Expression Of Cationic Amino Acid Transporter 1 Research Articles

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.

Gallic acid affects intestinal-epithelial-cell integrity and selected amino-acid uptake in porcine in vitro and ex vivo permeability models.

Gallic acid (GA) is widely used as a dietary supplement due to several health-promoting effects, although its effects on intestinal-epithelial-cell integrity and transport remain mostly unknown. The present study aims to clarify the effects of GA on tight junctions and intestinal nutrient uptake through in vitro and ex vivo models. Both intestinal porcine enterocyte cell line-J2 cells and porcine middle-jejunum segments were treated with 5 (T5), 25 (T25) and 50 (T50) µm GA and mounted in Ussing chambers to determine transepithelial resistance (TEER), claudin-1 (CLDN1), occludin (OCLN), zonula occludens-1 (ZO-1) protein (in tissues and cells) and mRNA (in cells) expression. In addition, uptake of l-glutamate (l-Glut), l-arginine (l-Arg), l-lysine (l-Lys) and l-methionine (l-Meth) together with cationic-amino-acid transporter-1 (CAT-1) and excitatory-amino-acid transporter-3 (EAAT3) expression was evaluated. No apoptosis was observed in GA-treated cells, but TEER and CLDN1 protein abundance was lower with T50 compared with untreated cells. l-Arg and l-Lys uptake was greater with T5 than with T25 and T50. Ex vivo, T50 decreased the TEER values and the protein levels of CLDN1, OCLN and ZO-1, whereas T5 and T25 only decreased CLDN1 protein expression compared with untreated tissues. Moreover, T25 increased l-Glut and l-Arg uptake, the latter confirmed by an increased protein expression of CAT-1. GA influences intestinal uptake of the tested cationic amino acids at low concentrations and decreases the intestinal-cell barrier function at high concentrations. Similarities were observed between in vitro and ex vivo, but different treatment times and structures must be considered.

Endothelial-specific overexpression of cationic amino acid transporter-1 prevents loss of kidney function in heart failure.

Heart failure (HF) is associated with impaired L-arginine transport. In the present study, we tested the hypothesis that augmented L-arginine transport prevents the loss of kidney function in HF. Renal function was assessed in wildtype mice (WT), transgenic mice with HF (dilated cardiomyopathy, DCM) and double transgenic mice (double transgenic mice with DCM and CAT-1 overexpression, HFCAT-1) with HF and endothelial-specific overexpression of the predominant L-arginine transporter, cationic amino acid transporter-1 (CAT-1) (n=4-8/group). Cardiac function was assessed via echocardiography and left ventricular catheterisation. Renal function was assessed via quantification of albuminuria and creatinine clearance. Plasma nitrate and nitrite levels together with renal fibrosis and inflammatory markers were also quantified at study end. Albumin/creatinine ratio was two-fold greater in DCM mice than in WT mice (P=0.002), and tubulointerstitial and glomerular fibrosis were approximately eight- and three-fold greater, respectively, in DCM mice than in WT mice (P≤0.02). Critically, urinary albumin/creatinine ratio and tubulointerstitial and glomerular fibrosis were less in HFCAT-1 mice than in DCM mice (P<0.05). Renal CAT-1 expression and plasma nitrate and nitrite levels were less in DCM mice compared with WT (P≤0.03) but was greater in HFCAT-1 mice than in DCM mice (P≤0.009). Renal expression of IL-10 was less in DCM mice compared with WT (P<0.001) but was greater in HFCAT-1 mice compared with DCM mice (P=0.02). Our data provide direct evidence that augmented L-arginine transport prevents renal fibrosis, inflammation and loss of kidney function in HF.

The circulating level of miR-122 is a potential risk factor for endothelial dysfunction in young patients with essential hypertension.

MicroRNAs are key molecules involved in the regulation of endothelial function. They are important risk factors and biomarkers for the development of hypertension related to endothelial dysfunction. However, the gene expression patterns associated with hypertension development related to endothelial dysfunction have not been fully elucidated. We conducted a case-control study of 65 patients with essential hypertension (EH) and 61 controls without EH. Plasma levels of miR-122 and its target protein high-affinity cationic amino acid transporter 1 (CAT-1) were measured by qRT-PCR and ELISA, respectively. miR-122 expression in plasma of patients with EH was significantly higher than that of the control group (p = 0.001), while CAT-1 expression in patients with EH was significantly lower than that in the control group (p = 0.018). miR-122 expression in plasma of young patients with EH was significantly higher than that in young people without EH (p = 0.0004), and CAT-1 expression in plasma of young patients with EH was also significantly lower than that of the control group (p = 0.002). CAT-1 expression in the plasma of young participants was significantly higher than that of individuals aged ≥40 years (p = 0.003), whereas miR-122 expression was significantly lower (p = 0.001). We showed that among patients with EH, the high expression of miR-122 contributed to endothelial dysfunction by suppressing the expression of the CAT-1 protein, which led to a decrease in CAT-1 expression in plasma. Therefore, high expression of miR-122 appears to be a risk factor for endothelial dysfunction in EH, especially in younger patients.

CAT1/SLC7A1 acts as a cellular receptor for bovine leukemia virus infection.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle, which is closely related to human T-cell leukemia viruses. BLV has spread worldwide and causes a serious problem for the cattle industry. The cellular receptor specifically binds with viral envelope glycoprotein (Env), and this attachment mediates cell fusion to lead virus entry. BLV Env reportedly binds to cationic amino acid transporter 1 (CAT1)/solute carrier family 7 member 1 (SLC7A1), but whether the CAT1/SLC7A1 is an actual receptor for BLV remains unknown. Here, we showed that CAT1 functioned as an infection receptor, interacting with BLV particles. Cells expressing undetectable CAT1 levels were resistant to BLV infection but became highly susceptible upon CAT1 overexpression. CAT1 exhibited specific binding to BLV particles on the cell surface and colocalized with the Env in endomembrane compartments and membrane. Knockdown of CAT1 in permissive cells significantly reduced binding to BLV particles and BLV infection. Expression of CAT1 from various species demonstrated no species specificity for BLV infection, implicating CAT1 as a functional BLV receptor responsible for its broad host range. These findings provide insights for BLV infection and for developing new strategies for treating BLV and preventing its spread.—Bai, L., Sato, H., Kubo, Y., Wada, S., Aida, Y. CAT1/SLC7A1 acts as a cellular receptor for bovine leukemia virus infection.

Transmissible gastroenteritis virus infection decreases arginine uptake by downregulating CAT-1 expression

Transmissible gastroenteritis virus (TGEV) is a coronavirus that causes severe diarrhea in suckling piglets. TGEV primarily targets and infects porcine intestinal epithelial cells, which play an important role in nutrient absorption. However, the effects of TGEV infection on nutrient absorption in swine have not yet been investigated. In this study, we evaluated the impact of TGEV infection on arginine uptake using the porcine small intestinal epithelial cell line IPEC-J2 as a model system. High performance liquid chromatography (HPLC) analyses showed that TGEV infection leads to reduced arginine uptake at 48 hours post-infection (hpi). Expression of cationic amino acid transporter 1 (CAT-1) was attenuated as well. TGEV infection induced activation of phospho-protein kinase C α (p-PKC α), phospho-epidermal growth factor receptor (p-EGFR), and enhanced the expression of caveolin-1, all of which appear to be involved in down-regulating arginine uptake and CAT-1 expression. These results illuminate the relationship between TGEV infection and nutrient absorption, and further our understanding of the mechanisms of TGEV infection.

Augmented endothelial-specific L-arginine transport prevents obesity-induced hypertension.

Hypertension is a major clinical complication of obesity. Our previous studies show that abnormal uptake of the nitric oxide precursor L-arginine, via the cationic amino acid transporter-1 (CAT1), contributes to endothelial dysfunction in cardiovascular disease. In this study, we tested the hypothesis that abnormal L-arginine transport may be a key mediator of obesity-induced hypertension. Mean arterial pressure (MAP) was monitored by telemetry in conscious wild-type (WT; n=13) mice, and transgenic mice with endothelial-specific overexpression of CAT1 (CAT+; n=14) fed a normal or a high fat diet for 20weeks. Renal angiotensin II (Ang II), CAT1 mRNA and plasma nitrate/nitrite levels were then quantified. In conjunction, plasma nitrate/nitrite levels were assessed in obese normotensive (n=15) and obese hypertensive subjects (n=15). Both genotypes of mice developed obesity when fed a high fat diet (P≤0.002). Fat fed WT mice had 13% greater MAP and 78% greater renal Ang II content, 42% lesser renal CAT1 mRNA levels and 42% lesser plasma nitrate/nitrite levels, than WT mice fed a normal fat diet (P≤0.02). In contrast, none of these variables were significantly altered by high fat feeding in CAT+ mice (P≥0.36). Plasma nitrate/nitrite levels were 17% less in obese hypertensives compared with obese normotensives (P=0.02). Collectively, these data indicate that obesity-induced down-regulation of CAT1 expression and subsequent reduced bioavailability of nitric oxide may contribute to the development of obesity-induced hypertension.

Altered growth and improved resistance of Arabidopsis against Pseudomonas syringae by overexpression of the basic amino acid transporter AtCAT1

Amino acid transporters in plants are crucial for distributing amino acids between plant organs and cellular compartments. The H(+)-coupled plasma membrane transporter CAT1 (cationic amino acid transporter 1) facilitates the high-affinity uptake of basic amino acids. The uptake of lysine (Lys) via the roots was not altered in loss-of-function mutants, in accordance with the minor expression of CAT1 in roots, but plants ectopically overexpressing CAT1 incorporated Lys at higher rates. Exogenous Lys inhibited the primary root of Arabidopsis, whereas lateral roots were stimulated. These effects were augmented by the presence or absence of CAT1. Furthermore, the total biomass of soil-grown plants ectopically overexpressing CAT1 was reduced and the time to flowering was accelerated. These effects were accompanied by only minor changes in the overall amino acid profile. Interestingly, CAT1 belongs to a specific small cluster of nitrogen-containing metabolite transporter genes that are rapidly up-regulated upon infection with Pseudomonas syringae and that may participate in the systemic response of plants to pathogen attack. The overexpression of CAT1 indeed enhanced the resistance to the hemibiotrophic bacterial pathogen P. syringae via a constitutively activated salicylic acid (SA) pathway, which is consistent with the developmental defects and the resistance phenotype.

Abstract 404: Effect of miR-122 and its target gene cationic amino acid transporter 1 on colorectal liver metastasis.

Abstract Background: Control of liver metastasis has been an important issue in the treatment of colorectal cancers. Although microRNA (miRNA) has been shown to be involved in the development of many cancers including colorectal cancers, only few studies have investigated the roles of miRNAs in the process of colorectal liver metastasis. In this study, we compared miRNA expression between primary colorectal cancers and their corresponding liver metastasis to identify miRNAs involved in colorectal liver metastasis and to detect novel molecular markers for prediction of the postoperative prognosis of colorectal cancer patients. Methods: Cancer cells were isolated from formalin-fixed paraffin-embedded (FFPE) tissues of primary colorectal cancers and their corresponding metastatic liver tumors in 6 patients using laser capture microdissection methods. miRNA expression was analyzed using TaqMan miRNA array. Protein levels of cationic amino acid transporter 1 (CAT1), a negative target gene of miR-122, in 132 primary colorectal cancers and 22 colorectal liver metastases were analyzed by immunohistochemical staining. Relationship between clinicopathological factors and CAT1 expression levels in 132 primary colorectal cancers were evaluated. Results: miR-122 was the most highly expressed in liver metastasis compared with primary tumor. Immunohistochemical analysis revealed that the expression levels of CAT1 were lower in liver metastases than primary tumors (P &amp;lt;0.001). Expression levels of CAT1 in 132 primary tumors were negatively correlated with the existence of synchronous liver metastasis (P=0.0333) and tumor stage (P &amp;lt;0.0001). In the analysis of 121 colon cancer patients without synchronous liver metastasis, patients with CAT1-low colon cancer had significantly shorter liver metastasis-free survival (P=0.0258) but not overall survival or disease-free survival. Univariate analysis, for clarifying the risk factors of metachronous colorectal liver metastasis after primary resection, revealed that the 5-year liver metastasis-free survival rate was negatively correlated with the existence of lymph node metastasis and positively correlated with expression levels of CAT1 protein in the primary tumors. Conclusions: Overexpression of miR-122 and concomitant suppression of CAT1 in the primary tumor appear to play important roles in the development of colorectal liver metastasis. CAT1 expression in the primary colorectal cancer has a potential to be a novel biomarker with which to predict the risk of postoperative liver metastasis of the patients with colorectal cancer. Citation Format: Ichirota Iino, Hirotoshi Kikuchi, Shinichiro Miyazaki, Yoshihiro Hiramatsu, Manabu Ohta, Kinji Kamiya, Takanori Sakaguchi, Mitsutoshi Setou, Hiroyuki Konno. Effect of miR-122 and its target gene cationic amino acid transporter 1 on colorectal liver metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 404. doi:10.1158/1538-7445.AM2013-404 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Effect of miR-122 and its target gene cationic amino acid transporter 1 on colorectal liver metastasis.

Control of liver metastasis is an important issue in the treatment of colorectal cancer (CRC). MicroRNAs have been shown to be involved in the development of many cancers, but little is known about their role in the process of colorectal liver metastasis. We compared miRNA expression between primary colorectal tumors and liver metastasis to identify those involved in the process of metastasis. Cancer cells were isolated from formalin-fixed paraffin-embedded primary CRC samples and their corresponding metastatic liver tumors in six patients using laser capture microdissection, and miRNA expression was analyzed using TaqMan miRNA arrays. The most abundant miRNA in liver metastasis compared with primary tumors was miR-122. Immunohistochemical analysis revealed that the expression levels of cationic amino acid transporter 1 (CAT1), a negative target gene of miR-122, were lower in liver metastases than primary tumors (P < 0.001). Expression levels of CAT1 in 132 primary tumors were negatively correlated with the existence of synchronous liver metastasis (P = 0.0333) and tumor stage (P < 0.0001). In an analysis of 121 colon cancer patients without synchronous liver metastasis, patients with CAT1-low colon cancer had significantly shorter liver metastasis-free survival (P = 0.0258) but not overall survival or disease-free survival. Overexpression of miR-122 and concomitant suppression of CAT1 in the primary tumor appears to play important roles in the development of colorectal liver metastasis. Expression of CAT1 in the primary CRC has the potential to be a novel biomarker to predict the risk of postoperative liver metastasis of CRC patients.

Arginine uptake is attenuated, through post-translational regulation of cationic amino acid transporter-1, in hyperlipidemic rats

Endothelial cell dysfunction (ECD) is a common feature of hypercholesterolemia. Defective nitric oxide (NO) generation due to decreased endothelial nitric oxide synthase (eNOS) activity is a crucial parameter characterizing ECD. l-arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate l-arginine uptake, cationic amino acid transporter-1 (CAT-1) acts as a specific arginine transporter for eNOS. Our hypothesis implies that CAT-1 is a major determinant of eNOS activity in hypercholesterolemia. We studied aortic arginine uptake, CAT-1 and CAT-2 mRNA expression, and CAT-1, and PKCα protein in: (a) control, untreated animals (CTL), (b) rats fed with 4% cholesterol + 1% cholate and 2% corn oil for 6 weeks (CHOL) and (c) rats with hypercholesterolemia treated orally with either atorvastatin (CHOL + ATORVA, 20 mg/kg BW/day) or arginine 1% (CHOL + ARG) in the drinking water (modalities which have been shown to enhance CAT-1 activity and improve endothelial function). Serum cholesterol levels significantly increased in cholesterol fed animals, an increase which was blocked by atorvastatin (CTL: 66.8 ± 15, CHOL: 133.9 ± 22, CHOL + ARG: 128.2 ± 20, CHOL + ATORVA: 77 ± 15 mg/dl). Arginine transport was significantly decreased in CHOL. Treatment with neither arginine nor atorvastatin had an effect. Using RT-PCR, we found no change in aortic CAT-1 and CAT-2 mRNA expression in CHOL as well as following arginine or atorvastatin administration. The abundance of CAT-1 protein was significantly augmented in cholesterol fed rats and was not affected by arginine or atorvastatin. PKCα protein content, which was previously shown to regulate CAT-1 activity, increased significantly in CHOL and was neither affected by atorvastatin nor arginine. In conclusion, aortic arginine uptake is attenuated in hypercholesterolemia, through post-translational modulation of CAT-1 protein, possibly via upregulation of PKCα.

Relief of microRNA-Mediated Translational Repression in Human Cells Subjected to Stress

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.

L-arginine import via cationic amino acid transporter CAT1 is essential for both differentiation and proliferation of erythrocytes

In the present study, we examined the role in hematopoiesis of cationic amino acid transporter 1 (CAT1), which transports L-arginine, L-lysine, L-ornithine, and L-histidine. The expression level of human CAT1 (hCAT1) mRNA in mononuclear cells (MNCs) fractionated according to lineage-selective markers was examined by reverse transcriptase-polymerase chain reaction. The expression of CAT1 in glycophorin A-positive erythroid cells was 8 times higher than in nonfractionated MNC (control) cells. Characteristics of L-arginine uptake by K562 cells, an established leukemic cell line used as an erythroid model, were similar to those of CAT1 in regards to saturation kinetics, sodium independence, and substantial inhibition of L-arginine uptake by N-ethylmaleimide, which is a specific inhibitor of system y+ amino acid transporter. Removal of L-arginine from the culture medium prevented both proliferation and differentiation of K562 cells, while removal of L-lysine or L-histidine had little effect on differentiation, though proliferation was blocked. Hematopoietic stem cells obtained from human cord blood failed to develop into erythroid cells in the absence of L-arginine in the culture medium. These findings indicate that hCAT1 is involved in erythroid hematopoiesis through its role in importing L-arginine, which appears to be essential for the differentiation of red blood cells.

Arginine uptake is attenuated through modulation of cationic amino-acid transporter-1, in uremic rats

Endothelial cell dysfunction (ECD) is a common feature of chronic renal failure (CRF). Defective nitric oxide (NO) generation due to decreased endothelial NO synthase (eNOS) activity is a crucial parameter characterizing ECD. L-arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate L-arginine uptake, cationic amino-acid transporter-1 (CAT-1) acts as the specific arginine transporter for eNOS. Our hypothesis implies that CAT-1 is a major determinant of eNOS activity in CRF. We studied glomerular and aortic arginine uptake, CAT-1, and CAT-2 messenger ribonucleic acid (mRNA) expression, and CAT-1 protein in: (a) rats 6 weeks following 5/6 nephrectomy (CRF), (b) sham-operated animals, and (c) rats with CRF treated orally with either atorvastatin or arginine in drinking water (modalities which have been shown to enhance eNOS activity and improve endothelial function). Both glomerular and aortic arginine transport were significantly decreased in CRF. Treatment with either arginine or atorvastatin abolished the decrease in arginine uptake in CRF rats. Using reverse transcriptase-polymerase chain reaction and Northern blotting, we found a significant increase in glomerular and aortic CAT-1 mRNA expression in CRF. Western blotting revealed that CAT-1 protein was decreased in CRF, but remained intact following arginine and atorvastatin administration. Renal and systemic arginine uptake is attenuated in CRF, through modulation of CAT-1 protein. These findings provide a possible novel mechanism to eNOS inactivation and endothelial dysfunction in uremia.

Augmented arginine uptake, through modulation of cationic amino acid transporter-1, increases GFR in diabetic rats

It is suggested that either arginine or its metabolites, nitric oxide and polyamines play a role in the renal hemodynamic alterations observed in the early stages of diabetes. Yet, the regulation of arginine transport in diabetic kidneys has never been studied. Arginine uptake was determined in glomeruli harvested from control rats; diabetic rats (2 weeks following an intraperitoneal injection of streptozotocin, 60 mg/kg body weight); rats, 4 days following left nephrectomy (a nondiabetic model of hyperfiltration); diabetes + lysine (0.5% in the drinking water to attenuate arginine uptake); and control + lysine. Glomerular arginine transport was significantly increased in diabetic rats, but remained unchanged following uninephrectomy. Lysine abolished the increase in arginine uptake in diabetic rats but had no effect in controls. The increase in creatinine clearance observed in diabetes was completely abolished by lysine. Using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and immunohistochemistry, we found a significant increase in glomerular cationic amino acid transporter-1 (CAT-1) expression in diabetic animals, which was unaffected by lysine. When human endothelial cells were incubated with arginine end products no effect on arginine transport was observed. However, only in the presence of 0.5 mM/L sodium nitroprusside (SNP) an augmented steady-state CAT-1 mRNA was demonstrated by RT-PCR. In a rat model of early diabetes, glomerular arginine uptake is elevated through modulation of CAT-1 expression, thus, contributing to the pathogenesis of hyperfiltration. Increased nitric oxide formation may play a role in this process.