Joan Bertran

The light subunit of system bo,+ is fully functional in the absence of the heavy subunit

The heteromeric amino acid transporters are composed of a type II glycoprotein and a non‐glycosylated polytopic membrane protein. System bo,+ exchanges dibasic for neutral amino acids. It is composed of rBAT and bo,+AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. bo,+AT‐reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the bo,+ substrates inside the liposomes. rBAT was essential for the cell surface expression of bo,+AT, but it was not required for reconstituted bo,+AT transport activity. No system bo,+ transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted bo,+AT showed kinetic asymmetry. Expressing the cystinuria‐specific mutant A354T of bo,+AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted bo,+AT activity. Thus, subunit bo,+AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters.

Arginine Transport via Cationic Amino Acid Transporter 2 Plays a Critical Regulatory Role in Classical or Alternative Activation of Macrophages

Arginine is processed by macrophages in response to the cytokines to which these cells are exposed. Th1-type cytokines induce NO synthase 2, which metabolizes arginine into nitrites, while the Th2-type cytokines produce arginase, which converts arginine into polyamines and proline. Activation of bone marrow-derived macrophages by these two types of cytokines increases l-arginine transport only through the y+ system. Analysis of the expression of the genes involved in this system showed that Slc7A1, encoding cationic amino acid transporters (CAT)1, is constitutively expressed and is not modified by activating agents, while Slc7A2, encoding CAT2, is induced during both classical and alternative activation. Macrophages from Slc7A2 knockout mice showed a decrease in l-arginine transport in response to the two kinds of cytokines. However, while NO synthase 2 and arginase expression were unmodified in these cells, the catabolism of arginine was impaired by both pathways, producing smaller amounts of nitrites and also of polyamines and proline. In addition, the induction of Slc7A2 expression was independent of the arginine available and of the enzymes that metabolize it. In conclusion, the increased arginine transport mediated by activators is strongly regulated by CAT2 expression, which could limit the function of macrophages.

Macrophages require distinct arginine catabolism and transport systems for proliferation and for activation

In murine macrophages, as a result of arginine catabolism during activation, citruline is produced under the effect of IFN‐γ and LPS, and ornithine and polyamines by IL‐4 and IL‐10. For proliferation, arginine is required from the extracellular medium and is used for protein synthesis. During activation, most arginine (>95% in 6 h) was metabolized, while under proliferation only half was incorporated into proteins. Under basal conditions, this amino acid was preferentially transported by y+L activity. During activation, arginine transport increased drastically (4–5‐fold) through y+ cationic amino acid transporter (CAT) activity. By contrast, M‐CSF induced only a modest increase in uptake (0.5‐fold). The increase in arginine transport during activation, but not proliferation, was mediated by the SLC7A2/Cat2 gene. SLC7A1/Cat1 is constitutively expressed, and is not modified by proliferating or activating agents. M‐CSF‐dependent proliferation was not affected in the macrophages of SLC7A2 knockout mice; however, these cells showed a drastic reduction in the production of citruline or ornithine and polyamines during activation. The data show that a large increase in a specific transport system (CAT2) is necessary for activation‐induced arginine metabolism, while arginine is in excess for the requirements of proliferation and a modest increase in transport occurs.

Neuregulin signaling on glucose transport in muscle cells

Neuregulin-1, a growth factor that potentiates myogenesis induces glucose transport through translocation of glucose transporters, in an additive manner to insulin, in muscle cells. In this study, we examined the signaling pathway required for a recombinant active neuregulin-1 isoform (rhHeregulin-β1, 177–244, HRG) to stimulate glucose uptake in L6E9 myotubes. The stimulatory effect of HRG required binding to ErbB3 in L6E9 myotubes. PI3K activity is required for HRG action in both muscle cells and tissue. In L6E9 myotubes, HRG stimulated PKBα, PKBγ, and PKCζ activities. TPCK, an inhibitor of PDK1, abolished both HRG- and insulin-induced glucose transport. To assess whether PKB was necessary for the effects of HRG on glucose uptake, cells were infected with adenoviruses encoding dominant negative mutants of PKBα. Dominant negative PKB reduced PKB activity and insulin-stimulated glucose transport but not HRG-induced glucose transport. In contrast, transduction of L6E9 myotubes with adenoviruses encoding a dominant negative kinase-inactive PKCζ abolished both HRG- and insulin-stimulated glucose uptake. In soleus muscle, HRG induced PKCζ, but not PKB phosphorylation. HRG also stimulated the activity of p70S6K, p38MAPK, and p42/p44MAPK and inhibition of p42/p44MAPK partially repressed HRG action on glucose uptake. HRG did not affect AMPKα1 or AMPKα2 activities. In all, HRG stimulated glucose transport in muscle cells by activation of a pathway that requires PI3K, PDK1, and PKCζ, but not PKB, and that shows cross-talk with the MAPK pathway. The PI3K, PDK1, and PKCζ pathway can be considered as an alternative mechanism, independent of insulin, to induce glucose uptake.

Heteromeric amino acid transporters explain inherited aminoacidurias.

In the past 5 years, the first genes responsible for aminoacidurias caused by defects in renal reabsorption transport mechanisms have been identified. These diseases are type I and non-type I cystinuria and lysinuric protein intolerance. This knowledge came from the molecular characterization of the first heteromeric amino acid transporters in mammals. In 1992, rBAT and 4F2hc (genes SLC3A1 and SLC3A2, respectively, in the nomenclature of the Human Genome Organization) were identified as putative heavy subunits of mammalian amino acid transporters. In 1994, it was demonstrated that mutations in SLC3A1 cause type I cystinuria. Very recently, several light subunits of the heteromeric amino acid transporters have been identified. In 1999, a putative light subunit of rBAT (the SLC7A9 gene; complementary DNA and protein termed bo,+AT) and a light subunit of 4F2hc (the SLC7A7 gene; cDNA and protein termed y+LAT-1) were shown to be the non-type I cystinuria and lysinuric protein intolerance genes, respectively. In this review, the characteristics of these heteromeric amino acid transporters and their role in these inherited aminoacidurias is described.

A New Age for Mammalian Plasma Membrane Amino Acid Transporters

A wealth of structural information on amino acid transport-related proteins has appeared during the last 4 years. Up to date, these proteins are structurally classified into five different families: c

y+ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages

Summary.This study has characterised the transport mechanism(s) for the novel and selective inhibitor of inducible nitric oxide synthase (iNOS), GW274150, in murine macrophage J774 cells. Transport of GW274150 was saturable (Km = 0.24 ± 0.01 mM and Vmax of 8.5 ± 0.12 pmol·µg protein−1 min−1), pH-insensitive and largely Na+-independent. Transport was also susceptible to trans-stimulation and was significantly inhibited by a 10-fold excess of L-arginine, L-lysine, L-leucine, L-methionine, L-glutamine and 6-diazo-5-oxo-L-norleucine but not by other amino acids or by N-ethylmaleimide. More importantly, the inhibitions caused by the neutral amino acids were critically dependent on Na+. These results strongly implicate system y+L in the transport of GW274150. Northern blot analysis confirmed this by revealing the presence of transcripts for y+LAT-1 but not y+LAT-2. Thus, taken together, our data show for the first time that J774 macrophages express y+LAT-1 transporters and that these carriers mediate transport of GW2741500 at least in these cells.

Effect of benzyl succinate on insulin receptor function and insulin action in skeletal muscle: further evidence for a lack of spare high-affinity insulin receptors.

Benzyl succinate inhibited insulin binding and tyrosine receptor kinase in a concentration-dependent manner in the partially purified insulin receptor preparation from rat skeletal muscle. Benzyl succinate lowered the apparent number of high-affinity insulin binding sites. We have made use of the inhibitory effect of benzyl succinate to investigate the possible presence of spare high-affinity insulin receptors in muscle. Benzyl succinate inhibited the effect of a supramaximal concentration of insulin on 3-O-methylglucose uptake, 2-(methylamino)isobutyric acid uptake and lactate production by the incubated muscle. Furthermore, the inhibitory effect of benzyl succinate on insulin binding in vitro closely correlated with its inhibitory effect on insulin action in vivo. These findings suggest the absence of spare high-affinity insulin receptors in skeletal muscle. In contrast to data obtained in skeletal muscle, benzyl succinate did not affect the maximally insulin-stimulated glucose transport, although it caused a marked decrease in insulin sensitivity in isolated rat adipocytes, for which the existence of spare insulin receptors is well documented.

Heteromeric amino acid transporters: cystinuria and lysinuric protein intolerance

Six families of plasma membrane amino acid transporters have been described in mammals, one of which has a heteromeric structure (Palacon et al. 1998; Chillaron et al. 2001). These heteromeric amino acid transporters (HATs) are composed of a heavy subunit and a ligh t subunit, linked by a disulfide bridge (Table 1, Figure 1). Two homologous heavy subunits (HSHATs) are known, rBAT (re lated to system bO,+ amino acid transport) and 4F2hc (heavy chain of the surface antigen 4F2, also referred to as CD98). Nine light subunits (LSHATs) have been identified. Six of them are partners of 4F2hc (LAT-l, LAT-2, y+LAT-l, y+LAT-2, asc-I, and xCT), one assembles with rBAT (bo,+AT), and two (asc-2 and AGT-l) seem to interact with as yet unknown heavy subunits (Kanai et al. 1998; Mastroberardi no et al. 1998; Torrents et al. 1998; Feliubadalo et al. 1999; Pineda et al. 1999; Rossier et al. 1999; Sato et al. 1999; Broer et al. 2000; Fukasawa et al. 2000; Chairoungdua et al. 2000; Matsuo et al. 2002).