Lysine deficiency and feed restriction independently alter cationic amino acid transporter expression in chickens (Gallus gallus domesticus)

The effect of a lysine-deficient diet on cationic amino acid transporter (CAT1-3) mRNA expression was determined in broiler chickens. Chicks consumed a lysine-adequate (LA; 1.3% lysine) or lysine-deficient (LD; 0.7% lysine) diet. Pair-fed chicks consumed the LA diet in an amount equal to that consumed by LD chicks during the previous day (PLA). CAT 1-3 mRNA expression in the liver, pectoralis and bursa of LD chicks were lower than that of LA and PLA chicks (P<0.05), and levels were not detectable in LD chick thymus. High affinity CAT mRNA expression in isolated bursacytes was 16-fold higher in LD chicks than that of LA chicks (P<0.001). Thymocyte high affinity CAT mRNA expression was 5-fold lower than that of LA chicks (P<0.05). The summed amount of high affinity CAT-1 and CAT-3 mRNA expression in chicks fed a lysine adequate diet was highly correlated (r2=0.51; P<0.001) to a tissue's growth during a lysine deficiency or feed restriction. In the thymus and bursa of LD chicks, CAT mRNA levels differed between resident lymphocytes and their surrounding tissues. By expressing high affinity CAT isoforms, developing lymphocytes may have a greater ability to obtain lysine than their surrounding tissue during a lysine deficiency.     

The acute phase response alters cationic amino acid transporter expression in growing chickens (Gallus gallus domesticus)

The effect of an acute phase response (APR) on cationic amino acid transporter (CAT1-3) mRNA expression in liver, muscle, bursa and thymus was determined in broiler strain chickens. The APR was initiated by injecting Salmonella typhimurium lipopolysaccharide subcutaneously (LPS; 1 mg/kg bw). In Experiment 1, CAT1-3 mRNA expression was determined at multiple time points following LPS administration. LPS increased bursa and liver total and high affinity CAT mRNA expression (P<0.05) and transiently increased pectoralis total CAT mRNA expression (P<0.05). Total CAT mRNA expression in the thymus decreased 7.7-fold from 0 to 8 h after LPS injection (P<0.05). In Experiment 2, fasted chicks were uninjected or LPS-injected. LPS increased total and high affinity CAT mRNA 2-fold in both the bursa and liver (P<0.05) and did not change thymus total and high affinity CAT mRNA expression (P>0.05). LPS increased liver weight only (P<0.05) and did not alter the plasma lysine and arginine concentration (P>0.05). In Experiments 3 and 4, thymocyte proliferation and total protein content were dependent upon the media lysine concentration (P<0.001). The inability of the thymus to compete for lysine and arginine during the APR may limit the ability of thymocytes to develop during infections.     

CATs,a family of three distinct mammalian cationic amino acid transporters

Summary Three related mammalian carrier proteins that mediate the transport of cationic amino acids through the plasma membrane have been identified in murine and human cells (CAT for cationic amino acid transporter). Models of the CAT proteins in the membrane suggest they have 12 or 14 transmembrane domains connected by short hydrophilic loops and intracellular N- and C-termini. The transport activity of the CAT proteins is sensitive to trans-stimulation and independent of the presence of sodium ions. These features agree with the behaviour of carrier proteins mediating facilitated diffusion. The three CAT proteins, CAT-1, CAT-2A and CAT-2(B) are encoded by two different genes (CAT-1 and CAT-2). CAT-1 and CAT-2(B) exhibit transport properties consistent with system y⁺, the principal mechanism for cellular uptake of cationic amino acids. In contrast, CAT-2A has tenfold lower substrate affinity, greater apparent maximal velocity and it is much less sensitive to trans-stimulation. In addition to structural and functional aspects, this review discusses the role of the CAT proteins for supplying substrate to NO synthases and the property of the rodent CAT-1 proteins to function as virus receptors.Abbreviations CAT cationic amino acid transporter - m mouse - h human - r rat - Tea T cell early activation protein - CAA cationic amino acids - TM transmembrane spanning domain - rBAT related to b^0,+ amino acid transporter - 4F2hc 4F2 heavy chain cell surface antigen - MuLV murine leukemia viruses - K_m Michaelis Menten constant     

Human cationic amino acid transporter hCAT-3 is preferentially expressed in peripheral tissues

At least five distinct carrier proteins form the family of mammalian cationic amino acid transporters (CATs). We have cloned a cDNA containing the complete coding region of human CAT-3. hCAT-3 is glycosylated and localized to the plasma membrane. Transport studies in Xenopus laevis oocytes revealed that hCAT-3 is selective for cationic L-amino acids and exhibits a maximal transport activity similar to other CAT proteins. The apparent substrate affinity and sensitivity to trans-stimulation of hCAT-3 resembles most closely hCAT-2B. This is in contrast to rat and murine CAT-3 proteins that have been reported to display a very low activity and to be inhibited by neutral and anionic L-amino acids as well as D-arginine (Hosokawa, H., et al. (1997) J. Biol. Chem. 272, 8717-8722; Ito, K., and Groudine, M. (1997) J. Biol. Chem. 272, 26780-26786). Also, in adult rat and mouse, CAT-3 has been found exclusively in central neurons. Human CAT-3 expression is not restricted to the brain, in fact, by far the highest expression was found in thymus. Also in other peripheral tissues, hCAT-3 expression was equal to or higher than in most brain regions, suggesting that hCAT-3 is not a neuron-specific transporter.     

Polyamines upregulate the mRNA expression of cationic amino acid transporter-1 in human retinal pigment epithelial cells

We previously showed that ornithine was mainly transported via cationic amino acid transporter (CAT)-1 in human retinal pigment epithelial (RPE) cell line, human telomerase RT (hTERT)-RPE, and that CAT-1 was involved in ornithine cytotoxicity in ornithine--aminotransferase (OAT)-deficient cell produced by a OAT specific inhibitor, 5-fluoromethylornithine (5-FMO). We showed here that CAT-1 mRNA expression was increased by ornithne in OAT-deficient RPE cells, which was reversed by an inhibitor of ornithine decarboxylase (ODC), -difluoromethylornithine (DFMO). Polyamines, especially spermine, one of the metabolites of ODC, also enhanced the expression of CAT-1 mRNA. ODC mRNA expression was also increased by ornithine and polyamines, and gene silencing of ODC by siRNA decreased ornithine transport activity and its cytotoxicity. In addition, the mRNA of nuclear protein c-myc was also increased in 5-FMO- and ornithine-treated hTERT-RPE cells, and gene silencing of c-myc prevented the induction of CAT-1 and ODC. Increases in expression of CAT-1, ODC, and c-myc, and the inhibition of these stimulated expression by DFMO were also observed in primary porcine RPE cells. These results suggest that spermine plays an important role in stimulation of mRNA expression of CAT-1, which is a crucial role in ornithine cytotoxicity in OAT-deficient hTERT-RPE cells. ornithine transport; ornithine decarboxylase; c-myc     

Arginase-1 overexpression induces cationic amino acid transporter-1 in psoriasis

Regulated uptake of extracellular l-arginine by cationic amino acid transporters (CATs) is required for inducible nitric oxide synthase and arginase activity. Both enzymes were recently recognized as important in the pathophysiology of psoriasis because of their contribution to epidermal hyperproliferation. We here characterize the expression pattern of CATs in psoriatic skin compared to healthy skin. CAT-1 mRNA expression was strongly upregulated in lesional and nonlesional areas of psoriatic skin compared to healthy skin, whereas expression of CAT-2A and the inducible isoform CAT-2B was unaltered in psoriatic skin. Furthermore, we tested the hypothesis that arginase-1 overexpression regulates CAT expression via intracellular l-arginine concentration. In in vitro experiments with arginase-1 overexpressing HaCaT cells, CAT-1 mRNA expression was increased. Likewise, this occurs in l-arginine-starved HaCaT cells. Both CAT-2 isoforms were not affected. Arginase-1 overexpression limits the synthesis of NO at physiological, but not supraphysiological, l-arginine levels. Plasma l-arginine concentration was diminished in psoriasis patients and the arginase product l-ornithine was significantly increased compared to healthy controls. In summary, arginase-1 overexpression leads to upregulated CAT-1 expression in psoriatic skin, which is due to lowered intracellular l-arginine levels and limits NO synthesis at physiological l-arginine concentrations.     

Expression and regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To study the molecular basis of tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we generated lines of transgenic mice carrying 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene (hGLUT4[2.4]-CAT). This reporter gene construct was specifically expressed in tissues that normally express GLUT4 mRNA, which include both brown and white adipose tissues as well as cardiac, skeletal, and smooth muscle. In contrast, CAT reporter activity was not detected in brain or liver, two tissues that do not express the GLUT4 gene. In addition, the relative levels of CAT mRNA driven by the human GLUT4 promoter in various tissues of these transgenic animals mirrored those of the endogenous mouse GLUT4 mRNA. Since previous studies have observed alterations in GLUT4 mRNA levels induced by fasting and refeeding (Sivitz, W. I., DeSautel, S. L., Kayano, T., Bell, G. I., and Pessin, J. E. (1989) Nature 340, 72-74), the regulated expression the hGLUT4[2.4]-CAT transgene was also assessed in these animals. Fasting was observed to decrease CAT activity in white adipose tissue which was super-induced upon refeeding. These alterations in CAT expression occurred in parallel to the changes in endogenous mouse GLUT4 mRNA levels. Although CAT expression in skeletal muscle and brown adipose tissue was unaffected, the endogenous mouse GLUT4 mRNA was also refractory to the effects of fasting/refeeding in these tissues. These data demonstrate that 2.4 kilobases of the 5'-flanking region of the human GLUT4 gene contain all the necessary sequence elements to confer tissue-specific expression and at least some of the sequence elements controlling the hormonal/metabolic regulation of this gene.     

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.     

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose (K(m) = 0.3 mM) and fructose (IC(50) = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 microM d-glucose was not inhibited by 200 microM phloretin or 100 microM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

The effect of early posthatch starvation on calpain mRNA levels

The calpain system is a family of calcium activated proteases that degrade myofibrillar protein. Male broiler chickens (Ross) were provided a standard starter diet top-dressed with Oasis((R)) nutritional supplement (fed; Novus International, St. Louis, MO, USA), or they were not provided any feed (starved) for the first 3 days posthatch. Subsequently, the standard starter diet was provided to all chickens between 3 and 7 days posthatch. RNA was extracted from the Pectoralis thoracicus, and skeletal muscle-specific n-calpain-1 (p94) calpain, mu-calpain, and m-calpain expression was evaluated using quantitative Northern analysis. Early posthatch starvation did not (P>0.05) affect calpain mRNA levels on each day examined. Similarly, there were no (P>0.05) changes in mu-calpain or m-calpain mRNA levels between 0 and 7 days posthatch in fed birds. However, p94 calpain mRNA levels were significantly (P<0.05) lower at 7 days posthatch compared to 0 or 2 days posthatch. Therefore, in the early posthatch chicken, it appears that the calpain system may not be affected by the presence of oral nutrition, and that there is an age-related downregulation of p94 calpain mRNA expression.     

Expression profiling of the solute carrier gene family in chicken intestine from the late embryonic to early post-hatch stages

Intestinal development during late embryogenesis and early post-hatch has a long-term influence on digestive and absorptive capacity in chickens. The objective of this research was to obtain a global view of intestinal solute carrier (SLC) gene family member expression from late embryogenesis until 2 weeks post-hatch with a focus on SLC genes involved in uptake of sugars and amino acids. Small intestine samples from male chicks were collected on embryonic days 18 (E18) and 20 (E20), day of hatch and days 1, 3, 7 and 14 post-hatch. The expression profiles of 162 SLC genes belonging to 41 SLC families were determined using Affymetrix chicken genome microarrays. The majority of SLC genes showed little or no difference in level of expression during E18–D14. A number of well-known intestinal transporters were upregulated between E18 and D14 including the amino acid transporters rBAT , y ⁺ LAT-2 and EAAT3 , the peptide transporter PepT1 and the sugar transporters SGLT1 , GLUT2 and GLUT5 . The amino acid transporters CAT-1 and CAT-2 were downregulated. In addition, several glucose and amino acid transporters that are novel to our understanding of nutrient absorption in the chicken intestine were discovered through the arrays ( SGLT6 , SNAT1 , SNAT2 and AST ). These results represent a comprehensive characterization of the expression profiles of the SLC family of genes at different stages of development in the chicken intestine and lay the ground work for future nutritional studies.     

Exofacial epitope-tagged glucose transporter chimeras reveal COOH- terminal sequences governing cellular localization

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti- HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.     

Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells

We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediated l-arginine transport in PAEC after a 1-h treatment and activated l-arginine uptake after an 18-h treatment of cells. These changes in l-arginine transport were not related to the changes in the expression of the CAT-1 transporter. The inhibitory effect of PMA on l-arginine transport was accompanied by a translocation of PKCalpha (a classical PKC isoform) from the cytosol to the membrane fraction, whereas the activating effect of PMA on l-arginine transport was accompanied by full depletion of the expression of PKCalpha in PAEC. A selective activator of Ca(2+)-dependent classical isoforms of PKC, thymeleatoxin (Thy; 100 nM; 1-h and 18-h treatments), induced the same changes in l-arginine uptake and PKCalpha translocation and depletion as PMA. The effects of PMA and Thy on l-arginine transport in PAEC were attenuated by a selective inhibitor of classical PKC isoforms Go 6976 (1 micro M). Phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP; 5 micro M), which activates novel PKC isoforms, did not affect l-arginine transport in PAEC after 1-h and 18-h treatment of cells. PIP (5 micro M; 1 h) induced the translocation of PKCepsilon (a novel PKC isoform) from the cytosolic to the particulate fraction and did not affect the translocation of PKCalpha. These results demonstrate that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. We suggest that translocation of PKCalpha to the plasma membrane induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCalpha after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of its activity.     

Association of L-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake

In this study, we investigated the possible interaction between the cationic amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin. Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited L-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in L-arginine transport activity in reconstituted proteoliposomes (3,291 +/- 117 vs. 8,101 +/- 481 pmol. mg protein(-1). 3 min(-1) in control). Incubation with anti-ankyrin antibodies had no effect on CAT-1 content or L-arginine transport in reconstituted proteoliposomes. These results demonstrate that CAT-1 arginine transporters in PAEC are associated with fodrin, but not with ankyrin, and that long-term hypoxia decreases L-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis.