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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 8 4089-4096
Copyright © 2004 by The Endocrine Society

Evidence for the Presence of Glucose Transporter 4 in the Endometrium and Its Regulation in Polycystic Ovary Syndrome Patients

Roberto Mioni, Silvia Chiarelli, Nadia Xamin, Laura Zuliani, Marnie Granzotto, Bruno Mozzanega, Pietro Maffei, Chiara Martini, Stella Blandamura, Nicola Sicolo and Roberto Vettor

Department of Medical and Surgical Sciences (R.M., N.X., L.Z., M.G., P.M., C.M., N.S., R.V.), Clinica Medica 3; Department of Oncological and Surgical Sciences (S.C., S.B.), Section of Pathology; and Department of Gynaecological and Human Reproduction Sciences (B.M.), University of Padova, 35128 Padova, Italy

Address all correspondence and requests for reprints to: Dr. Roberto Mioni, M.D., Ph.D., Dipartimento di Scienze Mediche e Chirurgiche, Clinica Medica 3, Università di Padova, Via Giustiniani 2, 35128 Padova, Italy. E-mail: robertomioni{at}libero.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Glucose transporter 4 (GLUT4) seems to be involved in the mechanism of insulin resistance in polycystic ovary syndrome (PCOS) patients (PCOSs) in both muscular and adipose tissue. The observation that insulin stimulates glucose oxidation in endometrial cells led us to investigate the presence of GLUT4 in this tissue and whether a defect of GLUT4 is present at the endometrial level in PCOSs. We also investigated whether body weight influences GLUT4 expression in this syndrome. GLUT4 mRNA content was examined by real-time quantitative RT-PCR and immunostaining reaction in the endometrial tissue of nine normal subjects, nine lean and eight obese hyperinsulinemic (h-INS), and eight lean and 10 obese normoinsulinemic (n-INS) PCOSs. GLUT4 mRNA and its positive immunostaining reaction were present in epithelial cell level in the endometrium of both normal and PCOS subjects. Significantly higher levels of GLUT4 were observed in normal and lean n-INS PCOSs in comparison with other groups. In both n-INS and h-INS obese PCOSs, GLUT4 was significantly lower than in lean subjects. However, obese n-INS and lean h-INS PCOSs showed a similar low GLUT4 expression, whereas obese h-INS PCOSs showed the lowest expression when compared with other groups. In conclusion, our data demonstrate that GLUT4 is present in the endometrium of normal and PCOS subjects and that hyperinsulinism and obesity seem to have a negative effect on endometrial GLUT4 expression in PCOS.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE POLYCYSTIC OVARY syndrome (PCOS) is one of the most common endocrinological disorders during reproductive age and, according to a recent study, 3–10% of women who present with hyperandrogenemia, chronic anovulation, and infertility are affected by PCOS (1, 2, 3). Underlying these clinical findings, several biochemical abnormalities have been described, including LH hypersecretion, hyperandrogenemia, acyclic estrogen production, decreased SHBG capacity, and hyperinsulinemia (4, 5).

Dunaif et al. (6) reported that PCOS patients (PCOSs) display abnormalities in the insulin secretory pattern and that most of them are affected by obesity, which is a well-known clinical picture associated with insulin resistance. However, it is now recognized that a significant proportion of hyperinsulinemic PCOS women are lean, and that obesity probably occurs in only 33–60% of these hyperandrogenic patients (4, 6, 7), strongly suggesting that the defect in the peripheral action of insulin is independent of body weight (8, 9, 10). Although insulin resistance has been well characterized both in vitro and in vivo, the mechanism by which insulin resistance plays a role in the pathophysiology of PCOS remains to be elucidated. In fact, several authors have shown that, in PCOS, a heterogeneous pattern of insulin sensitivity on different targets, such as adipose, muscle, skin, adrenal, or ovary tissues, exists, suggesting that insulin acts on cell functions with different effects. Moreover, improved insulin sensitivity, by weight loss or insulin-sensitizing drugs, has been shown to ameliorate hirsutism, gonadotropin secretion, menstrual disturbances, and infertility (11, 12, 13, 14). The latter symptoms in PCOS are most probably caused by abnormal ovarian steroid production and, in particular, increased amounts of both androgens and estrogens.

Because several findings have demonstrated that insulin induces excessive ovarian steroid secretion, it is suggested that insulin is directly involved in the pathogenetic mechanism of menstrual dysfunction. The endometrium is one of the major sites of estrogen action, and it is well demonstrated that an abnormal ovarian steroid milieu, such as that observed in PCOS anovulatory subjects, can induce several abnormalities in endometrial growth or function. Nevertheless, direct involvement of the insulin effect on endometrial cell function cannot be excluded. Previous in vitro studies have shown that insulin can stimulate glucose oxidation activity in the late luteal phase in the human endometrium, suggesting its involvement in the metabolic activities of endometrial tissue (15, 16). Moreover, insulin receptors are present at the endometrial level, reaching their maximal expression in the secretory phase, further supporting the hypothesis that insulin directly influences endometrial growth through its mitogenic and metabolic effects (17, 18). Recent data demonstrate the expression of glucose transporters, such as glucose transporter (GLUT) 1, at the endometrial level in animals and humans (19, 20), but so far little is known about the metabolic functions of insulin on the endometrium and whether the latter possesses all the machinery to be considered an insulin-regulated tissue.

Insulin is essential for appropriate maintenance of whole-body glucose homeostasis by increasing the rate of glucose uptake through the synthesis and translocation to the cell surface of the glucose transporter isoform GLUT4. Diminished adipocyte insulin responsiveness associated with decreased GLUT4 expression has been observed in obese patients and particularly in PCOSs (21), and it is probable that a similar defect may be observed in other tissues. Although the insulin dependence of the endometrium is still unknown, we aimed at determining whether endometrial cells contain GLUT4 and testing the hypothesis that GLUT4 is abnormally regulated in the endometrium of PCOSs. In these patients, the effects of body weight and/or insulin on GLUT4 at the endometrial cellular level were also investigated.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Thirty-five consecutive patients, aged 26–32 yr and affected by PCOS, were studied in our endocrine unit. PCOS was diagnosed with a combination of these clinical, biological, and ultrasonographic parameters: 1) oligomenorrhea (seven or fewer menstrual periods in the previous year); 2) hirsutism (hormonal score > 10 by the Ferriman-Gallwey modified method); 3) LH/FSH greater than 2.0; 4) testosterone and/or androstenedione greater than 2.5 and 9.0 nmol/liter, respectively; 5) presence of at least seven to 10 microcysts (< 8 mm in diameter) with peripheral distribution, by transvaginal pelvic ultrasound (AU5 Esaote-Biomedica, Firenze, Italy) with a 6.5-MHz probe.

Our PCOSs were divided into two groups, obese and lean, according to their body mass index (BMI), calculated as body weight in kilograms divided by the square of the height in meters. Obesity was defined as BMI more than 27 kg/m2, with normal range between 18 and 24.9. None of the patients presented thyroid disorders, Cushing’s syndrome, or hyperprolactinemia. Moreover, to avoid any confounding effect, other causes of insulin resistance such as acanthosis nigricans and history of type 2 diabetes mellitus in patients or their first-degree relatives were a priori excluded. None had taken any drug or hormonal medications, including contraceptive pills, for at least 3 months before the study. Alteration of adrenal enzymatic pathways were excluded by the ACTH stimulation test (0.250 mg iv; Synacthen-Novartis, Origgio, Italy). A group of nine lean healthy women, aged 28–32 yr, with BMI from 18.6 to 23.1 kg/m2 and regular menstrual cycles (more than 10 menstrual periods/yr), were recruited as controls.

After an overnight fast and 3 d of a 2000-kcal standardized diet containing 300 g of carbohydrate, a 3-h 75-g oral glucose tolerance test (OGTT) was performed on each subject. Blood samples were taken and changes in glucose and insulin plasma levels were evaluated every 30 min for 3 h; these data were then analyzed as the integrated secretory area under the curve (AUC), calculated by the trapezoidal rule and expressed as concentrations of glucose and insulin per 60 min. The insulin sensitivity index (ISI) of Matsuda and DeFronzo (22) was used to evaluate insulin sensitivity from data obtained from the OGTT. This index of whole-body insulin sensitivity [10,000/square root of (fasting glucose x fasting insulin) x (mean glucose x mean insulin during OGTT)] represents a composite of hepatic and peripheral tissue sensitivity to insulin. All subjects had normal glucose tolerance according to the report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus (23), whereas a normal insulin response (n-INS) to the OGTT was considered as the mean ± 2 SD of the insulin AUC obtained from more than 200 of our normal local controls. From our local controls, we considered as hyperinsulinemic (h-INS) subjects only those who presented an insulin response to the OGTT of more than the mean + 2 SD of insulin AUC. All subjects presented an ISI ranging from 1.0 to 10.0. Our h-INS PCOSs (lean and obese) showed ISI values less than 4.0, whereas n-INS PCOSs (lean and obese) and controls showed ISI values of more than 6.0. All hormonal and biochemical parameters were performed between d 1 and 4 of the last menstrual period, after certain and spontaneous menses, corresponding to the early follicular phase of controls, and none had vaginal, endometrial, or pelvic inflammatory diseases. Moreover, previous gynecological assessment excluded the presence of hyperplasia or neoplasia by endometrial cytology. Assessment of menstrual history, with recording of menses in the 12-month periods before and during the study, was carried out for each subject.

The procedures used were in accordance with the guidelines of the Helsinki Declaration on human experimentation. The investigational nature of the study was explained to all participants, and informed consent was obtained. The study was approved by the Committee of Ethics of Padova Medical Faculty.

Study design

Endometrial samples were obtained from each subject between d 12 and 14 of the last menstrual cycle. All women were in the equivalent phase of the menstrual cycle, corresponding to the late proliferative phase, as documented by the appearance of endometrial tissue at histology and serum hormonal parameters (17-ß-estradiol concentration of 200–300 pmol/liter, progesterone < 0.6 nmol/liter). Endometrial biopsy samples were obtained by aspiration (30-cc vacuum syringe) using sterile flexible feeding tubes (length 40 cm, caliber 2.0–2.7 mm) as previously described (24). After washing twice in Hanks’ balanced salt solution at 4 C, samples were snap frozen in liquid nitrogen and immediately stored at –80 C in Eppendorf tubes until real-time quantitative RT-PCR assay. Other samples were fixed in 10% buffered formalin phosphate and embedded in paraffin for histological evaluation and immunohistochemical studies.

Quantification of GLUT4 mRNA expression by real-time quantitative RT-PCR in endometrium

Total RNA was isolated from 30–32 mg endometrial tissue using the RNAzol method (Tel-Test, Inc., Friendswood, TX) following the supplier’s instructions. Two micrograms RNA were treated with Dnase treatment and removal reagents (Ambion, Inc., Austin, TX) and reverse transcribed for 1 h at 37 C in a 50-µl reaction containing 1 x reverse transcription buffer, 150 ng random hexamer primer, 0.5 mM deoxynucleotide triphosphates, 20 U RNAsin ribonuclease inhibitor, and 200 U Moloney murine leukemia virus reverse transcription (Promega Corp., Madison, WI). Oligonucleotide primers for GLUT 4-mRNA (5'-TGC AGT TTG GGT ACA ACA TTG G-3'and 5'-ATG AGG AAG GAG GAA ATC ATG C-3') and 18S-rRNA as reference (5'-CGG CTA CCA CAT CCA AGG AA-3' and 5'-GCT GGA ATT ACC GCG GCT-3') were designed using the Omiga 2.0 program (Oxford Molecular Ltd., Madison, WI); their amplification products were, respectively, 190 and 187 bp in size.

PCR was carried out in duplicate for each sample on DNA Engine Opticon 2 continuous fluorescence detection system (MJ Research, Waltham, MA), and all reactions were performed on at least three occasions. Each 30-µl reaction contained 5 µl first-strand cDNA, 15 µl 2x Syber Green PCR master mix (Applied Biosystems, Foster City, CA), 300 nM forward and 50 nM reverse 18S rRNA primer, or 300 nM of each forward and reverse GLUT4 primer. All reactions were carried out using the following cycling parameters: 95 C for 10 min, followed by 40 cycles at 95 C for 15 sec and 60 C for 1 min. Post-PCR melting curves confirmed the specificity of signal target amplification and the expression of GLUT4 relative to ribosomal 18 subunit (r18S). Standard curves were constructed using the PCR cDNA from strong positive samples of adipose tissue serially diluted (1:5) in Rnase-Dnase-free water by plotting values for log cDNA quantity (in arbitrary units) vs. cycle threshold (the cycle number at which the fluorescence signal exceeds background).

The cycle threshold for each unknown sample was then used to calculate the amount of several candidate and reference mRNA relative to the internal standard. For each sample, results were normalized by dividing the amount of candidate mRNA by the amount of reference (r18S).

Immunohistochemical staining

In all patients and controls, endometrial biopsy was processed for immunohistochemistry by polyclonal antibody GLUT4 (C20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). A 20-amino acid peptide sequence within the carboxy terminal, cytoplasmatic domain, of human GLUT4 was used for antibody production. Briefly, sections of 4 µm were deparaffinized, rehydrated in alcohol, rinsed in PBS, treated in citrate buffer in a microwave for 20 min at 750 W, and incubated with primary antibody (GLUT4 1:100 in PBS) in a moist chamber for 45 min at room temperature. Detection system Ultra-tek HRP antipolyvalent (ScyTek, Logan, UT), chromogen DAB system (Dako, Carpinteria, CA) and routine Mayer hematoxylin for 2 min were used. GLUT4 immunohistochemical positivity was evaluated by conventional light microscopy (calculating the area of endometrial tissue as number of fields at 10 x and calculating the percentage of stained cells with respect to 100). Negative control slides were prepared from the same tissue block. Rather than the primary antibody, a normal nonimmune serum supernatant was used. Positive control slides for GLUT4 were prepared from adipose tissue containing high GLUT4. The percentage of staining was assessed by two modalities: 1) light microscopy as absent (–) (less than 2% of positively stained cells), slight (+) (up to about 25%), moderate (2+) (up to about 70%), or strong (2++) (more than 70%); and 2) blindly, with the light microscope connected to a computer image analysis system used to control this semiquantitative evaluation (considering percentage of cytoplasm with positive immunostaining against total cytoplasmatic area and expressed as pixel per cellular area) on endometrial biopsies. Five to 10 fields were evaluated at x20 magnification. Measurements were made using a CI-RES workstation (Carl Zeiss, Jena, Germany), consisting of a light microscope (Zeiss model Axioscope) connected to a 3-CCD color video camera (model KY-F55BE, JVC, Kyoto, Japan). Köhler illumination was used to reduce stray light, and images were analyzed using a PC with frame grabber (Kontron, Eching, Germany). Results are usually expressed as intensity of staining, but in the present study, they are arbitrarily graded as absent (<5% of positively stained epithelial cells), slight (present in 6–25%), moderate (present in 26–70%), or strong (present in >70%).

Plasma assays

Blood samples were centrifuged as soon as they were obtained, and serum or plasma was stored at –20 C until assayed. Plasma glucose was measured by means of the glucose oxidase method (glucose analyzer, Beckman Instruments, Fullerton, CA); insulin by means of RIA methods (Techno Genetics, Milan, Italy; Linco Research, St. Charles, MO); and LH, FSH, estradiol, and testosterone by time-resolved immunofluorimetric assay (ACS: Centaur, Chiron Diagnostics, Los Angeles, CA). The intra- and interassay coefficients of variation (CVs) were 2.8 and 4.7% for LH, 3.8 and 5.0% for FSH, and less than 6.4 and 7.0% for estradiol and testosterone, respectively. Dehydroepiandrosterone sulfate and androstenedione, after celite column chromatographic separation, were assayed by RIA (Radim, Rome, Italy) and their intra- and interassay CVs were less than 8.0 and 10.0%, respectively. SHBG was measured by immunoradiometric assay based on coated tubes with monoclonal antibodies against distinct epitopes of the molecule of SHBG (Radim); its intra- and interassay CVs were less than 4.8 and 6.0%, respectively.

Statistical analysis

Results are expressed as means ± SD. Hormone data and the relative OD of the GLUT4 mRNA content of PCOS and normal groups were analyzed by two-way ANOVA with repeated measures and analysis of covariance. Post hoc testing was done with Tukey’s test. Comparisons of mean baseline values between PCOS and normal women were performed using independent Student’s t test. A stepwise multiple regression analysis was used to correlate GLUT4 expression, in arbitrary units, with ISI values and the frequency of menstruation. Correlations among variables were analyzed using the Pearson correlation coefficient method. All analyses were performed using the Statview statistical package (SAS Institute, Cary, NC). A level of significance of P < 0.05 was used for all data analyses.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical and endocrine metabolic characteristics

Clinical features, and baseline hormonal and metabolic parameters of patients are listed in Table 1Go. There was no significant difference in BMI or waist-hip ratio between n-INS and h-INS obese patients. Likewise, in lean PCOSs, no significant differences were found in these parameters between the n-INS and h-INS groups. As expected, all PCOSs had LH/FSH ratios and plasma androstenedione and testosterone levels significantly higher than controls. Plasma SHBG levels were significantly lower in both lean and obese h-INS PCOS groups with respect to lean and obese n-INS PCOSs and controls. Both fasting glucose levels and the glucose response area (AUC), after a 75-g oral glucose load, did not differ among the five groups. However, fasting insulin levels were significantly higher only in the obese h-INS PCOSs, when compared with those of the other groups, whereas after the oral glucose load, both maximal insulin peak and insulin response area (AUC) were significantly higher in the lean and obese h-INS PCOSs when compared with those of the other three groups. The whole-body insulin sensitivity from the OGTT by ISI showed values significantly lower in h-INS PCOSs when compared with the n-INS PCOS and control subjects. ISI values were significantly lower in obese than lean h-INS PCOSs.


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  		TABLE 1. Clinical and endocrine-metabolic characteristics of women with PCOS (n-INS and h-INS) and control subjects between d 1 and 4 of menstrual cycle

 
Endometrial GLUT4 mRNA expression

A real-time RT-PCR was set up to quantify precisely the mRNA level of human GLUT4 in an adipose tissue biopsy of a normoinsulinemic lean and normal subject. We used the cDNA of strong positive samples as internal standard. The good reaction efficiency of the standard curve was about 88%, and the location of the samples were within the standard dilution for GLUT4 mRNA.

The differences in GLUT4 mRNA content of the endometrial samples in controls and PCOSs are shown in Fig. 1Go. Significantly higher levels of GLUT4 mRNA were evidenced in controls and, unexpectedly, also in lean n-INS PCOSs when compared with the other groups. In both n-INS and h-INS PCOS groups, GLUT4 mRNA levels were significantly lower in obese than lean patients. However, lean h-INS PCOSs showed similar GLUT4 mRNA levels when compared with obese n-INS PCOSs. Conversely, obese h-INS PCOSs displayed the lowest GLUT4 mRNA content in endometrial cells, in comparison with all groups.



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  		FIG. 1. GLUT4 mRNA expression in endometrial samples of hyper- and normoinsulinemic PCOS and control subjects. Real-time RT-PCR products of three representative samples of each group were resolved on endometrial samples, obtained between d 12 and 14 of the last menstrual period. Levels of GLUT4 mRNA were normalized by dividing the amount of candidate mRNA by the amount of references (r18S), expressed by arbitrary units. PCOSs were subdivided into hyperinsulinemic (lean and obese) and normoinsulinemic (lean and obese) groups. As described in Study Design, the histology of the endometrium correspond to the late follicular phase. Differences in GLUT4 mRNA levels, expressed as means ± SD of arbitrary units, were compared by ANOVA and multiple comparisons (post hoc Tukey’s test).

 
Immunohistochemical GLUT4 staining

In endometrial samples, a significant proportion of GLUT4 immunoreactive endometrial cells was found in both normal and PCOSs (Fig. 2Go). In controls, a strong GLUT4 positive reaction was observed only in epithelial cells, as presented, at various magnifications, in Fig. 2Go, A (x40) and B (x80); stromal cells did not present any positive immunostaining. A strong GLUT4-positive reaction, like that of controls, was also observed in the lean n-INS PCOS group (Fig. 2CGo), whereas a significantly lower and moderate positive reaction occurred in obese n-INS PCOSs (Fig. 2DGo). In a diverse manner, only a weak GLUT4-positive reaction was observed in endometrial samples from both lean (Fig. 2EGo) and obese (Fig. 2FGo) h-INS PCOSs. Interestingly, in both controls and PCOSs, GLUT4 appeared to be mainly located at the plasma membrane and cytoplasmatic level of the epithelial cells. At higher magnification, particularly in controls, more intense positive staining was observed at plasma membrane level and in several intracellular compartments within the cytoplasma. When we analyzed endometrial cells by computer image analysis, we were able to calculate the different percentages of epithelial cells with a positive cytoplasmatic GLUT4 reaction in the endometrial samples of all groups (Fig. 3Go). The same method also yielded strong positive GLUT4 staining in controls and lean n-INS PCOSs when compared with that obtained in the other groups. A close correlation was found between cytometric and light microscopy findings for GLUT4 (r2 = 0.93: P = 0.002, data not shown).



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  		FIG. 2. Immunohistochemical staining of GLUT4 in endometrium of normal and PCOSs. Strong positive staining in epithelial cells of endometrium of control subjects (A), with negative staining of stromal cells (original magnification, x40). B, Same endometrial sample shows positive GLUT4 staining to both the plasma membrane and intracytoplasmatic storage site of epithelial cells (original magnification, x80). Strong (C) and moderate (D) positive staining in epithelial cells of endometrium of lean and obese n-INS PCOSs, respectively; moderate (E) and weak (F) positive staining in epithelial cells of endometrium of lean and obese h-INS PCOSs, respectively (original magnification, x40). Immunoreaction revealed by using diaminobenzidine as chromogen. Sections were counterstained with hematoxylin to highlight nucleus in blue.

 


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  		FIG. 3. Variation of GLUT4 immunoreactive endometrial cells in both normal and PCOSs. Intensity of staining evaluated by computer image analysis and arbitrarily graded as described in Subjects and Methods. In the PCOS group, GLUT4-positive cells were significantly lower in obese n-INS (a: P < 0.005), lean h-INS (b: P < 0.002), and obese h-INS (c: P < 0.0005) when compared with lean n-INS subjects. Obese h-INS PCOSs show the lowest percentage of GLUT4-expressing cells (d: P < 0.005) when compared with obese n-INS or lean h-INS PCOSs.

 
Correlation between GLUT4 mRNA expression and insulin sensitivity or menstrual cycles

GLUT4 expression was closely related to insulin sensitivity evaluated by the method of Matsuda and DeFronzo (22), as shown in Fig. 4Go. In fact, a significant positive correlation between GLUT4 expression and ISI values was obtained in our PCOSs and was also found when control subjects were added to the regression analysis (r = 0.77, P < 0.004: y = 12.281 + 2.856x; data not shown).



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  		FIG. 4. Correlation between GLUT4 expression, ISI values, and number of menstrual cycles per year. GLUT4 is expressed in arbitrary units. The ISI evaluates insulin sensibility from data obtained from OGTT and represents a composite of hepatic and peripheral tissue sensibility to insulin. Frequency of menstrual cycles calculated as number of cycles per year, recorded 1 yr before study in all subjects.

 
When we related the menstrual history obtained from self-made recordings of the menses of each subject, expressed as the number of cycles in 12-month periods before the study, with GLUT mRNA expression, a strong positive correlation was found (Fig. 4Go). In this study, ovulatory function, by serum progesterone assay in the luteal phase, was not assessed. When we also included body weight as a parameter, we found, as expected, a significant inverse correlation between GLUT4 expression and BMI (r = –0.58, P < 0.01: y = 9785 – 3789x; data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our data show for the first time that both GLUT4 mRNA and its protein are present in the endometrium of both normal and PCOSs. In the latter, GLUT4 seems to be regulated by body weight and insulin. The expression of insulin receptors and insulin binding has been well documented in human endometrial cells (20), and adequate endometrial glucose metabolism has been reported to be essential for cell differentiation (25). In fact, some authors demonstrated the presence of the noninsulin-regulated transporter GLUT1 in the endometrium, indicating that this glucose transporter is an important marker of endometrial metabolic activity (20, 26, 27). Although GLUT1 is an ubiquitous glucose transporter, it accounts for only 5–10% of total glucose carried in both skeletal muscle and adipose tissue (28, 29). Instead, GLUT4 is the most important glucose transporter isoform in insulin-dependent tissues, and, as far as we know, its presence in endometrial cells has never been demonstrated. In basal conditions, GLUT4 is mostly located in intracellular membranes and one or more intracellular compartments, and insulin acutely stimulates, in a reversible manner, its translocation to the cell surface, thus representing the major mechanism by which insulin stimulates glucose transport in muscle and adipose tissues (30, 31, 32). During the menstrual cycle, epithelial cells play a crucial role in the maturation of endometrial glandular tissue throughout the proliferative phase so that increased glucose metabolism should be expected, and an adequate glucose supply to epithelial cells must be assured.

Our data show that GLUT4 mRNA is highly expressed in endometrial cells, and the presence of GLUT4 protein was confirmed by immunohistochemistry to be exclusively located in epithelial cells at the plasma membrane and cytoplasmatic levels. These cells therefore possess all the machinery involved in insulin-mediated glucose uptake. Although specific insulin receptors and binding have been reported in stromal cells (25, 33), we observed that they have negative immunostaining for GLUT4 protein. Because several data have demonstrated that the insulin receptor and its expression are observed in stromal cells, peaking during the luteal phase (20), the lack of significant GLUT4 expression during the proliferative phase may indicate that, in this phase of the menstrual cycle, the metabolic effect of insulin is absent or minimally involved in these endometrial cells. However, it is important to remember that stromal tissue is physiologically involved in endometrial growth, and therefore insulin, like other growth factors (34, 35, 36), may preferentially act as a growth factor without any or only a small effect on glucose uptake.

In this study we evaluated whether a state of insulin resistance in PCOS may influence the insulin-dependent glucose transporter system at the endometrial level. In fact, in both skeletal muscle cells and adipocytes a significant decrease in GLUT4 expression or translocation has always been linked to insulin resistance (37, 38, 39, 40). Recently, Dunaif and colleagues (41) found a significant decrease in adipocyte GLUT4 glucose transporter content in PCOS subjects, regardless of degree of obesity and in the absence of metabolic derangement of glucose tolerance. Our data show that GLUT4 expression in the epithelial cells of the endometrium is significantly decreased in h-INS PCOSs when compared with n-INS PCOSs or controls. These results strongly support the relationship between hyperinsulinemic state and diminished GLUT4 content in endometrial cells. Because it is well known that insulin-stimulated glucose transport is mediated by GLUT4, the significant decrease in this transporter observed in h-INS PCOSs may account for impaired cellular metabolism at the endometrial level. However, on one hand, whether this reduced GLUT4 expression is associated with consequent impairment of endometrial cell function is still unknown. We did not measure glucose uptake in this new putative insulin target tissue and therefore cannot make any conclusive statement about the presence of a functional defect on glucose transport at the endometrial level in PCOSs. On the other hand, one of the most characteristic clinical signs of this hyperandrogenic syndrome is a disorder of the menstrual cycle and, although it has been clearly demonstrated that this alteration is mainly linked to an ovarian dysfunction, our data indicate that, in h-INS PCOSs, a local endometrial defect may exist. The positive correlation observed in our patients between number of cycles per year and endometrial GLUT4 expression supports this hypothesis because normoinsulinemic subjects exhibit slight disorders of their menstrual cycle when compared with hyperinsulinemic patients. However, the actions of insulin on the endometrium in vivo are difficult to separate from those of ovarian steroids because insulin and ovarian function are closely connected in PCOSs (37, 42).

PCOS and obesity often coexist, but their relative contribution toward inducing insulin resistance still remains a matter of debate (43). Some authors have demonstrated a decrease in GLUT4 expression in the adipocytes of obese subjects and noted that obese PCOSs are not characterized by a further reduction of GLUT4 (42, 43). In a different manner, in n-INS PCOSs, we observed that GLUT4 expression was significantly decreased only in obese patients when compared with lean ones. Moreover, no difference in GLUT4 expression or immunostaining was observed in endometrial cells between lean n-INS PCOSs and controls, thus indicating that the hyperandrogenic syndrome per se does not play a crucial role in the mechanism by which GLUT4 levels are regulated in the endometrium. This fact is further emphasized by the observation that, in the lean PCOS group, only h-INS subjects presented an important decrease in GLUT4, suggesting that alterations in insulin secretion and/or action are closely associated with GLUT4 levels in endometrial cells. The presence of a positive correlation between GLUT4 expression and insulin sensitivity index further confirms this close functional link between GLUT4 and insulin action. Lastly, we observed that the lowest endometrial GLUT4 expression was present in obese h-INS PCOSs, suggesting that insulin and obesity not only influence GLUT4 expression through independent mechanisms but seem to exert an additional impairment on the insulin-resistant state of the endometrium in PCOS subjects.

In conclusion, we provide evidence for the first time in humans that GLUT4 mRNA and protein are present in the endometrial cells of both PCOS and normal subjects. GLUT4 is demonstrated in the epithelial cells of the endometrium during the proliferative phase of the menstrual cycle. A significant decrease in GLUT4 content in endometrial cells is found only in obese and hyperinsulinemic PCOSs, indicating that obesity and insulin independently induce their effects on GLUT4 expression in the endometrium. We found a more significant decrease in GLUT4 content in the endometrium of hyperinsulinemic PCOSs, supporting the hypothesis that GLUT4 is involved in the endometrial mechanism of the insulin-resistant state. Lastly, a further decrease in GLUT4 expression, observed in hyperinsulinemic and obese PCOSs, suggests that hyperinsulinemia and obesity induce an additive reduction in GLUT4 expression at the endometrial level.


   Acknowledgments
 
We kindly thank Dr. Gabriella Milan, B.D., Dr. Luciano Giacomelli, Ph.D., and Mrs. Sonia Leandri for their expert technical assistance and Dr. Martin Edward Donach for his useful assistance with scientific English.


   Footnotes
 
This work was supported by a grant from the Ministero Istruzione Universita Ricerca (to R.V.).

Abbreviations: AUC, Area under the curve; BMI, body mass index; CV, coefficient of variation; GLUT, glucose transporter; ISI, insulin sensitivity index; n-INS, normal insulin response; h-INS, hyperinsulinemic; OGTT, oral glucose tolerance test; PCOS, polycystic ovary syndrome; PCOSs, PCOS patients; r18S, ribosomal 18 subunit.

Received November 20, 2003.

Accepted April 30, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

  1. Hull MGR 1987 Epidemiology of infertility and polycystic ovarian disease: endocrinological and demographic studies. Gynecol Endocrinol 1:235–245[Medline]
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