Amino Acid Transporter Isoforms Research Articles

Maternal, placental, and fetal Insulin-Like Growth Factor-I (IGF-1) and IGF Binding proteins (IGFBPs) in Diabetic pregnancies: Effects on fetal growth and birth size.

Introduction: During gestation, IGF1 secretion and availability in the maternal blood and at the maternal-fetal interface is mainly regulated by IGF-binding proteins (IGFBP) such as IGFBP-1 synthesized by the decidua. Data about the interaction between maternal, placental, and fetal IGF1/IGFBP in relation to fetal growth and newborn size during diabetic pregnancy (gestational Diabetes (GDM) and Type 1 DM (T1DM) is not clear. Aim of the study and Methods: We reviewed the research papers published in Pubmed, Google scholar, Research gate, and Scopus in the past 20 years on the relationship between maternal, placental, and fetal/infantile/ IGF1/IGFBP-1 in relation to birth size in pregnancies associated with maternal diabetes. Results: Twenty-eight research papers were selected and reviewed (patients’ number = 1902). In GDM pregnancies, higher maternal IGF1 levels and/or its availability due to lower IGFBP1 levels can increase the size (weight) and functions of the placenta. These include the upregulation of specific placental amino acid transporter isoforms and GLUT-1, stimulation of mTOR signaling which stimulates protein synthesis, increasing mitochondrial functions, and accelerating nutrient transport which significantly contributes to fetal growth and newborn birth size. On the other hand, in pregnant women with T1DM, lower maternal IGF1 is associated with subsequent underweight placenta and lower birthweight.

Insulin like growth factor 1 (IGF-1) and IGF binding proteins in obese pregnant women and their babies: Potential effects on placental function and fetal growth

Introduction: The placenta expresses significant amounts of insulin and IGF1 receptors at distinct locations on both fetal and maternal surfaces. This makes the IGF1 and the insulin receptor accessible to fetal and/or maternal insulin, IGF1 and IGF2. IGFs are involved in the receptor-mediated regulation of placental growth and transport, and placental angiogenesis. Maternal obesity during gestation mediates significant changes in the metabolism of mothers, placentas as well as fetal growth. Objectives: In obese women. the role of the insulin like growth factor system IGFs, IGF receptors, IGF-binding proteins (IGFBPs) and IGFBP proteases during gestation, and their effect on placental growth and fetal anthropometric changes need further clarification. In this update we reviewed the literature on the detected changes in the maternal and fetal IGFs in relation to placental growth and function and to fetal growth and newborn size in pregnant obese mothers. Eighteen research articles fitted the criteria of this update. Results: Twenty-three research papers were including 2817 pregnant obese and non-obese women (controls) and their babies were selected and reviewed. Results showed that obesity and excessive nutrient intake during gestation increase maternal IGF1and decreases IGBP1. Increased maternal IGF1 and/or its availability due to decreased IGFBP1 can increase the size (weight) and development of the placenta, stimulate mTOR signaling which stimulates protein synthesis, mitochondrial function, and upregulate specific placental amino acid transporter isoforms (amino acids transport), GLUT-1, (glucose transport) and possibly lipid transport to the fetus which can induce fetal IGF1 secretion and lead to overgrowth. Conclusions: In obese women during pregnancy, increased level of IGF1 and/or its availability due to decreased IGFBP1 can increase the size (weight) and development of the placenta, stimulate mTOR signaling which stimulates protein synthesis, mitochondrial function, and upregulate placental transport of amino acids, glucose and possibly fatty acids.

Down-regulation of placental Cdc42 and Rac1 links mTORC2 inhibition to decreased trophoblast amino acid transport in human intrauterine growth restriction.

Intrauterine growth restriction (IUGR) increases the risk for perinatal complications and metabolic and cardiovascular disease later in life. The syncytiotrophoblast (ST) is the transporting epithelium of the human placenta, and decreased expression of amino acid transporter isoforms in the ST plasma membranes is believed to contribute to IUGR. Placental mechanistic target of rapamycin Complex 2 (mTORC2) signaling is inhibited in IUGR and regulates the trafficking of key amino acid transporter (AAT) isoforms to the ST plasma membrane; however, the molecular mechanisms are unknown. Cdc42 and Rac1 are Rho-GTPases that regulate actin-binding proteins, thereby modulating the structure and dynamics of the actin cytoskeleton. We hypothesized that inhibition of mTORC2 decreases AAT expression in the plasma membrane and amino acid uptake in primary human trophoblast (PHT) cells mediated by down-regulation of Cdc42 and Rac1. mTORC2, but not mTORC1, inhibition decreased the Cdc42 and Rac1 expression. Silencing of Cdc42 and Rac1 inhibited the activity of the System L and A transporters and markedly decreased the trafficking of LAT1 (System L isoform) and SNAT2 (System A isoform) to the plasma membrane. mTORC2 inhibition by silencing of rictor failed to decrease AAT following activation of Cdc42/Rac1. Placental Cdc42 and Rac1 protein expression was down-regulated in human IUGR and was positively correlated with placental mTORC2 signaling. In conclusion, mTORC2 regulates AAT trafficking in PHT cells by modulating Cdc42 and Rac1. Placental mTORC2 inhibition in human IUGR may contribute to decreased placental amino acid transfer and reduced fetal growth mediated by down-regulation of Cdc42 and Rac1.

1,25-Dihydroxy vitamin D3 stimulates system A amino acid transport in primary human trophoblast cells

Vitamin D deficiency during pregnancy is linked to adverse perinatal outcomes such as small for gestational age infants. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. We tested the hypothesis that 1,25-dihydroxy vitamin D3 increases the gene expression of System A and L amino acid transporter isoforms and stimulates placental amino acid transport activity in cultured primary human trophoblast cells mediated by mTOR signaling. Treatment with 1,25-dihydroxy vitamin D3 significantly increased mRNA expression of the System A isoform SNAT2 and System A activity, but had no effect on System L and did not affect mTOR signaling. siRNA silencing of the vitamin D receptor prevented 1,25-dihydroxy vitamin D3-stimulated System A transport. In conclusion, 1,25-dihydroxy vitamin D3 regulates System A activity through increased mRNA expression of SNAT2 transporters. Effects on placental amino acid transport may be the mechanism underlying the association between maternal vitamin D status and fetal growth.

Down-Regulation of Placental Transport of Amino Acids Precedes the Development of Intrauterine Growth Restriction in Maternal Nutrient Restricted Baboons

Intrauterine growth restriction (IUGR) is an important risk factor for perinatal complications and adult disease. IUGR is associated with down-regulation of placental amino acid transporter expression and activity at birth. It is unknown whether these changes are a cause or a consequence of human IUGR. We hypothesized that placental amino acid transport capacity is reduced prior to onset of reduced fetal growth in baboons with maternal nutrient restriction (MNR). Pregnant baboons were fed either a control (n = 8) or MNR diet (70% of control diet, n = 9) from Gestational Day 30. At Gestational Day 120 (0.65 of gestation), fetuses and placentas were collected. Microvillous (MVM) and basal (BM) plasma membrane vesicles were isolated. System A and system L transport activity was determined in MVM, and leucine transporter activity was assessed in BM using radiolabeled substrates. MVM amino acid transporter isoform expression (SNAT1, SNAT2, and SNAT4 and LAT1 and LAT2) was measured using Western blots. LAT1 and LAT2 expression were also determined in BM. Maternal and fetal plasma amino acids concentrations were determined using mass spectrometry. Fetal and placental weights were unaffected by MNR. MVM system A activity was decreased by 37% in MNR baboon placentas (P = 0.03); however MVM system A amino acid transporter protein expression was unchanged. MVM system L activity and BM leucine transporter activity were not altered by MNR. Fetal plasma concentrations of essential amino acids isoleucine and leucine were reduced, while citrulline increased (P < 0.05) in MNR fetuses compared to controls. In this primate model of IUGR, placental MVM system A amino acid transporter activity is decreased prior to the onset of reduction in the fetal growth trajectory. The reduction in plasma leucine and isoleucine in MNR fetuses may be caused by reduced activity of MVM system A, which is strongly coupled with system L essential amino acid uptake. Our findings indicate that reduced placental amino acid transport may be a cause rather than a consequence of IUGR due to inadequate maternal nutrition.

Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.

Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear L-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The L-glutamate (2mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4.

Upregulation of excitatory amino acid transporters by coexpression of Janus kinase 3.

Janus kinase 3 (JAK3) contributes to cytokine receptor signaling, confers cell survival and stimulates cell proliferation. The gain of function mutation JAK3(A572V) is found in acute megakaryoplastic leukemia. Replacement of ATP coordinating lysine by alanine yields inactive JAK3(K855A). Most recent observations revealed the capacity of JAK3 to regulate ion transport. This study thus explored whether JAK3 regulates glutamate transporters EAAT1-4, carriers accomplishing transport of glutamate and aspartate in a variety of cells including intestinal cells, renal cells, glial cells, and neurons. To this end, EAAT1, 2, 3, or 4 were expressed in Xenopus oocytes with or without additional expression of mouse wild-type JAK3, constitutively active JAK3(A568V) or inactive JAK3(K851A), and electrogenic glutamate transport was determined by dual electrode voltage clamp. Moreover, Ussing chamber was employed to determine electrogenic glutamate transport in intestine from mice lacking functional JAK3 (jak3(-/-)) and from corresponding wild-type mice (jak3(+/+)). As a result, in EAAT1, 2, 3, or 4 expressing oocytes, but not in oocytes injected with water, addition of glutamate to extracellular bath generated an inward current (Ig), which was significantly increased following coexpression of JAK3. Ig in oocytes expressing EAAT3 was further increased by JAK3(A568V) but not by JAK3(K851A). Ig in EAAT3 + JAK3 expressing oocytes was significantly decreased by JAK3 inhibitor WHI-P154 (22 µM). Kinetic analysis revealed that JAK3 increased maximal Ig and significantly reduced the glutamate concentration required for half maximal Ig (Km). Intestinal electrogenic glutamate transport was significantly lower in jak3(-/-) than in jak3(+/+) mice. In conclusion, JAK3 is a powerful regulator of excitatory amino acid transporter isoforms.

Activation of Placental mTOR Signaling and Amino Acid Transporters in Obese Women Giving Birth to Large Babies

Babies of obese women are often large at birth, which is associated with perinatal complications and metabolic syndrome later in life. The mechanisms linking maternal obesity to fetal overgrowth are largely unknown. We tested the hypothesis that placental insulin/IGF-I and mammalian target of rapamycin (mTOR) signaling is activated and amino acid transporter activity is increased in large babies of obese women. Pregnant women were recruited prospectively for collection of placental tissue at a university hospital and academic biomedical center. Twenty-three Swedish pregnant women with first trimester body mass index ranging from 18.5 to 44.9 kg/m(2) and with uncomplicated pregnancies participated in the study. There were no interventions. We determined the phosphorylation of key signaling molecules (including Akt, IRS-1, S6K1, 4EBP-1, RPS6, and AMPK) in the placental insulin/IGF-I, AMPK, and mTOR signaling pathways. The activity and protein expression of the amino acid transporter systems A and L were measured in syncytiotrophoblast microvillous plasma membranes. Birth weights (range, 3025-4235 g) were positively correlated to maternal body mass index (P < 0.05). The activity of placental insulin/IGF-I and mTOR signaling was positively correlated (P < 0.001), whereas AMPK phosphorylation was inversely (P < 0.05) correlated to birth weight. Microvillous plasma membrane system A, but not system L, activity and protein expression of the system A isoform SNAT2 were positively correlated to birth weight (P < 0.001). Up-regulation of specific placental amino acid transporter isoforms may contribute to fetal overgrowth in maternal obesity. This effect may be mediated by activation of insulin/IGF-I and mTOR signaling pathways, which are positive regulators of placental amino acid transporters.

Chronic maternal infusion of full‐length adiponectin in pregnant mice down‐regulates placental amino acid transporter activity and expression and decreases fetal growth

Maternal adiponectin levels are inversely correlated to birth weight, suggesting that maternal adiponectin limits fetal growth. We hypothesized that full-length adiponectin (fADN) infusion in pregnant mice down-regulates placental amino acid transporters and decreases fetal growth. Starting at embryonic day (E) 14.5, fADN (0.62 ± 0.02 μg (g body weight)(−1) day(−1), n = 7) or vehicle (control, n = 9) were infused in pregnant C57/BL6 mice by mini-osmotic pump. At E18.5, dams were killed and placental homogenates and trophoblast plasma membrane (TPM) vesicles were prepared. Infusion of fADN elevated maternal serum fADN by 4-fold and decreased fetal weights by 18%. Adiponectin receptor 2, but not adiponectin receptor 1, was expressed in TPM. fADN infusion decreased TPM System A (–56%, P < 0.001) and System L amino acid transporter activity (–50%, P < 0.03). TPM protein expression of SNAT1, 2 and 4 (System A amino acid transporter isoforms) and LAT1 and LAT2, but not CD98, (System L amino acid transporter isoforms) was down-regulated by fADN infusion. To identify possible mechanisms underlying these changes we determined the phosphorylation of proteins in signalling pathways known to regulate placental amino acid transporters. fADN decreased phosphorylation of insulin receptor substrate-1 (Tyr-608), Akt (Thr-308 and Ser-473), S6 kinase 1 (Thr-389), eukaryotic initiation factor 4E binding protein 1 (Thr-37/46 and Thr-70) and ribosomal protein S6 (Ser-235/236) and increased the phosphorylation of peroxisome proliferator-activated receptor α (PPARα) (Ser-21) in the placenta. These data suggest that maternal adiponectin decreases fetal growth by down-regulation of placental amino acid transporters, which limits fetal nutrient availability. This effect may be mediated by inhibition of insulin/IGF-I and mTOR signalling pathways, which are positive regulators of placental amino acid transporters. We have identified a novel physiological mechanism by which the endocrine functions of maternal adipose tissue influence fetal growth.

Elevated ammonium levels: differential acute effects on three glutamate transporter isoforms

Increased ammonium (NH(4)(+)/NH(3)) in the brain is a significant factor in the pathophysiology of hepatic encephalopathy, which involves altered glutamatergic neurotransmission. In glial cell cultures and brain slices, glutamate uptake either decreases or increases following acute ammonium exposure but the factors responsible for the opposing effects are unknown. Excitatory amino acid transporter isoforms EAAT1, EAAT2, and EAAT3 were expressed in Xenopus oocytes to study effects of ammonium exposure on their individual function. Ammonium increased EAAT1- and EAAT3-mediated [(3)H]glutamate uptake and glutamate transport currents but had no effect on EAAT2. The maximal EAAT3-mediated glutamate transport current was increased but the apparent affinities for glutamate and Na(+) were unaltered. Ammonium did not affect EAAT3-mediated transient currents, indicating that EAAT3 surface expression was not enhanced. The ammonium-induced stimulation of EAAT3 increased with increasing extracellular pH, suggesting that the gaseous form NH(3) mediates the effect. An ammonium-induced intracellular alkalinization was excluded as the cause of the enhanced EAAT3 activity because 1) ammonium acidified the oocyte cytoplasm, 2) intracellular pH buffering with MOPS did not reduce the stimulation, and 3) ammonium enhanced pH-independent cysteine transport. Our data suggest that the ammonium-elicited uptake stimulation is not caused by intracellular alkalinization or changes in the concentrations of cotransported ions but may be due to a direct effect on EAAT1/EAAT3. We predict that EAAT isoform-specific effects of ammonium combined with cell-specific differences in EAAT isoform expression may explain the conflicting reports on ammonium-induced changes in glial glutamate uptake.