ORIGINAL ARTICLE Insulin stimulation of Akt/PKB phosphorylation in the placenta of preeclampsia patients Estimulação com insulina da fosforilação da Akt/PKB em placenta de pacientes com pré-eclâmpsia Gustavo Dias FerreiraI, Rafael Bueno OrcyII, Sérgio Hofmeister Martins-CostaIII, José Geraldo Lopes RamosIV, Ilma Simoni BrumV, Helena von Eye CorletaVI, Edison CappVII Master’s degree program in Biological Sciences (Physiology), Molecular, Endocrine and Tumor Biology Laboratory and Department of Gynecology and Obstetrics, Universidade Federal do Rio Grande do Sul (UFRGS), and Gynecology and Molecular Obstetrics Laboratory, Gynecology and Obstetrics Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil I MSc, Molecular, Endocrine and Tumor Biology Laboratory, Universidade Federal do Rio Grande do Sul (UFRGS), and PhD Student in Gynecology and Molecular Obstetrics Laboratory, Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil. II PhD. Physiologist, Molecular, Endocrine and Tumor Biology Laboratory, Universidade Federal do Rio Grande do Sul (UFRGS), and Researcher in Gynecology and Molecular Obstetrics Laboratory, Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil. III MD, PhD. Adjunct Professor, Gynecology and Obstetrics Service, Hospital de Clínicas de Porto Alegre, and Department of Gynecology and Obstetrics, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil. IV MD, PhD. Associate Professor, Gynecology and Obstetrics Service, Hospital de Clínicas de Porto Alegre, and Department of Gynecology and Obstetrics, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil. V MD, PhD. Associate Professor, Department of Physiology, Molecular, Endocrine and Tumor Biology Laboratory, Universidade Federal do Rio Grande do Sul (UFRGS), Gynecology and Molecular Obstetrics Laboratory, Research Center, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil. VI MD. Associate Professor, Gynecology and Obstetrics Service, Hospital de Clínicas de Porto Alegre, and Department of Gynecology and Obstetrics, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil. VII MD, PhD. Associate Professor, Department of Gynecology and Obstetrics, Universidade Federal do Rio Grande do Sul (UFRGS), and Coordinator of Master’s degree program in Biological Sciences (Physiology), Molecular, Endocrine and Tumor Biology Laboratory, UFRGS, Porto Alegre, Rio Grande do Sul, Brazil. KEY WORDS: Pre-eclampsia. Placenta. Insulin resistance. Receptor, insulin. Proto-oncogene proteins c-akt. ABSTRACT CONTEXT AND OBJECTIVE: Preeclampsia is a multi-systemic disease and one of the most frequent severe health problems during pregnancy. Binding of insulin triggers phosphorylation and activates cytoplasmic substrates such as phosphatidylinositol 3 kinase (PI3K). Phosphorylation of membrane phosphoinositide 2 (PIP2) to phosphoinositide 3 (PIP3) by PI3K starts Akt/PKB activation. Defects in phosphorylation of the insulin receptor and its substrates have an important role in insulin resistance. Studies have shown that insulin resistance is associated with preeclampsia and its pathophysiology. The aim here was to investigate insulin stimulation of the Akt/PKB pathway in the placenta, in normal and preeclampsia parturients. DESIGN AND SETTING: Cross-sectional study in a tertiary public university hospital. METHODS: Placentas were collected from 12 normal and 12 preeclampsia patients. These were stimulated and analyzed using Western blot to quantify the Akt/PKB phosphorylation. RESULTS: The insulin stimulation was conirmed through comparing the stimulated group (1.14 ± 0.10) with the non-stimulated group (0.91 ± 0.08; P < 0.001). The phosphorylation of Akt/PKB did not differ between the placenta of the normal patients (1.26 ± 0.16) and those of the preeclampsia patients (1.01 ± 0.11; P = 0.237). CONCLUSIONS: In vitro insulin stimulation of the human placenta has been well established. There was no diference in Akt/PKB phosphorylation, after stimulation with insulin, between placentas of normal and preeclampsia patients. Nevertheless, it cannot be ruled out that the Akt/PKB signaling pathway may have a role in the pathophysiology of preeclampsia, since the substrates of Akt/PKB still need to be investigated. RESUMO CONTEXTO E OBJETIVO: Pré-eclâmpsia (PE) é uma doença multissistêmica das mais frequentes e graves durante a gestação. A ligação da insulina inicia a fosforilação e ativação de substratos citoplasmáticos, tais como fosfatidil-inositol 3 quinase (PI3K). A fosforilação do fosfoinositol 2 (PIP2) da membrana em fosfoinosiltol 3 (PIP3) pela PI3K inicia a ativação da Akt/PKB. Defeitos na fosforilação do receptor de insulina e seus substratos têm papel importante na resistência à insulina. Estudos demonstraram que resistência à insulina está associada com pré-eclâmpsia e sua patoisiologia. O objetivo foi investigar a via de estimulação com insulina da Akt/PKB em placenta de parturientes normais e com pré-eclampsia. TIPO DE ESTUDO E LOCAL: Estudo do tipo transversal em um hospital universitário público de nível terciário. MÉTODOS: Vinte e quatro placentas (12 normais, 12 com PE) foram coletadas, estimuladas e analisadas por Western blot para quantiicar a fosforilação da Akt/PKB. RESULTADOS: A estimulação com insulina foi conirmada comparando os grupos estimulados (1,14 ± 0,10) e não estimulados (0.91 ± 0.08; P < 0.001). A fosforilação de Akt/PKB não foi diferente na placenta de pacientes normais (1,26 ± 0,16) e com PE (1,01 ± 0,11; P = 0,237). CONCLUSÕES: A estimulação in vitro da placenta humana com insulina foi bem estabelecida. Não houve diferença na fosforilação da Akt/PKB após estimulação em placentas de pacientes normais e PE. Contudo, não é possível descartar a participação desta via de sinalização na patoisiologia da PE, uma vez que os substratos da Akt/PKB ainda precisam ser investigados. PALAVRAS-CHAVE: Pré-eclâmpsia. Placenta. Resistência à insulina. Receptor de insulina. Proteínas proto-oncogênicas c-akt. Sao Paulo Med J. 2011; 129(6):387-91 387 ORIGINAL ARTICLE | Ferreira GD, Orcy RB, Martins-Costa SH, Ramos JGL, Brum IS, Corleta HVE, Capp E INTRODUCTION Preeclampsia is a multi-systemic disease and is considered to be one of the most signiicant health problems in pregnancy. It occurs mainly in nulliparous women, particularly ater the 20th week of gestation, and most frequently near delivery.1 It is diagnosed when the patient presents gestational hypertension associated with proteinuria, vasoconstriction of the maternal vascular bed and, consequently, increased vascular resistance.2-4 he incidence ranges from 2-5% to more than 10% of pregnancies in developing countries, where prenatal care is still inadequate.1 It can afect both maternal and fetal health,5 leading to fetal growth restriction, prematurity and, in severe cases, maternal and perinatal death.6-8 Studies have shown that insulin resistance is associated with preeclampsia and contributes towards its pathophysiology.9 he insulin receptor belongs to the family of tyrosine kinases (RTKs). he binding of insulin triggers phosphorylation and activates cytoplasmic substrates such as phosphatidylinositol 3 kinase (PI3K). Phosphorylation of membrane phosphoinositide 2 (PIP2) to phosphoinositide 3 (PIP3) by PI3K starts Akt/PKB activation.10,11 Defects in phosphorylation of the insulin receptor and its substrates, and non-activation of PI3K-Akt/PKB has an important role in developing insulin resistance.10,12 If not activated, the Akt/ PKB pathway will also not phosphorylate its substrates, which participate in various cell functions, such as control of metabolism, survival, glucose uptake, proliferation, growth and angiogenesis.13 OBJECTIVE he aims of this study were to achieve in vitro insulin stimulation of the human placenta and to investigate the expression of the protein Akt/PKB in the baseline state and ater stimulation, in the placentas of normal and preeclampsia patients. METHODS A cross-sectional study with control group was performed. Twenty-four women participated, including 12 patients who presented a medical diagnosis of preeclampsia and 12 normotensive pregnant women (control group). he samples were divided into four groups: stimulated controls, non-stimulated controls, stimulated preeclampsia patients and non-stimulated preeclampsia patients. Information such as age, use of hormonal medication, family history of diabetes mellitus, gynecological and obstetric history, fasting glucose, blood pressure, proteinuria, glucose tolerance test of the mother and gestational age was gathered before the birth. his study was submitted to and approved by the Research Ethics Committee of the Research and Postgraduate Program Group of Hospital de Clínicas de Porto Alegre (GPPG 08-124). Sample preparation Approximately 30 g of placenta were obtained immediately ater cesarean sections. he samples were washed with phosphate388 Sao Paulo Med J. 2011; 129(6):387-91 bufered saline (PBS) (4 °C) to remove excess blood and were taken to the laboratory for preparation and stimulation with insulin. he preparation of the placenta was performed in accordance with Klein et al.,14 with modiications. he tissue was weighed in the laboratory, separated from blood vessels and cut in slices. One gram of tissue was incubated in 5 ml of bovine serum albumin (BSA) bufer: 32 mM of HEPES, 195 mM of NaCl, 7.2 mM of KCl, 1.8 mM of KH2PO4, 8.3 mM of glucose and 1% albumin in distilled H2O. Briely, the placental tissue was digested with collagenase type I (Gibco, Invitrogen Corporation) and incubated at 37 °C under orbital stirring (100 rpm) for 45 minutes. Ater this, a 250 mm ilter was used to separate out and remove large particles and remaining ibrin. For cell separation, the sample was divided into two 50 ml tubes and centrifuged at 2000 x g for 10 minutes at 30 °C. Ater the supernatant had been discarded, a small portion of the sample was viewed under a microscope to observe the viability of the cells for stimulation. he cells were then homogenized with 0.5 ml of PBS. hese samples were transferred to two 1.5 ml tubes, which were incubated with 0.5 ml of a stimulation bufer containing 50 mM of Tris-HCl (pH 7.4), 0.01% BSA, 1 mM of ATP, 2 mM of MgCl2, 1 mM of EDTA, 5 mM of sodium pyrophosphate, 1 mM of sodium orthovanadate and 50 mM of sodium luoride, with and without insulin [10-7 M] for eight minutes at 37 °C.9 For analysis, placental pieces were pooled and homogenized. Protein concentrations were measured using Bradford’s method.15 he samples were analyzed using Western blot.16,17 Samples of 60 µg of protein (per lane) were loaded onto 10% acrylamide gel. he proteins were transferred to nitrocellulose membranes by means of a semidry system. Rabbit polyclonal antibodies (Santa Cruz, California, United States) were used: total anti-Akt (sc-8312) and phosphor-Akt (Ser473) (sc-7985). Statistical analysis Statistical analysis was performed by means of the Statistical Package for the Social Sciences (SPSS) 15.0 sotware. he data were tested using Student’s t test for paired samples and independent samples, for parametric variables, and were shown as means ± standard deviations (SD). he signiicance level was taken to be P < 0.05. RESULTS he patients with preeclampsia fulilled the diagnostic criteria, presenting proteinuria over 300 mg/dl and hypertension. In the normotensive group, there were no patients with proteinuria over 300 mg/dl. he normotensive and preeclampsia groups did not difer signiicantly in age (26.8 ± 4.1 versus 27.8 ± 8.2; P = 0.714), body mass index (BMI) (28.4 ± 3.5 versus 31.3 ± 4.1; Insulin stimulation of Akt/PKB phosphorylation in the placenta of preeclampsia patients | ORIGINAL ARTICLE P = 0.08) or number of pregnancies (2.5 ± 2.1 versus 1.8 ± 1.1; P = 0.303) (Table 1). he mean gestational age was approximately four weeks shorter in the preeclampsia group (35.6 ± 0.5 versus 39.1 ± 0.4; P < 0.001), while the fasting glucose levels (mg/dl) were signiicantly higher in the preeclampsia group (91.58 ± 2.83 versus 76.00 ± 2.39; P < 0.001). Diastolic and systolic blood pressures were signiicantly higher in the preeclampsia patients (P < 0.001) (Table 1). Table 1. Clinical characteristics of women with preeclampsia and control subjects Sample stimulation Sample stimulation was assessed by comparing the expression of phosphor-Akt (Ser473) in samples stimulated with insulin (+) and not stimulated (-), both in the control group and in the preeclampsia group. he values were normalized according to total anti-Akt protein expression (60 kDa) (Figure 1). In the control group (n = 12), the expression of phosphorAkt (Ser473) was 1.26 ± 0.16 for samples stimulated with insulin (+) and 0.93 ± 0.12 for samples not stimulated (-), with a signiicant diference of P < 0.001. In the PE group (n = 12), the expression was 1.01 ± 0.01 for samples stimulated with insulin and 0.89 ± 0.11 for samples not stimulated, with a signiicant difference of P = 0.001, thus conirming that the samples had been stimulated (Figure 2). PE = preeclampsia; BMI = body mass index. *Student’s t test. Age (years) BMI (kg/m²) Gestational age (weeks) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Number of pregnancies Fasting glucose (mg/dl) Normal 26.8 ± 4.1 28.4 ± 3.5 39.1 ± 0.4 118.5 ± 10 70.6 ± 6.4 2.5 ± 2.1 76.0 ± 2.3 PE 27.8 ± 8.2 31.3 ± 4.1 35.6 ± 0.5 154.8 ± 10 97.5 ± 5.9 1.8 ± 1.1 91.5 ± 2.8 P 0.714 0.080 < 0.001* < 0.001* < 0.001* 0.303 < 0.001* Figure 1. Representative Western blot analysis on placenta samples from patients with preeclampsia (PE) and normal controls (C): sample stimulated with insulin (+); sample not stimulated with insulin (-). Protein expression in the preeclampsia group and control group To compare the expression of Akt/PKB between the preeclampsia and control group, the analyses were performed in two states: baseline state (no stimulation with insulin) and stimulated state. In the baseline state, the expression of phosphor-Akt (Ser473) was 0.93 ± 0.13 in the control group (n = 12) and 0.89 ± 0.11 in the preeclampsia group (n = 12) (P = 0.82) (Figure 3A). Among the samples stimulated with insulin, the expression in the control group (n = 12) was 1.26 ± 0.16 and in the preeclampsia group (n = 12), it was 1.01 ± 0.11 (P = 0.23) (Figure 3B). here was no statistically signiicant diference between the preeclampsia group and the control group, either in the baseline state or in the stimulated state. DISCUSSION Preeclampsia occurs frequently during pregnancy, and it is a multisystem disease of unknown etiology.3,18 Insulin resistance and obesity have been deined as risk factors for the development of this disease.9,19,20 In the present study, age and BMI did not difer between the control and preeclampsia groups. Age seems to be a risk factor for preeclampsia, since women older than 40 years and younger than 20 years are at higher risk of preeclampsia. Being pregnant for the irst time, among young women, may also be a factor that increases the risk of preeclampsia among these patients.21 In the control group, six Figure 2. Expression of phosphor-Akt (Ser473) in stimulated and nonstimulated samples. In the control group (A), 0.93 ± 0.12 for the nonstimulated samples (-) and 1.26 ± 0.16 for the stimulated samples (+) (P < 0.001). In the preeclampsia group (B), 0.89 ± 0.11 for the non-stimulated samples (-) and 1.01 ± 0.11 for the stimulated samples (+) (P = 0.001). Figure 3. Expression of phosphorylation of Akt/PKB compared between the non-stimulated control group and preeclampsia group (A) (P = 0.828), and between the stimulated control group and preeclampsia group (B) (P = 0.237). Sao Paulo Med J. 2011; 129(6):387-91 389 ORIGINAL ARTICLE | Ferreira GD, Orcy RB, Martins-Costa SH, Ramos JGL, Brum IS, Corleta HVE, Capp E patients were overweight (BMI from 25 kg/m2 to 29.9 kg/m2) and four patients were obese (BMI ≥ 30.0 kg/m2), while in the preeclampsia group, ive patients were overweight and six were obese. Obesity is a major risk factor for preeclampsia, given that an increase in BMI of 7 kg/m2 doubles the risk of disease.22 Soonthornpun et al. demonstrated that even with no signiicant diference in BMI, women with preeclampsia had higher serum triglycerides and lower HDL levels than seen in the control group.23 Villa et al. investigated the fatty acid proile in women with preeclampsia and controls, and found that even without a diference in BMI, the preeclampsia group showed higher levels of oleic acid, linoleic acid and arachidonic acid, and increased insulin resistance.19 Preterm delivery (deined as gestational age < 37 weeks) is a common complication in preeclampsia patients (15-67%).3 As expected, in this study, the mean gestational age at delivery was lower in the preeclampsia group. However, this did not seem to inluence our results. Preeclampsia is one of the most important diseases during pregnancy, but its molecular mechanisms are not fully understood yet.24 Since the role of the placenta in the pathogenesis of preeclampsia is undisputed, and Akt/PBK action in insulin resistance is evident,25-28 we analyzed the expression of Akt/PKB at the baseline and with insulin stimulation, in placental tissue from preeclampsia and normal pregnancies. Ater insulin binding, the receptor is autophosphorylated, and it phosphorylates cytoplasmic substrates such as insulin receptor substrate 1 and PI3 kinase (PI3K). Ater phosphorylation, PI3K triggers phosphorylation of Akt/PKB at serine (ser473) and threonine (thr308) sites.29 Phosphorylated Akt/PKB translocates to the cytoplasm or to the nucleus, where it activates substrates with diferent actions on metabolism, growth and cell survival.13 Scioscia et al. showed that there was a decrease in tyrosine phosphorylation of IRS1, in insulin-stimulated preparations of human placenta from preeclampsia patients, compared with normal placenta, thus demonstrating that there is a lower level of insulin signaling in women with preeclampsia.9 In our study, sample stimulation was conirmed when the expression of phospho-Akt/PKB (p-Ser 473) was compared between insulin-stimulated and non-stimulated placenta cells. his signaling transduction pathway is an important milestone in the molecular mechanisms for insulin resistance syndrome in patients with preeclampsia.9 Data from Scoscia suggested that there was signiicantly higher serine phosphorylation of IRS1 and IRS2 in insulin-treated samples from preeclampsia patients. On the other hand, the insulin signal transduction signal was impaired in the controls,9 as also described in other insulin resistant states (diabetes type 2). Kunjara et al. demonstrated that accumulation of inositol phosphoglycan (P-IPG), a putative second insulin messenger, exerted several insulin-mimetic actions in placental tissue from preeclampsia patients and might be associated with insulin resistance.30 390 Sao Paulo Med J. 2011; 129(6):387-91 Orcy et al. previously studied the Akt/PKB expression pathway in the baseline state in the placenta, skeletal muscle and adipose tissue of preeclampsia patients. However, their indings did not show any signiicant diference in protein between the groups of patients.6 Similarly, our results also showed that there were no signiicant diferences in baseline protein expression of Akt/PKB between the placentas of preeclampsia patients and controls. Likewise, there was no diference in stimulated Akt/PKB phosphorylation. 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