Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

The organic zinc with moderate chelation strength enhances the expression of related transporters in the jejunum and ileum of broilers.

Our previous study demonstrated that the zinc (Zn) proteinate with moderate chelation strength (Zn-Prot M) enhanced the Zn absorption in the small intestine partially via increasing the expression of some Zn and amino acid transporters in the duodenum of broilers. However, it remains unknown whether the Zn-Prot M could also regulate the expression of related transporters in the jejunum and ileum of broilers in the above enhancement of Zn absorption. The present study was conducted to investigate the effect of the Zn-Prot M on the expression of related transporters in the jejunum and ileum of broilers compared to the Zn sulfate (ZnS). Zinc-deficient broilers (13-d-old) were fed with the Zn-unsupplemented basal diets (control) or the basal diets supplemented with 60 mg Zn/kg as ZnS or Zn-Prot M for 26 d. The results showed that in the jejunum, compared to the control, supplementation of the organic or inorganic Zn increased (P < 0.05) mRNA and protein expression of b0,+-type amino acid transporter (rBAT), Zn transporter 10 (ZnT10), and peptide-transporter 1 (PepT1) mRNA expression and Zn transporter 7 (ZnT7) protein expression on d 28, while y+L-type amino transporter 2 (y+LAT2) mRNA and protein expression, and protein expression of ZnT7 and ZnT10 on 28 d and zrt-irt-like protein 3 (ZIP3) and zrt-irt-like protein 5 (ZIP5) on d 39 were higher (P < 0.05) for Zn-Prot M than for ZnS. In the ileum, Zn addition regardless of Zn source up-regulated (P < 0.05) mRNA expression of Zn transporter 9 (ZnT9) and ZIP3, ZIP5, and y+LAT2 protein expression on d 28, and PepT1 mRNA and protein expression, ZIP3 and y+LAT2 mRNA expression and ZnT10 protein expression on d 39. Furthermore, Zn transporter 4 (ZnT4) and ZnT9 mRNA expression and Zn transporter 1 (ZnT1) protein expression on d 28, and y+LAT2 mRNA expression and ZnT10 and PepT1 protein expression on d 39 were higher (P < 0.05) for Zn-Prot M than for ZnS. It was concluded that the Zn-Prot M enhanced the expression of the ZnT1, ZnT4, ZnT9, ZnT10, ZIP3, ZIP5, y+LAT2, and PepT1 in the jejunum or ileum of broilers compared to the ZnS.

L-Theanine Regulates the Abundance of Amino Acid Transporters in Mice Duodenum and Jejunum via the mTOR Signaling Pathway.

The intestine is a key organ for the absorption of amino acids. L-theanine (LTA) is a structural analog of glutamine and a characteristic non-protein amino acid found in tea (Camellia sinensis) that regulates lipid and protein metabolism. The present study explored the role of LTA in intestinal amino acid absorption, protein synthesis, and its mechanisms. Overall, our findings suggest that LTA supplementation not only affects serum alkaline phosphatase (AKP), total protein (TP), and urea nitrogen (BUN) levels, but it also upregulates the mRNA and protein expression of amino acid transporters (EAAT3, EAAT1, 4F2hc, y+LAT1, CAT1, ASCT2, and B0AT1), and activates the mTOR signaling pathway. The downstream S6 and S6K1 proteins are regulated, and the expression of amino acid transporters is regulated. These findings suggest that LTA increases intestinal AA absorption, promotes protein metabolism, and increases nitrogen utilization by upregulating AAT expression, activating the mTOR signaling pathway, and phosphorylating the mTOR downstream proteins S6 and S6K1.

Arginine Regulates TOR Signaling Pathway through SLC38A9 in Abalone Haliotis discus hannai.

Arginine plays an important role in the regulation of the target of the rapamycin (TOR) signaling pathway, and Solute Carrier Family 38 Member 9 (SLC38A9) was identified to participate in the amino acid-dependent activation of TOR in humans. However, the regulations of arginine on the TOR signaling pathway in abalone are still unclear. In this study, slc38a9 of abalone was cloned, and the slc38a9 was knocked down and overexpressed to explore its function in the regulation of the TOR signaling pathway. The results showed that knockdown of slc38a9 decreased the expression of tor, ribosomal s6 protein kinase (s6k) and eukaryotic translation initiation factor 4e (eif4e) and inhibited the activation of the TOR signaling pathway by arginine. Overexpression of slc38a9 up-regulated the expression of TOR-related genes. In addition, hemocytes of abalone were treated with 0, 0.2, 0.5, 1, 2 and 4 mmol/L of arginine, and abalones were fed diets with 1.17%, 1.68% and 3.43% of arginine, respectively, for 120 days. Supplementation of arginine (0.5-4 mmol/L) increased the expressions of slc38a9, tor, s6k and eif4e in hemocytes, and abalone fed with 1.68% of dietary arginine showed higher mRNA levels of slc38a9, tor, s6k and eif4e and phosphorylation levels of TOR, S6 and 4E-BP. In conclusion, the TOR signaling pathway of abalone can be regulated by arginine, and SLC38A9 plays an essential role in this regulation.

The Amino Acid-mTORC1 Pathway Mediates APEC TW-XM-Induced Inflammation in bEnd.3 Cells.

The blood-brain barrier (BBB) is key to establishing and maintaining homeostasis in the central nervous system (CNS); meningitis bacterial infection can disrupt the integrity of BBB by inducing an inflammatory response. The changes in the cerebral uptake of amino acids may contribute to inflammatory response during infection and were accompanied by high expression of amino acid transporters leading to increased amino acid uptake. However, it is unclear whether amino acid uptake is changed and how to affect inflammatory responses in mouse brain microvascular endothelial (bEnd.3) cells in response to Avian Pathogenic Escherichia coli TW-XM (APEC XM) infection. Here, we firstly found that APEC XM infection could induce serine (Ser) and glutamate (Glu) transport from extracellular into intracellular in bEnd.3 cells. Meanwhile, we also shown that the expression sodium-dependent neutral amino acid transporter 2 (SNAT2) for Ser and excitatory amino acid transporter 4 (EAAT4) for Glu was also significantly elevated during infection. Then, in amino acid deficiency or supplementation medium, we found that Ser or Glu transport were involving in increasing SNAT2 or EAAT4 expression, mTORC1 (mechanistic target of rapamycin complex 1) activation and inflammation, respectively. Of note, Ser or Glu transport were inhibited after SNAT2 silencing or EAAT4 silencing, resulting in inhibition of mTORC1 pathway activation, and inflammation compared with the APEC XM infection group. Moreover, pEGFP-SNAT2 overexpression and pEGFP-EAAT4 overexpression in bEnd.3 cells all could promote amino acid uptake, activation of the mTORC1 pathway and inflammation during infection. We further found mTORC1 silencing could inhibit inflammation, the expression of SNAT2 and EAAT4, and amino acid uptake. Taken together, our results demonstrated that APEC TW-XM infection can induce Ser or Glu uptake depending on amino acid transporters transportation, and then activate amino acid-mTORC1 pathway to induce inflammation in bEnd.3 cells.

Rice amino acid transporter-like 6 (OsATL6) is involved in amino acid homeostasis by modulating the vacuolar storage of glutamine in roots.

Glutamine is a product of ammonium (NH4+ ) assimilation catalyzed by glutamine synthetase (GS) and glutamate synthase (GOGAT). The growth of NH4+ -preferring paddy rice (Oryza sativa L.) depends on root NH4+ assimilation and the subsequent root-to-shoot allocation of glutamine; however, little is known about the mechanism of glutamine storage in roots. Here, using transcriptome and reverse genetics analyses, we show that the rice amino acid transporter-like 6 (OsATL6) protein exports glutamine to the root vacuoles under NH4+ -replete conditions. OsATL6 was expressed, along with OsGS1;2 and OsNADH-GOGAT1, in wild-type (WT) roots fed with sufficient NH4 Cl, and was induced by glutamine treatment. We generated two independent Tos17 retrotransposon insertion mutants showing reduced OsATL6 expression to determine the function of OsATL6. Compared with segregants lacking the Tos17 insertion, the OsATL6 knock-down mutant seedlings exhibited lower root glutamine content but higher glutamine concentration in the xylem sap and greater shoot growth under NH4+ -replete conditions. The transient expression of monomeric red fluorescent protein-fused OsATL6 in onion epidermal cells confirmed the tonoplast localization of OsATL6. When OsATL6 was expressed in Xenopus laevis oocytes, glutamine efflux from the cell into the acidic bath solution increased. Under sufficient NH4+ supply, OsATL6 transiently accumulated in sclerenchyma and pericycle cells, which are located adjacent to the Casparian strip, thus obstructing the apoplastic solute path, and in vascular parenchyma cells of WT roots before the peak accumulation of GS1;2 and NADH-GOGAT1 occurred. These findings suggest that OsATL6 temporarily stores excess glutamine, produced by NH4+ assimilation, in root vacuoles before it can be translocated to the shoot.

Iron chelates hitch a ride on PAT1.

The nicotianamine-iron chelate [NA-Fe2+], which is found in many plant-based foods, has been recently described as a new form of bioavailable iron in mice and chickens. How NA-Fe2+ is assimilated from the diet, however, remains unclear. The current investigation by Murata et al. has identified the proton-coupled amino acid transporter 1 (PAT1) as the main mechanism by which NA-Fe2+ is absorbed in the mammalian intestine. Discovery of this new form of dietary iron and elucidation of its pathway of intestinal absorption may lead to the development of improved iron supplementation approaches.

Beta-alanine fails to improve on 5000 m running time despite increasing PAT1 expression in long-distance runners.

BACKGROUND: Beta-alanine has become a dietary supplement widely used by athletes due to its ergogenic effect. However, there is still no consensus on the performance benefit of beta-alanine on exercise lasting longer than ten minutes. The present study aimed to evaluate the effect of beta-alanine supplementation on running performance and the expression of TauT and PAT1. METHODS: This double-blind, randomized study enrolled 16 long-distance runners (37±8 years) who were randomly allocated to two groups: placebo (PLA) and beta-alanine (BA) (4.8 g/day 1) for four weeks. Maximal oxygen consumption, anthropometry, body composition, and food intake were determined. Before and after the intervention, the athletes undertook a 5000 m running time trial. Venous blood (TauT and PAT1 expressions) and ear lobe capillary blood (lactate) collected before and after exercise. Between tests, we monitored the training variables. RESULTS: The results were analyzed by t-tests and an ANOVA of repeated measures, with Sidak's post hoc (P<0.05). PLA exhibited lower body fat than BA (8.7±2.2 vs. 11.5±2.8%, P=0.04). After supplementation, there was an increase in PAT1 expression in BA when compared to PLA (1.17±0.47 vs. 0.77±0.18, P=0.04). No significant differences were shown for the 5000 m running time in PLA (PRE: 1128±72; POST: 1123±72s) and BA (PRE: 1107±95; POST: 1093±86s). CONCLUSIONS: Although beta-alanine supplementation increased PAT1 expression, there was no statistically significant improvement in 5000 m running performance. However, individual responses should be considered as the BA showed a higher delta than the PLA.

Characterization of In Vivo Function(s) of Members of the Plant Mitochondrial Carrier Family.

Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

Effects of lysine and leucine in free and different dipeptide forms on the growth, amino acid profile and transcription of intestinal peptide, and amino acid transporters in turbot (Scophthalmus maximus).

This study was conducted to evaluate the effects of different dipeptides (lysine-leucine, lysine-glycine, and leucine-glycine) and free amino acids (lysine and leucine) on the growth, gene expression of intestinal peptide and amino acid transporters, and serum free amino acid concentrations in turbot. Fish (11.98 ± 0.03 g) were fed four experimental diets supplementing with crystalline amino acids (CAA), lysine-leucine (Lys-Leu), lysine-glycine (Lys-Gly), and leucine-glycine (Gly-Leu). Fish protein hydrolysate (FPH) containing a mixture of free amino acids and small peptides was designed as a positive control diet. There was no significant difference in the growth and feed utilization among three dipeptide diets (Lys-Leu, Lys-Gly, and Gly-Leu). Compared with the CAA group, feed efficiency ratio was significantly higher in the Lys-Leu and Lys-Gly groups, and protein efficiency ratio was significantly higher in the Lys-Leu group. For peptide transporter, oligopeptide transporter 1 (PepT1) mRNA level was not affected by dietary treatments. For amino acid transporters, lower expression of B0 neutral amino acid transporter 1 (B0AT1) and proton-coupled amino acid transporter 1 (PAT1) were observed in fish fed the dipeptide and FPH diets compared with the CAA diet. In conclusion, juvenile turbot fed Lys-Leu, Gly-Leu, and Lys-Gly had a similar growth performance, whereas lysine and leucine in the Lys-Leu form can be utilized more efficiently for feed utilization than those in free amino acid from. In addition, compared to free amino acids, dipeptides and fish protein hydrolysate in diets may down-regulate the expression of amino acid transporters but did not affect the expression of PepT1.

Genome-wide identification, characterization and expression analysis of the amino acid permease gene family in tea plants (Camellia sinensis).

Amino acid permeases (AAPs) are involved in transporting a broad spectrum of amino acids and regulating physiological processes in plants. In this study, 19 AAP genes were identified from the tea plants genome database and named CsAAP1-19. Based on phylogenetic analysis, the CsAAP genes were classified into three groups, having significantly different structures and conserved motifs. In addition, an expression analysis revealed that most of CsAAP genes were specifically expressed in different tissues, especially CsAAP19 was expressed only in root. These genes also were significantly expressed in the Baiye 1 and Huangjinya cultivars. Nitrogen treatments indicated that the CsAAPs were obviously expressed in root. CsAAP2, -6, -12, -13 and - 16 were significantly expressed at 6 d after the glutamate treatment, while the expression trend at 24 h after contained the ammonium. These results improve our understanding of the CsAAP genes and their functions in nitrogen utilization in tea plants.

Dietary protein-bound or free amino acids differently affect intestinal morphology, gene expression of amino acid transporters, and serum amino acids of pigs exposed to heat stress.

Pigs exposed to heat stress (HS) increase body temperature in which can damage the intestinal epithelia and affect the absorption and availability of amino acids (AA). Protein digestion and metabolism further increase body temperature. An experiment was conducted with six pairs of pigs (of 47.3 ± 1.3 kg initial body weight) exposed to natural HS to assess the effect of substituting dietary protein-bound AA by free AA on morphology and gene expression of intestinal epithelial and serum concentration (SC) of free AA. Treatments were: high protein, 21.9% crude protein (CP) diet (HShp) and low protein, 13.5% CP diet supplemented with crystalline Lys, Thr, Met, Trp, His, Ile, Leu, Phe, and Val (HSaa). The HShp diet met or exceeded all AA requirements. The HSaa diet was formulated on the basis of ideal protein. Pigs were fed the same amount at 0700 and 1900 hours during the 21-d study. Blood samples were collected at 1700 hours (2.0 h before the evening meal), 2030 hours, and 2130 hours (1.5 and 2.5 h after the evening meal). At the end, all pigs were sacrificed to collect intestinal mucosa and a 5-cm section from each segment of the small intestine from each pig. Villi measures, expression of AA transporters (y+L and B0) in mucosa, and SC of AA were analyzed. Ambient temperature fluctuated daily from 24.5 to 42.6 °C. Weight gain and G.F were not affected by dietary treatment. Villi height tended to be larger (P ≤ 0.10) and the villi height:crypt depth ratio was higher in duodenum and jejunum of pigs fed the HSaa diet (P < 0.05). Gene expression of transporter y+L in jejunum tended to be lower (P < 0.10) and transporter B0 in the ileum was lower (P < 0.05) in HSaa pigs. Preprandial (1700 hours) SC of Arg, His, Ile, Leu, Thr, Trp, and Val was higher (P < 0.05), and Phe tended to be higher (P < 0.10) in HShp pigs. At 2030 hours (1.5 h postprandial), serum Lys, Met, and Thr were higher in the HSaa pigs (P < 0.05). At 2130 hours (2.5 h), Arg, His, Ile, Phe, and Trp were lower (P < 0.05); Met was higher (P < 0.05); and Lys tended to be higher (P < 0.10) in HSaa pigs. In conclusion, feeding HS pigs with low protein diets supplemented with free AA reduces the damage of the intestinal epithelia and seems to improve its absorption capacity, in comparison with HS pigs fed diets containing solely protein-bound AA. This information is useful to formulate diets that correct the reduced AA consumption associated with the decreased voluntary feed intake of pigs under HS.

Theanine transporters are involved in nitrogen deficiency response in tea plant (Camellia sinensis L.).

Nitrogen in soil directly influences the production and quality of tea. However, high nitrogen application in tea plantation leads to soil acidification and environmental pollution. Studies in model plants showed that plasma membrane localized amino acid transporter can regulate the distribution of amino acids to enhance nitrogen use efficiency. Our recent study identified six CsAAPs as transporters for theanine, a unique and most abundant non-proteinaceous amino acid in tea plant. In this work, we found these theanine transporters can also transport Glutamine, Glutamate, aspartate, alanine and γ-aminobutyric acid. Tissue-specific expression analyses showed that CsAAP1, CsAAP5 and CsAAP6 mainly expressed in leaves, CsAAP8 in root, CsAAP4 and CsAAP2 in stem. Furthermore, the expression of these CsAAPs was induced by nitrogen deficiency in a tissue-specific manner. Subcellular localization analyses showed that CsAAP1, CsAAP2 and CsAAP6 location were in the plasma membrane and endoplasmic reticulum. Taken together, these results suggested theanine transporters are involved in nitrogen deficiency response probably by mediating amino acid transport from roots to new shoots and from source to sink tissues in tea plants.

Effects of vitamin B6 on the growth performance, intestinal morphology, and gene expression in weaned piglets that are fed a low-protein diet1.

Vitamin B6 (VB6), which is an essential functional substance for biosome, plays an irreplaceable role in animal health. However, there are few studies that focus on the correlation between VB6 and intestinal health in weaned piglets. This study was conducted to investigate the effects of VB6 on the growth performance, intestinal morphology, and inflammatory cytokines and amino acid (AA) transporters mRNA expression in weaned piglets that are fed a low crude-protein (CP, 18%) diet. Eighteen crossbred piglets with initial body weights of 7.03 ± 0.17 kg (means ± SEM), weaned at 21-d age, were randomly assigned three diets with 0, 4, and 7 mg/kg VB6 supplementation, respectively. The experimental period lasted 14 days. Our results showed that there were no significant differences in growth performance, diarrhea rate, and biochemical parameters among the three treatments. In the jejunum, dietary VB6 supplementation did not affect the morphology and positive Ki67 counts. Dietary supplementation with 4 mg/kg VB6 decreased the mRNA expression of COX-2, IL-10, and TGF-ß (P < 0.05). Dietary supplementation with 7 mg/kg VB6 increased the mRNA expression of SLC7A1, SLC7A6, SLC16A14, and SLC38A5 (P < 0.05) and 4 or 7 mg/kg VB6 decreased SLC36A1 mRNA expression (P < 0.05). In the ileum, VB6 supplementation did not affect positive Ki67 counts but significantly decreased villus area (P < 0.05) and tended to decrease villus height (P = 0.093). Dietary supplementation with 4 mg/kg VB6 had significantly increased the mRNA expression of IL-1ß, TNF-α, COX-2, IL-10, and TGF-ß (P < 0.05). Dietary supplementation with 4 or 7 mg/kg VB6 had significantly decreased SLC6A20, SLC7A1, SLC7A6, SLC16A14, and SLC38A5 mRNA expression (P < 0.05). These findings suggest that dietary supplementation of VB6 mainly down-regulated inflammatory cytokines and up-regulated AA transporters mRNA expression in jejunum, while up-regulated (4 mg/kg) inflammatory cytokines and down-regulated AA transporters mRNA expression in ileum, which may provide a reference for the intestinal development of weaned piglets that are fed a low-CP diet.

Theanine transporters identified in tea plants (Camellia sinensis L.).

Theanine, a unique non-proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H+ -dependent. The tissue-specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root-to-bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root-to-shoot delivery in the tea plant.

Prenatal exercise reverses high-fat-diet-induced placental alterations and alters male fetal hypothalamus during late gestation in rats†.

Maternal high-fat (HF) diet negatively affects maternal metabolism and placental function. This study aimed to determine whether gestational exercise prevents the effect of HF diet on placental amino acid transporter expression and nutrient-sensing signaling and the fetal response. Pregnant Sprague-Dawley rats were either fed with a CHOW (13.5% fat) or HF (60% fat) diet during gestation and further divided into two subgroups: voluntary exercised and sedentary. Placentae were collected on gestational day (GD) 14 and GD20, and male placentae were used in this study. We found that gestational exercise ameliorated the detrimental effects of HF diet on dams' adiposity, plasma leptin, and insulin concentrations. Maternal exercise did not influence fetoplacental growth but affected male fetal hypothalamic Leprb, Stat3, Insr, Agrp, and Pomc expressions on GD20. Maternal HF diet decreased placental labyrinth thickness and increased system A amino acid transporter SNAT2 expression, while these changes were normalized by exercise. The activation of placental mechanistic target of rapamycin complex 1/4E-BP1 and LepRb/STAT3 signaling might contribute to the increased placental SNAT2 expression in HF-fed dams, which were reversed by exercise on GD20. These data highlight that gestational exercise reverses HF-diet-induced placental alterations during late gestation without influencing fetal growth. However, maternal exercise altered fetal hypothalamic gene expression, which may affect long-term offspring health.

Effects of low-protein diet supplementation with alpha-ketoglutarate on growth performance, nitrogen metabolism and mTOR signalling pathway of skeletal muscle in piglets.

Excessive protein levels in diets result in incomplete digestion of nitrogenous nutrients that are excreted from the body, causing environment pollution. Alpha-ketoglutarate (AKG) has been reported to decrease dietary protein levels, promote intestinal health in piglets and reduce environmental pollution. However, the underlying mechanisms of AKG are largely unknown. The objective of this study was to determine the effects of low-protein diet supplementation of AKG on the growth performance, nitrogen metabolism, relative expression of amino acid transporter genes and mTOR signalling pathway of skeletal muscle in piglets. Forty-eight piglets with an initial weight of 11.53 ± 0.04 kg were randomly divided into four groups. Each group had four replicates, and each replicate had three pigs. A low-protein (LP) diet (crude protein was 14.96%) served as the control without AKG, while 0.5%, 1.0% and 1.5% AKG were added to the LP diet for the other experimental groups. The trial period lasted for 28 days. Compared with the LP group, the LP + 1.0%A and LP + 1.5%A groups increased the growth performance (p < .05);increased the mRNA levels of amino acid transporters in the duodenum, anterior jejunum and posterior jejunum (p < .05); and reduced faecal nitrogen and urine nitrogen emissions (p < .05). They also showed greater mRNA levels and phosphorylated protein levels for S6 kinase beta (S6K) (p < .05), mammalian target of rapamycin (mTOR) (p < .05) and 4E-binding protein 1 (4EBP1) (p < .05) in skeletal muscle. An LP diet supplemented with AKG activated the mTOR signalling and promoted the ability of the small intestine to absorb protein, thereby increasing protein deposition. Taken together, an LP diet supplemented with AKG provides a theoretical basis for the promotion and application of AKG in piglet production.

Effect of l-theanine on growth performance, intestinal development and health, and peptide and amino acid transporters expression of broilers.

BACKGROUND: l-Theanine has multiple beneficial biological activities. However, there is little information about the use of l-theanine in broiler production. Therefore, this study investigated the effect of l-theanine on growth performance, intestinal development and health, and the mRNA levels of intestinal peptide and amino acid (AA) transporters of broilers. RESULTS: Body weight and average daily gain were increased by l-theanine, whereas feed to gain ratio was decreased (quadratic, P < 0.05). Notably, the relative weight of duodenum, jejunum and ileum, villus height, villus height to crypt depth ratio, the jejunal activities of glutathione peroxidase, total antioxidant capacity, catalase and total superoxide dismutase were increased linearly and/or quadratically by l-theanine (P < 0.05), whereas crypt depth, serum d-lactic acid, and jejunal protein carbonyls and malondialdehyde content were decreased linearly and/or quadratically (P < 0.05). Moreover, l-theanine enhanced the jejunal mRNA levels of occludin, claudin-1, E-cadherin, zona occludens-1, di- and tripeptide transporter, excitatory AA transporter 3, Na+ -independent cationic AA transporter 1, Na+ -independent cationic and zwitterionic AA transporter, Na+ - and Cl- -dependent neutral and cationic AA transporter, Na+ -independent cationic and Na+ -dependent neutral AA transporter (y+LAT) 1, y+LAT2, Na+ -independent branched-chain and aromatic AA transporter, and heavy chain corresponding to the b°,+ transport system (linear and/or quadratic, P < 0.05). CONCLUSIONS: l-Theanine beneficially affected the growth performance of broilers by improving intestinal development and health, and the intestinal mRNA levels of AA and peptide transporters. Therefore, l-theanine has the potential to be a promising feed additive for broilers. © 2020 Society of Chemical Industry.