Glucose ingestion stimulates atherothrombotic inflammation in polycystic ovary syndrome

Frank González; John P Kirwan; Neal S Rote; Judi Minium

doi:10.1152/ajpendo.00491.2012

. 2012 Dec 18;304(4):E375–E383. doi: 10.1152/ajpendo.00491.2012

Glucose ingestion stimulates atherothrombotic inflammation in polycystic ovary syndrome

Frank González ^1,^✉, John P Kirwan ², Neal S Rote ³, Judi Minium ³

PMCID: PMC3566504 PMID: 23249695

Abstract

Women with polycystic ovary syndrome (PCOS) have chronic low-grade inflammation that can increase the risk of atherothrombosis. We performed a cross-sectional study to examine the effect of glucose ingestion on markers of atherothrombotic inflammation in mononuclear cells (MNC) of 16 women with PCOS (8 lean, 8 obese) and 16 weight-matched controls. Activator protein-1 (AP-1) activation and the protein content of early growth response-1 (EGR-1), matrix matalloproteinases-2 (MMP2), and tissue factor (TF) were quantified from MNC obtained from blood drawn fasting and 2 h after glucose ingestion. Plasma MMP9 and C-reactive protein (CRP) were measured from fasting blood samples. Truncal fat was determined by DEXA. Lean women with PCOS exhibited greater AP-1 activation and MMP2 protein content after glucose ingestion and higher plasma MMP9 and CRP levels than lean controls. Obese women with PCOS exhibited greater EGR-1 and TF protein content after glucose ingestion, and plasma CRP levels were even higher compared with lean subjects regardless of PCOS status. Truncal fat correlated with MMP9 and CRP levels and glucose-stimulated increases in AP-1 activation and EGR-1 and TF protein content. Testosterone correlated with glucose-stimulated AP-1 activation, and androstenedione correlated with MMP9 and CRP levels and glucose-stimulated AP-1 activation. Thus, both PCOS and obesity contribute to an atherothrombotic state in which excess abdominal adiposity and hyperandrogenism may be specific risk factors for developing atherothrombosis.

Keywords: inflammation, glucose, atherosclerosis, thrombosis, abdominal adiposity

the polycystic ovary syndrome (PCOS) is the most common female endocrinopathy, affecting between 8 and 12% of reproductive-age women (24). The disorder is characterized by hyperandrogenism, chronic oligo- or anovulation, and polycystic ovaries, with two of three of these findings required to diagnose PCOS (37). Insulin resistance is another common finding in PCOS, that is thought to promote hyperandrogenism through compensatory hyperinsulinemia and is also associated with accelerated atherothrombosis (7, 28). In fact, women with PCOS have endothelial dysfunction, increased fibrinolytic activity, and a higher prevalence of coronary artery calcification reflecting a predisposition to atherothrombosis (23, 32, 39). Women with PCOS are often obese, which raises the risk of developing hyperglycemia and is characterized by chronic low-grade inflammation (8–13).

We demonstrated previously that in PCOS glucose serves as a dietary trigger capable of provoking inflammation from peripheral blood mononuclear cells (MNC) and that this response is independent of obesity (8–13). Indeed, oral glucose ingestion stimulates nuclear factor κB (NF-κB) activation in MNC of lean women with PCOS (11, 12). Regulation of gene transcription by NF-κB gives rise to a variety of proatherogenic inflammatory mediators not the least of which is C-reactive protein (CRP) (20, 36). Aside from its predictive value in cardiovascular disease, CRP may play a causal role by inducing the uptake of lipids into MNC-derived foamy macrophages within atherosclerotic plaques (46). Circulating CRP is elevated in PCOS independently of obesity and is the most reliable marker of chronic low-grade inflammation in the disorder (6, 22).

Activator protein-1 (AP-1) and early growth response-1 (EGR-1) are two additional proinflammatory transcription factors that participate in the development of atherothrombosis. AP-1 is a protein homo- or heterodimer encoded by the fos and jun gene families that regulates the transcription of a family of zinc-dependent matrix matalloproteinases (MMP) (33). MMP2 and MMP9 in particular are involved in extracellular matrix remodeling within the blood vessel wall (29). EGR-1 undergoes rapid transient activation to regulate the transcription of tissue factor (TF), the receptor for coagulation factor VII that induces thrombin generation to promote fibrin formation and platelet activation (5). MMP2, MMP9, and TF are produced by MNC-derived foamy macrophages and activated vascular smooth muscle cells within atherosclerotic plaque (27). Excessive extracellular matrix breakdown by MMP2 and MMP9 culminates in atherosclerotic plaque rupture, thereby exposing TF to the circulating blood. This in turn triggers thrombosis and culminates in blood vessel occlusion.

Glucose has been shown to increase the gene expression EGR-1 and the c-Fos and c-Jun subunits of AP-1 (18, 38). These glucose-stimulated gene products are biologically active and have increased DNA binding activity (41). Circulating levels of MMP2, MMP9, and TF are elevated in PCOS. We have previously reported failed circulating MMP2 suppression in response to hyperglycemia in lean women with PCOS (12, 14, 19, 21). These findings, coupled with the fact that activation of AP-1 and EGR-1 occurs in response to glucose ingestion, suggest that, in PCOS, inflammation triggered by diet may promote an atherothrombotic state (2).

We embarked on a study to examine the effect of glucose ingestion on MNC-derived AP-1 activation and the protein content of MMP2, EGR-1, and TF in women with PCOS. We hypothesized that, in response to glucose ingestion, activated AP-1 and the protein content of MMP2, EGR-1, and TF are increased in women with PCOS compared with body composition-matched controls and that these markers of atherothrombosis are related to abdominal adiposity, insulin sensitivity, and circulating levels of MMP9, CRP, and androgens.

MATERIALS AND METHODS

Subjects.

Sixteen women with PCOS (8 lean and 8 obese) 20–32 yr of age and 16 weight-matched control subjects (8 lean and 8 obese) 20–40 yr of age volunteered for study participation. Subjects in the present report had been previously involved in our studies on PCOS and insulin resistance, and some largely descriptive data from these subjects included herein have been presented in previous publications (8, 9). Obesity was defined as a BMI between 30 and 40 kg/m². Lean subjects had a BMI between 18 and 25 kg/m². The women with PCOS were diagnosed on the basis of oligoamenorrhea and hyperandrogenemia after nonclassic congenital adrenal hyperplasia, Cushing's syndrome, hyperprolactinemia, and thyroid disease were excluded. Polycystic ovaries were present on ultrasound in all subjects with PCOS. All control subjects had regular menses lasting 25–35 days and a luteal range serum progesterone level consistent with ovulation (>5 ng/ml). All control subjects exhibited normal circulating androgen levels and did not have any skin manifestations of androgen excess or polycystic ovaries on ultrasound.

Diabetes and inflammatory illnesses were excluded in all subjects. None of them smoked tobacco or used medications that could impact carbohydrate metabolism or immune function for a minimum of 6 wk before beginning the study. No subjects exercised regularly during the 6 mo before study participation. Written informed consent was obtained in all subjects according to Institutional Review Board guidelines for the protection of human subjects.

Study design.

All study subjects underwent an oral glucose tolerance test (OGTT) between days 5 and 8 following the onset of menstruation, and an overnight fast of ∼12 h. The women were provided with a healthy diet consisting of 50% carbohydrate, 35% fat, and 15% protein for 3 consecutive days before the test. Body composition was assessed immediately before the OGTT.

OGTT.

A 75-g glucose beverage was administered to all subjects. Blood samples were drawn during fasting and at 30, 60, 90, 120, and 180 min after ingestion of the glucose beverage to measure glucose and insulin. Plasma glucose concentrations were assayed immediately, and insulin measurements were performed later from plasma stored at −80°C. Additional plasma was isolated from the fasting blood samples and stored at −80°C until assayed for MMP9 and CRP. Insulin sensitivity was derived from the OGTT (IS_OGTT) using the following formula: 10,000 divided by the square root of (fasting glucose × fasting insulin) × (mean glucose × mean insulin) (25).

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. In addition, all subjects underwent dual-energy X-ray absorptiometry to determine percent total body fat and percent truncal fat using the QDR 4500 Elite model scanner (Hologic, Waltham, MA) as previously described (9, 40).

MNC isolation and processing.

MNC were isolated from blood samples obtained during the OGTT at 0 and 120 min (2 h). A 12-ml sample of Na-EDTA-anticoagulated blood was layered over 12 ml of polymorphonuclear cell separation media (1-Step Polymorphprep; Accurate Chemical and Scientific, Westbury, NY), and centrifuged at 450 g for 30 min at 18°C, resulting in separation of two bands above the red blood cell pellet. MNC present in the top band were harvested with a Pasteur pipette and washed repeatedly with Hank's buffered saline solution (HBSS).

Nuclear extracts of DNA-binding protein were prepared from a portion of MNC using a method described by Andrews et al. (3) and stored at −80°C until processing for oligonucleotide-based ELISA. The remaining MNC portion was suspended in phosphate-buffered saline (pH 7.4) and centrifuged at 13,000 g for 2 min at 18°C to yield a pellet that was flash-frozen in liquid nitrogen for storage at −80°C until processing for Western blotting.

Oligonucleotide-based ELISA.

Nuclear extract total protein concentrations were determined using the BCA protein assay (Pierce Chemical, Rockville, IL). Activated AP-1 components in the samples were detected by oligonucleotide-based ELISA (Active Motif, Carlsbad, CA). The nuclear extracts were added to wells of a 96-well microtiter plate precoated with an AP-1 consensus oligonucleotide sequence (5′-TGAGTCA-3′). The bound AP-1 c-Fos and c-Jun proteins were detected by specific antibodies and anti-rabbit-HRP-conjugated IgG. The c-Fos and c-Jun binding specificity was determined by binding comparisons with a known quantity of free consensus nucleotide or mutated nucleotide added to the reaction buffer. A hydrogen peroxide-3,3′,5,5′-tetramethylbenzidine chromogenic substrate was used for colorimetric readout. The reaction was stopped with 2 N sulfuric acid, and the optical density of the developed color was measured in an ELISA plate reader (BioTek ELx800, Winooski, VT) at 450 nm. The quantity of AP-1 in each sample was proportional to the developed color intensity. 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 6% and 10%, respectively.

Western blotting.

MNC lysates were prepared by adding 1 ml of boiling lysis buffer (1% SDS), 1 mM sodium orthovanadate, and 10 mM Tris (pH 7.4) to MNC pellets. Total protein concentrations were determined using the BCA protein assay. Samples (60 μg) were electrophoresed onto 10% gels (Bio-Rad Laboratories, Hercules, CA). The proteins were transferred to polyvinylidene difluoride membranes, which were then blocked for 1 h in 5% nonfat dry milk and incubated for 1 h with a polyclonal antibody against MMP2 (1:250) (Santa Cruz Biotechnology, Santa Cruz, CA) or EGR-1 (1:1,000) (Santa Cruz Biotechnology) and a monoclonal antibody against TF (1:250) (Calbiochem® EMD Biosciences, La Jolla, CA) or actin (1:1,000) (Santa Cruz Biotechnology). The membranes were washed, incubated in SuperSignal West Femto chemiluminescence substrate (Pierce Chemical), and exposed to X-ray film. Densitometry was performed on scanned films using Carestream Molecular Imaging software version 5.0.2.30 (Rochester, NY), and all values for MMP2, EGR-1, and TF were corrected for loading using those obtained for actin.

Plasma and serum measurements.

Plasma glucose was measured by the glucose oxidase method (YSI, Yellow Springs, OH), and plasma insulin was measured by double-antibody RIA (Linco Research, St. Charles, MO). Serum luteinizing hormone (LH), testosterone, androstenedione, and dehydroepiandrosterone-sulfate (DHEA-S) levels were measured by RIA (Diagnostic Products, Los Angeles, CA). Plasma MMP9 was measured by ELISA (R&D Systems, Minneapolis, MN). Plasma CRP was measured by high-sensitivity ELISA (Alpha Diagnostics International, San Antonio, TX). 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 software package (SAS Institute, Cary, NC) was used to perform the statistical analysis. Descriptive data and change from baseline in atherothrombotic inflammation markers were compared between groups using ANOVA for multiple-group comparisons. Detection of significance by ANOVA was followed by a post hoc analysis using Tukey's honestly significant difference test to identify the source of significance. Data are presented as means ± SE. Treatment effects on atherothrombotic inflammation markers were determined by calculating the percent change for each participant in view of interindividual variability. The Spearman rank correlation coefficient was used to estimate the correlation between parameters. Results were considered significant at a two-tailed α-level of 0.05.

RESULTS

Age, body composition, blood pressure, and lipids.

Obese women with PCOS were similar in age compared with obese controls, whereas lean women with PCOS were younger than lean controls (P < 0.2; Table 1). Weight, BMI, %total body fat, %truncal fat, and waist circumference were significantly (P < 0.5) greater in obese subjects compared with those who were lean whether or not they had PCOS. However, %truncal fat was also significantly (P < 0.05) greater in lean women with PCOS than in lean controls.

Table 1.

Age, body composition, blood pressure, and endocrine and metabolic parameters of subjects

	PCOS		CONTROL
	Lean	Obese	Lean	Obese
Age, yr	26 ± 1^*	25 ± 2^†	33 ± 2	30 ± 3
Height, cm	162.9 ± 3.8	165.3 ± 2.5	165.1 ± 1.0	163.9 ± 2.7
Body weight, kg	62.5 ± 2.1	96.3 ± 3.9^†^,^‡	60.3 ± 2.0	94.2 ± 4.1^§^,^‖
BMI, kg/m²	23.6 ± 0.8	35.2 ± 1.0^†^,^‡	22.0 ± 0.9	35.0 ± 1.0^§^,^‖
Total body fat, %	31.5 ± 1.9	42.8 ± 1.1^†^,^‡	29.1 ± 1.8	42.5 ± 1.0^§^,^‖
Truncal fat, %	30.8 ± 2.0^*	44.0 ± 1.1^†^,^‡	25.7 ± 2.5	42.3 ± 0.9^§^,^‖
Waist circumference, cm	78.0 ± 2.7	102.9 ± 2.9^†^,^‡	73.6 ± 3.0	99.2 ± 3.4^§^,^‖
Systolic blood pressure, mmHg	110 ± 3	128 ± 5^†^,^‡	104 ± 3	120 ± 5^§
Diastolic blood pressure, mmHg	70 ± 4	78 ± 4 b	60 ± 3	76 ± 3^§
Total cholesterol, mg/dl	163 ± 13	182 ± 10	163 ± 11	182 ± 10
Triglycerides, mg/dl	111 ± 33	107 ± 25	47 ± 5	110 ± 39
HDL-cholesterol, mg/dl	50 ± 4	45 ± 4	53 ± 4	48 ± 4
LDL-cholesterol, mg/dl	100 ± 11	119 ± 10	104 ± 9	119 ± 10
LH, mIU/ml	14.0 ± 1.4^*	9.5 ± 1.4^†^,^‡	3.7 ± 0.9	2.7 ± 0.4^‖^,^¶
Testosterone, ng/dl	78.9 ± 10.3^*	86.2 ± 11.1^†	43.5 ± 4.4	32.6 ± 4.8^‖^,^¶
Androstendione, ng/ml	3.2 ± 0.3^*	3.7 ± 0.2^†	1.6 ± 0.2	1.9 ± 0.2^‖^,^¶
DHEA-S, μg/dl	318 ± 43	308 ± 51	229 ± 28	173 ± 31^‖^,^¶
Fasting glucose, mg/dl	85 ± 2	89 ± 1	87 ± 1	84 ± 4
Fasting insulin, μiU/ml	10.9 ± 1.6	19.9 ± 3.3^†^,^‡	6.9 ± 1.0	13.4 ± 2.1^§^,^¶
IS_OGTT	4.2 ± 0.5^*	2.5 ± 0.5^†	8.7 ± 1.0	4.5 ± 1.1^§

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Values are means ± SE; n = 8. PCOS, polycystic ovary syndrome. Conversion factors to SI units: testosterone ×3.467, nmol/l, androstenedione ×3.492, nmol/l, DHEA-S ×0.002714, μmol/l, glucose ×0.0551, mmol/l, insulin ×7.175, pmol/l, IS_OGTT, Insulin sensitivity oral glucose tolerance test.

Lean PCOS vs. Lean Control, P < 0.05;

^§

Obese Control vs. Lean Control, P < 0.05;

^†

Obese PCOS vs. Lean Control, P < 0.03;

^‖

Obese Control vs. Lean PCOS, P < 0.008;

^‡

Obese PCOS vs. Lean PCOS, P < 0.02;

^¶

Obese Control vs. Obese PCOS, P < 0.05.

Systolic and diastolic blood pressures were significantly (P < 0.05) higher in obese individuals than in lean controls and modestly higher in obese women with PCOS compared with lean women with PCOS (P = 0.09), but mean values were in the normotensive range. All lipid levels were similar among groups.

Plasma hormone levels, glycemic status, and insulin resistance.

Circulating levels of LH, testosterone, and androstenedione were significantly (P < 0.05) elevated in women with PCOS compared with control subjects independently of body mass (Table 1). Circulating DHEA-S levels were significantly (P < 0.05) elevated in lean and obese women with PCOS compared with obese controls but only modestly higher in lean women with PCOS compared with lean controls.

Glucose levels while fasting and post-glucose ingestion were similar in women with PCOS compared with controls independently of body mass (Fig. 1). All subjects had a normal glucose response during the OGTT, with fasting glucose levels <100 mg/dl and 2-h glucose levels ranging between 68 and 138 mg/dl. Fasting insulin levels were significantly higher (P < 0.05) in obese subjects than in those who were lean and in obese women with PCOS compared with obese controls. Insulin levels post-glucose ingestion were significantly higher (P < 0.04) in obese women with PCOS compared with those in lean controls at 30 and 120 min and with all three of the other groups at 60 and 90 min. Compared with lean controls, insulin levels post-glucose ingestion were significantly higher (P < 0.05) in obese controls at 30 min and in lean women with PCOS at 60 min. IS_OGTT was significantly higher (P < 0.05) in obese subjects compared with lean controls and in lean women with PCOS compared with lean controls.

Fig. 1. — A: plasma glucose levels measured during the oral glucose tolerance test (OGTT). Levels were similar in all 4 groups throughout the OGTT. B: plasma insulin levels measured during the OGTT. *Levels in obese women with PCOS were significantly greater than those in lean controls at 30 and 120 min and with all 3 of the other groups at 60 and 90 min, P < 0.04. †Levels in obese controls were significantly greater than those of lean controls at 30 min, P < 0.05. ‡Levels in lean women with PCOS were significantly greater than those of lean controls at 60 min, P < 0.04.

Molecular markers of atherothrombosis.

In response to glucose ingestion, the %change in activated AP-1 and MMP2 protein content from MNC was significantly (P < 0.03) higher in both groups of women with PCOS compared with lean controls and in obese controls compared with lean controls (Fig. 2). In contrast, the %change in EGR-1 protein content was significantly (P < 0.02) higher in obese women with PCOS compared with either group of lean subjects (Fig. 3). The %change in TF protein content was significantly (P < 0.001) higher in obese subjects compared with those who were lean whether or not they had PCOS.

Fig. 2. — A: comparison among groups of the change from baseline (%) in activator protein-1 (AP-1) in nuclear extracts from mononuclear cells (MNC) collected during fasting and 2 h after glucose ingestion. *Response in lean women with PCOS was significantly greater than that of lean controls, P < 0.02. †Response in obese controls was significantly greater than that of lean controls, P < 0.0004. ‡Response in obese women with PCOS was significantly greater than that of lean controls, P < 0.004. B: representative Western blots from the 4 study groups showing the change in quantity of matrix matalloproteinase-2 (MMP2) and actin in MNC homogenates in samples collected pre- and post-glucose ingestion. Samples used to quantify proteins from both study groups were run on the same gel. C: Densitometric quantitative analysis comparing the change from baseline (%) in MNC-derived MMP2 protein content between fasting and 2 h post-glucose ingestion samples. *Response in lean women with PCOS was significantly greater than that of lean controls, P < 0.03. †Response in obese controls was significantly greater than that of lean controls, P < 0.03. ‡Response in obese women with PCOS was significantly greater than that of lean controls, P < 0.04.

Fig. 3. — Representative Western blots from the 4 study groups showing the change in quantity of (A) early growth response-1 (EGR-1) and actin and (C) tissue factor (TF) and actin in MNC homogenates in samples collected pre- and post-glucose ingestion. Samples used to quantify proteins from both study groups were run on the same gel. Densitometric quantitative analysis comparing the change from baseline (%) in MNC-derived (B) EGR-1 and (D) TF protein content between fasting and 2 h post-glucose ingestion samples. B: *Response in obese women with PCOS was significantly greater than that of lean controls, P < 0.02. †Response in obese women with PCOS was significantly greater than that of lean women with PCOS, P < 0.02. D: *Response in obese controls was significantly greater than that of lean women with PCOS and lean controls, P < 0.001. †Response in obese women with PCOS was significantly greater than that of lean women with PCOS and lean controls, P < 0.004.

Plasma markers of atherothrombosis.

Plasma MMP9 levels were significantly (P < 0.02) higher in both groups of women with PCOS compared with lean controls and in obese women with PCOS compared with obese controls (Fig. 4). Plasma CRP levels were significantly (P < 0.003) higher in obese subjects compared with those who were lean regardless of PCOS status and in lean women with PCOS compared with lean controls.

Fig. 4. — A: plasma levels of MMP2 measured in fasting blood samples. *Levels in lean women with PCOS were significantly greater than those of lean controls, P < 0.02. †Levels in obese women with PCOS were significantly greater than those of obese controls, P < 0.04. ‡Levels in obese women with PCOS were significantly greater than those of lean controls, P < 0.002. B: plasma levels of C-reactive protein (CRP) measured in fasting blood samples. *Levels in lean women with PCOS were significantly greater than those of lean controls, P < 0.003. †Levels in obese controls were significantly greater than those of lean women with PCOS and lean controls, P < 0.0009. ‡Levels in obese women with PCOS were significantly greater than those of lean women with PCOS and lean controls, P < 0.0003.

Correlations.

For the combined groups, the %change in activated AP-1, EGR-1, and TF protein content and plasma CRP were positively correlated with BMI, %body fat, %truncal fat, and waist circumference (Table 2). The %change in MMP2 protein content was positively correlated with BMI and waist circumference, and plasma MMP9 was positively correlated with %body fat and %truncal fat. Plasma CRP and the %change in MMP2 and TF protein content were also negatively correlated with IS_OGTT. The %change in activated AP-1 was positively correlated with the %change in TF protein content and plasma levels of MMP9 and CRP, and the %change in MMP2 protein content was positively correlated with the %change in EGR-1 protein content and plasma CRP.

Table 2.

Spearman rank correlations for the combined groups

	Activated AP-1, %change	MMP2 Protein, %change	EGR-1 Protein, %change	TF Protein, %change	Plasma MMP9, ng/ml	Plasma CRP, mg/l
BMI, kg/m²
r	0.448	0.381	0.579	0.671	0.288	0.639
P	0.018^*	0.037^*	0.006^*	0.0002^*	0.135	0.0004^*
Total body fat, %
r	0.464	0.297	0.591	0.682	0.370	0.729
P	0.014^*	0.104	0.005^*	0.0001^*	0.048^*	0.0001^*
Truncal fat, %
r	0.467	0.333	0.603	0.705	0.393	0.771
P	0.014^*	0.067	0.004^*	0.0001^*	0.041^*	0.0001^*
Waist circum., cm
r	0.475	0.380	0.514	0.681	0.230	0.581
P	0.014^*	0.037^*	0.016^*	0.0002^*	0.241	0.002^*
IS_OGTT
r	−0.422	−0.386	−0.278	−0.470	−0.278	−0.496
P	0.026^*	0.034^*	0.188	0.009^*	0.149	0.006^*
Activated AP-1, % change
r		0.322	0.408	0.465	0.417	0.459
P		0.094	0.068	0.014^*	0.041^*	0.015^*
MMP2 protein, % change
r			0.517	0.421	0.128	0.269
P			0.015^*	0.021^*	0.515	0.140
EGR-1 protein, % change
r				0.699	0.278	0.435
P				0.0008^*	0.225	0.037^*
TF protein, % change
r					0.351	0.633
P					0.068	0.0004^*
Plasma MMP9, ng/ml
r						0.359
P						0.062

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AP-1, activator protein-1; MMP2, matrix metalloprotease-2; EGR-1, early growth response-1; TF, tissue factor; MMP9, matrix metalloprotease-9; CRP, C-reactive protein; r, correlation coefficient; P, level of significance;

P < 0.05.

In women with PCOS, the %changes in EGR-1 and TF protein content and plasma CRP were positively correlated with BMI, %body fat, %truncal fat, and waist circumference (Table 3). There was also a negative correlation between plasma CRP and IS_OGTT. The %change in EGR-1 protein content was positively correlated with the %change in TF protein content and plasma CRP, and the %change in TF protein content and plasma CRP were positively correlated with each other for the combined groups and in women with PCOS.

Table 3.

Spearman rank correlations in women with PCOS

	Activated AP-1, %change	MMP2 Protein, %change	EGR-1 Protein, %change	TFProtein, %change	Plasma MMP9, ng/ml	Plasma CRP, mg/l
BMI, kg/m²
r	0.129	0.015	0.587	0.668	0.042	0.568
P	0.631	0.955	0.034^*	0.009^*	0.889	0.028^*
Total body fat, %
r	0.050	0.051	0.656	0.696	0.201	0.707
P	0.852	0.846	0.018^*	0.007^*	0.508	0.006^*
Truncal fat, %
r	0.104	−0.029	0.644	0.688	0.280	0.803
P	0.698	0.909	0.020^*	0.008^*	0.354	0.002^*
Waist circum., cm
r	0.293	−0.065	0.477	0.709	0.301	0.509
P	0.273	0.802	0.086	0.006^*	0.319	0.049^*
IS_OGTT
r	−0.229	0.156	−0.292	−0.444	0.098	−0.553
P	0.392	0.546	0.292	0.085	0.745	0.032^*
Activated AP-1, %change
r		0.104	−0.033	0.204	0.055	0.136
P		0.698	0.909	0.446	0.863	0.612
MMP2 protein, %change
r			0.451	0.282	−0.266	0.002
P			0.104	0.274	0.378	0.998
EGR-1 protein, %change
r				0.684	0.127	0.631
P				0.014^*	0.703	0.023^*
TF protein, %change
r					0.007	0.553
P					0.982	0.032^*
Plasma MMP9, ng/ml
r						0.196
P						0.516

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P < 0.05.

The %change in activated AP-1 was positively correlated with serum levels of testosterone (r = 0.38, P < 0.05) and androstendione (r = 0.40, P < 0.04), and plasma MMP9 was positively correlated with serum levels of LH (r = 0.37, P < 0.05) and androstendione (r = 0.57, P < 0.005) for the combined groups. There was also a positive correlation between the %change in TF protein content and LH (r = 0.57, P < 0.03) and plasma CRP and serum androstenedione (r = 0.51, P < 0.05) in women with PCOS.

DISCUSSION

Our data clearly show for the first time that, in PCOS, physiological hyperglycemia is capable of triggering molecular events collectively involved in atherothrombosis and that both PCOS and obesity make significant contributions to this proinflammatory, proatherothrombotic milieu. Our findings corroborate the known mechanisms for glucose-stimulated atherothrombotic inflammation by demonstrating an increase in the amount of translated gene products as well as an increase in transcription factor activation. Lean women with PCOS exhibit increases in activated AP-1 and MMP2 protein content in response to glucose ingestion, along with higher circulating MMP9 and CRP levels and lower insulin sensitivity, compared with lean controls. Obese women with PCOS exhibit similar alterations along with increases in EGR-1 and TF protein content, even greater circulating CRP elevations, higher systolic and diastolic blood pressures, and higher fasting insulin levels compared with lean subjects regardless of PCOS status. The inverse relationship between plasma CRP and the changes in MMP2 and TF protein content with insulin sensitivity lends additional support for this concept. Furthermore, lean women with PCOS have greater abdominal adiposity than lean controls, and multiple atherothrombotic inflammation markers are positively associated with abdominal adiposity and circulating androgens. These findings suggest that, in PCOS, excess abdominal adiposity and hyperandrogenism may be specific risk factors for developing atherothrombotic inflammation and subsequent cardiovascular events.

Lean women with PCOS may be at greater risk for rapid atherogenic development and ensuing atherosclerotic plaque rupture. They exhibit evidence of insulin resistance, a feature highly associated with atherosclerosis (30). The CRP elevations in lean women with PCOS compared with lean controls are milder (1–3 mg/l) than what is observed in obese subjects (>3 mg/l) but are nonetheless indicative of intermediate atherosclerotic cardiovascular risk (35). This finding is also in keeping with our previous reports of increased glucose-stimulated NF-κB activation from MNC and elevated circulating monocyte chemotactic protein-1 (MCP-1) levels in lean women with PCOS (11, 12). Moreover, CRP and MCP-1 both emanate from NF-κB gene regulation. MCP-1 can facilitate migration of MNC into the vascular interstitium, and CRP may subsequently promote the uptake of lipids into MNC-derived foamy macrophages within atherosclerotic plaques (20, 46). There is also increased glucose-stimulated AP-1 activation and MMP2 protein content as well as higher MMP9 levels in lean women with PCOS compared with lean controls. These findings corroborate our previous report of failed plasma MMP2 suppression in response to glucose ingestion in lean women with PCOS and may incite atherosclerotic plaque rupture (12). Additional support is provided by the direct relationship between the change in activated AP-1 and plasma levels of MMP9 and CRP and the inverse relationship between MMP2 protein content and insulin sensitivity. In contrast, lean controls exhibit suppression of AP-1 activation and MMP2 protein content, suggesting that this in vivo response to physiological hyperglycemia may be the norm for preservation of blood vessel integrity. We have previously reported a similar response pattern in MNC-derived NF-κB activation and associated mediators of inflammation in lean, healthy young women (8, 9, 11, 15, 16). Thus, women with PCOS display a distinct proinflammatory risk profile for atherogenesis that is independent of obesity and incited by glucose ingestion.

Obese women with PCOS may also be at greater risk for rapid atherogenic development and subsequent vascular thrombosis. The circulating CRP elevations observed in this group appear to be more the result of obesity than PCOS per se, because they are markedly higher (>3 mg/l) in obese subjects than in those who are lean regardless of PCOS status. We have previously reported similar marked elevations in both circulating IL-6 and CRP along with increases in glucose-stimulated MNC-derived NF-κB activation in obese women with PCOS (11, 12). This is important because NF-κB directly regulates gene transcription of IL-6, which in turn stimulates CRP synthesis in the liver and in adipose tissue of the obese (31). The increases in EGR-1 and TF protein content are also evident only in obese women with PCOS compared with lean subjects regardless of PCOS status. These findings in conjunction with the elevations in circulating PAI-1 that we previously reported in obese women with PCOS may promote vascular thrombosis (12). Furthermore, the greater degree of insulin resistance evident in obese women with PCOS compared with those who are lean is suggestive of a more severe atherothrombotic profile. This is corroborated by the inverse relationship of plasma CRP and TF protein content with insulin sensitivity. Thus, the combination of PCOS and obesity may be a more significant risk for atherothrombosis resulting from inflammation compared with PCOS or obesity alone.

In PCOS, there may be a connection between adiposity and atherothrombotic inflammation. Plasma MMP9 and CRP and glucose-stimulated atherothrombotic inflammation markers are positively associated with measures of adiposity, especially abdominal adiposity for the combined groups and in women with PCOS. In addition, excess abdominal adiposity is evident in lean women with PCOS and is consistent with previous studies (8, 17, 44). MNC-derived macrophages produce roughly one-half of the IL-6 in the expanded adipose mass of obese individuals (42). This suggests that inflamed adipose tissue of women with PCOS, particularly of abdominal origin, is a contributor to the increases in molecular and circulating mediators of atherothrombosis. Thus, these data provide the remarkable revelation that in PCOS excess adiposity sparks an early and possibly premature presence of an atherothrombotic environment.

Hyperandrogenism in PCOS may also play a role in the promotion of atherothrombotic inflammation. LH is positively associated with MMP9 levels and glucose-stimulated TF protein content. Testosterone is positively associated with glucose-stimulated AP-1 activation, and androstenedione is positively associated with MMP9 and CRP levels and glucose-stimulated AP-1 activation. This corroborates similar associations between androgens and measures of inflammation in our past reports (9–14). Although the association with LH suggests a central impact of androgen production on atherothrombotic inflammation, local effects are well described. Androgen exposure in vitro stimulates adhesion of MNC to vascular endothelium and increases oxidation of LDL by MNC-derived macrophages (26, 45). Induction of hyperandrogenism in cholesterol-fed female cynomolgus monkeys encourages the progression of atherosclerosis (1). Most importantly, induction of hyperandrogenism in normal reproductive-age women activates MNC and increases MNC sensitivity to glucose ingestion (15, 16). Thus, hyperandrogenism in PCOS may upregulate the transcription of atherothrombotic inflammation mediators from glucose-activated MNC to drive the increased risk of atherothrombosis.

The absence of a direct assessment of the effects of atherothrombosis in our study population to bolster the notion that our molecular findings point to atherothrombotic risk is a limitation of the study. Nevertheless, atherothrombotic effects such as impaired vascular flow and decreased whole blood viscosity have been previously reported in reproductive-age women with PCOS (4, 43). Furthermore, the CRP elevations evident in the women with PCOS in the current report strongly reflect atherothrombotic risk, since CRP is equally predictive of a cardiovascular event compared with the ATP III criteria for metabolic syndrome (34).

In conclusion, glucose ingestion stimulates atherothrombotic inflammation in PCOS. Activated AP-1 and MMP2 protein contents increase in response to glucose ingestion and circulating MMP9 and CRP are elevated in lean women with PCOS. Similar findings are evident in obese women with PCOS in addition to increases in EGR-1 and TF protein content and even greater elevations in circulating CRP. Thus, both PCOS and obesity contribute significantly to increases in atherothrombotic inflammation markers. The association of these markers with abdominal fat and circulating androgens also suggests that, in PCOS, excess abdominal adiposity and hyperandrogenism contribute to the promotion of atherothrombosis.

GRANTS

This research was supported by Grant HD-048535 to F. González from the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: F.G., J.P.K., and N.S.R. conception and design of research; F.G. and J.M. performed experiments; F.G. analyzed data; F.G., J.P.K., and N.S.R. interpreted results of experiments; F.G. prepared figures; F.G. drafted manuscript; F.G., J.P.K., and N.S.R. edited and revised manuscript; F.G., J.P.K., N.S.R., and J.M. approved final version of manuscript.

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[B1] 1.Adams MR, Williams JK, Kaplan JR. Effects of androgens on coronary artery atherosclerosis and atherosclerosis-related impairment of vascular responsiveness. Arterioscler Thromb Vasc Biol 15: 562–570, 1995 [DOI] [PubMed] [Google Scholar]

[B2] 2.Aljada A, Ghanim H, Mohanty P, Syed T, Bandyopadhyay A, Dandona P. Glucose intake induces an increase in activator protein 1 and early growth response 1 binding activities, in the expression of tissue factor and matrix metalloproteinase in mononuclear cells, and in plasma tissue factor and matrix metalloproteinase concentrations. Am J Clin Nutr 80: 51–57, 2004 [DOI] [PubMed] [Google Scholar]

[B3] 3.Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limited numbers of mammalian cells. Nucleic Acids Res 19: 2499, 1991 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Coksuer H, Koplay M, Oghan F, Haliloglu B, Keskin N. Evaluation of carotid wall thickness and vertebro-basilar system insufficiency in patients with obese polycystic ovary syndrome. J Obstet Gynaecol Res 37: 997–1003, 2011 [DOI] [PubMed] [Google Scholar]

[B5] 5.Cui MZ, Parry GC, Oeth P, Larson H, Smith M, Huang RP, Adamson ED, Mackman N. Transcriptional regulation of the tissue factor gene in human epithelial cells is mediated by Sp1 and EGR-1. J Biol Chem 271: 2731–2739, 1996 [DOI] [PubMed] [Google Scholar]

[B6] 6.Escobar-Morreale HF, Luque-Ramírez M, González F. Serum inflammatory markers in polycystic ovary syndrome: a systematic review and meta-analysis. Fertil Steril 95: 1048–1058, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Freeman MS, Mansfield MW, Barrett JH, Grant PJ. Insulin resistance: an atherothrombotic syndrome. Thromb Haemost 89: 161–168, 2003 [PubMed] [Google Scholar]

[B8] 8.González F, Rote NS, Minium J, Kirwan JP. Hyperglycemia alters tumor necrosis factor-α release from mononuclear cells in women with polycystic ovary syndrome. J Clin Endocrinol Metab 90: 5336–5342, 2005 [DOI] [PubMed] [Google Scholar]

[B9] 9.González F, Rote NS, Minium J, Kirwan JP. In vitro evidence that hyperglycemia stimulates tumor necrosis factor-α release in obese women with polycystic ovary syndrome. J Endocrinol 88: 521–529, 2006 [DOI] [PubMed] [Google Scholar]

[B10] 10.González F, Rote NS, Minium J, Kirwan JP. Reactive oxygen species-induced oxidative stress in the development of insulin resistance and hyperandrogenism in polycystic ovary syndrome. J Clin Endocrinol Metab 91: 336–340, 2006 [DOI] [PubMed] [Google Scholar]

[B11] 11.González F, Rote NS, Minium J, Kirwan JP. Increased activation of nuclear factor κB triggers inflammation and insulin resistance in polycystic ovary syndrome. J Clin Endocrinol Metab 91: 1508–1512, 2006 [DOI] [PubMed] [Google Scholar]

[B12] 12.González F, Rote NS, Minium J, Kirwan JP. Evidence of proatherogenic inflammation in polycystic ovary syndrome. Metabolism 58: 954–962, 2009 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.González F, Rote NS, Minium J, Kirwan JP. Elevated circulating levels of macrophage migration inhibitory factor in polycystic ovary syndrome. Cytokine 51: 240–244, 2010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.González F, Kirwan JP, Rote NS, Minium J. Elevated circulating levels of tissue factor in polycystic ovary syndrome. Clin Appl Thromb Hemost 19: 66–72, 2012 [DOI] [PubMed] [Google Scholar]

[B15] 15.González F, Nair KS, Daniels JK, Basal E, Schimke JM. Hyperandrogenism sensitizes mononuclear cells to promote glucose-induced inflammation in lean reproductive-age women. Am J Physiol Endocrinol Metab 302: E297–E306, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.González F, Nair KS, Daniels JK, Basal E, Schimke JM, Blair HE. Hyperandrogenism sensitizes leukocytes to hyperglycemia to promote oxidative stress in lean reproductive-age women. J Clin Endocrinol Metab 97: 2836–2843, 2012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17.Ibañez L, de Zegher F. Flutamide-Metformin plus an oral contraceptive (OC) for young women with polycystic ovary syndrome: switch from third- to fourth-generation OC reduces body adiposity. Hum Reprod 19: 1725–1727, 2004 [DOI] [PubMed] [Google Scholar]

[B18] 18.Josefsen K, Sorensen LR, Buschard K, Birkenbach M. Glucose induces early growth response gene (Egr-1) expression in pancreatic cells. Diabetologia 42: 195–203, 1999 [DOI] [PubMed] [Google Scholar]

[B19] 19.Lewandowski KC, Komorowski J, O'Callaghan CJ, Tan BK, Chen J, Prelevic GM, Randeva HS. Increased circulating levels of matrix metalloproteinase-2 and -9 in women with polycystic ovary syndrome. J Clin Endocrinol Metab 91: 1173–1177, 2006 [DOI] [PubMed] [Google Scholar]

[B20] 20.Libby P. Inflammation in atherosclerosis. Nature 420: 868–874, 2002 [DOI] [PubMed] [Google Scholar]

[B21] 21.Liu B, Cai LY, Lv HM, Xia L, Zhang YJ, Zhang HX, Guan YM. Raised serum levels of matrix metalloproteinase-9 in women with polycystic ovary syndrome and its association with insulin-like growth factor binding protein-1. Gynecol Endocrinol 24: 285–288, 2008 [DOI] [PubMed] [Google Scholar]

[B22] 22.Makedos A, Goulis DG, Arvanitidou M, Mintziori G, Papanikolaou A, Makedou A, Panidis D. Increased serum C-reactive protein levels in normal weight women with polycystic ovary syndrome. Hippokratia 15: 323–326, 2011 [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Mannerås-Holm L, Baghaei F, Holm G, Janson PO, Ohlsson C, Lönn M, Stener-Victorin E. Coagulation and fibrinolytic disturbances in women with polycystic ovary syndrome. J Clin Endocrinol Metab 96: 1068–1076, 2011 [DOI] [PubMed] [Google Scholar]

[B24] 24.March WA, Moore VM, Willson KJ, Phillips DIW, Normal RJ, Davies MJ. The prevalence of polycystic ovary syndrome in a community sample assessed under contrasting diagnostic criteria. Hum Reprod 25: 544–551, 2010 [DOI] [PubMed] [Google Scholar]

[B25] 25.Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28: 412–419, 1985 [DOI] [PubMed] [Google Scholar]

[B26] 26.McCrohon JA, Jessup W, Handelsman DJ, Celermajer DS. Androgen exposure increases human monocyte adhesion to vascular endothelium and endothelial cell expression of vascular adhesion molecule-1. Circulation 99: 2317–2322, 1999 [DOI] [PubMed] [Google Scholar]

[B27] 27.Moreno PR, Bernardi VH, Lopez-Cuellar J, Murcia AM, Palacios IF, Gold HK, Mehran R, Sharma SK, Nemerson Y, Fuster V, Fallon JT. Macrophages, smooth muscle cells, and tissue factor in unstable angina. Implications for cell-mediated thrombogenicity in acute coronary syndromes. Circulation 94: 3090–3097, 1996 [DOI] [PubMed] [Google Scholar]

[B28] 28.Nestler JE, Jakubowicz DJ, de Vargas AF, Brik C, Quintero N, Medina F. Insulin stimulates testosterone biosynthesis by human theca cells from women with polycystic ovary syndrome by activating its own receptor and using inositoglycan mediators as the signal transduction system. J Clin Endocrinol Metab 83: 2001–2005, 1998 [DOI] [PubMed] [Google Scholar]

[B29] 29.Newby AC. Dual role of matrix metalloproteases (matrixins) in intimal thickening and atherosclerotic plaque rupture. Physiol Res 85: 1–31, 2005 [DOI] [PubMed] [Google Scholar]

[B30] 30.Nigro J, Osman N, Dart AM, Little PJ. Insulin resistance and atherosclerosis. Endocr Rev 27: 242–259, 2006 [DOI] [PubMed] [Google Scholar]

[B31] 31.Ouchi N, Kihara S, Funahashi T, Nakamura T, Nishida M, Kumada M, Okamoto Y, Ohashi K, Nagaretani H, Kishida K, Nishizawa H, Maeda N, Kobayashi H, Hiraoka H, Matsuzawa Y. Reciprocal association of C-reactive protein with adiponectin in blood stream and adipose tissue. Circulation 107: 671–674, 2003 [DOI] [PubMed] [Google Scholar]

[B32] 32.Paradisi G, Steinberg HO, Hempfling A, Cronin J, Hook G, Shepard MK, Baron AD. Polycystic ovary syndrome is associated with endothelial dysfunction. Circulation 103: 1410–1415, 2001 [DOI] [PubMed] [Google Scholar]

[B33] 33.Radler-Pohl A, Gebel S, Sachsenmaier C, König H, Krämer M, Oehler T, Streile M, Ponta H, Rapp U, Rahmsdorf HJ, Cato ACB, Angel P, Herrlich P. The activation and activity control of AP-1 (Fos/Jun). Ann NY Acad Sci 684: 127–148, 1993 [DOI] [PubMed] [Google Scholar]

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PERMALINK

Glucose ingestion stimulates atherothrombotic inflammation in polycystic ovary syndrome

Frank González

John P Kirwan

Neal S Rote

Judi Minium

Abstract