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BACKGROUND: Malignant cells require increased amounts of amino acids, in particular glutamine and leucine, to support DNA and protein biosynthesis. Although plasma concentrations in the center of solid tumors can be much lower than normal circulating levels, it is still unknown how tumor cells can survive despite low amino acid levels. We examined the effects of glutamine or leucine deprivation on cell growth and amino acid transport activity in two human hepatoma cell lines, SK-Hep and HepG2. METHODS: We studied the transport of glutamine, leucine, alanine, and arginine. The carriermediated uptake of [sup.3]H-amino acids was determined in cells cultured in normal and amino acid-deprived media. RESULTS: The growth of both cell lines was dependent on the concentration of glutamine and leucine. In SK-Hep, there was a significant increase in initial rate glutamine transport activity in the glutamine-deprived group, attributable to an increase in transporter affinity (Km; 0.6 mmol/L [control], 385[+ or -]43 [mu]mol/L; 0.1 mmol/L, 221[+ or -]11[micro]mol/L; P

BACKGROUND: Hepatocytes possess a novel, Plasma-membrane, sodium ion ([Na.sup.+])-independent, glutamine transporter (system n), which functions to transport glutamine out of the cell into the blood. In the tumor-bearing rat, the activity of system n increases but its regulation is unknown. We hypothesized that the increase in system n that occurs in rats with cancer was related to a fall in the circulating glutamine concentration. METHODS: Ten male rats underwent flank implantation with a cube of methylcholanthrene-induced fibrosarcoma cells and 10 rats underwent a sham operation. After 9 days of standard diet, all rats were randomized to receive either a glutamine-enriched oral diet or an isonitrogenous diet without supplemental glutamine, for 1 week. Tumors and livers were harvested 16 days postimplantation. Arterial blood samples were obtained from all animals. Hepatic plasma membrane vesicles were prepared and the carrier-mediated, Na+-independent transport of glutamine was assayed. RESULTS: When compared to nontumor-bearing animals, tumor-hearing rats that were fed a control diet exhibited hypoglutaminemia and a 2.3-fold increase in the activity of system n. Glutamine dietary supplementation produced blood glutamine levels that were similar in both tumor-bearing and nontumor-bearing rats, apparently abrogating the increase in system n activity that was observed in tumor-hearing rats that were not fed supplemental glutamine. Tumor-bearing animals receiving supplemental glutamine had a decreased number of system n carriers ([V.sub.max]) in the hepatic plasma membrane compared to that of tumor-bearing animals receiving a control diet; this apparently abrogated the glutamine efflux rate. Glutamine feeding did not alter system n activity in nontumor-bearing controls. CONCLUSIONS: In the tumor-hearing animal model, system n is modulated by the circulating glutamine concentration. This is the first study that demonstrates the ability of specialized nutrition to 'downregulate' transport activity in vivo. Provision of glutamine-enriched diets to the host with cancer may maintain hepatic glutamine levels and prevent host glutamine depletion.

Neonatal PMN (polymorphonuclear neutrophils) exhibit altered inflammatory responsiveness and greater longevity compared with adult PMN; however, the involved mechanisms are incompletely defined. Receptors containing immunoreceptor tyrosine-based inhibitory motif (ITIM) domains promote apoptosis by activating inhibitory phosphatases, such as Srchomology domain 2-containing tyrosine phosphatase-1 (SHP-1), that block survival signals. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9, an immune inhibitory receptor with an ITIM domain, has been shown to induce cell death in adult PMN in association with SHP-1. To test our hypothesis that neonatal PMN inflammatory function may be modulated by unique Siglec-9 and SHP-1 interactions, we compared expression of these proteins in adult and neonatal PMN. Neonatal PMN exhibited diminished cellular expression of Siglec-9, which was phosphorylated in the basal state. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment decreased Siglec-9 phosphorylation levels in neonatal PMN but promoted its phosphorylation in adult PMN, observations associated with altered survival signaling. Although SHP-1 expression was also diminished in neonatal PMN, GM-CSF treatment had minimal effect on phosphorylation status. Further analysis revealed that Siglec-9 and SHP-1 physically interact, as has been observed in other immune cells. Our data suggest that age-specific interactions between Siglec-9 and SHP-1 may influence the altered inflammatory responsiveness and longevity of neonatal PMN.

Skeletal muscle serves as the body's major glutamine repository, and releases glutamine at enhanced rates during diabetes, but whether all muscles are equally affected is unknown. System Nmactivity mediates most trans-sarcolemmal glutamine movement, and although two System N (SN) isoforms have been identified (SN1/sodium-coupled neutral amino acid transporter or System N and A transporters [SNAT]-3; and SN2/SNAT5), their expression in skeletal muscle remains controversial. Here, the impact of Type I diabetes on glutamine uptake and System N transporter expression were examined in fast- and slow-twitch skeletal muscle from spontaneously diabetic (BB/Wor-DP) rats. Net glutamine uptake in fast-twitch fibers was decreased 75%-95%, but enhanced more than 2-fold in slow-twitch muscle from diabetic animals relative to nondiabetic controls. Both SNAT3 and SNAT5 mRNA were expressed in both muscle fiber types and their abundance was unaffected by diabetes. This represents the first report of differential fiber-specific effects of diabetes on skeletal muscle glutamine transport and the co-expression of distinct System N transporters in skeletal muscle.

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 vivoenvironment, 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.

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/B0-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.

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. FASEB J.14, 301–311 (2000)

Amino acid metabolism by the vascular endothelium is a complex process that often begins with the carrier-mediated uptake of circulating amino acids into the endothelial cytoplasm. Amino acids are essential for maintaining intact endothelial functions, which include cell proliferation, regulation of blood flow and vascular tone, coagulation and fibrinolysis, and metabolism of a variety of macromolecules. The disturbances in endothelial amino acid transport and metabolism that occur during infection and inflammation are due, in part, to changes in substrate availability and to the local and/or systemic elaboration of specific mediators. An improved understanding of endothelial amino acid metabolism will not only provide new knowledge regarding disease mechanisms and regulation, but may also lead to new treatment strategies that may include the clinical use of specific nutritional formulas.

During stress states, organismal glutamine production is augmented secondary to an increase in the activity of glutamine synthetase (GS) in the lung and skeletal muscle. Because glucocorticoids are key regulators of the metabolic response to stress, we undertook a survey of glucocorticoid induction of GS expression in rat organs in response to dexamethasone. Male adult rats were injected with glucocorticoid or vehicle and 4 hr later, 10 organs were assayed for GS messenger RNA and protein contents by Northern and Western blotting. We observed a 20-fold range of GS mRNA levels in organs of control animals. Blotting detected two GS RNA species of approximately 2.8- and 1.4-kb sizes in all tissue except testis, where an additional 2-kb RNA species was observed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mBNA levels were also assayed and used as a normalization factor. An approximately 10-fold range of GAPDH mRNA levels was observed. Four hours after dexamethasone injection, a nearly a 5-fold increase in glutamine synthetase mRNA levels in lung and muscle, as well as an approximately 2-fold increase in heart were observed. Relative to GAPDH mRNA, a significant decrease in GS mRNA levels was observed in the liver. A wide range of glutamine synthetase protein contents were observed in rat organs. Comparison of Northern and Western blotting results revealed a dichotomy in the ratio of relative GS mRNA and protein level in rat organs, suggesting that tissue-specific posttranscriptional processes determine GS protein levels. Four hours after dexamethasone injection, an apparent increase in GS protein was observed in the lung, muscle, and thymus; however, significant induction of GS protein was demonstrated only in lung. The differential response of GS mRNA and protein levels to dexamethasone suggests that GS expression is induced by glucocorticoids in a tissue-specific manner. Copyright 1995, 1999 Academic Press

The role of the glucocorticoid hormones in mediating the accelerated hepatic amino acid transport that is characteristic of endotoxemia was investigated. To determine the role of these steroid hormones, rats that received endotoxin (LPS) were pretreated with the glucocorticoid receptor antagonist RU38486. The activities of the Na⁺-dependent amino acid transport systems A, ASC, and N and the Na⁺-independent systems L, y⁺, n, b^0,+, and asc in hepatic plasma membrane vesicles were measured 4 hr after exposure to LPS. Endotoxin treatment resulted in time- and dose-dependent 5-fold (System A), 2.5-fold (System N), 2.6-fold (System ASC), and 2-fold (System y⁺ and b^0,+) increases in transport activity attributable to an increase in carrier V_max. The activities of L, asc, and n were unchanged by LPS administration. Pretreatment of endotoxemic animals with RU38486 attenuated the LPS-induced enhancement in transport activity by 20-60% by diminishing carrier V_max, with no effect on transport K_m. We conclude that the marked increase in hepatic amino acid transport activity that occurs during endotoxemia requires participation of the glucocorticoid hormones. Copyright 1995, 1999 Academic Press

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 hepatoctyes 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 hepatoctyes 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.

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 andde novobiosynthesis 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 μM) while 19% occurred via the Na+-independent System ASC (Vmax= 11 ± 2 pmole/mg/15 sec;Km= 141 ± 23 μM). 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.

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 β-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.

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.

14CO2capture 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.

The effects of endotoxin on the activities of the major Na-independent amino acid transporters in rat liver (Systems n, asc, L, bo,, and y) were studied using using hepatic plasma membrane vesicles (HPMVs). Rats were treated with a single dose of Escherichia coliendotoxin (E. colilipopolysaccharide 0127:B8 (LPS), 7.5, 15, or 30 mg/kg BW) and HPMVs were prepared by Percoll density gradient centrifugation at various timepoints after LPS administration. Vesicle purity and integrity was established by assay of enzyme markers and identical equilibrium uptakes. The activities of the Na-independent amino acid transport systems yand bo,(arginine), asc (alanine and cysteine), L (leucine), and n (glutamine) were evaluated by measuring the uptake of radiolabeled amino acids using a rapid mixing/filtration technique. Amino acid uptake by HPMVs consisted of saturable and nonsaturable components. Prior treatment with endotoxin did not alter the activities of Systems n, asc, or L but resulted in a time- and dose-dependent stimulation of saturable arginine transport. Arginine transport increased within 2 h of LPS administration and exhibited a return towards basal levels by 24 h. Nonsaturable uptake (diffusion) in HPMVs was unaltered by LPS treatment. Kinetic analysis of arginine transport demonstrated the presence of both a high affinity and a low affinity carrier. Treatment with LPS resulted in a 73 increase in the Vmaxof the high affinity carrier (System y) and a 25 increase in the Vmaxof the low affinity transporter (System bo,). The data indicate selective stimulation of Na-independent arginine transport in the liver during endotoxemia which may serve to support important arginine-dependent pathways during sepsis.