared for in accordance with the principles of the Guide to the Care
and Use of Experimental Animals (49a). The study was approved by
the Animal Ethics Committee of the University of Gothenburg.
Study Procedure
The study procedure has been described (35). In brief, at 21 days of
age, rats were randomly divided into two groups. The DHT-induced
PCOS rats (n ⫽ 36) were implanted subcutaneously in the neck with
90-day continuous-release pellets (Innovative Research of America,
Sarasota, FL) containing 7.5 mg of DHT (daily dose, 83 ␮g), which
induces metabolic disturbances and other characteristics of PCOS in
adulthood (34); controls (n ⫽ 13) received pellets containing 7.5 mg
of vehicle. Forty days later, to compensate for weight gain, an
additional pellet was implanted that released 3.5 mg of DHT or
placebo for 60 days (i.e., an additional daily dose of 58 ␮g). A
microchip (AVID, Norco, CA) with an identification number was
inserted along with the pellets. After 7 wk of DHT exposure, rats in
the PCOS group were randomly subdivided into three treatment
groups: PCOS (n ⫽ 12), PCOS plus exercise (n ⫽ 13), and PCOS plus
EA (n ⫽ 11). The treatments lasted 4 –5 wk. The rats were weighed
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R1125
weekly. The study was concluded after ⬃12 wk of DHT exposure,
when the rats were 15–16 wk of age.
EA. Low-frequency EA was administered to conscious rats every
second weekday for 4 –5 wk (12–14 treatments). The treatment
procedure has been described in detail previously (35). Each treatment
lasted 15 min during the first week, 20 min during weeks 2–3, and
25 min during weeks 4 –5. Acupuncture needles were inserted
bilaterally into the abdominal and hindlimb muscles, in somatic
segments corresponding to the innervation of the ovaries. The
intensity was adjusted to produce local muscle contractions and
varied from 0.8 –1.3 mA.
Before needle insertion, rats were lightly anesthetized with 2%
isoflurane (Isoba Vet; Schering-Plough, Stockholm, Sweden) in a 1:1
mixture of oxygen and air for 2–3 min. After needle insertion, the rats
were suspended in a fabric harness during EA treatment. To avoid
possible acute effects of EA, rats were not treated for 24 h before the
experiment was ended.
Physical exercise. Rats in the PCOS exercise group were placed
individually in cages with an exercise wheel and allowed to exercise
voluntarily for 4 –5 wk. The exercise wheels were locked 24 h before
the experiment was ended to avoid possible acute effects of physical
exercise.
Three times per week, rats in the PCOS group and the PCOS
exercise group were anesthetized, suspended in a harness, and handled
in the same way as rats in the PCOS EA group but without needle
insertion or EA stimulation. All rats were conscious during the
handling/treatment procedure.
Vaginal smears. The stage of cyclicity was determined by mi-
croscopic analysis of the predominant cell type in vaginal smears
obtained daily from rats at 11 wk of age to the end of the experi-
ment (38).
Tissue collection, RNA isolation and cDNA synthesis and real-
time RT-PCR. Rats were decapitated at 15–16 wk of age (i.e., after
4 –5 wk of treatment and 11–12 wk after pellet implantation). The
mesenteric fat depot was dissected, snap frozen in liquid nitrogen,
and stored at ⫺80°C for mRNA analyses. mRNA isolation and
real-time RT-PCR were performed as previously described in
detail by using a low-density array card (35). Primers and probes
for rat genes corresponding to the TaqMan Gene Expression Assay
numbers and GenBank accession numbers (Table 1). Gene expres-
sion values were calculated with the 2⫺⌬⌬Ct method (31), where Ct is
cycle threshold. The ovaries were excised, fixed in neutral buffered
4% formaldehyde for 24 h, placed in 70% ethanol, dehydrated, and
embedded in paraffin.
Ovarian morphology. The ovaries were longitudinally and serially
sectioned at 4 ␮m; every 20th section (n ⫽ 5 per ovary) was mounted
on a glass slide, stained with hematoxylin and eosin, and analyzed
under a conventional birefringence microscope by two persons
blinded to the origin of the sections. The slides were scanned with
ScanScope (Aperio Technologies, Vista, CA) and analyzed with
ImageScope virtual microscopy software (Aperio Technologies). The
diameter of the ovaries in the section with the largest ovarian cross
section was measured with a calibrated scale tool in the virtual
microscope. Antral follicles, distinguished by an antrum within the
granulosa cell layers enclosing the oocyte, were counted by two
persons (to avoid duplicate counting) and classified as atretic or
healthy. Follicles were considered atretic if at least two pyknotic
granulosa cells were observed or if the oocyte showed obvious signs
of degeneration (22). The thickness of theca interna cell layer was
measured in the largest healthy and atretic antral follicles. Corpora
lutea were noted but not counted.
Statistical Analyses
All statistical evaluations were performed with SPSS software
(version 13.0; SPSS, Chicago, IL). Values are reported as means ⫾
SE. The Kruskal-Wallis test was used for comparisons of all groups,
296 • APRIL 2009 • www.ajpregu.org
R1126 ACUPUNCTURE AND EXERCISE RESTORE SYMPATHETIC MARKERS IN PCOS

Table 1. Genes presented on the TaqMan low-density array with their respective TaqMan gene expression assay numbers
and GenBank accession numbers
Gene Symbol Gene Description TaqMan Gene Expression Assay No. GenBank Accession No.

Putative Reference Genes

18S 18S ribosomal RNA Hs99999901_s1 X03205
Actb ␤-Actin Rn00667869_m1 NM_012583.2
Hprt Hypoanthine guanine phosphoribosyl transferase Rn01527840_m1 NM_012583-2
Ppia Peptidylprolyl isomerase A Rn00690933_m1 NM_017101.1
Target Genes
Adrb3 Adrenergic receptor, beta 3 Rn00565393_m1 NM_013108.1
Ar Androgen receptor Rn00560747_m1 NM_012502.1
Ngfr Nerve growth factor receptor Rn00561634_m1 NM_012610.1
Ngf Nerve growth factor, beta Rn01533872_m1 XM_227525.3
Npy Neuropeptide Y Rn00561681_m1 NM_012614.1

and if significant, a Mann-Whitney U-test was performed between
individual groups. P ⬍ 0.05 was considered significant.
RESULTS
Exercise and EA Influence Ovarian Morphology
All control rats had a normal estrus cycle of 4 days. Rats in
the PCOS group were acyclic, while those in the PCOS
exercise and PCOS EA groups exhibited irregular cycles.
Ovarian weight and area were lower in the PCOS group than in
the controls, and neither variable was affected by exercise or
low-frequency EA (Table 2).
Morphologically, ovaries from control rats exhibited fol-
licles in various stages of development, ranging from pri-
mordial follicles to mature preovulatory follicles, as well as
several corpora lutea, many resulting from recent ovulations
(Fig. 1, A–B). Some antral follicles contained pyknotic granu-
losa cells.
In rats with DHT-induced PCOS, small follicles in early
development were observed, as were antral follicles with
various degrees of atresia: some follicles had nuclear pyk-
nosis in few cells, and others exhibited massive degenera-
tion and granulosa cells in the antrum, as well as degener-
ated cells containing pyknotic nuclei scattered throughout
the membrane granulosa (Fig. 1, C–D). Corpora lutea were
absent in all PCOS rats. Although the number of antral
follicles was similar in DHT-induced PCOS and control
rats, DHT-induced PCOS rats had a higher proportion of
atretic antral follicles (Fig. 2) in which the thickness of
theca interna was increased (Table 2).
Rats in the PCOS exercise and PCOS EA groups had a
lower proportion of atretic antral follicles (Fig. 2), and the
theca interna cell layer in those follicles was thinner than in
untreated DHT-induced PCOS rats (Table 2). However, both
the PCOS exercise and PCOS EA rats had still a signifi-
cantly higher proportion of atretic antral follicles compared
with controls (Fig. 2). In the PCOS EA group, fresh corpora
lutea were observed in five of eleven rats (45%) (Fig. 1,
E–F), indicating ovulation. In the PCOS exercise group,
healthy follicles were observed, but no fresh corpora lutea
(Fig. 1, G–H).
PCOS Increases Gene Expression of Sympathetic Markers in
Mesenteric Adipose Tissue
In mesenteric adipose tissue, expression of Adrb3, Ngf, and
Npy mRNA was higher in rats with DHT-induced PCOS than
controls (Fig. 3). Expression of Ar and Ngfr mRNA did not
differ in DHT-induced PCOS rats and controls (Fig. 3).
EA and Physical Exercise Influence the Expression of
Sympathetic Markers and AR in Mesenteric Adipose Tissue
Repeated low-frequency EA and physical exercise for
4 –5 wk each lowered DHT-induced changes in the expres-
sion of Ngf and Npy mRNA in mesenteric adipose tissue; EA
also lowered the expression of Adrb3 and Ar mRNA (Fig.
3). Neither exercise nor EA influenced Ngfr mRNA expres-
sion (Fig. 3). Furthermore, the expression of selected genes
after low-frequency EA and physical exercise were not
significantly different from the corresponding level in con-
trol rats.
DISCUSSION
Increased sympathetic activity may be an important etiolog-
ical factor in PCOS (15, 17, 19, 20, 48, 49, 58, 61). Like the

Table 2. Effects of physical exercise and electro-acupuncture on ovarian weight and area and thickness of the theca interna
of antral follicles
Variable Control PCOS PCOS Exercise PCOS EA Kruskal-Wallis

Average weight of one ovary, mg 82.64⫾3.04 35.39⫾2.17* 39.06⫾2.74* 37.39⫾3.27* P ⬍ 0.001

Ovarian area, mm2 24.00⫾1.43 7.74⫾0.43* 8.64⫾0.62* 6.61⫾1.13* P ⬍ 0.001
Thickness of TI, ␮m
Atretic antral follicles 23.40⫾2.45 31.71⫾1.34† 26.84⫾1.03‡ 22.86⫾1.65§ P ⬍ 0.001
Healthy antral follicles 19.95⫾1.80 18.53⫾0.98 19.23⫾1.02 16.77⫾1.18 NS
Values are means ⫾ SE, n ⫽ 10 –12/group. PCOS, polycystic ovary syndrome; TI, theca interna; NS, not significant. *P ⬍ 0.001 vs. control (Mann-Whitney
U-test); †P ⬍ 0.01 vs. control (Mann-Whitney U-test); ‡P ⬍ 0.05 vs. PCOS (Mann-Whitney U-test); §P ⬍ 0.01 vs. PCOS (Mann-Whitney U-test).

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 ACUPUNCTURE AND EXERCISE RESTORE SYMPATHETIC MARKERS IN PCOS R1127

Fig. 1. A: ovary from a normal cycling control

rat. B: high-power view shows a recently ovu-
lated collapsed follicle with the apical rupture
hole (broken arrow) closed by granulosa cells
and perifollicular edema (thin arrow) typical of
ovulation and (on the left) an antral follicle
containing macrophages (thick arrow) and a
trapped oocyte (long thick arrow). C: ovary
from a rat with dihydrotestosterone (DHT)-
induced polycystic ovary syndrome (PCOS)
with small early atretic follicles (EAF) and
antral follicles with various degrees of atresia.
D: high-power view shows degenerating py-
knotic granulosa cells in the antral part of the
membrane granulosa in EAF. E: ovary from a
rat in PCOS EA group with healthy follicles
(HF), atretic follicles (AF), and fresh corpora
lutea (FCL). F: high-power view shows FCL
and a HF. G: ovary from a rat in the PCOS
exercise group with HF, EAF, and old AF
(arrows). H: high-power view shows a HF and
an EAF.

metabolic syndrome, PCOS is characterized by insulin resis- is increased in mesenteric adipose tissue, a fat depot that
tance and obesity, which are associated with enhanced activity affects metabolic status by releasing free fatty acids to the liver
of the sympathetic nervous system (28, 59). Recently, it was via the portal vein. This finding supports the notion that
demonstrated that sympathetic activation is greater in subjects autonomic nervous system is involved in the pathogenesis of
with central obesity, a feature of PCOS, than in those with PCOS. Low-frequency EA and physical exercise each influ-
peripheral obesity (18). These findings underscore the impor- enced ovarian morphology and downregulated the expression
tance of evaluating new treatment strategies that attenuate of several markers of sympathetic nervous activity in mesen-
sympathetic activity in patients with PCOS. teric adipose tissue, indicating that these interventions exert
Using a rat model of DHT-induced PCOS, we show here their effects by modulating sympathetic outflow to the adipose
that the expression of several markers of sympathetic activity tissue and ovaries.
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R1128 ACUPUNCTURE AND EXERCISE RESTORE SYMPATHETIC MARKERS IN PCOS
Fig. 2. Ratio of healthy and atretic antral follicles, counted from 5 consecutive
slides. Values are means ⫾ SE, n ⫽ 10 –12/group. P ⬍ 0.001 (Kruskal Wallis),
†††
P ⬍ 0.001; ††P ⬍ 0.01 vs. control (Mann-Whitney U-test); *P ⬍ 0.05 vs.
PCOS (Mann-Whitney U-test).
Mechanisms Behind the Effects of Low-Frequency EA and
Physical Exercise
Most likely, low-frequency EA and physical exercise ex-
erted their effects in rats with DHT-induced PCOS by stimu-
lating ergoreceptors and somatic afferents in the muscles,
which results in modulation of spinal reflexes and central
sympathetic outflow. Previously, we showed that low-fre-
quency EA increases ovarian blood flow and that this effect is
mediated as a reflex response via ovarian sympathetic nerves,
which in turn is controlled via central nervous system pathways
(50). Furthermore, reduction in central sympathetic activity, as
reflected by a drop in blood pressure, after low-frequency
electrical muscle stimulation is mediated by the release of
␤-endorphin (24, 25). Neurons that express proopiomelanocor-
tin (the precursor of ␣-MSH and ␤-endorphin) and other
neuropeptides reside in the hypothalamic arcuate nucleus, a
site for the regulation of metabolism and reproduction (9).
Hypothetically, low-frequency EA and physical exercise could
each modulate sympathetic outflow, metabolism, and release of
gonatropin-releasing hormone and lutenizing hormone (LH)
through effects on ␤-endorphin and other neuropeptides (9,
51). Evidence that ␤-endorphin is involved in PCOS comes
from recent trials in which naltrexone, a ␮-receptor antagonist,
improved endocrine function (induced ovulation and decreases
in LH, LH-to-follicle-stimulating hormone ratio, and testoster-
Fig. 3. Relative expression of ␤3-adrenergic re-
ceptor (ADRB3), androgen receptor (AR), nerve
growth factor (NGF), NGF receptor (NGFR),
and neuropeptide Y (NPY) mRNA in mesenteric
adipose tissue in control rats and in the 3 PCOS
groups. Values are means ⫾ SE, n ⫽ 10/group.
The average cycle threshold (Ct) value for all
genes was 28.6 ⫾ 0.1 (range, 23.8 –34.6).
Kruskal-Wallis analysis: ADRB3, P ⬍ 0.01; AR,
P ⬍ 0.01; NGF, P ⬍ 0.01; NPY, P ⬍ 0.001.
†
P ⬍ 0.05, ††P ⬍ 0.01 vs. control (Mann-Whit-
ney U-test). ***P ⬍ 0.001 vs. PCOS (Mann-
Whitney U-test).
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one) and metabolic function (decreases in body mass index and
fasting serum insulin) in women with PCOS (1, 14).
Markers of Sympathetic Activity in Mesenteric Adipose
Tissue. Effect of Treatment?
The autonomic nervous system has a distinct organization in
different adipose tissue depots. The same set of neurons con-
trols the various intra-abdominal adipose tissue depots,
whereas subcutaneous adipose tissue located outside the ab-
dominal compartment receives input from other autonomic
neurons (45).
NPY. NPY, a phenotypic marker for sympathetic neurons, is
colocalized and coreleased with norepinephrine at peripheral
sites of action and is therefore considered a good indicator of
sympathetic activity (42). In coculture with adipocytes, sym-
pathetic neurons secrete increased amounts of NPY, suggesting
cross talk between these two cell types (60). Adipose tissue and
adipocytes both produce and secrete NPY (29), which is
important in adipose tissue remodeling that results in abdom-
inal obesity and metabolic syndrome (30). Consistent with
these findings, mesenteric expression of Npy mRNA was
higher in our DHT-induced rat PCOS model than in controls.
Furthermore, in women with PCOS, circulating levels of NPY
are elevated independently of body mass index (4). Both
low-frequency EA and physical exercise resulted in pro-
nounced downregulation of DHT-induced increase of in mes-
enteric Npy mRNA expression, indicating decreased sympa-
thetic activity. Moreover, the mesenteric Npy expression after
low-frequency EA and physical exercise were not significantly
different from the corresponding level in control rats.
NGF and NGFR. The development and maintenance of
sympathetic nerves are facilitated by the target-derived neuro-
trophin NGF and its low-affinity receptor (NGFR). NGF is
secreted from adipocytes and can therefore be considered to be
an adipokine (46), which may be involved in communication
between adipocytes and sympathetic neurons. NGF expression
is closely linked to inflammatory conditions, as TNF-␣ stim-
ulates NGF production by adipocyte (46). Overweight women
and women with metabolic syndrome have high levels of
circulating NGF (7), which are associated with insulin resis-
tance, as well as increased levels of IL-6 and leptin. The high
circulatory levels of NGF may reflect the increased levels of
NGF mRNA in adipose tissue in obese women (7). NGF
production in adipose tissue is also increased in genetic models
of obesity in animals (44).
296 • APRIL 2009 • www.ajpregu.org
 ACUPUNCTURE AND EXERCISE RESTORE SYMPATHETIC MARKERS IN PCOS
Ngf expression in mesenteric adipose tissue was higher in
rats with DHT-induced PCOS than in controls and was reduced
by both low-frequency EA and exercise. This result is in line
with the decrease we observed in the high levels of ovarian
NGF expression after 4 wk of low-frequency EA and exercise
in rats with EV-induced polycystic ovaries (36, 54, 56). Fur-
thermore, the Ngf expression level after low-frequency EA and
exercise was not different from the Ngf expression level in
control rats.
ADRB3. Lipolysis is under control of stimulatory ␤-ADR
and inhibitory ␣-ADR. Increased release of free fatty acids
from visceral fat cells, due to enhanced lipolysis, to the portal
venous system has been hypothesized to cause insulin resis-
tance and other metabolic disturbances. In lean women with
PCOS, lipolytic activity in visceral adipose tissue is higher
than in lean controls (12). This finding might partly explain the
increased prevalence of insulin resistance in lean women with
PCOS, although obesity has an additive role to insulin resis-
tance in PCOS (11, 41, 58). Moreover, a relationship between
upper-body obesity, its associated metabolic complications,
and increased visceral fat ADRB3 sensitivity has been reported
(26, 32).
In the present study, expression of Adrb3 mRNA in mesen-
teric adipose tissue was higher in rats with DHT-induced
PCOS than in controls, supporting the notion that androgens
enhance lipolytic activity in rodents (2). Low-frequency EA
downregulated Adrb3 gene expression compared with un-
treated PCOS rats, and the Adrb3 expression after low-fre-
quency EA was not different from the corresponding level in
control rats. Exercise had no effect on Adrb3 gene expression.
Lower Adrb3 expression in visceral adipose tissue might de-
crease lipolytic activity in this depot, a potential explanation
for the increased insulin sensitivity induced by EA in our
previous study (35).
AR. The presence of ARs in preadipocytes and adipocytes
suggests that androgens contribute to the control of adipose
development and regulation (10). The induction of PCOS in
our rat model by continuous administration of DHT did not
influence the expression of Ar mRNA in adipose tissue. How-
ever, after EA treatment, Ar gene expression in adipose tissue
was lower than in untreated rats with DHT-induced PCOS, and
did not differ from Ar gene expression in controls.
Exercise and EA Improve Ovarian Morphology
Ovarian morphology in DHT-induced PCOS rats has been
extensively described elsewhere (34). In evaluating the effects
of different treatment modalities, it is important to be aware
that the rat is a multiovulatory species. Therefore, the ovarian
morphology of our DHT-induced rat PCOS model cannot be
directly compared with human polycystic ovaries. For exam-
ple, the decreased ovarian size, as well as increased proportion
of atretic follicles, seen in this rat PCOS model, are not in line
with human polycystic ovaries. Nevertheless, both exercise and
low-frequency EA positively influenced ovarian morphology.
In the PCOS EA groups, we observed fresh corpora lutea in
45% of rats, indicating ovulation, and a decreased proportion
of atretic antral follicles, both novel findings. A lower propor-
tion of atretic antral follicles was also observed in the PCOS
exercise group. Previously, we showed that ovarian morpho-
logical changes are partly reversed by exercise (36) but not by
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R1129
EA (54, 56) in rats with EV-induced polycystic ovaries. A
reasonable explanation for this discrepancy might be that rats
in the present study were conscious during EA but were
anesthetized in our previous studies. Regarding the exercise,
rats with EV-induced polycystic ovaries ran longer distances
than rats with DHT-induced PCOS in the present study. We
found no differences in ovarian weight or area after EA or
exercise treatment. Since corpora lutea were observed in the
EA group, one would expect increases in ovarian weight and
area. However, the EA group had fewer antral follicles than
untreated rats with DHT-induced PCOS, which might explain
why weight and area were unaffected.
Furthermore, the theca interna cell layer was thinner in both
the PCOS EA and PCOS exercise groups than in PCOS rats.
Since the theca interna is innervated by sympathetic neurons
and is important in steroidogenesis (47), the reduced thickness
might reflect inhibition of ovarian sympathetic neurons. Previ-
ously, we showed that low-frequency EA increases ovarian
blood flow (50, 52). Transection of the ovarian sympathetic
nerves eliminated this response and also reversed the changes
in ovarian morphology induced by EV in rats with polycystic
ovaries (5). These findings confirm the role of the sympathetic
nervous system in the control of ovarian function. Further
supporting the involvement of sympathetic nervous system in
PCOS, women with the syndrome have significantly higher
NGF levels in the follicular fluid than controls (16).
Perspectives and Significance
Previously, we found that women with PCOS had increased
sympathetic nerve activity related to hormonal and metabolic
features and that both testosterone and cholesterol were inde-
pendent predictors of such activity, with testosterone having
the stronger impact (60). It is not yet clear whether increased
sympathetic activity is an etiologic factor in PCOS or a
consequence of hyperandrogenism. However, our findings sup-
port the theory that increased sympathetic activity contributes
to the development and maintenance of PCOS. Thus, therapies
aimed at reducing sympathetic nerve activity might alleviate
the signs and symptoms of PCOS.
Conclusion
In rats with DHT-induced PCOS, low-frequency EA and
exercise each reduced the expression of genes encoding mark-
ers of sympathetic activity in adipose tissue and had beneficial
effects on ovarian morphology, reflecting modulation of sym-
pathetic outflow to the adipose tissue and ovaries.
ACKNOWLEDGMENTS
We thank Malin Govik and Kristy McKamey-Blom for technical assistance.
We also thank the SWEGENE Göteborg Genomics Core Facility platform,
University of Gothenburg.
GRANTS
This study was supported by grants from the Swedish Medical Research
Council (project numbers: 2005-72VP-15445-01A, 2005-72VX-15276-01A,
K2007-54X-20325-01-3), Novo Nordisk Foundation, Wilhelm and Martina
Lundgrens’s Science Fund, Hjalmar Svensson Foundation, Tore Nilson Foun-
dation, Åke Wiberg Foundation, Herbert and Karin Jacobsson’s Foundation,
Swedish Diabetes Association Research Foundation, Adlerbert Research Foun-
dation, Ekhaga Foundation, and the Swedish federal government under the
LUA/ALF agreement ALFFGBG-10984 and Regional research and develop-
ment (FOU) agreement.
296 • APRIL 2009 • www.ajpregu.org
R1130 ACUPUNCTURE AND EXERCISE RESTORE SYMPATHETIC MARKERS IN PCOS
REFERENCES
1. Ahmed MI, Duleba AJ, El Shahat O, Ibrahim ME, Salem A. Naltrex-
one treatment in clomiphene resistant women with polycystic ovary
syndrome. Hum Reprod 23: 2564 –2569, 2008.
2. Arner P. Effects of testosterone on fat cell lipolysis. Species differences
and possible role in polycystic ovarian syndrome. Biochimie 87: 39 – 43,
2005.
3. Bai YH, Lim SC, Song CH, Bae CS, Jin CS, Choi BC, Jang CH, Lee
SH, Pak SC. Electro-acupuncture reverses nerve growth factor abundance
in experimental polycystic ovaries in the rat. Gynecol Obstet Invest 57:
80 – 85, 2004.
4. Baranowska B, Radzikowska M, Wasilewska-Dziubinska E, Kaplin-
ski A, Roguski K, Plonowski A. Neuropeptide Y, leptin, galanin and
insulin in women with polycystic ovary syndrome. Gynecol Endocrinol
13: 344 –351, 1999.
5. Barria A, Leyton V, Ojeda SR, Lara HE. Ovarian steroidal response to
gonadotropins and ␤-adrenergic stimulation is enhanced in polycystic
ovary syndrome: role of sympathetic innervation. Endocrinology 133:
2696 –2703, 1993.
6. Bartness TJ, Song CK. Thematic review series: Adipocyte biology.
Sympathetic and sensory innervation of white adipose tissue. J Lipid Res
48: 1655–1672, 2007.
7. Bullo M, Peeraully MR, Trayhurn P, Folch J, Salas-Salvado J. Cir-
culating nerve growth factor levels in relation to obesity and the metabolic
syndrome in women. Eur J Endocrinol 157: 303–310, 2007.
8. Chen BY, Yu J. Relationship between blood radioimmunoreactive ␤-en-
dorphin and hand skin temperature during the electro-acupuncture induc-
tion of ovulation. Acupunct Electrother Res 16: 1–5, 1991.
9. Crown A, Clifton DK, Steiner RA. Neuropeptide signaling in the
integration of metabolism and reproduction. Neuroendocrinology 86:
175–182, 2007.
10. Dieudonne MN, Pecquery R, Boumediene A, Leneveu MC, Giudicelli
Y. Androgen receptors in human preadipocytes and adipocytes: regional
specificities and regulation by sex steroids. Am J Physiol Cell Physiol 274:
C1645–C1652, 1998.
11. Dunaif A, Segal KR, Futterweit W, Dobrjansky A. Profound peripheral
insulin resistance, independent of obesity, in polycystic ovary syndrome.
Diabetes 38: 1165–1174, 1989.
12. Ek I, Arner P, Ryden M, Holm C, Thorne A, Hoffstedt J, Wahrenberg
H. A unique defect in the regulation of visceral fat cell lipolysis in the
polycystic ovary syndrome as an early link to insulin resistance. Diabetes
51: 484 – 492, 2002.
13. Fagius J. Sympathetic nerve activity in metabolic control—some basic
concepts. Acta Physiol Scand 177: 337–343, 2003.
14. Fruzzetti F, Bersi C, Parrini D, Ricci C, Genazzani AR. Effect of
long-term naltrexone treatment on endocrine profile, clinical features, and
insulin sensitivity in obese women with polycystic ovary syndrome. Fertil
Steril 77: 936 –944, 2002.
15. Garcia-Rudaz C, Armando I, Levin G, Escobar ME, Barontini M.
Peripheral catecholamine alterations in adolescents with polycystic ovary
syndrome. Clin Endocrinol 49: 221–228, 1998.
16. Garcia-Rudaz MC, Mayerhofer A, Ojeda SR, Dissen GA. An excessive
ovarian production of nerve growth factor (NGF) facilitates the develop-
ment of polycystic ovarian morphology in mice and is a discernible feature
ovarian syndrome in humans. Proc Endocrine Society’s 90th Annual
Meeting. San Francisco, CA, 2008.
17. Giallauria F, Palomba S, Manguso F, Vitelli A, Maresca L, Tafuri D,
Lombardi G, Colao A, Vigorito C, Orio F. Abnormal heart rate recovery
after maximal cardiopulmonary exercise stress testing in young over-
weight women with polycystic ovary syndrome. Clin Endocrinol (Oxf) 68:
88 –93, 2008.
18. Grassi G, Dell’Oro R, Facchini A, Quarti Trevano F, Bolla GB,
Mancia G. Effect of central and peripheral body fat distribution on
sympathetic and baroreflex function in obese normotensives. J Hypertens
22: 2363–2369, 2004.
19. Greiner M, Paredes A, Araya V, Lara HE. Role of stress and sympa-
thetic innervation in the development of polycystic ovary syndrome.
Endocrine 28: 319 –324, 2005.
20. Heider U, Pedal I, Spanel-Borowski K. Increase in nerve fibers and loss
of mast cells in polycystic and postmenopausal ovaries. Fertil Steril 75:
1141–1147, 2001.
21. Hendriks ML, Ket JCF, Hompes PGA, Homburg R, Lambalk CB.
Why does ovarian surgery in PCOS help? Insight into the endocrine
AJP-Regul Integr Comp Physiol • VOL
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (152.118.148.234) on January 15, 2018.
Copyright © 2009 American Physiological Society. All rights reserved.
implications of ovarian surgery for ovulation induction in polycystic ovary
syndrome. Hum Reprod Update 13: 249 –264, 2007.
22. Hirshfield AN, Midgley ARJ. Morphometric analysis of follicular devel-
opment in the rat. Biol Reprod 19: 597– 605, 1978.
23. Hoeger KM. Role of lifestyle modification in the management of poly-
cystic ovary syndrome. Best Pract Res Clin Endocrinol Metab 20: 293–
310, 2006.
24. Hoffmann P, Carlsson S, Skarphedinsson JO, Thoren P. Role of
different serotonergic receptors in the long-lasting blood pressure depres-
sion following muscle stimulation in the spontaneously hypertensive rat.
Acta Physiol Scand 139: 305–310, 1990.
25. Hoffmann P, Thoren P. Electric muscle stimulation in the hind leg of the
spontaneously hypertensive rat induces a long-lasting fall in blood pres-
sure. Acta Physiol Scand 133: 211–219, 1988.
26. Hoffstedt J, Wahrenberg H, Thorne A, Lönnqvist F. The metabolic
syndrome is related to ␤3-adrenoceptor sensitivity in visceral adipose
tissue. Diabetologia 39: 838 – 844, 1996.
27. Huber-Buchholz MM, Carey DGP, Norman RJ. Restoration of repro-
ductive potential by lifestyle modification in obese polycystic ovary
syndrome: role of insulin sensitivity and luteinizing hormone. J Clin
Endocrinol Metab 84: 1470 –1474, 1999.
28. Kaaja RJ, Poyhonen-Alho MK. Insulin resistance and sympathetic
overactivity in women. J Hypertens 24: 131–141, 2006.
29. Kos K, Harte AL, James S, Snead DR, O’Hare JP, McTernan PG,
Kumar S. Secretion of neuropeptide Y in human adipose tissue and its
role in maintenance of adipose tissue mass. Am J Physiol Endocrinol
Metab 293: E1335–E1340, 2007.
30. Kuo LE, Kitlinska JB, Tilan JU, Li L, Baker SB, Johnson MD, Lee EW,
Burnett MS, Fricke ST, Kvetnansky R, Herzog H, Zukowska Z. Neu-
ropeptide Y acts directly in the periphery on fat tissue and mediates stress-
induced obesity and metabolic syndrome. Nat Med 13: 803– 811, 2007.
31. Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 2⫺⌬⌬CT method. Methods 25:
402– 408, 2001.
32. Lönnqvist F, Thome A, Nilsell K, Hoffstedt J, Arner P. A pathogenic
role of visceral fat ␤3-adrenoceptors in obesity. J Clin Invest 95: 1109 –
1116, 1995.
33. Mancia G, Bousquet P, Elghozi JL, Esler M, Grassi G, Julius S, Reid
J, Van Zwieten PA. The sympathetic nervous system and the metabolic
syndrome. J Hypertens 25: 909 –920, 2007.
34. Mannerås L, Cajander S, Holmäng A, Seleskovic Z, Lystig T, Lönn
M,. Stener-Victorin E. A new rat model exhibiting both ovarian and
metabolic characteristics of polycystic ovary syndrome. Endocrinology
148: 3781–3791, 2007.
35. Mannerås L, Jonsdottir IH, Holmäng A, Lönn M, Stener-Victorin E.
Low frequency electro-acupuncture and physical exercise improve meta-
bolic disturbances and modulate gene expression in adipose tissue in rats
with dihydrotestosterone-induced polycystic ovary syndrome. Endocrinol-
ogy 149: 3559 –3568, 2008.
36. Manni L, Cajander S, Lundeberg T, Naylor AS, Aloe L, Holmäng A,
Jonsdottir IH, Stener-Victorin E. Effect of exercise on ovarian mor-
phology and expression of nerve growth factor and ␣1- and ␤2-adrenergic
receptors in rats with steroid-induced polycystic ovaries. J Neuroendocri-
nol 17: 846 – 858, 2005.
37. Manni L, Lundeberg T, Holmäng A, Aloe L, Stener-Victorin E. Effect
of electro-acupuncture on ovarian expression of ␣1- and ␤2-adrenoceptors,
and p75 neurotrophin receptors in rats with steroid-induced polycystic
ovaries. Reprod Biol Endocrinol 3: 21, 2005.
38. Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous
cycle phases of rats: Some helpful considerations. Braz J Biol 62: 609 –
614, 2002.
39. Middlekauff HR, Hui K, Yu JL, Hamilton MA, Fonarow GC, Morigu-
chi J, MacLellan WR, Hage A. Acupuncture inhibits sympathetic acti-
vation during mental stress in advanced heart failure patients. J Card Fail
8: 399 – 406, 2002.
40. Mo X, Li D, Pu Y, Xi G, Le X, Fu Z. Clinical studies on the mechanism
for acupuncture stimulation of ovulation. J Tradit Chin Med 13: 115–119,
1993.
41. Morales AJ, Laughlin GA, Butzow T, Maheshwari H, Baumann G,
Yen SS. Insulin, somatotropic, and luteinizing hormone axes in lean and
obese women with polycystic ovary syndrome: common and distinct
features. J Clin Endocrinol Metab 81: 2854 –2864, 1996.
42. Morris MJ, Russell AE, Kapoor V, Cain MD, Elliott JM, West MJ,
Wing LM, Chalmers JP. Increases in plasma neuropeptide Y concentra-
296 • APRIL 2009 • www.ajpregu.org
 ACUPUNCTURE AND EXERCISE RESTORE SYMPATHETIC MARKERS IN PCOS
tions during sympathetic activation in man. J Auton Nerv Syst 17: 143–
149, 1986.
43. Mueller PJ. Exercise training and sympathetic nervous system activity:
evidence for physical activity dependent neural plasticity. Clin Exp Phar-
macol Physiol 34: 377–384, 2007.
44. Nisoli E, Tonello C, Benarese M, Liberini P, Carruba MO. Expression
of nerve growth factor in brown adipose tissue: implications for thermo-
genesis and obesity. Endocrinology 137: 495–503, 1996.
45. Nogueiras R, Wilson H, Rohner-Jeanrenaud F, Tschöp MH. Central
nervous system regulation of adipocyte metabolism. Regul Pept 149:
26 –31, 2008.
46. Peeraully MR, Jenkins JR, Trayhurn P. NGF gene expression and
secretion in white adipose tissue: regulation in 3T3–L1 adipocytes by
hormones and inflammatory cytokines. Am J Physiol Endocrinol Metab
287: E331–E339, 2004.
47. Ricu M, Paredes A, Greiner M, Ojeda SR, Lara HE. Functional
development of the ovarian noradrenergic innervation. Endocrinology
149: 50 –56, 2008.
48. Semenova II. Adrenergic innervation of ovaries in Stein-Leventhal syn-
drome [in Russian]. Vestn Akad Med Nauk SSSR 24: 58 – 62, 1969.
49. Shoupe D, Lobo RA. Evidence for altered catecholamine metabolism in
polycystic ovary syndrome. Am J Obstet Gynecol 150: 566 –571, 1984.
49a.Statens jordbruksverks föreskrifter om uppfödning, förvaring, tillhanda-
hållande och användning m.m. av försöksdjur. L 50, DFS 2004: 15,
www.sjv.se.
50. Stener-Victorin E, Fujisawa S, Kurosawa M. Ovarian blood flow
responses to electroacupuncture stimulation depend on estrous cycle and
on site and frequency of stimulation in anesthetized rats. J Appl Physiol
101: 84 –91, 2006.
51. Stener-Victorin E, Jedel E, Mannerås L. Acupuncture in polycystic
ovary syndrome: current experimental and clinical evidence. J Neuroen-
docrinol 20: 290 –298, 2008.
52. Stener-Victorin E, Kobayashi R, Kurosawa M. Ovarian blood flow
responses to electro-acupuncture stimulation at different frequencies and
intensities in anaesthetized rats. Auton Neurosci 108: 50 –56, 2003.
AJP-Regul Integr Comp Physiol • VOL
Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (152.118.148.234) on January 15, 2018.
Copyright © 2009 American Physiological Society. All rights reserved.
R1131
53. Stener-Victorin E, Lindholm C. Immunity and ␤-endorphin concentra-
tions in hypothalamus and plasma in rats with steroid-induced polycystic
ovaries: effect of low-frequency electroacupuncture. Biol Reprod 70:
329 –333, 2004.
54. Stener-Victorin E, Lundeberg T, Cajander S, Aloe L, Manni L,
Waldenström U, Janson P. Steroid-induced polycystic ovaries in rats:
effect of electro-acupuncture on concentrations of endothelin-1 and nerve
growth factor (NGF), and expression of NGF mRNA in the ovaries, the
adrenal glands, and the central nervous system. Reprod Biol Endocrinol 1:
33, 2003.
55. Stener-Victorin E, Lundeberg T, Waldenström U, Bileviciute-Ljungar
I, Janson PO. Effects of electro-acupuncture on corticotropin-releasing
factor in rats with experimentally-induced polycystic ovaries. Neuropep-
tides 35: 227–231, 2001.
56. Stener-Victorin E, Lundeberg T, Waldenström U, Manni L, Aloe L,
Gunnarsson S, Janson PO. Effects of electro-acupuncture on nerve
growth factor and ovarian morphology in rats with experimentally induced
polycystic ovaries. Biol Reprod 63: 1497–1503, 2000.
57. Stener-Victorin E, Waldenström U, Tägnfors U, Lundeberg T, Lind-
stedt G, Janson PO. Effects of electro-acupuncture on anovulation in
women with polycystic ovary syndrome. Acta Obstet Gynecol Scand 79:
180 –188, 2000.
58. Sverrisdottir YB, Mogren T, Kataoka J, Janson PO, Stener-Victorin
E. Is polycystic ovary syndrome associated with high sympathetic nerve
activity and size at birth? Am J Physiol Endocrinol Metab 294: E576 –
E581, 2008.
59. Troisi RJ, Weiss ST, Parker DR, Sparrow D, Young JB, Landsberg L.
Relation of obesity and diet to sympathetic nervous system activity.
Hypertension 17: 669 – 677, 1991.
60. Turtzo LC, Marx R, Lane MD. Cross-talk between sympathetic neurons
and adipocytes in coculture. Proc Natl Acad Sci USA 98: 12385–12390, 2001.
61. Yildirir A, Aybar F, Kabakci G, Yarali H, Oto A. Heart rate variability
in young women with polycystic ovary syndrome. Ann Noninvasive
Electrocardiol 11: 306 –312, 2006.
296 • APRIL 2009 • www.ajpregu.org