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. Author manuscript; available in PMC: 2022 Jul 15.
Published in final edited form as: Mol Cell Endocrinol. 2021 May 12;532:111319. doi: 10.1016/j.mce.2021.111319

EFFECT OF TYPE 2 DIABETES MELLITUS ON PLACENTAL EXPRESSION AND ACTIVITY OF NUTRIENT TRANSPORTERS AND THEIR ASSOCIATION WITH BIRTH WEIGHT AND NEONATAL ADIPOSITY

Marisol Castillo-Castrejon 1,*, Kyohei Yamaguchi 1,2, Rachel L Rodel 3, Kathryn Erickson 1, Anita Kramer 1, Nicole M Hirsch 3, Kristy Heiss 3, Thomas Jansson 1, Linda A Barbour 3,4, Theresa L Powell 1,5
PMCID: PMC8206039  NIHMSID: NIHMS1703474  PMID: 33989714

Abstract

Aims:

Infants born to women with Type 2 Diabetes Mellitus (T2DM) are at risk of being born large for gestational age due to excess fetal fat accretion. Placental nutrient transport determines fetal nutrient availability, impacting fetal growth. The aims of the study were to evaluate the effect of T2DM on placental insulin signaling, placental nutrient transporters and neonatal adiposity.

Methods:

Placentas were collected from BMI-matched normoglycemic controls (NGT, n=9) and T2DM (n=9) women. Syncytiotrophoblast microvillous (MVM) and basal (BM) plasma membranes were isolated. Expression of glucose (GLUT1, −4), fatty acid (FATP2, −4, −6, FAT/CD36), amino acid (SNAT1, −2, −4, LAT1, −2) transporters, insulin signaling, and System A transporter activity was determined. Neonatal fat mass (%) was measured in a subset of neonates born to T2DM women.

Results:

GLUT1 protein expression was increased (p=0.001) and GLUT4 decreased (p=0.006) in BM from T2DM. MVM FATP6 expression was increased (p=0.02) and correlated with birth weight in both T2DM and NGT groups (r=0.65, p=0.02). BM FATP6 expression was increased (p=0.01) in T2DM. In MVM of T2DM placentas, SNAT1 expression was increased (p=0.05) and correlated with birth weight (r=0.84, p=0.004); SNAT2 was increased (p=0.01), however System A transporter activity was not different between groups. MVM LAT1 expression was increased (p=0.01) in T2DM and correlated with birth weight (r=0.59, p=0.04) and neonatal fat mass (r=0.76, p=0.06).

Conclusion:

In pregnancies complicated by T2DM placental protein expression of transporters for glucose, amino acids and fatty acids is increased, which may contribute to increased fetal growth and neonatal adiposity.

Keywords: fetal growth, trophoblast, maternal-fetal exchange, fatty acids, amino acids, human, pregnancy

1. INTRODUCTION

The epidemic of Type 2 Diabetes mellitus (T2DM) and its complications represent a major public health problem with significant economic consequences (Lascar, et al. 2018). The global prevalence of diabetes during pregnancy is estimated to be 14%, with gestational diabetes mellitus (GDM) accounting for more than 90% and T2DM approximately 8% of cases. Pregestational diabetes mellitus now affects 1% to 2% of pregnancies in the United States (Sugrue and Zera 2018), the prevalence of T2DM is even higher in high risk ethnic populations (Zhu and Zhang 2016), and increasingly seen in adolescent pregnancies (Klingensmith, et al. 2016). The prevalence of T2DM is increasing in parallel with the prevalence of obesity (Collaborators, et al. 2017; Zheng, et al. 2018) and due to the progression to T2DM from a GDM pregnancy, the latter affecting up to 25% in high risk populations (Valent and Barbour 2020). Overweight and obesity, which affects nearly 60% of women of child-bearing age in the US (Flegal, et al. 2016), represent the main driving factor of the global T2DM epidemic. Diabetes in pregnancy has been associated with short- and long-term adverse health outcomes for both mother and child (Berry, et al. 2016; Burlina, et al. 2019; Buschur 2018). Infants born to mothers with T2DM have an increased risk of prematurity, low Apgar, congenital abnormalities, asthma, fetal macrosomia, neonatal mortality, hypoglycemia and hyperinsulinemia at birth (Cnattingius, et al. 2017; Martinez, et al. 2020). Women with T2DM during pregnancy have higher risk to develop preeclampsia and gestational hypertension and to deliver by caesarean section (Guariguata, et al. 2014), and have higher risks of stillbirth at term (Browne, et al. 2019; Mackin, et al. 2019).

Excess maternal nutrient availability, a characteristic of obese and T2DM mothers, results in an increased risk for obesity in the offspring (Berry et al. 2016; Friedman 2018). Obesity in pregnancy accounts for the majority of large-for-gestational age infants with increased adiposity (2015; Dutton, et al. 2018). It has been proposed that exacerbated maternal availability of macronutrients and other signaling molecules to the fetal-placental unit, resulting in enhanced nutrient transfer, fetal overgrowth and changes in body composition (Durnwald, et al. 2004), linking maternal metabolic perturbations to fetal programing (Dimasuay, et al. 2016; Hernandez, et al. 2020).

Placental nutrient transport capacity is one key determinant of fetal growth. Increased expression and activity of placental nutrient transporters contributes to accelerated fetal growth in GDM (Castillo-Castrejon and Powell 2017). Nutrient transporters are localized in the plasma membranes of the syncytiotrophoblast (ST), the multinucleated transporting epithelium of the human placenta (Jansson 2018). The ST consists of two polarized plasma membranes: the maternal-facing microvillous plasma membrane (MVM) which is in direct contact with maternal blood; and the fetal-facing basal membrane (BM) adjacent to the fetal capillaries (James-Allan, et al. 2018).

Glucose is the primary energy source for the fetus as there is no significant fetoplacental gluconeogenesis. Glucose transport takes place by facilitated diffusion mediated by glucose transporters (GLUTs) localized in the MVM and BM. Lipids are essential for fetal brain development and adipose tissue accretion. Maternal lipids levels are strong predictors for higher fetal lipid levels, fetal growth and fat mass in the neonate (Wang, et al. 2018). In women with obesity, triglycerides may be a stronger predictor of neonatal adiposity than glucose and accounted for 50% of the variance in neonatal fat in one prospective trial (Barbour, et al. 2018). Lipids in the maternal circulation are hydrolyzed to non-esterified or “free” fatty acids (NEFAs) before being transferred across the MVM by transport proteins including fatty acid transport proteins (FATPs) and fatty acid translocase (FAT/CD36). Lipid metabolism and transport in the syncytiotrophoblast remains poorly understood, but metabolic changes associated with maternal obesity and diabetes are likely to affect placental transfer of lipids (Lewis and Desoye 2017). Amino acid transfer across the placenta is mediated by energy dependent processes, against a concentration gradient through accumulative transporters and exchangers in the MVM and BM (Cleal, et al. 2018). System A transporter is a sodium-dependent accumulative transporter (SNATs, sodium-coupled neutral amino acid transporter), which creates high intracellular concentrations of non-essential neutral amino acids. These are either transported to the fetus or exchanged for extracellular essential amino acids via System L (LATs). Therefore, System A and L work in concert to provide both essential and non-essential amino acids. Recent evidence indicates that placental mTOR signaling is a regulator of fetal nutrient availability by modulating placental growth and nutrient transport capacity (Diaz, et al. 2014). Growth factors such as insulin and IGF-1 activate mTOR, leading to greater amino acid transport capacity supporting accelerated fetal growth (Gupta and Jansson 2019; Vaughan, et al. 2017).

Diabetes during pregnancy is associated with an increased risk of fetal overgrowth, where placental nutrient transport expression may be altered. There is evidence of increased expression or activity of placental nutrient transporters in pregnancies complicated by maternal obesity, particularly in cases of fetal overgrowth. We have previously reviewed the placental nutrient transport in pregnancies complicated with GDM (Castillo-Castrejon and Powell 2017) and in maternal obesity (Kelly, et al. 2020). The vast majority of the literature that has been reported is on GDM complicated pregnancies, where T2DM pregnancies are not included in the analysis. Moreover, the analysis of the transporter expression has been done in placental homogenate or only one membrane fraction of the trophoblast. Very little is known about the placental macronutrient transport capacity in T2DM complicated pregnancies. The aims of the present study were to evaluate the effect of T2DM on placental insulin signaling and to present a comprehensive study of the effect of T2DM on polarized expression of glucose, fatty acid and amino acid transporters in the MVM and BM. To describe the associations between birth weight and neonatal adiposity and placental nutrient transporters in order to provide evidence into the causes underlying the development of T2DM associated pregnancy complications. We tested the hypothesis that expression and activity of placental nutrient transporters are increased and associated with birth weight and neonatal adiposity in T2DM pregnancies.

2. METHODS

2.1. Study subjects and tissue collection

Placental tissue was collected after informed written consent. Placentas from women with T2DM prior to pregnancy (n=9), and relevant clinical data were collected under approved protocol by the Colorado Multiple Institutional Review Board (COMIRB 17–0055) (Rodel, et al. 2019). Placentas from normoglycemic (NGT) BMI-matched women with uncomplicated healthy pregnancies (n=9) and relevant clinical data were selected from a bio/data repository collected with informed consent (COMIRB-14–1073).

Criteria for diagnosis of T2DM prior pregnancy or up to 24 weeks of gestation included A1c ≥ 6.5%, fasting plasma glucose ≥126 mg/dL, or a random plasma glucose concentration ≥200 mg/dL (American Diabetes 2018). All women with T2DM were taking insulin but had relatively mild disease given their mean A1C was 7.7 +/− 0.5. Exclusion criteria for T2DM and NGT included: tobacco use, age <20 or >45, BMI >40 and fetal malformations. Gestational-age-adjusted birth length and birth weight were determined based on INTERGROWTH-21st fetal growth charts and presented as z-scores. Clinical characteristics of the study participants are presented in Table 1.

Table 1.

Clinical characteristics of study participants

NGT T2DM P-Value
N 9 9
Mother Body mass index (kg/m2) 29.0 ± 1.40 28.6 ± 1.30 0.86
Age (years) 31.2 ± 1.90 28.1 ± 2.40 0.33
Ethnicity
(White/Black/Asian/Other) 		7/2/0/0 5/1/2/1
Gestational age 39.0 ± 0.20 37.5 ± 0.20 <0.001
Hemoglobin A1c, (%, 1st visit) -- 7.70 ± 0.57
Mode of delivery
 (vaginal/C-section) 		1/8 5/4 0.13#
Complications (n)
(Preeclampsia / preterm birth) 		0 1/1 >0.99#
Newborn Sex (male/female) 5/4 5/4 >0.99#
Birth weight (kg) 3.43 ± 0.20 3.36 ± 0.20 0.41
Birth weight z-score 0.66 ± 0.39 1.15 ± 0.48 0.22
Birth length (cm) 49.9 ± 0.68 49.9 ± 0.87 0.96
Birth length z-score 0.36 ± 0.26 1.01 ± 0.47 0.25
Large for gestational age (n) 0 1
Placenta weight (g) 664.2 ± 42.1 588.4 ± 43.8 0.23
Placental efficiency (BW/PW) 5.30 ± 0.39 5.80 ± 0.29 0.16
Fat mass (%) -- 12 ± 1.8
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Data are presented as mean ± S.E.M. NGT, Normoglycemic control; T2DM, Type 2 Diabetes Mellitus; BW, birth weight; PW, placental weight; Unpaired Student’s T test

#

Fisher’s exact Test.

Air displacement plethysmography in newborns of T2DM participants

Informed consent was obtained from participants to obtain infant percent fat mass measured by air displacement plethysmography (PeaPod) at approximately 2 weeks of age (n=5).

2.2. Tissue collection and processing

Placentas from NGT and T2DM women were obtained after vaginal or cesarean delivery. Approximately 100 g of villous tissue was collected from different areas of the placenta and pooled. Placental tissue was rinsed with physiological saline solution and homogenized with Polytron (15,000 rpm for 2 minutes) in ice-cold buffer D (250 mM sucrose, 10 mM HEPES, pH 7.4) with protease and phosphatase inhibitors. Homogenized tissue was snap-frozen and stored at −80°C until further analysis.

2.4. Isolation of syncytiotrophoblast plasma membranes and enrichment analysis

Syncytiotrophoblast maternal-facing microvillous plasma membrane (MVM) and fetal-facing basal membrane (BM) were isolated from the homogenate using differential ultracentrifugation and Mg2+-precipitation, as previously described in detail (James-Allan, et al. 2019). Isolated plasma membranes were snap frozen and stored at 80°C until further analysis. MVM enrichment (Bowers and McComb 1966) was determined by MVM/homogenate ratio of alkaline phosphatase activity (Sigma Aldrich A9226, N2770). Enrichment of the BM fraction was determined by Western blot using the BM/homogenate ratio of the protein expression of voltage-dependent anion-selective channel 1 (VDAC1), which is exclusively expressed in the BM. Alkaline phosphatase activity in MVM (NGT, 17±3.4; T2DM 14.3±1.3; p=0.79) and VDAC expression in BM (NGT, 66.5±8.3; T2DM 62.2±9.3; p=0.66) were not different between groups.

2.3. System A transporter activity in microvillous plasma membrane

System A transporter activity was determined by measuring Na+-dependent uptake of 14C-methyl-aminoisobutyric acid ([14C]MeAIB) using a modified protocol previously described (Mahendran, et al. 1993). MVM vesicles were loaded overnight at 4°C in incubation buffer (300mM mannitol, 10 mM HEPES-Tris, pH 7.4). MVM vesicles were incubated at 37°C with [14C]MeAIB (150 μM) in a sodium-containing buffer (150 mM NaCl, 10 mM HEPES, pH7.4). After 20 seconds of incubation, tracer uptake was stopped by removing vesicles from substrate by filtration on mixed ester filters (0.45 μm pore size, Millipore Corporation) and washed with ice-cold PBS. Total radiotracer accumulation within the vesicles was quantified by liquid scintillation counting. System A activity was determined by subtracting Na+-free (non-mediated) from total uptake (Na+-containing buffer) performed in triplicate and expressed as picomoles of MeAIB uptake per milligram of protein per second (pmol/mg/s).

2.4. Transporter protein expression

Total protein from placental homogenate (1μg), MVM and BM (0.5μg) were used to determine total protein expression and phosphorylation of cellular signaling proteins by automated capillary-based immunoassay (Protein Simple, San Jose, CA, catalog #SM-W004–1, #PS-ST01, #PN-009–050). Proteins were separated and probed using primary antibodies as listed and vinculin was used as a protein loading control to calculate relative abundance (Supplementary Table 1 and Supplementary Material 1). SNAT1, SNAT2 and SNAT4 antibodies were validated as described previously (Gu, et al. 2001; Jones, et al. 2010; Ling, et al. 2001; Rosario, et al. 2013). Secondary anti-rabbit HRP antibody conjugate (Protein Simple, San Jose, CA, USA, DM-001) was used to target the primary antibody. Densitometry analysis was performed by Compass for SW software (38).

Additional protein expression analysis was performed using traditional Western blot protocol. Briefly, proteins were separated by SDS-PAGE electrophoresis using Mini-Protean TGX precast gels (Bio-Rad, Hercules, CA), 10μg of total protein crude homogenate, MVM and BM were loaded and separated on Bis-Tris gels. An equalizer sample was used to correct for variation between gels. Proteins were transferred onto PVDF membrane (0.2 μm, BIO-RAD) overnight at 4°C. After transfer, all membranes were stained with Amido Black (Sigma-Aldrich) to control for differences in total protein loading and transfer. Blocking was carried out in 5% (w/v) bovine serum albumin fraction V (Fisher Scientific) or 5% (w/v) non-fat milk in Tris-buffered saline (TBS) plus 0.1 % Tween 20 (TBS-T) for 1 h at room temperature. Subsequently, membranes were incubated in primary antibody (Supplementary Table 1 and Supplementary Material 1) immunolabeling was visualized using enhanced chemiluminescence detection reagents (Thermo Fisher Scientific, Rockford, IL). Densitometry analysis was performed using G:BOX Chemi XT4 gel imaging system and GeneTools analysis software (Syngene, Cambridge, United Kingdom). For comparison purposes the average expression of placentas from NGT was arbitrarily assigned a value of 1.0.

2.5. Data presentation and statistical analysis

Data are presented as mean ± S.E.M. Normoglycemic controls and T2DM were compared using unpaired Student’s t test. Data largely complied with homoscedastic or normality assumptions and when data did not follow normality, groups were compared using Mann-Whitney U-test. Relationships between placental nutrient transporter expression and percentage neonatal fat mass were determined using Pearson’s or Spearman correlation coefficient. A P value <0.05 and α=0.05 was considered statistically significant. Because of the number of statistical tests performed in this study (less than 100) a small number of the more than 20 statistically significant findings may be due to chance alone.

3. RESULTS

3.1. Clinical characteristics of study participants by group

Demographic data for study participants by group is provided in Table 1. By design, BMI was not different among NGT and T2DM participants and both cohorts started pregnancy on average with an overweight BMI. There were no significant differences in maternal age, newborn sex, birth weight, birth length and placental weight. Placental efficiency, used as a parameter to estimate placental function and is defined as birth weight/placental weight ratio. In our study, placental efficiency was not different among studied groups. Although the frequency of C-section was not different among studied groups, placental mTOR signaling differences observed in this study are not influenced by the presence or absence of labor (Lager, et al. 2014). Gestational age was lower in the T2DM group, likely due to concerns about the risk of stillbirth at term in women with T2DM. One late-preterm delivery and one case of pre-eclampsia were included in this analysis. Gestational-age-adjusted birth weight and length z-scores were not different between groups. Insulin was given in all women in the T2DM group.

3.2. Effect of T2DM on placental insulin/ Akt /mTOR signaling

We determined protein expression and phosphorylation of the insulin and mTOR signaling pathway in placentas from NGT and T2DM pregnancies. MVM IR-β protein expression was increased in the T2DM group (+30%, p=0.02) when compared with NGT (Fig. 1A). Activity of Akt, determined by the phosphorylation of Akt (T308) / total Akt ratio, was decreased (−50%, p=0.01) in T2DM (Fig. 1C), but phosphorylation of Akt (S473)/total Akt ratio (Fig. 1B) was not different. The mTOR downstream readouts: phosphorylated rpS6 (S235/236)/ total rpS6 ratio was increased in T2DM (+48%, p=0.05), but the phosphorylated 4E-BP1(T37/46) / total 4E-BP1 ratio in T2DM group was not different from NGT controls (Fig. 1D, E). Total abundance of Akt, 4E-BP1 and rpS6 did not differ between groups (Supplementary Figure 1).

Figure 1.

Figure 1.

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Effect of T2DM on placental insulin/ Akt /mTOR signaling. Scatter plot represents protein expression as fold change of (A) IRβ protein expression in the maternal-facing microvillous plasma membrane; (B) Akt(S473)/Akt ratio; (C) Akt(T308)/Akt ratio; (D) 4E-BP1(T37/46)/4E-BP1 ratio and (E) rpS6(S235/236)/rpS6 ratio in placental homogenate, measured by Western blot. Vinculin protein expression or amido black staining was used as loading control. Representative Western Blot images are shown. Data are presented as mean ± S.E.M. Unpaired Student’s T used for (A), (B), (D) and (E); Mann-Whitney test for (C); n=9. NGT, Normoglycemic control; T2DM, Type 2 Diabetes Mellitus.

3.3. Effect of T2DM on glucose, fatty acid and amino acid transporters expression

MVM GLUT1 expression was similar in the two groups (Fig. 2A). GLUT1 expression in the BM was increased in placentas from T2DM pregnancies (+44%, p-0.001) when compared to placentas from NGT women (Fig. 2B). In contrast, BM GLUT4 expression was decreased in T2DM group (−36%, p=0.006) (Fig. 2C).

Figure 2.

Figure 2.

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Effect of T2DM on glucose transporters expression in syncytiotrophoblast plasma membranes. Scatter plot represents protein expression as fold change of (A) GLUT1 in syncytiotrophoblast maternal-facing microvillous membrane (MVM); (B) GLUT1 in fetal-facing basal plasma membrane (BM); (C) GLUT4 protein expression in BM, measured by Western blot. Representative images are shown. Amido black staining was used as loading control. Data are presented as mean ± S.E.M. Mann-Whitney test for (A) and Unpaired Student’s T for (B) and (C) or; n=9. NGT, Normoglycemic control; T2DM, Type 2 Diabetes Mellitus.

Placental expression of fatty acid transporters FATP2, FATP4 and FAT/CD36 in MVM or BM were similar in the two groups (Fig. 3 A–D, G, H). FATP6 expression in T2DM group was increased in MVM (+87%, p=0.02) and in BM (+92%, p=0.01) when compared with BMI-matched NGT control placentas (Fig. 3 E, F).

Figure 3.

Figure 3.

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Effect of T2DM on fatty acids transporter expression in syncytiotrophoblast plasma membranes. Scatter plot represents protein expression as fold change of FATP2 in MVM (A) and (B) in BM; FATP4 in MVM (C) and (D) in BM; FATP6 in MVM (E) and (F) in BM; FAT/CD36 in MVM (G) and (H) in BM, measured by Western blot. Amido black staining was used as loading control. Representative images are shown. Data are presented as mean ± S.E.M. Unpaired Student’s T; n=8–9. NGT, Normoglycemic control; T2DM, Type 2 Diabetes Mellitus.

System A amino acid transporter SNAT1 and SNAT2 expression in the MVM was increased in T2DM (+26%, p=0.05, +47%, p=0.01, respectively) when compared with placentas from NGT control women (Fig. 4 A, B). SNAT4 expression in MVM was similar in the two groups (Fig. 4C). MVM System A amino acid transporter activity was not different between NGT and T2DM women (Fig. 4D). System L amino acid transporter LAT1 expression in MVM was also increased in T2DM (+27%, p=0.01), but not in BM (Fig. 4E, F). Similarly, MVM, but not BM, LAT2 was increased in T2DM (+59%, p=0.01, Fig. 4G, H).

Figure 4.

Figure 4.

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Effect of T2DM on amino acid transporter expression in syncytiotrophoblast plasma membranes. Scatter plot represents protein expression as fold change of SNAT1 (A), SNAT2 (B) and SNAT3 (C) in MVM; LAT1 (E) and LAT2 (F) in MVM; LAT1 (G) and LAT2 (H) in BM; measured by Western blot. Representative images are shown. Vinculin protein expression was used as loading control System A activity in microvillous plasma membrane (MVM) (D). Data are presented as mean ± S.E.M. Unpaired Student’s T used in (B), (E), (F) and (G) or Mann-Whitney test used in (A), (C) and (H); n=9. NGT, Normoglycemic control; T2DM, Type 2 Diabetes Mellitus.

3.4. Correlation between placental nutrient transporter expression and birth weight or newborn fat mass

Correlations between nutrient transporter expression in syncytiotrophoblast plasma membranes and birth weight are shown for both groups separately in Table 2.

Table 2.

Correlation coefficient between placental nutrient transporter expression in syncytiotrophoblast plasma membranes with birth weight.

MVM BM
Nutrient Transporter NGT T2DM NGT T2DM
Glucose GLUT1 −0.36 −0.08 0.10 −0.07
GLUT4 0.37 −0.23
Fatty Acids FATP2 −0.52 0.05 −0.77* 0.79*
FATP4 0.39 0.05 0.17 −0.48
FATP6 −0.14 0.65* −0.50 −0.03
FAT/CD36 −0.29 −0.23 −0.26 −0.01
Amino Acids SNAT1 0.29 0.84**
SNAT2 −0.16 0.53
SNAT4 −0.72* −0.71*
LAT1 0.10 0.59* 0.15 −0.06
LAT2 −0.02 −0.03 −0.53 −0.41
Amino Acids transport activity System A 0.06 0.15
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Pearson’s or Spearman correlation coefficient (r). N=9 per group. NGT, Normoglycemic control; T2DM, Type 2 Diabetes Mellitus.

*

Statistical significance at the level of P<0.05

**

Statistical significance at the level of P<0.005.

Associations between GLUT1, FATP6 and LAT1 transporter expression with neonatal fat mass in a subset of infants born to T2DM mothers are shown in Fig. 5. Placental glucose transporters of NGT or T2DM were not correlated with birth weight. In contrast, placental MVM GLUT1 expression in T2DM women was negatively correlated with neonatal fat mass (r= −0.90, p=0.04; Fig. 5A).

Figure 5.

Figure 5.

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The relationship between nutrient transporters in the syncytiotrophoblast plasma membranes and neonatal fat mass born to T2DM women. Correlation between (A) GLUT1, (B) FATP6 (n=4) and (C) LAT1 protein expression and percentage of neonatal fat mass born to T2DM women. n=5, r= Pearson’s correlation coefficient. Arbitrary units, AU; Microvillous plasma membrane, MVM; basal plasma membrane, BM.

BM FATP2 expression was negatively correlated with birth weight in NGT controls (r= −0.77, p=0.01) and positively correlated with birth weight in T2DM (r=0.79, p=0.01). We found no association between MVM/BM FATP2 expression and neonatal fat mass (not shown). Placental FATP4 and FAT/CD36 expression were not correlated with birth weight nor with neonatal fat mass. MVM FATP6 expression in T2DM women was positively correlated with birth weight (r=0.65, p=0.02). BM FATP6 expression was also increased in placentas from T2DM women but was not correlated with birth weight. Although it did not reach statistical significance, FATP6 expression in both MVM and BM was positively correlated with neonatal fat mass (Fig. 5B, MVM r=0.74, p=0.12; BM r=0.66, p=0.11).

Placental SNAT1 expression in MVM of T2DM women was positively correlated with birth weight (r=0.84, p=0.004), but not with neonatal fat mass. SNAT2 expression in MVM was not correlated with birth weight in any group or fat mass in neonates born to T2DM women. In contrast, SNAT4 expression in MVM was negatively correlated with birth weight in NGT (r= −0.72, p=0.02) and in T2DM (r= −0.71, p=0.01). SNAT4 expression was not associated with neonatal adiposity. MVM LAT1 expression in the T2DM group was increased, correlated with birth weight (r=0.59, p=0.04) and neonatal fat mass (Figure 5 C, r=0.76, p=0.06). LAT2 expression in MVM or BM was not associated with birth weight nor neonatal fat mass. We found similar correlations when gestational age-adjusted birth weight (z-scores) were performed.

4. DISCUSSION

Using a comprehensive approach to study glucose, fatty acid and amino acid transporters in the syncytiotrophoblast plasma membranes isolated from T2DM compared to euglycemic pregnancies, we demonstrated increased protein expression of FATP6, SNAT1, SNAT2, LAT1 and LAT2 transporters in MVM and GLUT1 and FAPT6 in BM in pregnancies complicated with T2DM. Exploring their role in fetal growth, we found that MVM FATP6, SNAT1 and LAT1 protein expression and BM FATP2 expression in T2DM women are positively associated with birth weight. Additionally, MVM GLUT1 expression is negatively correlated with adiposity in a small cohort of neonates born to T2DM women.

Human pregnancy is characterized by metabolic adaptations to support fetal growth, which involves increased maternal insulin resistance and enhanced pancreatic insulin release. In T2DM, the metabolic adaptations of pregnancy are superimposed on the pre-existing insufficiency of β-cell insulin secretion, frequently on the background of insulin resistance and obesity. Commonly pregnancies complicated by T2DM are characterized by maternal hyperglycemia, hyperlipidemia and hyperinsulinemia if beta cell insulin production can compensate for the increasing peripheral insulin resistance or when women are given exogenous insulin (Sugrue and Zera 2018).

Insulin is proven safe in pregnancy, it does not cross the placenta, and oral agents have an extremely high failure rate in women with T2DM. Thus, insulin is the only agent approved for treatment of pregnant women with pre-gestational diabetes. Insulin therapy has beneficial effects such as lowering the incidence of macrosomia, and caesarean deliveries (Lopez-Tinoco, et al. 2019). In our study, the women had mild T2DM based on their A1C, insulin was used in all women with T2DM, and large for gestational age outcome was only observed in one case. While insulin is effective to control maternal glycemia, it also suppresses maternal lipolysis, reducing maternal triglycerides which can be hydrolyzed to free fatty acids by the placenta (Barbour and Hernandez 2018b). We have previously demonstrated that trophoblast cells and villous explants isolated from women with GDM are insulin sensitive to physiological concentrations of insulin determined by the activation of the Akt pathway and amino acid transport activity. Moreover, insulin stimulates placental mTOR activity resulting in translocation of nutrient transporters, such as System A to the plasma membrane to increase amino acid transfer capacity (Rosario, et al. 2016; Rosario et al. 2013). As we previously demonstrated in pregnancies complicated by GDM (Castillo-Castrejon, et al. 2019), in some pregnant women with T2DM where insulin is standard of care for glycemic control, exogenous insulin likely activates the insulin-mTOR-AKT pathway which increases amino acid transporters in MVM and may contribute to fetal overgrowth and neonatal adiposity.

Glucose is the macronutrient that crosses the placenta in greatest quantities (Illsley and Baumann 2020) and is the principal oxidative substrate used for energy by the fetus. Under normal conditions, fetal endogenous glucose production is absent and placental glucose delivery from the mother is required meet fetal glucose requirements (Fowden, et al. 1998). GLUT1 is the major glucose transporter in the apical, maternal facing MVM membrane of the human placenta and is not insulin sensitive. Higher maternal glucose will lead to greater placental uptake because these transporters operate by facilitated diffusion. Factors determining net glucose transfer include the maternal-fetal concentration gradient and the transport capacity of the exchange area determined by the density of GLUT1 transporters and membrane surface area. GLUT1 expression in the BM has been suggested to be the rate limiting step in transplacental transfer of glucose. Interestingly, the expression and activity of GLUT1 in BM, is increased in placentas of pregnancies complicated by obesity T1DM or GDM (Jansson, et al. 1999) (Borges, et al. 2019; Diaz, et al. 2017). Moreover, GLUT1 protein expression was significantly increased in placentas from glyburide treated GDM pregnancies in both MVM and BM (Diaz et al. 2017; Stanirowski, et al. 2017). Our finding that GLUT1 is increased in the BM in pregnancies complicated by T2DM is consistent with these previous observations (Acosta, et al. 2015) and may contribute to increased glucose flux to the fetus. Fetal glucose in excess of what is needed for energy production can be used for de-novo lipogenesis and fetal fat accretion (Herrera and Desoye 2016; Kim, et al. 2014). Studies in humans have demonstrated that the offspring of women with GDM or obesity have increased subcutaneous fat at birth and hepatic fat storage at 1–3 weeks of life (Brumbaugh, et al. 2013). In our study, MVM GLUT1 expression in T2DM pregnancies was negatively correlated with adiposity in a subset of neonates with body composition measurements at 2 weeks of age, suggesting that glucose uptake capacity from the mother is not contributing to fetal fat accretion in these women.

We have recently demonstrated that the human placenta expresses the insulin-sensitive isoform GLUT4 transporter exclusively in the BM and the expression increases throughout gestation. GLUT4 expression in BM is decreased in obese women delivering large babies (James-Allan et al. 2019). In agreement, we found a decrease of GLUT4 in BM of placentas from T2DM women. The localization of this transporter and the fact that it is insulin sensitive suggest that insulin may be regulating the expression of this transporter in the BM and delivery of glucose increases when maternal insulin is elevated post-prandially. The samples for this study were collected at delivery and whether the fasting state of the mother at the time of delivery influences these results is unclear.

The placenta expresses insulin receptor isoform beta (IRβ) across gestation predominantly in the maternal facing MVM, suggesting signaling is primarily by maternal rather than fetal insulin (James-Allan et al. 2019). The results from the current study show that MVM IRβ expression is increased in T2DM pregnancies. Interestingly, despite increased MVM IRβ protein placental Akt phosphorylation was decreased in placentas from T2DM women. This data suggest that T2DM may be associated with placental insulin resistance in contrast to GDM (Castillo-Castrejon et al. 2019). However, this needs to be confirmed in additional studies directly assessing placental insulin responsiveness in T2DM. Insulin sensitivity can be experimentally tested in cultured primary human trophoblast cells isolated from T2DM pregnancies and healthy controls. After syncytialization treating with increasing concentrations of insulin and measuring amino acid transport capacity, Akt and Erk activity as functional readouts will demonstrate changes in placental insulin sensitivity between groups. Insulin receptors isoform α and β are expressed in the foetoplacental endothelium in normal and gestational diabetes pregnancies (Subiabre, et al. 2020). Changes in IR abundance in the fetoplacental endothelium of T2DM pregnancies and their association with adverse pregnancy outcomes deserves further investigation.

The mTOR signaling pathway is a key regulator of placental nutrient transport, in particular amino acid transport (Roos, et al. 2009a; Roos, et al. 2009b). As fetal growth is strongly dependent on nutrient supply, changes in the expression and activity of placental nutrient transporters may directly contribute to altered fetal growth (Gupta and Jansson 2019; Rosario et al. 2013). We found a significantly greater phosphorylation of rpS6, a readout of mTOR signaling, indicating increased activity of the mTOR pathway in placentas from women with T2DM. Trophoblast mTOR signaling responds to an array of diverse nutritional and metabolic signals. For example, trophoblast mTOR is activated by insulin/IGF-1, glucose and amino acids (Roos et al. 2009b), fatty acids (Lager, et al. 2013; Rosario, et al. 2017a; Rosario, et al. 2017b) and inhibited by adiponectin (Aye, et al. 2015; Rosario, et al. 2012). The factors activating placenta mTOR signaling in T2DM remain to be established, however is unlikely to involve insulin/IGF-1 signaling given the observed decrease in Akt phosphorylation.

Maternal TG and/or FA levels may be more strongly correlated to excess fetal growth than for example maternal glucose concentrations (Barbour and Hernandez 2018a; Barbour et al. 2018; Vrijkotte, et al. 2011; Whyte, et al. 2013). Placental FATP4 mRNA levels have been reported to be decreased in pregnancies complicated by maternal overweight, obesity and GDM (Segura, et al. 2017). In contrast, BM FATP2 protein expression is increased in maternal obesity (Lager, et al. 2016). In our study there was no difference in the expression of FATP2 and FATP4 in either syncytiotrophoblast plasma membranes isolated from placentas of women with T2DM. In agreement with studies reporting increased placental mRNA expression of FATP6 and FAT/CD36 in GDM (Segura et al. 2017), we observed that increased FATP6 expression in MVM and BM. Moreover, MVM FATP6 protein expression was positively correlated with birth weight. Although it did not reach statistical significance, FATP6 was positively correlated with neonate fat mass, a better predictor of childhood obesity (Catalano, et al. 2009). In addition to glucose, maternal concentration of lipids specifically triglycerides and free fatty acids appear to be strong contributors to excess fetal fat accretion and adiposity at birth, in obese pregnancies (Barbour and Hernandez 2018b) (Heerwagen, et al. 2018) and in GDM (Herrera and Ortega-Senovilla 2018; Schaefer-Graf, et al. 2008). We propose that maternal dyslipidemia in T2DM and increased placental lipid transfer capacity enhances the transfer of lipids to the fetus, contributing in part to increased fetal adiposity and consequent risk of developing macrosomia.

MVM System A amino acid transporter expression and/or activity is increased in fetal overgrowth associated with maternal obesity, GDM and T2DM (Jansson, et al. 2013; Jansson, et al. 2002). In general agreement with these findings we report that the protein expression System A and System L amino acid transporter isoforms in the MVM are significantly increased in placentas from T2DM women. In addition, SNAT1 and LAT1 were also positively correlated with birth weight only in the T2DM group. Interestingly, and in spite of the small sample size, LAT1 was also positively correlated with neonatal fat mass. mTOR signaling is a positive regulator of placental amino acid transport (Roos et al. 2009a; Roos et al. 2009b) and it is possible that placental mTOR activation in T2DM, as evidenced by increased rp6 phosphorylation, is responsible for the observed increased expression of amino acid transporter isoforms without System A activity being unchanged.

One limitation of our study is the small sample size. In particular, the correlations between placental measurements and neonatal adiposity need to be confirmed in studies with larger sample sizes. Furthermore, our women with T2DM had mild diabetes and were not likely to have significant underlying vascular disease, given only one developed preeclampsia and delivered before term. Women with more advanced T2DM may have hypertension, renal disease, sleep apnea, or occult cardiovascular disease that could actually increase the risk of abnormal placentation and placental insufficiency. Changes in the placental morphology have been described in GDM, Type 1 diabetes (T1DM) and T2DM. Histological changes include increased volume of intervillous space, increased volume of the intermediate and terminal villi, syncytiotrophoblast number and syncytial knots, increased fibrinoid, glycogen deposits and collagen fibers, parenchymal infarcts, decidual vasculopathy, thrombosis of fetal vessels and chorangiosis, chorangioma, chorangiomatosis; which may result in functional changes thus limiting nutrient availability (Carrasco-Wong, et al. 2020). In general, macro and microscopic findings in placentas from diabetic pregnancies vary depending on the pathogenic mechanisms associated to T1DM and T2DM (Starikov, et al. 2014). Therefore, it is critical to characterize women with T2DM when studying fetal growth outcomes and placental modifications, especially with respect to the presence or absence of vascular disease.

5. CONCLUSION

The objective of this study was to provide a comprehensive approach of the polarized expression of placental nutrient transporters in the maternal-facing membrane (MVM) and fetal-facing membrane (BM) to provide a deeper understanding of the causes of increased birth weight and neonatal adiposity in T2DM. In conclusion, we propose that placental activation of mTOR signaling and increased protein expression of glucose, fatty acid and amino acids transporters contribute to enhanced delivery of macronutrients to the fetus in pregnant women with T2DM providing a mechanism to for accelerated fetal growth and increased neonatal adiposity in this pregnancy complication (Fig. 6).

Figure 6.

Figure 6.

Open in a new tab

Integrative model of the impact of Type 2 Diabetes on placental expression of nutrient transporters and their association with fetal growth and neonatal adiposity. Arrow show increased or decreased protein expression. Akt, protein kinase B; eIF4E, eukaryotic translation initiation factor 4E; FATP6, fatty acid transport protein-6 and −2; FAT/CD36, fatty acid translocase/CD36; GLUT1 and GLUT4, glucose transporter 1 and 4; IRβ, insulin receptor beta; IRS, insulin receptor substrate; LAT-1 and −2, System L amino acid transporter 1 and 2; mTORC1, mechanistic target of rapamycin complex 1; PI3K, phosphoinositide 3 kinase; rpS6, ribosomal protein S6; SNAT1, SNAT2 and SNAT4, sodium-coupled neutral amino acid transporter 1, 2 and 4; S6K1, ribosomal protein S6 kinase β1; 4E-BP1, eukaryotic translation initiation factor binding protein 1.

Supplementary Material

1

Highlights.

  • Type 2 diabetes and obesity are associated with fetal overgrowth and fetal adiposity

  • Placental nutrient transporter protein expression is increased in type 2 diabetes

  • Fatty and amino acid transporters correlated with birth weight and neonatal adiposity

  • Interventions must go beyond glucocentric strategies to improve fetal outcomes

Acknowledgements.

This work was supported by the NIH/NCRR Colorado CTSI Grant Number UL1 RR025780, National Institutes of Health Grant HD068370. Its contents are the authors’ sole responsibility and do not necessarily represent official view of the National Institutes of Health. Authors acknowledge the contributions of Lana Madi for technical support.

Abbreviations

Akt

protein kinase B

BMI

body mass index

FATPs

Fatty acid transport proteins

GDM

gestational diabetes mellitus

GLUTs

glucose transporters

T2DM

type 2 diabetes mellitus

IRβ

insulin receptor beta

mTOR

mechanistic target of rapamycin

OGTT

oral glucose tolerance test

SNATs

sodium-coupled neutral amino acid transporter

Footnotes

Duality of interest. The authors declare that there is no duality of interest associated with this manuscript.

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