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Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have promise in regenerative medicine because of their ability to differentiate into all 3 primary germ layers. This review describes recent advances in the understanding of the link between the metabolism of ESCs/iPSCs and their maintenance/differentiation in the cell culture setting, with particular emphasis on amino acid (AA) metabolism. ESCs are endowed with unique metabolic features with regard to energy consumption, metabolite flux through particular pathways, and macromolecular synthesis. Therefore, nutrient availability has a strong influence on stem cell growth, self-renewal, and lineage specification, both in vivo and in vitro. Evidence from several laboratories has documented that self-renewal and differentiation of mouse ESCs are critically dependent on proline metabolism, with downstream metabolites possibly serving as signal molecules. Likewise, catabolism of either threonine (mouse) or methionine (human) is required for growth and differentiation of ESCs because these AAs serve as precursors for donor molecules used in histone methylation and acetylation. Epigenetic mechanisms are recognized as critical steps in differentiation, and AA metabolism in ESCs appears to modulate these epigenetic processes. Recent reports also document that, in vitro, the nutrient composition of the culture medium in which ESCs are differentiated into embryoid bodies can influence lineage specification, leading to enrichment of a specific cell type. Although research designed to direct tissue specification of differentiating embryoid bodies in culture is still in its infancy, early results indicate that manipulation of the nutrient milieu can promote or suppress the formation of specific cell lineages.

This study addresses the mechanisms by which CHOP directs gene regulatory networks and determines cell fate. Transcriptional expression of ATF5 is activated by both CHOP and ATF4 in the integrated stress response. CHOP and ATF5 control a switch to activate apoptotic genes and decrease cell survival in response to loss of proteostatic control.

Mammals exhibit multiple adaptive mechanisms that sense and respond to fluctuations in dietary nutrients. Consumption of reduced total dietary protein or a protein diet that is deficient in 1 or more of the essential amino acids triggers wide-ranging changes in feeding behavior and gene expression. At the level of individual cells, dietary protein deficiency is manifested as amino acid (AA) deprivation, which activates the AA response (AAR). The AAR is composed of a collection of signal transduction pathways that terminate in specific transcriptional programs designed to catalyze adaptation to the nutrient stress or, ultimately, undergo apoptosis. Independently of the AAR, endoplasmic reticulum stress activates 3 signaling pathways, collectively referred to as the unfolded protein response. The transcription factor activating transcription factor 4 is one of the terminal transcriptional mediators for both the AAR and the unfolded protein response, leading to a significant degree of overlap with regard to the target genes for these stress pathways. Over the past 5 y, research has revealed that the basic leucine zipper superfamily of transcription factors plays the central role in the AAR. Formation of both homo- and heterodimers among the activating transcription factor, CCAAT enhancer-binding protein, and FOS/JUN families of basic leucine zipper proteins forms the nucleus of a highly integrated transcription factor network that determines the initiation, magnitude, and duration of the cellular response to dietary protein or AA limitation.

The neutral amino acid transport activity, System A, is enhanced by amino acid limitation of mammalian cells. Of the three gene products that encode System A activity, the one that exhibits this regulation is SNAT2 (sodium-coupled neutral amino acid transporter 2). Fibroblasts that are deficient in the amino acid response pathway exhibited little or no induction of SNAT2 mRNA. Synthesis of SNAT2 mRNA increased within 1–2 h after amino acid removal from HepG2 human hepatoma cells. The amino acid responsive SNAT2 genomic element that mediates the regulation has been localized to the first intron. Increased binding of selected members of the ATF (activating transcription factor) and C/EBP (CCAAT/enhancer-binding protein) families to the intronic enhancer was established both in vitro and in vivo. In contrast, there was no significant association of these factors with the SNAT2 promoter. Expression of exogenous individual ATF and C/EBP proteins documented that specific family members are associated with either activation or repression of SNAT2 transcription. Chromatin immunoprecipitation analysis established in vivo that amino acid deprivation led to increased RNA polymerase II recruitment to the SNAT2 promoter.

For animals, dietary protein is critical for the nutrition of the organism and, at the cellular level, protein nutrition translates into amino acid availability. Amino acid deprivation triggers the AAR (amino acid response) pathway, which causes enhanced transcription from specific target genes. The present results show that C/EBPβ (CCAAT/enhancer-binding protein β) mRNA and protein content were increased following the deprivation of HepG2 human hepatoma cells of a single amino acid. Although there was a modest increase in mRNA half-life following histidine limitation, the primary mechanism for the elevated steady-state mRNA was increased transcription. Transient transfection documented that C/EBPβ genomic fragments containing the 8451 bp 5′ upstream of the transcription start site did not contain amino-acid-responsive elements. However, deletion analysis of the genomic region located 3′ downstream of the protein coding sequence revealed that a 93 bp fragment contained an amino-acid-responsive activity that functioned as an enhancer. Exogenous expression of ATF4 (activating transcription factor 4), known to activate other genes through amino acid response elements, caused increased transcription from reporter constructs containing the C/EBPβ enhancer in cells maintained in complete amino acid medium. Chromatin immunoprecipitation demonstrated that RNA polymerase II is bound at the C/EBPβ promoter and at the 93 bp regulatory region in vivo, whereas ATF4 binds to the enhancer region only. Immediately following amino acid removal, the kinetics of binding for ATF4, ATF3, and C/EBPβ itself to the 93 bp regulatory region were similar to those observed for the amino-acid-responsive asparagine synthetase gene. Collectively the findings show that expression of C/EBPβ, which contributes to the regulation of amino-acid-responsive genes, is itself controlled by amino acid availability through transcription.

mRNA abundance for a number of genes is increased by amino acid limitation. From an array screening study in HepG2 human hepatoma cells, it was established that one set of genes affected by amino acid availability is the set associated with cell-cycle control. The present study describes the increased expression of both mRNA and protein for the cyclin-dependent kinase inhibitors p21 and p27 in response to deprivation of HepG2 cells for a single essential amino acid, histidine. The increase in p21 and p27 mRNA content depended on de novo protein synthesis and involved a post-transcriptional mRNA stabilization component. For p21, increase in mRNA by histidine depletion appeared to be independent of p53 transactivation, and the absolute level of p53 protein was unaffected by this treatment. Histidine limitation caused an increase in the phosphorylation of ERK1/ERK2 (extracellular-signal-regulated kinase), and inhibition of the ERK signal transduction pathway resulted in a reduction in the starvation-dependent increase in p21 mRNA. Blockade of the phosphoinositide 3-kinase and mTOR (mammalian target of rapamycin) pathways also blunted the increase in p21 mRNA content. These results document the amino acid-dependent control of the synthesis of specific cell-cycle regulators and help to explain the block at G1 phase after amino acid limitation.

The concept of insulin production afforded by hepatic gene therapy retains promise as a potential therapy for type 1 diabetes, but the approach has been limited by the need for strict transgene regulation in response to fluctuating levels of both glucose and insulin. Furthermore, while hepatocytes contain various glucose-responsive elements, they lack the appropriate regulated secretory system necessary for insulin release, thereby necessitating the requirement for transcriptional regulation of hepatic insulin production under the direction of a glucose-responsive promoter. To address this, we have evaluated several glucose-responsive promoters that may be used successfully for hepatic insulin production via recombinant adeno-associated virus (rAAV) therapy. Our results suggest that the human insulin promoter represents a strong candidate as a robust, glucose-responsive promoter for regulated hepatic insulin production.

Transcription from the human asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent nutrient-sensing pathways converge on the same set of genomic cis-elements, referred to as nutrient sensing-response elements (NSREs) 1 and 2, within the A.S. promoter. The present report uses single-nucleotide mutagenesis to confirm that both NSRE-1 and NSRE-2 are absolutely required for gene activation and to identify the boundaries of each binding site. The core sequence of the NSRE-1 site is contained within nucleotides −68 to −60 and the NSRE-2 core sequence is within nucleotides −48 to −43. Through insertion or deletion of 5–10 nucleotides in the intervening sequence between NSRE-1 and NSRE-2, transient transfection studies with an A.S. promoter/reporter gene construct showed that the 11 bp distance between these two elements is critical. These results document that the optimal configuration is with both binding sites on the same side of the DNA helix, only one helical turn away from each other and the data provide support for the hypothesis that a larger multi-protein complex exists between the binding proteins for NSRE-1 and NSRE-2. The data also illustrate that the combination of NSRE-1 and NSRE-2, referred to as the nutrient-sensing response unit (NSRU), has enhancer activity in that it functions in an orientation- and position-independent manner, and conveys nutrient-dependent transcriptional control to a heterologous promoter.

Glucose depletion results in cellular stress and reactive oxygen species (ROS) production, which evokes adaptive and protective responses. One such protective response is the induction of haem oxygenase 1 (HO-1), which catalyses the rate-limiting step in haem degradation, liberating iron, CO and biliverdin. The present study evaluated the role of ROS and the mitochondrial electron-transport chain in the induction of HO-1 by glucose deprivation in HepG2 hepatoma cells. Either N-acetylcysteine, an antioxidant, or deferoxamine, an iron chelator, resulted in a dose-dependent inhibition of HO-1 mRNA and protein induction during glucose deprivation, suggesting a redox- and iron-dependent mechanism. Inhibitors of electron-transport chain complex III, antimycin A and myxothiazol, the ATP synthase inhibitor oligomycin and ATP depletion with 2-deoxyglucose or glucosamine also blocked HO-1 induction. To address the involvement of ROS further, specifically H2O2, we showed that overexpression of catalase completely blocked HO-1 activation by glucose deprivation. In contrast, inhibition of nuclear factor κB, mitogen-activated protein kinase (MAPK), protein kinase A, protein kinase C, phosphoinositide 3-kinase, cyclo-oxygenase or cytosolic phospholipase A2, did not prevent HO-1 induction. These results demonstrate that activation of the HO-1 gene by glucose deprivation is mediated by a ‘glucose metabolic response’ pathway via generation of ROS and that the pathway requires a functional electron-transport chain.

A promoter element called the amino acid response element (AARE), which is essential for the induction of CHOP (a CCAAT/enhancer-binding protein-related gene) transcription by amino acid depletion, has been previously characterized. Conversely, the human asparagine synthetase (AS) promoter contains two cis-acting elements termed nutrient-sensing response elements (NSRE-1 and NSRE-2) that are required to activate the gene by either amino acid deprivation or the endoplasmic reticulum stress response. The results reported here document the comparison between CHOP and AS transcriptional control elements used by the amino acid pathway. We first establish that the AS NSRE-1 sequence shares nucleotide sequence and functional similarities with the CHOP AARE. However, we demonstrate that the CHOP AARE can function independently, whereas AS NSRE-1 is functionally weak by itself and instead requires the presence of NSRE-2. Furthermore, AS NSRE-2 can confer endoplasmic reticulum stress responsiveness to the CHOP AARE. Using activating transcription factor-2-deficient mouse embryonic fibroblasts, we also show that lack of this transcription factor does not abolish the amino acid inducibility of AS transcription, but this transcription factor is necessary to obtain the full AS response to amino acid starvation. Collectively, these results document that there are significant differences in the molecular mechanisms involved in the transcriptional activation of CHOP and AS by amino acid limitation.

Rat C6 glioma cells were used as a model system to study the biosynthesis, intracellular targeting, and degradation of the EAAC1 transporter, a sodium-dependent glutamate/aspartate transport protein that encodes System X−A,Gactivity. At steady state, nearly 70% of the EAAC1 transporter was located at the cell surface. The newly synthesized EAAC1 protein was co-translationallyN-glycosylated with high mannose oligosaccharide chains that were processed into complex-type sugar chains as the protein matured. The final maturation steps for EAAC1 protein coincided with its plasma membrane arrival, which was first detected at about 45 min after the initial synthesis. The newly synthesized EAAC1 protein was protected from degradation during the maturation and targeting process, as well as during the first 5 h after plasma membrane arrival. After this initial lag period, both the newly synthesized transporter and the total cellular EAAC1 pool were degraded by first order kinetics with a half-life of 6 h. These results represent the first analysis of the synthesis and degradation of the EAAC1 amino acid transporter.

Transcription from the asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within theA.S.promoter referred to as nutrient-sensing response elements (NSRE) 1 and 2, both of which are necessary for gene activation. The NSRE-1 sequence was used to screen ATF/CREB family members by electrophoresis mobility shift assays and supershift by specific antibodies. The results indicated that ATF4 binds to the NSRE-1 sequence and that the amount of the ATF4 complex was increased when extracts from amino acid-deprived or glucose-deprived cells were tested. Using electrophoresis mobility shift assay experiments and a probe that contained both NSRE-1 and NSRE-2, mutation of the NSRE-1 sequence completely prevented formation of the ATF4-containing complexes, whereas mutation of the NSRE-2 sequence did not. Overexpression of ATF4 increased A.S.promoter-driven transcription, whereas an inhibitory dominant negative ATF4 mutant blocked both basal and starvation-enhanced transcription. Collectively, the results provide both in vitroand in vivoevidence for a role of ATF4 in the transcriptional activation of theA.S.gene in response to nutrient deprivation.

The human asparagine synthetase (AS) gene responds to depletion of mammalian cells for either amino acids or carbohydrates. Five specific cis-elements have been implicated: three GC boxes (GC-I, GC-II and GC-III) and two nutrient-sensing response elements (NSRE-1, -2). This study shows that all three GC boxes are required to maintain basal transcription and to obtain maximal induction of the AS gene by amino acid limitation. However, there is not complete redundancy among the three GC boxes, and there is a hierarchy of importance with regard to transcription (GC-III > GC-II > GC-I). In vitro, two GC boxes formed protein-DNA complexes (GC-II and GC-III) with Sp1 and Sp3. Although transcription of the AS gene is elevated by nutrient limitation, the absolute amount of these protein-DNA complexes and the total pools of Sp1 and Sp3 did not increase. A small, but detectable portion of Sp1 was modified by phosphorylation following amino acid deprivation. In vivo, expression of Sp1 and Sp3 inDrosophilaSL2 cells increased AS promoter activity. Sp1 expression increased basal transcription but did not cause a further increase when SL2 cells were amino acid-deprived. Sp3 expression enhanced both the basal and the starvation-induced transcription.

Nutrients such as glucose regulate the expression of genes that are involved in plasma membrane transport, metabolic functions, and protein trafficking in the endoplasmic reticulum. Depletion of nutrients results in cellular stress, which evokes adaptive and protective responses, one of which is the induction of heme oxygenase-1 (HO-1), a 32-kDa endoplasmic reticulum enzyme that catalyzes the rate-limiting step in heme degradation. Incubation of HepG2 human hepatoma cells in glucose-free medium resulted in an increased HO-1 mRNA content, reaching a maximum of ∼25-fold over control cells after 12 h. The glucose-dependent induction of HO-1 mRNA was concentration-dependent (k½∼0.5 mm) and was attenuated by fructose, galactose, mannose, and 2-deoxyglucose, but not by the non-metabolizable glucose analog, 3-O-methylglucose. Tunicamycin, thapsigargin, or azetidine 2-carboxylate, each of which activates the unfolded protein response pathway, did not induce HO-1 mRNA expression, whereas glucose-regulated protein 78 mRNA was increased. These results demonstrate that glucose availability regulates transcription of the HO-1 gene via a pathway that is different from the unfolded protein response. The induction of HO-1 may serve as a protective response in hypoglycemic circumstances and underscores the importance of understanding nutrient control of the HO-1 gene.

Transcription from the human asparagine synthetase (AS) gene is increased in response to either amino acid (amino acid response) or glucose (unfolded protein response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the ASpromoter, which are referred to as nutrient-sensing response element (NSRE)-1 and -2, both of which are absolutely necessary for gene activation. The NSRE-1 sequence was used to identify the corresponding transcription factor by yeast one-hybrid screening. Based on those results, electrophoretic mobility shift assays for individual CCAAT/enhancer-binding protein-β (C/EBP) family members were performed to test for supershifting of complexes by specific antibodies. The results indicated that of all the family members, C/EBPβ bound to the NSRE-1 sequence to the greatest extent and that the absolute amount of this complex was increased when extracts from amino acid- or glucose-deprived cells were tested. Using electrophoretic mobility shift assays, mutation of the NSRE-1 sequence completely prevented formation of the C/EBPβ-containing complexes. In contrast, mutation of the NSRE-2 sequence did not block C/EBPβ binding. Overexpression in HepG2 hepatoma cells of the activating isoform of C/EBPβ increased ASpromoter-driven transcription, whereas the inhibitory dominant-negative isoform of C/EBPβ blocked enhanced transcription following amino acid or glucose deprivation. Collectively, the results provide both in vitroand in vivoevidence for a role of C/EBPβ in the transcriptional activation of the ASgene in response to nutrient deprivation.

Childhood acute lymphoblastic leukaemia is treated by combination chemotherapy with a number of drugs, almost always including the enzyme l-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance remain a problem. In vitro studies have demonstrated an adaptive increase in asparagine synthetase (AS) expression in ASNase-resistant cells, which is believed to permit ASNase-resistant human leukaemia cells to survive in vivo. The present results, obtained with ASNase-sensitive and -resistant human MOLT-4 leukaemia cell lines, illustrate that several other adaptive processes occur to provide sufficient amounts of the AS substrates, aspartate and glutamine, required to support this increased enzymic activity. In both cell populations, aspartate is derived almost exclusively from intracellular sources, whereas the necessary glutamine arises from both intracellular and extracellular sources. Transport of glutamine into ASNase-resistant cells is significantly enhanced compared with the parental cells, whereas amino acid efflux (e.g. asparagine) is reduced. Most of the adaptive change for the amino acid transporters, Systems A, ASC and L, is rapidly (12h) reversed following ASNase removal. The enzymic activity of glutamine synthetase is also enhanced in ASNase-resistant cells by a post-transcriptional mechanism. The results demonstrate that there are several sites of metabolic adaptation in ASNase-treated leukaemia cells that serve to promote the replenishment of both glutamine and asparagine.

Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme l-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate.

The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both amino acid and glucose deprivation of cells. The data reported here document that the human AS gene is induced following activation of the UnfoldedResponse Pathway (UPR), also known as theEndoplasmic Reticulum StressResponse (ERSR) in mammals. Increased AS transcription occurs in response to glucose deprivation, tunicamycin, or azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway. Previously identified ERSR target genes contain multiple copies of a single highly conserved cis-element. In contrast, the human AS gene does not contain the ERSR element, as it has been described for other responsive genes. Instead, AS induction requires an Sp1-like sequence, a sequence previously shown to be associated with amino acid control of transcription, and possibly, a third region containing no consensus sequences for known transcription factors. Oligonucleotides covering each of these regions form DNA-protein complexes in vitro, and for some the amount of these complexes is greater when nuclear extracts from glucose-starved cells are tested. These results document that a wider range of metabolic activities are activated by the UPR/ERSR pathway than previously recognized and that genomic elements other than those already described can serve to enhance transcription of specific target genes.

Transcription of the asparagine synthetase (AS) gene is induced by amino acid deprivation. The present data illustrate that this gene is also under transcriptional control by carbohydrate availability. Incubation of human HepG2 hepatoma cells in glucose-free medium resulted in an increased AS mRNA content, reaching a maximum of about 14-fold over control cells after approx. 12 h. Extracellular glucose caused the repression of the content of AS mRNA in a concentration-dependent manner, with a k1/2 (concentration causing a half-maximal repression) of 1 mM. Fructose, galactose, mannose, 2-deoxyglucose and xylitol were found to maintain the mRNA content of both AS and the glucose-regulated protein GRP78 in a state of repression, whereas 3-O-methylglucose did not. Incubation in either histidine-free or glucose-free medium also resulted in adaptive regulation of the AS gene in BNL-CL.2 mouse hepatocytes, rat C6 glioma cells and human MOLT4 lymphocytes, in addition to HepG2 cells. In contrast, the steady-state mRNA content of GRP78 was unaffected by amino acid availability. Transient transfection assays using a reporter gene construct documented that glucose deprivation increases AS gene transcription via elements within the proximal 3 kbp of the AS promoter. These results illustrate that human AS gene transcription is induced following glucose limitation of the cells.

Summary Improvements in the collagenase perfusion techniques have made isolated rat hepatocytes a popular model in which to study hepatic function. Our knowledge of hepatic amino acid transport has been advanced as a result of this methodology. Translocation across the hepatocyte plasma membrane can, in some instances, represent the rate-limiting step in the overall metabolism of certain amino acids. Furthermore, regulation of amino acid uptake by hepatocytes appears to play a role in diabetes, and perhaps in malignant transformation. Comparisons between normal adult hepatocytes and several hepatoma cell lines show basic differences in amino acids transport. There are at least eight distinct systems in normal hepatocytes for transport of the amino acids. One of these, System A, transports the small neutral amino acids most efficiently and responds to a wide variety of hormones. Systems A and N exhibit enhanced uptake rates after the cells have been maintained in the absence of extracellular amino acids, a phenomenon termed adaptive control. Further studies using isolated hepatocytes will increase our basic understanding of membrane transport processes and their regulation.

Given the central role of protein synthesis in cellular function, it is likely that intricate mechanisms exist to detect and respond to amino acid deprivation. However, the current understanding of amino acid‐dependent control of gene expression in mammalian cells is limited. A few examples of enzymes, transporters, and unidentified mRNA species subject to amino acid availability have been reported and some examples are summarized here, Each example chosen — asparagine synthetase, system A transport activity, and ribosomal protein L17 — are associated with different aspects of amino acid metabolism, and therefore reflect the spectrum of metabolic pathways influenced by substrate control. Most of the data accumulated thus far suggest that a general control response exists such that these various activities are induced when any one of several amino acids becomes limiting. Consistent with observations in yeast, it appears that the degree of tRNA acylation and its resultant effect on protein synthesis may play an important role in initiating the starvation signal. De novo protein synthesis is required for starvation‐dependent increases in several mRNA species, which suggests that the amino acid signaling pathway is composed of a series of intermediate steps before activation of specific structural genes.— Kilberg, M. S., Hutson, R. G., Laine, R. O. Amino acid‐regulated gene expression in eukaryotic cells. FASEB J.8: 13‐19; 1994.

Immunohistochemistry of porcine pulmonary artery endothelial cells (PAEC) with antibodies specific for caveolin, endothelial nitric-oxide synthase (eNOS), and the arginine transporter (CAT1) demonstrates that all of these proteins co-localize in plasma membrane caveolae. When incubated with solubilized PAEC plasma membrane proteins, eNOS-specific antibody immunoprecipitates CAT1-mediated arginine transport. These results document the existence of a caveolar complex between CAT1 and eNOS in PAEC that provides a mechanism for the directed delivery of substrate arginine to eNOS. Direct transfer of extracellular arginine to membrane-bound eNOS accounts for the “arginine paradox” and explains why caveolar localization of eNOS is required for optimal nitric oxide production by endothelial cells.

Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and XAG−, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNA-transfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein–Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.4×) and EAAC1 (39×) transport activity than does the vaccinia/HeLa expression system (2.6× and 22×, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X−AG, or A; and (iii) the endogenous transport velocities of Systems ASC and XAG−in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA-1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism.

The regulation of the high affinity cationic amino acid transporter Cat-1 in Fao rat hepatoma cells by amino acid availability has been studied. Cat-1mRNA level increased (3-fold) in 4 h in response to amino acid starvation and remained high for at least 24 h. This induction was independent of the presence of serum in the media and transcription and protein synthesis were required for induction to occur. When Fao cells were shifted from amino acid-depleted media to amino acid-fed media, the levels of the induced cat-1mRNA returned to the basal level. In amino acid-fed cells, accumulation of cat-1mRNA was dependent on protein synthesis, indicating that a labile protein is required to sustain cat-1mRNA level. No change in the transcription rate of the cat-1gene during amino acid starvation was observed, indicating thatcat-1is regulated at a post-transcriptional step. System y+ mediated transport of arginine was reduced by 50% in 1 h and by 70% in 24 h after amino acid starvation. However, when 24-h amino acid-starved Fao cells were preloaded with 2 mmlysine or arginine for 1 h prior to the transport assays, arginine uptake was trans-stimulated by 5-fold. This stimulation was specific for cationic amino acids, since alanine, proline, or leucine had no effect. These data lead to the hypothesis that amino acid starvation results in an increased cat-1mRNA level to support synthesis of additional Cat-1 protein. The following lines of evidence support the hypothesis: (i) the use of inhibitors of protein synthesis in starved cells inhibits the trans-zero transport of arginine; (ii) cells starved for 1–24 h exhibited an increase of trans-stimulated arginine transport activity for the first 6 h and had no loss of activity at 24 h, suggesting that constant replenishment of the transporter protein occurs; (iii) immunofluorescent staining of 24-h fed and starved cells for cat-1 showed similar cell surface distribution; (iv) new protein synthesis is not required for trans-stimulation of arginine transport upon refeeding of 24-h starved cells. We conclude that the increased level of cat-1mRNA in response to amino acid starvation support the synthesis of Cat-1 protein during starvation and increased amino acid transport upon substrate presentation. Therefore, the cat-1mRNA content is regulated by a derepression/repression mechanism in response to amino acid availability. We propose that the amino acid-signal transduction pathway consists of a series of steps which include the post-transcriptional regulation of amino acid transporter genes.