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Among the 20 proteinogenic amino acids, glutamine (GLN) and asparagine (ASN) represent a unique cohort in containing a terminal amide in their side chain, and share a direct metabolic relationship, with glutamine generating asparagine through the ATP-dependent asparagine synthetase (ASNS) reaction. Circulating glutamine levels and metabolic flux through cells and tissues greatly exceed those for asparagine, and "glutamine addiction" in cancer has likewise received considerable attention. However, historic and recent evidence collectively suggest that in spite of its modest presence, asparagine plays an outsized regulatory role in cellular function. Here, we present a unifying evidence-based hypothesis that the amides constitute a regulatory signaling circuit, with glutamine as a driver and asparagine as a second messenger that allosterically regulates key biochemical and physiological functions, particularly cell growth and survival. Specifically, it is proposed that ASNS serves as a sensor of substrate sufficiency for S-phase entry and progression in proliferating cells. ASNS-generated asparagine serves as a subsequent second messenger that modulates the activity of key regulatory proteins and promotes survival in the face of cellular stress, and serves as a feed-forward driver of S-phase progression in cell growth. We propose that this signaling pathway be termed the amide signaling circuit (ASC) in homage to the SLC1A5-encoded ASCT2 that transports both glutamine and asparagine in a bidirectional manner, and has been implicated in the pathogenesis of a broad spectrum of human cancers. Support for the ASC model is provided by the recent discovery that glutamine is sensed in primary cilia via ASNS during metabolic stress. [ABSTRACT FROM AUTHOR]

System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hepl cells led to >90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (roTOR complex-l; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by ~50%, while signaling from mTOR/rictor (roTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and roTOR function. amino acid transport; hepatocellular carcinoma; apoptosis; protein synthesis doi:10.1152/ajpcell.00330.2006

Amino acid transporter [B.sup.0]/ASC transporter 2 (AT[B.sup.0]/ASCT2) is responsible for most glutamine uptake in human hepatoma cells. Because this transporter is not expressed in normal hepatocytes, we hypothesized that its expression is necessary for growth of human liver cancer cells. To test this hypothesis, Sloan Kettering hepatoma (SK-Hep) cells were stably transfected with an inducible 1.3-kb AT[B.sup.0]/ASCT2 antisense RNA expression plasmid under the transcriptional control of mifepristone, a synthetic steroid. Induced antisense RNA expression in monolayer cultures decreased AT[B.sup.0]/ASCT2 mRNA levels by 73% and glutamine transport rates by 65% compared with controls after 24 h, leading to a 98% decrease in cell number after 48 h. Cellular death was attributable to apoptosis based on cellular blebbing, caspase-3 activation, vital dye and terminal deoxynucieotidyl transferase-mediated dUTP nick-end labeling staining, and poly-(ADP-ribose) polymerase (PARP) cleavage. Transporter knockdown also markedly increased activities of caspases-2 and -9, marginally enhanced caspase-8 activity, and dramatically increased ASCT1 mRNA levels, presumably as a futile compensatory response. Apoptosis elicited via transporter silencing was not attributable to the double-stranded RNA-dependent protein kinase R (PKR) pathway. For comparison, glutamine deprivation also caused apoptotic cell death but with slower temporal kinetics, stimulated caspases-2 and -3 but not caspases-8 or -9 activities, and led to considerable PARP cleavage. Thus ASCT2 suppression exerts proapoptotic effects transcending those of glutamine starvation alone. We conclude that AT[B.sup.0]/ASCT2 expression is necessary for SK-Hep cell growth and viability and suggest that it be further explored as a selective target for human hepatocellular carcinoma. caspase; glutamine; hepatocellular carcinoma

Human hepatoma cells take up glutamine at rates severalfold faster than the system N-mediated transport rates observed in normal human hepatocytes. Amino acid inhibition, kinetic, Northern blotting, RT-PCR, and restriction enzyme analyses collectively identified the transporter responsible in six human hepatoma cell lines as amino acid transporter [B.sup.O] (AT[B.sup.O]), the human ortholog of rodent ASCT2. The majority of glutamine uptake in liver fibroblasts and an immortalized human liver epithelial cell line (THLE-5B) was also mediated by AT[B.sup.O]. The 2.9-kb AT[B.sup.O] mRNA was equally expressed in all cell lines, whereas expression of the system A transporters ATA2 and ATA3 was variable. In contrast, the system N isoforms (SN1 and SN2) were expressed only in well-differentiated hepatomas. AT[B.sup.O] mRNA was also expressed in cirrhotic liver and adult and pediatric liver cancer biopsies but was not detectable in isolated human hepatocytes or fetal liver. Although the growth of all hepatomas was glutamine dependent, competitive inhibition of AT[B.sup.O]-mediated glutamine uptake blocked proliferation only in poorly differentiated cells lacking SN1 or SN2 expression and exhibiting low glutamine synthetase mRNA levels. amino acid transporter [B.sup.O]; ASCT2; SN1; SN2; hepatocellular carcinoma; transport; system A

The process by which glutamine is transported into sinusoidal endothelial cells was investigated by functionally characterizing the transport of L-glutamine in hepatic endothelial cells isolated from male rats. Functional analyses, kinetics, cation substitution and amino acid inhibition showed that a Na+-dependent carrier different from system N in parenchymal cells mediated most of the glutamine transport in hepatic endothelial cells.

Easson, Alexandra M., Timothy M. Pawlik, Craig P. Fischer, Jennifer L. Conroy, Dennis Sgroi, Wiley W. Souba, and Barrie P. Bode. Tumor-influenced amino acid transport activities in zonal-enriched hepatocyte populations. Am J Physiol Gastrointest Liver Physiol 279: G1209-G1218, 2000.--Cancer influences hepatic amino acid metabolism in the host. To further investigate this relationship, the effects of an implanted fibrosarcoma on specific amino acid transport activities were measured in periportal (PP)- and perivenous (PV)-enriched rat hepatocyte populations. [Na.sup.+]-dependent glutamate transport rates were eightfold higher in PV than in PP preparations but were relatively unaffected during tumor growth. System N-mediated glutamine uptake was 75% higher in PV than in PP preparations and was stimulated up to twofold in both regions by tumor burdens of 9 [+ or -] 4% of carcass weight compared with hepatocytes from pair-fed control animals. Excessive tumor burdens (26 [+ or -] 7%) resulted in hypophagia, loss of PV-enriched system N activities, and reduced transporter stimulation. Conversely, saturable arginine uptake was enhanced fourfold in PP preparations and was induced twofold only after excessive tumor burden. These data suggest that hepatic amino acid transporters are differentially influenced by cancer in a spatial and temporal manner, and they represent the first report of reciprocal zonal enrichment of system N and saturable arginine uptake in the mammalian liver. digitonin-collagenase; cancer; liver; glutamine; arginine

Pawlik, Timothy M., Rudiger Lohmann, Wiley W. Souba, and Barrie P. Bode. Hepatic glutamine transporter activation in burn injury: role of amino acids and phosphatidylinositol-3 kinase. Am J Physiol Gastrointest Liver Physiol 278: G532-G541, 2000.--Burn injury elicits a marked, sustained hypermetabolic state in patients characterized by accelerated hepatic amino acid metabolism and negative nitrogen balance. The transport of glutamine, a key substrate in gluconeogenesis and ureagenesis, was examined in hepatocytes isolated from the livers of rats after a 20% total burn surface area full-thickness scald injury. A latent and profound two- to threefold increase in glutamine transporter system N activity was first observed after 48 h in hepatocytes from injured rats compared with controls, persisted for 9 days, and waned toward control values after 18 days, corresponding with convalescence. Further studies showed that the profound increase was fully attributable to rapid posttranslational transporter activation by amino acid-induced cell swelling and that this form of regulation may be elicited in part by glucagon. The phosphatidylinositol-3-kinase (PI3K) inhibitors wortmannin and LY-294002 each significantly attenuated transporter stimulation by amino acids. The data suggest that PI3K-dependent system N activation by amino acids may play an important role in fueling accelerated hepatic nitrogen metabolism after burn injury. liver; glucagon; cell volume; signal transduction