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Thomas Jansson - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Placental Amino Acid Transport and Fetal Growth.
Progress in molecular biology and translational science, 2017Co-Authors: Or Vaughan, Fredrick J. Rosario, Theresa L Powell, Thomas JanssonAbstract:The fetus requires Amino Acids for the processes of protein synthesis, carbon accretion, oxidative metabolism, and biosynthesis, which ultimately determine growth rate in utero. The fetal supply of Amino Acids is critically dependent on the Transport capacity of the placenta. System A Amino Acid Transporters in the syncytiotrophoblast microvillous plasma membrane, directed toward maternal blood, actively accumulate Amino Acids, while system L exchangers mediate uptake of essential Amino Acids from the maternal circulation. The functional capacity and protein abundance of these Transporters in the placenta are related to fetal growth in both humans and experimental animals. Maternal nutritional and endocrine signals including insulin, insulin-like growth factors, adipokines, and steroid hormones regulate placental Amino Acid Transport, against the background of growth signals originating from the fetus. Anabolic signals of abundant maternal resource availability stimulate placental Amino Acid Transport to optimize offspring fitness, whereas catabolic signals reduce placental Amino Acid Transport in an attempt to ensure survival and long-term reproductive capacity of the mother when resources are scarce. These signals regulate placental Amino Acid Transport by controlling transcription, translation, plasma membrane trafficking, and degradation of Transporters. Adaptations in placental Amino Acid Transport capacity may underlie either under- or overgrowth of the fetus when maternal nutrient and hormone levels are altered as a result of altered maternal nutrition or metabolic disease. Strategies to modulate placental Amino Acid Transport may prove effective to normalize fetal growth in intrauterine growth restriction and fetal overgrowth.
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differential regulation of placental Amino Acid Transport by saturated and unsaturated fatty Acids
American Journal of Physiology-cell Physiology, 2014Co-Authors: Susanne Lager, Thomas Jansson, Theresa L PowellAbstract:Fatty Acids are critical for normal fetal development but may also influence placental function. We have previously reported that oleic Acid (OA) stimulates Amino Acid Transport in primary human trophoblasts (PHTs). In other tissues, saturated and unsaturated fatty Acids have distinct effects on cellular signaling, for instance, palmitic Acid (PA) but not OA reduces IκBα expression. We hypothesized that saturated and unsaturated fatty Acids differentially affect trophoblast Amino Acid Transport and cellular signaling. To test this hypothesis, PHTs were cultured in docosahexaenoic Acid (DHA; 50 μM), OA (100 μM), or PA (100 μM). DHA and OA were also combined to test whether DHA could counteract the OA stimulatory effect on Amino Acid Transport. The effects of fatty Acids were compared against a vehicle control. Amino Acid Transport was measured by isotope-labeled tracers. Activation of inflammatory-related signaling pathways and the mechanistic target of rapamycin (mTOR) pathway were determined by Western blot analysis. Exposure of PHTs to DHA for 24 h reduced Amino Acid Transport and phosphorylation of p38 MAPK, STAT3, mTOR, eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein (rp)S6. In contrast, OA increased Amino Acid Transport and phosphorylation of ERK, mTOR, S6 kinase 1, and rpS6. The combination of DHA with OA increased Amino Acid Transport and rpS6 phosphorylation. PA did not affect Amino Acid Transport but reduced IκBα expression. In conclusion, these fatty Acids differentially regulated placental Amino Acid Transport and cellular signaling. Taken together, these findings suggest that dietary fatty Acids could alter the intrauterine environment by modifying placental function, thereby having long-lasting effects on the developing fetus.
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interleukin 1β inhibits insulin signaling and prevents insulin stimulated system a Amino Acid Transport in primary human trophoblasts
Molecular and Cellular Endocrinology, 2013Co-Authors: Irving L M H Aye, Thomas Jansson, Theresa L PowellAbstract:Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated Amino Acid Transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10pg/ml) for 24h. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L Amino Acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A Transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth.
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Mammalian target of rapamycin signalling modulates Amino Acid uptake by regulating Transporter cell surface abundance in primary human trophoblast cells
Journal of Physiology, 2013Co-Authors: Fredrick J. Rosario, Theresa L Powell, Yoshikatsu Kanai, Thomas JanssonAbstract:Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental Amino Acid Transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental Amino Acid Transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate Amino Acid Transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L Amino Acid Transport activity but had no effect on growth factor-stimulated Amino Acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated Amino Acid Transport activity. In contrast, mTOR inhibition had no effect on serotonin Transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) Transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast Amino Acid Transporters by modulating the cell surface abundance of specific Transporter isoforms. This is the first report showing regulation of Amino Acid Transport by mTORC2. Because placental mTOR activity and Amino Acid Transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.
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full length adiponectin attenuates insulin signaling and inhibits insulin stimulated Amino Acid Transport in human primary trophoblast cells
Diabetes, 2010Co-Authors: Helen Jones, Thomas Jansson, Theresa L PowellAbstract:OBJECTIVE Maternal adiponectin levels are reduced and placental nutrient Transporters are upregulated in obesity and gestational diabetes mellitus; however, the effects of adiponectin on placental function are unknown. We hypothesized that adiponectin regulates placental Amino Acid Transport. RESEARCH DESIGN AND METHODS Human primary trophoblast cells were cultured and incubated with globular adiponectin (gAd) or full-length adiponectin (fAd) alone or in combination with insulin. System A and L Amino Acid Transport and SNAT1, SNAT2, and SNAT4 isoform expression was measured. The activity of the AMP-activated protein kinase (AMPK), phosphatidylinositol 3 kinase–AKT, and peroxisome proliferator–activated receptor-α (PPARα) signaling pathways was determined. RESULTS In the absence of insulin, gAd stimulated AMPK Thr172 phosphorylation, SNAT2 protein expression, and system A activity. This effect appeared to be mediated by interleukin-6 release and signal transducer and activator of transcription 3 (STAT3) signaling because gAd failed to stimulate system A in cells in which STAT3 had been silenced using small interfering RNA. fAd alone had no effect on system A activity or SNAT expression. Insulin increased AKT and insulin receptor substrate 1 (IRS-1) phosphorylation, system A activity, and SNAT2 expression. When combined with insulin, gAd did not affect system A activity or SNAT expression. In contrast, fAd abolished insulin-stimulated AKT Thr308 and IRS-1 Tyr612 phosphorylation, system A activity, and SNAT2 expression. Furthermore, fAd increased PPARα expression and PPARα (Ser21) phosphorylation. CONCLUSIONS In contrast to the insulin-sensitizing actions of adiponectin in liver and muscle reported in the literature, fAd attenuates insulin signaling in primary human trophoblast cells. As a result, fAd inhibits insulin-stimulated Amino Acid Transport, which may have important implications for placental nutrient Transport and fetal growth in pregnancy complications associated with altered maternal adiponectin levels.
C A R Boyd - One of the best experts on this subject based on the ideXlab platform.
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differential effect of cross linking the cd98 heavy chain on fusion and Amino Acid Transport in the human placental trophoblast bewo cell line
Biochimica et Biophysica Acta, 2007Co-Authors: Paola Dalton, Helen C Christian, C W G Redman, I L Sargent, C A R BoydAbstract:CD98 (otherwise known as 4F2) is an integral membrane protein with multiple functions including Amino Acid Transport, integrin activation, cell fusion and cell activation. The molecular mechanisms coordinating these multiple functions remain unclear. We have studied CD98 heavy chain (hc) function in a human placental trophoblast cell line (BeWo). We show that cross-linking of CD98hc by incubation of cells in the presence of functional monoclonal antibodies causes cellular re-distribution of the protein from the cytoplasm to the plasma membrane as measured by flow cytometry, western blotting and quantitative immuno-electron microscopy. The latter technique also indicated that CD98hc is trafficked between cell surface and cytoplasmic pools in vesicles. Increased cell surface CD98 correlates with increased cellular fusion in BeWo cells. In addition, we show reduced LAT 1 surface expression and neutral Amino Acid Transport in the presence of the CD98 mabs. The results thus suggest that the function of CD98 in cell fusion is distinct from its role in cellular nutrient delivery.
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cationic Amino Acid Transport through system y l in erythrocytes of patients with lysinuric protein intolerance
Pflügers Archiv: European Journal of Physiology, 2000Co-Authors: C A R Boyd, Rosa Deves, Robert Laynes, Yoshiki Kudo, Gianfranco SebastioAbstract:We test the hypothesis that lysinuric protein intolerance (LPI), a rare autosomal recessive defect of cationic Amino Acid Transport, results from the absence of the recently described y+L Amino Acid Transporter. We compare fluxes of lysine (1 µM) into erythrocytes of normal subjects with those of patients homozygous for the LPI mutation. No significant differences in fluxes through system y+L in normal or LPI cells were found, excluding the possibility that system y+L cannot be expressed in patients with LPI. Reasons for supposing that there may be tissue-specific processing of two recently described genes encoding the y+L Transporter are discussed. Polymerase chain reaction measurement of expression of these two genes in an erythroleukemic cell line suggests that alternatively there may be an as-yet-unidentified additional member of this gene family.
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heterodimeric Amino Acid Transporters expression of heavy but not light chains of cd98 correlates with induction of Amino Acid Transport systems in human placental trophoblast
The Journal of Physiology, 2000Co-Authors: Yoshiki Kudo, C A R BoydAbstract:Activity of Amino Acid Transport and relative abundance of mRNAs encoding related Transporters have been studied in parallel either before or following in vitro culture of explants of human placental chorionic villi. Amino Acid Transport activities through systems L (1.9-fold), y+L (2.6-fold) and y+ (3.2-fold) were markedly enhanced following culture for 48 h. Relative mRNA abundance (determined by reverse transcription-polymerase chain reaction) for the heavy chain of CD98 surface antigen and for the cationic Amino Acid Transporter-1 were similarly stimulated (2.8-fold and 2.6-fold, respectively). In contrast, none of the mRNA levels for light chains of CD98 (system L-Amino Acid Transporter-1, system L-Amino Acid Transporter-2, system y+L-Amino Acid Transporter-1 and system y+L-Amino Acid Transporter-2) studied nor for the cationic Amino Acid Transporter-2B were altered. The molecular basis of Amino Acid Transport in vertebrate cells is currently a topic of much research (Palacin et al. 1998). Particularly striking are recent observations showing that some Transporters are monomeric whereas others (a rapidly expanding group) are heterodimeric (Palacin, 1994). These include Transporters responsible for system L (Broer et al. 1998; Kanai et al. 1998; Mannion et al. 1998; Mastroberardino et al. 1998; Pineda et al. 1999), for system y+L (Broer et al. 1998; Mastroberardino et al. 1998; Torrents et al. 1998; Pfeiffer et al. 1999) and for system XC− (Sato et al. 1999). Such Transporters are composed of a common heavy chain (heavy chain of CD98 surface antigen (CD98hc)) and one of a family of related light chains (system L-Amino Acid Transporter-1 (LAT-1), system L-Amino Acid Transporter-2 (LAT-2), system y+L-Amino Acid Transporter-1 (y+LAT-1), system y+L-Amino Acid Transporter-2 (y+LAT-2) and system XC−-Amino Acid Transporter (xCT)). Mastroberardino and colleagues (Mastroberardino et al. 1998) have proposed that in Xenopus oocytes it is expression of the heavy chain that is necessary for maturation, Transport and/or surface residence of heterologous expressed light chain subunits; additionally, in epithelia one role of the heavy chain may be to determine the polarity of Transporter expression (Pfeiffer et al. 1999; Pineda et al. 1999). In contrast other Transporters (e.g. system y+) are composed of only a single chain (for system y+, a member of the cationic Amino Acid Transporter (CAT) gene family). In the placenta it has been known since the work of Smith and colleagues (Smith et al. 1973) that induction of Transport activity follows in vitro culture. For one heterodimeric Transporter (system y+L), Fei and colleagues (Fei et al. 1995) showed that following anti-sense depletion of the heavy chain of CD98 from total placental mRNA there was no longer expression of this Transport function. This experiment importantly shows that the heavy chain is necessary for heterodimeric function; it does not, however, show whether under normal conditions it is expression of the heavy or of the light chain or of both that is altered, and that which therefore may be rate limiting. We have now looked at the pattern of Transporter mRNA expression following in vitro culture of this tissue in order to discover for two heterodimeric Transporters (systems L and y+L) how subunit mRNA abundance relates to Transport function in this human tissue.
Stefan Broer - One of the best experts on this subject based on the ideXlab platform.
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Amino Acid Transport across the mammalian intestine
Comprehensive Physiology, 2018Co-Authors: Stefan Broer, Stephen J FairweatherAbstract:The small intestine mediates the absorption of Amino Acids after ingestion of protein and sustains the supply of Amino Acids to all tissues. The small intestine is an important contributor to plasma Amino Acid homeostasis, while Amino Acid Transport in the large intestine is more relevant for bacterial metabolites and fluid secretion. A number of rare inherited disorders have contributed to the identification of Amino Acid Transporters in epithelial cells of the small intestine, in particular cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria, and dicarboxylic AminoAciduria. These are most readily detected by analysis of urine Amino Acids, but typically also affect intestinal Transport. The genes underlying these disorders have all been identified. The remaining Transporters were identified through molecular cloning techniques to the extent that a comprehensive portrait of functional cooperation among Transporters of intestinal epithelial cells is now available for both the basolateral and apical membranes. Mouse models of most intestinal Transporters illustrate their contribution to Amino Acid homeostasis and systemic physiology. Intestinal Amino Acid Transport activities can vary between species, but these can now be explained as differences of Amino Acid Transporter distribution along the intestine. © 2019 American Physiological Society. Compr Physiol 9:343-373, 2019.
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Amino Acid Transport across mammalian intestinal and renal epithelia
Physiological Reviews, 2008Co-Authors: Stefan BroerAbstract:The Transport of Amino Acids in kidney and intestine is critical for the supply of Amino Acids to all tissues and the homeostasis of plasma Amino Acid levels. This is illustrated by a number of inherited disorders affecting Amino Acid Transport in epithelial cells, such as cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria, dicarboxylic AminoAciduria, and some other less well-described disturbances of Amino Acid Transport. The identification of most epithelial Amino Acid Transporters over the past 15 years allows the definition of these disorders at the molecular level and provides a clear picture of the functional cooperation between Transporters in the apical and basolateral membranes of mammalian epithelial cells. Transport of Amino Acids across the apical membrane not only makes use of sodium-dependent symporters, but also uses the proton-motive force and the gradient of other Amino Acids to efficiently absorb Amino Acids from the lumen. In the basolateral membrane, antiporters cooperate with facilitators to release Amino Acids without depleting cells of valuable nutrients. With very few exceptions, individual Amino Acids are Transported by more than one Transporter, providing backup capacity for absorption in the case of mutational inactivation of a Transport system.
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adaptation of plasma membrane Amino Acid Transport mechanisms to physiological demands
Pflügers Archiv: European Journal of Physiology, 2002Co-Authors: Stefan BroerAbstract:The molecular identification of almost all physiologically characterized Amino Acid Transporters in recent years has facilitated the functional analysis of this important class of Transport proteins. The picture that emerges from these studies is that antiport is the prevalent mode of Amino Acid Transport rather than a combination of uniporters and coTransporters. Mainly neurotransmitters and osmolytes are Transported by complex coTransport mechanisms that allow a high intracellular accumulation. Antiport mechanisms almost invariably include the nonessential Amino Acids alanine and glutamine, which are used as exchange substrates. The intracellular level of both Amino Acids is well regulated by Na(+)/Amino Acid coTransporters. Transport mechanisms are not conserved within families and may change with mutation of even a single Amino Acid residue in the Transport protein. Thus Transport mechanisms are easily adapted to physiological demands during evolution.
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expression of the surface antigen 4f2hc affects system l like neutral Amino Acid Transport activity in mammalian cells
Biochemical Journal, 1997Co-Authors: Stefan Broer, Angelika Broer, Bernd HamprechtAbstract:Mammalian cells possess a variety of Amino Acid-Transport systems with overlapping substrate specificity. System L is one of the major Amino Acid-Transport systems of non-epithelial cells. By expression cloning we have recently demonstrated that the surface antigen 4F2hc (CD98) is a necessary component for expression of system-L-like Amino Acid-Transport activity in C6-BU-1 rat glioma cells [Broer, Broer and Hamprecht (1995) Biochem. J. 312, 863-870]. 4F2hc mRNA was detected in CHO cells, COS cells, activated lymphocytes isolated from mouse spleen and primary cultures of astrocytes. In all these cell types, Na+-independent isoleucine Transport was mediated by system L. No contribution of system y+L to isoleucine or arginine Transport was detected in C6-BU-1 cells. In lymphocytes, both system-L-like Amino Acid-Transport activity and 4F2hc mRNA levels increased after treatment with phorbol ester plus ionomycin. Antisense oligonucleotides caused modest inhibition of Na+-independent isoleucine Transport in C6-BU-1 cells and primary cultures of astroglial cells, whereas arginine Transport was unaffected. Overexpression of 4F2hc cDNA in CHO cells resulted in an increase in Na+-independent isoleucine Transport.
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the 4f2hc surface antigen is necessary for expression of system l like neutral Amino Acid Transport activity in c6 bu 1 rat glioma cells evidence from expression studies in xenopus laevis oocytes
Biochemical Journal, 1995Co-Authors: Stefan Broer, Angelika Broer, Bernd HamprechtAbstract:Mammalian cells possess a variety of Amino Acid-Transport systems with overlapping substrate specificity. System L is one of the major Amino Acid-Transport systems in all non-epithelial cells. Its molecular structure is not known. To clone the neutral Amino Acid-Transporter system L, we followed an expression cloning strategy using Xenopus laevis oocytes. A cDNA library derived from C6-BU-1 rat glioma cells was used as a source, because high expression of system L activity could be demonstrated with polyadenylated RNA isolated from these cells, when injected into Xenopus laevis oocytes [Broer, Broer and Hamprecht (1994) Biochim. Biophys. Acta 1192, 95-100]. A single clone (ILAT) was identified, the sense cRNA of which, on injection into Xenopus laevis oocytes, stimulated sodium-independent isoleucine Transport by about 100-fold. Further characterization revealed that Transport of cationic Amino Acids was also stimulated. Sequencing of the cDNA showed that the identified clone is the heavy chain of the rat 4F2 surface antigen, a marker of tumour cells and activated lymphocytes. Uptake of neutral and cationic Amino Acids was not stimulated by the presence of Na+ ions. Antisense cRNA transcribed from this clone or antisense oligonucleotides, when co-injected with polyadenylated RNA from C6-BU-1 rat glioma cells, completely suppressed system L-like isoleucine-Transport activity. We conclude that ILAT is necessary for expression of system L-like Amino Acid-Transport activity by polyadenylated RNA from C6-BU-1 rat glioma cells.
Theresa L Powell - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Placental Amino Acid Transport and Fetal Growth.
Progress in molecular biology and translational science, 2017Co-Authors: Or Vaughan, Fredrick J. Rosario, Theresa L Powell, Thomas JanssonAbstract:The fetus requires Amino Acids for the processes of protein synthesis, carbon accretion, oxidative metabolism, and biosynthesis, which ultimately determine growth rate in utero. The fetal supply of Amino Acids is critically dependent on the Transport capacity of the placenta. System A Amino Acid Transporters in the syncytiotrophoblast microvillous plasma membrane, directed toward maternal blood, actively accumulate Amino Acids, while system L exchangers mediate uptake of essential Amino Acids from the maternal circulation. The functional capacity and protein abundance of these Transporters in the placenta are related to fetal growth in both humans and experimental animals. Maternal nutritional and endocrine signals including insulin, insulin-like growth factors, adipokines, and steroid hormones regulate placental Amino Acid Transport, against the background of growth signals originating from the fetus. Anabolic signals of abundant maternal resource availability stimulate placental Amino Acid Transport to optimize offspring fitness, whereas catabolic signals reduce placental Amino Acid Transport in an attempt to ensure survival and long-term reproductive capacity of the mother when resources are scarce. These signals regulate placental Amino Acid Transport by controlling transcription, translation, plasma membrane trafficking, and degradation of Transporters. Adaptations in placental Amino Acid Transport capacity may underlie either under- or overgrowth of the fetus when maternal nutrient and hormone levels are altered as a result of altered maternal nutrition or metabolic disease. Strategies to modulate placental Amino Acid Transport may prove effective to normalize fetal growth in intrauterine growth restriction and fetal overgrowth.
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differential regulation of placental Amino Acid Transport by saturated and unsaturated fatty Acids
American Journal of Physiology-cell Physiology, 2014Co-Authors: Susanne Lager, Thomas Jansson, Theresa L PowellAbstract:Fatty Acids are critical for normal fetal development but may also influence placental function. We have previously reported that oleic Acid (OA) stimulates Amino Acid Transport in primary human trophoblasts (PHTs). In other tissues, saturated and unsaturated fatty Acids have distinct effects on cellular signaling, for instance, palmitic Acid (PA) but not OA reduces IκBα expression. We hypothesized that saturated and unsaturated fatty Acids differentially affect trophoblast Amino Acid Transport and cellular signaling. To test this hypothesis, PHTs were cultured in docosahexaenoic Acid (DHA; 50 μM), OA (100 μM), or PA (100 μM). DHA and OA were also combined to test whether DHA could counteract the OA stimulatory effect on Amino Acid Transport. The effects of fatty Acids were compared against a vehicle control. Amino Acid Transport was measured by isotope-labeled tracers. Activation of inflammatory-related signaling pathways and the mechanistic target of rapamycin (mTOR) pathway were determined by Western blot analysis. Exposure of PHTs to DHA for 24 h reduced Amino Acid Transport and phosphorylation of p38 MAPK, STAT3, mTOR, eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein (rp)S6. In contrast, OA increased Amino Acid Transport and phosphorylation of ERK, mTOR, S6 kinase 1, and rpS6. The combination of DHA with OA increased Amino Acid Transport and rpS6 phosphorylation. PA did not affect Amino Acid Transport but reduced IκBα expression. In conclusion, these fatty Acids differentially regulated placental Amino Acid Transport and cellular signaling. Taken together, these findings suggest that dietary fatty Acids could alter the intrauterine environment by modifying placental function, thereby having long-lasting effects on the developing fetus.
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interleukin 1β inhibits insulin signaling and prevents insulin stimulated system a Amino Acid Transport in primary human trophoblasts
Molecular and Cellular Endocrinology, 2013Co-Authors: Irving L M H Aye, Thomas Jansson, Theresa L PowellAbstract:Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated Amino Acid Transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10pg/ml) for 24h. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L Amino Acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A Transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth.
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Mammalian target of rapamycin signalling modulates Amino Acid uptake by regulating Transporter cell surface abundance in primary human trophoblast cells
Journal of Physiology, 2013Co-Authors: Fredrick J. Rosario, Theresa L Powell, Yoshikatsu Kanai, Thomas JanssonAbstract:Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental Amino Acid Transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental Amino Acid Transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate Amino Acid Transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L Amino Acid Transport activity but had no effect on growth factor-stimulated Amino Acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated Amino Acid Transport activity. In contrast, mTOR inhibition had no effect on serotonin Transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) Transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast Amino Acid Transporters by modulating the cell surface abundance of specific Transporter isoforms. This is the first report showing regulation of Amino Acid Transport by mTORC2. Because placental mTOR activity and Amino Acid Transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.
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full length adiponectin attenuates insulin signaling and inhibits insulin stimulated Amino Acid Transport in human primary trophoblast cells
Diabetes, 2010Co-Authors: Helen Jones, Thomas Jansson, Theresa L PowellAbstract:OBJECTIVE Maternal adiponectin levels are reduced and placental nutrient Transporters are upregulated in obesity and gestational diabetes mellitus; however, the effects of adiponectin on placental function are unknown. We hypothesized that adiponectin regulates placental Amino Acid Transport. RESEARCH DESIGN AND METHODS Human primary trophoblast cells were cultured and incubated with globular adiponectin (gAd) or full-length adiponectin (fAd) alone or in combination with insulin. System A and L Amino Acid Transport and SNAT1, SNAT2, and SNAT4 isoform expression was measured. The activity of the AMP-activated protein kinase (AMPK), phosphatidylinositol 3 kinase–AKT, and peroxisome proliferator–activated receptor-α (PPARα) signaling pathways was determined. RESULTS In the absence of insulin, gAd stimulated AMPK Thr172 phosphorylation, SNAT2 protein expression, and system A activity. This effect appeared to be mediated by interleukin-6 release and signal transducer and activator of transcription 3 (STAT3) signaling because gAd failed to stimulate system A in cells in which STAT3 had been silenced using small interfering RNA. fAd alone had no effect on system A activity or SNAT expression. Insulin increased AKT and insulin receptor substrate 1 (IRS-1) phosphorylation, system A activity, and SNAT2 expression. When combined with insulin, gAd did not affect system A activity or SNAT expression. In contrast, fAd abolished insulin-stimulated AKT Thr308 and IRS-1 Tyr612 phosphorylation, system A activity, and SNAT2 expression. Furthermore, fAd increased PPARα expression and PPARα (Ser21) phosphorylation. CONCLUSIONS In contrast to the insulin-sensitizing actions of adiponectin in liver and muscle reported in the literature, fAd attenuates insulin signaling in primary human trophoblast cells. As a result, fAd inhibits insulin-stimulated Amino Acid Transport, which may have important implications for placental nutrient Transport and fetal growth in pregnancy complications associated with altered maternal adiponectin levels.
Vadivel Ganapathy - One of the best experts on this subject based on the ideXlab platform.
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involvement of Transporter recruitment as well as gene expression in the substrate induced adaptive regulation of Amino Acid Transport system a
Biochimica et Biophysica Acta, 2001Co-Authors: Ruan Ling, Mitsuru Sugawara, Puttur D Prasad, Frederick H Leibach, Christy C Bridges, Takuya Fujita, Vadivel GanapathyAbstract:We investigated the molecular mechanism involved in the adaptive regulation of the Amino Acid Transport system A, a process in which Amino Acid starvation induces the Transport activity. These studies were done with rat C6 glioma cells. System A activity in these cells is mediated exclusively by the system A subtype, Amino Acid Transporter A2 (ATA2). The other two known system A subtypes, ATA1 and ATA3, are not expressed in these cells. Exposure of these cells to an Amino Acid-free medium induces system A activity. This process consists of an acute phase and a chronic phase. Laser-scanning confocal microscopic immunolocalization of ATA2 reveals that the acute phase is associated with recruitment of preformed ATA2 from an intracellular pool to the plasma membrane. In contrast, the chronic phase is associated with an induction of ata2 gene expression as evidenced from the increase in the steady-state levels of ATA2 mRNA, restoration of the intracellular pool of ATA2 protein, and blockade of the induction by cycloheximide and actinomycin D. The increase in system A activity induced by Amino Acid starvation is blocked specifically by system A substrates, including the non-metabolizable alpha-(methylAmino)isobutyric Acid.
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structure and function of ata3 a new subtype of Amino Acid Transport system a primarily expressed in the liver and skeletal muscle
Biochimica et Biophysica Acta, 2000Co-Authors: Mitsuru Sugawara, Frederick H Leibach, Takeo Nakanishi, You Jun Fei, Robert G Martindale, Malliga E Ganapathy, Vadivel GanapathyAbstract:Abstract To date, two different Transporters that are capable of Transporting α-(methylAmino)isobutyric Acid, the specific substrate for Amino Acid Transport system A, have been cloned. These two Transporters are known as ATA1 and ATA2. We have cloned a third Transporter that is able to Transport the system A-specific substrate. This new Transporter, cloned from rat skeletal muscle and designated rATA3, consists of 547 Amino Acids and has a high degree of homology to rat ATA1 (47% identity) and rat ATA2 (57% identity). rATA3 mRNA is present only in the liver and skeletal muscle. When expressed in Xenopus laevis oocytes, rATA3 mediates the Transport of α-[ 14 C](methylAmino)isobutyric Acid and [ 3 H]alanine. With the two-microelectrode voltage clamp technique, we have shown that exposure of rATA3-expressing oocytes to neutral, short-chain aliphatic Amino Acids induces inward currents. The Amino Acid-induced current is Na + -dependent and pH-dependent. Analysis of the currents with alanine as the substrate has shown that the K 0.5 for alanine (i.e., concentration of the Amino Acid yielding half-maximal current) is 4.2±0.1 mM and that the Na + :alanine stoichiometry is 1:1.
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primary structure functional characteristics and tissue expression pattern of human ata2 a subtype of Amino Acid Transport system a
Biochimica et Biophysica Acta, 2000Co-Authors: Takahiro Hatanaka, Wei Huang, Haiping Wang, Mitsuru Sugawara, Puttur D Prasad, Frederick H Leibach, Vadivel GanapathyAbstract:We report here on the primary structure and functional characteristics of the protein responsible for the system A Amino Acid Transport activity that is known to be expressed in most human tissues. This Transporter, designated ATA2 for Amino Acid Transporter A2, was cloned from the human hepatoma cell line HepG2. Human ATA2 (hATA2) consists of 506 Amino Acids and exhibits a high degree of homology to rat ATA2. hATA2-specific mRNA is ubiquitously expressed in human tissues. When expressed in mammalian cells, hATA2 mediates Na+-dependent Transport of alpha-(methylAmino)isobutyric Acid, a specific model substrate for system A. The Transporter is specific for neutral Amino Acids. It is pH-sensitive and Li+-intolerant. The Na+:Amino Acid stoichiometry is 1:1.
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human lat1 a subunit of system l Amino Acid Transporter molecular cloning and Transport function
Biochemical and Biophysical Research Communications, 1999Co-Authors: Puttur D Prasad, Wei Huang, Haiping Wang, Frederick H Leibach, Ramesh Kekuda, Deva P Rajan, Vadivel GanapathyAbstract:We report here on the cloning and functional characterization of human LAT1, a subunit of the Amino Acid Transport system L. The hLAT1 cDNA, obtained from a human placental cDNA library, codes for a protein of 507 Amino Acids. When functionally expressed in mammalian cells together with the heavy chain of the rat 4F2 antigen (r4F2hc), hLAT1 induces the Transport of neutral Amino Acids. When expressed independently, neither hLAT1 nor r4F2hc was capable of Amino Acid Transport to any significant extent. Thus, the hLAT1-r4F2hc heterodimeric complex is responsible for the observed Amino Acid Transport. The Transport process induced by the heterodimer is Na+ independent and is not influenced by pH. It recognizes exclusively neutral Amino Acids with high affinity. LAT1-specific mRNA is expressed in most human tissues with the notable exception of the intestine.
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modulation of the activity of Amino Acid Transport system l by phorbol esters and calmodulin antagonists in a human placental choriocarcinoma cell line
Biochimica et Biophysica Acta, 1992Co-Authors: Sammanda Ramamoorthy, Frederick H Leibach, Virendra B Mahesh, Vadivel GanapathyAbstract:We investigated the regulation of the activity of Amino Acid Transport system L in the JAR human placental choriocarcinoma cell line by agents which are known to modulate the activities of three different classes of protein kinases, A-kinase, C-kinase and CaM-kinase. The system L activity was measured by determining the uptake of leucine in these cells, grown as confluent monolayers. Leucine uptake in these cells was predominantly Na(+)-independent, was stimulated by lowering the extracellular pH and was inhibited by hydrophobic neutral Amino Acids. These characteristics demonstrate that uptake of leucine in this cell line occurs primarily via system L. Treatment of the cells with cholera toxin and forskolin, agents which are known to elevate intracellular cAMP levels, did not have any effect on the activity of system L. 4 beta-Phorbol 12-myristate 13-acetate, an activator of C-kinase, but not the inactive analogue 4 alpha-phorbol 12,13-didecanoate, caused a significant stimulation of system L. The involvement of C-kinase in the phorbol ester-induced stimulation was supported by the finding that staurosporine, an inhibitor of C-kinase, effectively blocked the stimulation. Calmodulin antagonists, calmidazolium, W-7 and CGS 9343 B stimulated system L activity markedly. The potency of these antagonists was in the following order: calmidazolium greater than CGS 9343 B greater than W-7. This stimulatory effect was specific for system L because systems A and ASC were not stimulated by these agents. The stimulation caused by these agents was primarily due to an increase in the maximal velocity, the apparent Km of the system being only minimally affected. It is concluded that the activity of Amino Acid Transport system L in the JAR human placental choriocarcinoma cell line is stimulated by C-kinase, inhibited by CaM-kinase and unaffected by A-kinase.