Cationic Amino Acid Transporter Research Articles

OR12-03 The Pancreatic Islet Alpha Cell Arginine Transporter SLC7A2 Regulates Arginine-induced Glucagon Secretion and Glycemia

Abstract Disclosure: J.E. Stanley: None. A. Reuter: None. K. Sellick: None. A.D. Attie: None. M.P. Keller: None. E. Dean: None. Glucagon secreted from pancreatic islet α cells stimulates both hepatic glycogenolysis and gluconeogenesis resulting in the fractional extraction of amino acids from the blood. Thus, glucagon stimulates hepatic glucose output and raises blood glucose. When liver glucagon signaling is impaired, amino acid accumulation (hyperaminoacidemia) promotes subsequent α cell hyperplasia and hyperglucagonemia via this liver-α-cell axis, an endocrine feedback loop. We show that high levels of both glutamine and recently arginine are required to stimulate α cells to proliferate. Arginine also potently stimulates glucagon secretion, but the mechanisms behind this are not well defined. We find that the cationic amino acid transporter, SLC7A2/CAT2 is the most highly expressed amino acid transporter in human pancreatic α cells and three-fold higher in α cells than it is in β-cells in both mouse and human islets. We also find two SNPs in the human SLC7A2 locus are strongly associated with HbA1c (rs142010226 (A/G) - chr8: 17,367,112; p=9.11e-18; β=0.0113 and rs2517232 - chr8: 17,367,421; p=2.553e-14; β=-0.0283). Both SNPs are in the first intron of SLC7A2 but appear to exert opposite effect on HbA1c and associated with distinct haplotype blocks. ATAC-Seq analyses of human islets show that these SNPs are within 1 kb of binding sites for MAFB and FOXA2 transcription factors, both critical for α cell gene expression. To test if α cell expression of SLC7A2 plays a role in both glycemia and arginine-stimulated glucagon secretion, we generated α cell-specific Slc7a2-/- (α7A2KO) mice. Blood glucose and glucagon levels were not significantly different after a 6-hour fast in α7A2KO and 7A2WTCre+ mice. After a 6-hour fast, mice were administered an arginine bolus (2g/kg) via intraperitoneal injections and their blood glucose and glucagon levels were measured 15 minutes post-injection. α7A2KO mice had a significantly higher blood glucose level than 7A2WTcre+ mice after stimulation with arginine (arginine-stimulated blood glucose - 7A2WTCre+ 124.3 ± 26.7 mg/dl vs. α7A2KO 159.6 ± 32.1 mg/dl n=23-25 p=0.0019). However, glucagon secretion was significantly lower after stimulation with arginine in the α7A2KO mice compared to the 7A2WTCre+ mice (arginine-stimulated glucagon - 7A2WTCre+ 321.8 ± 171.9 pM vs. α7A2KO 74.6 ± 22.8 pM n=4-6; p<0.0001). Together, these data indicate that SLC7A2 mediates arginine’s effects in α cells and plays an important role in regulating glycemia. Presentation: Friday, June 16, 2023

Research progress on the role of cationic amino acid transporter (CAT) family members in malignant tumors and immune microenvironment.

Amino acids are essential for the survival of all living organisms and living cells. Amino acid transporters mediate the transport and absorption of amino acids, and the dysfunction of these proteins can induce human diseases. Cationic amino acid transporters (CAT family, SLC7A1-4, and SLC7A14) are considered to be a group of transmembrane transporters, of which SLC7A1-3 are essential for arginine transport in mammals. Numerous studies have shown that CAT family-mediated arginine transport is involved in signal crosstalk between malignant tumor cells and immune cells, especially T cells. The modulation of extracellular arginine concentration has entered a number of clinical trials and achieved certain therapeutic effects. Here, we review the role of CAT family on tumor cells and immune infiltrating cells in malignant tumors and explore the therapeutic strategies to interfere with extracellular arginine concentration, to elaborate its application prospects. CAT family members may be used as biomarkers for certain cancer entities and might be included in new ideas for immunotherapy of malignant tumors.

Effect of mannan-rich fractions and a mixture of sodium butyrate and zinc proteinate on performance, intestinal morphometry, and gene expression in broilers

This study evaluated the effects of isolated or combined prebiotics blended with sodium butyrate and zinc proteinate on performance, intestinal morphometry, and expression of genes associated with nutrient transport and immune response in broilers. 1,400 birds were randomly assigned to 5 treatments (Negative control (NC) without antibiotic; Positive control (PC) with antibiotic; Prebiotic (Prebio) composed of mannan fractions; Blend, composed of sodium butyrate, hydrolysed yeast cell wall and zinc proteinate; and the combination Prebio + Blend), with eight replications and 35 birds per experimental unit. The PC led to higher weight gain (WG) and lowered feed conversion (FCR) from 1 to 7 days of life than the other treatments. The use of prebio and blend resulted in high WG from 22 to 42 days (1773; 1768.9 g, respectively). However, the combination of prebio + blend was similar to PC and NC treatments. The additives resulted in longer duodenal villi when administered alone and in ileal villi (p = 0.001) combined, with no effect on jejunal villi. Crypt depth and duodenal villus: crypt ratio was higher for both additives types. A significant difference among treatments in the expression of the sodium-dependent glucose transporter gene (SGLT1) was noted, with greater expression in birds that received NC. There was higher expression of the neutral and cationic amino acid transporter gene (y + LAT-2) with the prebio and blend, either alone or in combination. The prebio blend with sodium butyrate and zinc proteinate additives used separately in broiler diets supported beneficial changes in intestinal morphometry, nutrient absorption, and performance, indicating their value in antibiotic-free broiler production systems.

Investigation of molecular details of a bacterial cationic amino acid transporter (GkApcT) during arginine transportation using molecular dynamics simulation and umbrella sampling techniques.

Cationic amino acid transporters (CATs) facilitate arginine transport across membranes and maintain its levels in various tissues and organs, but their overexpression has been associated with severe cancers. A recent study identified the alternating access mechanism and critical residues involved in arginine transportation in a cationic amino acid transporter from Geobacillus kaustophilus (GkApcT). Here, we used molecular dynamics (MD) simulation methods to investigate the transportation mechanism of arginine (Arg) through GkApcT. The results revealed that arginine strongly interacts with specific binding site residues (Thr43, Asp111, Glu115, Lys191, Phe231, Ile234, and Asp237). Based on the umbrella sampling, the main driving force for arginine transport is the polar interactions of the arginine with channel-lining residues. An in-depth description of the dissociation mechanism and binding energy analysis brings valuable insight into the interactions between arginine and transporter residues, facilitating the design of effective CAT inhibitors in cancer cells. The membrane-protein system was constructed by uploading the prokaryotic CAT (PDB ID: 6F34) to the CHARMM-GUI web server. Molecular dynamics simulations were done using the GROMACS package, version 5.1.4, with the CHARMM36 force field and TIP3P water model. The MM-PBSA approach was performed for determining the arginine binding free energy. Furthermore, the hotspot residues were identified through per-residue decomposition analysis. The characteristics of the channel such as bottleneck radius and channel length were analyzed using the CaverWeb 1.1 web server. The proton wire inside the transporter was investigated based on the classic Grotthuss mechanism. We also investigated the atomistic details of arginine transportation using the path-based free energy umbrella sampling technique (US).

A Cationic Amino Acid Transporter NtCAT1 Promotes Leaf Senescence by the Accumulation of ABA in Nicotiana tabacum

Leaf senescence is a comprehensive process performed by integrating various internal and external signals, followed by nutrient reallocation, especially via organic nitrogen (e.g., amino acids) mobilization. Amino acid (AA) transporters play an important role in crop growth and development by participating in the process of organic nitrogen remobilization. However, the biological functions and downstream effectors of amino acid transporters involved in leaf senescence are still poorly understood. In the present study, we cloned and characterized a cationic amino acid transporter gene, NtCAT1, from tobacco (K326). We found that NtCAT1 transcript levels were induced by age and abscisic acid (ABA). The NtCAT1 protein was highly localized in the plasma membrane. The overexpressing NtCAT1 line (OECAT1) showed early leaf senescence, accompanied with increased reactive oxygen species (ROS) and ABA content. By contrast, the NtCAT1 mutant (ntcat1-36) generated by the CRSPR/Cas9 system, showed a delayed-senescence phenotype with a decreased accumulation of ROS and ABA. Moreover, we discovered that the overexpression of NtCAT1 could downregulate the expression of the target of rapamycin (TOR) kinase gene and upregulate the transcript levels of ABA-related genes during leaf senescence compared with wild type (WT), while the expression of these genes in ntcat1-36 plants exhibited inverse trends. Furthermore, an analysis of the amino acid concentration demonstrated that NtCAT1 transgenic plants displayed dramatic changes in the amino acid profile during leaf senescence. In summary, our results suggest that NtCAT1 could promote leaf senescence via the increased biosynthesis of ABA, and our study provides new insights into the molecular mechanism of leaf senescence.

Plasma calprotectin is extremely high in patients with lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is a rare autosomal recessive disorder affecting the transport of cationic amino acids. Elevated plasma zinc concentrations have been described in patients with LPI. Calprotectin is a calcium- and zinc-binding protein, produced by polymorphonuclear leukocytes and monocytes. Both zinc and calprotectin have an important role in immune system. In this study, we describe plasma zinc and plasma calprotectin concentrations in Finnish LPI patients. Plasma calprotectin concentration was measured from 10 LPI patients using an enzyme-linked immunosorbent assay (ELISA) and it was remarkably high in all LPI patients (median: 622 338 μg/L) compared to that in healthy controls (608 μg/L). Plasma zinc concentration was measured by photometry and it was normal or only mildly elevated (median: 14.9 μmol/L). All the patients had decreased glomerular infiltration rate (median: 50 mL/min/1.73 m2). In conclusion, we observed extremely high plasma calprotectin concentration in patients with LPI. Mechanism of this phenomenon is unknown.

Loss of the mitochondrial protein Abcb10 results in altered arginine metabolism in MEL and K562 cells and nutrient stress signaling through ATF4

Abcb10 is a mitochondrial membrane protein involved in hemoglobinization of red cells. Abcb10 topology and ATPase domain localization suggest it exports a substrate, likely biliverdin, out of mitochondria that is necessary for hemoglobinization. In this study, we generated Abcb10 deletion cell lines in both mouse murine erythroleukemia and human erythroid precursor human myelogenous leukemia (K562) cells to better understand the consequences of Abcb10 loss. Loss of Abcb10 resulted in an inability to hemoglobinize upon differentiation in both K562 and mouse murine erythroleukemia cells with reduced heme and intermediate porphyrins and decreased levels of aminolevulinic acid synthase 2 activity. Metabolomic and transcriptional analyses revealed that Abcb10 loss gave rise to decreased cellular arginine levels, increased transcripts for cationic and neutral amino acid transporters with reduced levels of the citrulline to arginine converting enzymes argininosuccinate synthetase and argininosuccinate lyase. The reduced arginine levels in Abcb10-null cells gave rise to decreased proliferative capacity. Arginine supplementation improved both Abcb10-null proliferation and hemoglobinization upon differentiation. Abcb10-null cells showed increased phosphorylation of eukaryotic translation initiation factor 2 subunit alpha, increased expression of nutrient sensing transcription factor ATF4 and downstream targets DNA damage inducible transcript 3 (Chop), ChaC glutathione specific gamma-glutamylcyclotransferase 1 (Chac1), and arginyl-tRNA synthetase 1 (Rars). These results suggest that when the Abcb10 substrate is trapped in the mitochondria, the nutrient sensing machinery is turned on remodeling transcription to block protein synthesis necessary for proliferation and hemoglobin biosynthesis in erythroid models.

Identification of transport systems involved in eflornithine delivery across the blood-brain barrier.

Human African Trypanosomiasis (HAT) is a neglected parasitic disease that continues to persist in sub-Saharan Africa. It is fatal if untreated. The first stage of the disease is associated with the presence of the parasite in the periphery and the second stage with the presence of the parasites in the CNS. The treatment of CNS stage HAT requires the drugs to cross the blood-brain barrier (BBB). Eflornithine is an amino acid analogue that is used to treat second stage HAT gambiense both alone and in combination with nifurtimox. Recent studies have identified that accumulation of eflornithine into the parasites (trypanosomes) involves the amino acid transporter (Trypanosoma brucei AAT6). In this study we tested the hypothesis that eflornithine uses a cationic amino acid transport system to cross the BBB. We particularly focused on system-y+ and system-B0,+. To do this we utilized specialist databases to compare the physicochemical characteristics of relevant molecules and an in vitro model of the BBB to explore the mechanisms of eflornithine delivery into the CNS. Our results confirmed that eflornithine is related to the endogenous amino acid, ornithine. At pH 7.4, eflornithine is predominately (92.39%) a zwitterionic (dipolar) amino acid and ornithine is predominately (99.08%) a cationic (tripolar) amino acid. In addition, the gross charge distribution at pH 7.4 of eflornithine is much smaller (+0.073) than that of ornithine (+0.99). Further results indicated that eflornithine utilized a saturable transport mechanism(s) to cross the hCMEC/D3 cell membranes and that transport was inhibited by the presence of other amino acids including ornithine. Eflornithine transport was also sodium-independent and sensitive to a y+-system inhibitor, but not a B0,+-system inhibitor. Eflornithine transport was also inhibited by pentamidine, suggestive of transport by organic cation transporters (OCT) which are expressed in this cell line. We confirmed expression of the y+-system protein, CAT1, and the B0,+-system protein, ATB0,+, in the hCMEC/D3 cells. We conclude that eflornithine uses the cationic amino acid transporter, system y+, and OCT to cross the BBB. This research highlights the potential of system-y+ to deliver drugs, including eflornithine, across the BBB to treat brain diseases.

Cecal Microbial Succession and Its Apparent Association with Nutrient Metabolism in Broiler Chickens.

The chicken gut microbiota plays an influential role in nutrient absorption and metabolism. A clear picture of microbiota succession can enhance host nutrition and disease resistance. This study investigated the cecal microbiota succession of broilers between 3 and 42 days after hatching using 16S rRNA gene sequencing and analyzed its potential association with intestinal nutrient metabolism. Microbiota structure differed significantly at different time points depending on the microbiota alpha-diversity or beta-diversity. Proteobacteria and Bacteroidetes promoted succession on days 3 to 7 and days 28 to 35, respectively. Firmicutes and Tenericutes maintained homeostasis on days 7 to 28 and days 35 to 42. Shigella, [Ruminococcus], Erysipelotrichaceae_Clostridium, and Coprobacillus promoted succession on days 3 to 7; Faecalibacterium modified microbial composition on days 7 to 14; Faecalibacterium and Bacteroides regulated microbial structure from days 21 to 28. The microbiota structure was relatively stable on days 14 to 21 and days 28 to 35. Spearman's correlation analysis indicated a positive correlation between Lactobacillus and villus height and crypt depth (P < 0.01). Faecalibacterium and Shigella were correlated with propionate, butyrate, and valerate concentrations (P < 0.01). Ruminococcus was correlated with sodium-glucose cotransporters 1 and cationic amino acid transporter 1 expression (P < 0.05). Erysipelotrichaceae_Clostridium and Shigella were positively correlated with serum levels of total cholesterol, tryglucerides, and high- and low-density lipoprotein cholesterol (P < 0.01). Bacteroides, Parabacteroides, Lactobacillus, and Shigella were correlated with serum VB6 levels (P < 0.01). Bacteroides, Erysipelotrichaceae_Clostridium, and Coprobacillus were correlated with the moisture content of cecal contents (P < 0.05). The identification of the microbiota in correlation with nutrient metabolism will promote microbial nutrition through microbiota intervention or nutritional regulation. IMPORTANCE The poultry industry has become a global leader in livestock farming over the past few decades. Poultry production has a large consumer market as an integrated industry producing high-protein foods. Establishing the association between microbiota and nutrient metabolism processes provides fresh insights for precise nutrient regulation. This study aimed to describe the development of cecal microbiota in broiler chickens throughout the production cycle and to assess the correlation of nutrient metabolism phenotypes with temporal changes in the microbiota. The results suggested that changes in cecal microbes with age partly explain changes in gut nutrient metabolic processes, and numerous microbes were significantly associated with the processes. Therefore, this study attempts to further find efficient ways of improving poultry production. One is to promote nutrient metabolism by identifying potential candidates for probiotics, and another is to foster the dominant colonization of the microbiota by regulating nutrient metabolism.

Abstract 4879: Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid

Abstract Tranexamic Acid (TA) is an anti-fibrinolytic agent that inhibits Plasminogen activation and is used to control bleeding. Past studies have suggested that the ability of TA to block Plasminogen activation may be useful for ablating the growth or invasion of cancer. Based on the similarity of TA to the amino acid Lysine we hypothesize that TA might exhibit novel mechanisms of action independent of blocking Plasminogen activation. Analysis of a series of signaling pathways indicated that TA inhibits phosphorylation of S6K1 and STAT3 on sites required for their activation and reduces expression of the MYC oncogene in a concentration-dependent manner. Interestingly, MYC transformed MCF10A human mammary epithelial cells exhibited enhanced sensitivity to TA-mediated reduction of cell viability, suggesting that TA may exhibit selectivity for MYC overexpressing breast cancers. Treatment of breast tumor bearing mice with TA strongly blocked tumor growth and was associated with extensive cancer cell death compared with control tumors. Analysis of extracts from TA or vehicle treated tumors showed that TA blocked S6K1 phosphorylation in vivo in a statistically significant manner. Since S6K1 phosphorylation is regulated by amino acid levels, we hypothesized that due to the structural similarity between TA and Lysine and Arginine, TA may block Lysine or Arginine uptake by blocking cationic amino acid transporters (CATs). Molecular docking simulations utilizing the structure of the bacterial CAT predicted that TA binds to CAT with affinity similar to that of Lysine. Amino acid uptake experiments performed with radiolabeled Arginine and Lysine showed that TA blocked both Arginine and Lysine absorption by cancer cells in a concentration-dependent manner. Together, these results indicate a novel mechanism by which TA inhibits uptake of Arginine and Lysine, triggering loss of S6K1 activity in parallel with tumor growth inhibition. Initial studies suggest the feasibility of designing TA analogs with significantly improved potency for amino acid transporters, and with corresponding increases in selective toxicity to breast cancer cells. Based on these novel mechanisms of TA action, we expect TA to exhibit anti-cancer activity against a broad range of human malignancies. Citation Format: Brian K. Law, Mary E. Law, Amanda F. Ghilardi, Elham Yaaghubi, Brad J. Davis, Zaafir M. Dulloo, Mengxiong Wang, Olga A. Guryanova, Coy D. Heldermon, Stephan C. Jahn, Ronald K. Castellano. Inhibition of amino acid transporters as a novel mechanism of action of the repurposed anti-cancer agent tranexamic acid. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4879.

Glucose supplementation improves intestinal amino acid transport and muscle amino acid pool in pigs during chronic cold exposure.

Mammals in northern regions chronically suffer from low temperatures during autumn-winter seasons. The aim of this study was to investigate the response of intestinal amino acid transport and the amino acid pool in muscle to chronic cold exposure via Min pig models (cold adaptation) and Yorkshire pig models (non-cold adaptation). Furthermore, this study explored the beneficial effects of glucose supplementation on small intestinal amino acid transport and amino acid pool in muscle of cold-exposed Yorkshire pigs. Min pigs (Exp. 1) and Yorkshire pigs (Exp. 2) were divided into a control group (17°C, n=6) and chronic cold exposure group (7°C, n=6), respectively. Twelve Yorkshire pigs (Exp. 3) were divided into a cold control group and cold glucose supplementation group (8°C). The results showed that chronic cold exposure inhibited peptide transporter protein 1 (PepT1) and excitatory amino acid transporter 3 (EAAT3) expression in ileal mucosa and cationic amino acid transporter-1 (CAT-1) in the jejunal mucosa of Yorkshire pigs (P<0.05). In contrast, CAT-1, PepT1 and EAAT3 expression was enhanced in the duodenal mucosa of Min pigs (P<0.05). Branched amino acids (BCAA) in the muscle of Yorkshire pigs were consumed by chronic cold exposure, accompanied by increased muscle RING-finger protein-1 (MuRF1) and muscle atrophy F-box (atrogin-1) expression (P<0.05). More importantly, reduced concentrations of dystrophin were detected in the muscle of Yorkshire pigs (P<0.05). However, glycine concentration in the muscle of Min pigs was raised (P<0.05). In the absence of interaction between chronic cold exposure and glucose supplementation, glucose supplementation improved CAT-1 expression in the jejunal mucosa and PepT1 expression in the ileal mucosa of cold-exposed Yorkshire pigs (P<0.05). It also improved BCAA and inhibited MuRF1 and atrogin-1 expression in muscle (P<0.05). Moreover, dystrophin concentration was improved by glucose supplementation (P<0.05). In summary, chronic cold exposure inhibits amino acid absorption in the small intestine, depletes BCAA and promotes protein degradation in muscle. Glucose supplementation ameliorates the negative effects of chronic cold exposure on amino acid transport and the amino acid pool in muscle.

Arginine transportation mechanism through cationic amino acid transporter 1: insights from molecular dynamics studies

Metabolic and signaling mechanisms in mammalian cells are facilitated by the transportation of L-arginine (Arg) across the plasma membrane through cationic amino acid transporter (CAT) proteins. Due to a lack of argininosuccinate synthase (ASS) activity in various tumor cells such as acute myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, these tumor entities are arginine-auxotrophic and therefore depend on the uptake of the amino acid arginine. Cationic amino acid transporter-1 (CAT-1) is the leading arginine importer expressed in the aforementioned tumor entities. Hence, in the present study, to investigate the transportation mechanism of arginine in CAT-1, we performed molecular dynamics (MD) simulation methods on the modeled human CAT-1. The MM-PBSA approach was conducted to determine the critical residues interacting with arginine within the corresponding binding site of CAT-1. In addition, we found out that the water molecules have the leading role in forming the transportation channel within CAT-1. The conductive structure of CAT-1 was formed only when the water molecules were continuously distributed across the channel. Steered molecular dynamics (SMD) simulation approach showed various energy barriers against arginine transportation through CAT-1, especially while crossing the bottlenecks of the related channel. These findings at the molecular level might shed light on identifying the crucial amino acids in the binding of arginine to eukaryotic CATs and also provide fundamental insights into the arginine transportation mechanisms through CAT-1. Understanding the transportation mechanism of arginine is essential to developing CAT-1 blockers, which can be potential medications for some types of cancers.Communicated by Ramaswamy H. Sarma

Characterizing the Specific Recognition of Xanthurenic Acid by GEP1 and GEP1-GCα Interactions in cGMP Signaling Pathway in Gametogenesis of Malaria Parasites.

Gametogenesis is an essential step for malaria parasite transmission and is activated in mosquito by signals including temperature drop, pH change, and mosquito-derived xanthurenic acid (XA). Recently, a membrane protein gametogenesis essential protein 1 (GEP1) was found to be responsible for sensing these signals and interacting with a giant guanylate cyclase α (GCα) to activate the cGMP-PKG-Ca2+ signaling pathway for malaria parasite gametogenesis. However, the molecular mechanisms for this process remain unclear. In this study, we used AlphaFold2 to predict the structure of GEP1 and found that it consists of a conserved N-terminal helical domain and a transmembrane domain that adopts a structure similar to that of cationic amino acid transporters. Molecular docking results showed that XA binds to GEP1 via a pocket similar to the ligand binding sites of known amino acid transporters. In addition, truncations of this N-terminal sequence significantly enhanced the expression, solubility, and stability of GEP1. In addition, we found that GEP1 interacts with GCα via its C-terminal region, which is interrupted by mutations of a few conserved residues. These findings provide further insights into the molecular mechanism for the XA recognition by GEP1 and the activation of the gametogenesis of malaria parasites through GEP1-GCα interaction.

Lower SLC7A2 expression is associated with enhanced multidrug resistance, less immune infiltrates and worse prognosis of NSCLC

BackgroundSolute carrier family 7 member 2 (SLC7A2), a cationic amino acid transporter, is lowly expressed in ovarian and hepatocellular cancers, which is associated with their worse prognosis. However, its roles in the prognosis, drug resistance and immune infiltration in non-small-cell lung cancer (NSCLC) are unclear.MethodsWe chose SLC7A2 from RNA-Seq of paclitaxel/cisplatin-resistant A549 cells, then bioinformatics, cell lines construction, RT-qPCR, and CCK8 were performed to investigate SLC7A2 role.ResultWe analyzed the 223 differentially expressed genes (DEGs) from RNA-Seq of paclitaxel/cisplatin-resistant A549 cells and found that SLC7A2 expression was down-regulated in NSCLC. Lower SLC7A2 expression was associated with worse recurrence-free survival (RFS) in NSCLC. SLC7A2 silencing enhanced the proliferation of NSCLC cells and their insensitivity to paclitaxel, cisplatin, and gemcitabine in vitro. Activation of AMPK has up-regulated SLC7A2 expression and enhanced the sensitivity of NSCLC cells to anti-tumor drugs, which could be attributed to E2F1’s regulation. In addition, the levels of SLC7A2 expression were correlated to the numbers of infiltrated neutrophils, macrophages, dendritic cells and their marker genes, like CD86, HLA-DPA1 and ITGAM.ConclusionsSLC7A2 may act as a tumor suppressor to modulate drug sensitivity, immune infiltration and survival in NSCLC.5Lxg85-2rHuSUrkE_-2WDqVideo abstract

Effects of low-protein diet and feed restriction on mRNA expression of cationic amino acid transporters in porcine skeletal muscles.

We investigated the effects of a low-protein diet and feed restriction on the mRNA expression of cationic amino acid transporters (CATs) in the longissimus dorsi (LD), rhomboideus (RH), and biceps femoris (BF) muscles of pigs. Eighteen piglets were divided into three groups: a control (CP21%), low-protein diet (LP, CP16%), and feed-restricted diet (FR, CP21%, 76% feed intake of control pigs) groups. The expression levels of CAT-1 in the LD and BF muscles of LP pigs were higher than that of control pigs, whereas that of FR pigs showed no difference. The CAT-2A expression levels in the RH muscle of FR pigs were higher than that of control pigs. The free lysine concentrations in all muscles of LP and FR pigs were lower than that of control pigs. To examine the factors that affect CATs mRNA expression, we evaluated the effects of lysine, arginine, insulin-like growth factor-I, and dexamethasone on the expression of CATs in C2C12 myotubes. CAT-1 expression levels increased in lysine and/or arginine deprivation. We show that CAT-1 and CAT-2A expression levels in skeletal muscles differ in response to dietary treatments and CAT-1 expression in skeletal muscles appears to increase in response to low free lysine concentrations.

&lt;i&gt;Astragalus membranaceus&lt;/i&gt; and &lt;i&gt;Panax notoginseng&lt;/i&gt; saponins improves intestinal l-arginine absorption and protects against intestinal disorder &lt;i&gt;in vivo&lt;/i&gt;

Nutritional supplementation with L-arginine has been shown to promote cell growth and exercise performance, and is helpful to various disorders. Astragalus membranaceus and Panax notoginseng are traditional herbal medicines that display a wide range of beneficial activities. To understand their effects on L-arginine absorption in human intestinal cells, in vitro and in vivo experiments were performed using a standardized mixture of Astragalus membranaceus and Panax notoginseng saponins (APS). Our studies indicated that APS increased the expression of cationic amino acid transporter 1 (CAT1) and L-arginine transport in human Caco-2 cells. In addition, APS benefits TNBS-induced-colitis rats by increasing food intake, body weight, intestinal epithelial integrity, CAT1 gene expression, and blood L-arginine level. APS also reduces intestinal inflammation associated with increased myeloperoxidase activity in colitis rats. Further studies indicated that the plasma L-arginine level significantly increases in human subjects administered APS.

Evaluation of dietary arginine supplementation to increase placental nutrient transporters in aged mares.

Nine pregnant mares (18.2 ± 0.7 yr; 493.82 ± 12.74 kg body weight [BW]) were used to test the hypothesis that dietary supplementation of l-arginine would enhance placental vascularity and nutrient transport throughout gestation in aged mares. Mares were balanced by age, BW, and stallion pairing, and assigned randomly to dietary treatments of either supplemental l-arginine (50 mg/kg BW; n = 7) or l-alanine (100 mg/kg BW; n = 6; isonitrogenous control). Mares were individually fed concentrate top-dressed with the respective amino acid treatment plus ad libitum access to Coastal Bermudagrass hay. Treatments began on day 14 of gestation and were terminated at parturition. Mare BW, body condition score (BCS), and rump fat were determined, and body fat percentage was calculated every 28 d and concentrate adjusted accordingly. Doppler blood flow measurements including resistance index (RI) and pulsatility index for uterine artery ipsilateral to the pregnant uterine horn were obtained beginning on day 21 and continued every 7 d until day 154 of gestation, and prior to parturition. Parturition was attended with foaling variables and placental measures recorded. Placental tissue from the pregnant horn was analyzed histologically to assess cell-specific localization of vascular endothelial growth factor (VEGF) and cationic amino acid transporter 1 (SLC7A1) proteins. Semiquantitative analyses were performed using 10 nonoverlapping images per sample fixed in a 10× field (Fiji ImageJ v1.2). Mare performance data were analyzed using PROC MIXED in SAS and foaling and placental data were analyzed using PROC GLM. Gestation length at parturition was not influenced (P > 0.05) by supplemental arginine. Compared with arginine-supplemented mares, control mares had a thicker rump fat layer (P < 0.01) and greater percent body fat (P = 0.03), and BCS (P < 0.01) at parturition. Arginine-supplemented mares had a lower RI than control mares prior to parturition (P < 0.01). Body length, height, and BW of foals at birth, as well as placental weight and volume, and immunohistochemical staining for VEGF and SLC7A1 at parturition, were not affected (P > 0.05) by maternal arginine supplementation. These results indicate that dietary arginine supplementation (50 mg/kg BW) is safe for gestating mares. A larger number of mares is required to extend knowledge of effects of supplemental arginine on embryonic/fetal survival and growth in mares.

Abstract 1455: Determining the binding interaction between S. pombe cationic amino acid transporter Cat1 and alpha arrestin Any1 by mutational analysis

Abstract 1455: Determining the binding interaction between S. pombe cationic amino acid transporter Cat1 and alpha arrestin Any1 by mutational analysis

Comparative transcriptome analysis of Liriomyza trifolii (Burgess) and Liriomyza sativae (Blanchard) (Diptera: Agromyzidae) in response to rapid cold hardening.

The ability of insets to react efficiently to fluctuation in temperature is crucial for them to survive in variable surroundings. Rapid cold hardening (RCH) is a process that increase cold tolerance in most insect species. The molecular mechanisms of RCH remain largely unknown, and whether it is associated with transcriptional changes is unclear. In this study, we compared the transcriptomes of Liriomyza trifolii and L. sativae exposed to RCH to investigate the transcript abundance due to RCH in both species. RNA-seq revealed 93,166 assembled unigenes, and 34,303 of these were annotated in the L. trifolii and L. sativae transcriptome libraries. After a 4-h treatment at 1°C (RCH) compared with control, 268 and 606 unigenes were differentially expressed in L. trifolii and L. sativae, respectively. When comparing pupae exposed to 2h cold shock directly with pupae went through 4h acclimation prior to 2h cold shock, 60 and 399 unigenes were differentially expressed in L trifolii and L sativae, respectively. Genes that were commonly expressed in both L. trifolii and L. sativae, included cytochrome P450, cuticular protein, glucose dehydrogenase, solute carrier family 22 and cationic amino acid transporter. Additionally, several pathways including galactose metabolism and peroxisome were significantly enriched during RCH. Our results show that the transcriptional response is correlated with RCH in the pupal stage of the two Liriomyza species, but more transcriptional changes were identified in L sativae than in L. trifolii.

Distribution and characterization of extrachromosomal circular DNA in colorectal cancer

Extrachromosomal circular DNA (eccDNA) has been shown to play an important role in the amplification of tumor genes and the maintenance of intra-tumor genetic heterogeneity, although its complex functional mechanism still remains to be elucidated. As the top three common malignancies in the world, colorectal cancer (CRC) has been threatening human life and health, whose tumorigenesis and development may have elusive connection with eccDNAs. Here, we described the extensive distribution of eccDNAs in the CRC tissues using Circle-seq, which range in size from hundreds to thousands of base pairs (bp). The distribution in tumor tissues had aggregation and tendency compared with random in tumor-adjacent tissues, accompanied with smaller and more regular circle lengths. After sequencing and restoring, we found that the shedding sites of eccDNAs in CRC had similar tendency in chromosome distribution, and focused on tumor-associated genes. Meanwhile, we combined RNA sequencing to explore the correlation of eccDNA differential expression in the gene transcription and signaling pathways, confirming a connection between eccDNA and RNA somewhere. Subsequently, we validated eccDNAs in CRC cell lines and the potential consistency of the junction sites of eccDNAs in CRC tissues and cell lines. Using fragments of the cationic amino acid transporter SLC7A1 to synthesize eccDNAs, we discovered the role of eccDNAs in different regions within the gene.