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¹ Department of Reproductive Biology and
² Department of Medicine, Schwartz Center for Metabolism and Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44109, USA

(Requests for offprints should be addressed to F González, MetroHealth Medical Center, Department of Obstetrics and Gynecology, Hamann S4-44, 2500 MetroHealth Drive, Cleveland, Ohio 44109, USA; Email: fgonzalez{at}metrohealth.org)

	Abstract

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Women with polycystic ovary syndrome (PCOS) are often insulin resistant and have chronic low-level inflammation. The purpose of this study was to determine the effects of hyperglycemia in vitro on tumor necrosis factor (TNF)- release from mononuclear cells (MNC) in PCOS. Twelve reproductive-age women with PCOS (six lean, six obese) and 12 age-matched controls (six lean, six obese) were studied. Insulin sensitivity (IS_HOMA) was estimated from fasting levels of glucose and insulin and percent truncal fat was determined by dual energy absorptiometry (DEXA). TNF release was measured from MNC cultured under euglycemic and hyperglycemic conditions. IS_HOMA was higher in obese women with PCOS than in lean women with PCOS (student’s t-test; 73.7 ± 14.8 vs 43.1 ± 8.6, P < 0.05), but similar to that of obese controls. IS_HOMA was positively correlated with percent truncal fat (r=0.57, P < 0.04). Obese women with PCOS exhibited an increase in the percent change in TNF release from MNC in response to hyperglycemia compared with obese controls (10 mM, 649 ± 208% vs 133 ± 30%, P < 0.003; 15 mM, 799 ± 347% vs 183 ± 59%, P < 0.04). The TNF response directly correlated with percent truncal fat (r=0.45, P < 0.03) and IS_HOMA (r=0.40, P < 0.05) for the combined groups, and with plasma testosterone (r=0.60, P < 0.05) for women with PCOS. MNC of obese women with PCOS exhibit an increased TNF response to in vitro physiologic hyperglycemia. MNC-derived TNF release may contribute to insulin resistance and hyperandrogenism, particularly when the combination of PCOS and increased adiposity is present.

	Introduction

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Polycystic ovary syndrome (PCOS) is one of the most common female endocrinopathies, affecting 4–10% of reproductive-age women (Knochenhauer et al. 1998, Dunaif 1999). The disorder is characterized by hyper-androgenism, chronic oligo- or anovulation, and polycystic ovaries, with two out of these three findings required to diagnose PCOS (Rotterdam Group 2004a, 2004b). As many as 70% of women with PCOS exhibit insulin resistance, with the compensatory hyperinsulinemia considered to be the cause of the hyperandrogenism (Burghen et al. 1980, Nestler et al. 1998, Goodarzi & Korenman 2002, Rotterdam Group 2004a, 2004b). In addition, women with PCOS are often obese, a condition strongly associated with insulin resistance and hyperglycemia (Kolterman et al. 1980, Ciaraldi et al. 1981).

Hyperglycemia can contribute to the development of insulin resistance and impaired insulin secretion in a phenomenon known as ‘glucose toxicity’ (Rossetti et al. 1990, Yki-Jarvinen 1992). It is recognized that these effects may be the exaggeration of normal regulatory responses to increases in circulating glucose. We have shown that in PCOS, hyperglycemia causes an increase in reactive oxygen species (ROS) generation from peripheral blood mononuclear cells (MNC) (González et al. 2006). ROS-induced oxidative stress is a known activator of nuclear factor B (NFB), a proinflammatory transcription factor that promotes tumor necrosis factor- (TNF) gene transcription (Barnes & Karin 1997, Mohanty et al. 2000, Evans et al. 2002). TNF is an established mediator of insulin resistance (Hotamisligil et al. 1994). Thus, increased TNF release from MNC in response to hyperglycemia may be an underlying mechanism for insulin resistance in PCOS.

In vitro studies have shown that TNF can truncate insulin receptor signaling in all insulin-sensitive tissues (Feinstein et al. 1993, Hotamisligil et al. 1994, Del Aguila et al. 1999). In obesity-related diabetic syndromes, TNF is overexpressed in adipose tissue and causes increased serine phosphorylation of insulin receptor substrate-1 (IRS-1) (Hotamisligal et al. 1993, 1995, Rui et al. 2001). This leads to decreased expression of GLUT 4, the insulin-sensitive glucose transport protein (Stephens & Pekala 1991). Insulin resistance in PCOS is also a post-receptor defect, and increased serine phosphorylation is implicated as the cause of decreased insulin-stimulated IRS-1 activation and decreased GLUT 4 expression (Rosenbaum et al. 1993, Dunaif et al. 2001, Li et al. 2002, Corbould et al. 2005). Thus, the ability of TNF to stimulate increased serine phosphorylation makes it an ideal candidate for initiating these molecular events in PCOS.

We have previously reported that circulating levels of TNF are elevated in PCOS (González et al. 1999). A likely source of excess circulating TNF in obese women with PCOS is adipose tissue, but the source remains unknown in lean women with the disorder. MNC are known to migrate into adipose tissue to activate adipocyte TNF production (Weisberg et al. 2003, Wellen & Hotamisligil 2003). It is now clear that the major source of TNF in adipose tissue of the obese is MNC-derived macrophages present in the stromal-vascular compartment (Weisberg et al. 2003, Xu et al. 2003, Fain et al. 2004a, 2004b). Thus, MNC may be an additional source of excess circulating TNF in PCOS.

In the present study, we evaluated an in vitro model of hyperglycemia to determine the effect of direct exposure to hyperglycemia on TNF release from MNC of women with PCOS. We hypothesized that TNF release from MNC is increased in women with PCOS compared with weight-matched controls in response to hyperglycemic conditions, and that there is a relationship between measures of adiposity and MNC-derived TNF release.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subjects

Twelve women with PCOS (six lean and six obese) aged 21–34 years and 12 weight-matched control subjects (six lean and six obese) aged 20–38 years volunteered to participate in the study. The women with PCOS were diagnosed on the basis of oligoamenorrhea and hyper-androgenemia after excluding nonclassic congenital adrenal hyperplasia, Cushing’s syndrome, hyperprolactinemia and thyroid disease. Polycystic ovaries were present on ultrasound in all subjects with PCOS. All control subjects were ovulatory as evidenced by regular menses and a luteal phase serum progesterone level greater than 5 ng/ml. All control subjects exhibited normal circulating androgen levels and the absence of polycystic ovaries on ultrasound.

All subjects were screened for diabetes or inflammatory illnesses, and none were taking medications that affect carbohydrate metabolism or immune function for at least 6 weeks prior to study participation. None of the subjects were involved in any regular exercise program for at least 6 months before the time of testing. All of the subjects provided written, informed consent in accordance with the Case Western Reserve University and Metro-Health Medical Center guidelines for the protection of human subjects.

Study design

All study subjects underwent the oral glucose tolerance test (OGTT) on days 5 and 8 after the onset of menses. Before the OGTT, they were provided with a healthy diet consisting of 50% carbohydrate, 35% fat and 15% protein for 3 consecutive days (days 1–3) before the test. The test was performed on the morning of day 4 after an overnight fast of ~12 h. All subjects also underwent body composition assessment on the same day the OGTT was performed.

Oral glucose tolerance test (OGTT)

Fasting baseline blood samples (5 ml each) were drawn for glucose and insulin determination. A 75 g glucose beverage was subsequently ingested over 10 min. Blood samples (5 ml each) were again drawn for glucose and insulin determination 2 h after glucose ingestion. Upon completion of the test, subjects were fed a high-carbohydrate snack. Plasma glucose concentrations were assayed immediately from the blood samples collected. Additional plasma was isolated from the fasting blood samples and stored at –70 °C until assayed for C-reactive protein (CRP) and TNF. Glucose tolerance was assessed by the WHO criteria with normal glucose tolerance defined as a 2-h glucose-stimulated value less than 140 mg/dl, impaired glucose tolerance as a 2-h value of 140–199 mg/dl, and type 2 diabetes mellitus defined as a 2-h value of 200 mg/dl or greater (Modan et al. 1989). Insulin sensitivity was estimated by IS_HOMA by the following formula (Matthews et al. 1985): fasting glucose x fasting insulin/22.5.

Body composition assessment

Height without shoes was measured to the nearest 1.0 cm. Body weight was measured to the nearest 0.1 kg. Waist circumference was measured at the level of the umbilicus and used to estimate abdominal adiposity (Kohrt et al. 1993). In addition, all subjects underwent dual energy absorptiometry (DEXA) to determine percent total body fat and percent truncal fat with the QDR 4500 Elite model scanner (Hologic, Waltham, MA, USA). Truncal fat content was defined as the area between the dome of the diaphragm (cephalad limit) and the top of the greater trochanter (caudal limit) (Taylor et al. 1998).

Analytic methods

MNC isolation and culture were performed on a 20 ml fasting blood sample drawn before ingestion of the glucose beverage during the OGTT. The cells were isolated by Histopaque-1077 density gradient centrifugation (Boyam 1968), washed two times in pyrogen-free saline, re-suspended in RPMI (0.3 mg/ml L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin) with serum substitute TCH, and seeded in coated culture plates (2.510⁶ cells/ml). The culture medium was supplemented with D-glucose at varying concentrations to mimic a euglycemic (5 mM) or hyperglycemic (10 or 15 mM) environment. The cells were incubated (humidified, 5% CO₂, 37°C) for 24 h. Cell supernatants were subsequently collected (10 000 g for 2 min) and stored at –70 °C until analysis. Plasma glucose concentrations were measured by the glucose oxidase method (YSI, Yellow Springs, OH, USA), while plasma insulin concentrations were measured by double-antibody RIA (Linco Research, St Charles, MO, USA). Plasma CRP concentrations were measured by high-sensitivity ELISA (Alpha Diagnostics International, San Antonio, TX, USA). TNF concentrations were also measured by ELISA (BioSource International, Camarillo, CA, USA). All samples from each subject were measured in duplicate in the same assay. The interassay and intra-assay coefficients of variation for all assays were 7% and 12% respectively.

Statistics

The StatView statistical package (SAS Institute, Cary, NC, USA) was used for data analysis. The difference in values under either hyperglycemic condition (10 or 15 mM) and the euglycemic baseline (5 mM) for primary dependent variables, such as TNF release from MNC, was calculated to represent the incremental change. Descriptive data and the incremental change of variables were compared between groups by unpaired Student’s t-test or ANOVA for multiple-group comparisons followed by post hoc analysis. Differences between the incremental change of variables within groups were analyzed by paired Student’s t-test. Alterations in TNF release from MNC within groups were expressed as the percent change between either hyperglycemic condition (10 or 15 mM) and the euglycemic condition (5 mM, 100% baseline). Differences in the MNC-derived TNF response among the different glycemic conditions within groups were analyzed by repeated-measures ANOVA. Regression analyses used the Pearson (r) correlation for parametric data and the Spearman rank order () correlation for nonparametric data. All values are expressed as means ± S.E.M. An -level of 0.05 was used to determine statistical significance.

	Results

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Age and height were similar among groups (Table 1). Weight, body-mass index (BMI), percent total body fat and waist circumference were significantly (P < 0.01) greater in obese subjects than in those who were lean whether or not they had PCOS, but were similar when women with PCOS were compared with weight-matched controls. The obese also exhibited significantly (P < 0.01) greater percent truncal fat than lean subjects whether or not they had PCOS. However, percent truncal fat was significantly (P < 0.02) greater in lean women with PCOS than in lean controls.

TNF release from MNC under euglycemic conditions (5 mM) was similar in women with PCOS compared with controls (Table 3). After exposure of MNC to hyperglycemia at the 10 mM glucose concentration, the incremental change in TNF release of women with PCOS increased significantly (P < 0.03) compared with that of controls, which slightly declined (7.0 ± 3.3 vs –2.9 ± 2.2). When subjects were grouped by body mass, the incremental change in TNF release from MNC exposed to 10 mM glucose increased significantly in obese women with PCOS compared with either lean controls (P < 0.005) or obese controls (P < 0.05). In contrast, the incremental change in TNF release from MNC of lean women with PCOS was similar to lean controls after the 10 mM glucose exposure. There were no significant differences in the TNF response of women with PCOS compared with control subjects regardless of body weight after exposure of MNC to 15 mM glucose.

View this table: [in this window] [in a new window]   Table 3 TNF release from mononuclear cells (MNC) under euglycemic and hyperglycemic conditions in vitro

View larger version (21K): [in this window] [in a new window]   Figure 1 Percent change in TNF release from MNC under hyperglycemic conditions (10 and 15 mM) compared with the eugylcemic condition (5 mM, 100% baseline). : lean controls; : obese controls; : lean women with PCOS; : obese women with PCOS. *TNF response from MNC exposed to 10 mM glucose was significantly higher in obese women with PCOS than in either control group (P < 0.003). TNF response from MNC exposed to 15 mM glucose was significantly higher in obese women with PCOS than in either control group (P < 0.04).

View this table: [in this window] [in a new window]   Table 4 Spearman rank correlation analysis examining the relationship between the percent change in TNF release from mononuclear cells and BMI, percent total body fat, percent truncal fat, ISHOMA, testosterone and androstenedione

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Hyperglycemia within the physiologic range does not result in increased TNF release from MNC in normal circumstances. Lean controls in the present study exhibited no change in MNC-derived TNF release in response to either hyperglycemic culture condition. This is in contrast to a previous report of increases in TNF release from MNC of normal individuals exposed to hyperglycemia in vitro (Morohoshi et al. 1996). In this study, however, the glucose concentration required to achieve this response was above the physiologic range. Since TNF is a known mediator of insulin resistance (Hotamisligil et al. 1994, 1995, Del Aguila et al. 1999), the lack of increase in TNF release from MNC may be a physiologic benefit in the presence of hyperglycemia. Thus, facilitation of glucose disposal in lean controls may be due to the control of TNF release to optimize insulin signaling in the postprandial state.

In contrast, the MNC of obese women with PCOS have increased sensitivity to hyperglycemic conditions in the physiologic range. Obese women with PCOS are in a proinflammatory state, as shown by the elevations of plasma CRP observed in this group that are consistent with previous reports (Kelly et al. 1996, Bastard et al. 1999, Yudkin et al. 1999). MNC-derived TNF release increased in response to either hyperglycemic culture condition in obese women with PCOS compared with obese controls. Oral intake of glucose, lipid and protein has been noted to elicit similar proinflammatory responses in vivo (Mohanty et al. 2000, 2002, Aljada et al. 2004). It is possible that in obese women with PCOS, feeding results in increased TNF release from MNC in the postprandial state to promote the insulin resistance observed in these individuals. This concept is supported by the positive correlation between the TNF response and IS_HOMA. Previous reports of a reduction in oxidative stress and inflammatory mediators after caloric restriction in the obese, and after a 2-day fast in normal subjects provide further corroboration (Dandona et al. 1998, 2001a, 2001b).

Our data suggest a link between adiposity and MNC-derived TNF release in PCOS. There was a direct relationship between the change in TNF release from MNC under hyperglycemic culture conditions and abdominal adiposity. It is possible that the inflamed adipose tissue in the abdominal region of obese women with PCOS perpetuates the increased sensitivity of MNC to hyperglycemia manifested by the increased TNF release observed in culture. Our data also demonstrate a direct relationship between the degree of insulin resistance by IS_HOMA and abdominal adiposity. These findings are consistent with previous observations in young adults demonstrating that changes in insulin sensitivity are a function of abdominal adiposity (Kriktetos et al. 2004, Linne 2004). Thus, increased TNF release from MNC may promote the insulin resistance observed in obese women with PCOS.

The MNC of obese controls and lean women with PCOS exhibited only modest increases in TNF release in response to hyperglycemia. Both of these groups are in a proinflammatory state, as evidenced by the elevated CRP concentrations in accordance with previous observations (Kelly et al. 1996, Boulman et al. 2004). The lack of statistical significance in the increases in TNF release in either of these groups and the elevated CRP concentrations in lean women with PCOS may be due to the small sample size. Nevertheless, there is a stepwise increasing trend in the TNF response, with progressively higher hyperglycemic conditions when study subjects are grouped by body weight. It is possible that the increased abdominal adiposity observed in lean women with PCOS also perpetuates increases in hyperglycemia-induced TNF release from MNC to promote insulin resistance in this group. Nevertheless, the presence of PCOS in combination with a greater amount of adiposity may explain the higher TNF response and greater degree of insulin resistance evident in obese women with PCOS.

In PCOS, TNF release from MNC in response to hyperglycemia may be capable of directly stimulating hyperandrogenism. This is suggested by the direct correlation of the TNF response with plasma levels of testosterone and androstenedione in women with PCOS. We have also demonstrated direct correlations of ROS generation (González et al. 2006) and activated NFB (unpublished data) with these androgen levels. Infiltration of the ovary by MNC-derived macrophages has been previously reported (Best et al. 1996). Ovarian steroidogenic enzymes responsible for androgen production are stimulated by oxidative stress and inhibited by antioxidants, such as statins, in vitro (Piotrowski et al. 2005, Rzepczynska et al. 2005). Circulating androgen levels decline in women with PCOS in response to statin therapy in vivo (Banaszewska et al. 2005). TNF stimulates in vitro proliferation of androgen-producing theca cells (Spazynsky et al. 1999). Thus, it is attractive to consider that increased TNF release from glucose-activated MNC recruited into the polycystic ovary may be the result of a local inflammatory response that stimulates ovarian androgen production in women with PCOS.

In conclusion, MNC of obese women with PCOS exhibit increased TNF response when directly exposed to hyperglycemic conditions in vitro in the physiologic range. Our findings suggest that the increased abdominal adiposity in women with PCOS promotes a proinflammatory state, especially in those who are obese. The associations of the TNF response with measures of adiposity and androgen levels suggest that MNC-derived TNF release contributes to insulin resistance and hyper-androgenism, particularly when the combination of PCOS and increased adiposity is present.

	Acknowledgements

 
We thank the nursing staff of the General Clinical Research Center for supporting the implementation of the study and assisting with data collection.

	Funding

This research was supported by National Institutes of Health Grants HD-01273 (Women’s Reproductive Health Research Program) to the Department of Obstetrics and Gynecology at MetroHealth Medical Center and MO1 RR-000080 to the General Clinical Research Center. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received 17 November 2005
Accepted 25 November 2005

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