Recent molecular advances in mammalian glutamine transport.

Much has been learned about plasma membrane glutamine transporter activities in health and disease over the past 30 years, including their potential regulatory role in metabolism. Since the 1960s, discrimination among individual glutamine transporters was based on functional characteristics such as substrate specificity, ion dependence, and kinetic and regulatory properties. Within the past two years, several genes encoding for proteins with these defined activities (termed "systems") have been isolated from human and rodent cDNA libraries and found to be distributed among four distinct gene families. The Na(+)-dependent glutamine transporter genes isolated thus far are System N (SN1), System A (ATA1, ATA2), System ASC/B(0) (ASCT2 or ATB(0)), System B(0,+) (ATB(0,+)) and System y(+)L (y(+)LAT1, y(+)LAT2). Na(+)-independent glutamine transporter genes encoding for System L (LAT1, LAT2) and System b(0,+) (b(0,+)AT) have also been recently isolated, and similar to y(+)L, have been shown to function as disulfide-linked heterodimers with the 4F2 heavy chain (CD98) or rBAT (related to b(0,+) amino acid transporter). In this review, the molecular features, catalytic mechanisms and tissue distributions of each are addressed. Although most of these transporters mediate the transmembrane movement of several other amino acids, their potential roles in regulating interorgan glutamine flux are discussed. Most importantly, these newly isolated transporter genes provide the long awaited tools necessary to study their molecular regulation during the catabolic states in which glutamine is considered to be "conditionally essential."

Asparagine uptake in rat hepatocytes: resolution of a paradox and insights into substrate-dependent transporter regulation.

Extracellular asparagine has previously been shown to markedly stimulate both ornithine decarboxylase and System N-mediated glutamine transport activities in hepatocytes by a transport-dependent mechanism. However, as a weak substrate of its inferred transporter System N, the specific route of asparagine uptake has remained enigmatic. In this study, asparagine transport was studied in detail and shown to be Na+-dependent, Li+-tolerant, stereospecific, and inhibited profoundly by glutamine and histidine. Coupled with competitive inhibition by glutamine (Ki = 2.63+/-1.11 mM), the data indicated that asparagine was indeed slowly transported by System N in rat hepatocytes, albeit at rates an order of magnitude less than for glutamine. The differential substrate transport velocities were shown to be attributable to a low transporter asparagine affinity (Km = 9.3 - 17.5mM) compared to glutamine (Km approximately 1 mM). Consistent with its slow uptake, asparagine accumulated to a fivefold lesser degree than glutamine after 60 min, yet stimulated System N activity to the same extent as glutamine. The transaminase inhibitor aminooxyacetate and starvation of the donor animal each enhanced asparagine uptake twofold and augmented subsequent transporter activation. Conversely, asparagine-dependent System N stimulation was abrogated by hyperosmotic media and blunted 30%-40% by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Collectively, the data suggest that System N-mediated asparagine uptake serves an autostimulatory role, mediated by cellular swelling and in part by a PI3K-dependent signal transduction pathway.

Tumor-influenced amino acid transport activities in zonal-enriched hepatocyte populations.

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(+)-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 +/- 4% of carcass weight compared with hepatocytes from pair-fed control animals. Excessive tumor burdens (26 +/- 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.

Phorbol esters rapidly attenuate glutamine uptake and growth in human colon carcinoma cells.

BACKGROUND: The amino acid glutamine, while essential for gut epithelial growth, has also been shown to stimulate colon carcinoma proliferation and diminish differentiation. Human colon carcinomas are known to extract and metabolize glutamine at rates severalfold greater than those of normal tissues, but the regulation of this response is unclear. Previously we reported that phorbol esters regulate hepatoma System ASC/B(0)-mediated glutamine uptake and cell growth. As human colon carcinoma cells use this same transporter for glutamine uptake, the present studies were undertaken to determine whether similar regulation functions in colon carcinoma. MATERIALS AND METHODS: Human colon carcinoma cell lines (WiDr and HT29) were treated with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and initial-rate transport of glutamine and other nutrients was measured at specific times thereafter. Growth rates were monitored during culture +/- PMA or an excess of System ASC/B(0) substrates relative to glutamine. RESULTS: PMA treatment induced a rapid inhibition of glutamine uptake rates in WiDr and HT29 cells by 30 and 57%, respectively, after 1 h. Cycloheximide failed to block this response, indicating that the mechanism by which PMA exerts its effects is posttranslational. The inhibition of glutamine uptake by PMA was abrogated by the PKC inhibitor staurosporine, suggesting that this rapid System ASC/B(0) regulation may be mediated by a PKC-dependent pathway. PMA also significantly decreased transport via System y(+) (arginine) and System A (small zwitterionic amino acids). Chronic phorbol ester treatment inhibited WiDr cell growth, as did attenuation of System B(0)-mediated glutamine uptake with other transporter substrates. CONCLUSIONS: System ASC/B(0) uptake governs glutamine-dependent growth in colon carcinoma cell lines, and is regulated by a phorbol ester-sensitive pathway that may involve PKC. The results further establish the link between glutamine uptake and colon carcinoma cell growth, a relationship worthy of further investigation with the goal of discovering novel cancer therapeutic targets.

Amino acid uptake and regulation in multicellular hepatoma spheroids.

BACKGROUND: Cancer cells maintained in monolayer tissue culture are frequently used to study tumor biology and nutrient uptake, but there is a concern that this system may not fully reflect clinical tumor physiology. Because cells grown in a 3-D configuration more closely resemble an in vivo environment, a model was developed and characterized for the growth of SK-Hep human hepatoma cells in suspension as multicellular tumor spheroids (MTS). The measurement of nutrient uptake in such a system has never been established. MATERIALS AND METHODS: SK-Hep cultures were initiated as single cell suspensions and grown as MTS in siliconized spinner flasks. The transport of several individual amino acids (arginine, glutamate, leucine, alpha-(N-methylamino)isobutyric acid (MeAIB), and glutamine (GLN)) was measured in SK-Hep single cell suspensions and MTS (0. 50-0.60 mm diameter) by a radiotracer/rapid filtration technique, as was the regulation of glutamine uptake by phorbol esters. l-[(3)H]GLN uptake was also measured in larger spheroids (0.85-1.5 mm diameter). MTS cellularity was evaluated by histological examination, and single cell integrity after the transport assay was confirmed by scanning electron microscopy (SEM). RESULTS: SK-Hep MTS displayed gradients of cellular morphology and staining, with central necrosis visible at diameters >0.8 mm. Single cell suspensions endured the rapid filtration technique based on functional Na(+)-dependent uptake rates and SEM analysis. Of all amino acids tested, only GLN transport rates were visibly affected by growth format. In small MTS, Na(+)-dependent GLN uptake was diminished by 40%, but was 40-53% higher in MTS >1 mm displaying central necrosis, when compared to single cell suspensions. Likewise, slight parallel changes in glutamine transporter ATB(0) mRNA levels were observed in Northern blot analysis. Finally, phorbol ester-dependent GLN transport down-regulation (by 40-50%), previously established in SK-Hep monolayers, remained operative in all cell formats tested. CONCLUSIONS: The data suggest that the tumor microenvironment differentially impacts the uptake of specific nutrients despite the conservation of key regulatory pathways. This MTS technique may prove useful for further studies on the role of nutrient transport in nascent tumor growth.

Mapping and regulation of the tumor-associated epitope recognized by monoclonal antibody RS-11.

We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568-1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the alpha-helix 2B domain of keratin 18 in which two amino acids (Leu(366) and Lys(370)) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha.

Hepatic glutamine transporter activation in burn injury: role of amino acids and phosphatidylinositol-3-kinase.

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.

An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma.

Viruses used for gene therapy are usually genetically modified to deliver therapeutic transgenes and prevent viral replication. In contrast, replication-competent viruses may be used for cancer therapy because replication of some viruses within cancer cells can result in their destruction (oncolysis). Viral ribonucleotide reductase expression is defective in the HSV1 mutant hrR3. Cellular ribonucleotide reductase, which is scarce in normal liver and abundant in liver metastases, can substitute for its viral counterpart to allow hrR3 replication in infected cells. Two or three log orders more of hrR3 virions are produced from infection of colon carcinoma cells than from infection of normal hepatocytes in viral replication assays. This viral replication is oncolytic. A single intravascular administration of hrR3 into immune-competent mice bearing diffuse liver metastases dramatically reduces tumor burden. hrR3-mediated tumor inhibition is equivalent in immune-competent and immune-incompetent mice, suggesting that viral oncolysis and not the host immune response is the primary mechanism of tumor destruction. HSV1-mediated oncolysis of diffuse liver metastases is effective in mice preimmunized against HSV1. These results indicate that replication-competent HSV1 mutants hold significant promise as cancer therapeutic agents. Yoon, S. S., Nakamura, H., Carroll, N. M., Bode, B. P., Chiocca, E. A., Tanabe, K. K. An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma.

Glutamine transport and human hepatocellular transformation.

Among other functions, the liver serves to regulate both glucose and nitrogen economy in the body, and in humans, the amino acid glutamine is a major gluconeogenic substrate and the primary extrahepatic ammonia shuttle. Accordingly, the liver acinus possesses a unique heterogeneous metabolic architecture suited to carry out these functions with glutamine-consuming urea cycle and gluconeogenic enzymes in the periportal hepatocytes and a high capacity for glutamine synthesis in the perivenous hepatocytes, resulting in net glutamine balance across the hepatic bed under most conditions. Cytoplasmic levels of glutamine are significantly governed by the activity of the System N transporter in the plasma membrane of parenchymal cells; in this capacity, this glutamine carrier has been shown to represent a rate-limiting step in metabolism via glutaminase. The unique properties of System N allow it to rapidly adapt in support of the dynamic demands of whole body ammonia and glucose homeostasis. In contrast to System N in normal hepatocytes, human hepatoma cells take up glutamine at rates several-fold faster through a broad-specificity higher affinity transporter with characteristics of System ASC or B0. It is currently hypothesized that the expression of this high activity carrier by hepatoma cells combined with accelerated metabolism and tumor-induced derangements in hepatocellular architecture result in net glutamine consumption, and may underlie the diminished plasma glutamine levels observed in patients with hepatocellular carcinoma (HCC). The transport of glutamine through System ASC has been shown to regulate growth in some human hepatoma cells, which suggests this transporter may warrant consideration as a therapeutic target for HCC.

Rat liver endothelial cell glutamine transporter and glutaminase expression contrast with parenchymal cells.

Despite the central role of the liver in glutamine homeostasis in health and disease, little is known about the mechanism by which this amino acid is transported into sinusoidal endothelial cells, the second most abundant hepatic cell type. To address this issue, the transport of L-glutamine was functionally characterized in hepatic endothelial cells isolated from male rats. On the basis of functional analyses, including kinetics, cation substitution, and amino acid inhibition, it was determined that a Na+-dependent carrier distinct from system N in parenchymal cells, with properties of system ASC or B0, mediated the majority of glutamine transport in hepatic endothelial cells. These results were supported by Northern blot analyses that showed expression of the ATB0 transporter gene in endothelial but not parenchymal cells. Concurrently, it was determined that, whereas both cell types express glutamine synthetase, hepatic endothelial cells express the kidney-type glutaminase isozyme in contrast to the liver-type isozyme in parenchymal cells. This represents the first report of ATB0 and kidney-type glutaminase isozyme expression in the liver, observations that have implications for roles of specific cell types in hepatic glutamine homeostasis in health and disease.

Protein kinase C regulates nutrient uptake and growth in hepatoma cells.

BACKGROUND: Human hepatoma cells extract glutamine at rates severalfold greater than normal hepatocytes through a high-affinity transporter encoded by the ATB0 gene, which contains two putative phosphorylation sites for protein kinase C (PKC). The studies presented here were undertaken to determine whether System B0-mediated glutamine uptake regulates hepatoma growth and whether PKC regulates the activity of this transporter. METHODS: SK-Hep cells were treated with the PKC activator phorbol 12-myristate 13-acetate (PMA) and the initial-rate transport of glutamine and other nutrients measured at specific times thereafter. Growth rates were monitored during culture +/- PMA or an excess of system B0 substrates relative to glutamine. RESULTS: PMA treatment exerted a rapid (half-life approximately 15 minutes) concentration-dependent inhibition of glutamine uptake rates to 50% of control values via a posttranslational mechanism that decreased transporter maximum velocity. This effect persisted after 24 hours and was abrogated by the PKC inhibitor staurosporine. PMA also significantly decreased amino acid transport System y+ and System L activities but no System A. Chronic treatment with PMA (PKC depletion) inhibited SK-Hep growth, as did attenuation of System B0-mediated glutamine uptake with other B0 substrates. CONCLUSIONS: System B0-mediated glutamine uptake regulates hepatoma cell growth, whereas PKC influences both processes.

Effects of endotoxin challenge on hepatic amino acid transport during cancer.

BACKGROUND: The hepatic uptake of amino acids is increased in both sepsis and cancer, and this response appears to be both global and essential in the catabolic host. Because immunocompromised cancer patients are susceptible to episodes of gram-negative sepsis, we examined the capacity of hepatocytes from normal and tumor-influenced livers to respond to the additional challenge of endotoxemia via increases in the Na+-dependent uptake of glutamine and zwitterionic amino acids by System N and System A, respectively. MATERIALS AND METHODS: Fischer 344 rats were implanted with methylcholanthrene-induced fibrosarcomas. Control rats were sham-operated and pair-fed. Animal pairs (tumor burden = 8-32% carcass weight) were injected intraperitoneally with either Escherichia coli endotoxin (10 mg/kg) or PBS, and after 4 h, hepatocytes were isolated from the livers of the animals via collagenase perfusion and placed in primary culture. Three hours later, amino acid transport rates were measured using radiolabeled glutamine for System N and alpha-methylaminoisobutyric acid (MeAIB), a nonmetabolizable substrate specific for System A. RESULTS: Cancer-independent of tumor size-and endotoxin each elicited similar 1.5- to 2-fold inductions of System N activity. When combined, their effects were additive rather than synergistic. In contrast, endotoxin induced an insignificant increase in System A activity, whereas cancer stimulated this carrier 2-fold in either the absence or the presence of endotoxin. CONCLUSIONS: The primary glutamine and alanine carriers in hepatocytes are differentially influenced during catabolic states, and the tumor-influenced liver is competent to further increase glutamine uptake in response to additional catabolic insults.

Adaptive alterations in cellular metabolism with malignant transformation.

OBJECTIVE: The authors studied the differences between glutamine and glucose utilization in normal fibroblasts and in fibrosarcoma cells to gain insights into the metabolic changes that may occur during malignant transformation. SUMMARY BACKGROUND DATA: The process of malignant transformation requires that cells acquire and use nutrients efficiently for energy, protein synthesis, and cell division. The two major sources of energy for cancer cells are glucose and glutamine. Glutamine is also essential for protein and DNA biosynthesis. We studied glucose and glutamine metabolism in normal and malignant fibroblasts. METHODS: Studies were done in normal rat kidney fibroblasts and in rat fibrosarcoma cells. We measured glutamine transport across the cell membrane, breakdown of glutamine by the enzyme glutaminase (the first step in oxidation), glutamine and glucose oxidation rates to CO2, rates of protein synthesis from glutamine, and glutamine-dependent growth rates. RESULTS: Glutamine transport rates were increased more than sixfold in fibrosarcomas compared to normal fibroblasts. In fibroblasts, glutamine transport was mediated by systems ASC and A. In malignant fibrosarcomas, only system ASC was identifiable, and its Vmax was 15 times higher than that observed in fibroblasts. Despite an increase in transport, glutaminase activity was diminished and glutamine oxidation to CO2 was reduced in fibrosarcomas versus normal fibroblasts. In fibroblasts, glutamine oxidation was 1.8 times higher than glucose oxidation. In contrast, glucose oxidation was 3.5 times greater than glutamine oxidation in fibrosarcomas. Protein synthesis from glutamine transported by fibrosarcomas was threefold greater than that observed in normal fibroblasts. Despite marked increases in glutamine utilization and glucose oxidation in fibrosarcoma cells, growth rates were higher in the normal fibroblasts. CONCLUSIONS: The process of malignant transformation is associated with a marked increase in cellular glutamine transport, which is mediated by a single high-affinity, high-capacity plasma membrane carrier protein. In normal fibroblasts, the transported glutamine is used primarily for energy production via oxidation of glutamine carbons to CO2. In fibrosarcomas, glutamine oxidation falls and glutamine is shunted into protein synthesis; simultaneously, the malignant cell switches to a glucose oxidizer. The increased glutamine transport and glucose oxidation in fibrosarcomas appears to be related to the malignant phenotype and not merely to an increase in cell growth rates.

Multiwell 14CO2-capture assay for evaluation of substrate oxidation rates of cells in culture.

14CO2 capture is commonly used to evaluate the cellular oxidation rate of respiratory substrates. A modification of the established 14CO2-capture method was developed that enables the use of cells in adherent culture and easy analysis of multiple samples under different culture conditions. The use of commercially available culture and filter plates designed for use in a multiplate scintillation spectrophotometer enabled substrate oxidation rates to be evaluated for cells in a 24-well plate format without the need to dislodge the cells from the culture substrate as is required in traditional methods. Evaluation of radioactivity captured in potassium hydroxide-saturated filters was accomplished by adding scintillation fluid to the filter plate wells and counting. Alternatively, filters could be removed and placed in vials for evaluation in a conventional scintillation counter. This method was applied to the oxidation of 14C-glutamine by human breast cell lines and demonstrated concentration-dependent linear accumulation of captured counts.

Stimulation of rat hepatic amino acid transport by burn injury.

Burn injury accelerates hepatic amino acid metabolism, but the role of transmembrane substrate delivery in this response has not been investigated. We therefore studied the effects of cutaneous scald injury on the Na+-dependent transport of glutamine and alanine in isolated rat liver plasma membrane vesicles. Scald injury resulted in liver damage and a 1.4- to 2.3-fold and 1.5- to 2.8-fold stimulation of hepatic transport rates for glutamine and alanine, respectively, proportional to the total burned surface area (TBSA) after 24 hours. Enhanced uptake of glutamine and alanine was attributable to increases in the maximum velocity (Vmax) of system N and system A activities, respectively. Hepatic amino acid transport activity remained elevated in vesicles from burned animals after 72 hours, but the degree of stimulation (1.3- to 1.7-fold for glutamine and 1.3- to 1.6-fold for alanine) was less than that observed 24 hours after thermal injury. Liver function tests returned to control values after 72 hours as well, indicating rectification of hepatic damage. In contrast to the induction of hepatic system A and system N activity in catabolic states such as cancer and endotoxemia, further studies showed that tumor necrosis factor (TNF) failed to play a significant role in burn-stimulated amino acid transport rates. When combined with plasma liver enzyme profiles, early transient hepatic amino acid transporter stimulation may support amino acid-dependent pathways involved in the repair of burn-dependent hepatic damage.

Alterations in oxidative metabolism and glutamine transport support glucose production in the tumor-influenced hepatocyte.

Glutamine is the primary substrate whose hepatic transport is upregulated in the tumor-bearing host; however, the subsequent metabolism of transported glutamine is currently unknown. The purpose of this study was to determine if glutamine is an important oxidative fuel source for hepatocytes in cancer. Specifically we compare rates of glutamine transport and oxidation in hepatocytes from control and tumor-bearing animals. We also compare rates of glucose oxidation and rates of glucose production from glutamine in control hepatocytes versus those from tumor-bearing animals. Hepatocytes from rats bearing the MCA fibrosarcoma were isolated when tumors comprised 5 and 15% of total body weight and compared to sham-implanted and pair-fed control animals. [3H]GLN transport, GLN and glucose oxidation to CO2, and glucose production from glutamine were assayed. Tumor burden of 5% stimulated a 2.52-fold increase in hepatocyte glutamine transport and a 2-fold increase when tumor burden reached 15%. Rates of oxidation of glutamine were suppressed by 1.5-fold when tumors comprised 5% of body weight compared to sham animals and were equivalent to sham animals when tumors comprised 15% of body weight. Significant alterations in glucose oxidation were observed when tumors were both small and large-glucose oxidation was suppressed by 3.6- and 3.7-fold when tumors comprised 5 and 15% of body weight respectively compared to sham-implanted rats. Incubation of hepatocytes from tumor-bearing animals with glutamine as a gluconeogenic substrate induced a 1.84-fold increase in glucose production compared to sham hepatocytes. In conclusion, (i) despite a doubling of GLN transport by the tumor-influenced hepatocyte, GLN oxidation by hepatocytes was not increased. (ii) Glucose oxidation by hepatocytes from tumor-bearing animals was decreased compared to sham hepatocytes and, simultaneously, glucose production by tumor-influenced hepatocytes from glutamine was increased. The augmentation of hepatic glutamine transport and decreased glutamine oxidation seen in tumor-influenced hepatocytes appear to support hepatocyte gluconeogenesis from glutamine.

Characterization of glutamine and glutamate transport in rat lung plasma membrane vesicles.

Insufficient glutamine for the lungs during sepsis may contribute to an impairment in lung function. Lung glutamine metabolism is supported by both blood glutamine uptake and de novo biosynthesis using circulating glutamate as a precursor. Information regarding the specific plasma membrane carriers involved in this uptake is lacking. Furthermore, the effect of sepsis on amino acid transport in whole lung has not been studied. We isolated lung plasma membrane vesicles (LPMVs) from control and LPS-treated rats and assayed glutamine and glutamate transport activity in LPMVs. Vesicle purity and functionality were confirmed by time-dependent concentrative amino acid uptake in the presence of Na+, impoverishment of microsomal enzymes, and a 25-fold enrichment in the plasma membrane marker 5'-nucleotidase. Eighty percent of glutamine uptake in lung vesicles was mediated via the high affinity Na(+)-dependent carrier System ASC (Vmax = 80 +/- 10 pmole/mg protein/15 sec; Km = 224 +/- 30 microM) while 19% occurred via the Na(+)-independent System ASC (Vmax = 11 +/- 2 pmole/mg/15 sec; Km = 141 +/- 23 microM). Ninety percent of glutamate transport was mediated by the Na(+)-independent System XAG-. Treatment of rats with LPS resulted in a decrease in both glutamine and glutamate transport in LPMVs. LPMVs offer a novel method for characterizing lung amino acid transport and studying the effects of catabolic states on this activity. The effects of endotoxin on System ASC and XAG- activity may contribute to reduced lung glutamine availability during septic states which may impair cellular metabolism and function.

Hepatic glutaminase gene expression in the tumor-bearing rat.

UNLABELLED: Previous studies have documented an increase in hepatic plasma membrane glutamine transport in the tumor-bearing rat, but the effects of tumor burden on hepatic glutaminase expression have not been carefully studied. The purpose of this study was to examine the effects of tumor burden and food intake on hepatic glutaminase expression. Rats were implanted with syngeneic methylcholanthrene-induced fibrosarcoma tumor tissue; control rats were sham operated and pair-fed every 24 hr. Northern blotting was used to assay the effect of tumor burden and fasting on hepatic glutaminase mRNA levels, using beta-actin mRNA as a control. Hepatic glutaminase mRNA levels in livers of pair-fed controls were found to be 4-fold greater than levels in livers of tumor-bearing animals. Examination of food intake patterns in these animals indicated that pair-fed controls ate their allotted chow quickly while tumor-bearing rats ate small amounts throughout each 24 hr period. This observation suggested that the differences in glutaminase mRNA levels may be due to a period of fasting by pair-fed animals which was not experienced by the tumor-bearing group. Hepatic glutaminase mRNA levels rapidly increased in normal rats during acute fasting to levels 5.5-fold greater than fed animals. Glucose feeding and insulin injection rapidly reversed the effect of fasting on hepatic glutaminase mRNA levels in normal rats. Tumor-bearing rats also exhibited upregulation of hepatic glutaminase mRNA levels in response to fasting. CONCLUSIONS: (1) Tumor burden itself does not alter hepatic glutaminase expression, at least at the pre-translational level. Instead, differences in hepatic glutaminase mRNA content are due to differences in food intake patterns. (2) Hepatic glutaminase mRNA levels are rapidly upregulated in response to fasting, an effect which appears to be linked to a decrease in plasma insulin concentrations. Because tumor-bearing rats eat regularly over a 24 hr period (albeit in small increments), thereby maintaining the plasma insulin concentration, hepatic glutaminase mRNA may not rise as it does in pair-fed controls whose daily chow intake is complete within hours of food allocation. (3) This study indicates that differences in the timing of food intake between tumor-bearing rats and pair-fed controls can alter the expression of genes that are influenced by nutrient availability. These differences should be taken into account when designing studies which involve pair-feeding to control nutrient intake.

Induction of glutamine synthetase expression after major burn injury is tissue specific and temporally variable.

BACKGROUND: Major burn injury results in a translocation of amino acids from peripheral tissues to the abdominal viscera. Glutamine is a major participant in this event. Thermal injury causes a depletion of plasma and muscle glutamine pools as well as activation of proteolysis and release of glutamine from skeletal muscle. De novo synthesis of glutamine is regulated by the expression of the enzyme glutamine synthetase (GS). We studied the tissue-specific regulation of GS expression after thermal injury. METHODS: Burn injury of rats was produced by scalding of 25 or 40% of skin surface. In normal rats, four organs, including lung, muscle, kidney, and liver were assayed for relative GS messenger RNA content by Northern blotting 8 and 24 hours after 40% area burn. The effect of adrenalectomy on GS mRNA induction in muscle was assessed 24 hours after 25% area burn injury. RESULTS: GS mRNA levels were increased 2.3-fold in lung at 8 hours and 7.3-fold in muscle at 24 hours after burn injury. No appreciable increase in GS mRNA level was observed in kidney or liver. Muscle GS mRNA levels were lower than sham-operated controls in both burned and unburned adrenalectomized rats. However, adrenalectomy did not attenuate relative GS mRNA induction in muscle at 24 hours after burn injury. CONCLUSIONS: Burn injury causes an induction in GS mRNA levels in a tissue-specific fashion. Adrenalectomy greatly reduced GS mRNA levels, but did not completely block the induction of GS express in muscle after burn injury. This finding suggests that glucocorticoid hormones together with a unknown factor of nonadrenal origin influence this metabolic response to burn injury.

A sarcoma-derived protein regulates hepatocyte metabolism via autocrine production of tumor necrosis factor-alpha.

OBJECTIVE: The effects of conditioned media from the methylcholanthrene (MCA) fibrosarcoma on hepatocyte albumin production and amino acid transport were studied. The authors characterized a factor responsible for the observed effects and investigated the role of tumor necrosis factor-alpha (TNF-alpha) in these events. SUMMARY BACKGROUND DATA: Cancer cachexia is mediated in part by TNF-alpha. However, few tumors secrete TNF-alpha, implicating host production of this cytokine in response to as yet uncharacterized tumor-derived factors. Autocrine production of TNF-alpha recently has been described as a potent mechanism for orchestrating hepatic metabolism. METHODS: Conditioned media from the MCA fibrosarcoma was incubated with isolated primary rat hepatocytes. Albumin production and TNF-alpha production by hepatocytes was measured by enzyme-linked immunosorbent assay and amino acid transport assayed by tritium (3H)-labeled amino acid uptake. Dialysis membranes ranging from 3 kD to 100 kD were used to determine the size of the factor/factors responsible for the observed effects. RESULTS: Conditioned media from the MCA fibrosarcoma contained no TNF-alpha, whereas treatment of primary rat hepatocytes with the conditioned media resulted in a 53-fold increase in TNF-alpha production by hepatocytes compared with control. Treatment of hepatocytes with MCA fibrosarcoma-conditioned media resulted in decreases in hepatic albumin production of 46%, 61%, and 42% over 3 days of treatment, and these effects were reversible by the addition of antibody to TNF-alpha. Treatment of hepatocytes with MCA fibrosarcoma conditioned media resulted in increases in hepatocyte amino acid transport via inductions of System N (1.87 fold) and System A (1.93 fold). These effects were partially abrogated by the addition of antibody to TNF-alpha. Dialysis experiments determined the molecular weight of the factor or factors responsible for the observed effects to be greater than 100 kD. The effects of the MCA fibrosarcoma conditioned media were abolished by both trypsin treatment and heat inactivation, indicating the protein nature of the factor being studied. CONCLUSIONS: A tumor-derived protein has been isolated from the MCA fibrosarcoma. The protein inhibits hepatocyte albumin production and increases amino acid transport in vitro via the autocrine production of TNF-alpha.