- Email: [email protected]
Placental Amino Acid Transport and Placental Leptin Resistance in Pregnancies Complicated by Maternal Obesity
Placenta 31 (2010) 718e724
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Placental Amino Acid Transport and Placental Leptin Resistance in Pregnancies Complicated by Maternal Obesityq D.M. Farley*, J. Choi, D.J. Dudley, C. Li, S.L. Jenkins, L. Myatt, P.W. Nathanielsz Center for Pregnancy and Newborn Research, Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, University of Texas Health Science Center at San Antonio, Texas, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 9 June 2010
Hypothesis and study objectives: We hypothesized that maternal obesity is associated with increased placental amino acid transport and hyperleptinemia. Our objectives were to study placental amino acid transport and the effect of leptin on placental amino acid transport in vitro in the setting of maternal obesity. Materials and methods: Seven lean, BMI at entry 22.4, and seven obese, BMI at entry 31.5 (p < 0.001), pregnant women were studied at 39 weeks. We measured baseline and leptin-stimulated placental system A sodium-dependent neutral amino acid transporter (SNAT) activity, placental immunoreactive protein expression of SNAT, leptin and leptin receptor, and maternal and fetal plasma leptin concentrations, with significance set at p 0.05. The primary outcome measure was placental SNAT activity. Results: The obese group had decreased placental SNAT activity (p ¼ 0.005), maternal hyperleptinemia (p ¼ 0.01) and decreased syncytiotrophoblast expression of leptin receptor (p ¼ 0.01) and SNAT-4 (p < 0.001). Placental amino acid uptake was significantly stimulated by leptin in the lean group as compared to the obese group. Maternal weight gain and offspring birth weights were not different between groups. Conclusion: Maternal obesity was accompanied by decreased placental SNAT activity associated with maternal hyperleptinemia and placental leptin resistance in spite of appropriate maternal weight gain and normally grown neonates. These findings suggest altered placental function that may have clinical implications in obese pregnant women. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Pregnancy Obesity Leptin SNAT Placental dysfunction Amino acid transporter
1. Introduction The prevalence of pre-pregnancy obesity has increased and is estimated to be almost 30% among reproductive aged women [1e3]. Moreover the prevalence of obesity is more than 50% in some groups of Hispanic women [4]. Maternal obesity is associated with adverse pregnancy outcomes related to altered placental function (e.g. fetal macrosomia, hypertensive disease and fetal death) although pathogenic mechanisms are not understood [5e13]. Placental dysfunction, in turn, is associated with abnormal amino acid transport [14e16]. Investigation into the effects of obesity on placental amino acid transport is timely and needed.
q Study conducted in San Antonio, Texas, USA. * Corresponding author. Department of Obstetrics and Gynecology, University of Texas Health Science Center at San Antonio, Mail code 7836, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. Tel.: þ1 210 567 5035; fax: þ1 210 567 3013. E-mail address: [email protected] (D.M. Farley). 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.06.006
Fetal growth depends on placental amino acid transport. The system A sodium-dependent neutral amino acid transporter (SNAT) consists of three isoforms (SNAT 1, 2, 4) that are well characterized in the placenta in relation to fetal growth in various situations [16e20]. Decreased placental SNAT activity has been illustrated in pregnancies complicated by fetal growth restriction [21]. However, little is known about the effects of maternal obesity on placental SNAT activity. Leptin is a peptide hormone originally described as being produced by adipocytes with an increase noted in obese patients [22e25]. However, leptin has been found to increase during pregnancy due to placental production [26e30]. Leptin has been shown to increase placental SNAT activity in the first trimester and at term [20]. Also, leptin resistance is apparent in both obesity [22e25] and pregnancy [26e30]. Whether maternal obesity during pregnancy increases leptin resistance or to what extent if any leptin affects placental SNAT activity is not known. We hypothesized that maternal obesity would increase placental SNAT activity due to the association of obesity with fetal overgrowth and maternal hyperleptinemia. The primary outcome
D.M. Farley et al. / Placenta 31 (2010) 718e724
of our study was to evaluate placental SNAT activity in the setting of maternal obesity. Additionally, we studied the effect of leptin on placental SNAT activity in this setting. 2. Materials and methods This study was approved by the Institutional Review Boards of the University of Texas Health Science Center and University Hospital of San Antonio, Texas. After informed consent, blood samples and placentas were collected from 14 patients undergoing elective cesarean delivery at 39 weeks gestation from November 2007 to June 2009. The inclusion criteria were a first trimester entry body mass index (BMI) of 18.5e24.9 kg/m2 for lean and 30e40 kg/m2 for obese subjects [31]. Exclusion criteria were labor, ruptured membranes, and bleeding which were determined by history and physical examination upon admission for delivery. Women with medical complications such as hypertension, pre-gestational and gestational diabetes (abnormal screening and diagnostic glucose testing) were also excluded. Maternal data collected included height and weight at entry and delivery (measured on standard scales approved by the hospital system), pregnancy weight gain, estimated gestational age (by last menstrual period and ultrasound), blood pressure at delivery, and medical and prenatal history. Fasting maternal venous blood samples (EDTA-treated plasma) were obtained preoperatively the morning of delivery. Neonatal data included birth weight, height, ponderal index, and APGAR scores as determined by a neonatal nurse practitioner. Umbilical cord blood samples (EDTAtreated plasma) were taken after delivery. Arterial cord blood (heparin-treated tube) gas analysis was performed within 5 min of delivery (37 C, NICU lab GEM 300014633, internal QA). Blood samples (maintained on ice) and the placenta (not on ice so as to not affect estimation of amino acid transport) were processed in the medical school adjacent to the hospital, which involved minimal transport time (<5 min). Blood samples were centrifuged at 4 C (300 rpm, 90 min) and the plasma was snap frozen and immediately stored at 80 C. Within 30 min of delivery, the placenta, fetal membranes and umbilical cord were trimmed, weighed and measured. Random 1 1-cm full thickness sections of placenta were dissected using a wire grid (12 6-cm wire rectangle with 3 rows of 2 2 cm squares) that was laid on the placenta. Using a random numbering sequence, 10e15 biopsies from each placenta were taken for amino acid transporter analysis, paraffin embedding and frozen storage. Using 6e7 of these biopsies, villous tissue was dissected by removing the chorionic plate and decidual layers. Then, triplicate 5-mm3 ’fragments’ of villous tissue were dissected, hung on specially designed hooks and bathed continuously in fresh buffer (Dulbecco’s Modifed Eagle Medium and Tyrode’s solution, 1:3, pH 7.4, 37 C). 2.1. Placental SNAT activity analysis Placental SNAT activity was measured as an index of placental amino acid transport via uptake of the neutral non-metabolized synthetic amino acid, 14Cmethyl-amino-isobutyric acid (14C-MeAIB, 10 nM). Placental SNAT activity was estimated by amount of 14C-MeAIB uptake into the 5-mm3 villous ’fragments’ over 160 min measured as picomoles of 14C-MeAIB taken up per milligram of villous protein per minute (pmol mg-protein1 min1) [17,20]. The uptake analysis was done within 6 h of delivery so as to approximate in vivo activity as previously described [20]. Briefly, the ’fragments’ were hung on hooks and moved through a series of solutions including an uptake solution containing 14C-MeAIB. Two sets of triplicate hooks were used, one in Naþ-containing solution and one in Naþ-free solution, because system A is Naþ-dependent. The effect of leptin stimulation on placental SNAT activity was measured by incubating villous ’fragments’ in human recombinant leptin (Sigma Corp, 100 ng mL1 or 500 ng mLˉ1) for 60 min just before placing them into uptake solution over 40 min [20]. Leptin concentrations of 100 and 500 ng mL1, which are higher than physiologic levels, were used to study responsiveness of system A because the villous tissue was subject to hyperleptinemia of pregnancy and obesity, thus higher leptin concentrations would be needed to illustrate an effect on placental SNAT activity [20]. After ’fragments’ were in uptake solution, they were placed in chilled Tyrode’s buffer to stop SNAT uptake of 14 C-MeAIB. Then, fragments were incubated in distilled water for 18 h to lyse cell membranes releasing 14C-MeAIB, which was measured with a scintillation counter to estimate 14C counts per minute. Differences in ‘fragment’ size were accounted for by measuring denatured protein concentrations [mg mLˉ1] using the Bradford method. Using 14C-MeAIB standards, amino acid uptake was calculated [pmol mgproteinˉ1 minˉ1]. System A uptake activity is Naþ-dependent and was calculated from the difference of uptake between fragments in Naþ-free and Naþ-containing uptake solution. Transporter activity was quantified using area under the curve (AUC) and linear regression. The AUC was calculated between the 2 uptake curves [pmol mg-proteinˉ1] generated by fragments in Naþ-free and Naþ-containing uptake solution from 5 to 160 min, and AUC values were compared and used for correlations. We estimated the transport activity with linear regression to determine the line of best fit of uptake activity (linear to 120 min); the slope of this line was the uptake rate [pmol mg-proteinˉ1 minˉ1]. Placental SNAT activity was compared between groups. Effect of leptin stimulation on placental SNAT activity was estimated using the 20- and 40-min time points on the leptin-incubated fragments.
719
2.2. Placental immunohistochemistry Tissue sections were cut at 5-mm and 2 random sections from each placenta were analyzed. Antibodies were purchased from Santa Cruz Biotechnology Inc, Santa Cruz, CA. Syncytiotrophoblast expression of leptin (sc-842), leptin receptor (sc-1832, long-form, ObR-b), SNAT isoforms 1 (sc-33441), 2 (sc-33444), and 4 (sc-33448) was studied using standard immunohistochemical techniques [32]. Tissue sections from the lean and obese groups were processed in parallel and handled identically in terms of antibody exposure and processing times. Paraffin blocks were deparaffinized, rehydrated, and antigen retrieval was achieved by microwaving slides submersed in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 1.5% hydrogen peroxideemethanol solution. Sections were rinsed and incubated in 10% blocking serum (leptin e goat serum; leptin receptor, SNAT 1,2,4 e rabbit serum), then rinsed again and incubated overnight at 4 C in primary antibody (primary antibody for leptin - rabbit; primary antibody for leptin receptor, SNAT 1,2,4 e goat) at concentrations previously determined by titration studies (leptin 1:250; leptin receptor 1:50; SNAT 1 1:300; SNAT 2,4 1:100). Concurrent negative controls were treated with nonimmune serum (leptin e nonimmune rabbit serum; leptin receptor, SNAT 1,2,4 e nonimmune goat serum). Sections were incubated in biotinylated secondary antibody, rinsed, then incubated in avidin-biotin (Vector Labs, Burlingame CA) solution diluted in 1:333 Triton X-100. After rinsing with KPBS and 0.175 M sodium acetate, antigens were localized using 3,30 -diaminobenzidine chromagen-nickel sulfate solution. After final rinsing, sections were counterstained, dehydrated and mounted. One examiner (blinded to the source of tissue) recorded images using light microscopy. Twelve pictures were taken (20) of each slide with each image having >50% of the high-power field containing terminal villi. Quantification was performed using the Image J program (NIH) to estimate percent area stained (fraction ¼ area of slide immunostained divided by total area of slide 100 (%)) and the density of staining (arbitrary units). The ObR-b (leptin receptor) was present in both syncytiotrophoblast (threshold 100) and villous stroma (threshold 170), so the difference in fraction and density staining between the syncytiotrophoblast and villous stroma was used to estimate leptin receptor staining in the syncytiotrophoblast.
2.3. Maternal and fetal plasma analyses Maternal fasting plasma insulin levels were determined using the Coat-A-Count I solid-phase radioimmunoassay (Siemens, TKIN1) and the intra-assay coefficient of variation was <1%. Leptin was measured in the maternal and fetal venous plasma by sandwich ELISA (R&D systems, SLP00). All samples were analyzed in the same assay. Intra-assay coefficient of variation was 5.5% for umbilical cord and 10.9% for maternal samples. 125
2.4. Statistical analysis Differences between the two groups were analyzed with Student’s unpaired t test. The ManneWhitney U test was used to compare levels of leptin and insulin as these were not normally distributed and these data are reported as median (interquartile range). Linear regression was used to analyze the placental SNAT activity. Analysis of variance was used to compare the slopes of the regression line between the lean and obese groups. Correlation analysis among the entire study group was performed with linear regression and Pearson’s coefficient. Data are presented as mean SEM, lean data first, and statistical significance was set at p 0.05.
3. Results 3.1. Demographic data Maternal, neonatal, and placental characteristics are presented in Table 1. There were no differences between the two groups except for entry and delivery BMI; maternal weight gain during pregnancy was not different. One lean subject was non-Hispanic white and the remainder of the study population was Hispanic. There were no smokers in either group. Results of the 1-hour glucose screen and maternal blood pressure at delivery between the 2 groups were not different (Table 1). Overall neonatal birth weights were average for gestational age and similar between groups and the ponderal index was the same in each group (Table 1). Umbilical arterial cord pH measurements (7.2 0.1 vs. 7.2 0.1, p ¼ 0.90) and neonatal APGAR scores at 1 min (lean 9, obese 8) and 5 min (lean 9, obese 9) were not different. Other neonatal biographical parameters and placental morphometric measurements were not different between groups (Table 1).
720
D.M. Farley et al. / Placenta 31 (2010) 718e724
Table 1 Patient characteristics. Variable
Lean (n ¼ 7)
Obese (n ¼ 7)
P
Maternal entry BMI (kg mˉ2) Maternal entry weight (lb) Maternal height (in) Maternal delivery BMI (kg mˉ2) Maternal delivery weight (lb) Maternal weight gain (lb) Gestational age at entry (weeks) Gestational age at delivery (weeks) Placenta weight (g) Neonatal birth weight (g) Neonatal wt (g)/placental wt (g) Neonatal ponderal index (kg3 m1) Maternal glucose screen (mg dL1) Maternal insulin [mUnits mLˉ1]a Maternal leptin [ng mLˉ1]a Fetal leptin [ng mLˉ1]a Maternal systolic blood pressure at delivery (mmHg) Maternal diastolic blood pressure at delivery (mmHg)
22.4 0.6 120.6 5.1 60.7 0.8 26.0 0.8 138.4 5.5 17.9 2.1 10.4 1.4 38.9 0.2 529.4 56.1 3240.0 113.2 6.5 0.4 29.6 0.5 98.3 4.2 0.1 (0.0e5.1) 19.5 (16.4e28.2) 5.2 (4.4e9.4) 109.3 5.1 68.3 3.1
31.5 0.7 172.4 6.3 61.5 0.9 35.1 0.8 188.8 5.7 16.6 4.0 11.7 1.3 39.1 0.0 505.8 31.6 3213.0 104.3 6.5 0.4 30.1 0.3 108.7 5.4 3.9 (0.0e7.7) 31.8 (30.6e52.4) 7.8 (5.0e9.9) 115.6 4.5 66.4 3.9
p p p p p p p p p p p p p p p p p p
< < ¼ < < ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
0.001 0.001 0.53 0.001 0.001 0.77 0.52 0.35 0.73 0.86 0.94 0.34 0.16 0.46 0.01 0.38 0.37 0.72
Comparison between groups using unpaired 2-tailed student’s t test, data are mean SEM. a Comparison between groups using ManneWhitney U test, data are median (interquartile range).
3.2. Placental SNAT activity
3.3. Immunohistochemistry
Placental SNAT activity was decreased in the obese group (Fig. 1). Uptake of 14C-MeAIB by the placenta was lower in the obese group (AUC, 610.6 146.0 vs. 257.8 57.1 pmol mg-proteinˉ1, p ¼ 0.04). Leptin stimulation of placental SNAT activity was reduced although not significantly so at both 100 ng mLˉ1 (p ¼ 0.07) and 500 ng mLˉ1 (p ¼ 0.10), see Fig. 1B,C. Placental SNAT activity was inversely correlated to maternal BMI at entry (R ¼ 0.32, p ¼ 0.03) and maternal delivery BMI (R ¼ 0.30, p ¼ 0.04).
Leptin was expressed in both the syncytiotrophoblast and villous stroma and protein abundance was unchanged between groups (fraction, lean 28.4 1.7% vs. obese 26.4 2.2%, p ¼ 0.47; density, lean 8.6 0.5 vs. obese 8.0 0.7, p ¼ 0.52, Fig. 2AeC). Expression of the leptin receptor (ObR-b) was decreased in both the syncytiotrophoblast (fraction, lean 24.7 1.5% vs. obese 18.5 1.4%, p ¼ 0.01; density, lean 7.1 0.4 vs. obese 5.5 0.4, p ¼ 0.01) and villous stroma (fraction, lean 2.1 0.1% vs. obese 1.3 0.1%,
AA-uptake [pmol mg-protein-1]
A *
10
*
5
* 0
0
**
*
25
50
75
100
125
150
Minutes
AA-uptake [pmol mg-protein-1]
2
*
1
0
20 min 1
40 min 1
AA-uptake [pmol mg-protein-1]
C
B
* 2
1
0
20 min
40 min
Fig. 1. A. Placental SNAT activity (pmol mg-proteinˉ minˉ ), term placental villous fragments. Lean (C, n ¼ 7), obese (B, n ¼ 7). Data are mean SEM, *p 0.05 (20, 40, 80, 120 min time points e lean vs. obese), **p ¼ 0.005 (SNAT activity lean vs. obese), amino acid (AA). B, C. Effect of leptin stimulation on placental SNAT activity (pmol mg-proteinˉ1 minˉ1), at concentrations of 100 ng mLˉ1 (B, lean vs. obese p ¼ 0.07), 500 ng mLˉ1 (C, lean vs. obese p ¼ 0.10) in term placental villous fragments. Lean (A, n ¼ 7), obese (>, n ¼ 7), lean without leptin (C, n ¼ 7), obese without leptin (B, n ¼ 7). Data are mean SEM. *(A. p ¼ 0.004, B. p ¼ 0.047 lean with leptin, A vs. obese with leptin, >).
D.M. Farley et al. / Placenta 31 (2010) 718e724
721
Fig. 2. Immunohistochemistry for leptin (A,B) and leptin (long-form) receptor (D,E) protein expression in placenta terminal villi (60) at 39 weeks gestation (C and F are negative controls). Lean (A,D), obese (B,E). Area (G) and intensity (H) of immunostaining for leptin receptor were quantified. Data are mean SEM, *p ¼ 0.01.
p < 0.001; density, lean 0.3 0.0 vs. obese 0.2 0.0, p < 0.001) in the obese group (Fig. 2DeH). Negative control sections revealed no detectable staining (Fig. 3C,F). Syncytiotrophoblast expression of SNAT 1 (fraction, lean 19.5 1.5% vs. obese 16.8 2.0%, p ¼ 0.31; density, lean 6.5 0.5 vs. obese 5.7 0.7, p ¼ 0.30, Fig. 3AeC) and SNAT 2 (fraction, lean 17.9 1.4% vs. obese 14.0 2.0%, p ¼ 0.15; density, lean 5.5 0.4 vs. obese 4.4 0.6, p ¼ 0.14, Fig. 3DeF) were unchanged. Syncytiotrophoblast expression of the SNAT 4 isoform (fraction, lean 24.9 1.9% vs. obese 8.9 1.4%, p < 0.001; density, lean 8.3 0.6 vs. obese 3.1 0.5, p < 0.001, Fig. 3GeK) was decreased in the obese group. Negative control sections revealed no detectable staining (Fig. 3C,F,I). Placental syncytiotrophoblast leptin receptor (ObR-b) expression was directly correlated to placental SNAT 4 expression (R ¼ 0.29, p ¼ 0.048). 3.4. Plasma insulin and leptin concentrations Fasting maternal venous insulin levels were not different between groups, while leptin concentration was increased in the obese group (p ¼ 0.01) and fetal leptin levels were unchanged (Table 1). Maternal plasma leptin was directly correlated to
maternal entry BMI (R ¼ 0.32, p ¼ 0.04) and to maternal delivery BMI (R ¼ 0.47, p < 0.001). Maternal plasma leptin was inversely correlated to placental SNAT activity (R ¼ 0.39, p ¼ 0.02). 4. Discussion Contrary to our hypothesis placental amino acid transport at term was decreased in the setting of maternal obesity. Our finding of maternal hyperleptinemia in the obese group is consistent with previous reports [26e30]. The decreased placental amino acid transport appears related to maternal hyperleptinemia in keeping with previous data that increased maternal leptin levels are associated with placental dysfunction [33]. Our data support the idea of placental leptin resistance in maternal obesity based on syncytiotrophoblast down regulation of leptin receptor in the setting of maternal hyperleptinemia. Furthermore, syncytiotrophoblast leptin protein abundance was the same in our two groups despite the presence of maternal hyperleptinemia in the obese group which may have induced syncytiotrophoblast leptin receptor down regulation. Under the conditions used placental MeAIB uptake was stimulated by leptin in the lean but not obese group during 40 min
722
D.M. Farley et al. / Placenta 31 (2010) 718e724
Fig. 3. Immunohistochemistry for SNAT 1 (A,B), SNAT 2 (D,E) and SNAT 4 (G,H) protein expression in placenta terminal villi (60) at 39 weeks gestation (C, F, and I are negative controls). Lean (A,D,G), obese (B,E,H). Area (J) and intensity (K) of immunostaining for SNAT 4 were quantified. Data are mean SEM, *p < 0.001.
of incubation. The fact that leptin-stimulated AA uptake rate was not significantly different between groups was likely due to power and time of incubation which are recognized limitations. However, we speculate this may be a long-term consequence of maternal hyperleptinemia throughout pregnancy in the obese group.
Normal placentas can increase SNAT activity in a substratespecific and dose-dependent manner to optimize fetal growth in the presence of amino acid limitation and there is a degree of adaptive regulation in normal placentas [34]. Our findings that placental SNAT activity is decreased in maternal obesity may
D.M. Farley et al. / Placenta 31 (2010) 718e724
represent a placental adaptive regulation. However, the obese group gained the recommended amount of weight and we feel this contributed to the appropriately grown neonates. A limitation of our study is that we do not have maternal serum amino acid concentrations, which if decreased compared to the lean group when gaining the recommended amount of weight, may support our belief that decreased SNAT activity represents a degree of placental dysfunction, not adaptive regulation. We feel that the decrease in SNAT 4 expression is evidence that this isoform is likely to contribute most to the decrease in overall SNAT activity between these 2 groups as SNAT 1 and 2 expression were not different. The contribution of SNAT 4 to placental system A amino acid transport has been shown to be less at term [19], a time when it has been speculated that SNAT 1 and 2 may contribute more. However, SNAT 4 expression has been shown to be increased at term, whereas SNAT 1 and 2 expression was unchanged [35]. To determine what isoform contributes most to the decrease in system A activity that we found in maternal obesity is an area of future research. The SNAT 4 isoform is a major component of the placental system A amino acid transporter [19] and the decrease in this isoform in the absence of changes in other SNAT isoforms would indicate that SNAT 4 plays a major role in decreased amino acid transport in the setting of maternal obesity and potentially in placental dysfunction. Notably, SNAT 4 is an imprinted gene susceptible to regulation by levels of methylation [36]. Alterations in methylation may be one mechanism that explains the increased incidence of birth defects associated with maternal obesity [37e39]. Our goal was to enroll well defined lean and obese mothers at entry and delivery and exclude patients with medical co-morbidities to increase the likelihood that the changes we observed in amino acid transport were due to maternal hyperleptinemia and placental leptin resistance. The plasma leptin levels in our lean group were consistent with previous reports in pregnancy [26e30] as was the stimulatory effect of leptin on placental SNAT activity [20]. The lack of statistical significance when measuring the effect of leptin on placental SNAT activity was likely secondary to power with respect to this variable since it was not the primary outcome. However, placental SNAT activity was inversely correlated with maternal plasma leptin concentration. This finding indicates that the decreased placental amino acid transport we found in the setting of maternal obesity may be related to maternal hyperleptinemia and development of placental leptin resistance evidenced by leptin receptor down regulation in association with a decrease in SNAT 4 expression. Neither group had overt insulin resistance evidenced by normal antenatal screening glucose challenge tests and maternal fasting plasma insulin levels in the normal range on the day of delivery [40]. The absence of fetal overgrowth in our obese group may be explained as follows. Hyperleptinemia in the obese group may have attenuated the tendency of maternal obesity to increase fetal growth in some way. Leptin correlates negatively with insulin sensitivity in pregnancy [41] so the possibility of hyperleptinemia increasing insulin sensitivity and attenuating fetal growth is unlikely [42]. We speculate that maternal hyperleptinemia impairs placental amino acid transport. In addition, maternal weight gain in the obese group was consistent with current recommendations [43], but maternal weight gain in the lean group was not. Gaining the recommended amount of weight in the obese group may have prevented excessive fetal growth. Furthermore, if gaining the recommended amount of weight in the obese group prevents excessive fetal growth, then our finding of decreased placental amino acid transport signifies altered placental function despite normal fetal growth. Further research is needed to determine whether gaining the recommended amount of weight prevents excess fetal growth and the clinical significance of decreased placental amino acid transport in the setting of maternal obesity.
723
We conclude that maternal obesity and associated hyperleptinemia appear to have a negative impact on placental amino acid transport. Our data suggest a certain degree of altered placental function that may have clinical implications in obese pregnant women. Further studies on the overweight and morbidly obese maternal populations are needed to determine if the negative impact on placental amino acid transport seen in our study is part of a continuum. Acknowledgements We are grateful to Dr. Jocelyn Glazier, Dr. Colin Sibley and Ralf Wimmer for their help with setting up the amino acid fragment uptake method. A special thanks to Dr. Elly Xenakis for her intellectual input, and the labor and delivery staff at University Hospital for assistance in collecting specimens, Susan Carvajal for her help with the neonatal measurements, Greg Langone for histology expertise, Michelle Zavala for assistance with the ELISA technique, Phylis Eagen at the Texas Diabetes Institute, and the University of Texas Health Science Center at San Antonio’s radiation safety laboratory for use of their scintillation counter. This study was approved by University of Texas Health Science Center at San Antonio’s IRB and the ID number is HSC20070723H. This study was supported by HD 21350-17. References [1] Kim SY, Dietz PM, England L, Morrow B, Callaghan WM. Trends in pre-pregnancy obesity in nine states, 1993e2003. Obesity (Silver Spring) 2007;15 (4):986e93. [2] Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of overweight and obesity in the United States, 1999e2004. JAMA 2006;295:1549e55. [3] Artal R, Lockwood CJ, Brown HL. Weight gain recommendations in pregnancy and the obesity epidemic. Obstet Gynecol 2010;115(1):152e5. [4] Wang Y, Beydoun MA. The obesity epidemic in the United Statesdgender, age, socioeconomic, racial/ethnic, and geographic characteristics: a systematic review and meta-regression analysis. Epidemiol Rev 2007;29:6e28. [5] Cnattingius S, Bergstrom R, Lipworth L, Kramer MS. Prepregnancy weight and the risk of adverse pregnancy outcomes. N Engl J Med 1998;338:147e52. [6] Leddy MA, Power ML, Schulkin J. The impact of obesity on maternal and fetal health. Rev Obstet Gynecol 2008;1(4):170e8. [7] Villamor E, Cnattingius S. Interpregnancy weight change and risk of adverse pregnancy outcomes: a population-based study. Lancet 2006;368:1164e70. [8] Chu SY, Kim SY, Lau J, Schmid CH, Dietz PM, Callaghan WM, et al. Maternal obesity and risk of stillbirth: a metaanalysis. Am J Obstet Gynecol 2007;197 (3):223e8. [9] Crane JM, White J, Murphy P, Burrage L, Hutchens D. The effect of gestational weight gain by body mass index on maternal and neonatal outcomes. J Obstet Gynaecol Can 2009;31(1):28e35. [10] Nohr EA, Vaeth M, Bech BH, Henriksen TB, Cnattingius S, Olsen J. Maternal obesity and neonatal mortality according to subtypes of preterm birth. Obstet Gynecol 2007;110:1083e90. [11] Kristensen J, Vestergaard M, Wisborg K, Kesmodel U, Secher NJ. Pre-pregnancy weight and the risk of stillbirth and neonatal death. BJOG 2005;112 (4):403e8. [12] Bodnar LM, Catov JM, Klebanoff MA, Ness RB, Roberts JM. Prepregnancy body mass index and the occurrence of severe hypertensive disorders of pregnancy. Epidemiology 2007;18(2):234e9. [13] Bhattacharya S, Campbell DM, Liston WA, Bhattacharya S. Effect of Body Mass Index on pregnancy outcomes in nulliparous women delivering singleton babies. BMC Public Health 2007;7:168. [14] Roos S, Powell TL, Jansson T. Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation. Am J Physiol Regul Integr Comp Physiol 2004;287(4):R886e93. [15] Jansson T. Amino acid transporters in the human placenta. Pediatr Res 2001;49(2):141e7. [16] Kuruvilla AG, D’Souza SW, Glazier JD, Mahendran D, Maresh MJ, Sibley CP. Altered activity of the system A amino acid transporter in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women. J Clin Invest 1994;94(2):689e95. [17] Greenwood SL, Sibley CP. In vitro methods for studying human placental amino acid transport placental villous fragments. Methods Mol Med 2006;122:253e64. [18] Jones HN, Powell TL, Jansson T. Regulation of placental nutrient transportea review. Placenta 2007;28(8e9):763e74.
724
D.M. Farley et al. / Placenta 31 (2010) 718e724
[19] Desforges M, Mynett KJ, Jones RL, Greenwood SL, Westwood M, Sibley CP, et al. The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane. J Phys 2009;587.1:61e72. [20] Jansson N, Greenwood SL, Johansson BR, Powell TL, Jansson T. Leptin stimulates the activity of the system A amino acid transporter in human placental villous fragments. J Clin Endocrinol Metab 2003;88(3):1205e11. [21] Glazier JD, Cetin I, Perugino G, Ronzoni S, Grey AM, Mahendran D, et al. Association between the activity of the system A amino acid transporter in the microvillous plasma membrane of the human placenta and severity of fetal compromise in intrauterine growth restriction. Pediatr Res 1997;42 (4):514e9. [22] Havel PJ, Kasim-Karakas S, Mueller W, Johnson PR, Gingerich RL, Stern JS. Relationship of plasma leptin to plasma insulin and adiposity in normal weight and overweight women: effects of dietary fat content and sustained weight loss. J Clin Endocrinol Metab 1996;81(12):4406e13. [23] Ogier V, Ziegler O, Mejean L, Nicolas JP, Stricker-Krongrad A. Obesity is associated with decreasing levels of the circulating soluble leptin receptor in humans. Int J Obes Relat Metab Disord 2002;26(4):496e503. [24] Van Dielen FM, van’t Veer C, Buurman WA, Greve JW. Leptin and soluble leptin receptor levels in obese and weight-losing individuals. J Clin Endocrinol Metab 2002;87(4):1708e16. [25] Vendrell J, Broch M, Vilarrasa N, Molina A, Gomez JM, Gutierrrez C, et al. Resistin, adiponectin, ghrelin, leptin, and proinflammatory cytokines: relationships in obesity. Obes Res 2004;12(6):962e71. [26] Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, et al. Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 1997;3(9):1029e33. [27] Sattar N, Greer IA, Pirwani I, Gibson J, Wallace AM. Leptin levels in pregnancy: marker for fat accumulation and mobilization? Acta Obstet Gynecol Scand 1998;77(3):278e83. [28] Hauguel-de Mouzon S, Lepercq J, Catalano P. The known and unknown of leptin in pregnancy. Am J Obstet Gynecol 2006;194:1537e45. [29] Highman T, Friedman J, Huston L, Wong W, Catalano P. Longitudinal changes in maternal leptin serum concentrations, body composition and resting metabolic rate in pregnancy. Am J Obstet Gynecol 1998;178:1010e5. [30] Schubring C, Kiess W, Englaro P, Rascher W, Dotsch J, Hanitsch S, et al. Levels of leptin in maternal serum, amniotic fluid, and arterial and venous cord
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38] [39] [40] [41]
[42] [43]
blood: relation to neonatal and placental weight. J Clin Endocrinol Metab 1997;82(5):1480e3. Borecki IB, Higgins M, Schreiner PJ, Arnett DK, Mayer-Davis E, Hunt SC, et al. Evidence for multiple determinants of the body mass index: the National Heart, Lung, and Blood Institute Family Heart Study. Obes Res 1998;6 (2):107e14. Li C, Levitz M, Hubbard GB, Jenkins SL, Han V, Ferry Jr RJ, et al. The IGF axis in baboon pregnancy: placental and systemic responses to feeding 70% global Ad libitum diet. Placenta 2007;28(11e12):1200e10. Lepercq J, Guerre-Millo M, Andre J, Cauzac M, Hauguel-de Mouzon S. Leptin: a potential marker of placental insufficiency. Gynecol Obstet Invest 2003;55 (3):151e5. Parrott MS, Versen-Hoeynck Fv, Ness RB, Markovik N, Roberts J. System A amino acid transporter activity in term placenta is substrate specific and inversely related to amino acid concentration. Reprod Sci 2007;14(7):687e93. Desforges M, Lacey HA, Glazier JD, Greenwood SL, Mynett KJ, Speake PF, et al. SNAT4 isoform of system A amino acid transporter is expressed in human placenta. Am J Physiol Cell Physiol 2006;290:305e12. Smith RJ, Dean W, Konfortova G, Kelsey G. Identification of novel imprinted genes in a genome-wide screen for maternal methylation. Genome Res 2003;13:558e69. Waller DK, Shaw GM, Rasmussen SA, Hobbs CA, Canfield MA, Siega-Riz AM, et al. Prepregnancy obesity as a risk factor for structural birth defects. Arch Pediatr Adolesc Med 2007;161(8):745e50. Koleganova N, Piecha G, Ritz E. Prenatal causes of kidney disease. Blood Purif 2009;27(1):48e52. Henriksen T, Haugen G, Bollerslev J, Kolset SO, Drevon CA, Iverson PO, et al. Fetal nutrition and future health. Tidsskr Nor Laegeforen 2005;125(4):442e4. Laakso M. How good a marker is insulin level for insulin resistance? Am J Epidemiol 1993;137(9):959e65. McIntyre HD, Chang AM, Callaway LK, Cowley DM, Dyer AR, Radaelli T, et al. Hormonal and metabolic factors associated with variations in insulin sensitivity in human pregnancy. Diabetes Care 2010;33:356e60. Sagawa N, Yura S, Itoh H, Mise H, Kakui K, Korita D, et al. Role of leptin in pregnancy e a review. Placenta 2002;23(Suppl. A16):S80e6. Rasmussen KM, Yaktine AL, editors. Weight gain during pregnancy: reexamining the recommendations. Washington, DC: The National Academies Press; May 2009. Report brief.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Placental Amino Acid Transport and Placental Leptin Resistance in Pregnancies Complicated by Maternal Obesityq D.M. Farley*, J. Choi, D.J. Dudley, C. Li, S.L. Jenkins, L. Myatt, P.W. Nathanielsz Center for Pregnancy and Newborn Research, Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, University of Texas Health Science Center at San Antonio, Texas, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 9 June 2010
Hypothesis and study objectives: We hypothesized that maternal obesity is associated with increased placental amino acid transport and hyperleptinemia. Our objectives were to study placental amino acid transport and the effect of leptin on placental amino acid transport in vitro in the setting of maternal obesity. Materials and methods: Seven lean, BMI at entry 22.4, and seven obese, BMI at entry 31.5 (p < 0.001), pregnant women were studied at 39 weeks. We measured baseline and leptin-stimulated placental system A sodium-dependent neutral amino acid transporter (SNAT) activity, placental immunoreactive protein expression of SNAT, leptin and leptin receptor, and maternal and fetal plasma leptin concentrations, with significance set at p 0.05. The primary outcome measure was placental SNAT activity. Results: The obese group had decreased placental SNAT activity (p ¼ 0.005), maternal hyperleptinemia (p ¼ 0.01) and decreased syncytiotrophoblast expression of leptin receptor (p ¼ 0.01) and SNAT-4 (p < 0.001). Placental amino acid uptake was significantly stimulated by leptin in the lean group as compared to the obese group. Maternal weight gain and offspring birth weights were not different between groups. Conclusion: Maternal obesity was accompanied by decreased placental SNAT activity associated with maternal hyperleptinemia and placental leptin resistance in spite of appropriate maternal weight gain and normally grown neonates. These findings suggest altered placental function that may have clinical implications in obese pregnant women. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Pregnancy Obesity Leptin SNAT Placental dysfunction Amino acid transporter
1. Introduction The prevalence of pre-pregnancy obesity has increased and is estimated to be almost 30% among reproductive aged women [1e3]. Moreover the prevalence of obesity is more than 50% in some groups of Hispanic women [4]. Maternal obesity is associated with adverse pregnancy outcomes related to altered placental function (e.g. fetal macrosomia, hypertensive disease and fetal death) although pathogenic mechanisms are not understood [5e13]. Placental dysfunction, in turn, is associated with abnormal amino acid transport [14e16]. Investigation into the effects of obesity on placental amino acid transport is timely and needed.
q Study conducted in San Antonio, Texas, USA. * Corresponding author. Department of Obstetrics and Gynecology, University of Texas Health Science Center at San Antonio, Mail code 7836, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. Tel.: þ1 210 567 5035; fax: þ1 210 567 3013. E-mail address: [email protected] (D.M. Farley). 0143-4004/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2010.06.006
Fetal growth depends on placental amino acid transport. The system A sodium-dependent neutral amino acid transporter (SNAT) consists of three isoforms (SNAT 1, 2, 4) that are well characterized in the placenta in relation to fetal growth in various situations [16e20]. Decreased placental SNAT activity has been illustrated in pregnancies complicated by fetal growth restriction [21]. However, little is known about the effects of maternal obesity on placental SNAT activity. Leptin is a peptide hormone originally described as being produced by adipocytes with an increase noted in obese patients [22e25]. However, leptin has been found to increase during pregnancy due to placental production [26e30]. Leptin has been shown to increase placental SNAT activity in the first trimester and at term [20]. Also, leptin resistance is apparent in both obesity [22e25] and pregnancy [26e30]. Whether maternal obesity during pregnancy increases leptin resistance or to what extent if any leptin affects placental SNAT activity is not known. We hypothesized that maternal obesity would increase placental SNAT activity due to the association of obesity with fetal overgrowth and maternal hyperleptinemia. The primary outcome
D.M. Farley et al. / Placenta 31 (2010) 718e724
of our study was to evaluate placental SNAT activity in the setting of maternal obesity. Additionally, we studied the effect of leptin on placental SNAT activity in this setting. 2. Materials and methods This study was approved by the Institutional Review Boards of the University of Texas Health Science Center and University Hospital of San Antonio, Texas. After informed consent, blood samples and placentas were collected from 14 patients undergoing elective cesarean delivery at 39 weeks gestation from November 2007 to June 2009. The inclusion criteria were a first trimester entry body mass index (BMI) of 18.5e24.9 kg/m2 for lean and 30e40 kg/m2 for obese subjects [31]. Exclusion criteria were labor, ruptured membranes, and bleeding which were determined by history and physical examination upon admission for delivery. Women with medical complications such as hypertension, pre-gestational and gestational diabetes (abnormal screening and diagnostic glucose testing) were also excluded. Maternal data collected included height and weight at entry and delivery (measured on standard scales approved by the hospital system), pregnancy weight gain, estimated gestational age (by last menstrual period and ultrasound), blood pressure at delivery, and medical and prenatal history. Fasting maternal venous blood samples (EDTA-treated plasma) were obtained preoperatively the morning of delivery. Neonatal data included birth weight, height, ponderal index, and APGAR scores as determined by a neonatal nurse practitioner. Umbilical cord blood samples (EDTAtreated plasma) were taken after delivery. Arterial cord blood (heparin-treated tube) gas analysis was performed within 5 min of delivery (37 C, NICU lab GEM 300014633, internal QA). Blood samples (maintained on ice) and the placenta (not on ice so as to not affect estimation of amino acid transport) were processed in the medical school adjacent to the hospital, which involved minimal transport time (<5 min). Blood samples were centrifuged at 4 C (300 rpm, 90 min) and the plasma was snap frozen and immediately stored at 80 C. Within 30 min of delivery, the placenta, fetal membranes and umbilical cord were trimmed, weighed and measured. Random 1 1-cm full thickness sections of placenta were dissected using a wire grid (12 6-cm wire rectangle with 3 rows of 2 2 cm squares) that was laid on the placenta. Using a random numbering sequence, 10e15 biopsies from each placenta were taken for amino acid transporter analysis, paraffin embedding and frozen storage. Using 6e7 of these biopsies, villous tissue was dissected by removing the chorionic plate and decidual layers. Then, triplicate 5-mm3 ’fragments’ of villous tissue were dissected, hung on specially designed hooks and bathed continuously in fresh buffer (Dulbecco’s Modifed Eagle Medium and Tyrode’s solution, 1:3, pH 7.4, 37 C). 2.1. Placental SNAT activity analysis Placental SNAT activity was measured as an index of placental amino acid transport via uptake of the neutral non-metabolized synthetic amino acid, 14Cmethyl-amino-isobutyric acid (14C-MeAIB, 10 nM). Placental SNAT activity was estimated by amount of 14C-MeAIB uptake into the 5-mm3 villous ’fragments’ over 160 min measured as picomoles of 14C-MeAIB taken up per milligram of villous protein per minute (pmol mg-protein1 min1) [17,20]. The uptake analysis was done within 6 h of delivery so as to approximate in vivo activity as previously described [20]. Briefly, the ’fragments’ were hung on hooks and moved through a series of solutions including an uptake solution containing 14C-MeAIB. Two sets of triplicate hooks were used, one in Naþ-containing solution and one in Naþ-free solution, because system A is Naþ-dependent. The effect of leptin stimulation on placental SNAT activity was measured by incubating villous ’fragments’ in human recombinant leptin (Sigma Corp, 100 ng mL1 or 500 ng mLˉ1) for 60 min just before placing them into uptake solution over 40 min [20]. Leptin concentrations of 100 and 500 ng mL1, which are higher than physiologic levels, were used to study responsiveness of system A because the villous tissue was subject to hyperleptinemia of pregnancy and obesity, thus higher leptin concentrations would be needed to illustrate an effect on placental SNAT activity [20]. After ’fragments’ were in uptake solution, they were placed in chilled Tyrode’s buffer to stop SNAT uptake of 14 C-MeAIB. Then, fragments were incubated in distilled water for 18 h to lyse cell membranes releasing 14C-MeAIB, which was measured with a scintillation counter to estimate 14C counts per minute. Differences in ‘fragment’ size were accounted for by measuring denatured protein concentrations [mg mLˉ1] using the Bradford method. Using 14C-MeAIB standards, amino acid uptake was calculated [pmol mgproteinˉ1 minˉ1]. System A uptake activity is Naþ-dependent and was calculated from the difference of uptake between fragments in Naþ-free and Naþ-containing uptake solution. Transporter activity was quantified using area under the curve (AUC) and linear regression. The AUC was calculated between the 2 uptake curves [pmol mg-proteinˉ1] generated by fragments in Naþ-free and Naþ-containing uptake solution from 5 to 160 min, and AUC values were compared and used for correlations. We estimated the transport activity with linear regression to determine the line of best fit of uptake activity (linear to 120 min); the slope of this line was the uptake rate [pmol mg-proteinˉ1 minˉ1]. Placental SNAT activity was compared between groups. Effect of leptin stimulation on placental SNAT activity was estimated using the 20- and 40-min time points on the leptin-incubated fragments.
719
2.2. Placental immunohistochemistry Tissue sections were cut at 5-mm and 2 random sections from each placenta were analyzed. Antibodies were purchased from Santa Cruz Biotechnology Inc, Santa Cruz, CA. Syncytiotrophoblast expression of leptin (sc-842), leptin receptor (sc-1832, long-form, ObR-b), SNAT isoforms 1 (sc-33441), 2 (sc-33444), and 4 (sc-33448) was studied using standard immunohistochemical techniques [32]. Tissue sections from the lean and obese groups were processed in parallel and handled identically in terms of antibody exposure and processing times. Paraffin blocks were deparaffinized, rehydrated, and antigen retrieval was achieved by microwaving slides submersed in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 1.5% hydrogen peroxideemethanol solution. Sections were rinsed and incubated in 10% blocking serum (leptin e goat serum; leptin receptor, SNAT 1,2,4 e rabbit serum), then rinsed again and incubated overnight at 4 C in primary antibody (primary antibody for leptin - rabbit; primary antibody for leptin receptor, SNAT 1,2,4 e goat) at concentrations previously determined by titration studies (leptin 1:250; leptin receptor 1:50; SNAT 1 1:300; SNAT 2,4 1:100). Concurrent negative controls were treated with nonimmune serum (leptin e nonimmune rabbit serum; leptin receptor, SNAT 1,2,4 e nonimmune goat serum). Sections were incubated in biotinylated secondary antibody, rinsed, then incubated in avidin-biotin (Vector Labs, Burlingame CA) solution diluted in 1:333 Triton X-100. After rinsing with KPBS and 0.175 M sodium acetate, antigens were localized using 3,30 -diaminobenzidine chromagen-nickel sulfate solution. After final rinsing, sections were counterstained, dehydrated and mounted. One examiner (blinded to the source of tissue) recorded images using light microscopy. Twelve pictures were taken (20) of each slide with each image having >50% of the high-power field containing terminal villi. Quantification was performed using the Image J program (NIH) to estimate percent area stained (fraction ¼ area of slide immunostained divided by total area of slide 100 (%)) and the density of staining (arbitrary units). The ObR-b (leptin receptor) was present in both syncytiotrophoblast (threshold 100) and villous stroma (threshold 170), so the difference in fraction and density staining between the syncytiotrophoblast and villous stroma was used to estimate leptin receptor staining in the syncytiotrophoblast.
2.3. Maternal and fetal plasma analyses Maternal fasting plasma insulin levels were determined using the Coat-A-Count I solid-phase radioimmunoassay (Siemens, TKIN1) and the intra-assay coefficient of variation was <1%. Leptin was measured in the maternal and fetal venous plasma by sandwich ELISA (R&D systems, SLP00). All samples were analyzed in the same assay. Intra-assay coefficient of variation was 5.5% for umbilical cord and 10.9% for maternal samples. 125
2.4. Statistical analysis Differences between the two groups were analyzed with Student’s unpaired t test. The ManneWhitney U test was used to compare levels of leptin and insulin as these were not normally distributed and these data are reported as median (interquartile range). Linear regression was used to analyze the placental SNAT activity. Analysis of variance was used to compare the slopes of the regression line between the lean and obese groups. Correlation analysis among the entire study group was performed with linear regression and Pearson’s coefficient. Data are presented as mean SEM, lean data first, and statistical significance was set at p 0.05.
3. Results 3.1. Demographic data Maternal, neonatal, and placental characteristics are presented in Table 1. There were no differences between the two groups except for entry and delivery BMI; maternal weight gain during pregnancy was not different. One lean subject was non-Hispanic white and the remainder of the study population was Hispanic. There were no smokers in either group. Results of the 1-hour glucose screen and maternal blood pressure at delivery between the 2 groups were not different (Table 1). Overall neonatal birth weights were average for gestational age and similar between groups and the ponderal index was the same in each group (Table 1). Umbilical arterial cord pH measurements (7.2 0.1 vs. 7.2 0.1, p ¼ 0.90) and neonatal APGAR scores at 1 min (lean 9, obese 8) and 5 min (lean 9, obese 9) were not different. Other neonatal biographical parameters and placental morphometric measurements were not different between groups (Table 1).
720
D.M. Farley et al. / Placenta 31 (2010) 718e724
Table 1 Patient characteristics. Variable
Lean (n ¼ 7)
Obese (n ¼ 7)
P
Maternal entry BMI (kg mˉ2) Maternal entry weight (lb) Maternal height (in) Maternal delivery BMI (kg mˉ2) Maternal delivery weight (lb) Maternal weight gain (lb) Gestational age at entry (weeks) Gestational age at delivery (weeks) Placenta weight (g) Neonatal birth weight (g) Neonatal wt (g)/placental wt (g) Neonatal ponderal index (kg3 m1) Maternal glucose screen (mg dL1) Maternal insulin [mUnits mLˉ1]a Maternal leptin [ng mLˉ1]a Fetal leptin [ng mLˉ1]a Maternal systolic blood pressure at delivery (mmHg) Maternal diastolic blood pressure at delivery (mmHg)
22.4 0.6 120.6 5.1 60.7 0.8 26.0 0.8 138.4 5.5 17.9 2.1 10.4 1.4 38.9 0.2 529.4 56.1 3240.0 113.2 6.5 0.4 29.6 0.5 98.3 4.2 0.1 (0.0e5.1) 19.5 (16.4e28.2) 5.2 (4.4e9.4) 109.3 5.1 68.3 3.1
31.5 0.7 172.4 6.3 61.5 0.9 35.1 0.8 188.8 5.7 16.6 4.0 11.7 1.3 39.1 0.0 505.8 31.6 3213.0 104.3 6.5 0.4 30.1 0.3 108.7 5.4 3.9 (0.0e7.7) 31.8 (30.6e52.4) 7.8 (5.0e9.9) 115.6 4.5 66.4 3.9
p p p p p p p p p p p p p p p p p p
< < ¼ < < ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
0.001 0.001 0.53 0.001 0.001 0.77 0.52 0.35 0.73 0.86 0.94 0.34 0.16 0.46 0.01 0.38 0.37 0.72
Comparison between groups using unpaired 2-tailed student’s t test, data are mean SEM. a Comparison between groups using ManneWhitney U test, data are median (interquartile range).
3.2. Placental SNAT activity
3.3. Immunohistochemistry
Placental SNAT activity was decreased in the obese group (Fig. 1). Uptake of 14C-MeAIB by the placenta was lower in the obese group (AUC, 610.6 146.0 vs. 257.8 57.1 pmol mg-proteinˉ1, p ¼ 0.04). Leptin stimulation of placental SNAT activity was reduced although not significantly so at both 100 ng mLˉ1 (p ¼ 0.07) and 500 ng mLˉ1 (p ¼ 0.10), see Fig. 1B,C. Placental SNAT activity was inversely correlated to maternal BMI at entry (R ¼ 0.32, p ¼ 0.03) and maternal delivery BMI (R ¼ 0.30, p ¼ 0.04).
Leptin was expressed in both the syncytiotrophoblast and villous stroma and protein abundance was unchanged between groups (fraction, lean 28.4 1.7% vs. obese 26.4 2.2%, p ¼ 0.47; density, lean 8.6 0.5 vs. obese 8.0 0.7, p ¼ 0.52, Fig. 2AeC). Expression of the leptin receptor (ObR-b) was decreased in both the syncytiotrophoblast (fraction, lean 24.7 1.5% vs. obese 18.5 1.4%, p ¼ 0.01; density, lean 7.1 0.4 vs. obese 5.5 0.4, p ¼ 0.01) and villous stroma (fraction, lean 2.1 0.1% vs. obese 1.3 0.1%,
AA-uptake [pmol mg-protein-1]
A *
10
*
5
* 0
0
**
*
25
50
75
100
125
150
Minutes
AA-uptake [pmol mg-protein-1]
2
*
1
0
20 min 1
40 min 1
AA-uptake [pmol mg-protein-1]
C
B
* 2
1
0
20 min
40 min
Fig. 1. A. Placental SNAT activity (pmol mg-proteinˉ minˉ ), term placental villous fragments. Lean (C, n ¼ 7), obese (B, n ¼ 7). Data are mean SEM, *p 0.05 (20, 40, 80, 120 min time points e lean vs. obese), **p ¼ 0.005 (SNAT activity lean vs. obese), amino acid (AA). B, C. Effect of leptin stimulation on placental SNAT activity (pmol mg-proteinˉ1 minˉ1), at concentrations of 100 ng mLˉ1 (B, lean vs. obese p ¼ 0.07), 500 ng mLˉ1 (C, lean vs. obese p ¼ 0.10) in term placental villous fragments. Lean (A, n ¼ 7), obese (>, n ¼ 7), lean without leptin (C, n ¼ 7), obese without leptin (B, n ¼ 7). Data are mean SEM. *(A. p ¼ 0.004, B. p ¼ 0.047 lean with leptin, A vs. obese with leptin, >).
D.M. Farley et al. / Placenta 31 (2010) 718e724
721
Fig. 2. Immunohistochemistry for leptin (A,B) and leptin (long-form) receptor (D,E) protein expression in placenta terminal villi (60) at 39 weeks gestation (C and F are negative controls). Lean (A,D), obese (B,E). Area (G) and intensity (H) of immunostaining for leptin receptor were quantified. Data are mean SEM, *p ¼ 0.01.
p < 0.001; density, lean 0.3 0.0 vs. obese 0.2 0.0, p < 0.001) in the obese group (Fig. 2DeH). Negative control sections revealed no detectable staining (Fig. 3C,F). Syncytiotrophoblast expression of SNAT 1 (fraction, lean 19.5 1.5% vs. obese 16.8 2.0%, p ¼ 0.31; density, lean 6.5 0.5 vs. obese 5.7 0.7, p ¼ 0.30, Fig. 3AeC) and SNAT 2 (fraction, lean 17.9 1.4% vs. obese 14.0 2.0%, p ¼ 0.15; density, lean 5.5 0.4 vs. obese 4.4 0.6, p ¼ 0.14, Fig. 3DeF) were unchanged. Syncytiotrophoblast expression of the SNAT 4 isoform (fraction, lean 24.9 1.9% vs. obese 8.9 1.4%, p < 0.001; density, lean 8.3 0.6 vs. obese 3.1 0.5, p < 0.001, Fig. 3GeK) was decreased in the obese group. Negative control sections revealed no detectable staining (Fig. 3C,F,I). Placental syncytiotrophoblast leptin receptor (ObR-b) expression was directly correlated to placental SNAT 4 expression (R ¼ 0.29, p ¼ 0.048). 3.4. Plasma insulin and leptin concentrations Fasting maternal venous insulin levels were not different between groups, while leptin concentration was increased in the obese group (p ¼ 0.01) and fetal leptin levels were unchanged (Table 1). Maternal plasma leptin was directly correlated to
maternal entry BMI (R ¼ 0.32, p ¼ 0.04) and to maternal delivery BMI (R ¼ 0.47, p < 0.001). Maternal plasma leptin was inversely correlated to placental SNAT activity (R ¼ 0.39, p ¼ 0.02). 4. Discussion Contrary to our hypothesis placental amino acid transport at term was decreased in the setting of maternal obesity. Our finding of maternal hyperleptinemia in the obese group is consistent with previous reports [26e30]. The decreased placental amino acid transport appears related to maternal hyperleptinemia in keeping with previous data that increased maternal leptin levels are associated with placental dysfunction [33]. Our data support the idea of placental leptin resistance in maternal obesity based on syncytiotrophoblast down regulation of leptin receptor in the setting of maternal hyperleptinemia. Furthermore, syncytiotrophoblast leptin protein abundance was the same in our two groups despite the presence of maternal hyperleptinemia in the obese group which may have induced syncytiotrophoblast leptin receptor down regulation. Under the conditions used placental MeAIB uptake was stimulated by leptin in the lean but not obese group during 40 min
722
D.M. Farley et al. / Placenta 31 (2010) 718e724
Fig. 3. Immunohistochemistry for SNAT 1 (A,B), SNAT 2 (D,E) and SNAT 4 (G,H) protein expression in placenta terminal villi (60) at 39 weeks gestation (C, F, and I are negative controls). Lean (A,D,G), obese (B,E,H). Area (J) and intensity (K) of immunostaining for SNAT 4 were quantified. Data are mean SEM, *p < 0.001.
of incubation. The fact that leptin-stimulated AA uptake rate was not significantly different between groups was likely due to power and time of incubation which are recognized limitations. However, we speculate this may be a long-term consequence of maternal hyperleptinemia throughout pregnancy in the obese group.
Normal placentas can increase SNAT activity in a substratespecific and dose-dependent manner to optimize fetal growth in the presence of amino acid limitation and there is a degree of adaptive regulation in normal placentas [34]. Our findings that placental SNAT activity is decreased in maternal obesity may
D.M. Farley et al. / Placenta 31 (2010) 718e724
represent a placental adaptive regulation. However, the obese group gained the recommended amount of weight and we feel this contributed to the appropriately grown neonates. A limitation of our study is that we do not have maternal serum amino acid concentrations, which if decreased compared to the lean group when gaining the recommended amount of weight, may support our belief that decreased SNAT activity represents a degree of placental dysfunction, not adaptive regulation. We feel that the decrease in SNAT 4 expression is evidence that this isoform is likely to contribute most to the decrease in overall SNAT activity between these 2 groups as SNAT 1 and 2 expression were not different. The contribution of SNAT 4 to placental system A amino acid transport has been shown to be less at term [19], a time when it has been speculated that SNAT 1 and 2 may contribute more. However, SNAT 4 expression has been shown to be increased at term, whereas SNAT 1 and 2 expression was unchanged [35]. To determine what isoform contributes most to the decrease in system A activity that we found in maternal obesity is an area of future research. The SNAT 4 isoform is a major component of the placental system A amino acid transporter [19] and the decrease in this isoform in the absence of changes in other SNAT isoforms would indicate that SNAT 4 plays a major role in decreased amino acid transport in the setting of maternal obesity and potentially in placental dysfunction. Notably, SNAT 4 is an imprinted gene susceptible to regulation by levels of methylation [36]. Alterations in methylation may be one mechanism that explains the increased incidence of birth defects associated with maternal obesity [37e39]. Our goal was to enroll well defined lean and obese mothers at entry and delivery and exclude patients with medical co-morbidities to increase the likelihood that the changes we observed in amino acid transport were due to maternal hyperleptinemia and placental leptin resistance. The plasma leptin levels in our lean group were consistent with previous reports in pregnancy [26e30] as was the stimulatory effect of leptin on placental SNAT activity [20]. The lack of statistical significance when measuring the effect of leptin on placental SNAT activity was likely secondary to power with respect to this variable since it was not the primary outcome. However, placental SNAT activity was inversely correlated with maternal plasma leptin concentration. This finding indicates that the decreased placental amino acid transport we found in the setting of maternal obesity may be related to maternal hyperleptinemia and development of placental leptin resistance evidenced by leptin receptor down regulation in association with a decrease in SNAT 4 expression. Neither group had overt insulin resistance evidenced by normal antenatal screening glucose challenge tests and maternal fasting plasma insulin levels in the normal range on the day of delivery [40]. The absence of fetal overgrowth in our obese group may be explained as follows. Hyperleptinemia in the obese group may have attenuated the tendency of maternal obesity to increase fetal growth in some way. Leptin correlates negatively with insulin sensitivity in pregnancy [41] so the possibility of hyperleptinemia increasing insulin sensitivity and attenuating fetal growth is unlikely [42]. We speculate that maternal hyperleptinemia impairs placental amino acid transport. In addition, maternal weight gain in the obese group was consistent with current recommendations [43], but maternal weight gain in the lean group was not. Gaining the recommended amount of weight in the obese group may have prevented excessive fetal growth. Furthermore, if gaining the recommended amount of weight in the obese group prevents excessive fetal growth, then our finding of decreased placental amino acid transport signifies altered placental function despite normal fetal growth. Further research is needed to determine whether gaining the recommended amount of weight prevents excess fetal growth and the clinical significance of decreased placental amino acid transport in the setting of maternal obesity.
723
We conclude that maternal obesity and associated hyperleptinemia appear to have a negative impact on placental amino acid transport. Our data suggest a certain degree of altered placental function that may have clinical implications in obese pregnant women. Further studies on the overweight and morbidly obese maternal populations are needed to determine if the negative impact on placental amino acid transport seen in our study is part of a continuum. Acknowledgements We are grateful to Dr. Jocelyn Glazier, Dr. Colin Sibley and Ralf Wimmer for their help with setting up the amino acid fragment uptake method. A special thanks to Dr. Elly Xenakis for her intellectual input, and the labor and delivery staff at University Hospital for assistance in collecting specimens, Susan Carvajal for her help with the neonatal measurements, Greg Langone for histology expertise, Michelle Zavala for assistance with the ELISA technique, Phylis Eagen at the Texas Diabetes Institute, and the University of Texas Health Science Center at San Antonio’s radiation safety laboratory for use of their scintillation counter. This study was approved by University of Texas Health Science Center at San Antonio’s IRB and the ID number is HSC20070723H. This study was supported by HD 21350-17. References [1] Kim SY, Dietz PM, England L, Morrow B, Callaghan WM. Trends in pre-pregnancy obesity in nine states, 1993e2003. Obesity (Silver Spring) 2007;15 (4):986e93. [2] Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, Flegal KM. Prevalence of overweight and obesity in the United States, 1999e2004. JAMA 2006;295:1549e55. [3] Artal R, Lockwood CJ, Brown HL. Weight gain recommendations in pregnancy and the obesity epidemic. Obstet Gynecol 2010;115(1):152e5. [4] Wang Y, Beydoun MA. The obesity epidemic in the United Statesdgender, age, socioeconomic, racial/ethnic, and geographic characteristics: a systematic review and meta-regression analysis. Epidemiol Rev 2007;29:6e28. [5] Cnattingius S, Bergstrom R, Lipworth L, Kramer MS. Prepregnancy weight and the risk of adverse pregnancy outcomes. N Engl J Med 1998;338:147e52. [6] Leddy MA, Power ML, Schulkin J. The impact of obesity on maternal and fetal health. Rev Obstet Gynecol 2008;1(4):170e8. [7] Villamor E, Cnattingius S. Interpregnancy weight change and risk of adverse pregnancy outcomes: a population-based study. Lancet 2006;368:1164e70. [8] Chu SY, Kim SY, Lau J, Schmid CH, Dietz PM, Callaghan WM, et al. Maternal obesity and risk of stillbirth: a metaanalysis. Am J Obstet Gynecol 2007;197 (3):223e8. [9] Crane JM, White J, Murphy P, Burrage L, Hutchens D. The effect of gestational weight gain by body mass index on maternal and neonatal outcomes. J Obstet Gynaecol Can 2009;31(1):28e35. [10] Nohr EA, Vaeth M, Bech BH, Henriksen TB, Cnattingius S, Olsen J. Maternal obesity and neonatal mortality according to subtypes of preterm birth. Obstet Gynecol 2007;110:1083e90. [11] Kristensen J, Vestergaard M, Wisborg K, Kesmodel U, Secher NJ. Pre-pregnancy weight and the risk of stillbirth and neonatal death. BJOG 2005;112 (4):403e8. [12] Bodnar LM, Catov JM, Klebanoff MA, Ness RB, Roberts JM. Prepregnancy body mass index and the occurrence of severe hypertensive disorders of pregnancy. Epidemiology 2007;18(2):234e9. [13] Bhattacharya S, Campbell DM, Liston WA, Bhattacharya S. Effect of Body Mass Index on pregnancy outcomes in nulliparous women delivering singleton babies. BMC Public Health 2007;7:168. [14] Roos S, Powell TL, Jansson T. Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation. Am J Physiol Regul Integr Comp Physiol 2004;287(4):R886e93. [15] Jansson T. Amino acid transporters in the human placenta. Pediatr Res 2001;49(2):141e7. [16] Kuruvilla AG, D’Souza SW, Glazier JD, Mahendran D, Maresh MJ, Sibley CP. Altered activity of the system A amino acid transporter in microvillous membrane vesicles from placentas of macrosomic babies born to diabetic women. J Clin Invest 1994;94(2):689e95. [17] Greenwood SL, Sibley CP. In vitro methods for studying human placental amino acid transport placental villous fragments. Methods Mol Med 2006;122:253e64. [18] Jones HN, Powell TL, Jansson T. Regulation of placental nutrient transportea review. Placenta 2007;28(8e9):763e74.
724
D.M. Farley et al. / Placenta 31 (2010) 718e724
[19] Desforges M, Mynett KJ, Jones RL, Greenwood SL, Westwood M, Sibley CP, et al. The SNAT4 isoform of the system A amino acid transporter is functional in human placental microvillous plasma membrane. J Phys 2009;587.1:61e72. [20] Jansson N, Greenwood SL, Johansson BR, Powell TL, Jansson T. Leptin stimulates the activity of the system A amino acid transporter in human placental villous fragments. J Clin Endocrinol Metab 2003;88(3):1205e11. [21] Glazier JD, Cetin I, Perugino G, Ronzoni S, Grey AM, Mahendran D, et al. Association between the activity of the system A amino acid transporter in the microvillous plasma membrane of the human placenta and severity of fetal compromise in intrauterine growth restriction. Pediatr Res 1997;42 (4):514e9. [22] Havel PJ, Kasim-Karakas S, Mueller W, Johnson PR, Gingerich RL, Stern JS. Relationship of plasma leptin to plasma insulin and adiposity in normal weight and overweight women: effects of dietary fat content and sustained weight loss. J Clin Endocrinol Metab 1996;81(12):4406e13. [23] Ogier V, Ziegler O, Mejean L, Nicolas JP, Stricker-Krongrad A. Obesity is associated with decreasing levels of the circulating soluble leptin receptor in humans. Int J Obes Relat Metab Disord 2002;26(4):496e503. [24] Van Dielen FM, van’t Veer C, Buurman WA, Greve JW. Leptin and soluble leptin receptor levels in obese and weight-losing individuals. J Clin Endocrinol Metab 2002;87(4):1708e16. [25] Vendrell J, Broch M, Vilarrasa N, Molina A, Gomez JM, Gutierrrez C, et al. Resistin, adiponectin, ghrelin, leptin, and proinflammatory cytokines: relationships in obesity. Obes Res 2004;12(6):962e71. [26] Masuzaki H, Ogawa Y, Sagawa N, Hosoda K, Matsumoto T, Mise H, et al. Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med 1997;3(9):1029e33. [27] Sattar N, Greer IA, Pirwani I, Gibson J, Wallace AM. Leptin levels in pregnancy: marker for fat accumulation and mobilization? Acta Obstet Gynecol Scand 1998;77(3):278e83. [28] Hauguel-de Mouzon S, Lepercq J, Catalano P. The known and unknown of leptin in pregnancy. Am J Obstet Gynecol 2006;194:1537e45. [29] Highman T, Friedman J, Huston L, Wong W, Catalano P. Longitudinal changes in maternal leptin serum concentrations, body composition and resting metabolic rate in pregnancy. Am J Obstet Gynecol 1998;178:1010e5. [30] Schubring C, Kiess W, Englaro P, Rascher W, Dotsch J, Hanitsch S, et al. Levels of leptin in maternal serum, amniotic fluid, and arterial and venous cord
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38] [39] [40] [41]
[42] [43]
blood: relation to neonatal and placental weight. J Clin Endocrinol Metab 1997;82(5):1480e3. Borecki IB, Higgins M, Schreiner PJ, Arnett DK, Mayer-Davis E, Hunt SC, et al. Evidence for multiple determinants of the body mass index: the National Heart, Lung, and Blood Institute Family Heart Study. Obes Res 1998;6 (2):107e14. Li C, Levitz M, Hubbard GB, Jenkins SL, Han V, Ferry Jr RJ, et al. The IGF axis in baboon pregnancy: placental and systemic responses to feeding 70% global Ad libitum diet. Placenta 2007;28(11e12):1200e10. Lepercq J, Guerre-Millo M, Andre J, Cauzac M, Hauguel-de Mouzon S. Leptin: a potential marker of placental insufficiency. Gynecol Obstet Invest 2003;55 (3):151e5. Parrott MS, Versen-Hoeynck Fv, Ness RB, Markovik N, Roberts J. System A amino acid transporter activity in term placenta is substrate specific and inversely related to amino acid concentration. Reprod Sci 2007;14(7):687e93. Desforges M, Lacey HA, Glazier JD, Greenwood SL, Mynett KJ, Speake PF, et al. SNAT4 isoform of system A amino acid transporter is expressed in human placenta. Am J Physiol Cell Physiol 2006;290:305e12. Smith RJ, Dean W, Konfortova G, Kelsey G. Identification of novel imprinted genes in a genome-wide screen for maternal methylation. Genome Res 2003;13:558e69. Waller DK, Shaw GM, Rasmussen SA, Hobbs CA, Canfield MA, Siega-Riz AM, et al. Prepregnancy obesity as a risk factor for structural birth defects. Arch Pediatr Adolesc Med 2007;161(8):745e50. Koleganova N, Piecha G, Ritz E. Prenatal causes of kidney disease. Blood Purif 2009;27(1):48e52. Henriksen T, Haugen G, Bollerslev J, Kolset SO, Drevon CA, Iverson PO, et al. Fetal nutrition and future health. Tidsskr Nor Laegeforen 2005;125(4):442e4. Laakso M. How good a marker is insulin level for insulin resistance? Am J Epidemiol 1993;137(9):959e65. McIntyre HD, Chang AM, Callaway LK, Cowley DM, Dyer AR, Radaelli T, et al. Hormonal and metabolic factors associated with variations in insulin sensitivity in human pregnancy. Diabetes Care 2010;33:356e60. Sagawa N, Yura S, Itoh H, Mise H, Kakui K, Korita D, et al. Role of leptin in pregnancy e a review. Placenta 2002;23(Suppl. A16):S80e6. Rasmussen KM, Yaktine AL, editors. Weight gain during pregnancy: reexamining the recommendations. Washington, DC: The National Academies Press; May 2009. Report brief.