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Endocrinology Vol. 147, No. 7 3203-3210
Copyright © 2006 by The Endocrine Society

Expression of Adiponectin Receptors and Its Possible Implication in the Human Endometrium

Yuri Takemura, Yutaka Osuga, Toshimasa Yamauchi, Masaki Kobayashi, Miyuki Harada, Tetsuya Hirata, Chieko Morimoto, Yasushi Hirota, Osamu Yoshino, Kaori Koga, Tetsu Yano, Takashi Kadowaki and Yuji Taketani

Departments of Obstetrics and Gynecology (Y.Takem., Y.O., M.H., T.H., C.M., Y.H., O.Y., K.K., T.Yan., Y.Taket.) and Internal Medicine (T.Yam., M.K., T.K.), Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan

Address all correspondence and requests for reprints to: Yutaka Osuga, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan. E-mail: yutakaos-tky{at}umin.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adiponectin, a pleiotropic cytokine, exerts its effects via the specific receptors AdipoR1 and AdipoR2. Whereas circulating adiponectin concentrations decrease in women with endometriosis and endometrial cancer, possible effects of adiponectin and the presence of the receptors in the endometrium have not been determined. In this study, we examined the expression of adiponectin receptors AdipoR1 and AdipoR2 in the human endometrium and assessed effects of adiponectin in endometrial cells. Expression of AdipoR1 and AdipoR2 in endometrial tissues was evaluated by real-time quantitative PCR, in situ hybridization, and Western blotting. The effects of adiponectin on phosphorylation of AMP-activated protein kinase, a regulator of energy homeostasis, in cultured endometrial stromal cells (ESCs) and epithelial cells (EECs) were studied by Western blotting. The effects of adiponectin on IL-1ß-induced secretion of IL-6, IL-8, and monocyte chemoattractant protein 1 from cultured ESCs were determined using specific ELISAs. The expression of AdipoR1 and AdipoR2 was detected in the endometrium. The expression of both genes was increased in the midluteal phase, the period of embryo implantation. In situ hybridization revealed that both AdipoR1 and AdipoR2 appeared to be equally expressed in the epithelial cells and in the stromal cells. Adiponectin increased phosphorylation of AMP-activated protein kinase in ESCs and EECs. Adiponectin decreased IL-1ß-induced secretion of IL-6, IL-8, and monocyte chemoattractant protein 1 from ESCs. These findings suggest that adiponectin exerts energy-homeostatic and antiinflammatory effects in the endometrium, and these effects might be relevant to pathological and physiological endometrium-related events such as implantation and endometriosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPONECTIN IS A hormone that structurally belongs to the complement 1q family (1). It is highly expressed in differentiated adipocytes and circulates at high levels in the bloodstream (2, 3, 4, 5). Adiponectin levels both in adipose tissue and in circulation are reduced in obesity (2, 3). An increasing body of evidence indicates that adiponectin plays an important role in regulating energy metabolism and insulin sensitivity (3, 6). In addition to its antidiabetic effects, adiponectin has been shown to have pleiotropic activities such as antiinflammatory, antiangiogenic, and antiatherosclerotic effects (7, 8, 9).

Two adiponectin receptors (AdipoR1 and AdipoR2) have recently been identified (10). The receptors contain seven-transmembrane domains but are structurally and functionally distinct from G protein-coupled receptors. Activation of these receptors phosphorylates AMP-activated protein kinase (AMPK), a regulator of energy homeostasis of the cell, and stimulates fatty acid oxidation and glucose uptake. In mice, AdipoR1 is abundantly expressed in the skeletal muscle, whereas AdipoR2 is predominant in the liver (10). In humans, although the expression of the receptors has been reported in a few tissues and cells (11, 12, 13, 14, 15), the expression in reproductive organs has been poorly understood.

A growing body of evidence indicates that many adipokines, including adiponectin, have biological implications for female fertility (16). Interestingly, we and others have recently demonstrated that serum adiponectin levels are decreased in women with endometriosis (17) and endometrial cancer (18, 19). Given the diverse effects of adiponectin, these findings imply that adiponectin exerts some effects on the endometrium.

With these backgrounds, we surmised that the adiponectin receptors are expressed in the human endometrium and that adiponectin has possible effects therein. To address this thesis, we studied the presence of the adiponectin receptors in endometrial tissues, and adiponectin-induced activation of the receptors was examined by measuring AMPK phosphorylation of endometrial cells. In addition, effects of adiponectin on IL-1ß-induced secretion of IL-6, IL-8, and monocyte chemoattractant protein (MCP)-1 from the endometrial cells was determined, considering that these proinflammatory cytokines are important in pathology and physiology of the endometrium (20, 21, 22).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and materials
Type I collagenase and antibiotics (a mixture of penicillin, streptomycin, and amphotericin B) were purchased from Sigma Chemical Co. (St. Louis, MO). DMEM/Ham’s F12 (F-12) medium was from Life Technologies, Inc. (Grand Island, NY). Human recombinant adiponectin was obtained from R&D Systems (Minneapolis, MN) and generated in our laboratory (6). 5-Aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) was obtained from Toronto Research Chemicals (Toronto, Canada). Antihuman rabbit antibodies of AMPK-{alpha} (no. 2532) and phospho-AMPK-{alpha} (no. 2535) were from Cell Signaling (Beverly, MA). Rabbit antibodies for AdipoR1 (ADIPOR12-A) and AdipoR2 (ADIPOR22-A) were purchased from Alpha Diagnostic International (San Antonio, TX). Antirabbit horseradish peroxidase secondary antibody was from Amersham Biosciences (Little Chalfont, UK). Recombinant IL-1ß was purchased from Genzyme/Techne (Minneapolis, MN). Charcoal-stripped fetal bovine serum (FBS) was from HyClone (Logan, UT). Deoxyribonuclease I was from Invitrogen (Carlsbad, CA).

Collection of samples
Endometrial tissues were obtained from women undergoing hysterectomy for benign gynecological conditions. In total, 77 women aged 32–45 yr were recruited to the present study. All women had regular menstrual cycles, and none had received hormonal treatment for at least 6 months before surgery. The tissues collected under sterile conditions were processed for primary cell cultures. The phases of the menstrual cycles were determined and classified as early, mid, and late proliferative and secretory phases according to the last and next menstrual period, basal body temperature, ultrasound findings on the endometrium and ovarian follicles, and standard histological criteria by Noyes et al. (23). Subcutaneous fat tissues of the abdomen were obtained from three women during hysterectomy. The tissues for mRNA extraction and Western blot analysis were snap frozen in liquid nitrogen and stored at –80 C. The tissues for in situ hybridization were fixed overnight in 10% formalin neutral buffer solution, subsequently dehydrated with a series of ethanol washes, and embedded in paraffin. The tissues for cell culture experiment were provided for additional preparation.

The experimental procedures were approved by the institutional review board of the University of Tokyo, and signed informed consent for use of the sample was obtained from each woman.

Isolation and culture of human endometrial stromal and epithelial cells
The isolation and culture of human endometrial stromal cells (ESCs) and epithelial cells (EECs) were processed as described previously (20, 21, 24). Fresh endometrial biopsy specimens collected in a sterile medium were rinsed to remove blood cells. The tissues were minced into small pieces and incubated in DMEM/F-12, containing 0.25% type I collagenase and 15 U/ml deoxyribonuclease I, for 60 min at 37 C. The resultant dispersed endometrial cells were separated by filtration through a 40-µm nylon cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ). The endometrial epithelial glands that remained intact were retained by the strainer, whereas the dispersed ESCs passed through the strainer into the filtrate.

ESCs in the filtrate were collected by centrifugation and resuspended in phenol-red-free DMEM/F-12 containing 10% charcoal-stripped FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/ml amphotericin B. The ESCs were seeded in a 100-mm culture plate and kept at 37 C in a humidified 5% CO2/95% air atmosphere. At the first passage, the cells were plated into six- or 48-well culture plates (Becton Dickinson) at a density of 2 x 105 cells/ml. The cells reached confluence in 2 or 3 d and then were used for the experiments.

EECs were collected by backwashing the strainer with DMEM/F-12 containing 10% charcoal-stripped FBS, seeded in a 100-mm plate, and incubated at 37 C for 60 min to allow contaminated ESCs to attach to the plate wall. The nonattached EECs were recovered and cultured in the culture medium at a density of 2 x 105 cells per well into a 12-well culture plate. The cells that reached confluence in 3 or 4 d were used for the experiments.

The purity of both the stromal and epithelial cell preparations was more than 95%, as judged by positive cellular staining for vimentin and cytokeratin, respectively.

Treatment of the cells
When the ESCs and EECs were approaching confluence, the complete media were removed and replaced with fresh media and antibiotics, and the cells were cultured in serum-free media for an additional 12 h. To examine whether AdipoR1 and AdipoR2 functioned in ESCs and EECs, the cells were incubated with adiponectin for 0–60 min or 1 mM AICAR, an experimental tool to activate AMPK, for 1 h. To evaluate the effects of adiponectin on the IL-1ß-induced production of IL-6, IL-8, and MCP-1 in ESCs, the cells were incubated with or without 50 µg/ml adiponectin in serum-free media for 24 h and then stimulated with 5 ng/ml IL-1ß in serum-free media for 24 h, according to our previous study (22).

RNA extraction, RT, standard PCR, and real-time quantitative PCR of adiponectin, AdipoR1, and AdipoR2
Total RNA was extracted individually from the endometrial tissue, ESCs, and EECs using an RNeasy minikit (QIAGEN, Hilden, Germany). One microgram of total RNA was reverse transcribed in a 20-µl volume using ReverTra Ace-{alpha} (TOYOBO, Osaka, Japan). Standard PCR was performed using ReverTra Dash (TOYOBO) according to the manufacturer’s instructions. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (TOYOBO) were used to ensure the quality and amounts of RNA. For negative controls, RNA without RT was used. Adiponectin primers (sense, 5'-AACATGCCCATTCGCTTTAC-3'; antisense, 5'-ATTACGCTCTCCTTCCCCAT-3') were chosen to amplify a 288-bp fragment. AdipoR1 primers (sense, 5'-AAACTGGCAACATCTGGACC-3'; antisense, 5'-GCTGTGGGGAGCAGTAGAAG-3') were chosen to amplify a 300-bp fragment. AdipoR2 primers (sense, 5'-ACAGGCAACATTTGGACACA-3'; antisense, 5'-CCAAGGAACAAA- ACTTCCCA-3') were chosen to amplify a 267-bp fragment. Both AdipoR1 and AdipoR2 primers span introns. PCR conditions for amplifications of adiponectin, AdipoR1, and AdipoR2 were 30 cycles at 98 C for 10 sec, 60 C for 2 sec, and 74 C for 15 sec. PCR products were analyzed by agarose gel electrophoresis with ethidium bromide.

To assess adiponectin, AdipoR1, and AdipoR2 mRNA expression, real-time quantitative PCR and data analysis were performed using LightCycler (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s instructions. Expression of adiponectin, AdipoR1, and AdipoR2 mRNA was normalized to RNA loading for each sample using GAPDH mRNA, for which expression was substantially constant during the menstrual cycle, as an internal standard. The primers for adiponectin, AdipoR1, AdipoR2, and GAPDH were the same as those used for standard PCR. PCR conditions were as follows: for adiponectin, 40 cycles at 95 C for 15 sec, 65 C for 8 sec, and 72 C for 12 sec; for AdipoR1, 35 cycles at 95 C for 15 sec, 65 C for 8 sec, and 72 C for 12 sec; for AdipoR2, 35 cycles at 95 C for 15 sec, 65 C for 8 sec, and 72 C for 11 sec. All these PCR conditions were followed by melting curve analysis.

Each PCR product was purified with a QIAEX II gel extraction kit (QIAGEN), and their identities were confirmed using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).

In situ hybridization
To prepare the digoxigenin (DIG)-labeled RNA probes for adiponectin, AdipoR1, and AdipoR2, the 288-, 300-, and 267-bp fragments of the human adiponectin, AdipoR1, and AdipoR2 cDNA, obtained by RT-PCR with the primers described above, were subcloned into the appropriate restriction sites of the PCR II-TOPO vector (Invitrogen). After linearization of plasmid with an appropriate enzyme, the linearized vectors were used as templates for the synthesis of DIG-labeled RNA probes using SP6 or T7 RNA polymerase.

In situ hybridization was performed using an ISHR Starting kit (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. The paraffin-embedded specimens were sliced at a 6-µm thickness. These sections were mounted on poly-L-lysine-treated slides, deparaffinized, and rehydrated. They were further digested with 5 mg/ml proteinase K for 10 min at room temperature, treated with 0.17% acetic anhydrate, and then subjected to treatment with prehybridization solution containing 50% formamide and 2x standard saline citrate (SSC) (1x SSC consists of 0.15 M NaCl and 0.015 M sodium citrate) for 30 min at 42 C. The probe was diluted to a concentration of 0.5 µg/ml in hybridization buffer. Hybridization was carried out by applying the diluted probe (150 µl) to each slide section. Each section was incubated in a humidified chamber overnight at 42 C.

Slides were washed three times in washing solution (50% formamide and 2x SSC) for 20 min each at 42 C, treated with ribonuclease for 30 min at 37 C, and washed three times in 0.1x SSC for 20 min each at 42 C. After being blocked with blocking solution, the sections were incubated with an anti-DIG, alkaline phosphate-conjugated antibody (1:500; Roche) for 60 min at room temperature, and washed three times in washing buffer. Color development was carried out by overlaying them with 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (NBT-BCIP; Roche), and they were incubated in a humidified chamber in the dark for 12 h at room temperature. All sections were evaluated under light microscope. Sense probe hybridization was used as a control for background level.

Western blot analysis
Cultured cells and endometrial tissues were homogenized in the lysis buffer containing 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue. The lysates were further diluted with lysis buffer to give a final concentration of 1 mg total protein/ml. Samples of 20 µg protein per lane were resolved by 10% (for total AMPK-{alpha} and phospho-AMPK-{alpha}) and 14% (for AdipoR1 and AdipoR2) SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and incubated with antirabbit antibodies to AdipoR1 (5 µg/ml) and AdipoR2 (5 µg/ml), total AMPK-{alpha} (1:1000), or phospho-specific AMPK-{alpha} (1:1000) as primary antibodies and antirabbit horseradish peroxidase as a secondary antibody (1:1000). Immune complexes were visualized by use of the ECL Western blotting system (Amersham Biosciences).

Measurement of IL-8, IL-6, and MCP-1
Concentrations of IL-6, IL-8, and MCP-1 were measured using a specific ELISA kit (Quantikine; R&D Systems) according to the manufacturer’s protocol as described previously (22, 25). The sensitivities of the assays were 3.12, 15.6, and 31.2 pg/ml for IL-6, IL-8, and MCP-1, respectively. The intraassay and interassay coefficients of variation were less than 5% in these assays.

Statistical analysis
Data were checked for normal distribution using Bartlett test and evaluated using ANOVA with post hoc analysis (Fisher’s protected least significance) for multiple comparisons. P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of adiponectin, AdipoR1, and AdipoR2 mRNA in endometrial tissues, EECs, and ESCs
The expression of adiponectin, AdipoR1, and AdipoR2 mRNA in endometrial tissues, EECs, and ESCs were detected by standard RT-PCR analysis (Fig. 1Go). A signal of the same size was detected in control sc fat tissues.


Figure 1
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  		FIG. 1. Expression of adiponectin, AdipoR1, and AdipoR2 mRNA in the endometrium, as detected by standard RT-PCR. Total RNA was extracted from endometrial tissues, cultured EECs, and ESCs in the proliferative phase and the secretory phase. Subcutaneous fat tissues were used for positive controls. For negative controls, RNA without RT was used. Lane T, endometrial tissues; lane E, EECs; lane S, ESCs; lane F, sc fat tissues; lane M, DNA molecular weight standards. The data shown are representative of three different samples in each phase.

 
Expression of adiponectin, AdipoR1, and AdipoR2 mRNA in the endometrium throughout the menstrual cycle
Real-time quantitative PCR analysis showed adiponectin, AdipoR1, and AdipoR2 mRNA were expressed in endometrial tissues throughout the menstrual cycle (Fig. 2Go). The expression levels of adiponectin mRNA were significantly higher in the early proliferative phase compared with those in the late proliferative phase and the early and late secretory phases (Fig. 2AGo). The expression levels of AdipoR1 mRNA were significantly higher in the mid-secretory phase compared with the early, mid, and late proliferative phases and the early secretory phase (Fig. 2BGo). The expression levels of AdipoR2 mRNA were significantly higher in the mid-secretory phase compared with all other phases (Fig. 2CGo).


Figure 2
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  		FIG. 2. Expression of adiponectin (A), AdipoR1 (B), and AdipoR2 (C) mRNA in the endometrium throughout the menstrual cycle. Endometrial tissues were obtained from 28 women (early proliferative and mid-secretory phase, n = 4; mid and late proliferative and early and late secretory phase, n = 5). Total RNA isolated from the endometrial tissues was reverse transcribed and amplified by real-time PCR using primers for adiponectin, AdipoR1, or AdipoR2. The data were calculated by subtracting the signal threshold cycles (CT) of the internal standard (GAPDH) from the CT of adiponectin, AdipoR1, or AdipoR2. Values are the mean ± SEM. A, *, P < 0.01 vs. late proliferative and early and late secretory phase; B, *, P < 0.01 vs. early, mid, and late proliferative, and early secretory phase; C, *, P < 0.05 vs. all other groups.

 
In vivo expression of adiponectin, AdipoR1, and AdipoR2 mRNA in the endometrium
In situ hybridization demonstrated that AdipoR1 and AdipoR2 were all expressed in glandular and luminal epithelial cells and stromal cells in all phases of the menstrual cycle (Fig. 3Go). Expression levels of AdipoR1 and AdipoR2 mRNA in the mid-secretory phase seemed to be higher than those in the other phases. The expression levels of each molecule appeared slightly higher in glandular and luminal epithelial cells than stromal cells. Adiponectin mRNA was also detected in glandular and luminal epithelial cells and stromal cells (Fig. 4Go). No specific hybridization products were observed when using the sense riboprobes.


Figure 3
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  		FIG. 3. In situ hybridization for AdipoR1 (A) and AdipoR2 (B) in the human endometrium throughout the menstrual cycle. Endometrial tissues obtained from 24 women [early (EP), mid (MP), and late proliferative (LP) phase, n = 3; early (ES) and late secretory (LS) phase, n = 4; mid-secretory (MS) phase, n = 7] were analyzed. The endometrial sections were hybridized with DIG-labeled antisense (AS) or sense (S) riboprobes. The pictures shown are representatives in each phase (AS) and MP (S). Magnification, x100.

 

Figure 4
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  		FIG. 4. In situ hybridization for adiponectin in the human endometrium. Endometrial sections were stained with hematoxylin and eosin (HE) or hybridized with DIG-labeled antisense (AS) or sense (S) riboprobes. The sections are of proliferative and secretory phases of the menstrual cycle. Magnification, x100.

 
Expression of AdipoR1 and AdipoR2 proteins in endometrial tissues, EECs, and ESCs
Using Western blotting, the expression of AdipoR1 and AdipoR2 proteins were detected as a bond at 42.4 and 35.4 kDa, respectively, in endometrial tissues, EECs, and ESCs (Fig. 5Go).


Figure 5
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  		FIG. 5. Expression of AdipoR1 and AdipoR2 proteins in endometrial tissues, EECs, and ESCs. Expression of AdipoR1 and AdipoR2 proteins were examined by Western blotting in endometrial tissues, EECs, and ESCs. The result is representative of six (AdipoR1) or nine (AdipoR2) separate experiments using samples from different women. Lane T, endometrial tissues; lane E, EECs; lane S, ESCs.

 
Adiponectin-induced AMPK phosphorylation in ESCs and EECs
As shown in Fig. 6AGo, phosphorylation of AMPK was induced by adiponectin in ESCs in a dose-dependent manner. The phosphorylation of AMPK was apparent at 5 min and most prominent at 10 min (Fig. 6BGo). The phosphorylation levels were lower with 50 µg/ml adiponectin than with 1 mM AICAR, a positive control (Fig. 6CGo). Phosphorylation of AMPK was also induced by 50 µg/ml adiponectin in EECs (Fig. 6DGo).


Figure 6
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  		FIG. 6. Phosphorylation of AMPK by adiponectin in ESCs and EECs. Adiponectin-induced phosphorylation of AMPK was examined by Western blot analysis. Cell lysates of ESCs (A–C) and EECs (D) treated with the indicated doses of adiponectin for 10 min or for the indicated times with 50 µg/ml adiponectin underwent Western blotting using the specific antibodies for phospho-AMPK and total AMPK. AICAR was used as a positive control (C). The results are representative of at least four separate experiments using samples from different women.

 
Effects of adiponectin on IL-1ß-induced IL-6, IL-8, and MCP-1 production in ESCs
Time-course experiments were conducted to determine the effect of adiponectin on the production of IL-6, IL-8, and MCP-1 in ESCs (Fig. 7Go). The concentrations of IL-6, IL-8, and MCP-1 in all samples were above the lower limits of the assays. Preincubation with adiponectin significantly decreased IL-1ß-induced IL-6, IL-8, and MCP-1 production in ESCs with time, up to 24 h in culture, compared with the controls. Significant decreases were seen at 8 h for IL-8 and 12 h for IL-6 and MCP-1.


Figure 7
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  		FIG. 7. Effects of adiponectin on the production of IL-6 (A), IL-8 (B), and MCP-1 (C) induced by IL-1ß in ESCs. ESCs were incubated with or without adiponectin (50 µg/ml) for 24 h and then stimulated with IL-1ß (5 ng/ml) for the indicated time. At the end of the incubation period, the conditioned media were collected and assayed for concentrations of IL-6, IL-8, and MCP-1 by ELISA. Values are the mean ± SEM of quadruplicate cultures. *, P < 0.005 vs. control; **, P < 0.0001 vs. control. The results are representative of at least three separate experiments using samples from different women.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated the expression of AdipoR1 and AdipoR2 in the human endometrium for the first time. RT-PCR analysis of the endometrial tissues revealed that the gene expression of both AdipoR1 and AdipoR2 was significantly increased in the mid-secretory phase of the menstrual cycle. Histologically, the expression of both genes was equally observed in ESCs and in EECs. In cultured ESCs, adiponectin stimulated AMPK phosphorylation and suppressed IL-1ß-induced IL-6, IL-8, and MCP-1 secretion. Adiponectin also induced AMPK phosphorylation in EECs.

The regulation of adiponectin receptors may be complex and varies depending on the cells and tissues. To date, several hormones and drugs, such as insulin, GH, fenofibric acid, and troglitazone, have been shown to up- or down-regulate the expression of AdipoR1 and AdipoR2 (12, 26, 27, 28, 29). In the present study, both AdipoR1 and AdipoR2 expression in the endometrium was increased in the midluteal phase. The midluteal phase is a period when the endometrium is receptive for the embryo and thus is called the implantation period. A myriad of genes are specifically expressed in the endometrium during the implantation period (30, 31, 32). Taken together, the increase of AdipoR1 and AdipoR2 may be a part of endometrial change for implantation, and it is intuitively speculated that the increase is affected by ovarian hormones and various cytokines that alter characteristics of the endometrium during the period.

AMPK is a fuel sensor to regulate cellular energy balance, and it also mediates effects of adipokines in modulating food intake, body weight, and glucose and lipid homeostasis (33). Adiponectin stimulates phosphorylation and activation of AMPK in the skeletal muscle, and the activated AMPK subsequently induces fatty-acid oxidation and glucose uptake. Adiponectin also stimulates AMPK in the liver and promotes fatty-acid oxidation and suppresses gluconeogenesis (10). In view of these findings, the present finding that adiponectin induced phosphorylation of AMPK in ESCs and EECs suggests that adiponectin may regulate energy supply in ESCs and EECs for an endometrial function such as reception of the embryo.

In the present study, a considerably high concentration (50 µg/ml) of adiponectin induced apparently small levels of phosphorylation of AMPK. There remains the possibility that the responses observed were cross-ligand activations.

Adiponectin has been indicated to have antiinflammatory properties. Although adiponectin inhibits phagocytic activity and LPS-induced production of TNF-{alpha} and IL-6 in macrophages (9, 34), it increases the production of the antiinflammatory mediators IL-10 and IL-1RA in monocyte, macrophage, and dendritic cells (34, 35). Antiinflammatory function of adiponectin is also suggested by its inhibiting LPS-induced IL-6 production in adipocytes (36). The present finding that adiponectin suppressed IL-1ß-induced secretion of IL-6, IL-8, and MCP-1 in ESCs suggests antiinflammatory roles of adiponectin in the endometrium.

Whereas the process of implantation entails inflammation-like events (37, 38, 39, 40), exaggerated inflammatory responses may perturb the integrity of endometrial function and lead to pathological conditions, including abortion and complicated pregnancies, such as preeclampsia and underdevelopment of the fetus. To render the endometrium favorable for implantation and ensuing fetal development, inflammatory responses of endometrial cells are suggested to be spatiotemporally fine tuned (22). In this context, the increased expression of AdipoR1 and AdipoR2 in the mid-secretory phase may subserve implantation, augmenting the adiponectin action of suppressing the production of inflammatory cytokines. However, adiponectin does not seem indispensable for pregnancy, because adiponectin-deficient mice were shown to be fertile (41).

The antiinflammatory effect of adiponectin in ESCs may be relevant to the pathogenesis of endometriosis. We have recently shown that serum and peritoneal fluid adiponectin levels are decreased in women with endometriosis (17, 42). Inflammation associated with endometriosis is suggested to promote the development of the disease. In particular, IL-1ß-induced production of IL-6, IL-8, and MCP-1 in ESCs is suggested to be involved in the pathogenesis of the disease (22, 25). Therefore, our finding that adiponectin suppressed IL-1ß-induced production of the proinflammatory molecules may explain, in part, why adiponectin levels are decreased in women with endometriosis. The inverse association between blood adiponectin levels and endometrial cancer risk (18, 19) might also be relevant to the antiinflammatory effect of adiponectin, given that inflammation is a possible promoting factor of carcinogenesis (43).

In addition to the expression of AdipoR1 and AdipoR2, we detected the expression of adiponectin in endometrial tissue. The expression levels are highest in the early proliferative phase. In light of the antifibrotic properties of adiponectin reported in the liver (44), it can be speculated that the increase in the early proliferative phase might be a protective response for fibrosis-free repair of the endometrium after shedding. However, because the expression levels of adiponectin in the endometrium were far below those in the adipose tissue (data not shown), a physiological implication of locally produced adiponectin in the endometrium is uncertain. Nevertheless, the increase in AdipoR1 and AdipoR2 levels in the implantation period is suggested to enhance adiponectin actions in the endometrium, considering that serum adiponectin levels do not show significant variation during the menstrual cycle (17).

In summary, we detected the increased expression of AdipoR1 and AdipoR2 in the human endometrium in the implantation period. In addition, adiponectin induced AMPK phosphorylation and suppressed IL-1ß-induced secretion of IL-6, IL-8, and MCP-1 in cultured endometrial cells, suggesting that adiponectin may play physiological and pathological roles in the endometrium.


   Acknowledgments
 
We thank Emi Nose for her technical assistance.


   Footnotes
 
Y.Takem., Y.O., T.Yam., M.K., M.H., T.H., C.M., Y.H., O.Y., K.K., T.Yan., T.K., and Y.Taket. have nothing to declare.

First Published Online April 6, 2006

Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; AMPK, AMP-activated protein kinase; DIG, digoxigenin; EEC, endometrial epithelial cell; ESC, endometrial stromal cell; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MCP, monocyte chemoattractant protein; SSC, standard saline citrate.

Received November 29, 2005.

Accepted for publication March 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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