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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-1965
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 2 709-717
Copyright © 2006 by The Endocrine Society

Insulin Resistance in Human Preeclamptic Placenta Is Mediated by Serine Phosphorylation of Insulin Receptor Substrate-1 and -2

Marco Scioscia1, Khalid Gumaa1, Sirilaksana Kunjara, Malcolm A. Paine, Luigi E. Selvaggi, Charles H. Rodeck and Thomas W. Rademacher

Department of Molecular Pathology (M.S., S.K., M.A.P., T.W.R.), Molecular Medicine Unit, Royal Free and University College Medical School, London W1N 8AA, United Kingdom; Department of Obstetrics and Gynecology (C.H.R.), Royal Free and University College Medical School, London WC1E 6HX, United Kingdom; College of Medicine and Medical Sciences (K.G.), Arabian Gulf University, Manama, Kingdom of Bahrain; and Department of Obstetrics and Gynaecology (L.E.S.), University of Bari, 70125 Bari, Italy

Address all correspondence and requests for reprints to: Dr. Marco Scioscia, Department of Obstetrics and Gynaecology, University of Bari, Policlinico di Bari, Piazza Giulio Cesare 11, 70125 Bari, Italy. E-mail: marco.scioscia{at}tin.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Preeclampsia is a severe complication of human pregnancy often associated with maternal risk factors. Insulin resistance represents a major risk for developing preeclampsia during pregnancy.

Objective: A putative second messenger of insulin, inositol phosphoglycan P type (P-IPG), was previously shown to be highly increased during active preeclampsia. Its association with insulin resistance was investigated.

Design and Setting: A cross-sectional study was carried out in a referral center.

Patients: Nine preeclamptic (PE) and 18 healthy women were recruited and matched for maternal age, body mass index, parity, and ethnicity in a 1:2 ratio. Placental specimens were collected immediately after delivery.

Intervention: Placental tissue was incubated with insulin and P-IPG production assessed. Insulin signaling proteins were subsequently studied by immunoblotting.

Results: P-IPG extracted from human term placentas upon incubation with insulin was found to be far lower in those with preeclampsia than controls (P < 0.001). Immunoblotting studies revealed serine phosphorylation of insulin receptor substrate-1 and -2 in PE placentas (P < 0.001) with downstream impairment of insulin signaling. The activation of the p85 regulatory subunit of phosphatidylinositol 3- kinase was markedly decreased in PE samples (P < 0.001).

Conclusions: These findings highlight the importance of P-IPG in active preeclampsia and demonstrate a substantially different response to the insulin stimulus of human PE placentas. Acquired alterations in activation of proteins involved in insulin signaling may play a role in the complex pathogenesis of preeclampsia, probably as a consequence of the immunological dysfunction that occurs in this syndrome. These results seem to confirm an insulin-resistant state in PE placenta and shed a different light on its role in the pathogenesis of this disease with potential therapeutic implications.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
PREECLAMPSIA IS A major complication of human pregnancy associated with a substantial maternal and perinatal morbidity and mortality (1). Its frequency ranges between 2 and 7% in healthy nulliparous women worldwide, and it increases in women with risk factors. Obesity and insulin resistance were found to be definite risks for preeclampsia, although the exact mechanism by which they are associated with the disorder is not completely understood (2).

The molecular mechanisms involved in development of insulin resistance during pregnancy and its correlation with preeclampsia are incompletely characterized (3). Recently we found a strong correlation between a putative second messenger of insulin, inositol phosphoglycan P type (P-IPG), and active preeclampsia (4). In several in vitro and in vivo studies, IPGs were shown to exert insulin-mimetic activity on glucose and lipid metabolism inducing the activation of pyruvate dehydrogenase phosphatase, glycogen synthase phosphatase, and glycerol-3-phosphate acyltransferase (5, 6). Moreover, antiinositolglycan antibodies were reported to block the in vitro insulin-induced stimulation of pyruvate dehydrogenase (PDH) in intact BC3H1 myocytes (7). It was hypothesized that the reported increased generation of P-IPG during preeclampsia was associated with insulin resistance. Thus, we investigated it in human placenta from preeclamptic (PE) patients, and its relationship with P-IPG as the placenta seems to play a central role in the pathogenesis of preeclampsia (8). A cross-sectional study assessing the placental response to insulin treatment was carried out on human placentas from PE women. No conclusive data were available in the literature confirming the efficiency or otherwise of the insulin signal in human placenta before term, so we studied term placental specimens only.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
This prospective study took place at Elizabeth Garrett Anderson Hospital-University College London Hospital (UCLH) in London with the approval of the Joint University College of London/UCLH Ethics Committee (no. 00/0030), and a written informed consent was obtained from each woman.

Antibodies and specialized reagents

Human recombinant insulin was purchased from Lilly (Humulin R, Lilly Co., Indianapolis, IN). The PDH complex and the PDH phosphatase (metal-dependent form) were prepared from beef heart as described by Lilley et al. (9). PDH and PDH phosphatase assays were carried out using a double-beam Jasco V-560 UV/VIS spectrophotometer (Jasco Ltd., Dunmow, Essex, UK).

Rabbit polyclonal antibodies to insulin receptor ß-subunit, insulin receptor/IGF-I receptor (phospho-Y1162 + Y1163), insulin receptor substrate (IRS)-1, IRS-1 (phospho-Y896), IRS-1 (phospho-S312), IRS-2, IRS-2 (phospho-S731), and phosphatidylinositol 3 kinase (PI3-K) catalytic {alpha}-subunit were purchased from Abcam Ltd. (Cambridge, UK). Polyclonal antibodies against caveolin-1 (N-20), IRS-1 (phospho-Y465), and PI3-K p85 {alpha} (phospho-Y508) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Patients and placental samples

Placental tissue was obtained immediately after delivery from PE and healthy women who delivered at term. Preeclampsia was diagnosed if there was hypertension (≥140/90 mm Hg) and proteinuria (≥300 mg per 24 h or ≥ 1+ on dipstick) that developed later than 20 wk of gestation (10). No features of any other complications (hemolysis, elevated liver enzymes, and low platelet count syndrome, intrauterine growth restriction, diabetes, thyroid disease) were present at the moment of recruitment. Two healthy pregnant women were recruited for each PE subject as controls within 2 wk from the recruitment of the PE patient. They were selected as the first patients delivering in the ward and meeting the following matching criteria: maternal age (± 3 yr), body mass index (± 3 kg/m2), parity, and ethnicity. Matching was accounted for recruitment only, not for the statistical analysis.

One piece of apparently normal tissue was taken from each of the four quadrants of the chorionic plate of each placenta to obtain about 20 g of sample, which was carefully washed with ice-cold PBS. Trophoblast microvillous membranes were then prepared following a previously described method (11) and assessed for content of caveolin-1 antigen. Caveolae are specialized membrane microdomains present in high numbers in insulin-target cells in which insulin receptors are localized (12), and the expression level of caveolin-1 directly correlates with the GLUT4 content in 3T3-L1 adipocytes (13). Components of the insulin signaling cascade appear to be enriched in caveolin-rich membrane fractions, compared with those from cells with caveolin-poor plasma membranes (14). The resulting sample was assayed for protein concentration using the micro BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL) with BSA as a standard.

Insulin incubation and P-IPG activity assay

Placental membranes (100 µg protein per milliliter) were incubated with human recombinant insulin (1 µU/ml) in 50 mM Tris-HCl buffer (pH 7.4) containing 0.01% BSA, 1 mM ATP, and 2 mM MgCl2 at 37 C for 5 min, and the reaction was stopped by cooling in ice for 5 min. The effect of varying concentrations of insulin on the generation of P-IPG from placental plasma membranes was plotted, and the effect of 1 µU/ml of insulin was chosen as the most reproducible.

The extraction procedure was carried out as described by Suzuki et al. (15), and P-IPG was determined using a specific bioassay procedure (16), namely the activation of PDH phosphatase by a spectrophotometric variant of the two-stage system of Lilley et al. (9). The assay was based on the activation of inactive phosphorylated PDH by P-IPG measured as the formation of nicotinamide adenine dinucleotide hydroxide (NADH), which was followed using an absorbance at 340 nm for 5 min. The formation of NADH was related to P-IPG content in a dose-dependent manner (16). Each sample was assessed blind in triplicate and the mean value plotted.

Accuracy of the bioassay

To validate the accuracy of the PDH phosphatase assay, the coefficient of variation (CV) was calculated as intra- and interassay variation. The internal validation procedure of the assay using P-IPG extracted from rat liver showed a linear-positive trend in releasing NADH (r = 0.97) with an overall CV of 6.9%. The intraassay variation was remarkably lower for P-IPG concentrations 10 U or more (CV 3.3 vs. 10.6%). The interassay CV for P-IPG was 4.2 ± 5.5% with an interoperator CV less than 9.8%. The average intraassay CV for this study was 9.0%.

Western blot analysis

Insulin signaling proteins were studied by immunoblotting in equal amounts of homogenized microvilli preparations (100 µg protein) from control and PE placentas. Samples were incubated with or without insulin (1 µU/ml) in a suspension buffer [50 mM Tris-HCl buffer (pH 7.4), 0.01% BSA, 1 mM ATP, 2 mM MgCl2, 1 mM EDTA, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 50 mM sodium fluoride] for 5 min at 37 C, and the reaction was stopped on ice for 10 min. Western blotting was carried out as previously described (11) with some modifications. Briefly, samples were extracted with Laemmli buffer (1:3) and heated for 5 min at 92 C. Five microliters of each sample were electrophoresed on a Phastgel gradient 8–25 (Amersham Biosciences AB, Uppsala, Sweden). The separated proteins were electrophoretically transferred for 1 h at 4 C to nitrocellulose membranes (Hybond-ECL-RPN 2020D, Amersham Pharmacia Biotech UK Ltd., Little Chalfont, UK) using a transfer buffer containing 39 mM glycine, 48 mM Tris base, 0.037% sodium dodecyl sulfate, and 20% (vol/vol) methanol. Nonspecific binding sites on membranes were blocked by immersion in a solution of 3% nonfat dry milk in PBS containing 0.1% Tween 20 for 1 h at room temperature on an orbital shaker. After washing with PBS containing 0.1% Tween 20, membranes were incubated for 1 h with the indicated antibodies at room temperature, washed, and incubated with the horseradish peroxidase-labeled antirabbit IgG secondary antibody. After washing, the proteins were visualized by enhanced chemiluminescence (ECL plus WB detection system, Amersham Bioscience, Little Chalfont, UK).

Statistical analysis

Power analysis demonstrated that a sample size of four women for each group, with an expected difference of placental content of P-IPG of 0.5 between PE and control specimens with SD within groups of 0.25, was necessary to provide a beta of 0.9 with an alpha set at 0.05. These expected values were based on published data (4).

Categorical variables were compared using the two-tailed {chi}2 test with Yates correction or Fisher’s exact test, as appropriate. Continuous variables with a normal distribution were assessed by unpaired t test and presented as mean ± SD. Mann-Whitney U test was carried out to assess nonparametric continuous variables of P-IPG activity. Normal distribution of raw data was confirmed using Kolmogorov-Smirnov test. Data with normal distribution are presented as mean ± SD and data with no Gaussian distribution as median ± SE.

Data were analyzed using STATISTICA data analysis software system (version 6.0, StatSoft, Inc., Tulsa, OK), and P < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
We recruited for this study 10 PE and 18 healthy (C) women. One PE patient was excluded from the analysis because she developed thyroiditis early in the puerperium. The characteristics of our study population (nine PE and 18 controls) are reported in Table 1Go. No significant differences were found between patients and controls apart from blood pressure and proteinuria according to the entry criteria.


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  		TABLE 1. Patient characteristics (mean ± SD)

 
Figure 1Go shows the P-IPG extracted from placental tissue expressed as nanomoles NADH per min per 100 µg placental protein. PE samples revealed no release of P-IPG (ratio with blank was nearly 1), whereas a 55% activation of PDH was found in controls (P < 0.001). P-IPG production by normal insulin-sensitive human placental membranes on insulin incubation was previously reported (15).


Figure 1
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  		FIG. 1. Generation of the insulin mediator, P-IPG, from human placental plasma membranes incubated with insulin (1 µU/ml per 100 µg placental protein). Here we report the formation of NADH after activation of PDH phosphatase expressed as nanomoles per minute per 100 µg, percentage of PDH activation, compared with the blank and total PDH activity in PE and C samples. Plotted values represent the mean of each sample assessed in triplicate in a single assay.

 
Typical representative results for immunoblotting are shown in the following figures. Placental plasma membranes, collected as a pellet by a previously described extraction procedure (11), revealed a high content of caveolin-1 antigen (data not shown). The expression of the insulin receptor and its signal-related proteins was similar in both groups, although impaired cascade activation was observed on incubation with insulin. Insulin exposure increased tyrosine phosphorylation of ß-IR in both C and PE membranes (Fig. 2BGo) but failed to induce similar tyrosine phosphorylation of IRS-1 in PE (Fig. 3BGo). In fact, slightly weaker bands of phosphorylated ß-IR and a decreased tyrosyl phosphorylation of IRS-1 at residues 465 and 896 were observed by immunoblotting in PE samples (Fig. 2Go, B and C, P = 0.05 and P < 0.001, respectively). To test whether the poor Tyr phosphorylation of IRS-1 was due to a serine/threonine kinase activation, Ser312 and Ser731 were assessed by immunoblotting in IRS-1 and IRS-2 (Figs. 3DGo and 4BGo), respectively. Strikingly, serine phosphorylation of both mediators was clearly demonstrated in insulin-treated PE samples, whereas it was barely detectable in controls (P < 0.001). A lower expression of the native form of IRS-2 was also reported (Fig. 4Go, P = 0.010), a second messenger not directly associated with the release of P-IPG after insulin stimulus.


Figure 2
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  		FIG. 2. The insulin-stimulated phosphorylation of IR in human placenta. Homogenized membranes from C and PE placentas were incubated with or without a physiological concentration of insulin (1 µU/ml) for 5 min at 37 C before being lysed in Laemmli sample buffer. Proteins were separated by electrophoresis on continuous polyacrylamide gradient (8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 ) gels and analyzed by immunoblotting with anti-IR-ß chain antibody. The results are representative of paired samples for the native protein (A) and the phosphorylated IR (B). Graphs on the right show the mean + SD of PE/C paired samples in a 1:2 ratio (six and 12 samples, respectively). *, P = 0.005.

 

Figure 3
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  		FIG. 3. The insulin-stimulated phosphorylation of IRS-1 in human placenta assessed by immunoblotting with the indicated antibodies. In B and C, we tested the tyrosyl phosphorylation of IRS-1 (residues 896 and 465), whereas in D is demonstrated the serine phosphorylation at residue 312. Data plotted as mean + SD (C, n = 12; PE, n = 6). *, P < 0.001.

 

Figure 4
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  		FIG. 4. IRS-2 phosphorylation at serine 731 after incubation with insulin in human placental membranes assessed by immunoblotting. Data reported as mean + SD (C, n = 12; PE, n = 6). *, P = 0.010; **, P = 0.001.

 
We next examined the activation of PI3-K (Fig. 5Go). Insulin treatment of extracted fractions resulted in tyrosine phosphorylation of the p85 regulatory subunit of PI3-K in normal placenta, whereas, interestingly, it was markedly decreased in PE samples (Fig. 3BGo, P < 0.001). These data suggest that serine phosphorylation of IRS-1 in PE placenta inhibits tyrosine phosphorylation and impairs the subsequent insulin-stimulated cascade.


Figure 5
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  		FIG. 5. Activation of PI3-K reported as tyrosyl phosphorylation of the {alpha}-subunit (p85). Data expressed as mean + SD (C, n = 12; PE, n = 6). *, P < 0.001.

 
Furthermore, immunoblot analysis showed a significantly higher degree of phosphorylation IR in PE samples not treated with insulin (Fig. 2BGo, P = 0.005) than in controls and a subsequent higher basal activation of IRS-1 (Fig. 3BGo, P < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Preeclampsia is a heterogeneous multisystem disorder of unknown cause (1). Several observations suggest the possibility that insulin resistance may be involved in its pathogenesis (2). Preeclampsia was reported to be associated with marked hyperinsulinemia in the fasting state, after oral glucose ingestion, and for 2 months after delivery (17, 18).

The complexity and specificity of insulin action is determined by an integrated signaling network of independent intracellular pathways with several downstream effectors that have been shown to modulate the signal amplitude and frequency for both short- and long-term effects (19). Inositol phosphoglycans (IPGs) have been demonstrated to exert partial insulin-mimetic activity being involved in glucose and lipid metabolism (20). A wide array of insulin effects in intact cells and cell-free assays, which can be induced directly or indirectly by IPGs, has been carefully reviewed by Varela-Nieto et al. (5). The activation of PI3-K and protein kinase B (Akt/PKB) and the inhibition of glycogen synthase kinase-3 represent the most important findings, given their pivotal role in glucose/lipid metabolism (6). We recently reported that the P-IPG represents a marker of preeclampsia because its content is far higher in the placenta (4), amniotic fluid, and maternal urine of PE women than matched controls (21). Moreover, under physiological conditions, the concentration of P-IPG in the amniotic fluid is higher than in urine samples from both healthy nonpregnant and pregnant women (22). Raised urinary P-IPG levels in pregnant compared with nonpregnant women may indicate a fetal/placental origin for these molecules. The accumulation of IPG in the fetal compartment (and subsequent overflow into maternal circulation) during preeclampsia may be a compensatory mechanism to the increased insulin resistance because these molecules mimic insulin action.

Here we report the first demonstration of a dichotomous behavior after insulin stimulation of normal and PE human placentas. The complete absence of P-IPG release from PE samples seems to confirm the presence of insulin resistance in placental tissue of PE patients. Based on our previous studies (4, 21), this finding was very surprising because we expected a higher production/release of P-IPG from PE placentas, compared with normal. A disturbed insulin signaling could explain, at least in part, the poor response to insulin stimuli leading to the no release of P-IPG. In fact, further investigations revealed an impaired insulin signal with low activation of IRS-1 (through multiple tyrosine phosphorylations) and evidence of phosphorylation of serine residues in both IRS-1 and IRS-2 (Fig. 6Go). This process, which is a common finding in insulin-resistant states and diabetes type 2 (23), can be induced by fatty acids, IGFs, cellular stress, or diverse inflammatory stimuli, including TNF{alpha}, IL-1ß, or lipopolysaccharide (24). Chronic treatment of cells with TNF{alpha} was reported to induce serine/threonine phosphorylation of IRS-1 and impair insulin action mainly through c-Jun N-terminal kinase (JNK) (25, 26). There is no consensus about the role of TNF{alpha} in the pathogenesis of preeclampsia (1), whereas a decreased phosphorylation of JNK was found in placental samples from preeclampsia and intrauterine growth restriction (27, 28). These findings seem to rule out TNF{alpha} from this scenario though another pathway [TNF{alpha}/PI3-K/Akt/mammalian target of rapamycin (mTOR)] has been proposed (26). Besides, insulin resistance in preeclampsia precedes the clinical onset of the disease and is not related to elevated levels of TNF{alpha} (29).


Figure 6
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  		FIG. 6. Basic signaling pathways of insulin are here schematically reported in a target cell. Insulin binds its receptor (IR), which is activated through a tyrosine autophosphorylation process (Y). Active IR induces the phosphorylation on tyrosine residues (Y) of the IRS-1 and -2, which serve as links to downstream signaling proteins (45 ). A complex network of signals leads to many metabolic effects. Serine/threonine phosphorylations (S) result in desensitization of insulin action, thus inducing insulin resistance (35 ). PHAS, Initiation factor 4E-binding protein; CAP, c-Cbl-associated protein; TCGAP, TC10/cdc42 GTPase activating protein; GSK, glycogen synthase kinase; SHP, Src homology 2 domain tyrosine phosphatase; MAP, mitogen-activated protein; SOS, exchange factor Son of sevenless; MEK, MAPK/ERK kinase.

 
Recently, some forkhead gene mutations, a family of transcription factors, were found in Dutch PE women (30). Missense mutations of STOX1 gene (also known as C10orf24), related to the activation of the PI3-K/Akt/forkhead pathway, were reported to be strongly associated with PE phenotype. Phosphorylation of these factors by Akt/PKB results in inhibition of transcription playing a critical role in regulation of proliferation, apoptosis, and control of oxidative stress mainly through immune cells (31). These findings highlight once more the key role of Akt/PKB in this disease.

An impairment of IRS-1 and PI3-K activity was found in rats after infusion of a lipid emulsion in a time-dependent manner through an activation of protein kinase C{theta} and I{kappa}B kinase, which phosphorylate IRS-1 at Ser307, decreasing tyrosine phosphorylation (32). Similar observations were reported in muscle biopsies from healthy human subjects infused with fatty acid (33).

Distinct kinases seem to converge at IRS-1-Ser307 (mTOR, I{kappa}B kinase, MAPK/ERK kinase, JNK-1, and protein kinase C{theta}) (34), whereas the inhibition of IRS-1 remains not completely clarified yet. In fact, a delicate balance between activated Tyr- and inactivated Ser-phosphorylation of IRS-1 has been pointed out over the past decade (35), and an important role seems to be played by insulin itself. After insulin stimulation IRS-1 is Tyr-phosphorylated. It is also phosphorylated on serine residues by PKB/Akt, which is activated downstream of PI3-K (24). Phosphorylation of Ser307 in response to insulin involves the mTOR signaling pathway in muscle, adipocytes, and hepatocytes (24, 36). This negative-feedback mechanism is used by insulin under physiological conditions to terminate its own action, whereas it can be activated chronically during chronic hyperinsulinemia (37). Moreover, the hyperinsulinemic state during preeclampsia (18) can probably justify the increased basal phosphorylation of IR found in PE samples.

The assessment of the pathways downstream of PI3-K was beyond the scope of this research. Further investigations are needed to elucidate the relative role of TNF{alpha} and PKB/Akt in inhibiting this pathway, given the number of publications reporting their interaction with insulin pathway (as previously argued).

The accumulation of P-IPG in human placenta during preeclampsia may be a counterregulatory mechanism to the local insulin resistance because these molecules mimic insulin action. Moreover, in a proportion of women, elevated values for the urinary ELISA correlated with the onset of clinical symptoms and diagnosis (Rademacher, T. W., unpublished observations). A subset of women had elevated ELISA values up to 40 d before clinical diagnosis of preeclampsia. All women with overt preeclampsia had positive urinary ELISA tests. Low concentrations of IPG were reported to trigger GLUT4 translocation to the plasma membrane promoting activation of glucose transport (6). Analysis of the relationship of structure and activity of IPGs revealed the pivotal role of the glycan core consisting of a backbone of mannose residues, glucosamine, and inositol moieties. On the other hand, chronic exposure to glucosamine was shown to decrease GLUT4 translocation (38) as well as high concentrations of synthetic IPG compounds can induce insulin resistance (Gumaa, K. A., unpublished observations).

Glycogen accumulation in villous syncytiotrophoblast was reported in PE placentas with a 16-fold higher activity of glycogen synthase, compared with healthy placental specimens (39). Insulin activates glycogen synthase inducing dephosphorylation at sites phosphorylated by glycogen synthase kinase-3, which is acutely inactivated by Akt (40). P-IPG was shown to enhance the dephosphorylation of glycogen synthase and pyruvate dehydrogenase (41).

Thus, there appear to be several distinct mechanisms to inhibit insulin signaling. Insulin resistance is a feature of normal pregnancy and can be enhanced by an inflammatory state like preeclampsia (8). Our findings confirm the presence of an impaired insulin signal mediated by serine phosphorylation. It may be a consequence of the immunological dysfunction that occurs in preeclampsia (1), which is temporized during sperm exposure and cohabitation (42), with acquired alterations in gene expression (30) that may play a role in the complex pathogenesis of this maternal syndrome. Correlation between the increase of P-IPG, preeclampsia, and impaired activation of immune cells was widely speculated in our recent article (21). On the other hand, the accumulation of P-IPG in placental tissue as a compensatory mechanism can induce an inhibitory effect flooding the insulin signal itself. As for the clinical relevance of these results, the pharmacological use of salicylates as inhibitory factors for serine/threonine kinases can improve glucose tolerance and perhaps reduce the risk of developing preeclampsia in women at risk (previous preeclampsia, intrauterine growth retardation). Aspirin was shown to protect IRS proteins from serine phosphorylation induced by several kinases (43). Controversial results were achieved in preventing preeclampsia with low-dose aspirin, although no homogeneous data were surveyed and, most important, different subject inclusion criteria were adopted (1). It was thought that aspirin action was on the imbalanced thromboxane A2 to prostacyclin ratio, and there are no available data on its effect on insulin signaling.

Additionally, the pathophysiology of preeclampsia developing before 34 wk gestation could differ from that developing at term, during labor, or postpartum (44). Unfortunately, we were forced to recruit patients at term because we failed to find enough data in the literature on the efficiency of the insulin signaling pathway preterm.

The number of patients enrolled may appear as a limitation of this study, whereas statistically we reached a beta value higher than 0.90. Moreover, the reproducibility of the bioassay was satisfactory, as mentioned above (CV < 10%), and the study was carried out in term placentas to ensure potentially working transduction of the insulin signal using a standardized tissue sampling procedure.

In conclusion, we have provided evidence of insulin resistance in PE human placentas. In addition, PI3-K, a pivotal signaling mediator, is not properly activated on insulin stimulation, and P-IPG may represent the key of this impaired response, either as cause or effect.


   Footnotes
 
First Published Online December 6, 2005

1 M.S. and K.G. contributed equally to this work. Back

Abbreviations: Akt, Activation of PI3-K; C, healthy control; CV, coefficient of variation; IPG, inositol phosphoglycan; IR, insulin receptor; ß-IR, ß-chains of the insulin receptor; IRS, insulin receptor substrate; JNK, c-Jun N-terminal kinase; mTOR, mammalian target of rapamycin; NADH, nicotinamide adenine dinucleotide hydroxide; PDH, pyruvate dehydrogenase; PE, preeclamptic; PI3-K, phosphatidylinositol 3-kinase; P-IPG, inositol phosphoglycan P type; PKB, protein kinase B.

Received September 1, 2005.

Accepted November 28, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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