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K+-ATPase Activity and Expression in Syncytiotrophoblast Plasma Membranes in Pregnancies Complicated by Diabetes
Placenta (2002), 23, 386–391 doi:10.1053/plac.2002.0807, available online at http://www.idealibrary.com on
Na + /K + -ATPase Activity and Expression in Syncytiotrophoblast Plasma Membranes in Pregnancies Complicated by Diabetes A. Persson, M. Johansson, T. Jansson and T. L. Powell Perinatal Center, Department of Physiology and Pharmacology, Go¨teborg University, Box 432, s-405 30 Go¨teborg, Sweden Paper accepted 22 January 2002
Many of the transport processes across the syncytiotrophoblast (ST), such as amino acid transport, are Na + -coupled. The maintenance of a low intracellular Na + concentration by Na + /K + -ATPase is therefore crucial for placental transport of nutrients and consequently, foetal growth. In pregnancies complicated by diabetes foetal growth is often accelerated despite rigorous glycemic control of the mother, however the underlying mechanisms are not fully understood. We tested the hypothesis that Na + /K + -ATPase in ST plasma membranes is up-regulated in diabetic pregnancies associated with accelerated growth. ST microvillous (MVM) and basal (BM) plasma membranes were purified from term placentas of normal pregnancies (control, n=13) and pregnancies complicated by insulin-dependent diabetes mellitus (n=7) or gestational diabetes (n=6). All mothers with diabetes gave birth to large for gestational age babies. The Na + /K + -ATPase 1-subunit protein expression (Western blot) in MVM and BM was unaltered by diabetes. Na + /K + -ATPase activity (K + -stimulated, ouabain-sensitive phosphatase activity) in ST plasma membranes was not affected by diabetes. This is the first study of Na + /K + -ATPase in ST membranes of the human placenta in diabetes. Our data show that accelerated foetal growth in diabetic pregnancies is not associated with elevated ST 2002 Published by Elsevier Science Ltd. Na + /K + -ATPase protein expression or activity. Placenta (2002), 23, 386–391
INTRODUCTION In pregnancies complicated by diabetes accelerated foetal growth (macrosomia) still constitutes a significant clinical problem (Spellacy et al., 1985; Garner, 1995). Delivery of a large for gestational age baby is associated with complications such as traumatic birth injury and asphyxia as well as high rate of operative delivery. In general, high birthweight has been linked to an increased incidence of breast cancer later in life (Ekbom et al., 1992). Further, hyperglycaemia of the mother during pregnancy is associated with long-term effects in the offspring such as obesity and diabetes (Pettitt et al., 1993) as well as gestational diabetes (Pettitt et al., 1991). As proposed by Pedersen (1954), a primary mechanism for the accelerated foetal growth is maternal hyperglycaemia, which, due to the facilitated nature of transplacental glucose transport, results in an elevation of foetal glucose levels. Foetal hyperglycaemia increases secretion of insulin, which stimulates foetal growth. However, accelerated foetal growth also occurs in pregnancies complicated by diabetes in which maternal glycemic control has been strict (Garner, 1995), suggesting that the relationship between maternal metabolic disturbance and foetal growth rate is complex and goes beyond maternal glucose levels. One possibility is that alterations in placental transport function may explain the weak correlation between indices of maternal glycemic control and birthweight. 0143–4004/02/$-see front matter
Compatible with this hypothesis is the demonstration that placental glucose transporter expression and activity are up regulated in pregnancies complicated by maternal insulindependent diabetes and foetal overgrowth (Jansson et al., 1999). The concentration of K + is typically 10–20 times higher inside cells than outside, whereas the reverse is true of Na + . The Na + /K + -ATPase is found in the plasma membrane of virtually all animal cells and maintains these concentration differences, which are crucial to cell volume regulation, pH regulation and nerve and muscle excitability. The Na + gradient is also crucial to drive transport of sugars and amino acids into the cell. Na + /K + -ATPase is usually uniformly distributed over the surface of the cells with the exception of most transporting epithelial cells, where Na + /K + -ATPase is polarized and exclusively localized to the basolateral membrane of the cell. However, recent findings have shown a different distribution of Na + /K + -ATPase in the syncytiotrophoblast, the transporting epithelium of the human placenta. In this cell, the activity of Na + /K + -ATPase, as well as the protein expression, is higher in the microvillous plasma membrane than in the basal plasma membrane (Johansson et al., 2000a). The activity of Na + /K + -ATPase is, in general, reduced in diabetes and these alterations have been implicated to be involved in the development of complications in this disease 2002 Published by Elsevier Science Ltd.
Persson et al.: Na + /K + -ATPase Activity and Expression
(Greene et al., 1987). Previous studies on either purified Na + /K + -ATPase or a crude membrane fraction from placentas in IDDM have shown a reduced enzyme activity (Zolese et al., 1997; Rabini et al., 1998). However, since the placenta consists of a number of different cell types, these studies do not address whether the activity of this transporter is altered in the plasma membrane of the syncytiotrophoblasts, the barrier for nutrient transport. In the placental syncytiotrophoblast, the Na + -gradient is the driving force for the transport of many nutrients, and Na + /K + -ATPase alone is responsible for maintaining this gradient. Consequently, a change in activity of Na + /K + -ATPase may directly affect the supply of nutrients to the foetus. In particular, the active transport of amino acids cross the placenta is dependent on the Na + -gradient across the syncytiotrophoblast plasma membranes. Since amino acids are potent stimulators of foetal insulin release (Milner and Hill, 1984) alterations in syncytiotrophoblast Na + /K + -ATPase activity may affect amino acid delivery to the foetus and subsequently foetal growth. Syncytiotrophoblast plasma membrane Na + /K + -ATPase activity and expression has been shown to be reduced in pregnancies complicated by IUGR (Johansson et al., 2000b). In the present study, we tested the hypothesis that Na + /K + ATPase activity is up-regulated in the transporting epithelium of the placenta in diabetic pregnancies associated with accelerated foetal growth. On-line Na + /K + -ATPase activity assays were performed on isolated syncytiotrophoblast MVM and BM fractions, from control, IDDM and GDM subjects, respectively. The protein expression of Na + /K + -ATPase 1-subunit in all three groups was analysed by Western blotting.
MATERIALS AND METHODS Placental material Human placentas were obtained immediately after vaginal delivery or caesarean section from 11 women with uncomplicated pregnancies, seven women affected by IDDM and six women who developed GDM. The collection of placental material was approved by the Committee for Research Ethics at Go¨teborg University. IDDM patients were classified as diabetes White D, defined as onset of disease prior to 10 years of age or a history of diabetes longer than 20 years, or White C, defined as onset of disease at 10–19 years of age or duration of 10–19 years (White, 1954). All IDDM patients received insulin in a 5-dose regime and had no other major complications. Screening for GDM was carried out routinely in all pregnancies by measurements of capillary blood glucose in the non-fasting state on at least four occasions starting in weeks 8–12 of gestation. If non-fasting blood-glucose was d7.0 mmol/l, patients were referred to a 75 g oral glucose tolerance test (OGTT) after overnight fasting. GDM was defined as a fasting blood glucose d6.1 mmol/l or blood glucose d9.0 mmol/l 2 h after OGTT in a pregnant woman
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without prior known diabetes. Patients with gestational diabetes were treated with diet adjustments only (Diabetes White A), or insulin administered in a 5-dose regime (Diabetes White AB). Criteria for initiating insulin therapy were elevated blood glucose levels (fasting glucose d6.1 and/or postprandial blood glucose d8.0) despite consistent dietary adjustments. All pregnant women with diabetes attended the obstetric antenatal clinic at Sahlgren’s University Hospital, Go¨teborg. Estimated date of delivery was determined from the last menstrual period and confirmed by ultrasound at 16–18 week of gestation. All women included in the diabetic groups gave birth to babies that were large for gestational age (LGA), defined as having a birthweight greater than the mean birthweight plus two standard deviations at that gestational age using intrauterine growth curves based on ultrasonically estimated foetal weight (Marsal et al., 1996).
Isolation of membrane vesicles The microvillous membrane (MVM) and the basal membrane (BM) of the syncytiotrophoblast was isolated from human placenta as described previously (Illsley et al., 1990) with some modifications (Johansson et al., 2000a). All preparation steps were carried out at +4C. In brief, the placenta was dissected and the amniotic sac, chorionic plate and decidua were removed. Placental tissue (100 g) was rinsed in cold physiological saline. Cold buffer D [sucrose (250 m), HEPES-Tris (10 m) and protease inhibitors; antipain (0.8 ), pepstatin (0.7 ), aprotinin (80 n) and EDTA] was added (2.5 ml/g tissue). Tissue was homogenized and the homogenate was then centrifuged for 15 min at 10 000g. The supernatant was collected and the pellet resuspended in buffer D and again homogenized and centrifuged for 10 min (10 000g). The combined supernatant was centrifuged at 125 000g for 30 min. Subsequently, the pellet (post nuclear fraction; P2) was resuspended in buffer D, MgCl2 (12 m) was added and the suspension was stirred slowly for 20 min. The suspension was then centrifuged at 2500g for 10 min. The supernatant, containing the MVM vesicles, was collected and centrifuged at 125 000g for 30 min and the pelleted MVM vesicles were homogenized in buffer D and placed on ice. The pellet, containing the precipitated BM vesicles, was homogenized in buffer D and purified in a sucrose gradient centrifugation step at 144 000g for 60 min. BM vesicles were collected and homogenized in buffer D and placed on ice. Finally, BM and MVM collections were then centrifuged at 125 000g for 30 min and the pellets were homogenized in buffer D, aliquoted, snap-frozen in liquid nitrogen and stored at 80C until use. The protein concentration of MVM and BM fractions was determined using the Bradford Assay. Using alkaline phosphatase as a MVM marker and adenylate cyclase as a BM marker, the enrichments of MVM and BM vesicles were tested by activity assays for alkaline phosphatase (Bowers and McComb, 1966) and adenylate cyclase (Schultz and Jakobs, 1984).
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Western blot analysis
Table 1. Clinical data
Membrane proteins from MVM and BM were separated by SDS-PAGE as previously described (Johansson et al., 2000a). In brief, vesicle preparations were thawed on ice and diluted with buffer D and sample buffer to a final concentration of 1 g/l. Electrophoresis was carried out at 200 V with a 7 per cent SDS-polyacrylamide gel onto which 10 g protein/well was loaded. A pre stained SDS molecular weight marker with a range from 26.6–180 kDa (Sigma) was used. After electrophoresis the gels were equilibrated in transfer buffer and mounted with a nitrocellulose transfer membrane, and blotted over night at 30 V. The membrane was then blocked in 5 per cent blotto buffer. For detection of Na + /K + -ATPase, the membrane was incubated with a monoclonal 1-specific antibody (Developmental Studies Hybridoma Bank, diluted 1 : 1000). The secondary horseradish peroxidase labelled sheep anti-mouse antibody (Amersham, diluted 1 : 1000) was used in conjunction with enhanced chemiluminiscence to visualize 1-subunit bands on autoradiographic film. Quantification of the 1-subunit signal was done with IP Lab Gel (Signal Analytics). The mean density for control (MVM and BM respectively) was arbitrarily assigned a value of one and the mean densities of the IDDM and GDM groups respectively, were calculated relative to the control group.
Placental weight (g)
Foetal weight (g)
64434 103322* 90272*
3351104 4528143* 4546127*
Na + /K + -ATPase assay Na + /K + -ATPase activity in MVM and BM vesicles was measured by a fluorometric method (Hill et al., 1968) with a few modifications (Johansson et al., 2000a). The method is based on the on-line detection of a change in fluorescence due to the cleavage of 3-O-methylfluorescein phosphate (3-OMFP) to 3-O-methylfluorescein (3-O-MF) by Na + /K + ATPase. In brief, fluorescence was assayed using a fluorometer, with the assay mixture in a thermostatically controlled (+37C) cuvette. Excitation wavelength was set to 470 nm and emission wavelength to 510 nm where the emission intensity was proportional to the concentration of 3-O-MF. A standard curve was created by using varying concentrations of 3-O-MF (0.05 –25 ). The medium for the assay contained 3-O-MFP, creatine phosphate, MgCl2, EGTA and Tris HCl pH 7.2. All assays were carried out under vigorous stirring at +37C. A baseline of background fluorescence was recorded for 30 s. Thereafter, approximately 100 g of protein of MVM or BM vesicles were added to the medium and fluorescence was recorded for additional 90 s. After this initial 120 s, KCl was added in order to activate Na + /K + -ATPase. On-line recording of fluorescence changes continued for another 90 s. The experiments were repeated with assay medium containing the Na + /K + -ATPase inhibitor ouabain (3 m). The activity of Na + /K + -ATPase was calculated as the slope difference in fluorescence recordings, before and after K + addition, with and without ouabain in the assay medium. Calculated data were related to protein content in
Group
n
Gestational age (weeks)
Control IDDM GDM
13 7 6
39.10.4 38.00.4 39.50.7
Values are given as means. *P<0.05 compared to control group.
Table 2. Glycosylated haemoglobin (HbA1C) in pregnancies complicated by IDDM Trimester
HbA1C
95 per cent c.i.
1st 2nd 3rd
6.600.49* 5.410.28 5.610.16
5.34–7.86 4.73–6.08 3.67–5.99
Values are given as means and 95 per cent confidence intervals (n=7). HbA1C value of IDDM patients was significantly different from the normal range of HbA1C in uncomplicated pregnancies (3.90–5.30), *P<0.05.
vesicle preparations and the final expression of Na + /K + ATPase activity was given as nmol liberated PO4/min/mg protein. We have previously shown that Na + /K + -ATPase activity measured in frozen vesicles is not significantly different from activities measured in freshly prepared plasma membranes (Johansson et al., 2000a). Data presentation and statistical presentation Analysis of variance (ANOVA) followed by Student– Newman–Keuls test was used to evaluate differences between groups statistically. Values are presented as means and standard error of mean (). RESULTS Study group characterization Gestational age was not significantly different between the three groups under study (Table 1). Foetal weights were higher in the IDDM and GDM groups. The placentas in pregnancies complicated by diabetes were also larger than placentas of the control group. The glycosylated haemoglobin (HbA1C) in pregnancies complicated by IDDM (no data was available on GDM patients) was increased in the 1st trimester but within normal range in the second and third trimester (Table 2). Purity of syncytiotrophoblast membrane vesicles Marker enzyme activity for homogenate and MVM fractions, post nuclear membrane pellet (P2) and BM fractions are shown
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Table 3. Marker enzyme activities Alkaline phosphatase (nmol PO4/smg)
Control
n 10
IDDM
7
GDM
6
MVM 69.112.3 (19.6) 87.021.8 (20.0) 50.417.4 (22.1)
BM 7.01.0 (2.0) 9.01.6 (2.1) 5.70.8 (2.5)
Adenylate cyclase (pmol/minmg) Hom 3.50.5
n 6
4.40.7
6
2.30.4
6
BM 43079.5 (31.5) 34728.1 (29.3) 43067.3 (32.8)
P2 13.69.1 11.85.4 13.12.7
Values are given as means. Enzyme enrichments (in parentheses) were calculated as vesicle enzyme specific activity relative to that for the homogenate (alkaline phosphatase) or the post nuclear membrane pellet (P2; adenylate cyclase).
Figure 1. Western blot of the Na + /K + -ATPase 1-subunit in MVM of control patients (C) and from pregnancies complicated by gestational diabetes (GDM).
in Table 3. Alkaline phosphatase has a high abundance in the MVM, which was demonstrated by a 20-fold enrichment in control MVM. Adenylate cyclase that served as the BM marker was enriched 32-fold in the BM isolated from control placentas. Enzyme activities in vesicles isolated from IDDM/ GDM placentas were not significantly different from control (Table 3). Protein expression of Na + /K + -ATPase Western blot analysis of the syncytiotrophoblast MVM and BM isolated from placentas of control, IDDM and GDM subjects, showed the presence of the 1-subunits of Na + /K + ATPase at a molecular weight of approximately 96 kDa (shown for controls and GDM in Figure 1). The relative density of the 1-subunit protein was not significantly different between groups in MVM [Figure 2(A)] or BM [Figure 2(B)]. Na + /K + -ATPase activity Na + /K + -ATPase activity, as measured as K + -stimulated, ouabain inhibitable phosphatase activity, in MVM and BM isolated from control, GDM and IDDM groups are shown in Figure 3. There was no significant alteration in MVM or BM Na + /K + -ATPase activity in diabetes associated with accelerated foetal growth. DISCUSSION In pregnancies complicated by diabetes mellitus, foetal growth is often accelerated. Pedersen proposed in 1954 that the
increased foetal growth in these pregnancies is directly related to maternal hyperglycaemia, which will result in an increased delivery of glucose to the foetus. Foetal hyperglycaemia stimulates the secretion of insulin, the most important growthpromoting hormone during foetal life. However, despite rigorous maternal glycemic control in modern management of the diabetic pregnancy, the incidence of large-for gestational age babies remains high. This fact suggests a more complicated model for the mechanisms underlying accelerated foetal growth than originally proposed by Pedersen. One possibility that has been explored during recent years is that diabetes in pregnancy alters placental transport function. Indeed, it has been shown that placental glucose transporter (Gaither et al., 1999; Jansson et al., 1999) as well as the system A amino acid transporter (Jansson et al., 2001) are up-regulated in pregnancies with diabetes mellitus. These findings may provide one explanation for the accelerated foetal growth despite ‘normal’ maternal glucose levels. In this study we tested the hypothesis that the activity/ expression of the syncytiotrophoblast plasma membrane Na + / K + -ATPase is increased in pregnancies with diabetes and associated with accelerated foetal growth. The rationale for this hypothesis is as follows. First, the transport of many nutrients, in particular amino acids, across the placenta is dependent on the Na + gradient across the microvillous plasma membrane of the syncytiotrophoblast. Since this gradient is maintained by Na + /K + -ATPase, any alteration in the expression/activity of this transporter may affect amino acid transport and consequently, foetal growth. Second, it has recently been shown that in pregnancies complicated by IUGR, the activity and expression of syncytiotrophoblast plasma membrane Na + /K + ATPase is reduced (Johansson et al., 2000b). This suggests a relationship between the activity of this transporter in the transporting epithelium of the placenta and foetal growth. However, in this study we have shown that the protein expression as well as the activity of Na + /K + -ATPase is not altered in the full term placental MVM or BM of diabetic mothers that have given birth to babies large for gestational age. It is therefore unlikely that changes in sodium pump activity in the placental barrier contribute to foetal overgrowth in diabetic pregnancies.
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Figure 3. Na + /K + -ATPase activity in the placental syncytiotrophoblast MVM and BM (control n=11, IDDM n=7 and GDM n=7). Activity was defined as K + -stimulated, ouabain-inhibitable release of phosphate. Activity data is presented as nmol PO4/min/mg protein. There was no significant difference in activity between groups in neither BM nor MVM. Values are given as means.
Figure 2. The relative density of Na + /K + -ATPase 1-subunit protein expression as measured by Western blot analysis in syncytiotrophoblast microvillous (A) and basal plasma membranes (B) isolated from uncomplicated pregnancies (control n=12–13) and pregnancies complicated by IDDM (n=6–7) or GDM (n=6). The protein expression in pregnancies complicated by diabetes (IDDM and GDM) was not significantly different from controls. The mean density for control MVM and BM, respectively was arbitrarily assigned a value of one and the mean densities of IDDM and GDM calculated relative to the control group. Values are given as means.
In general, diabetes is associated with a reduced activity of Na + /K + -ATPase in tissues such as neurons (Greene and
Mackway, 1986), retina (MacGregor and Matschinsky, 1986), renal glomerulus (Cohen et al., 1985) and the aorta (Simmons et al., 1986). This study clearly demonstrates that the syncytiotrophoblast plasma membranes are not affected in a similar way. In studies of crude membrane fractions as well as purified Na + /K + -ATPase obtained from human placenta, Na + /K + -ATPase activity has been shown to be reduced (Zolese et al., 1997; Rabini et al., 1998). The apparent discrepancies between previous studies and our study can be resolved by considering the possibility that a crude membrane fraction from the whole placenta may not be representative for the syncytiotrophoblast plasma membranes. Since it is well known that erythrocyte Na + /K + -ATPase activity is reduced in IDDM (Finotti and Palatini, 1986; Raccah et al., 1992; De La Tour et al., 1998), erythrocyte membranes in the crude preparations may provide an explanation for the reduction in the studies of Zolese and co-workers. Term placental tissue were used in the current study and the finding of unchanged Na + /K + -ATPase activity in the placental transport barrier in association with diabetes does not preclude alterations in this transporter earlier in gestation. With respect to the IDDM group late first and early second trimester represents a period of gestation that is characterized by a suboptimal glucose control even in patients with successful management during the second half of pregnancy. This is illustrated by the elevated HbA1C values of the IDDM patients in first trimester in our study. Indeed, in some studies first trimester HbA1C has been identified as the only index of maternal glucose control that correlates to birthweight suggesting that important regulation of for example placental nutrient
Persson et al.: Na + /K + -ATPase Activity and Expression
transporters may take place early in pregnancy. Studies of placental transporters such as glucose and amino acid transporters as well as Na + /K + -ATPase in early pregnancy may provide important information about mechanisms underlying accelerated foetal growth in diabetes.
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In conclusion, we have extended previous studies on placental Na + /K + -ATPase activity in diabetes and demonstrated that the activity and protein expression of this transporter is unaltered in the transport barrier of the placenta in diabetic pregnancies associated with accelerated foetal growth.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (10838), the Swedish Diabetes Association, the Ragnar and Torsten So¨derberg Foundation, Frimurare-Barnhus-direktionen, the A r hlens Foundation, and the Willhelm & Martina Lundgrens Foundation.
REFERENCES Bowers GN Jr & McComb RB (1966) A continuous spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clin Chem, 12, 70–89. Cohen MP, Dasmahapatra A & Shapiro E (1985) Reduced glomerular sodium/potassium adenosine triphosphatase activity in acute streptozocin diabetes and its prevention by oral sorbinil. Diabetes, 34, 1071–1074. De La Tour DD, Raccah D, Jannot MF, Coste T, Rougerie C & Vague P (1998) Erythrocyte Na/K ATPase activity and diabetes: relationship with C-peptide level. Diabetologia, 41, 1080–1084. Ekbom A, Trichopoulos D, Adami HO, Hsieh CC & Lan SJ (1992) Evidence of prenatal influences on breast cancer risk. Lancet, 340, 1015– 1018. Finotti P & Palatini P (1986) Reduction of erythrocyte (Na+-K+)ATPase activity in type 1 (insulin-dependent) diabetic subjects and its activation by homologous plasma. Diabetologia, 29, 623–628. Gaither K, Quraishi AN & Illsley NP (1999) Diabetes alters the expression and activity of the human placental GLUT1 glucose transporter. J Clin Endocrinol Metab, 84, 695–701. Garner P (1995) Type I diabetes mellitus and pregnancy. Lancet, 346, 157–161. Greene DA & Mackway AM (1986) Decreased myo-inositol content and Na+-K+-ATPase activity in superior cervical ganglion of STZ-diabetic rat and prevention by aldose reductase inhibition. Diabetes, 35, 1106–1108. Greene DA, Lattimer SA & Sima AA (1987) Sorbitol, phosphoinositides, and sodium potassium-ATPase in the pathogenesis of diabetic complications. N Engl J Med, 316, 599–606. Hill HD, Summer GK & Waters MD (1968) An automated fluorometric assay for alkaline phosphatase using 3-O-methylfluorescein phosphate. Anal Biochem, 24, 9–17. Illsley NP, Wang ZQ, Gray A, Sellers MC & Jacobs MM (1990) Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. Biochim Biophys Acta, 1029, 218–226. Jansson T, Wennergren M & Powell TL (1999) Placental glucose transport and GLUT 1 expression in insulin-dependent diabetes. Am J Obstet Gynecol, 180, 163–168. Jansson T, Ekstrand Y, Bjo¨rn C & Powell TL (2001) Diabetes is associated with an increased activity of placental system A. Abstract. Placenta, 22, A55. Johansson M, Jansson T & Powell TL (2000a) Na(+)-K(+)-ATPase is distributed to microvillous and basal membrane of the syncytiotrophoblast in human placenta. Am J Physiol Regul Integr Comp Physiol, 279, R287– 294.
Johansson M, Jansson T & Powell TL (2000b) Human placental Na + /K + ATPase activity and protein expression are reduced in microvillous membranes from IUGR placentas. J Soc Gynecol Investig, 7(Supplement 1), 139A. MacGregor LC & Matschinsky FM (1986) Altered retinal metabolism in diabetes. II. Measurement of sodium-potassium ATPase and total sodium and potassium in individual retinal layers. J Biol Chem, 261, 4052–4058. Marsal K, Persson PH, Larsen T, Lilja H, Selbing A & Sultan B (1996) Intrauterine growth curves based on ultrasonically estimated foetal weights. Acta Paediatr, 85, 843–848. Milner RD & Hill DJ (1984) Foetal growth control: the role of insulin and related peptides. Clin Endocrinol (Oxf), 21, 415–433. Pedersen J (1954) Weight and length at birth of infants of diabetic mothers. Acta Endocrinol (Copenh), 16, 1554–1562. Pettitt DJ, Bennett PH, Saad MF, Charles MA, Nelson RG & Knowler WC (1991) Abnormal glucose tolerance during pregnancy in Pima Indian women: long-term effects on offspring. Diabetes, 40(Suppl 2), 126–130. Pettitt DJ, Nelson RG, Saad MF, Bennett PH & Knowler WC (1993) Diabetes and obesity in the offspring of Pima Indian women with diabetes during pregnancy. Diabetes Care, 16(Suppl 1), 310–314. Rabini RA, Fumelli P, Zolese G, Amler E, Salvolini E, Staffolani R, Cester N & Mazzanti L (1998) Modifications induced by plasma from insulin-dependent diabetic patients and by lysophosphatidylcholine on human Na+,K(+)-adenosine triphosphatase. J Clin Endocrinol Metab, 83, 2405–2410. Raccah D, Gallice P, Pouget J & Vague P (1992) Hypothesis: low Na/K-ATPase activity in the red cell membrane, a potential marker of the predisposition to diabetic neuropathy. Diabete Metab, 18, 236–241. Schultz G & Jakobs K (1984) Adenylate cyclase. In Methods of Enzymatic Analysis 3rd Edition (Ed.) Bergmeyer H, pp. 369–378. Weinheim: Verlag Chemie. Simmons DA, Kern EF, Winegrad AI & Martin DB (1986) Basal phosphatidylinositol turnover controls aortic Na+/K+ ATPase activity. J Clin Invest, 77, 503–513. Spellacy WN, Miller S, Winegar A & Peterson PQ (1985) Macrosomiamaternal characteristics and infant complications. Obstet Gynecol, 66, 158– 161. White P (1954) Pregnancy Complicating Diabetes. American Journal of Medicine, 7, 609–616. Zolese G, Rabini RA, Fumelli P, Staffolani R, Curatola A, Kvasnicka P, Kotyk A, Cester N & Mazzanti L (1997) Modifications induced by insulin-dependent diabetes mellitus on human placental Na+/K+adenosine triphosphatase. J Lab Clin Med, 130, 374–380.
Na + /K + -ATPase Activity and Expression in Syncytiotrophoblast Plasma Membranes in Pregnancies Complicated by Diabetes A. Persson, M. Johansson, T. Jansson and T. L. Powell Perinatal Center, Department of Physiology and Pharmacology, Go¨teborg University, Box 432, s-405 30 Go¨teborg, Sweden Paper accepted 22 January 2002
Many of the transport processes across the syncytiotrophoblast (ST), such as amino acid transport, are Na + -coupled. The maintenance of a low intracellular Na + concentration by Na + /K + -ATPase is therefore crucial for placental transport of nutrients and consequently, foetal growth. In pregnancies complicated by diabetes foetal growth is often accelerated despite rigorous glycemic control of the mother, however the underlying mechanisms are not fully understood. We tested the hypothesis that Na + /K + -ATPase in ST plasma membranes is up-regulated in diabetic pregnancies associated with accelerated growth. ST microvillous (MVM) and basal (BM) plasma membranes were purified from term placentas of normal pregnancies (control, n=13) and pregnancies complicated by insulin-dependent diabetes mellitus (n=7) or gestational diabetes (n=6). All mothers with diabetes gave birth to large for gestational age babies. The Na + /K + -ATPase 1-subunit protein expression (Western blot) in MVM and BM was unaltered by diabetes. Na + /K + -ATPase activity (K + -stimulated, ouabain-sensitive phosphatase activity) in ST plasma membranes was not affected by diabetes. This is the first study of Na + /K + -ATPase in ST membranes of the human placenta in diabetes. Our data show that accelerated foetal growth in diabetic pregnancies is not associated with elevated ST 2002 Published by Elsevier Science Ltd. Na + /K + -ATPase protein expression or activity. Placenta (2002), 23, 386–391
INTRODUCTION In pregnancies complicated by diabetes accelerated foetal growth (macrosomia) still constitutes a significant clinical problem (Spellacy et al., 1985; Garner, 1995). Delivery of a large for gestational age baby is associated with complications such as traumatic birth injury and asphyxia as well as high rate of operative delivery. In general, high birthweight has been linked to an increased incidence of breast cancer later in life (Ekbom et al., 1992). Further, hyperglycaemia of the mother during pregnancy is associated with long-term effects in the offspring such as obesity and diabetes (Pettitt et al., 1993) as well as gestational diabetes (Pettitt et al., 1991). As proposed by Pedersen (1954), a primary mechanism for the accelerated foetal growth is maternal hyperglycaemia, which, due to the facilitated nature of transplacental glucose transport, results in an elevation of foetal glucose levels. Foetal hyperglycaemia increases secretion of insulin, which stimulates foetal growth. However, accelerated foetal growth also occurs in pregnancies complicated by diabetes in which maternal glycemic control has been strict (Garner, 1995), suggesting that the relationship between maternal metabolic disturbance and foetal growth rate is complex and goes beyond maternal glucose levels. One possibility is that alterations in placental transport function may explain the weak correlation between indices of maternal glycemic control and birthweight. 0143–4004/02/$-see front matter
Compatible with this hypothesis is the demonstration that placental glucose transporter expression and activity are up regulated in pregnancies complicated by maternal insulindependent diabetes and foetal overgrowth (Jansson et al., 1999). The concentration of K + is typically 10–20 times higher inside cells than outside, whereas the reverse is true of Na + . The Na + /K + -ATPase is found in the plasma membrane of virtually all animal cells and maintains these concentration differences, which are crucial to cell volume regulation, pH regulation and nerve and muscle excitability. The Na + gradient is also crucial to drive transport of sugars and amino acids into the cell. Na + /K + -ATPase is usually uniformly distributed over the surface of the cells with the exception of most transporting epithelial cells, where Na + /K + -ATPase is polarized and exclusively localized to the basolateral membrane of the cell. However, recent findings have shown a different distribution of Na + /K + -ATPase in the syncytiotrophoblast, the transporting epithelium of the human placenta. In this cell, the activity of Na + /K + -ATPase, as well as the protein expression, is higher in the microvillous plasma membrane than in the basal plasma membrane (Johansson et al., 2000a). The activity of Na + /K + -ATPase is, in general, reduced in diabetes and these alterations have been implicated to be involved in the development of complications in this disease 2002 Published by Elsevier Science Ltd.
Persson et al.: Na + /K + -ATPase Activity and Expression
(Greene et al., 1987). Previous studies on either purified Na + /K + -ATPase or a crude membrane fraction from placentas in IDDM have shown a reduced enzyme activity (Zolese et al., 1997; Rabini et al., 1998). However, since the placenta consists of a number of different cell types, these studies do not address whether the activity of this transporter is altered in the plasma membrane of the syncytiotrophoblasts, the barrier for nutrient transport. In the placental syncytiotrophoblast, the Na + -gradient is the driving force for the transport of many nutrients, and Na + /K + -ATPase alone is responsible for maintaining this gradient. Consequently, a change in activity of Na + /K + -ATPase may directly affect the supply of nutrients to the foetus. In particular, the active transport of amino acids cross the placenta is dependent on the Na + -gradient across the syncytiotrophoblast plasma membranes. Since amino acids are potent stimulators of foetal insulin release (Milner and Hill, 1984) alterations in syncytiotrophoblast Na + /K + -ATPase activity may affect amino acid delivery to the foetus and subsequently foetal growth. Syncytiotrophoblast plasma membrane Na + /K + -ATPase activity and expression has been shown to be reduced in pregnancies complicated by IUGR (Johansson et al., 2000b). In the present study, we tested the hypothesis that Na + /K + ATPase activity is up-regulated in the transporting epithelium of the placenta in diabetic pregnancies associated with accelerated foetal growth. On-line Na + /K + -ATPase activity assays were performed on isolated syncytiotrophoblast MVM and BM fractions, from control, IDDM and GDM subjects, respectively. The protein expression of Na + /K + -ATPase 1-subunit in all three groups was analysed by Western blotting.
MATERIALS AND METHODS Placental material Human placentas were obtained immediately after vaginal delivery or caesarean section from 11 women with uncomplicated pregnancies, seven women affected by IDDM and six women who developed GDM. The collection of placental material was approved by the Committee for Research Ethics at Go¨teborg University. IDDM patients were classified as diabetes White D, defined as onset of disease prior to 10 years of age or a history of diabetes longer than 20 years, or White C, defined as onset of disease at 10–19 years of age or duration of 10–19 years (White, 1954). All IDDM patients received insulin in a 5-dose regime and had no other major complications. Screening for GDM was carried out routinely in all pregnancies by measurements of capillary blood glucose in the non-fasting state on at least four occasions starting in weeks 8–12 of gestation. If non-fasting blood-glucose was d7.0 mmol/l, patients were referred to a 75 g oral glucose tolerance test (OGTT) after overnight fasting. GDM was defined as a fasting blood glucose d6.1 mmol/l or blood glucose d9.0 mmol/l 2 h after OGTT in a pregnant woman
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without prior known diabetes. Patients with gestational diabetes were treated with diet adjustments only (Diabetes White A), or insulin administered in a 5-dose regime (Diabetes White AB). Criteria for initiating insulin therapy were elevated blood glucose levels (fasting glucose d6.1 and/or postprandial blood glucose d8.0) despite consistent dietary adjustments. All pregnant women with diabetes attended the obstetric antenatal clinic at Sahlgren’s University Hospital, Go¨teborg. Estimated date of delivery was determined from the last menstrual period and confirmed by ultrasound at 16–18 week of gestation. All women included in the diabetic groups gave birth to babies that were large for gestational age (LGA), defined as having a birthweight greater than the mean birthweight plus two standard deviations at that gestational age using intrauterine growth curves based on ultrasonically estimated foetal weight (Marsal et al., 1996).
Isolation of membrane vesicles The microvillous membrane (MVM) and the basal membrane (BM) of the syncytiotrophoblast was isolated from human placenta as described previously (Illsley et al., 1990) with some modifications (Johansson et al., 2000a). All preparation steps were carried out at +4C. In brief, the placenta was dissected and the amniotic sac, chorionic plate and decidua were removed. Placental tissue (100 g) was rinsed in cold physiological saline. Cold buffer D [sucrose (250 m), HEPES-Tris (10 m) and protease inhibitors; antipain (0.8 ), pepstatin (0.7 ), aprotinin (80 n) and EDTA] was added (2.5 ml/g tissue). Tissue was homogenized and the homogenate was then centrifuged for 15 min at 10 000g. The supernatant was collected and the pellet resuspended in buffer D and again homogenized and centrifuged for 10 min (10 000g). The combined supernatant was centrifuged at 125 000g for 30 min. Subsequently, the pellet (post nuclear fraction; P2) was resuspended in buffer D, MgCl2 (12 m) was added and the suspension was stirred slowly for 20 min. The suspension was then centrifuged at 2500g for 10 min. The supernatant, containing the MVM vesicles, was collected and centrifuged at 125 000g for 30 min and the pelleted MVM vesicles were homogenized in buffer D and placed on ice. The pellet, containing the precipitated BM vesicles, was homogenized in buffer D and purified in a sucrose gradient centrifugation step at 144 000g for 60 min. BM vesicles were collected and homogenized in buffer D and placed on ice. Finally, BM and MVM collections were then centrifuged at 125 000g for 30 min and the pellets were homogenized in buffer D, aliquoted, snap-frozen in liquid nitrogen and stored at 80C until use. The protein concentration of MVM and BM fractions was determined using the Bradford Assay. Using alkaline phosphatase as a MVM marker and adenylate cyclase as a BM marker, the enrichments of MVM and BM vesicles were tested by activity assays for alkaline phosphatase (Bowers and McComb, 1966) and adenylate cyclase (Schultz and Jakobs, 1984).
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Western blot analysis
Table 1. Clinical data
Membrane proteins from MVM and BM were separated by SDS-PAGE as previously described (Johansson et al., 2000a). In brief, vesicle preparations were thawed on ice and diluted with buffer D and sample buffer to a final concentration of 1 g/l. Electrophoresis was carried out at 200 V with a 7 per cent SDS-polyacrylamide gel onto which 10 g protein/well was loaded. A pre stained SDS molecular weight marker with a range from 26.6–180 kDa (Sigma) was used. After electrophoresis the gels were equilibrated in transfer buffer and mounted with a nitrocellulose transfer membrane, and blotted over night at 30 V. The membrane was then blocked in 5 per cent blotto buffer. For detection of Na + /K + -ATPase, the membrane was incubated with a monoclonal 1-specific antibody (Developmental Studies Hybridoma Bank, diluted 1 : 1000). The secondary horseradish peroxidase labelled sheep anti-mouse antibody (Amersham, diluted 1 : 1000) was used in conjunction with enhanced chemiluminiscence to visualize 1-subunit bands on autoradiographic film. Quantification of the 1-subunit signal was done with IP Lab Gel (Signal Analytics). The mean density for control (MVM and BM respectively) was arbitrarily assigned a value of one and the mean densities of the IDDM and GDM groups respectively, were calculated relative to the control group.
Placental weight (g)
Foetal weight (g)
64434 103322* 90272*
3351104 4528143* 4546127*
Na + /K + -ATPase assay Na + /K + -ATPase activity in MVM and BM vesicles was measured by a fluorometric method (Hill et al., 1968) with a few modifications (Johansson et al., 2000a). The method is based on the on-line detection of a change in fluorescence due to the cleavage of 3-O-methylfluorescein phosphate (3-OMFP) to 3-O-methylfluorescein (3-O-MF) by Na + /K + ATPase. In brief, fluorescence was assayed using a fluorometer, with the assay mixture in a thermostatically controlled (+37C) cuvette. Excitation wavelength was set to 470 nm and emission wavelength to 510 nm where the emission intensity was proportional to the concentration of 3-O-MF. A standard curve was created by using varying concentrations of 3-O-MF (0.05 –25 ). The medium for the assay contained 3-O-MFP, creatine phosphate, MgCl2, EGTA and Tris HCl pH 7.2. All assays were carried out under vigorous stirring at +37C. A baseline of background fluorescence was recorded for 30 s. Thereafter, approximately 100 g of protein of MVM or BM vesicles were added to the medium and fluorescence was recorded for additional 90 s. After this initial 120 s, KCl was added in order to activate Na + /K + -ATPase. On-line recording of fluorescence changes continued for another 90 s. The experiments were repeated with assay medium containing the Na + /K + -ATPase inhibitor ouabain (3 m). The activity of Na + /K + -ATPase was calculated as the slope difference in fluorescence recordings, before and after K + addition, with and without ouabain in the assay medium. Calculated data were related to protein content in
Group
n
Gestational age (weeks)
Control IDDM GDM
13 7 6
39.10.4 38.00.4 39.50.7
Values are given as means. *P<0.05 compared to control group.
Table 2. Glycosylated haemoglobin (HbA1C) in pregnancies complicated by IDDM Trimester
HbA1C
95 per cent c.i.
1st 2nd 3rd
6.600.49* 5.410.28 5.610.16
5.34–7.86 4.73–6.08 3.67–5.99
Values are given as means and 95 per cent confidence intervals (n=7). HbA1C value of IDDM patients was significantly different from the normal range of HbA1C in uncomplicated pregnancies (3.90–5.30), *P<0.05.
vesicle preparations and the final expression of Na + /K + ATPase activity was given as nmol liberated PO4/min/mg protein. We have previously shown that Na + /K + -ATPase activity measured in frozen vesicles is not significantly different from activities measured in freshly prepared plasma membranes (Johansson et al., 2000a). Data presentation and statistical presentation Analysis of variance (ANOVA) followed by Student– Newman–Keuls test was used to evaluate differences between groups statistically. Values are presented as means and standard error of mean (). RESULTS Study group characterization Gestational age was not significantly different between the three groups under study (Table 1). Foetal weights were higher in the IDDM and GDM groups. The placentas in pregnancies complicated by diabetes were also larger than placentas of the control group. The glycosylated haemoglobin (HbA1C) in pregnancies complicated by IDDM (no data was available on GDM patients) was increased in the 1st trimester but within normal range in the second and third trimester (Table 2). Purity of syncytiotrophoblast membrane vesicles Marker enzyme activity for homogenate and MVM fractions, post nuclear membrane pellet (P2) and BM fractions are shown
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Table 3. Marker enzyme activities Alkaline phosphatase (nmol PO4/smg)
Control
n 10
IDDM
7
GDM
6
MVM 69.112.3 (19.6) 87.021.8 (20.0) 50.417.4 (22.1)
BM 7.01.0 (2.0) 9.01.6 (2.1) 5.70.8 (2.5)
Adenylate cyclase (pmol/minmg) Hom 3.50.5
n 6
4.40.7
6
2.30.4
6
BM 43079.5 (31.5) 34728.1 (29.3) 43067.3 (32.8)
P2 13.69.1 11.85.4 13.12.7
Values are given as means. Enzyme enrichments (in parentheses) were calculated as vesicle enzyme specific activity relative to that for the homogenate (alkaline phosphatase) or the post nuclear membrane pellet (P2; adenylate cyclase).
Figure 1. Western blot of the Na + /K + -ATPase 1-subunit in MVM of control patients (C) and from pregnancies complicated by gestational diabetes (GDM).
in Table 3. Alkaline phosphatase has a high abundance in the MVM, which was demonstrated by a 20-fold enrichment in control MVM. Adenylate cyclase that served as the BM marker was enriched 32-fold in the BM isolated from control placentas. Enzyme activities in vesicles isolated from IDDM/ GDM placentas were not significantly different from control (Table 3). Protein expression of Na + /K + -ATPase Western blot analysis of the syncytiotrophoblast MVM and BM isolated from placentas of control, IDDM and GDM subjects, showed the presence of the 1-subunits of Na + /K + ATPase at a molecular weight of approximately 96 kDa (shown for controls and GDM in Figure 1). The relative density of the 1-subunit protein was not significantly different between groups in MVM [Figure 2(A)] or BM [Figure 2(B)]. Na + /K + -ATPase activity Na + /K + -ATPase activity, as measured as K + -stimulated, ouabain inhibitable phosphatase activity, in MVM and BM isolated from control, GDM and IDDM groups are shown in Figure 3. There was no significant alteration in MVM or BM Na + /K + -ATPase activity in diabetes associated with accelerated foetal growth. DISCUSSION In pregnancies complicated by diabetes mellitus, foetal growth is often accelerated. Pedersen proposed in 1954 that the
increased foetal growth in these pregnancies is directly related to maternal hyperglycaemia, which will result in an increased delivery of glucose to the foetus. Foetal hyperglycaemia stimulates the secretion of insulin, the most important growthpromoting hormone during foetal life. However, despite rigorous maternal glycemic control in modern management of the diabetic pregnancy, the incidence of large-for gestational age babies remains high. This fact suggests a more complicated model for the mechanisms underlying accelerated foetal growth than originally proposed by Pedersen. One possibility that has been explored during recent years is that diabetes in pregnancy alters placental transport function. Indeed, it has been shown that placental glucose transporter (Gaither et al., 1999; Jansson et al., 1999) as well as the system A amino acid transporter (Jansson et al., 2001) are up-regulated in pregnancies with diabetes mellitus. These findings may provide one explanation for the accelerated foetal growth despite ‘normal’ maternal glucose levels. In this study we tested the hypothesis that the activity/ expression of the syncytiotrophoblast plasma membrane Na + / K + -ATPase is increased in pregnancies with diabetes and associated with accelerated foetal growth. The rationale for this hypothesis is as follows. First, the transport of many nutrients, in particular amino acids, across the placenta is dependent on the Na + gradient across the microvillous plasma membrane of the syncytiotrophoblast. Since this gradient is maintained by Na + /K + -ATPase, any alteration in the expression/activity of this transporter may affect amino acid transport and consequently, foetal growth. Second, it has recently been shown that in pregnancies complicated by IUGR, the activity and expression of syncytiotrophoblast plasma membrane Na + /K + ATPase is reduced (Johansson et al., 2000b). This suggests a relationship between the activity of this transporter in the transporting epithelium of the placenta and foetal growth. However, in this study we have shown that the protein expression as well as the activity of Na + /K + -ATPase is not altered in the full term placental MVM or BM of diabetic mothers that have given birth to babies large for gestational age. It is therefore unlikely that changes in sodium pump activity in the placental barrier contribute to foetal overgrowth in diabetic pregnancies.
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Figure 3. Na + /K + -ATPase activity in the placental syncytiotrophoblast MVM and BM (control n=11, IDDM n=7 and GDM n=7). Activity was defined as K + -stimulated, ouabain-inhibitable release of phosphate. Activity data is presented as nmol PO4/min/mg protein. There was no significant difference in activity between groups in neither BM nor MVM. Values are given as means.
Figure 2. The relative density of Na + /K + -ATPase 1-subunit protein expression as measured by Western blot analysis in syncytiotrophoblast microvillous (A) and basal plasma membranes (B) isolated from uncomplicated pregnancies (control n=12–13) and pregnancies complicated by IDDM (n=6–7) or GDM (n=6). The protein expression in pregnancies complicated by diabetes (IDDM and GDM) was not significantly different from controls. The mean density for control MVM and BM, respectively was arbitrarily assigned a value of one and the mean densities of IDDM and GDM calculated relative to the control group. Values are given as means.
In general, diabetes is associated with a reduced activity of Na + /K + -ATPase in tissues such as neurons (Greene and
Mackway, 1986), retina (MacGregor and Matschinsky, 1986), renal glomerulus (Cohen et al., 1985) and the aorta (Simmons et al., 1986). This study clearly demonstrates that the syncytiotrophoblast plasma membranes are not affected in a similar way. In studies of crude membrane fractions as well as purified Na + /K + -ATPase obtained from human placenta, Na + /K + -ATPase activity has been shown to be reduced (Zolese et al., 1997; Rabini et al., 1998). The apparent discrepancies between previous studies and our study can be resolved by considering the possibility that a crude membrane fraction from the whole placenta may not be representative for the syncytiotrophoblast plasma membranes. Since it is well known that erythrocyte Na + /K + -ATPase activity is reduced in IDDM (Finotti and Palatini, 1986; Raccah et al., 1992; De La Tour et al., 1998), erythrocyte membranes in the crude preparations may provide an explanation for the reduction in the studies of Zolese and co-workers. Term placental tissue were used in the current study and the finding of unchanged Na + /K + -ATPase activity in the placental transport barrier in association with diabetes does not preclude alterations in this transporter earlier in gestation. With respect to the IDDM group late first and early second trimester represents a period of gestation that is characterized by a suboptimal glucose control even in patients with successful management during the second half of pregnancy. This is illustrated by the elevated HbA1C values of the IDDM patients in first trimester in our study. Indeed, in some studies first trimester HbA1C has been identified as the only index of maternal glucose control that correlates to birthweight suggesting that important regulation of for example placental nutrient
Persson et al.: Na + /K + -ATPase Activity and Expression
transporters may take place early in pregnancy. Studies of placental transporters such as glucose and amino acid transporters as well as Na + /K + -ATPase in early pregnancy may provide important information about mechanisms underlying accelerated foetal growth in diabetes.
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In conclusion, we have extended previous studies on placental Na + /K + -ATPase activity in diabetes and demonstrated that the activity and protein expression of this transporter is unaltered in the transport barrier of the placenta in diabetic pregnancies associated with accelerated foetal growth.
ACKNOWLEDGEMENTS This study was supported by grants from the Swedish Medical Research Council (10838), the Swedish Diabetes Association, the Ragnar and Torsten So¨derberg Foundation, Frimurare-Barnhus-direktionen, the A r hlens Foundation, and the Willhelm & Martina Lundgrens Foundation.
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