Abstract
Appropriate foetal growth and development is dependent on adequate placental glucose uptake. Oxidative stress regulates glucose uptake in various tissues. The effect of oxidative stress on placental glucose transport is not known. Thus, the aim of this study was to determine the effect of oxidative stress on glucose uptake and glucose transporters (GLUTs) in human placenta. Human placenta was incubated in the absence or presence of 0.5 mM hypoxanthine+15 mU/ml xanthine oxidase (HX/XO) for 24 h. Gene and protein expressions of the GLUTs were analysed by quantitative RT-PCR and western blotting respectively. Glucose uptake was measured using radiolabelled (14C) glucose. HX/XO significantly decreased GLUT1 gene and protein expression and resultant glucose uptake. There was no effect of the antioxidants N-acetylcysteine, catalase and superoxide dismutase or the NF-κB inhibitor BAY 11-0782 on HX/XO-induced decrease in glucose uptake. However, HX/XO treatment significantly decreased both gene and protein expression of SIRT1. In the presence of the SIRT1 activator resveratrol, the decrease in GLUT1 expression and glucose uptake mediated by HX/XO was abolished. Collectively, the data presented here demonstrate that oxidative stress reduces placental glucose uptake and GLUT1 expression by a SIRT1-dependent mechanism.
Introduction
Placental glucose uptake and transport is paramount for foetal growth and development (Hay 2006, Jansson & Powell 2007). In the absence of significant foetal gluconeogenesis (Kalhan & Parimi 2000), for appropriate growth and development the foetus entirely depends on glucose from maternal circulation. To accommodate this, maternal metabolism shifts into an insulin-resistant state, resulting in decreased glucose uptake by insulin target tissues of the mother. This increase in extracellular glucose levels enables placental uptake and transfer of glucose to the foetus, mediated by glucose transporters (GLUTs; Hauguel-de Mouzon et al. 1997, Xing et al. 1998, Illsley 2000, Colomiere et al. 2010). Glucose uptake and transport by the endothelial, epithelial and trophoblast cells of the placenta are mediated by GLUTs, with GLUT1 and -3 being the most abundant in human term placenta (Hauguel-de Mouzon et al. 1997, Xing et al. 1998, Illsley 2000, Colomiere et al. 2010). Their respective cellular localisations suggest that GLUT1 is responsible for supplying glucose for use as a placental fuel and that GLUT3 is important for glucose transfer to the foetus (Hauguel-de Mouzon et al. 1997, Illsley 2000). Certainly, a number of significant pregnancy complications, including gestational diabetes mellitus (GDM) and pre-eclampsia (Hubel 1999, Myatt & Cui 2004, Siddiqui et al. 2010), are associated with altered placental glucose metabolism (Osmond et al. 2001, Jansson & Powell 2006). However, the molecular mechanisms of human placental glucose transport and its regulation remain to be fully elucidated.
The mechanisms that regulate placental glucose uptake are largely unknown, yet they are fundamental to understand how the placenta directly affects foetal growth and development. In non-gestational tissues, oxidative stress impairs glucose uptake, though the effects of oxidative stress on glucose uptake are not consistent. For example, reactive oxygen species (ROS) increases glucose transport in skeletal muscle (Kozlovsky et al. 1997a,b, Toyoda et al. 2004, Higaki et al. 2008) and M07 human leukaemic megakaryocytic cell lines (Fiorentini et al. 2001). On the other hand, in 3T3-L1 adipocytes, induction of oxidative stress inhibits insulin-stimulated glucose transport (Tirosh et al. 1999, Regazzetti et al. 2009).
In pregnancies complicated by diabetes (both pre-existing and GDM), there is an overproduction of free radicals, placental oxidation reactions are accelerated and the radical scavenger function mechanisms are impaired (Coughlan et al. 2004, Lappas et al. 2010, 2011a). Similarly, in pre-eclampsia, the placenta also displays evidence of excessive oxidative stress (Hubel 1999, Myatt & Cui 2004, Siddiqui et al. 2010). GDM and pre-eclampsia are two complications of pregnancy that are associated with placental insulin resistance (Scioscia et al. 2006, Colomiere et al. 2010). It is not known what effects oxidative stress has on glucose transport in human placenta. Thus, the aim of this study was to determine the effect of oxidative stress on glucose transport in human placenta. Hypoxanthine (HX) and xanthine oxidase (XO) were used to induce ROS as we have previously described (Coughlan et al. 2004, Lappas et al. 2010). Using this system, we have previously demonstrated an increase in placental release of 8-isoprostane, a marker of oxidative stress. Of note, the level of 8-isoprostane produced by HX/XO was similar to the release of 8-isoprostane produced by the GDM placenta (Coughlan et al. 2004, Lappas et al. 2010).
Materials and Methods
Tissue collection
Approval for this study was obtained from the Mercy Hospital for Women's Research and Ethics Committee, and informed consent was obtained from all participating subjects. Human placenta was obtained from normal glucose-tolerant women at the time of term (37–40 weeks of gestation) Caesarean section before the onset of labour. Indications for Caesarean section included repeat Caesarean section or breech presentation. Women with any adverse underlying medical condition (i.e. asthma, diabetes, pre-eclampsia and intrauterine growth restriction) were excluded.
Placental explant incubation
Placental lobules (cotyledons) were obtained from various locations of the placenta; the basal plate and chorionic surface were removed from the cotyledon, and villous tissue was obtained from the middle cross-section. Placental tissues were blunt dissected to remove visible connective tissue and calcium deposits. The dissected tissues were placed in DMEM (containing 5 mM glucose, 100 U/ml penicillin G and 100 μg/ml streptomycin) at 37 °C in a humidified atmosphere of 5% CO2 and 8% O2 for a pre-incubation period of 1 h. Explants were blotted dry on sterile filter paper and transferred to 24-well tissue culture plates (50 mg wet weight per well). The tissues were then incubated in 1 ml DMEM with and without treatments for 24 h as detailed below. Each treatment was performed in duplicate. Initial explants were incubated in the absence or presence of 0.5 mM HX and 15 mU/ml XO (HX/XO; n=7); 0.25, 0.5 and 1 mM H2O2 (n=4); and 0.5 mM HX (n=4). The concentration of HX/XO used in this study was based on our previously published studies (Coughlan et al. 2004, Lappas et al. 2010). Additional explants were pre-treated with 300 U/ml polyethylene glycol (PEG)–superoxide dismutase (SOD), 1000 U/ml PEG-catalase, 5 mM N-acetylcysteine (NAC), 100 mM thiourea, 100 mM mannitol, 1% DMSO, 50 μM BAY 11-7082, 50 μM resveratrol and 10 μM SRT1720 for 30 min before incubation with HX/XO (n=3–6 per treatment). HX, XO, PEG–SOD, PEG-catalase, NAC, thiourea, mannitol, DMSO and resveratrol were purchased from Sigma. After the 24 h incubation, medium and tissue were collected separately and stored at −80 °C until assayed as detailed below.
Oxidative stress assays
For the measurement of hydrogen peroxide in the incubation buffer in the presence or absence of HX/XO, a colorimetric hydrogen peroxide assay kit was used (BioVision, Inc., Mountain View, CA, USA). The calculated coefficients of variation were all <10%.
Western blotting
Assessment of tissue protein expression was analysed by western blotting as described previously (Colomiere et al. 2009, 2010). Rabbit polyclonal anti-IR-β (0.4 μg/ml), rabbit polyclonal anti-IRS1 (0.8 μg/ml), rabbit polyclonal anti-GLUT1 (0.5 μg/ml) and mouse monoclonal anti-GLUT3 (0.5 μg/ml) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were viewed and analysed using the Chemi-Doc system (Bio-Rad). Quantitative analysis of the relative density of the bands in western blots was performed using Quantity One 4.2.1 image analysis software (Bio-Rad). Data were corrected for background and then normalised to β-actin expression.
RNA extraction and RT-PCR
Total RNA was extracted from ∼100 mg tissue using Tri Reagent according to the manufacturer's instructions (Sigma–Aldrich). RNA concentrations were quantified using a spectrophotometer (Smart Spec; Bio-Rad). RNA quality was determined via the A260/A280 ratio. RNA (1 μg) was converted to cDNA using the SuperScript VILO cDNA synthesis kit (Invitrogen) according to the manufacturer's instructions. The cDNA was diluted tenfold, and 2 μl cDNA was used to perform RT-PCR using Sensimix Plus SYBR green (Quantace, Alexandria, NSW, Australia) and 100 nM QuantiTect Primer Assays (Qiagen). Pre-validated primers for IR-β (QT00082810), IRS1 (QT00074144), PI3K p85α (QT01005984), GLUT1 (QT00068957), GLUT3 (QT00047124), GLUT4 (QT00097902) and SIRT1 (QT00051261) were purchased from Qiagen. The syncytin primer has previously been described (Paiva et al. 2011). Average gene CT values were normalised to the average GAPDH values of the same cDNA sample. Of note, there was no effect of experimental treatment on GAPDH gene expression. There was equal efficiency of PCR amplification of target and reference mRNA (average between 90 and 110%). The specificity of the product was assessed from melting curve analysis. RNA without reverse transcriptase during cDNA synthesis as well as PCR reactions using water instead of template showed no amplification. Gene expression levels were determined using the comparative threshold cycle (Ct) method.
Glucose uptake
Placental explants were performed as detailed above. After 24 h incubation with treatment, tissues were pre-incubated in the absence or presence of 20 μM cytochalasin B (Sigma) in Krebs buffer for 5 min. 2-doxy-d-glucose (2DG) uptake was measured by adding 3 μCi/ml [14C]-2DG (Perkin Elmer) and 1 mM 2DG to Krebs buffer containing 0.1% BSA (fatty acid free). Glucose transport was measured at 37 °C for 20 min and was stopped by washing three times in ice-cold PBS. Tissues were solubilised with 1 M NaOH, neutralised with 1 M HCl and then centrifuged at 15 000 g for 5 min to pellet insoluble material. The supernatant was added to 3 ml liquid scintillation fluid, and samples were counted for radioactivity in a liquid scintillation counter. GLUT-specific glucose uptake was measured by subtracting values for [14C]-2DG uptake in the presence of cytochalasin B. The rate of [14C]-2DG transport was expressed in nanomoles per minute per milligram tissue.
Statistical analysis
Statistical analyses were performed using a commercially available statistical software package (Statgraphics Plus version 3.1; Statistical Graphics Corp., Rockville, MD, USA). Student's t-test was used to assess statistical significance between normally distributed data; otherwise, the nonparametric Mann–Whitney U (Wilcoxon) test was used. Statistical difference was indicated by a P value of <0.05. Data are expressed as mean±s.e.m.
Results
Effect of HX/XO on GLUT expression in human placenta
GLUT1, GLUT3 and GLUT4 have all been localised in human placenta (Hauguel-de Mouzon et al. 1997, Xing et al. 1998, Illsley 2000, Colomiere et al. 2010) and thus may be responsible for glucose uptake in this tissue. Thus, our next aim was to determine the effect of HX/XO on GLUT expression. The effect of 24 h treatment of human placenta with HX/XO on GLUT mRNA and protein expression is shown in Fig. 1. HX/XO treatment significantly decreased GLUT1 mRNA and protein expression. On the other hand, there was no effect of HX/XO on GLUT3 and GLUT4 mRNA and protein expression.
Placenta was incubated in the absence or presence of 0.5 mM HX+15 mU/ml XO for 24 h (n=7). (A) GLUT gene expression is expressed as mean±s.e.m. (B) Protein expression is expressed as mean (normalised to β-actin)±s.e.m. A representative GLUT1 and GLUT3 western blot is also shown. *P<0.05 vs basal GLUT1 expression.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0355
Effect of HX/XO on glucose uptake in human placenta
To investigate the effects of oxidative stress on glucose uptake, an ex situ tissue explant incubation model was used. Human placenta was incubated in the absence (basal) and presence of HX/XO for 24 h (n=7). When compared with basal uptake, glucose uptake was significantly decreased with HX/XO treatment in placenta (Fig. 2A). As placenta is a source of XO (Biri et al. 2006), control experiments were also performed in the presence of HX alone. There was, however, no effect of HX on placental glucose uptake (Fig. 2B). The insulin signalling pathway regulates glucose uptake; however, in this study, there was no effect of HX/XO on IR-β, IRS1 and PI3K mRNA and protein expression (data not shown).
Placenta was incubated in the absence or presence of (A) 0.5 mM HX+15 mU/ml XO (n=7) for 24 h, (B) 0.5 mM HX (n=4) for 24 h or (C) 0.5 mM HX+15 mU/ml XO (n=4) for 4 h. Data are expressed as mean 2DG uptake (nmol/min per mg protein)±s.e.m. *P<0.05 vs basal 2DG uptake. (D) Placenta was incubated in the absence or presence of 0.5 mM HX+15 mU/ml XO for 24 h (n=7). Syncytin gene expression is expressed as mean±s.e.m.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0355
HX/XO treatment is not associated with syncytial damage
In this study, all treatments were performed for 24 h. Thus, it is possible that the effects of HX/XO on 2DG uptake may be due to damage and loss of the syncytium. Thus, we also determined the effect of HX/XO on 2DG uptake after 4 h. As shown in Fig. 2C, HX/XO significantly decreased placental glucose uptake after 4 h (Fig. 2C). In addition, there was no effect of HX/XO on syncytin gene expression (Fig. 2D).
Superoxide and hydrogen peroxide do not regulate glucose uptake in human placenta
Catalytic oxidation of HX by XO produces H2O2 and superoxide anions. To determine whether this occurs in our system, we measured H2O2 concentration in the incubation buffer containing HX/XO. As shown in Fig. 3A, there was a significant increase in H2O2 concentration in the presence of HX/XO. We then determined whether the increase in H2O2 and/or superoxide anions was responsible for the decrease in glucose uptake. Experiments were carried out by pre-incubating placental tissue explants with the superoxide scavenger SOD (300 U/ml) or the H2O2 scavenger catalase (1000 U/ml) for 30 min, before incubation with HX/XO for 24 h. Figure 3B shows that catalase and SOD treatment did not restore the decrease in glucose uptake induced by HX/XO. Further, there was no effect of 0.25, 0.5 and 1 mM H2O2 on placental glucose uptake (Fig. 3C). Collectively, these data show that superoxide and H2O2 are not involved in the regulation of glucose uptake by HX/XO in this system.
(A) Placenta was incubated in the absence or presence of 0.5 mM HX+15 mU/ml XO for 24 h (n=7). Data are expressed as mean H2O2 release (μM/mg protein)±s.e.m. *P<0.05 vs basal H2O2 release. (B) Placenta was incubated in the absence or presence of 0.5 mM HX+15 mU/ml XO with or without 1000 U/ml catalase-PEG (n=4), 300 U/ml SOD–PEG (n=4), 100 mM mannitol (n=3), 1% DMSO (n=3), 5 mM NAC (n=3) and 50 μM BAY 11-7082 (n=4) for 24 h. Data are expressed as mean 2DG uptake (nmol/min per mg protein)±s.e.m. *P<0.05 vs basal 2DG uptake. (C) Placenta was incubated in the absence or presence of 0.25, 0.5 and 1 mM H2O2 for 24 h (n=4). Data are expressed as mean 2DG uptake (nmol/min per mg protein)±s.e.m.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0355
Hydroxyl radical scavengers do not restore the decrease in glucose uptake induced by HX/XO
H2O2 can readily pass through cell membranes and interact with intracellular iron to form highly reactive oxidants such as the hydroxyl radical (•OH). Thus, we assessed the effect of •OH scavengers on placental glucose uptake in the presence of HX/XO. As shown in Fig. 3B, there was no effect of thiourea, mannitol, DMSO or NAC on glucose uptake in the presence of HX/XO.
Inhibition of NF-κB does not restore the decrease in glucose uptake induced by HX/XO
There are multiple mechanisms that regulate glucose uptake, and in recent years NF-κB has been suggested to be a key signalling protein regulating insulin-independent glucose uptake. Previous studies from our laboratory have shown that in response to HX/XO treatment, placenta increases NF-κB DNA-binding activity (Coughlan et al. 2004). Thus, in order to elucidate whether NF-κB is involved in the HX/XO regulation of glucose uptake, placenta was incubated with the NF-κB inhibitor BAY 11-7082 for 24 h, in the presence of HX/XO. There was, however, no effect of BAY 11-7082 on placental glucose uptake in the presence of HX/XO (Fig. 3B).
Resveratrol restores the decrease in glucose uptake induced by HX/XO
In non-gestational tissues, recent studies have shown that SIRT1 regulates glucose uptake (Yoshizaki et al. 2009). The natural photochemical, resveratrol, is an activator of SIRT1 (Bai et al. 2008, Csiszar et al. 2008, Backesjo et al. 2009, Kennedy et al. 2009). Similarly, we have previously shown that, in human placenta, resveratrol inhibits lipopolysaccharide (LPS)-induced inflammation via SIRT1 (Lappas et al. 2011b). Thus, we sought to investigate whether resveratrol can restore the decrease in glucose uptake induced by HX/XO. First, we determined the effect of HX/XO on SIRT1 gene and protein expression. As shown in Fig. 4, SIRT1 mRNA gene and protein expression was significantly decreased in the presence of HX/XO. Co-incubation with resveratrol was associated with an increase in SIRT1 gene and protein expression.
Placenta was incubated in the absence or presence of 0.5 mM HX+15 mU/ml XO with or without 50 μM resveratrol for 24 h (n=6). (A) SIRT1 gene expression is expressed as mean±s.e.m. *P<0.05 vs HX/XO SIRT1 mRNA expression. (B) SIRT1 protein expression is expressed as mean (normalised to β-actin)±s.e.m. *P<0.05 vs HX/XO SIRT1 protein expression. Two representative western blots are shown.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0355
Then, we determined the effect of resveratrol on glucose uptake. Placenta was incubated with HX/XO in the absence or presence of 50 μM resveratrol for 24 h. The effect of resveratrol on glucose uptake is shown in Fig. 5A. Resveratrol significantly attenuated the decrease in placental glucose uptake induced by HX/XO. In addition, co-incubation with resveratrol significantly attenuated the HX/XO-induced decrease in GLUT1 mRNA (Fig. 5B) and protein expression (Fig. 5C).
(A) Placenta was incubated in the absence or presence of 0.5 mM HX+15 mU/ml XO with or without 50 μM resveratrol for 24 h (n=6). Data are expressed as mean 2DG uptake (nmol/min per mg protein)±s.e.m. *P<0.05 vs HX/XO 2DG uptake. (B) Placenta was incubated in the absence or presence of 0.5 mM HX+15 mU/ml XO with or without 50 μM resveratrol for 24 h (n=6). GLUT1 gene expression is expressed as mean±s.e.m. *P<0.05 vs HX/XO GLUT1 mRNA expression. (C) Placenta was incubated in the absence or presence of 0.5 mM HX+15 mU/ml XO with or without 50 μM resveratrol for 24 h (n=6). GLUT1 protein expression is expressed as mean (normalised to β-actin)±s.e.m. *P<0.05 vs HX/XO GLUT1 protein expression. A representative western blot image is shown.
Citation: Journal of Endocrinology 213, 1; 10.1530/JOE-11-0355
Discussion
In this study, we have demonstrated that HX/XO-induced oxidative stress decreases placental GLUT1 expression and glucose uptake via SIRT1. Specifically, we have shown that oxidative stress decreases SIRT1 gene and protein expression in human placenta, and the SIRT1 activator resveratrol attenuates the decrease in GLUT1 expression and glucose uptake mediated by HX/XO.
HX/XO treatment significantly reduced GLUT1 expression that was associated with a concomitant decrease in glucose uptake. GLUT1 is located at both the maternal-facing microvillous trophoblast membrane and the foetal-facing basal trophoblast membrane, thereby allowing bidirectional transport across the placenta (Illsley 2000). In this study, we found that there was no effect of HX/XO on GLUT3 and GLUT4 expression. GLUT3 is mainly localised to the vascular endothelium (Hauguel-de Mouzon et al. 1997, Illsley 2000) and therefore cannot be involved in placental glucose uptake from the maternal circulation; it may act specifically in the transport of glucose to the foetus. GLUT4 also has been found in the human placenta (Xing et al. 1998), and insulin mediates glucose uptake in first-trimester chorionic villous explants (Kniss et al. 1994), but its role in term placenta is controversial (Challier et al. 1986). In this study, though we could detect low levels of GLUT4 at the mRNA level, we were unable to detect GLUT4 protein expression in the placenta. There was also no effect of HX/XO on total expression levels of proteins of the insulin signalling pathways, namely IR-β, IRS1 and PI3K, though activation of this pathway was not examined.
The molecular mechanism responsible for this decrease is not known but is not attributable to ROS. In this study, we used various antioxidants, including NAC, SOD and catalase, that quench the production of superoxide anion and hydrogen peroxide produced by the HX and XO reaction; however, there was no effect of these antioxidants on the reduction of placental glucose uptake-induced HX/XO. It is possible that the concentration of antioxidants used in this study was not optimal or that a combination of various antioxidants is required to see an effect. A previous study has, however, reported that the overexpression of antioxidant thioredoxin increases placental GLUT1 in mice (Umekawa et al. 2008). Previous studies from our laboratory have shown that cytokine response to HX/XO may be mediated by the oxidant-responsive transcription factor NF-κB (Coughlan et al. 2004). However, in this study, the NF-κB inhibitor was unable to restore the decrease in placental glucose uptake in the presence of oxidative stress.
HX/XO reduced SIRT1 gene and protein expression in human placenta. Likewise, oxidative stress decreases SIRT1 expression in the hippocampus and cerebral cortex (Wu et al. 2006), and human umbilical vein endothelial cells exposed to cigarette smoke extract and hydrogen peroxide showed decreased SIRT1 levels and activity (Arunachalam et al. 2010). More recently, it has been shown that direct modification of SIRT1 by ROS or alkylating agents can mediate its degradation (Caito et al. 2010). Given that HX/XO decreased SIRT1 expression, we sought to determine the effect of the SIRT1 activator, resveratrol (Bai et al. 2008, Csiszar et al. 2008, Backesjo et al. 2009, Kennedy et al. 2009). Treatment with resveratrol ameliorated the HX/XO-induced impairment in glucose uptake and this coincided with significant increases in the expression of GLUT1. This suggests that SIRT1 is responsible for the regulation of glucose uptake mediated by HX/XO in human placenta. This is in keeping with our previous studies in human placenta, whereby resveratrol inhibits inflammation by a SIRT1-dependent mechanism (Lappas et al. 2011b). Of note, these studies showed that resveratrol had no effect on NF-κB and MAPK pathways in human placenta. Our data is in keeping with other studies; in muscle cells (Breen et al. 2008) and adipocytes (Fischer-Posovszky et al. 2010), resveratrol elevates glucose uptake via SIRT1 and improves insulin sensitivity in adipocytes (Yoshizaki et al. 2009).
Our previous studies demonstrate that the pro-inflammatory cytokines tumour necrosis factor α (TNFα) and interleukin 1β (IL1β) decrease SIRT1 expression in human placenta (Lappas et al. 2011b). Further, in recent studies in our laboratory, we showed that both TNFα and IL1β down-regulate glucose uptake and GLUT1 expression (R Lim and M Lappas 2010, unpublished observations). Whether these cytokines are also regulating glucose transport via SIRT1 is not known at this stage; however, it is tempting to speculate that TNFα and IL1β may also decrease placental glucose uptake via SIRT1-dependent mechanisms.
It is known that incubation of placental tissue causes significant syncytial damage and loss after ∼6 h of incubation (Palmer et al. 1997). In this study, all treatments were performed for 24 h. Thus, it is possible that the effects of HX/XO on 2DG uptake may be due to damage and loss of the syncytium. However, we also determined the effect of HX/XO on 2DG uptake after 4 h. HX/XO was able to significantly decrease placental glucose uptake. In addition, we also assessed the general morphology of the placental tissue after incubation using hematoxylin and eosin (H&E). No difference was observed between the basal and the HX/XO-treated tissues (data not shown). There was also no effect of HX/XO on PI3K and syncytin–proteins that are specifically localised to the syncytiotrophoblast of the placenta. We have also previously reported no effect of HX/XO on placental release of lactate dehydrogenase, a marker of tissue viability (Coughlan et al. 2004, Lappas et al. 2010). Collectively, this suggests that the effects of HX/XO on glucose transport are not due to damage or loss of the syncytium.
Our original hypothesis was that ROS, generated by the HX/XO system, would regulate glucose uptake in human placenta. However, the data presented in this paper suggest that this is not the case; the antioxidants, including SOD, catalase and NAC, had no effect on HX/XO-induced decreases in glucose uptake and GLUT1 expression. However, we did find that resveratrol, a SIRT1 activator, was able to restore the effects of HX/XO on glucose uptake and GLUT1 expression. Though the underlying mechanism/pathway that elicited these changes is not known, it is possible that HX/XO, via its effects on SIRT1, is inducing chromatin modifications. SIRTs possess NAD+-dependent histone deacetylase (HDAC) activity (North & Verdin 2004). Though there are no studies that have investigated the effect of HX/XO on histone acetylation, GLUT1 expression and glucose uptake, at least in part, are regulated by changes in deacetylation. For example, in myeloma cells, a number of different HDAC inhibitors induced a decrease in glucose uptake and GLUT1 expression (Wardell et al. 2009). Likewise, glucose uptake was inhibited in colorectal adenocarcinomas cells (HT29) treated with HDAC inhibitors (Boren et al. 2003). Future studies are required to determine whether HX/XO regulates glucose uptake by interfering with or promoting histone acetylation in human placenta.
There are much data showing that the placenta, by transferring nutrients to the foetus, plays a major role in the regulation of foetal metabolism and growth (Fowden et al. 2009). Alterations in the amounts of proteins, carbohydrates and fat that are supplied to the foetus may have long-term consequences (Jansson & Powell 2007). Changes in glucose metabolism (Osmond et al. 2001) and increased oxidative stress are characteristics of pregnancies complicated with diabetes (Lappas et al. 2011a). Excess uptake and transfer of maternal glucose across the placenta would lead to foetal hyperglycaemia with subsequent hyperinsulinaemia and its associated detrimental sequelae (Nold & Georgieff 2004). Thus, in order to circumvent and/or control the effects of aberrant nutrient transfer and thus harmful effects on foetal development, it is of clinical significance to identify modulators of nutrient transport within the placenta. In this study, we provide evidence that the reduction in placental glucose uptake and GLUT1 expression induced by HX/XO is mediated by SIRT1.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
Dr M L was a recipient of a National Health and Medical Research Council (NHMRC) RD Wright Fellowship (grant no. 454777). The work described in this manuscript was, in part, funded by a project grant from NHMRC (grant no. 454310). Funding for the iQ5 multicolour RT-PCR machine (in-part), Chemi-Doc system and xMark Microplate Absorbance Spectrophotometer was provided by the Medical Research Foundation for Women and Babies.
Acknowledgements
The authors thank Gillian Barker for her assistance with the experiments; research midwives Gabrielle Fleming and Astrid Tiefholz for collection of clinical samples; and obstetrics and midwifery staff of the Mercy Hospital for Women for their co-operation.
References
Arunachalam G, Yao H, Sundar IK, Caito S & Rahman I 2010 SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: role of resveratrol. Biochemical and Biophysical Research Communications 393 66–72. doi:10.1016/j.bbrc.2010.01.080.
Backesjo CM, Li Y, Lindgren U & Haldosen LA 2009 Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells. Cells, Tissues, Organs 189 93–97. doi:10.1159/000151744.
Bai L, Pang WJ, Yang YJ & Yang GS 2008 Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes. Molecular and Cellular Biochemistry 307 129–140. doi:10.1007/s11010-007-9592-5.
Biri A, Onan A, Devrim E, Babacan F, Kavutcu M & Durak I 2006 Oxidant status in maternal and cord plasma and placental tissue in gestational diabetes. Placenta 27 327–332. doi:10.1016/j.placenta.2005.01.002.
Boren J, Lee WN, Bassilian S, Centelles JJ, Lim S, Ahmed S, Boros LG & Cascante M 2003 The stable isotope-based dynamic metabolic profile of butyrate-induced HT29 cell differentiation. Journal of Biological Chemistry 278 28395–28402. doi:10.1074/jbc.M302932200.
Breen DM, Sanli T, Giacca A & Tsiani E 2008 Stimulation of muscle cell glucose uptake by resveratrol through sirtuins and AMPK. Biochemical and Biophysical Research Communications 374 117–122. doi:10.1016/j.bbrc.2008.06.104.
Caito S, Rajendrasozhan S, Cook S, Chung S, Yao H, Friedman AE, Brookes PS & Rahman I 2010 SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress. FASEB Journal 24 3145–3159. doi:10.1096/fj.09-151308.
Challier JC, Hauguel S & Desmaizieres V 1986 Effect of insulin on glucose uptake and metabolism in the human placenta. Journal of Clinical Endocrinology and Metabolism 62 803–807. doi:10.1210/jcem-62-5-803.
Colomiere M, Permezel M, Riley C, Desoye G & Lappas M 2009 Defective insulin signaling in placenta from pregnancies complicated by gestational diabetes mellitus. European Journal of Endocrinology 160 567–578. doi:10.1530/EJE-09-0031.
Colomiere M, Permezel M & Lappas M 2010 Diabetes and obesity during pregnancy alter insulin signalling and glucose transporter expression in maternal skeletal muscle and subcutaneous adipose tissue. Journal of Molecular Endocrinology 44 213–223. doi:10.1677/JME-09-0091.
Coughlan MT, Permezel M, Georgiou HM & Rice GE 2004 Repression of oxidant-induced nuclear factor-kappaB activity mediates placental cytokine responses in gestational diabetes. Journal of Clinical Endocrinology and Metabolism 89 3585–3594. doi:10.1210/jc.2003-031953.
Csiszar A, Labinskyy N, Podlutsky A, Kaminski PM, Wolin MS, Zhang C, Mukhopadhyay P, Pacher P, Hu F & de Cabo R et al. 2008 Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations. American Journal of Physiology. Heart and Circulatory Physiology 294 H2721–H2735. doi:10.1152/ajpheart.00235.2008.
Fiorentini D, Hakim G, Bonsi L, Bagnara GP, Maraldi T & Landi L 2001 Acute regulation of glucose transport in a human megakaryocytic cell line: difference between growth factors and H(2)O(2). Free Radical Biology & Medicine 31 923–931. doi:10.1016/S0891-5849(01)00678-5.
Fischer-Posovszky P, Kukulus V, Tews D, Unterkircher T, Debatin KM, Fulda S & Wabitsch M 2010 Resveratrol regulates human adipocyte number and function in a Sirt1-dependent manner. American Journal of Clinical Nutrition 92 5–15. doi:10.3945/ajcn.2009.28435.
Fowden AL, Sferruzzi-Perri AN, Coan PM, Constancia M & Burton GJ 2009 Placental efficiency and adaptation: endocrine regulation. Journal of Physiology 587 3459–3472. doi:10.1113/jphysiol.2009.173013.
Hauguel-de Mouzon S, Challier JC, Kacemi A, Cauzac M, Malek A & Girard J 1997 The GLUT3 glucose transporter isoform is differentially expressed within human placental cell types. Journal of Clinical Endocrinology and Metabolism 82 2689–2694. doi:10.1210/jc.82.8.2689.
Hay WW Jr 2006 Placental–fetal glucose exchange and fetal glucose metabolism. Transactions of the American Clinical and Climatological Association 117 321–339.discussion 339–340
Higaki Y, Mikami T, Fujii N, Hirshman MF, Koyama K, Seino T, Tanaka K & Goodyear LJ 2008 Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway. American Journal of Physiology. Endocrinology and Metabolism 294 E889–E897. doi:10.1152/ajpendo.00150.2007.
Hubel CA 1999 Oxidative stress in the pathogenesis of preeclampsia. Proceedings of the Society for Experimental Biology and Medicine 222 222–235. doi:10.1046/j.1525-1373.1999.d01-139.x.
Illsley NP 2000 Glucose transporters in the human placenta. Placenta 21 14–22. doi:10.1053/plac.1999.0448.
Jansson T & Powell TL 2006 IFPA 2005 Award in Placentology Lecture. Human placental transport in altered fetal growth: does the placenta function as a nutrient sensor? – a review. Placenta 27 S91–S97. doi:10.1016/j.placenta.2005.11.010.
Jansson T & Powell TL 2007 Role of the placenta in fetal programming: underlying mechanisms and potential interventional approaches. Clinical Science 113 1–13. doi:10.1042/CS20060339.
Kalhan S & Parimi P 2000 Gluconeogenesis in the fetus and neonate. Seminars in Perinatology 24 94–106. doi:10.1053/sp.2000.6360.
Kennedy A, Overman A, Lapoint K, Hopkins R, West T, Chuang CC, Martinez K, Bell D & McIntosh M 2009 Conjugated linoleic acid-mediated inflammation and insulin resistance in human adipocytes are attenuated by resveratrol. Journal of Lipid Research 50 225–232. doi:10.1194/jlr.M800258-JLR200.
Kniss DA, Shubert PJ, Zimmerman PD, Landon MB & Gabbe SG 1994 Insulin-like growth-factors – their regulation of glucose and amino-acid-transport in placental trophoblasts isolated from first-trimester chorionic villi. Journal of Reproductive Medicine 39 249–256.
Kozlovsky N, Rudich A, Potashnik R & Bashan N 1997a Reactive oxygen species activate glucose transport in L6 myotubes. Free Radical Biology & Medicine 23 859–869. doi:10.1016/S0891-5849(97)00069-5.
Kozlovsky N, Rudich A, Potashnik R, Ebina Y, Murakami T & Bashan N 1997b Transcriptional activation of the Glut1 gene in response to oxidative stress in L6 myotubes. Journal of Biological Chemistry 272 33367–33372. doi:10.1074/jbc.272.52.33367.
Lappas M, Mitton A & Permezel M 2010 In response to oxidative stress, the expression of inflammatory cytokines and antioxidant enzymes are impaired in placenta, but not adipose tissue, of women with gestational diabetes. Journal of Endocrinology 204 75–84. doi:10.1677/JOE-09-0321.
Lappas M, Hiden U, Desoye G, Froehlich J, Mouzon SH & Jawerbaum A 2011a The role of oxidative stress in the pathophysiology of gestational diabetes mellitus. Antioxidants & Redox Signaling 15 3061–3100. doi:10.1089/ars.2010.3765.
Lappas M, Mitton A, Lim R, Barker G, Riley C & Permezel M 2011b SIRT1 is a novel regulator of key pathways of human labor. Biology of Reproduction 84 167–178. doi:10.1095/biolreprod.110.086983.
Myatt L & Cui X 2004 Oxidative stress in the placenta. Histochemistry and Cell Biology 122 369–382. doi:10.1007/s00418-004-0677-x.
Nold JL & Georgieff MK 2004 Infants of diabetic mothers. Pediatric Clinics of North America 51 619–637. doi:10.1016/j.pcl.2004.01.003.
North BJ & Verdin E 2004 Sirtuins: Sir2-related NAD-dependent protein deacetylases. Genome Biology 5 224 doi:10.1186/gb-2004-5-5-224.
Osmond DT, King RG, Brennecke SP & Gude NM 2001 Placental glucose transport and utilisation is altered at term in insulin-treated, gestational-diabetic patients. Diabetologia 44 1133–1139. doi:10.1007/s001250100609.
Paiva P, Whitehead C, Saglam B, Palmer K & Tong S 2011 Measurement of mRNA transcripts of very high placental expression in maternal blood as biomarkers of preeclampsia. Journal of Clinical Endocrinology and Metabolism 96 E1807–E1815. doi:10.1210/jc.2011-1233.
Palmer ME, Watson AL & Burton GJ 1997 Morphological analysis of degeneration and regeneration of syncytiotrophoblast in first trimester placental villi during organ culture. Human Reproduction 12 379–382. doi:10.1093/humrep/12.2.379.
Regazzetti C, Peraldi P, Gremeaux T, Najem-Lendom R, Ben-Sahra I, Cormont M, Bost F, Le Marchand-Brustel Y, Tanti JF & Giorgetti-Peraldi S 2009 Hypoxia decreases insulin signaling pathways in adipocytes. Diabetes 58 95–103. doi:10.2337/db08-0457.
Scioscia M, Gumaa K, Kunjara S, Paine MA, Selvaggi LE, Rodeck CH & Rademacher TW 2006 Insulin resistance in human preeclamptic placenta is mediated by serine phosphorylation of insulin receptor substrate-1 and -2. Journal of Clinical Endocrinology and Metabolism 91 709–717. doi:10.1210/jc.2005-1965.
Siddiqui IA, Jaleel A, Tamimi W & Al Kadri HM 2010 Role of oxidative stress in the pathogenesis of preeclampsia. Archives of Gynecology and Obstetrics 282 469–474. doi:10.1007/s00404-010-1538-6.
Tirosh A, Potashnik R, Bashan N & Rudich A 1999 Oxidative stress disrupts insulin-induced cellular redistribution of insulin receptor substrate-1 and phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. A putative cellular mechanism for impaired protein kinase B activation and GLUT4 translocation. Journal of Biological Chemistry 274 10595–10602. doi:10.1074/jbc.274.15.10595.
Toyoda T, Hayashi T, Miyamoto L, Yonemitsu S, Nakano M, Tanaka S, Ebihara K, Masuzaki H, Hosoda K & Inoue G et al. 2004 Possible involvement of the α1 isoform of 5′AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle. American Journal of Physiology. Endocrinology and Metabolism 287 E166–E173. doi:10.1152/ajpendo.00487.2003.
Umekawa T, Sugiyama T, Kihira T, Murabayashi N, Zhang L, Nagao K, Kamimoto Y, Ma N, Yodoi J & Sagawa N 2008 Overexpression of thioredoxin-1 reduces oxidative stress in the placenta of transgenic mice and promotes fetal growth via glucose metabolism. Endocrinology 149 3980–3988. doi:10.1210/en.2007-1682.
Wardell SE, Ilkayeva OR, Wieman HL, Frigo DE, Rathmell JC, Newgard CB & McDonnell DP 2009 Glucose metabolism as a target of histone deacetylase inhibitors. Molecular Endocrinology 23 388–401. doi:10.1210/me.2008-0179.
Wu A, Ying Z & Gomez-Pinilla F 2006 Oxidative stress modulates Sir2alpha in rat hippocampus and cerebral cortex. European Journal of Neuroscience 23 2573–2580. doi:10.1111/j.1460-9568.2006.04807.x.
Xing AY, Challier JC, Lepercq J, Cauzac M, Charron MJ, Girard J & Hauguel-de Mouzon S 1998 Unexpected expression of glucose transporter 4 in villous stromal cells of human placenta. Journal of Clinical Endocrinology and Metabolism 83 4097–4101. doi:10.1210/jc.83.11.4097.
Yoshizaki T, Milne JC, Imamura T, Schenk S, Sonoda N, Babendure JL, Lu JC, Smith JJ, Jirousek MR & Olefsky JM 2009 SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes. Molecular and Cellular Biology 29 1363–1374. doi:10.1128/MCB.00705-08.
