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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 7 3585-3594
Copyright © 2004 by The Endocrine Society

Repression of Oxidant-Induced Nuclear Factor-{kappa}B Activity Mediates Placental Cytokine Responses in Gestational Diabetes

Melinda T. Coughlan, Michael Permezel, Harry M. Georgiou and Gregory E. Rice

Mercy Perinatal Research Centre, University of Melbourne, Mercy Hospital for Women, Melbourne 3002, Australia

Address all correspondence and requests for reprints to: Melinda T. Coughlan, Danielle Alberti Memorial Centre for Diabetic Complications, Baker Heart Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne 8008, Victoria, Australia. E-mail: melinda.coughlan{at}baker.edu.au.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Although oxidative stress has been implicated in the pathogenesis of type 2 diabetes, limited data are available regarding its role in gestational diabetes mellitus (GDM), a disease of similar pathophysiology. The proinflammatory cytokines TNF{alpha}, IL-6, and IL-8 are released from the placenta at term and have been implicated in and/or associated with various metabolic events, including decreased insulin sensitivity. Previously we reported differences in the ex situ release of proinflammatory cytokines from placental and adipose tissues obtained from women with and without GDM. We proposed that these differences reflect preexposure and/or adaptation to oxidative stress by GDM tissues. In this study, we tested the hypothesis that placental tissue from women with GDM is less responsive to oxidative stress than tissue from normal women. Under basal conditions, release of TNF{alpha}, IL-6, and IL-8 was similar in both control and GDM groups. However, 8-isoprostane release was 2-fold greater in the GDM group (P < 0.01). In response to oxidative stress, TNF{alpha} and 8-isoprostane release and nuclear factor-{kappa}B (NF-{kappa}B) DNA-binding activity were significantly increased in normal tissues (20-fold, 2-fold, and 35%, respectively, P < 0.01). In contrast, the response of GDM tissues to oxidant stress was blunted, with no change in 8-isoprostane release, a 4-fold increase in TNF{alpha} release, and a 40% reduction in NF-{kappa}B DNA-binding activity. These data support the hypothesis that placentae from women with GDM display a reduced capacity, mediated by repression of NF-{kappa}B activity, to respond to oxidative stress.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GESTATIONAL DIABETES MELLITUS (GDM) is a glucose intolerance of varying severity with onset or first recognition during pregnancy (1) that complicates approximately 2–4% of pregnancies. It is a significant cause of fetal macrosomia, perinatal mortality, and long-term maternal risk of developing type 2 diabetes (2). GDM is characterized by hyperglycemia, insulin resistance, and hyperlipidemia, biochemical abnormalities that are common to type 2 diabetes. Gestational diabetes mellitus has been considered a prediabetic state (3), and the pathophysiology of the two are clearly related. The role of oxidative stress in the pathogenesis of type 2 diabetes is well documented (4, 5); however, there is a paucity of data concerning its role in GDM.

There is a large body of evidence demonstrating that reactive oxygen species (ROS) are involved in the regulation of important physiological functions (6). ROS have been implicated in the activation of a variety of transcription factors. Among the various transcription factors whose activity can be influenced by oxidative stress, nuclear factor-{kappa}B (NF-{kappa}B) is one of the most important and is recognized as central to regulating the expression of oxidative stress-responsive genes (7). NF-{kappa}B regulates the expression of multiple immune and inflammatory genes, including proinflammatory cytokines and the proteins and enzymes involved in ROS generation (8).

In addition to performing a physiological role in the fetoplacental unit during pregnancy, cytokines may also play a pathophysiological role if expressed in abnormal amounts or sites. TNF{alpha}, IL-6, and IL-8 are released from the placenta at term (9) and have been linked to various states of insulin resistance (10, 11). NF-{kappa}B is involved in the regulation of proinflammatory cytokines in gestational tissues, including the placenta (12). This regulation is at least partly mediated by oxidative stress. For example, the antioxidant sulfasalazine has been demonstrated to inhibit both lipopolysaccharide (LPS)-induced proinflammatory cytokine release from the placenta and NF-{kappa}B DNA-binding activity (12).

Previously we reported differences in the release of proinflammatory cytokines from placental and adipose tissue obtained from women with and without GDM in response to high-glucose concentrations (a putative oxidative challenge) (13). In this study, to further elucidate the mechanism(s) that mediate GDM-induced differences in placental response to oxidative stress, we tested the hypothesis that placental tissues obtained from women with GDM are less responsive to exogenous oxidative stress than tissues obtained from normal pregnant women. This hypothesis is based on evidence that GDM tissues have been preexposed and/or adapted to oxidative stress and thus may be better equipped to accommodate an oxidative challenge (e.g. increased intracellular antioxidant capacity), have a different capacity to release oxidant-induced effectors (e.g. proinflammatory cytokines), or may be a combination of both processes.

To test this hypothesis, an ex situ placental tissue explant incubation model was used in which tissues were incubated in the absence (control) and presence of the superoxide generating xanthine/xanthine oxidase (X/XO) system. The experimental end points assessed included the release (i.e. the 24-h accumulation of analyte in incubation medium) of immunoreactive IL-6, IL-8, TNF{alpha}, the arachidonic acid derivative 8-isoprostane (8-epi prostaglandin F2{alpha} or 15-F2t-IsoP), and NF-{kappa}B DNA-binding activity.


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

Placentae were obtained from a total of 32 pregnant women (16 healthy pregnant women and 16 with GDM) at the time of term cesarean section before the onset of labor. Indications for cesarean section included repeat cesarean section (n = 25) or breech presentation (n = 7). Women with any adverse underlying medical condition (i.e. asthma, arthritis, preeclampsia, pregestational diabetes, and macrovascular complications) were excluded; and all participants were nonsmokers. All pregnant women were screened for GDM, and women participating in the normal group had a negative screen. Women with GDM were diagnosed according to the current criteria of the Australasian Diabetes in Pregnancy Society by either a fasting venous plasma glucose level of 5.5 mmol/liter or more (99 mg/dl) glucose, and/or 8.0 mmol/liter or more (144 mg/dl) glucose 2 h after a 75-g oral glucose load at approximately 28 wk gestation (14). Nine women with GDM were clinically managed by diet alone, and seven women were prescribed insulin in addition to dietary management. The majority of the participants (22) were Caucasian, whereas a smaller number were from various ethnic groups including Asia (five), India (two), and the Mediterranean (two). One participant was of Indigenous Australian heritage. Approval for this study was obtained from the Mercy Hospital for Women’s Human Research and Ethics Committee, and informed consent was obtained from all participating subjects.

Tissue collection and explant culture

Placentae were collected from term elective cesarean sections performed before the onset of labor. Tissue processing commenced within 15 min of delivery and involved two rinses in PBS (37 C) (pH 7.4) and RPMI 1640 (pH 7.2) (37 C). Placental harvesting involved the removal of a cotyledon (placental lobule) from a 4-cm2 area from the central region of the placenta. The fetal membranes and chorionic surface were removed from the cotyledon along with the basal plate, and villous tissue was obtained from the middle cross-section. Placental villous tissue explants were obtained by blunt dissection and removal of visible connective tissue, vessels, and calcium deposits. Approximately 200 mg (wet weight per well) of placental tissue were placed into a 12-well tissue culture plate (Becton Dickinson Labware, Lincoln Park, NJ) with 2 ml of RPMI 1640 (pH 7.2) supplemented with 5 mmol/liter D-glucose. The tissue was incubated at 37 C in a humidified atmosphere of carbogen (95% O2/5% CO2) in a shaking water bath for 1 h. At 1 h the medium was replaced with RPMI 1640 supplemented with appropriate experimental treatments and incubated for 24 h. All incubations were performed in duplicate.

To induce oxidative stress, placental explants were exposed to 0.5 mmol/liter hypoxanthine (HX, Sigma, St. Louis, MO) and 15 mU/ml XO (grade III, from buttermilk, Sigma) in RPMI 1640 (pH 7.2). Several explants were performed in the presence of 10 µg/ml LPS (Sigma) to facilitate the production of proinflammatory mediators. To further explore the relationship between oxidative stress and placental cytokine release, various pharmacological agents were supplemented to the explant incubation medium, including 5 mmol/liter {alpha}-lipoic acid (DL-6, 8-thioctic acid) dissolved in 0.5% vol/vol NaOH and adjusted to pH 7.4 and 100 µmol/liter 2-methoxyestradiol (2-Me) dissolved in 0.3% vol/vol dimethylsulfoxide (DMSO) (Sigma); 100 µmol/liter troglitazone dissolved in 0.03% vol/vol DMSO (Sankyo Co. Ltd., Tokyo, Japan); 5 mmol/liter gliclazide dissolved in 0.4% vol/vol DMSO (Servier Laboratories, Neuilly, France); and 100 µmol/liter BAY 11–7082 dissolved in 0.08% DMSO (Biomol Research Laboratories, Plymouth Meeting, PA). The concentrations used were based on the results of previously performed dose-response inhibition experiments (data not shown). Concentrations chosen were IC80 or more. At the completion of the 24-h incubation period, the conditioned medium and tissue explants were frozen at –80 C until required for analysis, except for tissue from six placentae that was used for NF-{kappa}B DNA-binding activity, in which nuclear protein was extracted immediately as described below.

Total protein determination

For the measurement of total protein in the explants, placental tissue was prepared as described previously (13). Total protein content of the tissue was determined using the bicinchoninic acid method (Pierce, Rockford, IL) following the microwell plate protocol as described by the manufacturer. The absorbance was quantified using a 96-well microtiter plate reader at 595 nm (Bio-Rad Laboratories, Hercules, CA). The intra- and interassay coefficients of variation were 5.0 and 5.5% (over eight assays), respectively.

Tissue viability

To assess tissue viability during in vitro incubation, the release of the intracellular enzyme lactate dehydrogenase (LDH) into the incubation medium was determined as described previously (15). The absorbance was quantified using a 96-well microtiter plate reader at 340 nm (Bio-Rad Laboratories). LDH release was expressed as a percent of total tissue control, which was calculated as LDH activity in the medium divided by total tissue LDH activity multiplied by 100.

Cytokine ELISA

Quantification of TNF{alpha}, IL-6, and IL-8 in the conditioned explant incubation medium was performed by sandwich ELISA (Biosource International, Camarillo, CA). The TNF{alpha} assay used two monoclonal mouse IgG antibodies at a concentration of 1 and 0.4 µg/ml for the capture and detecting antibodies, respectively. The concentration of the IL-6 and IL-8 capture antibodies was 5 and 0.6 µg/ml, respectively. The detection antibodies were conjugated to peroxidase at an unspecified concentration and diluted according to the manufacturer’s instructions. Incubation medium samples were diluted with RPMI 1640 as necessary within a range of neat to 1:30 for the TNF{alpha} ELISA and from 1:50 to 1:1000 for the IL-6 and IL-8 ELISA, depending on the tissue sample and treatment. The absorbance was quantified using a 96-well microtiter plate reader at 450 nm (Bio-Rad Laboratories). The limit of detection of the assays (defined as 2 SDs from the zero standard) were 15 pg/ml for the TNF{alpha} ELISA, 40 pg/ml for the IL-6, and 13 pg/ml for the IL-8 ELISA. The intra- and interassay coefficients of variation were 4.4 and 11.4%, respectively, for the TNF{alpha} assay (over 20 assays), 2.7 and 10.7% for the IL-6 ELISA (over six assays), and 4.9 and 14.3% for the IL-8 ELISA (over six assays).

8-Isoprostane determination

8-Isoprostane is a member of the family of F2 isoprostanes and is produced by free radical-catalyzed peroxidation of arachidonic acid (16). Determination of 8-isoprostane accumulation in the explant incubation medium was performed using a competitive enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI) according to the manufacturer’s specifications. Samples were diluted with RPMI 1640 as necessary within a range of neat to 1:30, depending on the tissue sample and treatment. The absorbance was quantified using a 96-well microtiter plate reader at 405 nm (Bio-Rad Laboratories). The limit of detection of the assay was 5 pg/ml. The intra- and interassay coefficients of variation were 7.2 and 18.5%, respectively (over six assays).

Nuclear protein extraction

Immediately after the 24-h incubation, placental tissue (200 mg/wet weight) was homogenized using a metal blade homogenizer in 1.0 ml (5 vol, wt/vol) of ice-cold Tris-buffered saline (150 mmol/liter NaCl and 50 mmol/liter Tris). All subsequent steps were performed at 4 C. The homogenate was centrifuged for 10 min at 240 x g; the resulting tissue pellet was washed with 1 ml Tris-buffered saline and centrifuged at 11,000 x g for 15 sec. The pellet was suspended in 800 µl buffer A [10 mmol/liter HEPES (pH 7.8), 10 mmol/liter KCl, 2 mmol/liter MgCl2, 1.0 mmol/liter dithiothreitol (DTT), 0.1 mmol/liter EDTA, 0.1 mmol/liter 4-(2-aminoethyl)benzene sulfonyl fluoride (Pefabloc SC), 4 µg/ml leupeptin] and incubated for 15 min. Fifty microliters of a 10% (vol/vol) solution of nonionic detergent (Igepal, Sigma) was added, vortexed for 30 sec, and centrifuged for 15 sec at 11,000 x g. The pellet was resuspended in 50 µl buffer B [50 mmol/l HEPES (pH 7.8), 50 mmol/liter KCl, 300 mmol/liter NaCl, 1.0 mmol/liter DTT, 0.1 mmol/liter EDTA, 0.1 mmol/liter 4-(2-aminoethyl)benzene sulfonyl fluoride, 4 µg/ml leupeptin, 10% (vol/vol) glycerol], mixed for 20 min, and then centrifuged for 5 min at 11,000 x g. Supernatants containing nuclear proteins were collected, snap frozen in liquid nitrogen, and stored at –80 C. Protein quantitation was performed using the bicinchoninic acid protein assay (Pierce) as described above.

EMSA

NF-{kappa}B activity was determined by EMSA. The oligonucleotide used as a probe for EMSA was a 23-bp double-stranded construct (5'-AGT TGA GGG GAC TTT CCC AGG C-3') that was end labeled with [{gamma}-32P] ATP (3000 Ci/mmol at 10 mCi/ml, Amersham Pharmacia Biotech, Uppsala, Sweden) and T4 polynucleotide kinase, according to the manufacturer’s specifications (Promega gel shift assay systems, Promega, Madison, WI). The removal of unincorporated nucleotides from the DNA probe was performed using a commercially available kit (QIAquick nucleotide removal kit, Qiagen, Valencia, CA) in accordance with the manufacturer’s specifications. The DNA-binding reactions were prepared using 12 µg nuclear protein extract and 2 µl gel shift binding buffer [20% glycerol, 5 mmol/l MgCl2, 2.5 mmol/l EDTA, 2.5 mmol/l DTT, 250 mmol/l NaCl, 50 mmol/l Tris-HCl (pH 7.5), 0.25 mg/ml poly dI-dC]. After the addition of the 32P-labeled oligonucleotide probe, the reactions were incubated at room temperature for 20 min. Samples were subjected to electrophoresis through a 4% nondenaturing polyacrylamide gel with 0.5 x Tris/borate/EDTA buffer at a constant 130 V. Autoradiograms were developed by exposing vacuum-dried gels to x-ray film (X-OMAT AR, Kodak, Rochester, NY) at –80 C with intensifying screens for 25–48 h. Autoradiographic results were evaluated quantitatively using the Bio-Rad Molecular Imager FX Pro Plus with Quantity One analysis software (version 4.2, Bio-Rad Laboratories).

For competition studies, an excess of unlabeled NF-{kappa}B consensus oligonucleotide was prepared plus an excess of unrelated unlabeled oligonucleotide spanning the activator protein-2 (AP-2) binding site (5'-GAT CGA ACT GAC CGC CCG CGG CCC GT-3'). For studies verifying NF-{kappa}B binding activity by supershift analysis, 2 µg of anti-p50 antibody or anti-p65 antibody (Transcruz gel supershift reagent, Santa Cruz Biotechnology, Santa Cruz, CA) was added after completion of the binding reaction, and the incubation was continued for 60 min at 4 C. A positive control containing 2 µl HeLa nuclear cell extract and a negative control were also prepared.

Statistical analysis

Statistical computations were performed using commercially available statistics analysis software (Statgraphics Plus for Windows, version 3.1, Statistical Graphics Corp., Rockville, MD). Student’s t test was used to assess statistical significance between normally distributed data; otherwise, the nonparametric Mann-Whitney U (Wilcoxon) test was used. Where appropriate, Bartlett’s test was used to assess the homogeneity of variance of the data. In cases in which Bartlett’s test was significant (P < 0.05), data were transformed logarithmically, and homogeneity of variance was examined and subsequently analyzed by a one-way ANOVA, if confirmed. Comparisons between groups were performed using Newman Keuls multiple-range tests. Statistical significance was assumed when P < 0.05. Data are expressed as mean ± SEM unless stated otherwise.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Participants

Demographic data of all participants involved in the investigation are summarized in Table 1Go. Fasting and 2-h plasma glucose concentrations at oral glucose tolerance test were significantly greater (P < 0.001) in women with GDM, compared with healthy pregnant women. Although six women with GDM were obese [body mass index (BMI) ≥ 30 kg/m2], there was no statistically significant difference in BMI between the two groups of women, due largely to the large variance, with two women with GDM having a BMI greater than 50. For all measured experimental parameters, there was no significant difference in the release from placental explants in women with GDM who were managed by dietary modification alone, compared with women who were treated with insulin.


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  		TABLE 1. Characteristics of all participants involved in the study

 
Tissue viability and function

To determine the effect of experimental treatments on cell membrane integrity, the release of LDH into the incubation medium over 24 h was determined for all explant incubations. Experimental treatments did not significantly affect LDH release into the incubation medium, indicating that the working concentrations did not adversely affect cell viability (data not shown).

No statistical effect of any vehicle used in this study on the experimental end point determined was identified (data not shown).

The effect of an oxidative challenge on cytokine release

Control, oxidant (XO), and LPS-stimulated TNF{alpha}, IL-6, and IL-8 release into the incubation medium over 24 h is presented in Fig. 1Go, A–C. There was no difference in the release of TNF{alpha}, IL-6, and IL-8 from normal or GDM placentae under basal conditions. Placental explants that were exposed to an oxidative challenge (X/XO) released significantly more TNF{alpha}, compared with basal release in both normal (P < 0.0001, n = 11) and GDM placental explants (P < 0.004, n = 11, Fig. 1AGo). Tissues obtained from women with GDM, however, were approximately 3-fold less responsive to an oxidative challenge than tissue from normal women (P < 0.0002, n = 11). The oxidative challenge did not affect the release of either IL-6 or IL-8 from placental tissue. When placental tissue was exposed to a LPS challenge, there was no significant difference identified in cytokine release from tissue from both normal and GDM women. The concentration of cytokines post LPS treatment was, however, up to 40-fold more TNF{alpha} and up to 3-fold more IL-6 and IL-8, compared with untreated placental explants from both normal and GDM women (P < 0.001, n = 11 for both groups).



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  		FIG. 1. Effect of an exogenous oxidative and LPS challenge on TNF{alpha} (A), IL-6 (B), IL-8 (C), and 8-isoprostane (D) release from the placenta. Human term placental explants obtained from both normal (open bars) and GDM (closed bars) women were stimulated with 0.5 mmol/liter HX + 15 mU/ml XO or 10 µg/ml LPS for 24 h. Each bar represents the mean ± SEM, n = 11. *, P < 0.02 vs. control (basal) release from normal placentae; {dagger}, P < 0.004 vs. control release from GDM placentae; {ddagger}, P < 0.02 vs. X/XO-treated tissue from GDM; §, P < 0.02 vs. X/XO-treated tissue from the same participant group.

 
The effect of an oxidative challenge on 8-isoprostane release

Figure 1DGo illustrates the basal, oxidant, and LPS-stimulated 8-isoprostane release over 24 h from placental explants from both groups of participants. Unlike TNF{alpha}, IL-6, and IL-8, basal release of 8-isoprostane from the placentae of women with GDM was significantly greater (2-fold), compared with normal women (P < 0.0005, n = 11). The induction of oxidative stress significantly increased the release of 8-isoprostane from normal placental explants, compared with basal levels (P < 0.001, n = 11). Oxidative challenge did not alter the already elevated 8-isoprostane release from the placentae of women with GDM. Normal tissue (but not GDM) treated with LPS secreted more 8-isoprostane into the incubation medium, compared with basal (P < 0.0003, n = 11).

The effect of an antioxidant

The effect of {alpha}-lipoic acid (LA) on oxidant-induced TNF{alpha} and 8-isoprostane release from placental explants is illustrated in Fig. 2Go. Coincubation with the free radical scavenger LA resulted in total suppression of oxidant-induced TNF{alpha} release from the placentae of both normal pregnant women (P < 0.0001, n = 5) and women with GDM (P < 0.004, n = 5, Fig. 2AGo). Oxidant-induced 8-IP release from normal placentae was also significantly attenuated with the addition of LA (P < 0.001, n = 5, Fig. 2BGo). LA also significantly inhibited LPS-stimulated TNF{alpha} and 8-isoprostane release from both groups of placentae (P < 0.004, n = 5, data not shown).



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  		FIG. 2. The effect of LA on oxidant-induced TNF{alpha} (A) and 8-isoprostane release (B). Human term placental explants obtained from both normal (open bars) and GDM (closed bars) women were stimulated with 0.5 mmol/liter HX + 15 mU/ml XO with or without the presence of 5 mmol/liter LA for 24 h. Each bar represents the mean ± SEM (n = 5). *, P < 0.02 vs. control release from normal placentae; {dagger}, P < 0.004 vs. control release from GDM placentae; §, P < 0.004 vs. LA-treated tissue for the same participant group; {ddagger}, P < 0.03 vs. X/XO-treated tissue for GDM.

 
The effect of antidiabetic agents

The effect of gliclazide (GL) (Fig. 3Go, A and B) or troglitazone (TRO) (Fig. 3Go, C and D) on oxidant-induced TNF{alpha} and 8-isoprostane release from placental explants was investigated. Coincubation with either GL or TRO significantly inhibited oxidant-induced TNF{alpha} release (P < 0.02 for normal women and P < 0.003 for GDM, n = 3, Fig. 3Go, A and C). The addition of GL or TRO abolished the oxidant-stimulated 8-isoprostane release from normal placentae (P < 0.02, n = 3, Fig. 3Go, B and D) and GL inhibited 8-isoprostane release from in the women with GDM (P < 0.004, n = 3, Fig. 3BGo).



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  		FIG. 3. The effect of GL (A and B) and TRO (C and D) on oxidant-induced TNF{alpha} (A and C) and 8-isoprostane (B and D) release. Human term placental explants obtained from both normal (open bars) and GDM (closed bars) women were stimulated with 0.5 mmol/liter HX + 15 mU/ml XO with or without the presence of 5 mmol/liter GL or 100 µM TRO for 24 h. Each bar represents the mean ± SEM (n = 3). *, P < 0.05 vs. control release from normal placentae; {dagger}, P < 0.004 vs. control release from GDM placentae; §, P < 0.02 vs. TRO or GL-treated tissue for the same participant group; {ddagger}, P < 0.04 vs. X/XO-treated tissue for GDM.

 
The effect of an estrogen metabolite

The effect of 2-Me on oxidant-induced TNF{alpha} and 8-isoprostane release was determined (Fig. 4Go). In both groups of placentae, the addition of 2-Me to the explant incubation medium abolished TNF{alpha} release (P < 0.0003, n = 3, Fig. 4AGo). Coincubation with 2-Me inhibited placental 8-isoprostane release from both groups (P < 0.04, n = 3, Fig. 4BGo).



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  		FIG. 4. Effect of 2-Me on oxidant-induced TNF{alpha} (A) and 8-isoprostane (B) release. Human term placental explants obtained from both normal (open bars) and GDM (closed bars) women were stimulated with 0.5 mmol/liter HX + 15 mU/ml XO with or without the presence of 100 µM 2-Me for 24 h. Each bar represents the mean ± SEM (n = 3). *, P < 0.05 vs. control release from normal placentae; {dagger}, P < 0.04 vs. control release from GDM placentae; §, P < 0.04 vs. 2-Me-treated tissue for the same participant group; {ddagger}, P < 0.05 vs. X/XO-treated tissue for GDM.

 
The effect of an NF-{kappa}B inhibitor

The effect of BAY 11–7082 on oxidant-induced TNF{alpha} and 8-isoprostane release from placental explants was assessed (Fig. 5Go, A and B). Oxidant-induced TNF{alpha} release was abolished by the addition of BAY 11–7082 to the incubation medium in placental explants of both normal and GDM women (P < 0.0001, n = 5). X/XO-treated placental release of 8-isoprostane was inhibited by BAY 11–7082 (P < 0.0001, n = 5).



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  		FIG. 5. Effect of BAY 11–7082 on oxidant-induced TNF{alpha} (A) and 8-isoprostane (B) release. Human term placental explants obtained from both normal (open bars) and GDM (closed bars) women were stimulated with 0.5 mmol/liter HX + 15 mU/ml XO with or without the presence of 100 µM BAY 11–7082 for 24 h. Each bar represents the mean ± SEM (n = 5). *, P < 0.02 vs. control release from normal placentae; {dagger}, P < 0.0001 vs. control release from GDM placentae; §, P < 0.0001 vs. BAY 11–7082-treated tissue for the same participant group; {ddagger}, P < 0.04 vs. X/XO-treated tissue for the normal group.

 
NF-{kappa}B DNA-binding activity

To investigate the role of the NF-{kappa}B signaling pathway in oxidative stress-mediated TNF{alpha} release and confirm that treatment with the BAY 11–7082-inhibited NF-{kappa}B activity, placental NF-{kappa}B DNA-binding activity was examined in nuclear extracts prepared from the untreated explants (control), oxidant-stimulated explants, and those coincubated with BAY 11–7082. Quantitation of NF-{kappa}B DNA-binding activity was determined by densitometry and represented as percent of untreated control (Fig. 6AGo). Normal placental tissue exposed to oxidative stress demonstrated a significant increase in NF-{kappa}B DNA-binding activity, compared with untreated tissue (P < 0.002, n = 3, Fig. 6AGo), and treatment with BAY 11–7082 significantly suppressed NF-{kappa}B DNA-binding activity (P < 0.002, n = 3) to below control levels. In placenta from women with GDM, treatment with X/XO significantly suppressed NF-{kappa}B DNA-binding activity, compared with untreated tissue (P < 0.01, n = 3), and treatment with BAY 11–7082 inhibited NF-{kappa}B DNA-binding activity further (P < 0.01, n = 3). Under conditions of oxidative stress, placentae from women with GDM demonstrated a 2-fold reduction in NF-{kappa}B DNA-binding activity, compared with placental explants from normal women (P < 0.01, n = 3).



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  		FIG. 6. Placental explants from healthy pregnant women (n = 3) and women with GDM (n = 3) were incubated for 24 h with 0.5 mmol/liter HX and 15 mU/ml XO in the absence or presence of BAY 11–7082, and NF-{kappa}B binding activity was monitored in EMSA and quantified by densitometry. A, NF-{kappa}B DNA-binding activity was significantly inhibited by BAY 11–7082. Each bar represents the mean ± SEM (n = 3). *, P < 0.002 vs. untreated control for normal group; {dagger}, P < 0.01 vs. untreated control for GDM; §, P < 0.01 vs. BAY 11–7082-treated tissue for the same participant group; {ddagger}, P < 0.04 vs. X/XO-treated tissue for GDM. B, A representative NF-{kappa}B EMSA from a single placenta obtained from a healthy pregnant woman (left) and a woman with GDM (right). EMSA of placental nuclear proteins demonstrates minimal constitutive activation in untreated tissue cultured for 24 h (control). Treating normal placental tissue with X/XO for 24 h caused NF-{kappa}B activation (XO, left) that was inhibited by coincubating the tissue with 100 µM BAY 11–7082 (XO + BAY, left). Treating GDM placenta with X/XO for 24 h inhibited NF-{kappa}B DNA-binding activity (XO, right), whereas coincubation with 100 µM BAY 11–7082 (XO + BAY, right) inhibited DNA-binding activity still further. Characterization of the NF-{kappa}B subunits contributing to the observed shift was performed by adding 2 µg anti-NF-{kappa}B p-50 or anti-NF-{kappa}B p-65 antibodies into binding reactions with nuclear extracts resulting in an upward shift (supershift) of the band. The sequence specificity of the protein-DNA interaction was determined using a specific unlabeled competitor oligonucleotide for the NF-{kappa}B binding site (SC) and a nonspecific unlabeled competitor oligonucleotide for the AP-2 binding site (NSC). Lane 1 (positive) represents HeLa protein nuclear extract as a positive control, and lane 2 (negative) represents the radiolabeled oligonucleotide without nuclear extract.

 
Figure 6BGo depicts a representative NF-{kappa}B EMSA from a single placenta obtained from a healthy pregnant woman (left) and a woman with GDM (right). EMSA of placental nuclear proteins demonstrates minimal constitutive activation in untreated tissue cultured for 24 h (control). Treating normal placenta with X/XO for 24 h caused NF-{kappa}B activation (XO, left) that was markedly inhibited by coincubating the tissue with 100 µmol/liter BAY 11–7082 (XO + BAY, left). Treating GDM placenta with X/XO for 24 h inhibited NF-{kappa}B DNA-binding activity (XO, right), whereas coincubation with 100 µmol/liter BAY 11–7082 (XO + BAY, right) inhibited NF-{kappa}B DNA-binding activity still further. Characterization of the NF-{kappa}B subunits involved in the observed shift was performed by adding 2 µg anti-NF-{kappa}B p-50 or anti-NF-{kappa}B p-65 antibodies into binding reactions with nuclear extracts, resulting in an upward shift (supershift) of the band. The oxidant-induced DNA-binding complex was identified as the p50/p65 heterodimer. Previous studies in our laboratory determined that NF-{kappa}B p-50 and p-65 are the predominant heterodimers activated in the placenta (our unpublished observations). The sequence specificity of the protein-DNA interaction was confirmed by the loss of detectable binding with unlabeled (cold) competition with the NF-{kappa}B (specific competitor) but not AP-2 consensus oligonucleotides (nonspecific competitor). A positive control (positive) and a negative control (negative) were run in parallel.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
When challenged by oxidative stress, cells employ a repertoire of responses to restore intracellular redox status. The induction of glutathione and antioxidative enzymes represent primary responses to counter elevated concentrations of ROS. Previous studies established that the ability of cells to accommodate oxidative stress may be enhanced by preexposure or preconditioning to a mild oxidative challenge, thus inducing resistance to subsequent oxidative stress (17).

Chronic hyperglycemia associated with diabetes induces intracellular oxidative stress that is characterized by increased formation of ROS and reduced antioxidant capacity (18). In GDM, a mild form of glucose intolerance, evidence of oxidative stress has been documented (19, 20). In contrast to type 2 diabetes, however, antioxidative enzymes are induced in tissues from women with GDM (e.g. superoxide dismutase) (20). GDM thus may represent a mild oxidative challenge to which cells can accommodate by increasing antioxidant defenses. If this hypothesis is correct, then tissue obtained from women with GDM should display a resistance to subsequent oxidative challenge when compared with tissue obtained from normal pregnant women.

The data obtained in this study are consistent with increased in vivo oxidative stress in women with GDM when compared with normal pregnant women, as evidenced by a more than 2-fold increase in the basal release of 8-isoprostane from GDM placental tissue (8-isoprostane representing the best available biomarker of marker of lipid peroxidation in vivo and is also induced by hyperglycemia) (21, 22). Furthermore, placental tissue obtained from women with GDM is less responsive to subsequent exogenous oxidative stress than placental tissue obtained from normal pregnant women. When placental tissue from normal pregnant women was subjected to oxidative stress (i.e. the superoxide forming X/XO system), TNF{alpha} release increased by 20-fold and 8-isoprostane release increased by 2-fold. This is in marked contrast to the response of placental tissue from GDM women, in whom oxidative stress induced only a 4-fold increase in TNF{alpha} release and 8-isoprostane release was unaffected.

The blunted TNF{alpha} response to oxidative stress by GDM tissues may be a consequence of increased antioxidative capacity (e.g. superoxide dismutase activity) that limits activation of oxidant-responsive transcription factors that induce TNF{alpha} expression; decreased capacity to synthesize and release cytokines; reduced tissue viability; or a combination of these factors. The failure of oxidant stress to increase 8-isoprostane release from GDM tissue may be similarly a consequence of the increased antioxidative defenses, that arachidonic acid peroxidation is already maximal in these tissues, reduced tissue viability, or a combination of these processes.

Placental tissues from both GDM and normal women display similar LPS-induced cytokine release (i.e. TNF{alpha}, IL-6, and IL-8); thus, the possibility of impaired TNF{alpha}, IL-6, and IL-8 synthesis and release in GDM is negated. LPS stimulated the release of 8-isoprostane from normal placentae but not from GDM placentae. These data indicate that LPS represents an additional oxidative challenge (as evidenced by increased 8-isoprostane in normal tissues and inhibition by LA), but, either as a consequence of increased antioxidative capacity or saturation of arachidonic acid peroxidation, no further increase in 8-isoprostane release of GDM tissue could be achieved.

The possibility that the attenuated response of the placenta from women with GDM was due to loss of cell viability or function as a result of the oxidative challenge is not supported by the observation that there was no effect of the oxidative challenge on the release of lactate dehydrogenase, a marker of cell membrane integrity.

The adaptive response to oxidative stress observed in GDM placentae may represent a protective mechanism to limit the oxidant-induced production of proinflammatory effectors, e.g. TNF{alpha}. There is a large body of evidence indicating that TNF{alpha} may contribute to glucose intolerance because it correlates negatively with insulin sensitivity, and greater circulating TNF{alpha} concentrations are observed in both type 2 diabetes (23) and GDM (24). During late gestation, insulin resistance is significantly correlated with changes in circulating TNF{alpha}, the primary source of which appears to be the placenta (25). In addition, TNF{alpha} inhibits insulin receptor signaling (26, 27).

The current data are consistent with a recent study by Malek et al. (28), who exposed normal human term placental explants to the X/XO system and LPS and found the release of TNF{alpha} to be increased, with the stimulatory effect of LPS on TNF{alpha} significantly more pronounced than that of oxidative stress. Similarly, the absence of an oxidative effect on IL-6 release from the placenta was also verified. In the current study, both IL-6 and IL-8 release was not significantly affected by the oxidative challenge. The effect of oxidative stress on these cytokines, however, may be cell specific, e.g. treatment of myotubes with X/XO exhibited an increase in IL-6 release, whereas incubation of myoblasts and epithelial cells did not produce a response under the same experimental conditions (29).

To further elucidate the mechanisms by which exogenous oxidative challenge affects cellular responses, the effects of known antioxidants, free radical scavengers, and inhibitors of NF-{kappa}B were assessed. Two antidiabetic (antioxidants, free radical scavengers) agents were used: GL, a sulfonylurea, and TRO, a thiazolidinedione, in addition to a classical antioxidant, LA, and a newly characterized antioxidant, 2-Me. All agents demonstrated antioxidative capacity, inhibiting placental 8-isoprostane release and attenuating TNF{alpha} release from both normal and GDM placentae.

The antioxidant activity of both GL and TRO has been previously documented (30, 31). Furthermore, other antioxidants have been shown to elicit improvements in insulin sensitivity, namely LA (32). The suppression of placental TNF{alpha} release by both GL and TRO is consistent with earlier studies, which demonstrated inhibition of circulating TNF{alpha} concentrations in patients with type 2 diabetes (33, 34) and attenuation of TNF{alpha} synthesis and release by TRO from various cells and tissues, including the placenta (35). TRO is thought to mediate most of its effects by acting as a ligand for the nuclear receptor peroxisome proliferator activated receptor-{gamma} (reviewed in Ref.36), which is highly expressed in adipose tissue (37) and has also been identified in the placenta (38).

The antioxidant 2-Me is an endogenous metabolite of 17ß-estradiol, formed by the breakdown of 17ß-estradiol, which is metabolized to 2-hydroxyestradiol and 2-Me by catechol-O-methyltransferase. It is now established that some estrogens possess either or both pro- and antioxidant properties, and 2-Me has recently been confirmed as an antioxidant (39). The finding that 2-Me has antioxidant effects in the placenta is novel. Estradiol has been shown to inhibit placental macrophage- and trophoblast-mediated low-density lipoprotein oxidation and cytotoxicity (40).

The differential responsiveness of placenta from GDM and normal women to oxidative stress observed in this study may be mediated by the oxidant-responsive transcription factor NF-{kappa}B. An oxidative challenge stimulated the activation of NF-{kappa}B in placentae from healthy pregnant women. Gel shift and supershift analysis showed activation of both p50 and p65 subunits of NF-{kappa}B. Although the complex appears to have predominantly contained p50/p65, the possibility of involvement of other subunits that may contribute to the complex remains to be elucidated. Conversely, in placentae from women with GDM, the oxidative challenge attenuated the activation of NF-{kappa}B, resulting in a 2-fold difference in NF-{kappa}B activity between normal and GDM placentae. The specific NF-{kappa}B inhibitor BAY 11–7082 suppressed TNF{alpha} and 8-isoprostane release in both normal and GDM tissues and significantly attenuated the oxidant-induced NF-{kappa}B DNA-binding activity in nuclear extracts prepared from placentae of normal pregnant women. BAY 11–7082 selectively and irreversibly inhibits the TNF{alpha} inducible phosphorylation of inhibitory {kappa}B{alpha}, which prevents proteasome-mediated inhibitory {kappa}B{alpha} degradation and in turn translocation of free NF-{kappa}B to the nucleus (41), thereby inhibiting NF-{kappa}B DNA-binding activity. Activation of the NF-{kappa}B pathway appears to be required for oxidative stress-mediated TNF{alpha} release in normal placentae.

It is of interest to note that suppression of NF-{kappa}B DNA-binding activity was associated with a reduction in 8-isoprostane release. This may indicate a phospholipase A2 (PLA2) requirement for the release of 8-isoprostane. Secretory PLA2, an NF-{kappa}B-inducible gene product, is abundant in human placenta (42). Suppression of its activity may limit the liberation of arachidonic acid (both native and peroxidated forms) from the cell membrane. Previous studies demonstrated that inhibition of NF-{kappa}B DNA-binding activity suppresses PLA2 expression (43).

In conclusion, this study establishes that placentae from women with GDM display an attenuated response to an in vitro oxidative challenge with respect to the release of the proinflammatory cytokine, TNF{alpha}, and a marker of lipid peroxidation, 8-isoprostane. In addition, we have demonstrated redox regulation of NF-{kappa}B in the placenta, which is reduced in placentae from women with GDM, suggesting that GDM placentae may be preconditioned by transient intracellular oxidative stress, which attenuates responsiveness to a further oxidative insult. Further studies evaluating end points upstream of NF-{kappa}B in relation to an oxidative challenge need to be investigated to further elucidate this mechanism.


   Acknowledgments
 
The authors gratefully acknowledge Servier Laboratories for supplying the GL and the assistance of clinical research nurses Lyn Tuttle, Angie Denning, Valerie Briant, Joanna McKay, and Melissa Ryan and the obstetric and midwifery staff at the Mercy Hospital for Women for their cooperation. This manuscript is dedicated to the memory of friend and colleague Lyn Tuttle in recognition of her unfailing achievements in midwifery and research.


   Footnotes
 
This work was supported by the Medical Research Foundation for Women and Babies, Australia, and the National Health and Medical Research Council of Australia (G.E.R.).

Abbreviations: AP-2, Activator protein-2; BMI, body mass index; DMSO, dimethylsulfoxide; DTT, dithiothreitol; GDM, gestational diabetes mellitus; GL, gliclazide; HX, hypoxanthine; LA, {alpha}-lipoic acid; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; 2-Me, 2-methoxyestradiol; NF-{kappa}B, nuclear factor-{kappa}B; PLA2, phospholipase A2; ROS, reactive oxygen species; TRO, troglitazone; X, xanthine; XO, xanthine oxidase.

Received November 11, 2003.

Accepted April 5, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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