Specific Amino Acid Transporters Research Articles

Sexually Dimorphic Gene Expression in Bovine Conceptuses at the Initiation of Implantation.

In cattle, maternal recognition of pregnancy occurs on Day 16 via secretion of interferon tau (IFNT) by the conceptus. The endometrium can distinguish between embryos with different developmental competencies. In eutherian mammals, X-chromosome inactivation (XCI) is required to ensure an equal transcriptional level of most X-linked genes for both male and female embryos in adult tissues, but this process is markedly different in cattle than mice. We examined how sexual dimorphism affected conceptus transcript abundance and amino acid composition as well as the endometrial transcriptome during the peri-implantation period of pregnancy. Of the 5132 genes that were differentially expressed on Day 19 in male compared to female conceptuses, 2.7% were located on the X chromosome. Concentrations of specific amino acids were higher in the uterine luminal fluid of male compared to female conceptuses, while female conceptuses had higher transcript abundance of specific amino acid transporters (SLC6A19 and SLC1A35). Of note, the endometrial transcriptome was not different in cattle gestating a male or a female conceptus. These data support the hypothesis that, far from being a blastocyst-specific phenomenon, XCI is incomplete before and during implantation in cattle. Despite differences in transcript abundance and amino acid utilization in male versus female conceptuses, the sex of the conceptus itself does not elicit a different transcriptomic response in the endometrium.

Biological evaluation of new [18F]F‐labeled synthetic amino acid derivatives as oncologic radiotracers

The present study evaluated the tumoral uptake of the novel synthetic amino acid positron emission tomography (PET) tracers (S)-2-amino-3-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)propanoic acid (AMC-101), (S)-2-amino-4-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)butanoic acid (AMC-102), and (S)-2-amino-5-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)pentanoic acid (AMC-103), all of which are (S)-2-amino-(4-([(18) F]fluoromethyl)-1H-1,2,3-triazol-1-yl)alkyl acids. In vitro cellular uptake was investigated using the rat glioma cell lines 9L and C6. In vitro competitive inhibition tests were performed to identify the involvement of specific amino acid transporters. In vivo dynamic PET images of 9L xenograft tumor-bearing model mice were acquired over 2 h after AMC administration. [(18) F]FDOPA PET studies were performed with and without S-carbidopa pretreatment for comparison. All three AMCs exhibited good in vitro cell uptake through the L and alanine-serine-cysteine transporters and enabled clear tumor visualization on PET, leaving the brain devoid of the tracer. Thirty minutes after injection, the mean tumor standardized uptake values were 1.59 ± 0.05, 1.89 ± 0.27, and 1.74 ± 0.13 for AMC-101, AMC-102, and AMC-103, respectively. Although the tumor uptake values of AMCs were lower than that of [(18) F]FDOPA with S-carbidopa pretreatment, AMCs enabled higher contrast images with lower background activity compared with [(18) F]FDOPA with S-carbidopa pretreatment. Our results indicate the potential uses of these new synthetic amino acids as oncologic radiotracers.

Effect of Copper on l-Cysteine/l-Cystine Influx in Normal Human Erythrocytes and Erythrocytes of Wilson's Disease.

Wilson's disease is a disease of abnormal copper metabolism in which free serum copper level is raised. The objective of the study was to determine, whether in Wilson disease, l-cysteine/l-cystine influx into RBC was decreased or not and the specific amino acid transporter affected by copper in normal human RBC. For l-cysteine/l-cystine influx, ten untreated cases, ten treated cases and ten age and sex matched healthy controls were recruited. To study the effect of copper on l-cysteine/l-cystine influx in RBC, 15 healthy subjects were selected. RBC GSH and l-cysteine/l-cystine influx were estimated by Beautler's and Yildiz's method respectively. In untreated cases, l-cysteine/l-cystine influx and erythrocyte GSH level were decreased showing that elevated level of free copper in serum or media decreased l-cysteine/l-cystine influx in human RBC. Copper treatment inhibited L amino acid transporter in normal RBC specifically.

Abstract A41: Leveraging metabolic dependencies in cancer: Cysteine addiction in pancreatic cancer cells

Abstract Pancreatic cancer is a devastating and lethal malignancy, estimated to kill over 40,000 Americans per year. Little has changed in the treatment of this disease over the past half-century, highlighting the need for an improved understanding of the molecular mechanisms driving tumor development and maintenance. The malignant behavior of tumor cells is underwritten by alterations to their cellular metabolism, which is optimized to meet the catabolic needs of a rapidly dividing cell. One of the consequences of this oncogenic growth is increased generation of reactive oxygen species (ROS), a potentially toxic byproduct of many metabolic reactions. Interestingly, oncogenes such as Kras, the dominant driver of pancreatic cancer, can counteract ROS toxicity through activation of an antioxidant program, creating a highly reducing intracellular environment. Cysteine is the rate-limiting precursor for the synthesis of glutathione (GSH), the preeminent antioxidant of the cell, and as such may represent a key metabolite in the maintenance of redox balance in tumor cells. In this study we explore the importance of cysteine metabolism in the viability of pancreatic cancer cells and test the effects of cysteine deprivation and the pharmacological inhibition of cysteine uptake on a variety of pancreatic cancer cell lines. To explore the role of cysteine in pancreatic cancer growth, we cultured a panel of human and murine pancreatic cancer cell lines in the absence of cysteine. We found that cysteine withdrawal rapidly induced a peculiar form of cell death called ferroptosis, which can result from the catastrophic accumulation of certain lipid peroxides. Cystine, the oxidized form of cysteine, is the predominant extracellular source of cysteine in most cells, and it is normally imported into the cell by a specific amino acid transporter known as system xc-. We show that pharmacological inhibition of sytem xc- causes rapid cell death in most pancreatic cancer cells through ferroptosis. We find, however, that some cells do exhibit baseline resistance to cysteine deprivation or system xc- inhibition. We hypothesized that these cells utilize methionine to synthesize cysteine through transsulfuration. We show that these cells can be sensitized to system xc- inhibition when co-treated with propargyglycine (PPG), an irreversible inhibitor of gamma-cystathionase, an enzyme required for transsulfuration. These findings imply that cysteine is an important substrate for appropriate redox homeostasis in these cells. To further explore if GSH synthesis is the key mediator of cysteine-deprivation induced cell death, we treated pancreatic tumor cells with buthionine sulfoxamine (BSO), a potent and specific inhibitor of GSH synthesis. To our surprise, cells treated with BSO do not exhibit any of the ill-effects we see in cysteine-deprived pancreatic cancer cells. Therefore, it is unlikely that the cell death seen in cells deprived of cysteine is due solely to GSH depletion, but possibly due to the activity of other antioxidant pathways that require cysteine. In conclusion, we find that pancreatic cancer cells are highly sensitive to cysteine-deprivation and to treatment with system xc- inhibitors, and that the cell death caused by this stress is an iron-dependent, oxidative form of cell death called ferroptosis. Furthermore, it appears that GSH, the primary antioxidant molecule of the cell, is not solely responsible for mediating this cell death, arguing that cysteine may play other important roles in maintaining redox homeostasis in these cells. Given that previous studies show that global system xc- knockout mice do not exhibit any changes in cellular GSH content, these findings may represent a targetable metabolic dependency in pancreatic cancer cells. Citation Format: Michael A. Badgley, Carmine F. Palermo, Stephen A. Sastra, Brent R. Stockwell, Kenneth P. Olive. Leveraging metabolic dependencies in cancer: Cysteine addiction in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A41.

Placental amino acid transport may be regulated by maternal vitamin D and vitamin D-binding protein: results from the Southampton Women's Survey.

Both maternal 25-hydroxyvitamin D (25(OH)D) concentrations during pregnancy and placental amino acid transporter gene expression have been associated with development of the offspring in terms of body composition and bone structure. Several amino acid transporter genes have vitamin D response elements in their promoters suggesting the possible linkage of these two mechanisms. We aimed to establish whether maternal 25(OH)D and vitamin D-binding protein (VDBP) levels relate to expression of placental amino acid transporters. RNA was extracted from 102 placental samples collected in the Southampton Women's Survey, and gene expression was analysed using quantitative real-time PCR. Gene expression data were normalised to the geometric mean of three housekeeping genes, and related to maternal factors and childhood body composition. Maternal serum 25(OH)D and VDBP levels were measured by radioimmunoassay. Maternal 25(OH)D and VDBP levels were positively associated with placental expression of specific genes involved in amino acid transport. Maternal 25(OH)D and VDBP concentrations were correlated with the expression of specific placental amino acid transporters, and thus may be involved in the regulation of amino acid transfer to the fetus. The positive correlation of VDBP levels and placental transporter expression suggests that delivery of vitamin D to the placenta may be important. This exploratory study identifies placental amino acid transporters which may be altered in response to modifiable maternal factors and provides a basis for further studies.

Are intact peptides absorbed from the healthy gut in the adult human?

For over 100 years it was believed that dietary protein must be completely hydrolysed before its constituent amino acids could be absorbed via specific amino acid transport systems. It is now known that the uptake of di- and tripeptides into the enterocyte is considerable, being transported across the intestinal endothelium by the PepT1 H+/peptide co-transporter. There is also evidence that some di- and tripeptides may survive cytosolic hydrolysis and be transported intact across the basolateral membrane. However, other than antigen sampling, the transport of larger intact macromolecules across the intestinal endothelium of the healthy adult human remains a controversial issue as there is little unequivocal in vivo evidence to support this postulation. The aim of the present review was to critically evaluate the scientific evidence that peptides/proteins are absorbed by healthy intestinal epithelia and pass intact into the hepatic portal system. The question of the absorption of oliogopeptides is paramount to the emerging science of food-derived bioactive peptides, their mode of action and physiological effects. Overall, we conclude that there is little unequivocal evidence that dietary bioactive peptides, other than di- and tripeptides, can cross the gut wall intact and enter the hepatic portal system in physiologically relevant concentrations.

Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

Lysosomal Amino Acid Efflux Assay

As the cellular “recycling” organelle, lysosomes break down proteins into amino acids, which are then transported into cytosol for reuse by various amino acid transporters. The transport rate of an amino acid is presumably regulated by cellular conditions such as organelle pH, membrane potential and metabolic states. Because of their intracellular localization and the relative inaccessibility, lysosomal amino acid transporters have been studied largely via indirect measurements. Using lysosome purification and 14C-labeled amino acids, this protocol provides a method to measure the efficiency of specific amino acid transporters on lysosomes., 作为细胞"再循环"细胞器，溶酶体将蛋白质分解成氨基酸，然后将其转运到细胞质中以供各种氨基酸转运蛋白重复使用。 氨基酸的转运速率可能通过细胞条件如细胞器pH，膜电位和代谢状态来调节。 由于它们的细胞内定位和相对不可接近性，溶酶体氨基酸转运蛋白主要通过间接测量进行研究。 使用溶酶体纯化和14 C标记的氨基酸，该协议提供了一种测量特定氨基酸转运蛋白在溶酶体上的效率的方法。

Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

The L-type amino acid transporter-1 (LAT1, SLC7A5) is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[18F]fluoroethyl)-L-tyrosine (FET) that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [89Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g) at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [18F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [89Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.

Transcriptional and functional regulation of the intestinal peptide transporter PEPT1

Dietary proteins are cleaved within the intestinal lumen to oligopeptides which are further processed to small peptides (di- and tripeptides) and free amino acids. Although the transport of amino acids is mediated by several specific amino acid transporters, the proton-coupled uptake of the more than 8000 different di- and tripeptides is performed by the high-capacity/low-affinity peptide transporter isoform PEPT1 (SLC15A1). Its wide substrate tolerance also allows the transport of a repertoire of structurally closely related compounds and drugs, which explains their high oral bioavailability and brings PEPT1 into focus for medical and pharmaceutical approaches. Although the first evidence for the interplay of nutrient supply and PEPT1 expression and function was described over 20 years ago, many aspects of the molecular processes controlling its transcription and translation and modifying its transporter properties are still awaiting discovery. The present review summarizes the recent knowledge on the factors modulating PEPT1 expression and function in Caenorhabditis elegans, Danio rerio, Mus musculus and Homo sapiens, with focus on dietary ingredients, transcription factors and functional modulators, such as the sodium-proton exchanger NHE3 and selected scaffold proteins.

Arginine Cools the Inflamed Gut

Maintenance of immunophysiological homeostasis and regulation of gut barrier function are essential for the defense of the host. Severe alterations, including infections and chronic inflammation, have been associated with increased intestinal permeability, leading to deregulation of gut function and homeostasis. To establish and reinforce this crucial balance, the intestinal metabolismplaysakeyroleandhastobetightlyregulatedsincein the human intestinal mucosa, the protein fractional synthesis rate is approximately 50% per day. This value is higher than that of other major metabolically active tissues, such as liver and muscle, and depends on the accessibility of the metabolic precursor pool (1). The amino acid L-arginine (L-Arg) is a central intestinal metabolite, both as a constituent of protein synthesis and as a regulatory molecule limiting intestinal alterations and maintaining immunophysiologicalfunctions(1,2).Infection-associated L-Arg deficiency has been shown to contribute to immunopathology, andclinicaltrialsinvolving L-Argadministrationhaveshownsubstantial decreases in inflammation and infectious complications (3). In this issue, Chau and colleagues demonstrate that malariaassociated hypoargininemia impairs intestinal barrier function and predisposes the host to coinfection with Salmonella. Increasingbioavailabilityof L-Argthroughoralsupplementationameliorates intestinal inflammation and pathology, demonstrating that pharmacological intervention at the metabolic-precursor level can be utilized to regulate mucosal immunohomeostasis (4). L-Arg is derived from the diet, turnover of proteins, and endogenous production through synthesis from L-citrulline (L-Cit) and successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), the third and fourth enzymes of the urea cycle. The major site of L-Arg metabolism is the liver, where L-Arg generated in the urea cycle is rapidly converted to urea and ornithine by arginases, however, with no net synthesis of L-Arg. Although synthesis of L-Arg from L-Cit can occur in many cell types, a major part of endogenous synthesis occurs via “the intestinal-renal axis,” a postnatally established collaboration between epithelial cells of the small intestine and proximal tubule cells of the kidney. In adult animals, L-Cit is produced primarily by intestinal epithelial cells from NH3 ,C O2, and ornithine by carbamylphosphate synthetase I and ornithine transcarbamylase, the first two enzymes of the urea cycle, and is supplied to the kidney and probably to other tissues for synthesis of L-Arg (2, 5, 6). L-Arg is a crucial amino acid that serves to modulate immune responses through conversion by several intracellular classes of enzymes, with isoforms of arginase and nitric oxide synthase (NOS) being the two major enzyme families exerting key immunological functions (7). However, catabolism of extracellular LArg requires active and regulated uptake via specific cationic amino acid transporters (CAT) or heteromeric amino acid transporters (HAT) that act as H-coupled symporters or antiporters

EFFECTS OF DIFFERENT NITROGEN SOURCES ON GROWTH AND LIPID ACCUMULATION OF A HETEROTROPHIC MICROALGAE-CHORELLA VULGARIS

Chlorella strains have great potential as a resource for production of biofuels due to their relatively fast growth rate and easy cultivation.Heterotrophic cultivation of Chlorella has been developed due to higher biomass concentration and accumulation of much higher lipid content than classical photoautotrophic culture.Instead of using glucose,using low cost materials,such as starch or cellulose-hydrolyzed solution,is a good strategy to reduce the cost of culture medium and the cost of microalgae-based biofuels from heterotrophic fermentation of Chlorella.Potato starch is relatively cheap material in comparison with cornstarch or cassava starch,hence it could be an ideal carbon source for cultivating Chlorella.In a number of oil-producing microalgal species,Chlorella has the capacity of accumulating much higher proportion of fatty acids by nitrogen starvation.The objective of this study was to investigate the effects of various nitrogen resources on cell growth and lipid accumulation in heterotrophic Chlorella vulgaris using potato starch hydrolysate(PSH) as the sole carbon source.In order to illustrate the influence of various nitrogen sources on biomass and lipid content in C.vulgaris,the effects of these nitrogen source including inorganic and organic nitrogen source such as alanine or tyrosine on cell growth and lipid accumulation of C.vulgaris were examined.As the sole carbon source PSH was added to SE medium for algal cultivation prior to addition of all nitrogen sources.NaNO3 and alanine were added to yield starting concentrations of 1.5 mmol /L,3.0 mmol/L and 6.0 mmol/L,respectively.Tyrosine was added to yield starting concentrations of 0.75 mmol/L,1.5 mmol /L and 3.0 mmol/L,respectively.Continuous cultivation of C.vulgaris was carried out under dark condition for 10 days.The cell number,special growth rate,cellular chlorophyll content,neutral lipid content and total lipid content of C.vulgaris under present cultural condition were determined throughout the entire experimental period.The results showed that:(1) Higher chlorophyll content and biomass production were observed with higher nitrate-nitrogen concentration,although low concentrations of nitrate-nitrogen can achieve high lipid accumulation in C.vulgaris.The highest biomass productivity was obtained at 1.5 mmol/L sodium nitrate,the biomass of 2.65 g/L,lipid concentration of 51.21% and the total lipid production of 1.36 g/L were achieved.(2) When alanine was used as the sole nitrogen source,it was also observed that the cell growth and total chlorophyll content increased but the lipid content reduced.(3) A low level of biomass production was observed while adding tyrosine as a nitrogen source;however,cell enlargement and high chlorophyll content were achieved under this condition,resulting in high lipid content of 38.78%—47.02%.Based on above results,it was concluded that the unicellular alga C.vulgaris was able to respond to nitrogen nutrient changes by inducing specific amino acid transport systems,and it seemed to have developed more specific mechanisms of amino-acid uptake and lipid production along with subtle regulatory phenomena such as induction by nitrogen starvation.Furthermore,tyrosine,as the part of the third uptake system induced by glucose transport system,can also enhance continually cellular lipid accumulation in C.vulgaris.

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.

Aging differentially affects human skeletal muscle amino acid transporter expression when essential amino acids are ingested after exercise

Amino acid transporters have been proposed as regulators of protein synthesis. The primary aim of this study was to determine whether amino acid transporter expression is increased in human muscle following resistance exercise (RE) coupled with essential amino acid (EAA) ingestion, and whether a differential response occurs with aging. Secondly, we aimed to compare this response to a previous study examining RE alone. Young (n=7, 30±2yr) and older men (n=6, 70±2yr) ingested EAA 1h after RE. Muscle biopsies were obtained at rest and 3 and 6h post exercise to examine amino acid transporter mRNA and protein expression. In both age groups, RE+EAA increased mRNA of L-type amino acid transporter 1 (LAT1)/solute linked carrier (SLC)7A5, sodium-coupled neutral amino acid transporter 2 (SNAT2)/SLC38A2, and cationic amino acid transporter 1/SLC7A1 (p<0.05). SNAT2 protein increased in young at 3 and 6h (p<0.05), whereas old maintained higher LAT1 protein (p<0.05). Compared to RE alone, RE+EAA enhanced amino acid transporter expression only in young (p<0.05). RE increases muscle amino acid transporter expression in young and older adults, however, post exercise EAA ingestion enhances amino acid transporter expression only in young indicating that aging may influence the function of specific amino acid transporters.

SLC6A14 (ATB0,+) Protein, a Highly Concentrative and Broad Specific Amino Acid Transporter, Is a Novel and Effective Drug Target for Treatment of Estrogen Receptor-positive Breast Cancer

SLC6A14, also known as ATB(0,+), is an amino acid transporter with unique characteristics. It transports 18 of the 20 proteinogenic amino acids. However, this transporter is expressed only at low levels in normal tissues. Here, we show that the transporter is up-regulated specifically in estrogen receptor (ER)-positive breast cancer, demonstrable with primary human breast cancer tissues and human breast cancer cell lines. SLC6A14 is an estrogen/ER target. The transport features of SLC6A14 include concentrative transport of leucine (an activator of mTOR), glutamine (an essential amino acid for nucleotide biosynthesis and substrate for glutaminolysis), and arginine (an essential amino acid for tumor cells), suggesting that ER-positive breast cancer cells up-regulate SLC6A14 to meet their increased demand for these amino acids. Consequently, treatment of ER-positive breast cancer cells in vitro with α-methyl-DL-tryptophan (α-MT), a selective blocker of SLC6A14, induces amino acid deprivation, inhibits mTOR, and activates autophagy. Prolongation of the treatment with α-MT causes apoptosis. Addition of an autophagy inhibitor (3-methyladenine) during α-MT treatment also induces apoptosis. These effects of α-MT are specific to ER-positive breast cancer cells, which express the transporter. The ability of α-MT to cause amino acid deprivation is significantly attenuated in MCF-7 cells, an ER-positive breast cancer cell line, when SLC6A14 is silenced with shRNA. In mouse xenograft studies, α-MT by itself is able to reduce the growth of the ER-positive ZR-75-1 breast cancer cells. These studies identify SLC6A14 as a novel and effective drug target for the treatment of ER-positive breast cancer.

Oocyte growth in vitro: potential model for studies of oocyte-granulosa cell interactions.

Various factors such as gonadotrophins, growth factors, and steroid hormones play important roles in the regulation of oocyte/follicular growth in mammalian ovaries. In addition to these factors, there is a bidirectional interaction between oocytes and granulosa cells that is essential for achieving optimal oocyte developmental competence. Oocytes play a key role in this interaction by secreting paracrine factors that alter the activities of neighboring cumulus cells, such as the expression of a specific amino acid transporter, cholesterol biosynthesis, and levels of glycolysis in the cumulus cells. Among the known oocyte-derived factors, growth differentiation factor 9 (GDF9) is the dominant factor mediating the regulation by oocytes leading to cumulus expansion and granulosa cell proliferation. GDF9 frequently interacts with other oocyte-derived factors in a synergistic manner. It seems reasonable to speculate that oocytes growing in vitro require interactions similar to those in vivo. Some of the oocyte-mediated regulations have been confirmed in vitro, providing evidence of the usefulness of culture systems as a strong tool for such studies. This review discusses in vitro culture of growing oocytes in terms of oocyte-granulosa cell interactions.

Dietary protein effects on growth performance, carcass traits and expression of selected jejunal peptide and amino acid transporters in broiler chickens

The effect of dietary protein on growth, carcass traits and some specific intestinal intestinal peptide and amino acid transporters in broiler chickens was studied. Birds received a common pre-test diet, and were subsequently fed either a standard positive control diet (PC) or a reduced CP diet (RCP) from 21 to 42 d of age. Growth was negatively impacted with feeding of RCP as manifested by an increase in feed consumption and feed conversion ratio. Carcass traits also showed evidence of negative effects of feeding the RCP diet, leading to a reduction in carcass and breast meat yield and an increase in abdominal fat percentage. Blood plasma total protein was reduced when the broilers were fed the RCP diet. Expression of mRNA for one peptide (PepT1) and four AA intestinal transporters (b o,+ AT; CAT2; y + LAT2; EAAT3) was measured from the jejunum. Quantified mRNA for the AA transporters y + LAT2 and EAAT3 showed that they were upregulated in chickens fed the RCP-diet. The transport systems PepT1, b o,+ AT, and CAT2, were not affected by the dietary treatment imposed. The live and processing data validated the in vivo portion of the study and elucidated the negative impact of feeding the RCP diet, while the responses observed with the expression of the various transporters may help provide some insight on the physiological consequences and adaptations that birds endure when provided diets too low in CP for abnormally extended periods of time.

Characterization of improved L‐tryptophan FRET biosensor for measurement of real time transport in live cells

The transmembrane flux and exchange of amino acids is mediated by various cooperating transporters that control cell and body amino acid homeostasis. To characterize the function of specific amino acid transporters and their cooperation in live cells, we aim at measuring subcellular changes in amino acid concentration in real time. To generate a highly sensitive biosensor, we modified a previously described L-tryptophan FRET biosensor composed of CFP and YFP linked together by a bacterial L-tryptophan binding protein and expressed this new CTY in wild type COS-7 cells. These cells display high endogenous levels of the amino acid antiporter LAT1-4F2 (SLC7A5-SLC3A2), a high affinity exchanger of L-tryptophan and of other neutral amino acids. Additionally, we expressed the low affinity aromatic amino acid uniporter TAT1 (SLC16A10) that can cooperate with Lat1-4F2hc. As a low affinity facilitated diffusion pathway, TAT1 can also be used for the control of intracellular L-tryptophan concentration and sensor calibration. Using a microplate reader and live cell imaging FRET, we show that the new biosensor displays a much improved FRET efficiency compared with the previously described CTYT. Thus, the new CTY biosensor allows us to understand better how the cooperation of amino acid exchangers (antiporters) and facilitated diffusion pathways (uniporters) can together control cellular amino acid homeostasis. Supported by Swiss NSF grant 31-130471 to FV

Essential amino acid sensing, signaling, and transport in the regulation of human muscle protein metabolism

To highlight the recent research pertaining to the cellular mechanisms linking amino acid availability, mTORC1 signaling, and muscle protein metabolism. Activation of the mTORC1 pathway in response to amino acids may be dependent upon cellular relocalization of mTORC1, a process that appears to involve the Rag GTPases. Recent studies have also identified other intracellular proteins, such as hVps34 and MAP4K3, and specific amino acid transporters as necessary links between amino acid availability and mTORC1. In human skeletal muscle, it appears that mTORC1 activity increases the expression of several amino acid transporters, which may be an important adaptive response to sensitize muscle to a subsequent increase in amino acid availability. The precise cellular mechanisms linking amino acids to mTORC1 signaling and muscle protein metabolism are currently not well understood. More defined cellular mechanisms are beginning to emerge suggesting a role for several intracellular proteins including hVps34, MAP4K3, and Rag GTPases. Additionally, specific amino acid transporters may have a role both upstream and downstream of mTORC1. Continued investigation into the precise cellular mechanisms linking amino acid availability and muscle protein metabolism will help facilitate improvements in existing therapies for conditions of muscle wasting.

The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes

BackgroundThe branched-chain amino acid (BCAA) leucine has been identified to be a key regulator of skeletal muscle anabolism. Activation of anabolic signalling occurs via the mammalian target of rapamycin (mTOR) through an undefined mechanism. System A and L solute carriers transport essential amino acids across plasma membranes; however it remains unknown whether an exogenous supply of leucine regulates their gene expression. The aim of the present study was to investigate the effects of acute and chronic leucine stimulation of anabolic signalling and specific amino acid transporters, using cultured primary human skeletal muscle cells.ResultsHuman myotubes were treated with leucine, insulin or co-treated with leucine and insulin for 30 min, 3 h or 24 h. Activation of mTOR signalling kinases were examined, together with putative nutrient sensor human vacuolar protein sorting 34 (hVps34) and gene expression of selected amino acid transporters. Phosphorylation of mTOR and p70S6K was transiently increased following leucine exposure, independently to insulin. hVps34 protein expression was also significantly increased. However, genes encoding amino acid transporters were differentially regulated by insulin and not leucine.ConclusionsmTOR signalling is transiently activated by leucine within human myotubes independently of insulin stimulation. While this occurred in the absence of changes in gene expression of amino acid transporters, protein expression of hVps34 increased.