Placental lipoprotein lipase activity is positively associated with newborn adiposity

Placenta 64 (2018) 53e60

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Placental lipoprotein lipase activity is positively associated with newborn adiposity Margaret J.R. Heerwagen a, b, 1, Diane L. Gumina a, Teri L. Hernandez c, d, Rachael E. Van Pelt e, Anita W. Kramer a, Rachel C. Janssen b, Dalan R. Jensen c, Theresa L. Powell a, b, Jacob E. Friedman b, c, f, Virginia D. Winn a, 2, Linda A. Barbour a, c, * a

Department of Obstetrics and Gynecology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Department of Pediatrics, Division of Neonatology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Colorado Anschutz Medical Campus, Aurora, CO, USA d College of Nursing, University of Colorado Anschutz Medical Campus, Aurora, CO, USA e Department of Medicine, Division of Geriatric Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA f Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 November 2017 Received in revised form 16 February 2018 Accepted 4 March 2018

Introduction: Recent data suggest that in addition to glucose, fetal growth is related to maternal triglycerides (TG). To reach the fetus, TG must be hydrolyzed to free fatty acids (FFA) and transported across the placenta, but regulation is uncertain. Placental lipoprotein lipase (pLPL) hydrolyzes TG, both dietary chylomicron TG (CM-TG) and very-low density lipoprotein TG (VLDL-TG), to FFA. This may promote fetal fat accretion by increasing the available FFA pool for placental uptake. We tested the novel hypothesis that pLPL activity, but not maternal adipose tissue LPL activity, is associated with newborn adiposity and higher maternal TG. Methods: Twenty mothers (n ¼ 13 normal-weight; n ¼ 7 obese) were prospectively recruited. Maternal glucose, insulin, TG (total, CM-TG, VLDL-TG), and FFA were measured at 14e16, 26e28, and 36e37 weeks, and adipose tissue LPL was measured at 26e28 weeks. At term delivery, placental villous biopsies were immediately analyzed for pLPL enzymatic activity. Newborn percent body fat (newborn %fat) was assessed by skinfolds. Results: Placental LPL activity was positively correlated with birthweight (r ¼ 0.48;P ¼ 0.03) and newborn %fat (r ¼ 0.59;P ¼ 0.006), further strengthened by correcting for gestational age at delivery (r ¼ 0.75;P ¼ 0.0001), but adipose tissue LPL was not. Maternal TG and BMI were not correlated with pLPL activity. Additionally, pLPL gene expression, while modestly correlated with enzymatic activity (r ¼ 0.53;P < 0.05), was not correlated with newborn adiposity. Discussion: This is the first study to show a positive correlation between pLPL activity and newborn %fat. Placental lipase regulation and the role of pLPL in pregnancies characterized by nutrient excess and fetal overgrowth warrant further investigation. © 2018 Published by Elsevier Ltd.

Keywords: Fetal growth Fetal fat accretion Triglycerides Lipoprotein lipase Placenta Newborn adiposity

Abbreviations: BW, birthweight; CM-TG, chylomicrons; EL, endothelial lipase; FA, fatty acids; FFA, free fatty acid; GDM, gestational diabetes mellitus; IUGR, intrauterine growth restriction; LPL, lipoprotein lipase; newborn %fat, newborn percent body fat; NW, normal weight; pLPL, placental LPL; TG, triglyceride; VLDLTG, very low-density lipoprotein. * Corresponding author. Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Colorado Anschutz Medical Campus, 12801 East 17th Ave, Mail Stop 8106, Aurora, CO 80045, USA. E-mail address: [email protected] (L.A. Barbour). 1 Current address. Margaret (Heerwagen) Magill-Collins, Department of Obstetrics and Gynecology, University of New Mexico, Albuquerque, NM, USA. 2 Current address. Department of Obstetrics and Gynecology, Stanford University Medical Campus, Stanford, CA, USA. https://doi.org/10.1016/j.placenta.2018.03.001 0143-4004/© 2018 Published by Elsevier Ltd.

1. Introduction The concept that intrauterine exposures can impact an offspring's future metabolic health is a well-established paradigm. However, the mechanism between exposure and outcome, including the critical role of the placenta, remains unclear. Developmental programming of offspring obesity and cardiometabolic disease is of high interest with rising maternal obesity and gestational diabetes (GDM) rates [1,2]. However, newborn body composition, rather than birthweight (BW), appears to be a better predictor of childhood obesity and, in a

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cohort of 89 maternal/infant dyads, increased newborn adiposity rather than BW was associated with increased adiposity at 9 years [3]. While glucose and amino acids contribute to infant growth, studies also support maternal triglycerides (TG) and non-esterified free fatty acids (FFA) as important determinants of newborn percent body fat (newborn %fat), given the limited capacity of the fetus for de novo lipogenesis [4e8]. Humans are born with one of the highest percent body fat of all terrestrial species [9,10], and most of this fat is deposited in the 3rd trimester, coinciding with fetal adipose tissue development [7,8,11,12]. Pregnancy is normally associated with a 2e3-fold increase in maternal TG [13e17], largely due to increasing very lowdensity lipoprotein (VLDL-TG) synthesis stimulated by estrogen, coupled with increasing maternal insulin resistance [17,18], resulting in enhanced lipolysis in maternal adipose tissue in late pregnancy [7,8]. However, this simple supply-and-demand scheme between maternal TG and fetal fat accretion overlooks a crucial point of regulation at the level of the placenta [7,8,19e21]. To cross the placenta, TG in the form of both liver-derived VLDLTG and dietary chylomicrons (CM-TG), must first be hydrolyzed to FFA. Two major placental lipases have been identified for FFA release: placental lipoprotein lipase (pLPL) and endothelial lipase (EL). Placental EL has phospholipase activity needed for liberating polyunsaturated fatty acids (FA) from phospholipids, and less TG hydrolase activity [22]. Maternal LPL is the major TG hydrolase in adipose tissue and likely plays a role in the placenta [15]. In maternal adipose tissue, LPL hydrolyzes VLDL-TG and CM-TG to FFA for uptake of FA into adipocytes for storage in early pregnancy, but its activity diminishes in late gestation to divert fuel to the growing fetus [7]. In the placenta, hydrolysis of TG to FFA allows them to be taken up by the syncytiotrophoblast, where they can be stored, metabolized, oxidized or transported into fetal circulation [23]. Both FA uptake from the mother and their release from placental lipid pools might provide FA for delivery to the fetus but regulation of this process is unclear, as is the role of pLPL [7,8]. Data suggest that pLPL can act as a gatekeeper molecule [15], facilitating the liberation of FFA from maternal TG, and contributing to the pool of FFA available for placental lipid uptake and transport into fetal circulation [11]. To date, studies in humans have demonstrated disparate results regarding the impact of maternal obesity and GDM on pLPL [24e28], but most measure pLPL gene expression rather than enzymatic activity, an important distinction given that LPL is posttranscriptionally regulated [29]. When pLPL activity was measured, it was increased in a small cohort of obese mothers [24] and decreased with intrauterine growth restriction (IUGR) [28], suggesting that pLPL may be a point of regulation in fetal growth. In an earlier study by Kaminsky et al., two separate placental lipase activities were measured, and findings supported increased placental lipolytic activity in pregnancies complicated by diabetes, and a positive association with birthweight [30]. However, the optimal pH activity of the lipase associated with birthweight was 4 rather than 8, which is the optimal activity for LPL, therefore it was unlikely to be LPL. Additionally, while implied, no studies have directly measured pLPL activity in conjunction with newborn fat adiposity estimates. Here, we test the hypothesis that human pLPL activity, but not adipose tissue LPL activity, is positively associated with newborn adiposity and that pLPL activity is influenced by maternal metabolism and TG availability. 2. Methods 2.1. Patient recruitment Written informed consent was obtained according to the

protocol approved by the Colorado Multiple Institutional Review Board (COMIRB). Healthy normal-weight (NW; n ¼ 13) and obese (n ¼ 7) pregnant women were recruited at 12e14 weeks gestation as part of a larger prospective, NIH-funded cohort study. Exclusion criteria included: age <18 or >35 years, multiple gestation, delivery <37 weeks, and pre-existing diabetes or GDM by ACOG criteria [31]. To exclude most risk factors for growth restriction, patients with chronic medical problems or a history of IUGR, stillbirth, or placental abruption were excluded. 2.2. Maternal serum measurements Fasting maternal blood samples were collected at 14e16, 26e28, and 36e37 weeks gestation. Fasting blood glucose, insulin, FFA, and TG were measured and HOMA-IR values calculated as previously described [32,33]. Postprandial TG were measured at 14e16 and 26e28 weeks following a liquid breakfast containing 30% of total estimated daily caloric needs (50% carbohydrate; 35% fat; 15% protein), with TG subsequently assessed at 10 time points over 4 h. Postprandial CM-TG and VLDL-TG lipoprotein sub-fractions were isolated from fresh plasma using a sequential flotation, ultracentrifugation method, as previously described [34,35]. Serum and plasma were spun promptly and frozen at 80  C until batchprocessed. 2.3. Maternal adipose tissue and placental tissue collection Subcutaneous adipose tissue was biopsied from the upper gluteal/flank region of fasting mothers at 26e28 weeks gestation, using our established protocol [36]. All placentas analyzed were delivered at term (37 weeks gestation) and were labored deliveries, with n ¼ 16 vaginal and n ¼ 4 cesarean deliveries following labor for obstetric or fetal indications. Immediately after delivery, placentas were weighed and placed on ice. Villous biopsies were obtained from central regions of the placenta after dissecting away the basal plate including decidua [15,37]. For gene expression analyses, villous samples were rinsed in ice-cold sterile 1X PBS to remove all visible blood before flash-frozen. For lipase activity, adipose tissue and villous biopsies were rinsed separately in icecold Krebs Ringer phosphate (KRP) buffer (pH 7.4), all visible vessels and connective tissue were dissected away, and tissues were minced to 1e2 mm3 pieces. Tissues were quickly dried on filter paper prior to weighing. LPL activity was then run in triplicate from three separate samples from this minced pool. 2.4. Placental and adipose tissue LPL activity LPL activity was determined using the methods described previously [38]. Since LPL, unlike other lipases, is displaced from proteoglycans by heparin, tissue-bound LPL was released by incubating 50 mg of minced adipose tissue or 100 mg of minced placental villous tissue in 0.1 or 0.5 mL, respectively, of KRP buffer containing 15 mg/mL heparin sulfate for 45 min at 37  C with agitation. A 100 mL aliquot of supernatant containing the heparin-released enzyme was then incubated with 100 mL of a 14C-triolein phosphatidylcholine-stabilized substrate containing ApoC2 for an additional 45 min at 37  C with agitation. The reaction was optimized for LPL activity by using neutral pH of 7.4 and inclusion of human serum ApoC2, a requisite cofactor for LPL activity. The reaction was then stopped by addition of 3.4 mL of Belfrage solution containing chloroform, methanol, and heptane, and the aqueous phase extracted by addition of 0.96 mL of bicarbonate buffer and agitation. The 14C-labeled FA were partitioned by centrifugation at 4  C. An aliquot of the resulting aqueous supernatant containing lipase-released 14C-labeled FFA was counted by b-scintillation

M.J.R. Heerwagen et al. / Placenta 64 (2018) 53e60

(Beckman-Coulter, Brea, CA). A separate 14C-oleic acid reaction was used to control for extraction efficiency; individual results were normalized to a heparinized rat plasma internal activity standard, and with final LPL activity expressed as an average of replicates. Placental lipase activity was calculated as nmol FFA released/minute/gram of tissue. All results were corrected for extraction efficiency and normalized to the internal activity standard. To ensure specificity of the lipase activity being analyzed, initial test reactions included two additional conditions as negative controls. First, a reaction with addition of 2 mL of a mouse monoclonal antihuman LPL-specific inhibitory antibody (kind gift from John Brunzell, University of Washington, Seattle, WA) was performed [39]. Second, a reaction was performed in which ApoC2, a required LPL cofactor, was excluded during substrate preparation. Both specificity conditions reduced measurable LPL activity to background levels. 2.5. RNA extraction and qRT-PCR RNA was extracted from frozen central villous samples using the RNeasy Mini kit (Qiagen, Valencia, CA), per manufacturer's instructions, and analyzed using the Experion System to test integrity (Bio-Rad, Hercules, CA). We performed validation experiments to demonstrate that efficiencies of target and reference genes were approximately equal. Relative gene expression of LPL (forward: 50 AAGCTATCCGCGTGATTGCAGAGA-30 , reverse: 50 -TGCACCTG0 TAGGCCTTACTTGGAT-3 ) was determined using iTaq SYBR Green Supermix (Bio-Rad) after cDNA synthesis (iScript kit; Bio-Rad). Reactions were run in duplicate on an iQ5 Real-Time PCR Detection System (Bio-Rad) and normalized to UBC (forward: 50 -TTATATAAGGACGCGCCGGG-30 , reverse: 50 0 GCATTGTCAAGTGACGATCACAG-3 ) and RPL13A (forward: 50 CCTGGAGGAGAAGAGGAAAGAGA-30 , reverse: 50 -TTGAGGACCTCTGTGTATTTGTCAA-30 ) using the comparative threshold cycle method. Expression of both reference genes were not different between groups. 2.6. Newborn body composition by anthropometry

of

Newborn %fat was determined by skinfold thickness within 48 h delivery, with the same individual performing all

55

anthropometric measurements. Triceps and subscapular skinfold measurements were performed in triplicate using Lange calipers (Beta Technology, St. Albans, UK). The sum of triceps and subscapular skinfold measurements and sex of the child were used to estimate newborn %fat according to the Slaughter equation [40]. Additionally, a second, gestational age-adjusted value for newborn %fat was calculated, which accounted for the approximate 0.1% per day increase in %fat that occurs from 37 to 40 weeks gestation as well as infant sex, as previously described [41e43]. 2.7. Data analysis Pearson correlations were performed using Prism 5.0 (GraphPad Software, La Jolla, CA). Differences between groups were determined by a 2-tailed Student's t-test for independent groups. All data are expressed as the mean ± standard error of mean, and significant differences are defined as P < 0.05. No prior studies examining the relationship between placental lipase activity and newborn size for sample size estimation were
et al. demonstrated a located. However, previous results from Dube significant difference in pLPL activity in 12 mothers (6 obese versus 6 NW, P ¼ 0.02) [24]. We estimated that in our total cohort of 20 mothers, we would have 80% power (alpha at 0.05) to detect a moderate to strong correlation between pLPL activity and newborn %fat [44]. 3. Results 3.1. Maternal characteristics and pregnancy outcomes By design, maternal BMI was approximately 10 points higher in the obese versus NW mothers, without significant differences in age, ethnicity, or parity (Table 1). All women delivered at term and labored, although there were four cesarean deliveries for failed induction. Obese mothers demonstrated mild, but significant, elevations in 36-week fasting insulin, glucose, and HOMA-IR relative to NW (P ¼ 0.01, 0.004, and 0.01 respectively; Table 2). Fasting TG were also higher in obese women at 14e16 and 26e28 weeks (both P ¼ 0.03) as were postprandial TG excursions (both P ¼ 0.01), with both VLDL-TG and CM-TG sub-fractions reflecting this pattern.

Table 1 Maternal, placenta and infant characteristics.

Maternal characteristics BMI (kg/m2) Age (years) Ethnicity Parity Gestational weight gain (kg) Mode of delivery: CS/NSVD Adipose tissue LPL activity# Placenta and infant characteristics Gestational age (weeks) Placenta weight (g) Infant sex (M/F) Birthweight (g) Birth length (cm) Ponderal index Newborn % body fat Placental LPL activity#

Normal weight (n ¼ 13)

Obese (n ¼ 7)

P-value

22.7 ± 0.4 30.5 ± 0.7 12 Non-Hispanic White 1 Non-Hispanic Black 0.5 ± 0.2 6.3 ± 0.6 3/10 33.87 ± 5.35

32.6 ± 1.0 29.7 ± 1.0 7 Non-Hispanic White

*<0.0001 0.98 e

0.4 ± 0.3 7.4 ± 0.9 1/6 33.31 ± 6.29

0.91 0.17 e 0.95

39.7 ± 0.4 534 ± 23 6/7 3213 ± 114 50.4 ± 0.4 2.50 ± 0.05 7.68 ± 0.51 2.78 ± 0.28

40.0 ± 0.6 576 ± 57 5/2 3549 ± 225 51.6 ± 0.8 2.55 ± 0.08 7.98 ± 0.89 2.60 ± 0.34

0.48 0.42 e 0.15 0.15 0.56 0.76 0.70

Values are reported as mean ± SEM; *P < 0.05. #nmol free fatty acid/min/g of tissue. BMI, body mass index; CS, cesarean section; NSVD, normal spontaneous vaginal delivery.

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Table 2 Maternal serum measures. Normal weight (n ¼ 13) Gestation (wks): Fasting Insulin (mU/mL) Glucose (mg/dL) TG (mg/dL) FFA (mEq/L) HOMA-IR Postprandial TG (AUCy) VLDL-TG (AUCy) CM-TG (AUCy)

Obese (n ¼ 7)

P-value

14e16

26e28

36e37

14e16

26e28

36e37

8±1 75 ± 1 86 ± 4 646 ± 52 1.4 ± 0.2

10 ± 1 75 ± 2 129 ± 8 469 ± 39 1.9 ± 0.2

13 ± 1 74 ± 2 200 ± 19 406 ± 56 2.5 ± 0.2

10 ± 1 ^80 ± 2 ^105 ± 8 587 ± 75 1.9 ± 0.3

13 ± 2 79 ± 2 # 165 ± 15 565 ± 51 2.5 ± 0.3

*22 ± 5 *86 ± 1 244 ± 19 488 ± 68 *4.6 ± 1

*0.01 ^0.02;*0.004 ^0.03; #0.03

23692 ± 1327 7985 ± 1125 30400 ± 2870

32425 ± 1485 18189 ± 1729 54684 ± 4158

N/A N/A N/A

^31599 ± 2633 ^16440 ± 1857 ^54806 ± 5848

#

N/A N/A N/A

^0.01; #0.01 ^0.001; #0.007 ^0.001; #0.002

42201 ± 3735 28042 ± 2885 # 80161 ± 550 #

*0.01

Values are reported as mean ± SEM; ^P < 0.05 at 14e16 weeks versus NW, #P < 0.05 at 26e28 weeks versus NW, *P < 0.05 at 36e37 weeks gestation versus NW. y4-h AUC calculated using the trapezoidal method. AUC, area under the curve; CM, chylomicron; FFA, non-esterified free fatty acids; NW, normal weight; TG, triglycerides; VLDL, very low-density lipoprotein.

B 40

90

35

80

Adipose Tissue LPL Activity (nmol FFA/min/g)

LPL Activity (nmol FFA/min/g)

A

30 25 20 15 10

***

5

r = 0.00009 P = 0.97

70 60 50 40 30 20

NW Obese

10

0

0

Adipose Tissue LPL

5

Placenta LPL

10

15

Newborn % Body Fat

Fig. 1. Adipose tissue LPL activity compared to placental LPL activity and correlation with newborn body composition. A) Mean difference between adipose tissue LPL and placental LPL activity compared in the same cohort (n ¼ 19; ***P < 0.0001) and B) absence of relationship between adipose tissue LPL and newborn % body fat from normal-weight (NW, n ¼ 13; open circles) and obese mothers (n ¼ 7; closed circles).

A

B

800

Placenta Weight (g)

Placenta Weight (g)

800

r = 0.62 P = 0.003

NW Obese

600

400

200

r = 0.40 P = 0.08

NW Obese

600

400

200 3000

4000

5000

Birthweight (g)

5

10

15

Newborn % Body Fat

Fig. 2. Placental weight and newborn birthweight and body composition. Correlation of placental weight with (A) birthweight and (B) newborn % body fat from normal-weight (NW, n ¼ 13; open circles) and obese mothers (n ¼ 7; closed circles).

Although BW trended higher in obese women, this did not reach statistical significance. In addition, no statistical differences in gestational age, placenta weight, newborn %fat, or pLPL activity were found between groups, likely due to limited numbers when each group was analyzed separately (Table 1).

3.2. Adipose tissue LPL activity is markedly higher than pLPL activity, but is not associated with newborn adiposity To confirm the specificity of LPL activity, both adipose tissue LPL and pLPL activity were assayed in an identical manner on fresh

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adipose tissue (28 weeks) and term placenta. As expected, adipose tissue LPL activity was about 10-fold more robust than pLPL activity (Fig. 1A). Furthermore, adipose tissue LPL activity, which hydrolyzes TG to FFA for uptake into maternal fat, was not correlated with BW or newborn adiposity (Fig. 1B). There was no difference in adipose tissue LPL activity between groups (Table 1). 3.3. Placental LPL activity is significantly correlated with BW and newborn adiposity but not LPL gene expression As expected, placenta weight and BW were significantly related (r ¼ 0.62, P ¼ 0.003; Fig. 2A), but placental weight with newborn % fat approached significance (P ¼ 0.08; Fig. 2B). Placental LPL activity was positively correlated with BW (r ¼ 0.48, P ¼ 0.03; Fig. 3A), and even more strongly associated with newborn %fat (r ¼ 0.59, P ¼ 0.006; Fig. 3B). Interestingly, when we corrected newborn %fat for gestational age (0.1% per day for 37e40 weeks) [41,42], the correlation between pLPL activity and newborn %fat was further strengthened (r ¼ 0.75, P ¼ 0.0001; Fig. 3C). Notably, there was no separate association between pLPL activity and gestational age (Fig. S1), which would serve as a potential confounder to the above relationship. Given the difficulty of directly analyzing pLPL activity immediately after delivery, we sought to verify the utility of LPL gene expression as a viable alternative. Placental LPL gene expression was modestly correlated with pLPL activity (r ¼ 0.53, P < 0.05; Fig. 4A) but pLPL gene expression was not associated with newborn %fat (Fig. 4B). 3.4. Maternal determinants of pLPL activity We examined whether pLPL activity was correlated with maternal BMI, gestational weight gain, and fasting serum glucose, insulin, TG and FFA (at all gestational ages) but did not find significant correlations. Although there were significant differences in fasting TG and postprandial total TG, VLDL-TG, and CM-TG between groups (Table 2), we found no association between TG at any gestational age and pLPL activity. 4. Discussion This study is the first to demonstrate a positive correlation between pLPL activity and newborn adiposity. Interestingly, although the correlation between pLPL activity was significant in this cohort of term (37 weeks) infants (r ¼ 0.59; P ¼ 0.006), the correlation was even stronger (r ¼ 0.75; P ¼ 0.0001) when adjusted for the expected ~0.1% increase in %fat that has been estimated to occur between 37 and 40 weeks [41,42]. We did not identify significant maternal metabolic correlations with pLPL activity at any stage of gestation; however, maternal lipids and other metabolic measures were not collected at delivery when pLPL activity was assessed. Further, although our cohort was larger than other studies that measured pLPL activity [15,24,28], it was modest and all of our mothers were relatively metabolically healthy with no differences in BW, newborn %fat, or pLPL activity between groups. The regulatory mechanism(s) for pLPL remain to be established in vivo. Data from Magnusson et al. showed increased villous pLPL activity in vitro with insulin plus hyperglycemic medium [15] and an inverse relationship of decreased cytotrophoblast LPL activity with increasing intralipid concentrations [45]. These authors also found increased pLPL activity in women with type 1 diabetes, yet
et al. decreased activity in GDM [15,28]. Additionally, Dube observed an increase in pLPL activity in a small cohort of obese compared with NW mothers [24]. Together, these studies suggest a possible increase in pLPL activity in obese/diabetic pregnancies and

Fig. 3. Placental lipoprotein lipase activity and newborn birthweight and body composition. Correlation of placental lipoprotein lipase (pLPL) activity with (A) birthweight, (B) newborn % body fat, and (C) newborn % body fat after adjustment for gestational age at delivery, from normal-weight (NW, n ¼ 13; open circles) and obese mothers (n ¼ 7; closed circles).

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B

A

0.4

r = 0.53 P = 0.03

pLPL Gene Expression (AU)

pLPL Gene Expression (AU)

0.4

0.3

0.2

0.1

r = 0.25 P = 0.33

0.3

0.2

0.1

0.0

0.0 1

2

3

4

5

pLPL Activity (nmol FFA/min/g)

0

5

10

15

Newborn % Body Fat

Fig. 4. Placental lipoprotein lipase gene expression/activity and infant adiposity. Correlation of placental lipoprotein lipase (LPL) gene expression (n ¼ 18 mothers) with (A) placental LPL (pLPL) enzymatic activity and (B) newborn % body fat.

reduced activity in vitro with TG excess. Our data show an increase in pLPL activity with newborn adiposity in the pooled cohort of 20 mothers, but no correlations with pLPL activity and maternal fasting or postprandial TG. These obese mothers were relatively metabolically healthy, given chronic medical conditions and risk factors for preterm labor or placental dysfunction were excluded. Compared with NW, they had only mildly elevated HOMA-IR and fasting glucose or TG. Without strong delineation between NW and obese metabolic phenotypes, perhaps it's not surprising that we observed no difference in BW, newborn %fat, placental weight, or pLPL activity between groups, especially given the limited sample size of the individual groups. Interestingly, if pLPL activity is indeed inhibited by high TG concentrations [45], one might speculate that lower pLPL activity attenuates the availability of FFA to the fetus, restraining the impetus for a marked excess in adiposity in infants born to obese or GDM mothers. Given previous data suggesting that pLPL can have variable affinity for TG hydrolysis based on lipoprotein subtype [46], we sought, for the first time, to fractionate VLDL-TG from diet-derived CM-TG. However, our comprehensive analysis of postprandial TG and lipoprotein sub-fractions at 14e16 and 26e28 weeks showed no correlation with pLPL activity at term. Notably, since all mothers labored, our measured pLPL activity reflects more of a fasted rather than postprandial state. Our assay was optimized to detect LPL activity, by neutral buffer pH, heparin pre-incubation, and inclusion of ApoC2 as a cofactor. Initial test studies using a LPL inhibitory antibody and ApoC2deficient negative controls reduced measured LPL activity to background levels. We confirmed our pLPL activity technique by using the same measurement methods in maternal adipose tissue in which adipose tissue LPL activity was 10-fold higher than pLPL activity, as would be expected. Although we are confident we were measuring LPL activity specifically, we acknowledge the potential contribution of other proposed placental lipases [7,8,11,23,27,47e49]. While LPL is considered the major lipase for maternal TG hydrolysis, data suggest that placental EL might hydrolyze TG, particularly illustrated in cases of maternal LPL deficiency [49,50]. While a number of studies report differential gene expression of placental lipases in maternal obesity and GDM [26,27,49,50], we observed only a modest association between LPL gene expression and pLPL enzymatic activity, and no association between gene expression and newborn %fat. Immunoblotting to assess pLPL

protein expression was attempted. Three antibodies specific to two epitopes (5D2, a gift; 5D2, Abcam; LPL.4, Abcam) were tested in placental villous samples. Each antibody produced banding patterns that differed from previously published works. To assess binding specificity, we performed a competition assay using purified LPL protein incubated with the antibody prior to immunoblotting. This assay did not reduce antibody binding at specific bands. Therefore, poor discrimination by the antibody, possibly due to denaturing of the active LPL dimer, precluded our ability to reproducibly measure pLPL protein expression [51]. Recently, Barrett et al. showed no differences in placental gene expression of LPL, EL, or hormone-sensitive lipase between normal versus GDM pregnancies [27]. Given the significant post-translational modification of LPL, measurements beyond gene expression are needed, although complicated by the timing of delivery and labile enzyme activity. Given the unpredictability of labor and delivery, flashfreezing and batch-processing tissue samples [24,25,28,45] could alleviate some of these challenges; however, in our hands, we observed a significant loss in activity and discriminatory values when we compared frozen tissue with fresh. Studying placentas from elective cesarean deliveries might circumvent this problem. Because the placenta ages over the course of gestation beginning at sites most distal from cord insertion [52], with term placentas showing evidence of villous thinning, syncytial knots, and apoptosis [53], we chose to sample the central villous region for enzyme activity. Our use of skinfolds to estimate adiposity allowed for assessment within 48 h of delivery, before significant postnatal nutritional influences. We have shown significant correlations between skinfolds and dual-energy X-ray absorptiometry at 2 weeks (r ¼ 0.70) [54]. In summary, this is the first study to demonstrate that pLPL activity is positively correlated with newborn adiposity. In this modest cohort of relatively healthy mothers, we did not identify any maternal metabolic factors in early or later pregnancy that might drive this activity at term. While these preliminary findings are novel, a larger cohort, elective cesarean deliveries, and more severe maternal metabolic pathology might help to clarify whether increased pLPL activity is an important placental mechanism contributing to fetal fat accretion. Conflicts of interest The authors report no conflict of interest associated with this manuscript.

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