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Endocrinology, doi:10.1210/en.2008-0053
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Endocrinology Vol. 149, No. 7 3559-3568
Copyright © 2008 by The Endocrine Society

Low-Frequency Electro-Acupuncture and Physical Exercise Improve Metabolic Disturbances and Modulate Gene Expression in Adipose Tissue in Rats with Dihydrotestosterone-Induced Polycystic Ovary Syndrome

Louise Mannerås, Ingibjörg H. Jonsdottir, Agneta Holmäng, Malin Lönn and Elisabet Stener-Victorin

Institute of Neuroscience and Physiology, Department of Physiology (L.M., A.H., E.S.-V.), and Institute of Medicine, Wallenberg Laboratory (M.L.), Sahlgrenska Academy, University of Gothenburg, SE-40530 Gothenburg, Sweden; and Institute of Stress Medicine, (I.H.J.), SE-41230 V-Frölunda, Sweden

Address all correspondence and requests for reprints to: Elisabet Stener-Victorin, Institute of Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, University of Gothenburg, Box 434, SE-40530 Gothenburg, Sweden. E-mail: elisabet.stener-victorin{at}neuro.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, abdominal obesity, and insulin resistance. Pharmacotherapy is often unsatisfactory. This study evaluates the effects of low-frequency electro-acupuncture (EA) and physical exercise on metabolic disturbances and adipose tissue mRNA expression of selected genes in a rat PCOS model characterized by insulin resistance and adiposity. Dihydrotestosterone (inducing PCOS) or vehicle (control) was administrated continuously, beginning before puberty. At age 10 wk, PCOS rats were randomly divided into three groups; PCOS, PCOS EA, and PCOS exercise. PCOS EA rats received 2-Hz EA (evoking muscle twitches) three times/wk during 4–5 wk. PCOS exercise rats had free access to a running wheel for 4–5 wk. EA and exercise improved insulin sensitivity, measured by clamp, in PCOS rats. Exercise also reduced adiposity, visceral adipocyte size, and plasma leptin. EA increased plasma IGF-I. Real-time RT-PCR revealed increased expression of leptin and IL-6 and decreased expression of uncoupling protein 2 in visceral adipose tissue of PCOS rats compared with controls. EA restored the expression of leptin and uncoupling protein 2, whereas exercise normalized adipose tissue leptin and IL-6 expression in PCOS rats. Thus, EA and exercise ameliorate insulin resistance in rats with PCOS. This effect may involve regulation of adipose tissue metabolism and production because EA and exercise each partly restore divergent adipose tissue gene expression associated with insulin resistance, obesity, and inflammation. In contrast to exercise, EA improves insulin sensitivity and modulates adipose tissue gene expression without influencing adipose tissue mass and cellularity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) is the most common endocrine disorder in women of reproductive age and the most frequent cause of hyperandrogenism and oligoovulation (1). PCOS is also strongly associated with abdominal obesity, and women with PCOS have a higher risk of developing hyperinsulinemia, insulin resistance, and type 2 diabetes (2).

The pathophysiology of PCOS is largely unknown but has been attributed to defects in various organ systems (1, 3) Uncontrolled ovarian steroidogenesis with a thickened thecal layer that secretes excessive amounts of androgens is thought to be a primary abnormality of PCOS (3). Furthermore, PCOS is associated with defects in insulin action and secretion that lead to hyperinsulinemia and insulin resistance, neuroendocrine defects with exaggerated LH pulsatility, and altered adrenal androgen production (1). Adiposity is also important in the pathogenesis of PCOS, and visceral fat, which is associated with hyperandrogenemia (4), tends to accumulate in women with PCOS (2).

Due to the heterogeneity of PCOS, it is difficult to create a single animal model that expresses the main PCOS characteristics. Previously, we used an estradiol valerate-induced rat PCO model to study the effects of low-frequency electro-acupuncture (EA) (i.e. electrical stimulation of acupuncture needles that activates afferent nerve fibers) and physical exercise (5, 6, 7, 8, 9). But although rats with estradiol valerate-induced PCO become anovulatory and present an ovarian morphology similar to that in PCOS, they lack the typical metabolic disturbances of human PCOS (10). Recently, our group developed a new rat PCOS model that incorporates ovarian and metabolic characteristics of the syndrome. After continuous exposure to dihydrotestosterone (DHT), a nonaromatizable androgen, from prepuberty until adult age, the rats have typical PCO with an increased number of apoptotic follicles (11). Moreover, the rats develop obesity accompanied by enlarged adipocyte size and insulin resistance, indicating that high levels of androgens induce alterations in body composition and reduced insulin sensitivity in this PCOS model (11).

Many women with PCOS require prolonged treatment, and one of the primary goals of therapy is normalization of androgen levels and restoration of reproductive function. In addition, because the metabolic disturbances present in PCOS women not only involve an increased risk for cardiovascular disease but also may worsen many of the typical PCOS symptoms, they have become an important target for therapy. Pharmacological approaches are usually effective but have adverse effects (12). Therefore, new nonpharmacological treatment strategies such as acupuncture and physical exercise need to be evaluated (13).

Repeated acupuncture treatment in women with PCOS and women with undefined ovulatory dysfunction was shown to exert long-lasting beneficial effects on endocrinological parameters and anovulation with no negative side effects (14, 15, 16). But neither the influence on metabolism nor the underlying mechanisms of the acupuncture effect were investigated in these studies. Human (17, 18, 19, 20) and animal (21, 22, 23) studies demonstrate EA’s beneficial effects on obesity in general and on obesity-related disturbances. However, the mechanisms behind these effects remain uncertain (13).

Physical exercise has been shown to reduce the waist-to-hip ratio (a risk factor for cardiovascular disease and type 2 diabetes) and homocysteine levels (an indicator of cardiovascular risk) in overweight PCOS women (24). Lifestyle modification including a 6-month diet and an exercise program was found to reduce central fat and improve insulin sensitivity and ovulatory function in overweight women with PCOS (25).

Thus, physical exercise appears to have beneficial effects on endocrine and metabolic features of the syndrome. And although EA has a documented beneficial effect on anovulation in women with PCOS, EA’s influence on metabolic disturbances seen in PCOS has not been investigated (13).

The primary aim of this study was to evaluate whether repeated low-frequency (2-Hz) EA treatment and voluntary physical exercise improve metabolic disturbances in rats with PCOS induced by continuous administration of DHT that was begun prepubertally. The effects of EA and physical exercise on mRNA expression of genes related to insulin resistance, obesity and inflammation were investigated in the visceral adipose tissue of these rats. Control rats were compared with the experimental PCOS rats throughout the study to confirm the model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Six Wistar dams, each with eight to nine female pups, were purchased from Charles River (Sulzfeld, Germany). Pups were raised with a lactating dam until 21 d of age and then housed four to five per cage under controlled conditions (21–22 C, 55–65% humidity, 12-h light, 12-h dark cycle). Rats were fed commercial chow (18.7% protein, 4.7% fat, 63% carbohydrates, vitamins, and minerals; B&K Universal, Sollentuna, Sweden) and tap water ad libitum. Accepted standards of animal care were used. The Animal Ethics Committee (University of Gothenburg) approved this study.

Study procedure
At 21 d of age, rats were randomly divided into two experimental groups (PCOS, n = 36; and control, n = 13) and implanted sc with 90-d continuous-release pellets (Innovative Research of America, Sarasota, FL) containing 7.5 mg DHT (daily dose, 83 µg) or 7.5 mg vehicle, respectively. The DHT dose was the same used in our previous study that resulted in PCOS characteristics including metabolic disturbances at adult age (11). A microchip (AVID, Norco, CA) with an identification number was inserted sc in the neck along with the pellets. After 40 d, a second pellet that released 3.5 mg DHT or vehicle during 60 d (i.e. an additional daily dose of 58 µg) was implanted in the PCOS and control rats to compensate for weight gain. The control pellets were identical to the DHT pellets but without the bioactive molecule. All rats were weighed weekly from 21 d of age.

At 69 d of age, 7 wk after onset of DHT exposure, treatment started and the rats in the PCOS group were randomly subdivided into 1) PCOS (n = 12), 2) PCOS exercise (n = 13), and 3) PCOS EA (n = 11). The study was concluded after 12–13 wk of DHT exposure, which included 4–5 wk of physical exercise and EA (i.e. when the rats were 15–16 wk of age). Figure 1Go shows a flow chart of the study design.


Figure 1
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  		FIG. 1. Flow chart of the study design.

 
Treatment
EA.
Low-frequency EA was given to conscious rats every second weekday for 4–5 wk (12–14 treatments total). Treatment duration was 15 min in wk 1, 20 min wk 2–3, and 25 min thereafter. Stimulation acupuncture points were in the rectus abdominis [i.e. stomach (ST) 29] and in the triceps surae muscles [i.e. spleen (SP) 6] bilaterally, in somatic segments that correspond to the innervation of the ovaries (i.e. from the level of thoracic spine 10 to lumbar spine 2 and at sacral level). The needles (HEGU Svenska AB, Landsbro, Sweden) were inserted to depths of 0.5–0.8 cm and then attached bilaterally to an electric stimulator (CEFAR ACU II; Cefar-Compex Scandinavia AB, Malmö, Sweden). The points were electrically stimulated with a low frequency of 2 Hz with burst pulses (a burst length of 0.1 sec and a burst frequency of 80 Hz). Intensity was adjusted to produce local muscle contractions and varied from 0.8–1.3 mA.

Before needle insertion, the rats were lightly anesthetized with isoflurane (2% in 1:1 mixture of oxygen and air; Isoba vet; Schering-Plough AB, Stockholm, Sweden) for about 2–3 min. One investigator inserted all needles. After needle insertion, the rats were placed in a fabric harness and suspended above the desk during EA treatment. To avoid potential acute effects of EA, no treatment was performed 24 h before examinations and blood sampling.

Physical exercise.
Rats in the PCOS exercise group had free access to a wheel (22.5 cm in diameter) in their own cage. Each rat was allowed to exercise voluntarily for 4–5 wk. Customized computer software registered all wheel rotations.

Three times/wk, 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 insertion of needles and electrical stimulation. All rats were conscious during handling and treatment.

The running wheels were locked 24 h before examinations and blood sampling to avoid potential acute effects of physical exercise.

Vaginal smears
The stage of cyclicity was determined by microscopic analysis of the predominant cell type in vaginal smears obtained daily from 11 wk of age to the end of the experiment (26).

Blood sampling
At 10 wk of age (before treatment start, 6 wk after pellet implantation) and at 14 wk of age (after 4 wk of treatment, 10 wk after pellet implantation), tail blood was obtained to assess corticosterone, leptin, and IGF-I concentrations. Plasma samples were stored at –20 C.

Body composition
Body composition was analyzed by dual-emission x-ray absorptiometry (DEXA) at 14 wk of age (i.e. after 4 wk of treatment, 10 wk after pellet implantation) with a whole-body DEXA instrument (QDR-1000/W, Hologic Inc., Waltham, MA). Rats were anesthetized by inhalation of isoflurane (Abbott Scandinavia AB, Solna, Sweden; 2% in 1:1 mixture of oxygen and air) before scanning. Body fat, lean body mass (LBM), and bone mineral content (BMC) were determined for each rat.

Euglycemic-hyperinsulinemic clamp
At 15–16 wk of age (i.e. 5 wk of treatment, 11–12 wk after pellet implantation), rats were subjected to a euglycemic-hyperinsulinemic clamp (27) during the estrous phase. Rats were anesthetized with thiobutabarbital sodium (130 mg/kg ip; Inactin; Sigma Chemical Co., St. Louis, MO). Body temperature was maintained at 37 C with a heating blanket. Catheters were inserted into the left carotid artery for blood sampling and into the right jugular vein for glucose and insulin infusions, and a tracheotomy was performed.

Blood glucose samples were analyzed (10 µl) with a B-glucose analyzer (HemoCue AB, Dronfield, Derbyshire, UK). Insulin (100 U/ml Actrapid; Novo Nordisk, Bagsvaerd, Denmark) together with 0.2 ml albumin and 10 ml physiological saline was infused at 24, 16, and 12 mU/min·kg for 1, 2, and 3 min, respectively, followed by 8 mU/min·kg for the rest of the clamp. To maintain plasma glucose at a euglycemic level (6.0 mM), a 20% glucose in saline solution was administered. The glucose infusion rate (GIR) was guided by glucose concentration measurements every 5 min. At steady state (after 50–70 min), mean GIR was normalized to body weight, and blood samples were taken to determine plasma insulin concentrations.

Upon completion of the clamp, the rats were decapitated. The adrenal glands, the hind limb muscles [extensor digitorum longus (EDL) and soleus], and the parametrial, retroperitoneal, inguinal, and mesenteric fat depots were dissected and weighed. Mesenteric adipose tissue was snap-frozen in liquid nitrogen and stored at –80 C for mRNA expression analysis.

Computerized determination of adipocyte size
Mesenteric adipose tissue was cut into small pieces and treated with collagenase (type A; Roche, Mannheim, Germany) in MEM (1.05 mg/ml; Invitrogen, Carlsbad, CA) containing 5.5 mM glucose, 25 mM HEPES, 4% bovine albumin (fraction V; Sigma), and 0.15 µM adenosine (pH 7.4) for 50 min at 37 C in a shaking water bath. After filtration through a 250-µm nylon mesh, adipocytes were washed three times and suspended in fresh medium. Mean cell size and size distribution were determined by computerized image analysis (KS400 software; Carl Zeiss, Oberkochen, Germany) (28). In brief, the cell suspension was placed between a siliconized glass slide and a coverslip and transferred to the microscope stage. Nine random visual fields were photographed with a CCD camera (Axiocam; Carl Zeiss). Relevant surface areas were measured automatically, and diameters of the corresponding circles were calculated. Uniform microspheres (diameter 98.00 µm; Bangs Laboratories, Fishers, IN) served as a reference.

Analytical methods
Plasma concentration of corticosterone was determined with a double-antibody RIA kit (catalog no. 07-120102; MP Biomedicals, Irvine, CA). Leptin plasma concentration was determined with an ELISA kit (EZRL-83K; Linco Research, St. Charles, MO). IGF-I plasma concentration was determined with an ELISA kit (mouse/rat IGF-I ELISA DSL-10-29200; Diagnostic System Laboratories, Inc., Webster, TX). Human insulin, given during the clamp, was measured with a human insulin ELISA kit (10-1113-01; Mercodia, Uppsala, Sweden). All samples were run in the same assay. The intraassay coefficients of variation and assay sensitivities were 3.2% and 7.7 ng/ml (corticosterone, n = 82 in duplicates), 4.2% and 0.04 ng/ml (leptin, n = 86 in duplicates), 2.9% and 1.3 ng/ml (IGF-I, n = 78 in duplicates), and 4.3% and 1 mU/liter (human insulin, n = 45 in duplicates).

RNA isolation and cDNA synthesis
Total RNA was extracted from mesenteric adipose tissue using RNeasy Lipid Tissue Mini Kit according to the manufacturer’s protocol (QIAGEN, Hilden, Germany). A deoxyribonuclease I (QIAGEN) digestion step was included to eliminate DNA contamination. The RNA concentration was determined spectrophotometrically with a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE), and RNA integrity was checked using the Agilent Bioanalyzer 2100 together with the RNA 6000 Nano LabChip Kit (Agilent Technologies, Santa Clara, CA).

First-strand cDNA was synthesized from 1 µg total RNA using High-Capacity cDNA Reverse Transcription Kits (PE Applied Biosystems, Stockholm, Sweden), according to the manufacturer’s protocol.

Real-time RT-PCR using low-density array (LDA) card
Real-time RT-PCR analysis was performed using custom TaqMan LDA cards (Applied Biosystems). Primers and probes for rat genes corresponding to the TaqMan gene expression assay numbers and GenBank accession numbers listed in Table 1Go were spotted onto a 384-well card. Eight samples were randomly analyzed in duplicates per card in one run, and 75 ng cDNA mixed with TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems) in a total volume of 100 µl was loaded per sample loading port. Thermal cycling and florescence detection was performed on an ABI Prism 7900HT Sequence Detection System with ABI Prism 7900HT SDS software version 2.1 (Applied Biosystems). Thermal cycling was carried out for 2 min at 50 C and 10 min at 94.5 C, followed by 40 cycles of 30 sec at 97 C and 1 min at 59.7 C.


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  		TABLE 1. Genes on the TaqMan LDA with their TaqMan Gene expression assay numbers and GenBank accession numbers

 
The NormFinder algorithm was used to calculate the expression stability of four putative reference genes (18S rRNA, β-actin, peptidylprolyl isomerase A, and hypoxanthine guanine phosphoribosyl transferase) for normalization (29). According to the NormFinder algorithm, peptidylprolyl isomerase A had the lowest intragroup and intergroup variability in mesenteric adipose tissue. Gene expression values were calculated using the 2–{Delta}{Delta}Ct method (30), because the LDA cards meet the assumptions necessary to use this equation (31). The {Delta} cycle threshold ({Delta}Ct) value of each sample was determined by subtracting the average Ct value of the reference gene from the average Ct value of the target gene. The {Delta}{Delta}Ct value was then calculated by subtracting the {Delta}Ct of the sample with highest expression, that is, that with lowest {Delta}Ct value, from the {Delta}Ct value of the sample. The target gene expression level relative to the sample with highest expression was then estimated as 2–{Delta}{Delta}Ct.

Statistical analyses
All statistical evaluations were performed with SPSS software (version 13.0; SPSS, Chicago, IL). Values are reported as mean ± SEM. The effects of EA or exercise on body weight gain were analyzed by repeated-measures Friedman test followed by Mann-Whitney U test at each time point. Effects on tissue weight, insulin sensitivity, body composition, adipocyte size, corticosterone concentrations, leptin concentrations, IGF-I concentrations, and mRNA expression in mesenteric adipose tissue were analyzed with the Mann-Whitney U test between PCOS and control rats and with the Kruskal-Wallis in the PCOS groups. Differences between PCOS groups were tested with Mann-Whitney U test. Correlation analyses were performed using Spearman rank correlation coefficient (Rs) in bivariate analyses. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Decreased body weight after physical exercise in PCOS rats
The PCOS group had gained more weight than the control group at 42 d of age, that is, after 3 wk of DHT exposure, and this difference remained throughout the study (Fig. 2Go, A and B). One week after treatment start (i.e. at 77 d of age) and throughout the experimental period, the PCOS exercise group gained significantly less weight than the PCOS group (Fig. 2Go, A and B). EA did not influence PCOS body weight.


Figure 2
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  		FIG. 2. Growth curves. A, Rats were exposed to placebo (control) or DHT (PCOS groups) from 21 d of age until the end of the experiment. At 69 d of age, PCOS rats began treatment; the PCOS group was subdivided into PCOS, PCOS exercise, and PCOS EA groups. B, Detailed view of the growth curve during the treatment period (70–98 d of age). Values are mean ± SEM. The effects of EA or exercise on body weight gain were analyzed by repeated-measures Friedman test (P < 0.001) followed by Mann-Whitney U test at each time point: {dagger}{dagger}, P < 0.01; {dagger}{dagger}{dagger}, P < 0.001 vs. control; *, P < 0.05; **, P < 0.01 PCOS exercise vs. PCOS.

 
Physical exercise
The PCOS exercise rats reached maximal running activity after 3 wk of free access to the exercise wheel (wk 1, 1.14 ± 0.14 km/d; wk 2, 2.25 ± 0.41 km/d; wk 3, 2.56 ± 0.42 km/d; and wk 4, 2.00 ± 0.38 km/d).

Reduced adiposity and adipocyte size after exercise in PCOS rats
DEXA.
When DEXA measurements were performed at 14 wk of age, PCOS rats were heavier and had more body fat, LBM, and BMC than control rats. But in relation to body weight, body fat and LBM were not affected, whereas BMC was lower in PCOS rats compared with control rats. PCOS exercise rats weighed less and had less body fat and BMC than PCOS rats (Table 2Go). In relation to body weight, the PCOS exercise group had decreased body fat and increased LBM compared with the PCOS group. DEXA-measured body composition of PCOS EA and PCOS rats was similar.


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  		TABLE 2. Effects of physical exercise and EA on body composition estimated by DEXA

 
Adipose tissue depots.
Weights of inguinal, parametrial, and retroperitoneal adipose tissue depots were higher in PCOS rats compared with controls (Table 3Go). In relation to body weight, the inguinal and the parametrial depots were higher in the PCOS group compared with controls. Exercise decreased the weight of all individual adipose tissue depots in PCOS rats, including in relation to body weight (Table 3Go). EA did not affect the weight of individual adipose tissue depots in PCOS rats in absolute or relative terms.


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  		TABLE 3. The effect of physical exercise and EA on dissected individual fat depots

 
Muscles.
In absolute terms, soleus muscle weight did not differ between the groups. But in relation to body weight, soleus weight was lower in PCOS rats compared with controls and higher in PCOS EA and PCOS exercise rats compared with PCOS. EDL weight did not differ between groups in absolute or relative terms (Table 3Go).

Mesenteric adipocyte size.
Mean mesenteric adipocyte size differed nonsignificantly between control and PCOS rats (control, 66.2 ± 1.67 µm; PCOS, 71.0 ± 2.46 µm), but physical exercise reduced mean mesenteric adipocyte size in PCOS rats (PCOS exercise, 57.1 ± 2.59 µm, P < 0.01). Low-frequency EA treatment of PCOS rats did not affect mean mesenteric adipocyte size (PCOS EA, 67.7 ± 2.25 µm).

Improved insulin sensitivity after physical exercise and EA treatment in PCOS rats
GIR, determined by a euglycemic-hyperinsulinemic clamp, was lower in PCOS rats than in control rats, indicating peripheral insulin resistance (Fig. 3Go). PCOS EA rats and PCOS exercise rats had higher GIR compared with PCOS rats (Fig. 3Go). At steady state, plasma glucose levels were about 6 mM, and the mean plasma insulin level was 285 ± 11 mU/liter. Insulin sensitivity correlated negatively with mesenteric adipocyte size in pooled groups (Rs = 0.60; P < 0.01; n = 22) and in the PCOS (Rs = 0.90; P < 0.05; n = 5) and PCOS EA (Rs = 0.89; P < 0.05; n = 6) groups.


Figure 3
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  		FIG. 3. GIR in control rats and in the three experimental PCOS rat groups: PCOS (no physical exercise or EA), PCOS exercise (after 4–5 wk free access to a wheel), and PCOS EA (after 4–5 wk EA). Comparisons were made between control vs. PCOS rats and between PCOS exercise and PCOS EA vs. PCOS rats. Values are mean ± SEM. {dagger}, P < 0.05 vs. control (Mann-Whitney); P < 0.01 (Kruskal-Wallis between PCOS groups); *, P < 0.05; ***, P < 0.001 vs. PCOS (Mann-Whitney).

 
Reduced leptin concentrations after exercise and increased IGF-I after EA treatment in PCOS rats
Before treatment, leptin concentrations were higher in PCOS rats compared with control rats (control, 5.3 ± 0.49 ng/ml, vs. PCOS, 9.0 ± 0.57 ng/ml, P < 0.001). After 4 wk of treatment, leptin concentrations were significantly decreased in the exercise group and unaffected in the EA group (Fig. 4AGo). A positive correlation was observed between plasma leptin concentrations and body fat estimated by DEXA in pooled groups (Rs = 0.82; P < 0.001) and in the various PCOS groups (PCOS, Rs = 0.77 and P < 0.01; PCOS exercise, Rs = 0.74 and P < 0.01; and PCOS EA, Rs = 0.88 and P < 0.001).


Figure 4
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  		FIG. 4. Plasma leptin (A) and IGF-I (B) concentration in control rats and in the three experimental PCOS rat groups: PCOS (no physical exercise or EA), PCOS exercise (after 4–5 wk free access to a wheel), and PCOS EA (after 4–5 wk EA). Comparisons were made between control vs. PCOS rats and between PCOS exercise and PCOS EA vs. PCOS rats. Values are mean ± SEM. {dagger}, P < 0.05 vs. control (Mann-Whitney); P < 0.001 (leptin, Kruskal-Wallis between PCOS groups); P < 0.06 (IGF-I, Kruskal-Wallis between PCOS groups); *, P < 0.05; ***, P < 0.001 vs. PCOS (Mann-Whitney).

 
Before treatment, plasma IGF-I concentrations differed nonsignificantly (control, 1574 ± 102 ng/ml; PCOS, 1421 ± 31 ng/ml). After 4 wk of treatment, PCOS rats had significantly lower IGF-I concentrations than control rats and PCOS EA rats had significantly higher IGF-I concentrations than PCOS rats, whereas PCOS exercise rats differed nonsignificantly from PCOS rats (Fig. 4BGo).

No effect of the intervention on the plasma levels of DHT was expected, due to continuous administration of DHT, and was therefore not analyzed. Furthermore, biological response in the DHT-exposed rats indicates that pellet dose was high enough to evoke metabolic disturbances seen in women with PCOS.

Treatment was not stressful
Plasma corticosterone concentrations were measured before and after 4 wk of treatment to evaluate potential stress induced by treatment. Before treatment, corticosterone concentrations were lower in the PCOS group compared with the control group (control, 845 ± 210 ng/ml vs. PCOS, 253 ± 25 ng/ml; P < 0.001). Four weeks of treatment did not affect corticosterone concentrations (control, 702 ± 108 ng/ml; PCOS, 229 ± 35 ng/ml; PCOS exercise, 221 ± 32 ng/ml; PCOS EA, 160 ± 34 ng/ml). Furthermore, the weight of the adrenal glands was lower in the PCOS than in the control group (control, 91.4 ± 5.0 mg, vs. PCOS control, 45.0 ± 2.1 mg, P < 0.001), whereas no difference was observed between the PCOS groups (PCOS exercise, 49.1 ± 1.2 mg; PCOS EA, 48.6 ± 1.6 mg). This shows that handling and treatments were not stressful for the rats.

Gene expression in mesenteric adipose tissue in PCOS vs. control rats
Higher mRNA expression of leptin and IL-6 and a lower expression of uncoupling protein 2 (UCP2) in mesenteric adipose tissue were observed in PCOS rats compared with controls (Fig. 5Go). No difference in the expression of TNF-{alpha} and peroxisome proliferator-activated receptor {gamma} (PPAR-{gamma}) was observed between control and PCOS rats (Fig. 5Go).


Figure 5
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  		FIG. 5. Gene expression of leptin, IL-6, TNF-{alpha}, PPAR-{gamma}, and UCP2 in mesenteric adipose tissue in control rats and in the three experimental PCOS rat groups: PCOS (no physical exercise or EA), PCOS exercise (after 4–5 wk free access to a wheel), and PCOS EA (after 4–5 wk EA). Comparisons were made between control vs. PCOS rats and between PCOS exercise and PCOS EA vs. PCOS rats. Data are expressed as mean 2–{Delta}{Delta}Ct ± SEM (see Materials and Methods); average Ct values for all genes were 26.3 ± 0.2 (range, 21.0–33.5). {dagger}{dagger}, P < 0.01, and {dagger}{dagger}{dagger}, P < 0.001 vs. control (Mann-Whitney); leptin, P < 0.001; IL-6, P < 0.05; PPAR-{gamma}, P < 0.01; and UCP2, P < 0.01 (Kruskal-Wallis between PCOS groups). **, P < 0.01; ***, P < 0.001 vs. PCOS (Mann-Whitney).

 
Effect of EA and physical exercise on gene expression in mesenteric adipose tissue
Repeated low-frequency EA and voluntary exercise for 4–5 wk down-regulated the expression of leptin in mesenteric adipose tissue (Fig 5Go). Exercise also down-regulated the expression of IL-6, and a trend (P = 0.075) toward down-regulation of IL-6 gene expression was also observed after EA treatment compared with untreated PCOS rats (Fig. 5Go). After EA treatment, mesenteric UCP2 mRNA expression was up-regulated and mRNA expression of PPAR-{gamma} was down-regulated compared with untreated PCOS rats (Fig. 5Go). Neither exercise nor EA influenced TNF-{alpha} gene expression (Fig. 5Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The novel finding in this study is that repeated low-frequency EA, like voluntary physical exercise for 4–5 wk, improves insulin sensitivity measured by the clamp method in PCOS rats. Voluntary physical exercise for 4–5 wk also reduces adiposity, visceral adipocyte size, and plasma leptin concentrations in PCOS rats. Amelioration of the divergent visceral adipose tissue gene expression related to insulin resistance, obesity, and inflammation in mesenteric adipose tissue might partly explain the beneficial effect of low-frequency EA and physical exercise on insulin sensitivity. Table 4Go summarizes the main findings of the study.


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  		TABLE 4. Summary table of main findings between PCOS vs. controls and PCOS vs. EA and exercise

 
Physiological basis for acupuncture
Physiological responses to acupuncture depend on a number of parameters employed during its application. The placement and stimulation of the acupuncture needles activate muscle afferents to the spinal cord and the central nervous system. In EA, low-frequency (1–15 Hz) electrical stimulation excites ergoreceptors in the muscles, which are physiologically activated during muscle contractions. Low-frequency EA causes release of a large number of neuropeptides, which seem to be essential for inducing functional changes in different organ systems (13). In this respect, we have recently demonstrated that EA at low (2–10 Hz) but not EA at high (80–100 Hz) frequency with needle placed in abdomen or in combination abdomen/hind limb significantly increases ovarian blood flow responses and that the response was mediated via ovarian sympathetic nerves, via the central nervous system (32, 33). These studies demonstrate that low-frequency electrical stimulation, which activates somatic afferents (A-{delta}- and C-fibers), modulate sympathetic output to the ovaries. Furthermore, these studies demonstrate that the strongest response was when needles were placed in abdominal muscles in combination with hind limb muscles. Therefore, in the present study, we placed acupuncture needles in both abdominal and hind limb muscles and stimulated with low-frequency EA.

Improved metabolic disturbances after low-frequency EA and voluntary physical exercise
Low-frequency EA and voluntary physical exercise each improved insulin sensitivity in PCOS rats. Interestingly, the effect of low-frequency EA was exerted without influencing adiposity. Previously, low-frequency EA was shown to enhance insulin sensitivity by raising glucose tolerance and lowering plasma glucose levels in diabetic rats (22). The authors attributed EA’s effect to an increase in plasma β-endorphin and insulin levels, which could interfere with the release of serotonin and induce hypoglycemia (22, 23).

Circulating IGF-I and locally derived skeletal muscle IGF-I has important metabolic effects. Low IGF-I levels have been shown to be independently related to impaired insulin sensitivity (34). IGF-I stimulates glucose transport in skeletal muscle as potently as insulin (35) and is involved in skeletal muscle remodeling (36). In the present study, the circulating levels of IGF-I were increased after low-frequency EA in PCOS rats but not after exercise. Whether the increased circulating IGF-I levels after EA is due to an increased production in the contracting skeletal muscle, as seen after exercise (35), or an increased production elsewhere remains to be elucidated, but it may be speculated that the increased IGF-I levels have contributed to the improved insulin sensitivity.

Another plausible explanation of the increased insulin sensitivity might be that soleus muscle mass increased after both low-frequency EA and physical exercise, thus indicating that EA, which evokes muscle twitches, has a local effect in the muscle, similar to the post-exercise effect. Muscle contractions during low-frequency EA and physical exercise most likely stimulate glucose uptake via an insulin-independent pathway. This is especially interesting in insulin-resistant states because the contractile-induced mechanisms are still functional. The mechanism by which muscle contraction activates glucose transport has been attributed to roles for cytosolic calcium, AMP-activated protein kinase activity, and nitric oxide (37). Chronic low-frequency electric stimulation and exercise has also been shown to affect insulin signal transduction, via changes in protein expression of key genes in the insulin-signaling pathway in skeletal muscle of both rats (38, 39) and humans (40). Furthermore, exercise may enhance insulin sensitivity by affecting the oxidative capacity of the skeletal muscle (37), muscle fiber type distribution, and muscular capillarization (41). Whether these mechanisms are involved in the observed improved insulin sensitivity after low-frequency EA and exercise in our PCOS model is a task for future studies.

The marked effects on body weight, adiposity, adipocyte size, and leptin concentrations most likely explain the positive effects of exercise on insulin sensitivity in this study; others concur (42, 43). The reduced adiposity seen after exercise was accompanied by reduced adipocyte size in mesenteric adipose tissue in PCOS rats in our study. A previous study demonstrated that weight loss due to exercise efficiently reduces adipocyte size (44).

Low-frequency EA therapy in obese women (17, 18, 19) and rats (21) has previously been shown to have beneficial effects on metabolic parameters such as body weight, leptin levels, lipid profile, and insulin levels. In our study, repeated low-frequency EA improved insulin sensitivity in PCOS rats without affecting body weight or adiposity. One reasonable explanation for the discrepant results regarding effects on body weight and fat mass after EA therapy might be that EA was given three times/wk in our study compared with daily in previous studies (17, 18, 19, 21).

Restored gene expression in adipose tissue after low-frequency EA and physical exercise
Abdominal adiposity may contribute to hyperandrogenism by mechanisms independent of insulin resistance (45), and abnormal protein and gene expression in PCOS intraabdominal adipose tissue (46, 47) highlights the importance of adipose tissue in the pathogenesis of PCOS. There seems to be a cross talk between adipose tissue and muscles, where adipokines secreted from the expanded adipose tissue mass acts in an endocrine manner and causes insulin resistance in skeletal muscle (48). Although improved insulin sensitivity by exercise, and most likely low-frequency EA, is mainly attributed to the skeletal muscles, it has been shown that exercise also enhances insulin-stimulated glucose uptake in intraabdominal adipose tissues (42). To elucidate a possible contribution of adipose tissue to the improved insulin sensitivity after EA and exercise, we chose mesenteric adipose tissue for gene expression analysis. Genes related to insulin resistance, obesity, and inflammation were selected.

Adiposity and circulating leptin levels are strongly correlated (49), which was evident in the present study. Hyperleptinemia is thought to indicate leptin resistance, which may contribute to the pathogenesis of obesity (50) and is strongly connected to insulin resistance (51). Whether women with PCOS have increased levels of leptin compared with healthy women matched for body fat is not known, but obese PCOS women clearly have elevated leptin levels compared with nonobese (52). Leptin, exclusively secreted by adipose tissue, may contribute to the ovulatory dysfunction of PCOS by exerting direct effects on the ovary (45, 53, 54, 55).

Interestingly, repeated low-frequency EA in PCOS rats decreased leptin mRNA in mesenteric adipose tissue, despite no effect on adiposity or plasma leptin concentrations. The discrepant effect of EA on mesenteric adipose tissue leptin gene expression and circulating levels of leptin probably reflects uninfluenced leptin production from other adipose tissue depots. Leptin is a major autocrine/paracrine factor known to influence insulin sensitivity, and therefore a locally decreased expression of leptin may be of importance.

As expected, due to reduced adiposity, physical exercise decreased circulating leptin and leptin mRNA expression in mesenteric adipose tissue compared with untreated PCOS rats. Voluntary wheel running was previously shown to reduce circulating leptin levels and leptin mRNA expression in epididymal adipose tissue in obese and nonobese male rats (56).

Adipose tissue leptin gene expression was previously reported to be lower in women with PCOS compared with controls (57); in this study, the expression of leptin was higher in sc tissue than intraabdominal tissue.

Given that proinflammatory cytokines are implicated in the pathogenesis of insulin resistance (58), several studies were conducted to investigate this relationship in PCOS. In this study, IL-6 gene expression was up-regulated in mesenteric adipose tissue, whereas TNF-{alpha} gene expression was unaffected in PCOS rats. After physical exercise, IL-6 gene expression in mesenteric adipose tissue was down-regulated and IL-6 expression tended to be restored after low-frequency EA, in line with increased insulin sensitivity. We did not observe effects on TNF-{alpha} gene expression by intervention. Improved insulin sensitivity via diet and exercise were shown to be associated with decreased expression of proinflammatory cytokines and reduced infiltration of macrophages in adipose tissue (59). But others demonstrated that improved insulin sensitivity by exercise in obese humans is not associated with modifications of plasma levels and adipose tissue gene expression of IL-6 and TNF-{alpha} (60, 61).

UCPs are mitochondrial proteins that have been proposed to play a prominent role in the regulation of energy balance and potentially contribute to the pathogenesis of obesity (62). However the correlation between UCP2 gene expression in adipose tissue and obesity is contradictory in humans and rats (63, 64, 65, 66, 67). To our knowledge, gene expression of UCP2 in adipose tissue has not been investigated in women with PCOS. PCOS rats in the present study were obese and exhibited decreased levels of UCP2 mRNA in mesenteric adipose tissue compared with controls. Moreover, low-frequency EA restored UCP2 expression in PCOS rats without affecting body fat. In a recent study, UCP2 levels in adipose tissue were suggested to influence systemic metabolism and insulin sensitivity via modifications in the release of adipokines such as adiponectin by adipose tissue (68). Unfortunately, plasma adiponectin levels were not measured in the present study.

PPAR-{gamma} is expressed mainly in adipose tissue and is involved in lipid and glucose metabolism. In this study, PPAR-{gamma} gene expression in PCOS rats was unaffected in mesenteric adipose tissue compared with controls. Low-frequency EA decreased PPAR-{gamma} gene expression in the mesenteric adipose tissue, whereas voluntary exercise in PCOS rats had no effect on mesenteric adipose tissue PPAR-{gamma} gene expression. This is in line with previous studies that found long-term physical exercise in rats to have no effect on PPAR-{gamma} protein in adipose tissue (69, 70).

Breaking the vicious circle after low-frequency EA and voluntary exercise
A vicious circle of androgen excess that favors abdominal accumulation of adipose tissue, which in turn promotes the release of adipokines that directly or indirectly via insulin resistance stimulate androgen production from ovaries, the adrenals, or both has been proposed for PCOS (45). Increased insulin sensitivity after low-frequency EA and physical exercise in PCOS rats, restored expression of leptin and UCP2 after EA, and leptin and IL-6 after exercise suggest that these interventions might be able to, at least partly, break this vicious circle.

Conclusions
The major finding in this study is that 12–14 treatments of low-frequency EA improves insulin sensitivity in rats with DHT-induced PCOS to the same extent as after 4–5 wk physical exercise. This beneficial effect may involve regulation of adipose tissue metabolism and production because EA and exercise each partly restore divergent adipose tissue gene expression associated with insulin resistance, obesity, and inflammation. In contrast to exercise, low-frequency EA improves insulin sensitivity and modulates adipose tissue gene expression without influencing adipose tissue mass and cellularity.


   Acknowledgments
 
We thank Birgitta Oden, Jessica Hansson, Mirjana Peternel, Effie Tagaras, and Malin Govik for technical assistance. We also thank the SWEGENE Göteborg Genomics Core Facility platform. DEXA measurements were performed at the Center for Mouse Physiology and Bio-Imaging, University of Gothenburg.


   Footnotes
 
This study was supported by grants from the Swedish Medical Research Council (Project No. 12206, 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, Magnus Bergwall Foundation, Tore Nilson Foundation, Åke Wiberg Foundation, Swedish Diabetes Association Research Foundation, Adlerbert Research Foundation, Ekhaga Foundation, and the Swedish Federal Government under the LUA/ALF agreement.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 3, 2008

Abbreviations: Ct, Cycle threshold; DHT, dihydrotestosterone; DEXA, dual-emission x-ray absorptiometry; EA, electro-acupuncture; EDL, extensor digitorum longus; GIR, glucose infusion rate; LDA, low-density array; PCOS, polycystic ovary syndrome; PPAR-{gamma}, peroxisome proliferator-activated receptor {gamma}; Rs, Spearman rank correlation coefficient; UCP2, uncoupling protein 2.

Received January 11, 2008.

Accepted for publication March 27, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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