SNAT2-mediated regulation of estrogen and progesterone in the proliferation of goat mammary epithelial cells.

The development of the goat mammary gland is mainly under the control of ovarian hormones particularly estrogen and progesterone (P4). Amino acids play an essential role in mammary gland development and milk production, and sodium-coupled neutral amino acid transporter 2 (SNAT2) was reported to be expressed in the mammary gland of rats and bovine mammary epithelial cells, which may affect the synthesis of milk proteins or mammary cell proliferation by mediating prolactin, 17ß-estradiol (E2) or methionine function. However, whether SNAT2 mediates the regulatory effects of E2 and P4 on the development of the ruminant mammary gland is still unclear. In this study, we show that E2 and P4 could increase the proliferation of goat mammary epithelial cells (GMECs) and regulate SNAT2 mRNA and protein expression in a dose-dependent manner. Further investigation revealed that SNAT2 is abundantly expressed in the mammary gland during late pregnancy and early lactation, while knockdown and overexpression of SNAT2 in GMECs could inhibit or enhance E2- and P4-induced cell proliferation as well as mammalian target of rapamycin (mTOR) signaling. We also found that the accelerated proliferation induced by SNAT2 overexpression in GMECs was suppressed by the mTOR signaling pathway inhibitor rapamycin. This indicates that the regulation of GMECs proliferation mediated by SNAT2 in response to E2 and P4 is dependent on the mTOR signaling pathway. Finally, we found that the total content of the amino acids in GMECs changed after knocking-down and overexpressing SNAT2. In summary, the results demonstrate that the regulatory effects of E2 and P4 on GMECs proliferation may be mediated by the SNAT2-transported amino acid pathway. These results may offer a novel nutritional target for improving the development of the ruminant mammary gland and milk production.

Bacterial over-production of the functionally active human SLC38A2 (SNAT2) exploiting the mistic tag: a cheap and fast tool for testing ligands.

BACKGROUND: SLC38A2 is a ubiquitously expressed Na+-dependent transporter specific for small and medium neutral amino acids. It is involved in human pathologies, such as type II diabetes and cancer. Despite its relevance in human physio-pathology, structure/function relationship studies and identification of ligands with regulatory roles are still in infancy. METHODS AND RESULTS: The cDNA coding for SLC38A2 was cloned in the pET-28-Mistic vector, and the BL21 codon plus RIL strain was transformed with the recombinant construct. 0.5% glucose and oxygen availability were crucial for protein expression. The over-expressed hSNAT2-Mistic chimera was cleaved on column and purified by nickel-chelating affinity chromatography, with a yield of about 60 mg/Liter cell culture. The purified hSNAT2 was reconstituted in proteoliposomes in an active form with a right-side-out orientation with respect to the native membrane. CONCLUSIONS: The addition of a Mistic tag at the N-terminus of the SNAT2 protein was crucial for its over-expression and purification. The purified protein was functionally active, representing a powerful tool for performing structure/function studies and testing ligands as inhibitors and/or activators.

CHOP upregulation and dysregulation of the mature form of the SNAT2 amino acid transporter in the placentas from small for gestational age newborns.

BACKGROUND: The placentas from newborns that are small for gestational age (SGA; birth weight < -2 SD for gestational age) may display multiple pathological characteristics. A key determinant of fetal growth and, therefore, birth weight is placental amino acid transport, which is under the control of the serine/threonine kinase mechanistic target of rapamycin (mTOR). The effects of endoplasmic reticulum (ER) stress on the mTOR pathway and the levels of amino acid transporters are not well established. METHODS: Placentas from SGA and appropriate for gestational age (AGA) newborns and the human placental BeWo cell line exposed to the ER stressor tunicamycin were used. RESULTS: We detected a significant increase in the levels of C/EBP homologous protein (CHOP) in the placentas from SGA newborns compared with those from AGA newborns, while the levels of other ER stress markers were barely affected. In addition, placental mTOR Complex 1 (mTORC1) activity and the levels of the mature form of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) were also reduced in the SGA group. Interestingly, CHOP has been reported to upregulate growth arrest and DNA damage-inducible protein 34 (GADD34), which in turn suppresses mTORC1 activity. The GADD34 inhibitor guanabenz attenuated the increase in CHOP protein levels and the reduction in mTORC1 activity caused by the ER stressor tunicamycin in the human placental cell line BeWo, but it did not recover mature SNAT2 protein levels, which might be reduced as a result of defective glycosylation. CONCLUSIONS: Collectively, these data reveal that GADD34A activity and glycosylation are key factors controlling mTORC1 signaling and mature SNAT2 levels in trophoblasts, respectively, and might contribute to the SGA condition. Video Abstract.

Sodium-Dependent Neutral Amino Acid Transporter 2 Can Serve as a Tertiary Carrier for l-Type Amino Acid Transporter 1-Utilizing Prodrugs.

Membrane transporters are the key determinants of the homeostasis of endogenous compounds in the cells and their exposure to drugs. However, the substrate specificities of distinct transporters can overlap. In the present study, the interactions of l-type amino acid transporter 1 (LAT1)-utilizing prodrugs with sodium-coupled neutral amino acid transporter 2 (SNAT2) were explored. The results showed that the cellular uptake of LAT1-utilizing prodrugs into a human breast cancer cell line, MCF-7 cells, was mediated via SNATs as the uptake was increased at higher pH (8.5), decreased in the absence of sodium, and inhibited in the presence of unselective SNAT-inhibitor, (α-(methylamino)isobutyric acid, MeAIB). Moreover, docking the compounds to a SNAT2 homology model (inward-open conformation) and further molecular dynamics simulations and the subsequent trajectory and principal component analyses confirmed the chemical features supporting the interactions of the studied compounds with SNAT2, which was found to be the main SNAT expressed in MCF-7 cells.

SNAT2 is responsible for hyperosmotic induced sarcosine and glycine uptake in human prostate PC-3 cells.

Solute carriers (SLC) are important membrane transport proteins in normal and pathophysiological cells. The aim was to identify amino acid SLC(s) responsible for uptake of sarcosine and glycine in prostate cancer cells and investigate the impact hereon of hyperosmotic stress. Uptake of 14C-sarcosine and 3H-glycine was measured in human prostate cancer (PC-3) cells cultured under isosmotic (300 mOsm/kg) and hyperosmotic (500 mOsm/kg) conditions for 24 h. Hyperosmotic culture medium was obtained by supplementing the medium with 200 mM of the trisaccharide raffinose. Amino acid SLC expression was studied using RT-PCR, real-time PCR, and western blotting. siRNA knockdown of SNAT2 was performed. Experiments were conducted in at least 3 independent cell passages. The uptake of Sar and Gly was increased approximately 8-ninefold in PC-3 cells after 24 h hyperosmotic culture. PAT1 mRNA and protein could not be detected, while SNAT2 was upregulated at the mRNA and protein level. Transfection with SNAT2-specific siRNA reduced Vmax of Sar uptake from 2653 ± 38 to 513 ± 38 nmol mg protein-1 min-1, without altering the Km value (3.19 ± 0.13 vs. 3.42 ± 0.71 mM), indicating that SNAT2 is responsible for at least 80% of Sar uptake in hyperosmotic cultured PC-3 cells. SNAT2 is upregulated in hyperosmotic stressed prostate cancer cells and SNAT2 is responsible for cellular sarcosine and glycine uptake in hyperosmotic cultured PC-3 cells. Sar is identified as a substrate for SNAT2, and this has physiological implications for understanding cellular solute transport in prostate cancer cells.

SNAT2/SLC38A2 Confers the Stemness of Gastric Cancer Cells via Regulating Glutamine Level.

BACKGROUND: Glutamine (Gln) is essential for cancer progression, however, few studies have been conducted to investigate the roles of Gln transporters in gastric cancer stem cells (CSCs). AIMS: This work aims to explore the roles of Gln transporters in gastric cancer cell stemness. METHODS: We collected spheres formed by gastric cancer (GC) cells through a 3-dimensional (3D) semisolid culture system which has been shown to hold CSC-like traits. Lentivirus package was used to construct GC cells with SNAT2 overexpression. Analysis of sphere-formation, stemness marker expression, ALDH activity were used to detect the effects of Gln transporters on GC cell stemness. Determination of reactive oxygen species (ROS) and Gln consumption combined with the methods analyzing cell stemness were performed to explore the underlying mechanisms. RESULTS: Gln consumption was upregulated in GC spheres compared to the parental GC cells. The Gln transporter SNAT2 was highly expressed in GC spheres compared to that in the parental GC cells. SNAT2 overexpression significantly increased the Gln consumption in GC cells and increased the expression of stemness markers, sphere-formation ability and ALDH activity. Notably, SNAT2-mediated promoting effects on GC cell stemness were rescued by Gln deprivation. What's more, high expression of SNAT2 was associated with a poor GC patient survival through different online datasets. CONCLUSIONS: SNAT2 can promote the stemness of GC cells in a Gln-dependent manner.

Sodium-coupled neutral amino acid transporter SNAT2 counteracts cardiogenic pulmonary edema by driving alveolar fluid clearance.

The constant transport of ions across the alveolar epithelial barrier regulates alveolar fluid homeostasis. Dysregulation or inhibition of Na+ transport causes fluid accumulation in the distal airspaces resulting in impaired gas exchange and respiratory failure. Previous studies have primarily focused on the critical role of amiloride-sensitive epithelial sodium channel (ENaC) in alveolar fluid clearance (AFC), yet activation of ENaC failed to attenuate pulmonary edema in clinical trials. Since 40% of AFC is amiloride-insensitive, Na+ channels/transporters other than ENaC such as Na+-coupled neutral amino acid transporters (SNATs) may provide novel therapeutic targets. Here, we identified a key role for SNAT2 (SLC38A2) in AFC and pulmonary edema resolution. In isolated perfused mouse and rat lungs, pharmacological inhibition of SNATs by HgCl2 and α-methylaminoisobutyric acid (MeAIB) impaired AFC. Quantitative RT-PCR identified SNAT2 as the highest expressed System A transporter in pulmonary epithelial cells. Pharmacological inhibition or siRNA-mediated knockdown of SNAT2 reduced transport of l-alanine across pulmonary epithelial cells. Homozygous Slc38a2-/- mice were subviable and died shortly after birth with severe cyanosis. Isolated lungs of Slc38a2+/- mice developed higher wet-to-dry weight ratios (W/D) as compared to wild type (WT) in response to hydrostatic stress. Similarly, W/D ratios were increased in Slc38a2+/- mice as compared to controls in an acid-induced lung injury model. Our results identify SNAT2 as a functional transporter for Na+ and neutral amino acids in pulmonary epithelial cells with a relevant role in AFC and the resolution of lung edema. Activation of SNAT2 may provide a new therapeutic strategy to counteract and/or reverse pulmonary edema.

ChREBP downregulates SNAT2 amino acid transporter expression through interactions with SMRT in response to a high-carbohydrate diet.

Carbohydrate responsive element-binding protein (ChREBP) has been identified as a primary transcription factor that maintains energy homeostasis through transcriptional regulation of glycolytic, lipogenic, and gluconeogenic enzymes in response to a high-carbohydrate diet. Amino acids are important substrates for gluconeogenesis, but nevertheless, knowledge is lacking about whether this transcription factor regulates genes involved in the transport or use of these metabolites. Here, we demonstrate that ChREBP represses the expression of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) in response to a high-sucrose diet in rats by binding to a carbohydrate response element (ChoRE) site located -160 bp upstream of the transcriptional start site in the SNAT2 promoter region. Additionally, immunoprecipitation assays revealed that ChREBP and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) interact with each other, as part of the complex that repress SNAT2 expression. The interaction between these proteins was confirmed by an in vivo chromatin immunoprecipitation assay. These findings suggest that glucogenic amino acid uptake by the liver is controlled by ChREBP through the repression of SNAT2 expression in rats consuming a high-carbohydrate diet.NEW & NOTEWORTHY This study highlights the key role of carbohydrate responsive element-binding protein (ChREBP) in the fine-tuned regulation between glucose and amino acid metabolism in the liver via regulation of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) expression after the consumption of a high-carbohydrate diet. ChREBP binds to a carbohydrate response element (ChoRE) site in the SNAT2 promoter region and recruits silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor to reduce SNAT2 transcription. This study revealed that ChREBP prevents the uptake of glucogenic amino acids upon the consumption of a high-carbohydrate diet.

Ablation of the ASCT2 (SLC1A5) gene encoding a neutral amino acid transporter reveals transporter plasticity and redundancy in cancer cells.

The neutral amino acid transporter solute carrier family 1 member 5 (SLC1A5 or ASCT2) is overexpressed in many cancers. To identify its roles in tumors, we employed 143B osteosarcoma cells and HCC1806 triple-negative breast cancer cells with or without ASCT2 deletion. ASCT2ko 143B cells grew well in standard culture media, but ASCT2 was required for optimal growth at <0.5 mm glutamine, with tumor spheroid growth and monolayer migration of 143B ASCT2ko cells being strongly impaired at lower glutamine concentrations. However, the ASCT2 deletion did not affect matrix-dependent invasion. ASCT2ko 143B xenografts in nude mice exhibited a slower onset of growth and a higher number of small tumors than ASCT2wt 143B xenografts, but did not differ in average tumor size 25 days after xenotransplantation. ASCT2 deficiency was compensated by increased levels of sodium neutral amino acid transporter 1 (SNAT1 or SLC38A1) and SNAT2 (SLC38A2) in ASCT2ko 143B cells, mediated by a GCN2 EIF2α kinase (GCN2)-dependent pathway, but this compensation was not observed in ASCT2ko HCC1806 cells. Combined SNAT1 silencing and GCN2 inhibition significantly inhibited growth of ASCT2ko HCC1806 cells, but not of ASCT2ko 143B cells. Similarly, pharmacological inhibition of l-type amino acid transporter 1 (LAT1) and GCN2 significantly inhibited growth of ASCT2ko HCC1806 cells, but not of ASCT2ko 143B cells. We conclude that cancer cells with reduced transporter plasticity are more vulnerable to disruption of amino acid homeostasis than cells with a full capacity to up-regulate redundant transporters by an integrated stress response.

Amino Acid Transporters as Targets for Cancer Therapy: Why, Where, When, and How.

Amino acids are indispensable for the growth of cancer cells. This includes essential amino acids, the carbon skeleton of which cannot be synthesized, and conditionally essential amino acids, for which the metabolic demands exceed the capacity to synthesize them. Moreover, amino acids are important signaling molecules regulating metabolic pathways, protein translation, autophagy, defense against reactive oxygen species, and many other functions. Blocking uptake of amino acids into cancer cells is therefore a viable strategy to reduce growth. A number of studies have used genome-wide silencing or knock-out approaches, which cover all known amino acid transporters in a large variety of cancer cell lines. In this review, these studies are interrogated together with other databases to identify vulnerabilities with regard to amino acid transport. Several themes emerge, such as synthetic lethality, reduced redundancy, and selective vulnerability, which can be exploited to stop cancer cell growth.

Amino Acid Transporters Are a Vital Focal Point in the Control of mTORC1 Signaling and Cancer.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. Dysregulation in the mTORC1 network underlies a wide array of pathological states, including metabolic diseases, neurological disorders, and cancer. Tumor cells are characterized by uncontrolled growth and proliferation due to a reduced dependency on exogenous growth factors. The genetic events underlying this property, such as mutations in the PI3K-Akt and Ras-Erk signaling networks, lead to constitutive activation of mTORC1 in nearly all human cancer lineages. Aberrant activation of mTORC1 has been shown to play a key role for both anabolic tumor growth and resistance to targeted therapeutics. While displaying a growth factor-independent mTORC1 activity and proliferation, tumors cells remain dependent on exogenous nutrients such as amino acids (AAs). AAs are an essential class of nutrients that are obligatory for the survival of any cell. Known as the building blocks of proteins, AAs also act as essential metabolites for numerous biosynthetic processes such as fatty acids, membrane lipids and nucleotides synthesis, as well as for maintaining redox homeostasis. In most tumor types, mTORC1 activity is particularly sensitive to intracellular AA levels. This dependency, therefore, creates a targetable vulnerability point as cancer cells become dependent on AA transporters to sustain their homeostasis. The following review will discuss the role of AA transporters for mTORC1 signaling in cancer cells and their potential as therapeutic drug targets.

Membrane topology of rat sodium-coupled neutral amino acid transporter 2 (SNAT2).

Sodium-coupled neutral amino acid transporter 2 (SNAT2) is a subtype of the amino acid transport system A that is widely expressed in mammalian tissues. It plays critical roles in glutamic acid-glutamine circulation, liver gluconeogenesis and other biological pathway. However, the topology of the SNAT2 amino acid transporter is unknown. Here we identified the topological structure of SNAT2 using bioinformatics analysis, Methoxy-polyethylene glycol maleimide (mPEG-Mal) chemical modification, protease cleavage assays, immunofluorescence and examination of glycosylation. Our results show that SNAT2 contains 11 transmembrane domains (TMDs) with an intracellular N terminus and an extracellular C terminus. Three N-glycosylation sites were verified at the largest extracellular loop. This model is consistent with the previous model of SNAT2 with the exception of a difference in number of glycosylation sites. This is the first time to confirm the SNAT2 membrane topology using experimental methods. Our study on SNAT2 topology provides valuable structural information of one of the solute carrier family 38 (SLC38) members.

Skeletal muscle amino acid transporter and BCAT2 expression prior to and following interval running or resistance exercise in mode-specific trained males.

Endurance (END)- and resistance (RES)-trained males performed interval running or resistance exercise during three consecutive days (bouts 1-3). Muscle biopsies were obtained at baseline, 2 h post-bout 1, and 72 h post-bout 3. Amino acid transporter SNAT2 mRNA was 75% greater in END (group p = 0.008), and increased ~ 70% 2 h post in both groups (time p = 0.023). Amino acid transporter PAT1 mRNA was 2.7-fold greater in RES (group p = 0.002). Baseline protein levels of the mitochondrial aminotransferase BCAT2 were 79% greater in END (p = 0.015).

Melatonin is involved in skotomorphogenesis by regulating brassinosteroid biosynthesis in rice plants.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis catalyzing the conversion of serotonin into N-acetylserotonin. In plants, SNAT is encoded by 2 isogenes of which SNAT1 is constitutively expressed and its overexpression confers increased yield in rice. However, the role of SNAT2 remains to be clarified. In contrast to SNAT1, the diurnal rhythm of SNAT2 mRNA expression peaks at night. In this study, transgenic rice plants in which SNAT2 expression were suppressed by RNAi technology showed a decrease in melatonin and a dwarf phenotype with erect leaves, reminiscent of brassinosteroids (BR)-deficient mutants. Of note, the dwarf phenotype was dependent on the presence of dark, suggesting that melatonin is involved in dark growth (skotomorphogenesis). In support of this suggestion, SNAT2 RNAi lines exhibited photomorphogenic phenotypes such as inhibition of internodes and increased expression of light-inducible CAB genes in the dark. The causative gene for the melatonin-mediated BR biosynthetic gene was DWARF4, a rate-limiting BR biosynthetic gene. Exogenous melatonin treatment induced several BR biosynthetic genes, including DWARF4, D11, and RAVL1. As expected from the erect leaves, the SNAT2 RNAi lines produced less BR than the wild type. Our results show for the first time that melatonin is a positive regulator of dark growth or shade outgrowth by regulating BR biosynthesis in plants.

Proteasomal modulation of cellular SNAT2 (SLC38A2) abundance and function by unsaturated fatty acid availability.

Expression and activity of the System A/SNAT2 (SLC38A2) amino acid transporter is up-regulated by amino acid starvation and hypertonicity by a mechanism dependent on both ATF4-mediated transcription of the SLC38A2 gene and enhanced stabilization of SNAT2 itself, which forms part of an integrated cellular stress response to nutrient deprivation and osmotic stress. Here we demonstrate that this adaptive increase in System A function is restrained in cells subjected to prior incubation with linoleic acid (LOA, an unsaturated C18:2 fatty acid) for 24 h. While fatty acid treatment had no detectable effect upon stress-induced SNAT2 or ATF4 gene transcription, the associated increase in SNAT2 protein/membrane transport activity were strongly suppressed in L6 myotubes or HeLa cells preincubated with LOA. Cellular ubiquitination of many proteins was increased by LOA and although the fatty acid-induced loss of SNAT2 could be attenuated by proteasomal inhibition, the functional increase in System A transport activity associated with amino acid starvation/hypertonicity that depends upon processing/maturation and delivery of SNAT2 to the cell surface could not be rescued. LOA up-regulated cellular expression of Nedd4.2, an E3-ligase implicated in SNAT2 ubiquitination, but shRNA-directed Nedd4.2 gene silencing could not curb fatty acid-induced loss of SNAT2 adaptation. However, expression of SNAT2 in which seven putative lysyl-ubiquitination sites in the cytoplasmic N-terminal domain were mutated to alanine protected SNAT2 against LOA-induced proteasomal degradation. Collectively, our findings indicate that increased availability of unsaturated fatty acids can compromise the stress-induced induction/adaptation in SNAT2 expression and function by promoting its degradation via the ubiquitin-proteasome system.

Prolactin and the dietary protein/carbohydrate ratio regulate the expression of SNAT2 amino acid transporter in the mammary gland during lactation.

The sodium coupled neutral amino acid transporter 2 (SNAT2/SAT2/ATA2) is expressed in the mammary gland (MG) and plays an important role in the uptake of alanine and glutamine which are the most abundant amino acids transported into this tissue during lactation. Thus, the aim of this study was to assess the amount and localization of SNAT2 before delivery and during lactation in rat MG, and to evaluate whether prolactin and the dietary protein/carbohydrate ratio might influence SNAT2 expression in the MG, liver and adipose tissue during lactation. Our results showed that SNAT2 protein abundance in the MG increased during lactation and this increase was maintained along this period, while 24 h after weaning it tended to decrease. To study the effect of prolactin on SNAT2 expression, we incubated MG explants or T47D cells transfected with the SNAT2 promoter with prolactin, and we observed in both studies an increase in the SNAT2 expression or promoter activity. Consumption of a high-protein/low carbohydrate diet increased prolactin concentration, with a concomitant increase in SNAT2 expression not only in the MG during lactation, but also in the liver and adipose tissue. There was a correlation between SNAT2 expression and serum prolactin levels depending on the amount of dietary protein/carbohydrate ratio consumed. These findings suggest that prolactin actively supports lactation providing amino acids to the gland through SNAT2 for the synthesis of milk proteins.

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.

Postprandial nutrient-sensing and metabolic responses after partial dietary fishmeal replacement by soyabean meal in turbot (Scophthalmus maximus L.).

In this study, we chose a carnivorous fish, turbot (Scophthalmus maximus L.), to examine its nutrient-sensing and metabolic responses after ingestion of diets with fishmeal (FM), or 45% of FM replaced by soyabean meal (34·6% dry diet) balanced with or without essential amino acids (EAA) to match the amino acid profile of FM diet for 30 d. After a 1-month feeding trial, fish growth, feed efficiency and nutrient retention were markedly reduced by soyabean meal-incorporated (SMI) diets. Compared with the FM diet, SMI led to a reduction of postprandial influx of free amino acids, hypoactivated target of rapamycin signalling and a hyperactivated amino acid response pathway after refeeding, a status associated with reduced protein synthesis, impaired postprandial glycolysis and lipogenesis. These differential effects were not ameliorated by matching an EAA profile of soyabean meal to that of the FM diet through dietary amino acid supplementation. Therefore, this study demonstrated that the FM diet and SMI diets led to distinct nutrient-sensing responses, which in turn modulated metabolism and determined the utilisation efficiency of diets. Our results provide a new molecular explanation for the role of nutrient sensing in the inferior performance of aquafeeds in which FM is replaced by soyabean meal.

GSK3-mediated raptor phosphorylation supports amino-acid-dependent mTORC1-directed signalling.

The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser(859). We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation.

System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts.

Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [(14)C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (Km) of betaine uptake by human and rat SNAT2 were estimated to be 5.3 mM and 4.6 mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.