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research-article |
Laboratoire de Physiologie Materno-F
tale,3 and Centre de Recherche BioMed,4 Université du Québec à Montréal, Montréal, Québec, Canada H3C 3P8
ABSTRACT
Low birth weight is observed in rabbit offspring when maternal hypercholesterolemia is induced during gestation, but the related etiology is still unknown. Glucose is one of the most important substances during fetal development, and defect in glucose supply to fetus was related to pathophysiological mechanisms in intrauterine growth restriction. Thus, the aim of this work was to evaluate the impact of maternal hypercholesterolemia during rabbit gestation on the glucose metabolism and the routing of glucose transporters (SLC2 and SLC5 [previously known as GLUT and SGLT]) in placenta. In this study, maternal and offspring serum levels of glucose and insulin were evaluated for control and hypercholesterolemic groups, and the mRNA and protein expressions of placental SLCs were quantified by real-time RT-PCR and Western immunoblot, respectively. Our data demonstrate that maternal hypercholesterolemia during gestation: 1) induces offspring hypoglycemia; 2) does not modify the genetic and protein expressions of SLC2A1 and SLC2A4 (previously GLUT1 and GLUT4) in total placental extract; 3) downregulates the placental SLC5A1 (previously SGLT1) protein expression without affecting its mRNA levels; 4) impairs the translocation of SLC2A1 but not SLC2A4 from cytoplasmatic pool to the cell membrane surface. Then we assume that reduction of offspring birth weight in presence of maternal hypercholesterolemia may be related to the offspring's hypoglycemia and the reduction of the cell surface expression of placental SLC2A1.
glucose, hypercholesterolemia, insulin, placenta, transporters
Glucose is the primary source of energy for metabolism and development for the fetus and placental tissue. The fetal consumption of glucose increases rapidly toward term due to the almost 20-fold increase in fetal weight during the second half of pregnancy [1]. In utero, the human fetal plasma glucose concentration correlates with the maternal one [2], and maternal basal plasma glucose concentration tends to decrease with the progression of pregnancy, even in the presence of a 2-fold increase of insulin level [3, 4]. In spite of the presence of insulin receptor in placenta [5], the effects of insulin, the major regulator of glucose level, have still not been clarified in the human placenta. Since insulin receptors are preferentially localized on the fetal side of the human term placenta, it has been suggested that fetal insulin can play a major role in regulating insulin-dependent processes.
Since the fetus is not capable of producing appreciable amounts of glucose until late in gestation, it is critically dependent on the net transfer of glucose across the placenta [6]. The high fetal demand for glucose, especially during the third trimester, necessitates the presence of a rapid, high-volume system for maternal-fetal glucose transfer. This is regulated by several factors: glucose supply, placental glucose metabolism, and placental glucose transporter density [7]. Placental glucose transport is a stereoscopic, saturable, and carrier-mediated process of facilitated diffusion [8], involving a family of membrane-spanning glycoproteins, the glucose transporters (SLC2) [9], and the Na+-dependent active glucose transporter (SLC5) [10]. SLC2A1 represents the major glucose transporter in human placenta that controls glucose transport from the maternal to the fetal compartment under physiological conditions, and its expression is maximal at delivery. In rodent and sheep, placental SLC2A3 (previously GLUT3) mRNA and protein levels increase as gestation advances, whereas SLC2A1 abundance is unaffected or decreased toward term [11, 12]. SLC2A4, an insulin-responsive glucose transporter, is present in placental stromal cells and may be important for transporting glucose and conversion to glycogen in these cells in response to insulin in the fetal circulation [13].
Although it is known that glucose is the primary substrate for fetal oxidative metabolism, the regulatory mechanisms and function of glucose transporters in placenta are still not fully understood.
Fetal metabolism and, consequently, fetal growth, directly depend on an adequate supply of oxygen and essential nutrients derived from the maternal circulation. Macrosomia and selective organomegaly are present in the excess of glucose of diabetic pregnant women, both insulin dependent and gestational [14]. In the opposite case, maternal malnutrition during pregnancy causes the intrauterine-grown restriction (IUGR). IUGR is a frequently occurring and serious complication of pregnancy. This etiology complication is multifactorial, but a reduced transfer of nutriment and oxygen to the fetus is likely the final common pathway in many cases. Measurements of fetal plasma glucose have consistently shown a decreased concentration of glucose in IUGR fetuses [15, 16]. A decreased expression of glucose transporters has been suggested as one possible mechanism by which fetal plasma glucose concentration is reduced. However, studies investigating placental transporter expression and activity in IUGR are limited, and their conclusions often are discordant [17–21]. Taken together, these observations indicate that placental SLC2 protein abundance is responsive to environmental conditions and/or the concomitant changes in fetal growth that these conditions induce.
Thus, to study the impact of a control and a specific diet, such as an enriched-cholesterol diet (ECD), on maternal and fetal metabolism, we developed an animal model of pregnant rabbit [22]. Rabbit atherogenic pattern is known to show similarity with that of the human.
In the previous study we reported that a 0.2% ECD during rabbit gestation induced maternal and fetal hypercholesterolemia and, consequently, significantly reduced offspring birth weight by 15.5% compared with control diet [22]. In the present study, we hypothesized that the reduction of offspring birth weight could be related to a modification of both glucose metabolism and placental glucose exchange. We also hypothesized that hypercholesterolemia changes the distribution pattern of SLC2s between the plasma membrane and intracellular sites in rabbit placental tissue. Thus, we evaluated fetal and maternal glucose levels, insulin levels, and expression of placental SLC2A1, SLC2A3, SLC2A4, and SLC5A1 in order to evaluate the impact of hypercholesterolemia on maternal and fetal glucose metabolism and placental glucose transfer.
New Zealand adult white virgin female rabbits weighing 3 to 4 kg were purchased from Charles River (Saint-Constant, QC, Canada). The rabbits were treated according to the criteria for the care and use of laboratory animals with the recommendation of the Canadian Council on Animal Care. During the 2-wk acclimatization period, all rabbits were fed 150 g/day of a standard rabbit chow diet (Ralston Purina, Drummondville, QC, Canada) and water ad libitum. To conduct this study, female rabbits were divided in two groups: 1) the control group, which consisted of rabbits fed with 150 g/day of the standard diet throughout their gestation (30 days) and which had follow-up beginning on the mating day (Day 0 up to Day 30); and 2) the ECD group, which was fed 150 g/day of standard diet enriched with 0.2% (w/w) cholesterol 7 days before mating and which had follow-up beginning with the administration of the diet throughout gestation.
Maternal blood was collected by an ear artery puncture, following the subcutaneous injection of acepromazine maleate (Atravet; 0.6 mg/kg), before mating and at the end of pregnancy. An additional blood sampling was done on rabbits determined to be part of the ECD group prior the beginning of the diet. The last blood collection was done at term (Day 30) by an intracardiac puncture, following a subcutaneous injection of Atravet (0.6 mg/kg, Ayerst, Montreal, Quebec, Canada) and an intramuscular injection of ketamine chlorhydrate (40 mg/kg) and xylazine (5 mg/kg). Thereafter, the dams were killed by a lethal intracardiac injection of Euthanyl (0.5 ml/kg, MTC, Cambridge, Ontario, Canada), and their abdomens were opened. Both horns of the uterus were excised, and the offspring were removed from the uterus with their respective intact placenta. Placentas were frozen immediately in liquid nitrogen and kept at –80°C until used. Blood was obtained from the offspring by intracardiac puncture, and they were killed by an intracardiac injection of Euthanyl (0.5 ml/kg). Blood samples were centrifuged for 5 min at 12 000 x g, and serum samples were kept at –80°C until analysis.
Glucose and Insulin Analysis in Rabbit Serum
The glucose and the insulin concentration were measured in the serum of dams and offspring of both control and ECD groups. The glucose assay was performed using a QuantiChrom Glucose Assay Kit (BioAssay System, Hayward, CA) according to the manufacturer's instructions, and the optical density was read at 640 nm. The insulin level was assayed using Mercodia Ultrasensitive Insulin ELISA kit (Mercodia AB, Uppsala, Sweden) with recombinant human insulin as a standard. The optical density was read at 450 nm.
RNA Extraction and Real-Time RT-PCR Experiments
Total RNA was isolated from 100 mg placental tissue using the RNeasy Lipid Tissue Mini Kit according to the manufacturer's instructions (Qiagen, Mississauga, ON, Canada). The cDNA synthesis was carried out using Omniscript RT kit (Qiagen) with 2 µg total RNA at 37°C for 1 h in a 20-µl final volume. Oligonucleotide primers were synthesized by Qiagen. Each amplification reaction (12.5 µl) was performed in the presence of 0.2 µM deoxynucleoside triphosphate, 0.5 µM of each primer, 2.5 units Taq DNA polymerase (Taq PCR core kit; Qiagen) and a dilution of 1:12.5 of the cDNA synthesis reaction. Fragments of cDNA were amplified using the followings primers: SLC2A1 sense (S) 5'-TGCCCTGCATGTCCTATCTGA-3' and SLC2A1 antisense (AS) 5'-TGAAATTCGAGGTCCAGTTGG-3'; SLC2A3 (S) 5'-CGTCATCTTTGCCGTCTTC-3' and SLC2A3 (AS) 5'-ACATGGGTGGTGGTCTCAA-3'; SLC2A4 (S) 5'-TGGTCTCGGTGTTCTTGGTG-3' and SLC2A4 (AS) 5'-CTCAAAGAACGCCACAAAGC-3'; and SLC5A1 (S) 5'-TCCTCACCCTTTGGTACTGG-3' and SLC5A1 (AS) 5'-ACAGGATACGGCTCACCATC-3'. The annealing temperature was 58°C for SLC2A1 and SLC2A3, and it was 60°C for SLC2A4 (40 cycles). After PCR, 10 µl of each sample was migrated in 1.5% agarose gels and visualized by ethidium bromide staining. Real-time RT-PCR experiments were performed using the LightCycler System (Roche Biochemicals, Laval, QC, Canada) in 10 µl reaction containing 1 µl of 1x SYBR Green according to the manufacturer's instructions (Takara Bio Inc., Otsu, Japan). HPRT1 was used as reference gene for normalization. The PCR cycling regimen was 5 min at 95°C, 40 iterations of a three-step temperature series (5 sec at 95°C, 20 sec at the optimal annealing temperature for each pair of primers, and 15 sec at 72°C). Cycling conditions for SLC5A1 were 2 min at 94°C; 1 min at 95°C for 35 cycles, 1 min at 55°C, 1 min at 72°C and then 10 min at 72°C. Amplification of the HPRT1 cDNA was used to control the integrity of the cDNA and as internal control to quantify the expression of a given gene in real-time PCR. For quantification studies, the comparative method or 
CT method of relative quantification was used. To compare the amplification efficiency of two target genes (A and B), a dilution series for each gene from a reference cDNA was prepared, and the CT values of target A were subtracted from the CT values of target B. The difference in CT values was then plotted against the logarithm of the dilution series. The slope of the resulting straight line was always <0.1. The relative quantification of the mRNA level of each gene was evaluated by comparison with the mRNA expression of HPRT1 using RelQuant 1.01 software (Roche).
Placental tissue samples (200 mg) were homogenized using a Polytron PT 3000 (Brinkmann Instruments, Mississauga, Ontario, Canada) in 1 ml ice-cold extraction buffer (125 mM Tris-HCl, pH 8.0; 2 mM CaCl2; l.4% (v/v) Triton X-100; 2 µg/ml aprotinin; 1 µg/ml pepstatin; 4 µM leupeptin; and 1 mM PMSF). Homogenates were kept on ice for 30 min and then centrifuged at 10 000 x g for 25 min at 4°C, and the supernatants were collected and stored at –80°C until used.
The fractionation of placental plasma membrane and cytosolic proteins was carried out from 500 mg placental tissue using the Plasma Membrane Protein Extraction Kit according to the manufacturer's instructions (BioVision, Mountain View, CA). The protein concentration was determined using the BCA kit (Pierce, Rockford, IL). SLC2A1 and SLC2A4 in both the plasma membrane and the cytosol fractions were measured by Western blotting.
Total, plasma membrane, and cytosolic proteins were solubilized in sample buffer (4% SDS, 30 mM dithiotheitol, 0.25 M sucrose, 0.01 M EDTA-Na2, and 0.075% bromophenol blue), resolved using a 10% SDS-PAGE, and electroblotted into PVDF membranes (Millipore, Cambridge, ON, Canada) at 1.7 mA/cm2. Thereafter, membranes were incubated in blocking buffer (Tris-buffered saline supplemented with 0.1% Tween-20 [TBS-T] containing 5% skim milk) for 1 h at room temperature, immunoblotted overnight at 4°C with the primary antibody SLC2A1 (1:500), SLC2A4 (1:1000), or SLC5A1 (1:300; all from Alpha Diagnostic International, San Antonio, TX), and diluted with the blocking buffer. Membranes were washed three times in TBS-T, and the probes were incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody IgG (1:2500; Chemicon International Inc., Temecula, CA) in blocking buffer for 1 h at room temperature. Blots then were washed three times with TBS-T, and the detection was performed using the BM Chemiluminescence system (Roche Biochemicals) and visualized by autoradiography (Amersham Bioscience, Little Chalfont, U.K.).
The loading of placental proteins was monitored by the detection of the glyceraldehyde phosphate dehydrogenase (GAPDH) protein (for total and cytosolic proteins) and of the folate binding protein (FBP; for plasma membrane faction). The blotting membrane were stripped and reblotted with a GAPDH or with FBP antibodies. Briefly, nitrocellulose membranes were incubated in a stripping buffer (100 mM 2-mercaptoethanol; 2% SDS; and 62.5 mM Tris–HCl, pH 6.7) for 10 min at room temperature. Thereafter, membranes were washed in washing buffer (1 M Na2HPO4; 1 M NaH2PO4; and 9 g/l NaCl, pH 7.2) for 5 min, blocked in blocking buffer for 1 h at room temperature, and reblotted with the primary monoclonal anti-GAPDH (1:2500; Chemicon International Inc.) or anti-FBP (1:5000; Abcam Inc., Cambridge, MA) for 1 h at room temperature. Membranes were washed three times with TBS-T, and probes were incubated with secondary anti-mouse peroxidase-linked IgG (1:2500; Chemicon International Inc.) or anti-goat peroxidase-linked IgG (1:5000; Chemicon International Inc.) in blocking buffer for 1 h at room temperature. Blots then were washed three times with TBS-T, and the detection was performed using the BM Chemiluminescence system (Roche Biochemicals) and visualized by autoradiography (Amersham Biosciences).
The band intensity was analyzed using Image analysis software (Quantity One software; Bio-Rad Laboratories, Mississauga, Ontario, Canada).
Results were reported as mean ± SEM. The concentration of glucose, insulin, relative mRNA, and protein expression levels were evaluated using the unpaired Student t-test. A P value <0.05 was considered statistically significant.
Maternal Body and Placenta Weight
The maternal body weights of two groups were equivalent at the start of gestation and were 3521.0 ± 42.8 g (n = 38) for control and 3498.2 ± 48.8 g (n = 16) for ECD groups of dams. Also, at Day 30 of gestation period the weights were not significantly different and represented 4036.4 ± 151.6 g and 4220.8 ± 171.2 g for control and ECD groups, respectively (Table 1). Moreover, no difference was observed in the weights of the placentas from the control and ECD groups (3.54 ± 0.12 g [n = 284] for control group and 3.48 ± 0.10 g [n = 139] for ECD group).
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Concentration of Glucose in Dams and Offspring Serum
The impact of maternal cholesterol level during pregnancy was studied on maternal and offspring serum glucose concentrations. The concentration of serum glucose was determined in dams for the control group before mating (Day 0) and for the ECD group before the beginning of treatment (Day –7). Our results demonstrated no significant differences between control and treated groups of rabbits before gestation and at term (Fig. 1A). In addition, the glycemia was not significantly different between both control and ECD dam groups. In contrast, the glucose concentrations in offspring were significantly decreased compared with dam concentration (Fig. 1B). Moreover, glycemia of offspring from the ECD dams was significantly lower compared with offspring from the control group.
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Serum Insulin Level in Dams and Offspring
The serum insulin level also was evaluated for dams and offspring for both control and ECD groups. Our data demonstrated that the serum insulin levels were significantly decreased in both groups of dams at term (Day 30) compared with before gestation (Fig. 2A). The hypercholesterolemia during gestation did not affect the insulin metabolism in hypercholesterolemic mothers. In addition, the insulinemia in offspring from both control and ECD groups was not modified (Fig. 2B).
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Placental SLC2As and SLC5A1 mRNA Expression
The analysis of mRNA expression for SLC2s and SLC5A1 was necessary to determine whether the ECD modified expression levels in placenta. Our RT-PCR study demonstrated the presence of SLC2A1, SLC2A3, and SLC2A4 in rabbit placentas at term (Fig. 3A). In order to quantitatively evaluate the specific expression of glucose transporter in placentas for both groups, real-time RT-PCR was used. No significant difference was observed in GLUT and SLC5A1 mRNA expressions between placentas from the control and ECD groups (Fig. 3B)
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Since mRNA expression level is not necessary correlated with protein levels, we performed Western blot experiments in placentas from control and ECD groups. For SLC2A1 and SLC2A4 proteins, the expected size of the band was 55 kDa (Figs. 4A and 5A), whereas the band for SLC5A1 was 75 kDa (Fig. 6A). The immunoblot analysis of SLC2As expression in placental total protein extract shows that neither SLC2A1 nor SLC2A4 expression levels were affected by maternal hypercholesterolemia (Figs. 4B and 5B). In contrast, in the placental total protein extract obtained from hypercholesterolemic dams, the expression level of SLC5A1 was significantly lower (P < 0.001) than it was in control dams (Fig. 6B).
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Since it is known that SLC2 protein expression on cell membrane surface is regulated by lipid environment, we investigated whether maternal hypercholesterolemia modified the routing of SLC2 to the plasma membrane of placenta. Thus, for this study we determined the expression level of SLC2A1 in plasma membrane (Fig. 7A) and cytosolic fraction (Fig. 8A) isolated from placentas of both groups (control and ECD). The FBP was used as a housekeeping gene for plasma membrane proteins, and it shows a single band at approximately 60 kDa, as described by Malm et al. [23]. Our results demonstrate that in cytosolic fraction a significant increase in SLC2A1 protein in placentas was observed for the hypercholesterolemic group (under ECD) compared with control group (Fig. 7B). In contrast, SLC2A1 obtained from the placental plasma membrane fraction from the ECD group was significantly decreased compared with the control group (Fig. 8B). However, the content of SLC2A4 protein in these placental fractions was similar from both groups, whereas no difference was observed for the SLC2A4 from cytosolic (Fig. 9B) and plasma membrane (Fig. 10B) fractions isolated from the placentas of both groups (control and ECD).
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This is the first study reporting on the influence of hypercholesterolemia during gestation regarding the characterization of the placental glucose transporters and glucose-insulin metabolism in the rabbit. The present paper demonstrates for the first time the presence of SLC5A1 in the placenta of rabbit and its downregulation in presence of hypercholesterolemia. In addition, we demonstrate that maternal hypercholesterolemia affects the routing of SLC2As to the plasma membrane of placenta.
A previous study conducted in our laboratory [22] showed that maternal hypercholesterolemia induced by a maternal diet (ECD) provoked an important increase in serum cholesterol and triglyceride level in the offspring in association with the reduction of their birth weight. Moreover, placentas from the hypercholesterolemic dams have an important lipid accumulation [22] but, in addition to the maternal body weight, the weights of the placentas from the control and hypercholesterolemic dams were not different. It is well established that glucose is a vital substrate for human fetal development and growth, and it has been demonstrated that 1) its placental transfer increases linearly with maternal glycemia, and 2) it is mainly dependent on the concentration of glucose in the maternal circulation [24]. It was not surprising to observe that low-birth weight offspring show decreased serum glucose concentration. Many studies reported that IUGR human fetuses are hypoglycemic and hypoinsulinemic in utero compared with normal birth weight fetuses and that alteration of the fetal availability for the glucose could explain the reduction of the birth weight [16, 25, 26].
One of the most important outcomes of this study was that hypercholesterolemia downregulated the placental content of SLC5A1 protein. Six main isoforms of SLC5A have been demonstrated in the small intestine, heart, and kidney [27], and their function, binding affinity, and capacity have been intensively studied. However, there is little information available on the sodium-glucose cotransporter and its function in the placenta. The presence of the SLC5A2 isoform has been shown in human trophoblast cells in culture. The SLC5A1 is a high-affinity, low-capacity sodium-glucose integral membrane protein that mediates the transport of glucose and, to a much lower affinity, galactose across the plasma membrane by active transport mechanisms involving the cotransport of glucose molecules and sodium ions, with a sodium-to-glucose coupling ratio of 2:1 [27]. Although the expression of SLC5A1 in placenta has not been previously reported, previous studies implied that sodium-related glucose transporters might be present in placental tissues [28–30]. Our results showed for the first time the presence of SLC5A1 in rabbit placenta and indicated that maternal hypercholesterolemia downregulates the expression of SLC5A1 protein without affecting its mRNA levels. This may suggest that the regulation occurs at a posttranscriptional level. Indeed, a study using a renal epithelial cell line showed that posttranscriptional mechanisms play an important role in regulating levels of SLC5A1 following cell differentiation [31]. Thus, additional studies are necessary to evaluate whether SLC5s are involved in placental glucose transport under pathological conditions.
It is also well established that the circulating glucose concentration is the primary determinant for fetal insulin secretion [32]. Then, since insulin is the most important stimulating growth hormone during fetal life, fetal hypoinsulinemia may be a primary mechanism causing restricted fetal growth in the presence of pregnancy complications. Most studies demonstrated decreased plasma insulin level in growth-retarded human and rat pregnancies [25, 33–35]. However, Spencer et al. [26] reported similar values for insulin, proinsulin, and des 31, 32 proinsulin in the cord blood of small for gestational age (SGA) babies. Similarly, we were unable to show significant differences in insulin level in control and ECD offspring. Moreover, the insulinemia was not different for both control and ECD dams. The question of whether insulin regulates placental glucose uptake and transport has been addressed quite extensively and remains controversial. Most studies [36–38], but not all [39], show that insulin does not affect placental glucose transport at term. In our study, the absence of difference in maternal insulinemia for both control and ECD groups confirms the hypothesis that placental glucose transport and metabolism are not sensitive to maternal plasma insulin level.
Proper fetal development is dependent on sufficient oxygen and essential nutrient supply to the fetus. Since substrate transport, including glucose, is directed from the maternal to the fetal circulation under physiological conditions, the placenta's ability to facilitate this transfer is of critical importance to the development of a healthy fetus [7]. Thus, despite the importance of a high-volume flow of glucose for fetal growth and development, there are few data on the expression and activity of glucose transporters in pathological conditions. SLC2A1 is the primary transporter mediating facilitated glucose transfer across the placental barrier in the second half of pregnancy. Also, glucose movement across the basal plasma membrane appears to be the rate-limiting step. Our studies demonstrate that the weight reduction in ECD offspring is not associated with an alteration in the gene expressions of placental SLC2A1, SLC2A3, and SLC2A4. However, it has been shown previously that IUGR caused by maternal epidermal growth factor deficiency results in a reduced mRNA expression of SLC2A3, with no effect in SLC2A1 mRNA expression in placenta [21]. Moreover, decreased levels of SLC2A8 mRNA and protein in placental tissue have been observed in an ovine IUGR model [18]. Placental SLC2A3 gene expression also was reduced under the conditions of food restriction during the last week of gestation in the rats [40]. However, the quantification of the SLC2A3 mRNA expression provides little functional information about protein expression, especially in the placenta, where tissue of both maternal and fetal origin is present. In addition, the relationship between SLC2A3 mRNA and protein expression appears to be inconsistent, implying that posttranscriptional regulation occurs [41]. As a specific antibody against rabbit SLC2A3 is not commercially available, it was not possible to measure its abundance in our placental extract. Therefore, we investigated the expression levels of SLC2A1 and SLC2A4 protein in the placental tissues. The protein expression levels of both SLC2A1 and SLC2A4 in placentas of ECD offspring were not significantly different from those of the control group using total protein extract. Similar results regarding SLC2A1 protein expression and activity were reported in human IUGR placenta [17]. In contrast, Pickard et al. [20] showed a correlation between placental abundance of SLC2A1 protein level and fetal growth retardation, whereas the placental abundance of SLC2A3 protein increased in hypothyroxinemic pregnancy. The reason for the different findings in these works may be due to differences in methods to induce IUGR.
A key mechanism in the regulation of glucose uptake involves the translocation of transporters from the intracellular pool to the plasma membrane through vesicular trafficking [42]. Thus, we investigated whether the presence of an enriched-cholesterol environment could differentially regulate this trafficking into placental cells. This paper reports on the diminution of SLC2A1 protein expression in the placental plasma membrane fraction of ECD dams with the concomitant increase in the cytosolic fraction, whereas any modification of the SLC2A4 protein distribution was observed between these cellular components. Interestingly, under normal conditions Hahn et al. [43] demonstrated that SLC2A1 is predominantly expressed in the plasma membrane of cultured trophoblast cells compared with SLC2A4, whereas SLC2A1 is translocated from the plasma membranes to the intracellular compartment in response to hyperglycemia. In contrast, the intracellular pool of SLC2A1 translocates to the plasma membrane in rat cardiac myocytes [44] and adipocytes [45] after short-term insulin stimulation, and in L6 skeletal muscle cells [46] after long-term insulin treatment. In contrast to SLC2A1, under basal nonstimulated conditions the majority of SLC2A4 is stored in an intracellular section [44]. Insulin is able to translocate SLC2A4 to the cell surface primarily by stimulation of exocytosis [47]. Insulin also decreases the rate of SLC2A4 endocytosis approximately 2- to 3-fold in adipose cells [48]. In our study we found similar levels of insulin for dams of control and ECD groups. Therefore, modification of translocation of SLC2A1 seems to not be regulated by maternal insulinemia. Thus, other factors, such as the membrane lipid environment, could be involved in the regulation of the translocation of SLC2A1 in the presence of maternal hypercholesterolemia. Effectively, morphological observations indicated that the placentas of ECD dams showed an important accumulation of lipids [22]. Moreover, several studies also showed that a high concentration of cholesterol in membranes is correlated with a reduction of membrane fluidity and vice versa [49]. Thus, hypoglycemia in low-birth weight offspring described in the present study could be related to a reduction in SLC2A1 in plasma membrane.
In summary, our study showed that maternal hypercholesterolemia during gestation induces hypoglycemia in offspring without any modification of the maternal glucose, and only reduction of the translocation of the SLC2A1 to the placental plasma membrane. In addition, we reported for the first time the presence of SLC5A1 protein in rabbit placenta and its downregulation by hypercholesterolemia. All these data suggest that the offspring hypoglycemia and low birth weight could be related to a defect in the routing of SLC2A1 according to the lipid cell environment.
ACKNOWLEDGMENTS
We are grateful to Dr. Alain Montoudis for his assistance in the housing rabbits.
FOOTNOTES
1Supported by the National Sciences and Engineering Research of Canada (NSERC). 
Correspondence:
2Julie Lafond, Laboratoire de Physiologie Materno-Ftale, Université du Québec à Montréal, Département des Sciences Biologiques, C.P. 8888, Succ. centre-ville, Montréal, Canada H3C 3P8. FAX: 514 987 4647; e-mail: Lafond.julie{at}uqam.ca
Received: 4 July 2006.
First decision: 23 August 2006.
Accepted: 28 November 2006.
REFERENCES
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  A. N. Sferruzzi-Perri, J. A. Owens, P. Standen, R. L. Taylor, J. S. Robinson, and C. T. Roberts Early Pregnancy Maternal Endocrine Insulin-Like Growth Factor I Programs the Placenta for Increased Functional Capacity throughout Gestation Endocrinology, September 1, 2007; 148(9): 4362 - 4370. [Abstract] [Full Text] [PDF]
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