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Am J Physiol Endocrinol Metab 293: E24-E30, 2007. First published February 13, 2007; doi:10.1152/ajpendo.00571.2006
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Effect of maternal triglycerides and free fatty acids on placental LPL in cultured primary trophoblast cells and in a case of maternal LPL deficiency

Anne Liese Magnusson-Olsson,1,* Susanne Lager,1,* Bo Jacobsson,2,3 Thomas Jansson,1,4 and Theresa L. Powell1,4

1Perinatal Center, Institute of Neuroscience and Physiology, 2Department of Obstetrics and Gynecology, Göteborg University, Gothenburg, Sweden, 3Department of Epidemiology, Institute of Public Health, University of Aarhus, Aarhus, Denmark and 4Department of Obstetrics and Gynecology, University of Cincinnati, Cincinnati, Ohio

Submitted 24 October 2006 ; accepted in final form 2 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Maternal hypertriglyceridemia is a normal condition in late gestation and is an adaptation to ensure an adequate nutrient supply to the fetus. Placental lipoprotein lipase (LPL) is involved in the initial step in transplacental fatty acid transport as it hydrolyzes maternal triglycerides (TG) to release free fatty acids (FFA). We investigated LPL activity and protein (Western blot) and mRNA expression (real-time RT-PCR) in the placenta of an LPL-deficient mother with marked hypertriglyceridemia. The LPL activity was fourfold lower, LPL protein expression 50% lower, and mRNA expression threefold higher than that of normal, healthy placentas at term (n = 4–7). To further investigate the role of maternal lipids in placental LPL regulation, we isolated placental cytotrophoblasts from term placentas and studied LPL activity and protein and mRNA expression after incubation in Intralipid (as a source of TG) and oleic, linoleic, and a combination of oleic, linoleic, and arachidonic acids as well as insulin. Intralipid (40 and 400 mg/dl) decreased LPL activity by {approx}30% (n = 10–14, P < 0.05) and 400 µM linoleic and linoleic-oleic-arachidonic acid (n = 10) decreased LPL activity by 37 and 34%, respectively. No major changes were observed in LPL protein or mRNA expression. We found no effect of insulin on LPL activity or protein expression in the cultured trophoblasts. To conclude, the activity of placental LPL is reduced by high levels of maternal TG and/or FFA. This regulatory mechanism may serve to counteract an excessive delivery of FFA to the fetus in conditions where maternal TG levels are markedly increased.

fetus; placenta; fatty acid transfer; lipoprotein lipase

DURING THE LAST TRIMESTER OF PREGNANCY, there is a high fetal demand for fatty acids (FA), as they are critical for normal neural and vascular development. The fetus is able to synthesize saturated and monounsaturated FAs from glucose and ketone bodies (35) but depends entirely on placental transport for its supply of the essential fatty acids (EFA) linoleic and {alpha}-linolenic acid. The EFAs and their derivatives the long-chain polyunsaturated free fatty acids (LC-PUFAs) arachidonic (ARA) and docosahexaoneic (DHA) are essential constituents of membranes, act as precursors of cellular signaling molecules, and are particularly important for the developing brain and retina (16).

Triglycerides (TG) are transported in the bloodstream as lipoproteins (22). TGs must be hydrolyzed into free fatty acids (FFA) before they can be transferred across the syncytiotrophoblast of the human placenta. This transporting epithelium is a multinucleated syncytium, polarized with the apical or microvillous plasma membrane (MVM), bathed in maternal blood, and the basal plasma membrane (BM), oriented toward the fetal capillary. At least two types of TG hydrolases have been identified in MVM, a placenta-specific hydrolase and lipoprotein lipase (LPL) (58, 59). LPL has been suggested to be the most important hydrolase in the initial step for transfer of TG-derived FA, as the placenta-specific TG hydrolase is inhibited by serum (57, 58, 59). Subsequent to TG hydrolysis, the FFA are available for transport, and once they reach the syncytial cytoplasm they are bound to intracellular cardiac or liver fatty acid binding proteins (C- and L-FABPs) (11). Thereafter, the FFA are guided to various intracellular sites for esterification, beta-oxidation, and prostaglandin production or they are directly transported to the fetal circulation (14) by facilitated diffusion across the BM or through production of lipoproteins in the syncytiotrophoblast (40), which are subsequently secreted into fetal blood.

In other tissues, such as adipose, skeletal, and heart muscle, LPL is synthesized by underlying parenchymal cells and then transported to the luminal surface of the capillary endothelium. LPL is anchored to heparan sulfate proteoglycans (HSPG) (24) and is easily displaced by heparin (3). LPL is regulated in a complex manner, either at the transcriptional or at the posttranscriptional level, and mechanisms of LPL regulation include effects on mRNA stability, glycosylation, interaction with activating proteins (apo-CII), and monomer/dimer activation (5, 19, 43). LPL is regulated in a tissue-specific manner by fasting, feeding, and insulin (38), and alterations in LPL activity appear to correlate with local tissue requirements of FFA (22). FFA in excess have been shown to lower LPL activity due to competition with the LPL/HSPG-binding site (54), whereas in mice FFA and TG deficiency results in a higher LPL activity in adipose and muscle tissue (26). However, mechanisms responsible for placental LPL regulation remain unclear (9, 43).

We have previously demonstrated that LPL activity in the MVM was upregulated in pregnancies complicated by type 1 diabetes and associated with fetal overgrowth, whereas a decreased placental LPL activity was observed in preterm pregnancies complicated by intrauterine growth restriction (IUGR) (41). These data are compatible with the hypothesis that alterations in placental LPL activity contribute to changes in FFA delivery to the fetus and, as a consequence, fetal growth (41). Therefore, information on the mechanisms of regulation of placental LPL will advance our understanding of the pathophysiology of common pregnancy complications.

Insulin, TG, and FFA have been shown to regulate LPL in other tissues and are altered in pregnancies complicated by diabetes (34, 45, 47, 50, 53) and IUGR (13, 18, 49, 52), respectively. Therefore, the objective of the present study was to investigate the effect of insulin, TG, and FFA on LPL activity and expression in cytotrophoblast cells isolated from human term placenta of uncomplicated pregnancies. LPL activity and expression in placental tissue from a patient suffering from LPL deficiency and giving birth to normal sized healthy babies near term was also investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The monoclonal LPL antibody (5D2) was kindly provided by Dr. John Brunzell (University of Washington, Seattle, WA). The L-FABP monoclonal antibody (Abcam, Cambridge, UK) cross-reacts with human and rat L-FABP. All chemicals were purchased from Sigma, unless otherwise noted.

LPL-negative patient. A 36-yr-old woman with primary LPL deficiency was followed rigorously during two pregnancies and was delivered at 36 wk of gestation by cesarean section in both. The condition of this patient has been described in detail (21) and represents typical chylomicronemia due to lack of significant extracellular LPL activity. The LPL defect in this patient has been characterized as a compound heterozygote carrying one allele that does not produce LPL mRNA and another that results in synthesis of a catalytically active but defectively transported form of LPL (21).

Tissue collection. The collection of human placental tissue was approved by the Göteborg University Committee for Research Ethics and conducted with informed consent. Villous tissue was dissected from the placenta and rinsed in physiological saline before further processing.

MVM preparation. MVM vesicles were prepared according to methods previously described (31), with minor modifications (41) and stored at –80°C until use. Alkaline phosphatase activity 10-fold greater than homogenate was used as an enrichment marker for MVM. Protein concentrations were determined by the Bradford method (8).

Isolation and maintenance of cytotrophoblasts. Isolation of cytotrophoblast cells from term human placenta was carried out using a method described previously (25, 36). Placental villous tissue (50 g) was dissected and washed in saline and then digested three times in a HEPES-buffered salt solution containing 0.25% trypsin (Roche Diagnostics Scandinavia) and 0.2 mg/ml DNAse. Tissue was shaken at 37°C for 30 min. The cytotrophoblast cells were separated on a Percoll gradient and resuspended in standard cell culture medium ({approx}16 mM glucose, 44.5% DMEM, 44.5% Ham's-F12, and 10% fetal calf serum supplemented with gentamicin, benzyl penicillin, streptomycin and L-glutamine). The cells were plated in 25-cm2 flasks at a density of ~10 x 106 cells. The cell culture medium was changed daily.

Harvesting of cells. Cells were harvested at 18, 42, 66, or 90 h after plating. After removal of cell culture medium, 75 µl of buffer D (250 mM sucrose, 0.7 µM pepstatin A, 1.6 µM antipain, 80 µM aprotinin, 1 mM EDTA, 10 mM HEPES-Tris, pH 7.4, at 4°C) was added, and cells were collected with a rubber spatula. Cell samples for Western blot were stored at –80°C. Protein concentration measurement and LPL assay were performed directly.

Intralipid-supplemented cell culture medium. After 18 h in culture, the cell culture medium was replaced with medium supplemented with Intralipid (Fresenius Kabi) as a soluble TG source. Intralipid is based on emulsified soybean oil (200 mg/ml), egg phospholipids, and glycerol and the FA composition is 53% linoleic acid, 8% {alpha}-linoleneic acid, 24% oleic acid, 4% palmitic acid, and 11% stearic acid. The cells were incubated with 4, 40, or 400 mg/dl Intralipid for 24 h. Cells were swiftly and carefully washed three times with standard culture medium to ensure the complete removal of all Intralipid before being harvested. Because residual Intralipid would alter LPL activity measurements, we performed initial experiments in which the trophoblast cells were exposed to Intralipid for only 15 min to minimize potential alterations in activity. The washing technique was monitored and modified to ensure that LPL activity returned to control levels. These experiments demonstrated that the use of three washes was sufficient to remove all Intralipid, even at high concentrations.

FFA-supplemented cell culture medium. Cell culture medium was supplemented with linoleic acid, oleic acid, or a combination of linoleic, oleic, and arachidonic acid (100, 200, or 400 µM) containing 100 mg/ml bovine serum albumin (BSA). The supplemented medium was added to cell cultures 18 h after plating and incubated for 24 h. Control cells were incubated in medium containing an equivalent volume of 100 mg/ml FA-free BSA. All cultures were quickly and carefully washed twice with standard cell culture medium before harvesting of cells.

Insulin-supplemented cell culture medium. At 24 and 3 h prior to LPL assay, cell cultures were incubated with insulin (final concentrations 36.25, 145, or 290 ng/ml)-supplemented cell culture medium. The insulin was dissolved in a stock buffer consisting of 0.1% BSA and 1.6% glycerol in sterile water. Control cells were given an equal amount of insulin stock buffer. The LPL activity was determined after 90 h in culture.

LPL activity measurement. The activity of LPL was measured by hydrolysis of [3H]trioleate incorporated into Intralipid micelles as described previously (42, 58, 59), with minor modifications. The LPL assay solution is optimized at pH 8 and contains 0.5 mg/ml FA-free BSA (dissolved in 10 mM Tris·HCl, pH 7.0), 1 mM glycerol tri-[9,10(n)-3H]oleate (final concentration 0.5 µl/ml), and 7.5% fetal calf serum. Cytotrophoblasts (150 µg total protein) were combined with the assay buffer and incubated for 60 min at 37°C. LPL activity in fresh villous tissue was measured after heparin release, as described in detail previously (42). Termination of the reaction (for both cytotrophoblasts and villous tissue) was accomplished by the addition of 3.25 ml of stop solution (methanol-chloroform-heptane, 1.4:1.25:1.0 vol/vol/vol). One milliliter of borate-carbonate (pH 10.2) was added, and the samples were forcefully vortexed for 30 s and centrifuged at 2,200 rpm at 4°C for 20 min. The upper aqueous phase was removed and transferred to scintillation vials for counting. Individual standard curves were made for all assays. LPL activity is expressed as picomoles mono[9,10(n)-3H]oleate released per milligram of total cell protein per minute or picomoles per milligram wet weight per minute for fresh villous tissue.

Western blotting. Western blotting was performed according to a previously published protocol (32). Ten micrograms of MVM protein or 15 µg of trophoblast cell homogenate protein was loaded and separated on a 10% SDS-PAGE for LPL detection. Rat cardiac muscle served as a positive control. For L-FABP, 40 µg of total protein from placental homogenates were loaded onto a 12% SDS-PAGE. Rat liver was used as positive control. Primary antibodies (5D2, 1:250, or L-FABP, 1:100) were incubated for 1 h and peroxidase-labeled horse-anti-mouse IgG (1:1,000) for 1 h. The immunolabeling was made visible using ECL detection (Amersham Biosciences).

Quantification of mRNA expression. Total RNA was extracted using RNA STAT-60 (Tel-Test, Friendswood, TX) according to the manufacturerrs protocol followed by reverse transcription (RT) using a SuperScript II Reverse Transcriptase kit (Invitrogen, San Diego, CA) as previously described (6). Relative quantification of LPL mRNA expression was carried out on a LightCycler (Roche Diagnostics, Mannheim, Germany) using SYBR Green I as described in detail previously (42). TATA box-binding protein (TBP) was selected as an endogenous control, as it has been shown to be stable in the placenta (44), to correct for potential variation in RNA loading or efficiency in the amplification reaction. Primers for LPL and TBP were designed according to previously published work (37, 44) and purchased from Cybergene (Huddinge, Sweden).

Statistical analysis. The LPL expression and activity data are presented as means ± SE, except in comparing the data from the single LPL-negative patient with controls where data in the control group are expressed as means and 95% confidence intervals. Differences between groups were evaluated statistically by one-way ANOVA or repeated-measures ANOVA and Dunnett's test or paired t-test. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lipid status and placental LPL activity and protein and mRNA expression in the LPL-deficient mother. During both pregnancies, the patient was placed on a fat-restricted diet with additional {omega}-3 fish oil tablets. The clinical management of the first pregnancy also included plasmaphoresis performed repeatedly to reduce the plasma TG levels, which varied between 820 and 4,200 mg/dl. This resulted in a transient reduction in TG levels. During the last four months of the second pregnancy TG values varied between 2,000 and 9,000 mg/dl.

The results from the placentas from the LPL-deficient mother and term controls are given in Table 1. The LPL activity was fourfold lower in villous tissue from the first placenta of the LPL-deficient mother and 50% lower in the second placenta compared with mean LPL activity in control placentas at term (n = 7). The placental expression of LPL protein in MVM was 50% lower in the LPL-deficient mother than in term control placentas (n = 6). One band at 37 kDa was detected in Western blot, and the results are shown in Table 1. The L-FABP expression in placental homogenates was 23% lower in the LPL-deficient mother compared with controls (n = 8). The protein migrated at 15 kDa, as previously described (41). When the protein expression data were being analyzed, the values for the control samples were arbitrarily assigned a density value of 1 to simplify comparisons between groups. The mRNA expression was threefold higher in the placenta from the LPL-deficient mother compared with normal term controls (n = 4).


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  		Table 1. LPL activity and expression in fresh villous tissue from LPL-deficient vs. normal-term women

 
LPL activity and protein and mRNA expression in cytotrophoblasts. No detectable LPL activity was measured in the cell culture medium when randomly tested in all treatment groups (data not shown).

Assay efficiency for LPL activity in cytotrophoblasts. The efficiency of oleic acid extraction for all LPL assays performed was 71.9 ± 0.27% (n = 663, total no. of test tubes) and the intra-assay coefficient of variation was 0.98 ± 0.06%. The coefficient of variation for interassay efficiency was 5.92%.

Time in culture. LPL activity was present at all of the times. No significant difference in LPL activity was observed between the time points (Fig. 1, n = 6, except at 90 h n = 5). We measured a significant rise in human chorionic gonadotropin production, a marker for syncytialization, between 48 and 66 h in culture (data not shown). One band at 37 kDa was detected by Western blot for LPL. No significant differences were detected in LPL protein expression (18 h: 1.00 ± 0.11; 42 h: 0.93 ± 0.05; 66 h: 0.97 ± 0.10; 90 h: 0.80 ± 0.04; 18 h was arbitrarily assigned a value of 1; n = 6 for all groups) by Western blot or in LPL mRNA expression (18 h: 0.56 ± 0.20; 42 h: 0.32 ± 0.15; 66 h: 0.24 ± 0.11; 90 h: 0.31 ± 0.12; relative values, n = 6) by real-time PCR.


Figure 1
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  		Fig. 1. LPL activity (pmol oleic acid·mg total protein–1·min–1) in human placental cytotrophoblasts measured after 18, 42, 66, and 90 h in culture (n = 6, except at 90 h n = 5).

 
Intralipid treatment. Cytotrophoblast cells incubated in 40 and 400 mg/dl Intralipid for 24 h showed a 28–39% decrease in LPL activity compared with control (n = 10–14, P < 0.05, one-way ANOVA; Fig. 2A). LPL protein was observed as a single band at 37 kDa, and a 16% higher expression was seen after incubation with 400 mg/dl Intralipid compared with control (n = 6, P < 0.05, t-test; Fig. 2B). LPL mRNA expression was measured after 24-h Intralipid incubation, but no significant differences were detected compared with controls (control: 0.74 ± 0.19; 4 mg/ml: 0.30 ± 0.13; 40 mg/ml: 0.18 ± 0.04; 400 mg/ml: 1.03 ± 0.45; control n = 10, Intralipid-treated groups n = 5).


Figure 2
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  		Fig. 2. A: LPL activity (pmol oleic acid·mg total protein–1·min–1; n = 10–14) in human placental cytotrophoblasts after incubation with Intralipid (4, 40, or 400 mg/dl)-supplemented medium for 24 h (*P < 0.05, ANOVA). B: LPL protein expression after 24-h incubation with 400 mg/dl Intralipid. C, Control; In, Intralipid; n = 6.

 
FFA treatment. Incubation with 400 µM linoleic acid and 400 µM combined linoleic-oleic-arachidonic acid for 24 h reduced LPL activity by 37 and 34%, respectively (n = 10, P < 0.05, repeated-measures ANOVA; Fig. 3). LPL protein was detected as a single band at 37 kDa, and no significant alterations in LPL protein expression (control: 1.0 ± 0.08; 400 µM linoleic acid: 0.98 ± 0.10; 400 µM linoleic-oleic-arachidonic acid: 0.86 ± 0.02; 400 µM oleic acid: 1.03 ± 0.09; the control group was arbitrarily assigned a value of 1; n = 6 for all groups) were observed after 24 h of incubation with FFA.


Figure 3
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  		Fig. 3. LPL activity (pmol oleic acid·mg total protein–1·min–1; n = 10) in human placental cytotrophoblasts after 24-h treatment with single or combined free fatty acids (FFA; *P < 0.05, repeated-measures ANOVA).

 
Insulin treatment. We found no change in LPL activity or protein expression after 3 or 24 h of incubation with various concentrations of insulin (Table 2) in the trophoblast cultures. Similarly, no effect was found on LPL protein expression after 24 h of incubation (control: 1.00 ± 0.11; 290 ng/ml insulin: 0.87 ± 0.13; n = 4).


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  		Table 2. LPL activity after 3-h (36.25, 145, and 290 ng/ml) and 24-h (290 ng/ml) incubation with insulin

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
There are several novel findings in the present study. First, we found that LPL activity was reduced in placenta of an LPL-deficient mother with hypertriglcyeridemia in pregnancy, as expected if the developing fetus has inherited the nonfunctional allele. Furthermore, we demonstrate that TG and/or FFA may be regulators of human placental LPL activity. We show for the first time that LPL is expressed and active in cultured cytotrophoblast cells. These findings strongly support that placental LPL is produced by the trophoblast cells.

Lipases in the apical membrane of the placenta provide a mechanism for release of FFA from maternal lipoproteins. Placental LPL prefers hydrolyzing circulating TG-containing FFA, which are more unsaturated (48). In the third trimester of pregnancy, maternal plasma LC-PUFAs are mainly esterified and associated with circulating lipoproteins rather than bound to albumin (27). Therefore, the major source of LC-PUFAs transported to the fetus is derived from maternal circulating lipoproteins, further supporting the importance of LPL in placental lipid transport and fetal LC-PUFA supply.

In an extreme example of hypertriglyceridemia experienced by a pregnant woman with nonfunctional extracellular LPL, two babies (different pregnancies) of normal weight were delivered near term. We demonstrated that the activity and protein expression of placental LPL were markedly reduced and mRNA expression was threefold higher compared with control placentas at term. In a previously published paper, this patient was carefully characterized and found to possess one allele that probably does not produce LPL mRNA and another that results in synthesis of a catalytically active but defectively transported form of LPL (21). The fetuses and placentas are therefore likely to be heterozygotic for LPL if the mutations in the mother did not affect only somatic cells. Our data suggest that the reduction in placental LPL activity is likely due to inheritance of one functional allele from the father and one defective allele from the mother. In one case, the placenta LPL activity met the expected 50% reduction, whereas in the other it was lower than predicted (25% of control). Protein expression was in the expected range predicted by an obligate heterozygote. We also found that the expression of L-FABP was decreased, suggesting a reduction in FFA handling capacity in the cytoplasm of the syncytiotrophoblast. Whether this is a regulatory step to reduce transfer of FFA to the fetus or in response to the reduction in LPL activity is currently unknown.

The LPL-deficient patient in our study gave birth to babies of normal birth weight, in agreement with previously reported pregnancies (1, 33, 56), indicating that placental FFA transfer was sufficient to allow for normal fetal growth. Placental FFA transfer in the LPL-negative patient may have been maintained in two ways despite a markedly decreased MVM LPL activity. In addition to LPL, placenta-specific lipase (58, 59) provides an additional mechanism for hydrolysis of TG to release FFA. The protein has not been cloned, and little is known about its source, regulation, or in vivo function. Since the pH optimum of the placental specific lipase is 6.0, the activity of this lipase was not assessed in our experiments. In a recent study, Lindegaard et al. (37) demonstrated mRNA and protein expression of endothelial lipase (EL) in syncytiotrophoblasts and mainly in endothelial cells along with LPL in placental syncytiotrophoblasts. In other tissues, EL mainly hydrolyzes phospholipids in high-density lipoproteins (23). A study in mice showed that EL can compensate for a lack of LPL in adipose tissue and mediates some of the FFA taken up by the tissue (39). Indeed, a stronger expression of EL was detected in the placenta from LPL-deficient mice (37). EL may be upregulated in the placentas of the LPL-deficient patient.

The source of placental LPL has not been definitively established. LPL production by fetal macrophages has been suggested (7), and recently, using in situ hybridization, LPL RNA was found in trophoblast cells (37). To our knowledge, this study is the first to demonstrate LPL activity and protein and mRNA expression in isolated cytotrophoblasts. The activity and expression were relatively stable over 90 h in culture. These data suggest that both cytotrophoblasts and syncytiotrophoblasts are able to synthesize LPL. In adipose and muscle, LPL is produced in underlying parenchymal cells and is transported, by mechanisms that are not completely clear, to the capillary endothelial surface where it is bound to HSPG and has its primary function. The mechanism in which LPL is translocated to MVM in syncytiotrophoblast is not clear and the syncytial nature of this epithelium, lacking intercellular spaces, would make transfer of LPL from underlying cytotrophoblasts a complex process. Furthermore, the number of cytotrophoblasts present in the placenta decreases, and the syncytiotrophoblast mass increases continuously during the second half of pregnancy, a time when placental LPL expression and activity increases (29, 42). Therefore, we propose that the syncytiotrophoblast is the primary source of placental LPL.

To test the hypothesis that maternal lipid status regulates placental LPL activity, we isolated cytotrophoblasts and measured LPL activity and expression in response to incubation with TG, FFA, or insulin. We found that LPL activity was significantly inhibited by incubation with 40 or 400 mg/dl Intralipid, concentrations that are within the physiological range in late pregnancy (204–265 mg/dl) (2, 10, 30). Intralipid was chosen as a VLDL/chylomicron substitute, as it contains a good source of EFAs (61%). The finding that physiological levels of TG are inhibitory to LPL activity was not expected. We predicted that pathological levels as were seen in the LPL-deficient patient would be inhibitory. Because residual Intralipid associated with the cells would alter the results, we carefully monitored LPL activity after short incubations with Intralipid to ensure that an adequate washing procedure was developed. However, the possibility remains that residual Intralipid alters the findings of the assay. Another possible explanation is that LPL was eluted from the cells and bound to Intralipid during the 24-h incubation. We did not test the medium after Intralipid incubation for LPL activity. However, control medium was periodically tested and no LPL activity was detected.

We were interested in whether FFA alone or in combination would be sufficient to alter LPL activity in cultured trophoblast cells. We found an inhibiting effect of 400 µM FFA. The total plasma FFA content in late pregnancy is in the range of 300–600 µM (30, 55). In other tissues LPL is known to respond to rapid changes in nutrient status and is regulated by fasting/feeding and exercise/rest in muscle and adipose tissues (4, 20, 38, 51). Much of this appears to be due to differential responses to metabolic hormones such as insulin, cortisol, and epinephrine (9, 19). In a recent study (42), we demonstrated that hyperglycemic medium together with insulin increased LPL activity in freshly isolated placental villous tissue after 3 h of incubation. In the current study, we found no effect of insulin on LPL activity or expression in cultured trophoblasts. Whether this was due to the fact that placental LPL does not respond to insulin or to the relatively low insulin receptor number in late pregnancy (15) is not clear. In a recent study in human placental choriocarcinoma (BeWo) cells where cells were incubated with various FFA and insulin, no insulin effect on FFA uptake was seen (17).

As LPL is a key enzyme in lipoprotein metabolism, it has been implicated in many pathological conditions involving hypertriglyceridemia, including chylomicronemia, obesity, insulin resistance, and diabetes (43). Any of these conditions during pregnancy, which is normally associated with insulin resistance and a fourfold increase in maternal plasma TG (28, 29), could lead to pathological plasma lipid concentrations. Obesity is strongly correlated with elevated TG and VLDL in pregnancy, both in the mother and in the macrosomic newborn (46). A better understanding of the mechanisms whereby placental lipid transfer is regulated in pregnancy is critically needed.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from Swedish Research Council (14555), The Novo Nordisk Foundation, the Sven Jerring Foundation, the Åhlens Foundation, the Swedish Diabetes Association, and Frimurare- Barnhus-direktionen.


   ACKNOWLEDGMENTS
 
We thank Ellen Samuelsson at the Department of Obstetrics and Gynecology, who helped us with the logistics around placental collection and transport.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. L. Magnusson-Olsson, Perinatal Center, Institute of Neuroscience and Physiology, Göteborg University, Box 432, S-405 30 Gothenburg, Sweden (e-mail: Anneliese.Olsson{at}fysiologi.gu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. L. Magnusson-Olsson and S. Lager contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Al-Shali K, Wang J, Fellows F, Huff MW, Wolfe BM, Hegele RA. Successful pregnancy outcome in a patient with severe chylomicronemia due to compound heterozygosity for mutant lipoprotein lipase. Clin Biochem 35: 125–130, 2002.[CrossRef][Web of Science][Medline]
  2. Alvarez JJ, Montelongo A, Iglesias A, Lasuncion MA, Herrera E. Longitudinal study on lipoprotein profile, high density lipoprotein subclass, and postheparin lipases during gestation in women. J Lipid Res 37: 299–308, 1996.[Abstract]
  3. Bengtsson-Olivecrona G, Olivecrona T. Binding of active and inactive forms of lipoprotein lipase to heparin. Effects of pH. Biochem J 226: 409–413, 1985.[Web of Science][Medline]
  4. Bergo M, Olivecrona G, Olivecrona T. Diurnal rhythms and effects of fasting and refeeding on rat adipose tissue lipoprotein lipase. Am J Physiol Endocrinol Metab 271: E1092–E1097, 1996.[Abstract/Free Full Text]
  5. Bergo M, Olivecrona G, Olivecrona T. Forms of lipoprotein lipase in rat tissues: in adipose tissue the proportion of inactive lipase increases on fasting. Biochem J 313: 893–898, 1996.[Web of Science][Medline]
  6. Blomgren K, Hallin U, Andersson AL, Puka-Sundvall M, Bahr BA, McRae A, Saido TC, Kawashima S, Hagberg H. Calpastatin is up-regulated in response to hypoxia and is a suicide substrate to calpain after neonatal cerebral hypoxia-ischemia. J Biol Chem 274: 14046–14052, 1999.[Abstract/Free Full Text]
  7. Bonet B, Brunzell JD, Gown AM, Knopp RH. Metabolism of very-low-density lipoprotein triglyceride by human placental cells: the role of lipoprotein lipase. Metabolism 41: 596–603, 1992.[CrossRef][Web of Science][Medline]
  8. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][Web of Science][Medline]
  9. Braun JE, Severson DL. Regulation of the synthesis, processing and translocation of lipoprotein lipase. Biochem J 287: 337–347, 1992.[Web of Science][Medline]
  10. Brizzi P, Tonolo G, Esposito F, Puddu L, Dessole S, Maioli M, Milia S. Lipoprotein metabolism during normal pregnancy. Am J Obstet Gynecol 181: 430–434, 1999.[CrossRef][Web of Science][Medline]
  11. Campbell FM, Bush PG, Veerkamp JH, Dutta-Roy AK. Detection and cellular localization of plasma membrane-associated and cytoplasmic fatty acid-binding proteins in human placenta. Placenta 19: 409–415, 1998.[Web of Science][Medline]
  12. Campbell FM, Gordon MJ, Dutta-Roy AK. Preferential uptake of long chain polyunsatured fatty acids by isolated human placental membranes. Mol Cell Biochem 155: 77–83, 1996.[CrossRef][Web of Science][Medline]
  13. Caruso A, Paradisi G, Ferrazzani S, Lucchese A, Moretti S, Fulghesu AM. Effect of maternal carbohydrate metabolism on fetal growth. Obstet Gynecol 92: 8–12, 1998.[CrossRef][Web of Science][Medline]
  14. Das T, Sa G, Mukherjea M. Characterization of cardiac fatty-acid-binding protein from human placenta. Comparison with placenta hepatic types. Eur J Biochem 211: 725–730, 1993.[Web of Science][Medline]
  15. Desoye G, Hartmann M, Jones CJ, Wolf HJ, Kohnen G, Kosanke G, Kaufmann P. Location of insulin receptors in the placenta and its progenitor tissues. Microsc Res Tech 38: 63–75, 1997.[CrossRef][Web of Science][Medline]
  16. Dutta-Roy AK. Transport mechanisms for long-chain polyunsaturated fatty acids in the human placenta. Am J Clin Nutr 71: 315S–322S, 2000.[Abstract/Free Full Text]
  17. Duttaroy AK, Jorgensen A. Insulin and leptin do not affect fatty acid uptake and metabolism in human placental choriocarcinoma (BeWo) cells. Prostaglandins Leukot Essent Fatty Acids 72: 403–408, 2005.[CrossRef][Web of Science][Medline]
  18. Economides DL, Crook D, Nicolaides KH. Investigation of hypertriglyceridemia in small for gestational age fetuses. Fetal Ther 3: 165–172, 1988.[Medline]
  19. Enerback S, Gimble JM. Lipoprotein lipase gene expression: physiological regulators at the transcriptional and post-transcriptional level. Biochim Biophys Acta 1169: 107–125, 1993.[Medline]
  20. Erskine JM, Jensen DR, Eckel RH. Macronutrient regulation of lipoprotein lipase is posttranslational. J Nutr 124: 500–507, 1994.[Abstract/Free Full Text]
  21. Fager G, Semb H, Enerback S, Olivecrona T, Jonasson L, Bengtsson-Olivecrona G, Camejo G, Bjursell G, Bondjers G. Hyperlipoproteinemia type I in a patient with active lipoprotein lipase in adipose tissue and indications of defective transport of the enzyme. J Lipid Res 31: 1187–1197, 1990.[Abstract]
  22. Fielding BA, Frayn KN. Lipoprotein lipase and the disposition of dietary fatty acids. Br J Nutr 80: 495–502, 1998.[CrossRef][Web of Science][Medline]
  23. Fuki IV, Blanchard N, Jin W, Marchadier DH, Millar JS, Glick JM, Rader DJ. Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway. J Biol Chem 278: 34331–34338, 2003.[Abstract/Free Full Text]
  24. Goldberg IJ. Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis. J Lipid Res 37: 693–707, 1996.[Abstract]
  25. Greenwood SL, Brown PD, Edwards D, Sibley CP. Patch clamp studies of human placental cytotrophoblast cells in culture. Trophoblast Res 7: 53–68, 1993.
  26. Haemmerle G, Zimmermann R, Strauss JG, Kratky D, Riederer M, Knipping G, Zechner R. Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle. J Biol Chem 277: 12946–12952, 2002.[Abstract/Free Full Text]
  27. Herrera E. Implications of dietary fatty acids during pregnancy on placental, fetal and postnatal development—a review. Placenta 23, Suppl A: S9–S19, 2002.
  28. Herrera E. Metabolic adaptations in pregnancy and their implications for the availability of substrates to the fetus. Eur J Clin Nutr 54: S47–S51, 2000.[Web of Science][Medline]
  29. Herrera E, Lasuncion MA, Gomez-Coronado D, Aranda P, Lopez-Luna P, Maier I. Role of lipoprotein lipase activity on lipoprotein metabolism and the fate of circulating triglycerides in pregnancy. Am J Obstet Gynecol 158: 1575–1583, 1988.[Web of Science][Medline]
  30. Herrera E, Ortega H, Alvino G, Giovannini N, Amusquivar E, Cetin I. Relationship between plasma fatty acid profile and antioxidant vitamins during normal pregnancy. Eur J Clin Nutr 58: 1231–1238, 2004.[CrossRef][Web of Science][Medline]
  31. Illsley NP, Wang ZQ, Gray A, Sellers MC, Jacobs MM. Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. Biochim Biophys Acta 1029: 218–226, 1990.[Medline]
  32. Johansson M, Glazier JD, Sibley CP, Jansson T, Powell TL. Activity and protein expression of the Na+/H+ exchanger is reduced in syncytiotrophoblast microvillous plasma membranes isolated from preterm intrauterine growth restriction pregnancies. J Clin Endocrinol Metab 87: 5686–5694, 2002.[Abstract/Free Full Text]
  33. Keilson L, Vary C, Sprecher D, Renfrew R. Hyperlipidemia and pancreatitis during pregnancy in two sisters with a mutation in the lipoprotein lipase gene. Ann Int Med 124: 425–428, 1996.[Abstract/Free Full Text]
  34. Kilby MD, Neary RH, Mackness MI, Durrington PN. Fetal and maternal lipoprotein metabolism in human pregnancy complicated by type I diabetes mellitus. J Clin Endocrinol Metab 83: 1736–1741, 1998.[Abstract/Free Full Text]
  35. Kimura RE. Fatty acid metabolism in the fetus. Semin Perinatol 13: 202–210, 1989.[Web of Science][Medline]
  36. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss JF 3rd. Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 118: 1567–1582, 1986.[Abstract/Free Full Text]
  37. Lindegaard ML, Olivecrona G, Christoffersen C, Kratky D, Hannibal J, Petersen BL, Zechner R, Damm P, Nielsen LB. Endothelial and lipoprotein lipases in human and mouse placenta. J Lipid Res 46: 2339–2346, 2005.[Abstract/Free Full Text]
  38. Lithell H, Boberg J, Hellsing K, Lundqvist G, Vessby B. Lipoprotein-lipase activity in human skeletal muscle and adipose tissue in the fasting and the fed states. Atherosclerosis 30: 89–94, 1978.[CrossRef][Web of Science][Medline]
  39. Ma K, Cilingiroglu M, Otvos JD, Ballantyne CM, Marian AJ, Chan L. Endothelial lipase is a major genetic determinant for high-density lipoprotein concentration, structure, and metabolism. Proc Natl Acad Sci USA 100: 2748–2753, 2003.[Abstract/Free Full Text]
  40. Madsen EM, Lindegaard ML, Andersen CB, Damm P, Nielsen LB. Human placenta secretes apolipoprotein B-100-containing lipoproteins. J Biol Chem 279: 55271–55276, 2004.[Abstract/Free Full Text]
  41. Magnusson AL, Waterman IJ, Wennergren M, Jansson T, Powell TL. Triglyceride hydrolase activities and expression of fatty acid binding proteins in the human placenta in pregnancies complicated by intrauterine growth restriction and diabetes. J Clin Endocrinol Metab 89: 4607–4614, 2004.[Abstract/Free Full Text]
  42. Magnusson-Olsson AL, Hamark B, Ericsson A, Wennergren M, Jansson T, Powell T. Gestational and hormonal regulation of human placental lipoprotein lipase. J Lipid Res 47: 2551–2561, 2006.[Abstract/Free Full Text]
  43. Mead JR, Irvine SA, Ramji DP. Lipoprotein lipase: structure, function, regulation, and role in disease. J Mol Med 80: 753–769, 2002.[CrossRef][Web of Science][Medline]
  44. Meller M, Vadachkoria S, Luthy DA, Williams MA. Evaluation of housekeeping genes in placental comparative expression studies. Placenta 26: 601–607, 2005.[CrossRef][Web of Science][Medline]
  45. Merzouk H, Madani S, Korso N, Bouchenak M, Prost J, Belleville J. Maternal and fetal serum lipid and lipoprotein concentrations and compositions in type 1 diabetic pregnancy: relationship with maternal glycemic control. J Lab Clin Med 136: 441–448, 2000.[CrossRef][Web of Science][Medline]
  46. Merzouk H, Meghelli-Bouchenak M, Loukidi B, Prost J, Belleville J. Impaired serum lipids and lipoproteins in fetal macrosomia related to maternal obesity. Biol Neonate 77: 17–24, 2000.[Web of Science][Medline]
  47. Moore TR. Fetal growth in diabetic pregnancy. Clin Obstet Gynecol 40: 771–786, 1997.[CrossRef][Web of Science][Medline]
  48. Mortimer BC, Holthouse DJ, Martins IJ, Stick RV, Redgrave TG. Effects of triacylglycerol-saturated acyl chains on the clearance of chylomicron-like emulsions from the plasma of the rat. Biochim Biophys Acta 1211: 171–180, 1994.[Medline]
  49. Munoz A, Uberos J, Molina A, Valenzuela A, Cano D, Ruiz C, Molina Font JA. Relationship of blood rheology to lipoprotein profile during normal pregnancies and those with intrauterine growth retardation. J Clin Pathol 48: 571–574, 1995.[Abstract/Free Full Text]
  50. Peterson CM, Jovanovic-Peterson L, Mills JL, Conley MR, Knopp RH, Reed GF, Aarons JH, Holmes LB, Brown Z, Van Allen M, et al. The Diabetes in Early Pregnancy Study: changes in cholesterol, triglycerides, body weight, and blood pressure The National Institute of Child Health and Human Development—the Diabetes in Early Pregnancy Study. Am J Obstet Gynecol 166: 513–518, 1992.[Web of Science][Medline]
  51. Richelsen B. Effect of growth hormone on adipose tissue and skeletal muscle lipoprotein lipase activity in humans. J Endocrinol Invest 22: 10–15, 1999.[Medline]
  52. Roberts A, Nava S, Bocconi L, Salmona S, Nicolini U. Liver function tests and glucose and lipid metabolism in growth-restricted fetuses. Obstet Gynecol 94: 290–294, 1999.[CrossRef][Web of Science][Medline]
  53. Rovamo LM, Taskinen MR, Kuusi T, Raivio KO. Postheparin plasma lipoprotein and hepatic lipase activities in hyperinsulinemic infants of diabetic mothers and in large-for-date infants at birth. Pediatr Res 20: 623–627, 1986.[Web of Science][Medline]
  54. Saxena U, Witte LD, Goldberg IJ. Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids. J Biol Chem 264: 4349–4355, 1989.[Abstract/Free Full Text]
  55. Sivan E, Homko CJ, Chen X, Reece EA, Boden G. Effect of insulin on fat metabolism during and after normal pregnancy. Diabetes 48: 834–838, 1999.[Abstract]
  56. Suga S, Tamasawa N, Kinpara I, Murakami H, Kasai N, Onuma T, Ikeda Y, Takagi A, Suda T. Identification of homozygous lipoprotein lipase gene mutation in a woman with recurrent aggravation of hypertriglyceridaemia induced by pregnancy. J Intern Med 243: 317–321, 1998.[CrossRef][Web of Science][Medline]
  57. Thomas CR, Lowy C, St Hillaire RJ, Brunzell JD. Studies of the placental hydrolysis and transfer of lipids to the fetal guinea pig. In: Fetal Nutrition, Metabolism and Immunology: The role of the placenta. Proceedings of the 9th Rochester Trophoblast Conference, edited by Miller RK and Thiede HA. New York: Plenum Medical Book, vol 1, p. 135–146, 1984.
  58. Waterman IJ, Emmison N, Dutta-Roy AK. Characterisation of triacylglycerol hydrolase activities in human placenta. Biochim Biophys Acta 1394: 169–176, 1998.[Medline]
  59. Waterman IJ, Emmison N, Sattar N, Dutta-Roy AK. Further characterization of a novel triacylglycerol hydrolase activity (pH 60 optimum) from microvillous membranes from human term placenta. Placenta 21: 813–823, 2000.[CrossRef][Web of Science][Medline]



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