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Augmented Placental Production of Leptin in Preeclampsia: Possible Involvement of Placental Hypoxia¹

Department of Gynecology and Obstetrics (H.Mi., N.S., T.M., S.Y., H.N., H.I., T.M.), Department of Medicine and Clinical Science (H.Ma., K.H., Y.O., K.N.), Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan

Address all correspondence and requests for reprints to: Norimasa Sagawa M.D., Ph.D., Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: fetus{at}kuhp.kyoto-u.ac.jp

	Abstract

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Preeclampsia (PE) is a hypertensive disorder, which develops in late pregnancy and is usually associated with placental hypoxia and dysfunction. We have recently demonstrated that leptin is a novel placenta-derived hormone in humans and suggested its significance in human pregnancy (see Ref. 19). To explore the changes in the leptin production in placenta in PE, we measured the plasma leptin level and placental leptin messenger RNA expression in pregnant women with PE. Plasma leptin levels in preeclamptic women were elevated significantly, compared with gestational age- and body mass index-matched normal pregnant women (P < 0.0001). Plasma leptin levels in the severe PE group were significantly higher than those in the mild PE group (P < 0.0001). Plasma leptin levels in preeclamptic women were reduced, soon after the placental delivery, to those expected for their body mass indices. Northern blot analysis revealed that leptin messenger RNA levels are increased in the placentas from preeclamptic women, compared with normal pregnant women. Leptin secretion was increased significantly in a human trophoblastic cell line (BeWo cells) cultured under hypoxic conditions (5% O₂), compared with those cultured under standard conditions (20% O₂; P < 0.01). The present study demonstrated that placental production of leptin is augmented in severe PE, probably because of placental hypoxia, thereby suggesting the possible significance of leptin as a marker of placental hypoxia in severe PE.

	Introduction

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LEPTIN is a novel hormone that is expressed abundantly and specifically in the adipose tissue (1, 2). It decreases food intake and body weight via its cognate receptor (Ob-R) in the hypothalamus (3, 4, 5, 6). We and others previously demonstrated that leptin synthesis and secretion are markedly increased in several models of rodent obesity and human obesity in proportion to the disease severity (1, 2, 7, 8, 9). Clinically, leptin may serve as a useful marker of adiposity, reflecting the total lipid content in humans (7, 9). On the other hand, accumulated evidence suggests that leptin acts also as a peripherally-produced metabolic signal to the neuroendocrine and reproductive systems (10, 11, 12, 13, 14, 15, 16, 17, 18).

We have recently demonstrated, for the first time, nonadipose tissue production of leptin (19). In pregnant women, leptin is synthesized in and secreted from placental trophoblasts into the maternal circulation at considerable amounts, comparable with those in nonpregnant obese women (19). Leptin is also produced by a cultured human choriocarcinoma cell line, BeWo cells (19). Furthermore, in BeWo cells, leptin synthesis and secretion are increased during the course of forskolin-induced differentiation from cytotrophoblasts into syncytiotrophoblasts. Plasma leptin levels are also markedly elevated in patients with hydatidiform mole and choriocarcinoma, indicating that gestational trophoblastic neoplasms are leptin-producing tumors (19). Indeed, leptin-like immunohistochemistry is detected in the trophoblast cells of hydatidiform mole, and leptin messenger RNA (mRNA) expression is augmented in molar tissues (20). These findings, taken together, suggest that leptin is a novel placenta-derived hormone in humans. However, the pathophysiologic roles of placenta-derived leptin in pregnancy-associated disorders still remain to be determined.

Preeclampsia (PE), which affects approximately 5–10% of all pregnant women (21), is one of the most common pregnancy-associated disorders. Hypertension with arteriolar vasoconstriction is its major clinical manifestation, which causes a reduction in uteroplacental blood flow, thus leading to placental hypoxia, as well as fetal growth retardation (22). It has been recognized that oxygen tension regulates a set of several placental genes that are critical to the proliferation and differentiation of cytotrophoblasts, which is proposed to contribute to the pathogenesis of PE (22, 23, 24, 25).

In the present study, we measured the plasma leptin level and placental leptin mRNA expression in pregnant women with PE. We also examined the effects of hypoxia on leptin secretion from placental trophoblasts using BeWo cells, an in vitro model with which to access the regulation of leptin synthesis and secretion (19).

	Materials and Methods

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Human subjects

The present study included 93 normal pregnant women (28.7 ± 0.5 yr old, mean ± SEM, 29–41 weeks of gestation) and 32 pregnant women with PE (30.4 ± 0.9 yr old, 27–41 weeks of gestation). PE was diagnosed and classified according to the technical bulletin of the American College of Obstetricians and Gynecologists (26) and National High Blood Pressure Education Program Working Group Report on High Blood Pressure in Pregnancy (27). The mild and severe PE groups were defined as follows: mild, 140/90 mm Hg blood pressure < 160/110 mm Hg and 0.3 g/day proteinuria < 2.0 g/day; and severe, blood pressure 160/110 mm Hg and proteinuria 2.0 g/day. Clinical features of the preeclamptic women studied were summarized in Table 1. Because gestational age is different between the mild and severe PE groups, their respective gestational age-matched groups were used as controls (control 1 and control 2). The body mass index (BMI) is not different among groups. In the present study, cesarean section was performed in 17 of 18 severe cases because of fetal distress, but only in 2 of 14 mild cases. The present study was conducted with informed consent.

In all pregnant women, before the delivery, blood was withdrawn after an overnight fast. In four cases, at cesarean section, before the onset of labor, blood was sampled 1 h before and 2 h, 4 h, and 24 h after the delivery. Blood samples thus obtained were transferred immediately to chilled siliconized glass tubes containing Na₂EDTA (1 mg/mL), which were centrifuged and then stored at -20 C until use.

Tissue preparations

The placental tissue was obtained at the time of vaginal delivery or cesarean section, at term (37–41 weeks of gestation). Chorionic tissues were sampled from four different parts of the placenta, from which amnionic membrane and maternal decidual tissue were removed and combined before use. Tissues were frozen and stored at -70 C until use.

Cell culture

BeWo cells, a human choriocarcinoma cell line, were obtained from the American Type Culture Collection and were cultured at a concentration of 2 x 10⁵ cells/mL in RPMI 1640 (GIBCO BRL, Gaithersburg, MD), as described (19). According to the previous reports (25, 28), we used 20% and 5% O₂ conditions as models of normoxic and hypoxic placenta conditions, respectively. We also examined the effect of a 10% O₂ condition on the leptin secretion from cultured BeWo cells. At confluency, the culture medium was removed, and cells were incubated for another 48 h in 10 mL fresh medium containing 10% FCS, under 20% O₂ condition, with 20 µmol/L forskolin (Sigma Chemical Co., St. Louis, MO). Subsequently, hypoxic stimulation was produced by exposure to 5% or 10% O₂ with the balance of N₂ in an O₂-CO₂ incubator (Model CPO₂-171, Hirasawa Co., Ltd., Tokyo, Japan), which resulted in a decrease in P_O2 from 120 ± 5 mm Hg to 35 ± 3 mm Hg within 1 h (n = 3). P_CO2 (38 ± 3 mm Hg) and pH (7.34 ± 0.05) of the bath solution remained unchanged. Hypoxic stimuli were applied for 72 h, during which 1 mL of conditioned media was obtained every 24 h for the RIA for human leptin.

Hormone assays

Human leptin levels and human CG (hCG) levels in plasma and culture media from BeWo cells were determined by use of the RIA for human leptin (29) and enzyme immunoassay for hCG (Johnson and Johnson Clinical Diagnostics, Rochester, NY), respectively.

RNA extraction and Northern blot analysis

Total RNA was extracted from the placental tissue, as described (2). Northern blot analysis was performed with the ³²P-labeled full-length human leptin complementary DNA as a probe (19). Autoradiographs were done for 60 h at -70 C with intensifying screens and were quantitated by densitometric scanning using BAS-2500 and Image Reader version 1.4J, (FUJI Photo Film Co. Ltd., Tokyo, Japan).

Statistical analysis

Statistical analysis was performed by Student’s t test or ANOVA with Fisher’s least-significance difference test, where applicable. All values were expressed as the mean ± SEM.

	Results

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Plasma leptin levels in PE

Plasma leptin levels were elevated significantly in pregnant women with PE (n = 32), compared with BMI- and gestational age-matched normal pregnant women (n = 93; 73.8 ± 10.2 ng/mL vs. 35.5 ± 3.0 ng/mL; P < 0.0001). In the present study, plasma leptin levels in normal pregnant women increased significantly compared with those in BMI- and age-matched nonpregnant women, as reported previously (data not shown) (19). No significant differences in plasma leptin levels were observed between the mild PE group and its control group (control 1) (38.2 ± 4.8 ng/mL, n = 14 vs. 33.8 ± 3.7 ng/mL, n = 54) (Fig. 1A). By contrast, plasma leptin levels in the severe PE group (101.5 ± 14.9 ng/mL, n = 18) were approximately 3-fold higher than those in its control group (control 2) (37.8 ± 5.0 ng/mL, n = 39) (P < 0.0001). Plasma leptin levels in the severe PE group were also significantly higher than those in the mild PE group (P < 0.0001). No significant differences in plasma hCG levels were noted between the mild PE group and control 1 (25,675 ± 3,660 mIU/mL vs. 38,534 ± 5,684 mIU/mL) and between the severe PE group and control 2 (31,153 ± 4,482 mIU/mL vs. 38,925 ± 5,214 mIU/mL).

View larger version (23K): [in this window] [in a new window]   Figure 1. A, Plasma leptin levels in pregnant women with mild and severe PE. *, P < 0.0001 vs. gestational age- and BMI-matched control 2 group. B, Time course of plasma leptin levels, determined consecutively before and after the placental delivery at cesarean section, in PE and normal pregnancy. Plasma leptin levels in four severe preeclamptic women (A-D) are indicated. Shaded area, The mean ± SEM of the changes in plasma leptin levels in four cases of normal pregnancy, as reported previously (19 ).

Placental leptin mRNA expression in PE

Northern blot analysis identified in the placental tissue a single leptin mRNA species of the same size ( 4.5 kb) as in mature adipocytes (19, 30) (Fig. 2A). Leptin mRNA expression was markedly augmented in the placental tissue from preeclamptic women, compared with gestational age-matched normal pregnant women (Fig. 2A). In the present study, placental leptin mRNA levels were roughly parallel to plasma leptin levels in all the preeclamptic women examined (Fig. 2B). Leptin mRNA expression in the placenta of severe preeclamptic women was significantly higher than that in normal pregnant women (P < 0.05, Fig. 2C). Leptin mRNA expression in the placenta of mild preeclamptic women was slightly higher than that in normal pregnant women, but the difference was not significant (P = 0.565, Fig. 2C).

View larger version (23K): [in this window] [in a new window]   Figure 2. A, Northern blot analysis of leptin mRNA in the placental tissues from the mild and severe preeclamptic women. From left to right on the gel, leptin mRNA expression was examined in mature adipocytes obtained from sc abdominal fat pads (lane 1), the placental chorionic tissues from normal pregnant women (lanes 2–4), and those with mild (lanes 5 and 6) and severe PE (lane 7). The adipocyte mRNA was obtained at the time of operation from a female patient with gastric cancer (19 ). Total RNA (20 µg/lane) was analyzed. The ribosomal RNA bands visualized with ethidium bromide are shown in the bottom panel. B, Plasma leptin levels in normal pregnant and preeclamptic women examined by Northern blot analysis (lanes 2–7 in Fig. 2A). C, Comparison of leptin mRNA expression in placentas from five normal pregnant women, four mild PE women, and three severe PE women, using densitometry analysis (normalized by 28 S expression) of Northern blot.

Up to 48 h, no significant differences in leptin levels in the culture media were observed between BeWo cells cultured under hypoxic conditions (5% O₂) and those cultured under standard conditions (20% O₂) (Fig. 3A). In BeWo cells cultured for 72 h under 5% O₂, leptin secretion was increased approximately 3-fold, relative to those cultured under 20% O₂ (17.2 ± 0.7 ng/mL under 5% O₂ vs. 6.9 ± 1.2 ng/mL under 20% O₂, P < 0.01). By contrast, hCG levels in the culture media from BeWo cells cultured for 24–72 h under 5% O₂ were decreased significantly, compared with those cultured under 20% O₂ (Fig. 3B), which is consistent with a previous report that hCG secretion from cultured trophoblasts is decreased under hypoxic conditions (31). Leptin secretion from BeWo cells cultured under 10% O₂ condition was not increased significantly, compared with those cultured under 20% O₂ condition (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 3. Leptin secretion (A) and hCG secretion (B) from BeWo cells cultured under hypoxic conditions. After confluency, BeWo cells were incubated for 48 h under 20% O2 with 20 µmol/L forskolin. Subsequently, hypoxic stimulation was produced by exposure to 5% O2. Hypoxia was applied for 72 h, during which 1 mL of conditioned media was sampled every 24 h for the RIA for human leptin. Hormone levels in BeWo cells cultured under 5% O2 and 20% O2 are indicated by closed and open circles, respectively. Values are the mean ± SEM of three independent experiments performed in triplicate dishes. The results were confirmed by three more independent series. *, P < 0.01 vs. the values of controls (20% O2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study demonstrates, for the first time, that plasma leptin levels are elevated significantly in pregnant women with PE. Furthermore, plasma leptin levels in the severe PE group are significantly higher than those in the mild PE group.

The present study demonstrates the augmentation of placental leptin mRNA expression in PE. In addition, placental leptin mRNA levels are roughly proportional to plasma leptin levels in all the preeclamptic women examined. Furthermore, plasma leptin levels in preeclamptic women are decreased, soon after the placental delivery, to levels expected for their BMIs. These observations strongly suggest that elevated plasma leptin levels in preeclamptic women are caused mostly by the augmentation of placental production of leptin.

The present study also demonstrates that leptin secretion is increased in BeWo cells under hypoxic conditions. These observations suggest that, in severe PE, placental production of leptin is increased in response to hypoxia, thereby supporting the notion that augmented plasma leptin levels in severe PE reflect placental hypoperfusion and/or hypoxia. Because hypoxia induces a set of several placental genes in trophoblasts (22, 23, 24, 25, 28), augmented placental production of leptin may represent one of the generalized hypoxic responses of trophoblasts in PE. Therefore, leptin may serve as a placenta-derived marker of PE, possibly reflecting placental hypoxia associated with severe PE.

We have recently observed that leptin secretion is increased in parallel with hCG secretion during the course of forskolin-induced differentiation from cytotrophoblasts into syncytiotrophoblasts (19). In the present study, however, leptin secretion is increased, whereas hCG secretion is decreased, in BeWo cells cultured under hypoxic conditions. These findings suggest that leptin production is regulated differently from hCG production in trophoblasts.

The functional significance of increased placental leptin production in PE is unclear at present. The satiety effect of elevated plasma leptin may be expected to affect fetal growth in severe PE. However, pregnant women with elevated plasma leptin levels have usually normal appetite. Thus, in pregnant women, there must be some mechanism of resistance to leptin. We are now investigating the leptin transport to brain and leptin binding protein in plasma of pregnant women. On the other hand, leptin administration increases the otherwise decreased metabolic rate, body temperature, and locomotor activity in ob/ob mice (3). This is compatible with the hypermetabolic state of pregnant women. Leptin also causes an increase in noradrenaline turnover to the brown adipose tissue (32), suggesting that leptin increases the sympathetic outflow. It is also known that preeclamptic women have increased sympathetic activity (33). Therefore, increased leptin secretion might contribute to increased sympathetic activity in PE. Moreover, intracerebroventricular or chronic (over 1 week) iv infusion of leptin increases arterial pressure in rats (34, 35), suggesting the possible involvement of elevated plasma leptin in the development of hypertension in severe PE. However, the functional role of leptin in the development of hypertension is still controversial (36). Further investigations are required to confirm this possibility. Because Ob-R is expressed in the placenta (37), it is tempting to speculate that placental leptin also plays some roles as a local regulator in PE.

In conclusion, the present study demonstrates that placental production of leptin is augmented in PE, probably because of placental hypoxia. The data of the present study suggest the pathophysiologic significance of placental leptin in PE.

	Acknowledgments

 
We thank Dr. T. Inoue of the Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine; Dr. A. Takahashi and Dr. M. Hasegawa of Kurashiki Central Hospital; Dr. F. Kobayashi of Hyogo Prefectural Amagasaki Hospital; Dr. T. Miyamoto of Otsu Red Cross Hospital; and Dr. M. Iwata and Dr. M. Kinoshita of Osaka Red Cross Hospital for their assistance in plasma and tissue samplings.

	Footnotes

 
¹ This work was supported, in part, by research grants from the Japanese Ministry of Education, Science, and Culture; the Japanese Ministry of Health and Welfare; the Yamanouchi Foundation for Research on Metabolic Disorders; the Uehara Memorial Foundation; the Smoking Research Foundation; the T. Nanba Memorial Health Care Foundation; and the Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan).

Received November 5, 1997.

Accepted June 11, 1998.

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				A. Grosfeld, J. Andre, S. Hauguel-de Mouzon, E. Berra, J. Pouyssegur, and M. Guerre-Millo Hypoxia-inducible Factor 1 Transactivates the Human Leptin Gene Promoter J. Biol. Chem., November 1, 2002; 277(45): 42953 - 42957. [Abstract] [Full Text] [PDF]

				Y. Takahashi, Y. Yokoyama, I. Kawabata, S. Iwasa, and T. Tamaya Leptin as an Acute Stress-Related Hormone in the Fetoplacental Circulation Obstet. Gynecol., October 1, 2002; 100(4): 655 - 658. [Abstract] [Full Text] [PDF]

				G. Ambrosini, A. K. Nath, M. R. Sierra-Honigmann, and J. Flores-Riveros Transcriptional Activation of the Human Leptin Gene in Response to Hypoxia. INVOLVEMENT OF HYPOXIA-INDUCIBLE FACTOR 1 J. Biol. Chem., September 6, 2002; 277(37): 34601 - 34609. [Abstract] [Full Text] [PDF]

				T. Reimer, D. Koczan, B. Gerber, D. Richter, H.J. Thiesen, and K. Friese Microarray analysis of differentially expressed genes in placental tissue of pre-eclampsia: up-regulation of obesity-related genes Mol. Hum. Reprod., July 1, 2002; 8(7): 674 - 680. [Abstract] [Full Text] [PDF]

				R. A. Ehrhardt, A. W. Bell, and Y. R. Boisclair Spatial and developmental regulation of leptin in fetal sheep Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2002; 282(6): R1628 - R1635. [Abstract] [Full Text] [PDF]

				F. M. Reis, D. D'Antona, and F. Petraglia Predictive Value of Hormone Measurements in Maternal and Fetal Complications of Pregnancy Endocr. Rev., April 1, 2002; 23(2): 230 - 257. [Abstract] [Full Text] [PDF]

				L. Gambling, Z. Charania, L. Hannah, C. Antipatis, R. G. Lea, and H. J. McArdle Effect of Iron Deficiency on Placental Cytokine Expression and Fetal Growth in the Pregnant Rat Biol Reprod, February 1, 2002; 66(2): 516 - 523. [Abstract] [Full Text] [PDF]

				S.M. Laird, N.D. Quinton, B. Anstie, T.C. Li, and A.I.F. Blakemore Leptin and leptin-binding activity in women with recurrent miscarriage: correlation with pregnancy outcome Hum. Reprod., September 1, 2001; 16(9): 2008 - 2013. [Abstract] [Full Text] [PDF]

				T. Laml, O. Preyer, B. W. Hartmann, E. Ruecklinger, G. Soeregi, and P. Wagenbichler Decreased Maternal Serum Leptin in Pregnancies Complicated by Preeclampsia Reproductive Sciences, April 1, 2001; 8(2): 89 - 93. [Abstract] [PDF]

				C. G. Solomon and E. W. Seely Brief Review: Hypertension in Pregnancy : A Manifestation of the Insulin Resistance Syndrome? Hypertension, February 1, 2001; 37(2): 232 - 239. [Abstract] [Full Text] [PDF]

				T. O Scholl, T P. Stein, and W. K Smith Leptin and maternal growth during adolescent pregnancy Am. J. Clinical Nutrition, December 1, 2000; 72(6): 1542 - 1547. [Abstract] [Full Text] [PDF]

				M. C. Henson and V. D. Castracane Leptin in Pregnancy Biol Reprod, November 1, 2000; 63(5): 1219 - 1228. [Abstract] [Full Text]

				R. F. Gariano, A. K. Nath, D. J. D’Amico, T. Lee, and M. R. Sierra–Honigmann Elevation of Vitreous Leptin in Diabetic Retinopathy and Retinal Detachment Invest. Ophthalmol. Vis. Sci., October 1, 2000; 41(11): 3576 - 3581. [Abstract] [Full Text]

				M. Castellucci, R. De Matteis, A. Meisser, R. Cancello, V. Monsurro, D. Islami, R. Sarzani, D. Marzioni, S. Cinti, and P. Bischof Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion Mol. Hum. Reprod., October 1, 2000; 6(10): 951 - 958. [Abstract] [Full Text] [PDF]

				N. YOSHIMITSU, T. DOUCHI, M. KAMIO, and Y. NAGATA Differences in Umbilical Venous and Arterial Leptin Levels by Mode of Delivery Obstet. Gynecol., September 1, 2000; 96(3): 342 - 345. [Abstract] [Full Text] [PDF]

				N. Anim-Nyame, S.R. Sooranna, P.J. Steer, and M.R. Johnson Longitudinal analysis of maternal plasma leptin concentrations during normal pregnancy and pre-eclampsia Hum. Reprod., September 1, 2000; 15(9): 2033 - 2036. [Abstract] [Full Text] [PDF]

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