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Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGMENT
LITERATURE CITED
ABSTRACT 
Dietary glutamine supplementation and exercise have been reported independently to enhance immune function and reduce tumor growth. We study the effect of both of these interventions on the growth of the Morris Hepatoma 7777, implanted in 59 female Sprague-Dawley Buffalo rats. Rats were fed a nutritionally complete, purified diet with or without L-glutamine 20 g/kg diet and randomized to swim 3 h/d or to remain sedentary. After 14 d, the mean tumor weight of glutamine-supplemented rats was lower (P < 0.0001) than that of unsupplemented rats (5.8 ± 0.4 vs. 8.7 ± 0.5 g, respectively). Exercise did not alter tumor growth. Glutamine supplementation increased [3H] thymidine incorporation by splenocytes incubated with Concanavalin A and the proportion of natural killer cells in spleen, but not cytotoxic activity against YAC-1 cells. Glutamine supplementation did not alter glutamine concentrations in plasma (691 ± 12 µmol/L) or soleus muscle (5328 ± 102 pmol/mg) but resulted in higher (P < 0.004) plasma concentrations of leucine, isoleucine and valine, precursors of glutamine. Splenocytes from exercised rats had a higher (P < 0.001) mitogen response than those from sedentary rats. Isolated tumor cells demonstrated high rates of non-oxidative glucose and glutamine metabolism and consumption of glutamine, tryptophan and methionine. However, neither diet nor exercise significantly affected glucose or glutamine metabolism by tumor cells. The precise mechanism of tumor growth suppression by oral glutamine supplementation is not clear but may be related to changes in substrate availability, improved tumor-directed natural killer cytotoxic activity or a faster response to an immune challenge.
Key words: tumor, natural killer cells, glutamine, exercise, rats.INTRODUCTION
Anti-cancer immune defense declines progressively with tumor growth (Shewchuk et al. 1996b
). Therefore, one approach in anticancer therapy is to impose nutritional, metabolic and pharmacologic strategies to support and enhance immune function. Two such strategies include dietary supplementation with the amino acid glutamine and regular aerobic exercise. Glutamine depletion in blood and tissue free pools occurs during the growth of a variety of tumors (LeBricon et al. 1995, Mares-Perlman and Shrago 1988, Souba 1993). This amino acid occupies a central role in metabolic processes in rapidly proliferating cells (Felig 1975, LeBricon et al. 1995, Mares-Perlman and Shrago 1988, Sauer and Dauchy 1983, Souba 1993), including cells of the immune system (Field 1995, Shewchuk et al. 1996a). Glutamine-depleted states are associated with impaired immune function (Alverdy et al. 1992). In catabolic states including surgery, trauma or sepsis, glutamine-enriched diets have been shown to replete the free glutamine pool in skeletal muscle and to improve immune function as well as host nitrogen balance (Alverdy et al. 1992). In studies in tumor bearing animals, glutamine-enriched enteral (Klimberg et al. 1990) or total parenteral nutrition (Austgen et al. 1992) was reported to replete glutamine pools without stimulating tumor growth. However, the effects of glutamine on anticancer defense have not yet been explored.
Exercise has also been demonstrated to alter the course of malignancy (Baracos 1989, Jadeski and Hoffman-Goetz 1996). The mechanism for this effect has not been established, but could relate in part to positive effects of exercise on immune function (Hoffman-Goetz and Pedersen 1994). Because exercise is reported to decrease glutamine release from muscle (Rennie et al. 1981), there is a potential for interaction between exercise and dietary glutamine. To test these relationships, tumor-bearing rats were exposed to regular aerobic exercise or remained sedentary, and were fed a nutritionally complete diet containing either 20 g/kg added L-glutamine or an isonitrogenous control diet containing L-glycine. Immune cell phenotypes, mitogen-stimulated proliferation of lymphocytes, natural killer (NK)5 cell cytotoxicity and tumor growth were determined. A 14-d experimental period was selected because we have demonstrated previously that by 2 wk after implantation the Morris Hepatoma 7777 (MH 7777) significantly suppresses the host's immune system (Shewchuk et al. 1996b), and 2 wk of exercise produce a significant training effect in nontumor-bearing rats (Shewchuk et al. 1996a).
MATERIALS AND METHODS
-ketoglutaric acid were purchased from Boehringer-Mannheim Canada (Laval, QC, Canada). Fluorescein isothiocyanate-conjugated goat anti-mouse IgG, with no cross-reaction to rat IgG, was obtained from Organon Teknika (Scarborough, ON, Canada). All diet ingredients (except glucose, glutamine and fats) were purchased from Teklad (Madison, WI).
Experimental design.
The experimental protocol was reviewed by the University Animal Policy and Welfare Committee and was conducted in accordance with the guidelines of the Canadian Council on Animal Care. In total, 59 female Sprague-Dawley rats (197 ± 2 g) of the Buffalo strain, from a colony maintained at the University of Alberta, were randomly assigned to one of two groups and given free access to water and a nutritionally complete purified diet supplemented with or without 20 L-glutamine g/kg diet. The diets were made isonitrogenous and isoenergetic by the addition of glycine (40 g/kg diet) to the unsupplemented diet (
gln diet) and cornstarch (20 g/kg diet) to the supplemented diet (+gln diet). The remaining composition of the diet was (per kg diet): 257 g high protein casein, 190 g starch, 198 g glucose, 48 g non-nutritive cellulose, 2.5 g choline, 6 g inositol, 2.5 g L-methionine, 48 g Bernhart-Tomarelli mineral mix and 9.5 g AOAC vitamin mix. The composition of the mineral and vitamin mix has been previously reported (Clandinin and Yamashiro 1980). To make the fat content of the diets representative of the human diet, both diets contained 200 g/kg fat, providing a polyunsaturated to saturated fatty acid ratio of 0.9 (as determined by gas liquid chromatography, Field et al. 1990). Rats were housed individually in wire-mesh cages in a temperature-controlled room, maintained on a 12-h light:dark cycle. After 1 wk of feeding, all rats were implanted with the MH 7777. A 20-µL subcutaneous injection of finely chopped tumor tissue, freshly harvested from a rat implanted 2 wk earlier, was injected into each flank of all experimental rats. Prior to implantation, the nonnecrotic tumor tissue from the donor rat was finely chopped in a sterile hood. Rats in each diet group were randomly assigned to one of two exercise groups and trained progressively to swim 3 h/d (6 d/wk) or remained sedentary for 14 d, as previously described in detail (Baracos 1989). Sedentary rats were matched in duration of water exposure to stand 3 h/d (6 d/wk) in 10 cm of 35°C water. Body weight and food intake were recorded every 3 d. Twenty-four hours following the last bout of exercise, rats were killed at the end of the dark cycle by CO2 asphyxiation. At necropsy, arterial blood (by cardiac puncture), tumours, spleen and soleus muscle were collected for the measurements described below. Because all variables could not be performed on each animal, the number of rats used for each assay is indicated below. Plasma (n = 59) was separated from arterial blood by centrifugation at 350 × g for 20 min and stored at 70°C for subsequent analysis of amino acids. The frozen muscle (n = 35) was homogenized in trichloroacetic acid (20 g/L). Amino acids (plasma, muscle and incubation media) were separated using a Varian 5000 HPLC (Varian Instruments, Georgetown, ON, Canada) with a fluorochrome detector (Le Bricon et al. 1995). Samples were injected onto a Supelcosil 3-µm LC-18 reverse-phase column (4.6 × 150 mm; Supelco, Bellafonte, CA), and peak area integrations were calculated using a Shimadzu Ezchrom Chromatography Data System (Shimadzu Scientific Instruments, Columbia, MD).
Succinate dehydrogenase activity.
For analysis of succinate dehydrogenase activity, one soleus muscle from each rat (n = 32) was homogenized in 0.3 mol/L phosphate buffer (pH 7.2) and analyzed by the method of Cooperstein and co-workers as previously described (Shewchuk et al. 1996a).
Isolation of tumor cells.
Hepatoma cells were isolated from rats (n = 35) by a modification of the method described by Mares-Perlman and Shrago (1988). At necropsy, tumors were carefully excised and the necrotic tissue removed. One gram of tissue was finely minced and suspended in 25 mL cold Ca++/Mg++ free Hanks' balanced salt solution (pH 7.4) with bovine serum albumin (50 g/L) and collagenase (2 × 105 units/L). Flasks were gassed with 95% O2 and incubated while shaking for 15 min. The suspension was filtered through a 250-µm mesh, resuspended in the collagenase buffer and incubated for an additional 60 min. The suspension was filtered through a 100-µm mesh and pelleted (200 × g for 10 min). The pellet was resuspended in the balanced salt solution with albumin and separated by density centrifugation using Histopaque® as previously described for peripheral mononuclear cells (Shewchuk et al. 1996a). Cell viability was determined by trypan blue exclusion and was greater than 90% for all treatments.
). All metabolism studies were performed on freshly isolated cells within 4 h after necropsy. Preliminary experiments were performed to determine the optimal cell concentration and incubation conditions. This was done by determining metabolite production in which isotope flux rates were linear over the duration of the incubation and were proportional to the concentration of cells in the incubation. Cells (0.5 × 109/L) were incubated in the Krebs' Ringer HEPES buffer, supplemented with 5 g/L bovine serum albumin) and 4 mmol/L glucose (containing 7.4 MBq/L glucose) with or without 1 mmol/L glutamine. Tubes were incubated while shaking for 2 h at 37°C, the reaction terminated by the injection of 100 µL of HClO4 (1.5 mol/L), and shaking continued for another hour to trap evolved CO2. 14CO2 was trapped in benzethonium hydroxide and its radioactivity measured by liquid scintillation spectrophotometry in a Beckman 5000 beta counter (LS 5801© Beckman Instruments, Mississauga, ON, Canada). The incubation media (extracts plus cells) were stored at 20°C for later spectrophotometric determination of lactate and, after neutralization, for pyruvate (Field 1995). To measure [14C] glucose conversion to fatty acids, lipids were extracted from the media (containing cell extracts) and quantified by liquid scintillation spectrophotometry (Field et al. 1990). The conversion of glutamine to glutamate, aspartate and CO2 was determined by incubating cells in 1 mmol/L glutamine (containing 18.5 MBq/L L-[U-14C] glutamine) with or without 4 mmol/L glucose in the incubation conditions described above. Upon termination of the reaction, trapped 14CO2 was collected and counted as described for glucose. The incubation media containing cell extract were neutralized and 14C-glutamine and 14C-aspartate separated on a Dowex AG1-X8 (200-400 mesh, acetate form) column as previously described (Field 1995). All metabolic assays were performed in triplicate and expressed per 106 cells. To determine the utilization of amino acids by MH 7777 cells, a third incubation was performed. Freshly isolated tumor cells from the sedentary gln rats (n = 7) were incubated in RPMI media with or without 1 mmol/L glutamine. Amino acid concentrations were analyzed as described above. Tubes (n = 3) containing culture media without cells were incubated to serve as controls.
) in Krebs' Ringer HEPES buffer supplemented with bovine serum albumin (5 g/L). For the mitogen response and NK cell assays, cells were prepared and cultured in RPMI containing fetal calf serum (50 g/L), 2-mercaptoethanol (2.5 µmol/L), glutamine (4 mmol/L), penicillin (1 × 105 U/L), streptomycin (100 mg/L), amphotericin B as Fungizone® (0.25 g/L) and HEPES (25 mmol/L). Cell viability was assessed by trypan blue exclusion and was greater than 90% for all groups.
Mononuclear cell phenotyping.
Lymphocyte subsets from spleen (n = 35) were characterized by an immunofluorescence assay using supernatants from hybridoma-secreting mouse monoclonal antibodies specific for the different rat mononuclear cell subsets (kindly provided by Dr. A. Rabinovitch, Edmonton, AB, Canada and 3.2.3 purchased from Cedarlane, Hornby, ON, Canada). OX19 recognizes a glycoprotein on the surface of thymocytes and T-lymphocytes (CD5); W3/25 recognizes a surface glycoprotein found on rat T helper cells (CD4); OX8 recognizes T-cytotoxic/suppressor lymphocytes (CD8) and NK cells; OX42 reacts with a receptor found on most monocytes, granulocytes and macrophages; OX12 recognizes a determinant on the rat kappa chain of immunoglobulins on B lymphocytes; and 3.2.3 reacts with rodent NK cells. An aliquot of 2-5 × 105 cells was incubated for 30 min at 4°C with each antibody and then washed three times in 200 µL of PBS containing fetal calf serum (40 g/L) and incubated for another 30 min at 4°C in 50 µL of a 1:300 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG. The cells were then washed three times and fixed in PBS containing paraformaldehyde (10 g/L) and analyzed on a FACScan® (Becton Dickinson, Sunnyvale, CA) according to relative fluorescence intensity. Resulting percentages were corrected for background fluorescence (5-10%), determined by incubating cells with fluorescein isothiocyanate-conjugated goat anti-mouse IgG only.
Natural killer cell cytotoxicity.
Sodium chromate [51Cr]-labeled NK cell sensitive YAC-1 cells (a gift of Dr. A. Rabinovitch, Department of Medicine, University of Alberta) were incubated for 4 h with splenocytes (n = 24) to achieve effector:target ratios between 5:1 and 100:1 and 51Cr release measured, as previously described (Field 1995). Spontaneous release was determined from target cells incubated in the absence of effector cells. Maximum release was determined from detergent lysis of labeled target cells. Cytotoxic activity was calculated as: % specific lysis = 100 × (experimental release-spontaneous release)/(maximum release-spontaneous release). Results were also calculated on a per cell basis using the number of NK cells present, as determined by 3.2.3 monoclonal antibody binding. This was expressed as lytic units with one lytic unit being equal to the number of effector cells (× 10
3) required to cause 15% lysis of target cells.
Splenocyte mitogen response.
Splenocytes (2.5 × 109 cells/L from 24 rats) were cultured with or without either Concanavalin A (Con A; 5 mg/L) or phorbol myristate acetate (PMA; 30 µg/L) plus ionomycin (Iono; 0.75 µmol/L) for 48 and 72 h as previously described (Shewchuk et al. 1996b). Preliminary experiments using cells from nontumor-bearing rats determined that 72 h incubation were required to produce the maximum [3H] thymidine response to both mitogens. Eighteen hours prior to harvesting the cells, each well was pulsed with 37 kBq of [3H] thymidine. Assays were performed in quadruplicate, and stimulation indices were calculated for each condition as [amount of [3H] thymidine (kBq/min) incorporated by the stimulated cells the amount of [3H] thymidine (kBq/min) incorporated by the unstimulated cells]/amount of [3H] thymidine (kBq/min) incorporated by unstimulated cells.
Statistical analysis.
Analysis was performed using SAS (Version 6.02, SAS Institute, Cary, NC). The effects of diet and exercise on variables were determined using a two-way ANOVA. Significant (P < 0.05) differences among groups were identified by Duncan's multiple-range test (Steele and Torrie 1980). Body weight changes, dietary intake and NK cytotoxic activity were compared among groups by a two-way split-plot (repeated measures) ANOVA. The effect of adding glucose or glutamine in the incubation media on tumor cell energy metabolism was determined using a paired t test. The change in amino acid concentration in the media from tumor cells was compared to the blank tubes by a t test. All data are expressed as means ± SEM.
RESULTS
gln diet compared with the +gln diet (Fig. 1). Exercised rats had smaller (P < 0.002) spleens (Table 1). However, the number of splenocytes isolated per spleen did not differ among groups. Neither exercise nor diet significantly affected soleus muscle weight (65 ± 1 mg, n = 35). However, soleus muscles from rats fed the +gln diet demonstrated lower succinate dehydrogenase activity than those from rats fed the gln diet (Table 1).
|
Table 1. Effect of exercise and diet on organ weight, tumor weight and muscle succinate dehydrogenase activity (SDH) in rats implanted with the Morris Hepatoma 77771 |
gln, n = 11). 
Indicates a significant (P < 0.05) effect of diet and 
a significant (P < 0.05) effect of exercise at time (d) after tumor implantation, as determined by two-way ANOVA procedures. When a significant (P < 0.05) interaction was found, means at a given time that do not share a letter are significantly (P < 0.05) different.
Plasma amino acid concentration. Plasma glutamine levels (691 ± 12 µmol/L, n = 59) were not significantly different among groups. Exercise, compared with the sedentary condition, resulted in a significantly lower (P < 0.02) plasma concentration of threonine (
18%), leucine (16%), valine (8%) and histidine (21% for the gln diet only; Table 2). Rats fed the +gln diet compared with the gln diet, had a significantly higher (P < 0.02) plasma concentration of aspartate (+25%), threonine (+25%), citrulline (+24%), leucine (+22%), isoleucine (+13% for the exercising rats and +24% for the sedentary), ornithine (+14% for the exercising rats and +66% for the sedentary) and valine (+20%). Feeding the +gln diet to exercised but not sedentary rats produced a higher tryptophan (+22%) and histidine (+19%) concentration in plasma (Table 2). Feeding the +gln compared with gln diet resulted in a lower (P < 0.0001) plasma concentration of glycine (65%) and serine (41%; Table 2). Feeding the gln (glycine-supplemented) diet resulted in a higher (P < 0.001) plasma concentration of glycine and serine (Table 2).
|
Table 2. Plasma amino acid concentrations altered by diet and/or exercise in rats implanted with the Morris Hepatoma 77771 |
18%) concentrations of histidine, lysine and threonine in rat soleus muscle (Table 3). Supplementation with glutamine resulted in a higher concentration of aspartate (+45% in exercising rats and +18% in sedentary rats). Feeding the glycine-supplemented diet produced higher muscle concentrations of glycine and serine (Table 3). Exercise and diet did not significantly affect the mean concentration (pmol/mg tissue, n = 35) of alanine (1610 ± 44), arginine (440 ± 28), asparagine (270 ± 10), citrulline (120 ± 4), glutamine (5328 ± 102), glutamate (1250 ± 36), isoleucine (70 ± 4), leucine (120 ± 8), methionine (50 ± 3), ornithine (30 ± 1), phenylalanine (90 ± 4), taurine (2650 ± 62), tryptophan (40 ± 1), tyrosine (90 ± 4) or valine (110 ± 6).
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Table 3. Amino acid concentrations in soleus muscle altered by diet and/or exercise in rats implanted with the Morris Hepatoma 77771 |
Table 4.
Accumulation of glucose carbons in media by
Morris Hepatoma 7777 cells1
Table 5.
Accumulation of glutamine carbons in media by
Morris Hepatoma 7777 cells1
Table 6.
Effect of exercise and diet on [3H]thymidine incorporation by splenocytes from rats implanted with the Morris Hepatoma 77771,2
DISCUSSION gln diet incubated in RPMI 1640 culture media supplemented with 1 mol/L glutamine utilized (P < 0.05) tryptophan, glutamine, methionine, isoleucine, asparagine, leucine, serine, histidine, threonine, valine, aspartate, phenylalanine and glycine and released (P < 0.05) glutamate, alanine, ornithine and tyrosine into the media (Fig. 2). There was no significant change in the concentration of arginine, lysine and taurine in the culture media.
Fig. 2.
Change in the amino acid balance of Morris Hepatoma 7777 cells incubated in culture media. Bars represent the mean ± SEM of tumor cells isolated from rats (n = 7) as determined by HPLC. For all of the amino acids illustrated, there was a significant (P < 0.05) change in the concentration after incubation with tumor cells. The RPMI culture media contained glucose (10 mmol/L) and amino acids in the following concentrations (mmol/L): 0.49 L-arginine, 0.32 L-asparagine, 0.14 L-aspartic acid, 0.14 L-glutamic acid, 1.14 glutamine, 0.12 glycine, 0.10 L-histidine, 0.15 L-hydroxyproline, 0.33 L-isoleucine, 0.31 L-leucine, 0.17 L-lysine·HCL, 0.14 L-methionine, 0.07 L-phenylalanine, 0.17 L-proline, 0.25 L-serine, 0.15 L-threonine, 0.15 tryptophan, 0.08 L-tyrosine and 0.14 L-valine.
[View Larger Version of this Image (29K GIF file)]
gln, 6.8 ± 1.0% (n = 10); sedentary +gln, 7.6 ± 1.2% (n = 10); sedentary gln, 4.6 ± 0.6% (n = 5). However, the percentage of specific lysis (NK activity) was not significantly affected by diet across the five effector:target ratios measured (data not illustrated). Lytic units, the number of cells (× 103) required to induce 15% lysis of target cells, were not affected by diet or exercise (33 ± 4, n = 24). There was no effect of diet or exercise on any of the other phenotypes measured in spleen [OX19+ (CD5), 56 ± 1%; OX8+ (CD8 + NK cells), 18 ± 1%; W3/25+ (CD4), 33 ± 1%; OX12+ (B cells), 33 ± 1%; OX42+ (macrophages) 21 ± 1% and the CD4/CD8 ratio, 2.2 ± 0.1%, n = 35].
gln groups was lower than that of the other three groups (Fig. 3).
Fig. 3.
The effect of diet and exercise on the mitogenic response of splenocytes from rats implanted with the Morris Hepatoma 7777. Bars represent the mean ± SEM (n = 6/group). Mitogenic response is expressed as the stimulation index = (amount of [3H] thymidine incorporated by stimulated cells amount of [3H] thymidine incorporated by unstimulated cells)/amount of [3H] thymidine incorporated by unstimulated cells. Cells were incubated for 72 h with Concanavalin A (Con A) or with phorbol myristate acetate + ionomycin (PMA + Iono). For each culture condition, bars that do not share a common letter are significantly different (P < 0.05).
[View Larger Version of this Image (35K GIF file)]
). The reason for not seeing an effect of exercise may be related to the macronutrient content of the diets fed in the present study. Previous studies demonstrating an effect of exercise on tumor growth used nonpurified rodent diets. In the present study, we formulated a basal diet providing 20 g/kg fat, and high fat diets have been shown to increase the growth of many experimental rodent tumors (Welsch 1994). The major finding of this study is that providing a purified diet supplemented with 20 g/kg L-glutamine reduced the growth of the MH 7777 in both sedentary and exercise-trained rats. Despite reports that low to moderate exercise decreases the growth of some experimental tumors (Jadeski and Hoffman-Goetz 1996), including the MH 7777 (Baracos 1989), exercise did not significantly alter tumor growth. The MH is a very rapidly growing tumor that results in host death within 3-4 wk (LeBricon et al. 1995). For lung tumors, it was recently reported that moderate exercise is effective in reducing the growth of only less aggressive tumors (Jadeski and Hoffman-Goetz 1996).
, LeBricon et al. 1995, Souba 1993), and the presence of a tumor is associated with an increase in muscle glutamine synthetase activity (Chen et al. 1993) and glutamine transport from the liver (Inoue et al. 1995). The extent of host depletion appears to be related to tumor size because a small tumor burden (similar to that in the present study) in contrast to a large burden (2-6% body weight) did not alter muscle glutamine concentration (Chen et al. 1993). In the present study, glutamine was supplemented at levels recommended for total parenteral nutrition and demonstrated to minimize muscle glutamine depletion in catabolic states (Alverdy et al. 1992, Swails et al. 1992). This represents a considerable percentage of total protein (7% w/w) and total energy intake (2%). Despite this high intake of glutamine, plasma and muscle glutamine concentrations did not differ among groups. Plasma concentrations of leucine, valine, isoleucine, glutamate and aspartate, all potential precursors of glutamine, were higher in glutamine-supplemented rats. This suggests a "sparing" of glutamine precursors in these rats. In support of this concept, it was recently demonstrated that providing dietary glutamine to tumor-bearing rats down-regulated hepatic glutamine transport (Inoue et al. 1995). The unsupplemented diet contained added glycine and resulted in a higher concentration of glycine (threefold) and serine (twofold) in both plasma and soleus muscle. We reported a similar effect of these diets on plasma concentrations when fed to nontumor-bearing rats (Shewchuk et al. 1996a). The present study differs from earlier studies (Austgen et al. 1992, Klimberg et al. 1990) that demonstrated an effect of supplemental glutamine on plasma and muscle glutamine concentrations, in that the unsupplemented diet was not glutamine free. Casein contains 8-13 protein-bound glutamine g/100 kg (Swails et al. 1992). Thus both diets provided ~2-3 protein-bound glutamine g/100 kg. To our knowledge, there have not been any studies examining whether protein-bound glutamine provides the same beneficial effects as those associated with free glutamine.
). The higher food intake in glutamine-supplemented rats by d 7 (Fig. 1) likely reflects the smaller tumor burden and decreased tumor-induced anorexia. Body composition was not measured in the current study, but based on body and tumor weights, the tumor-free body weight of the glutamine-supplemented rats was greater (P < 0.05) than that of the nonsupplemented rats.
). The tumor appears to act as a glutamine trap, leading to proteolysis and the subsequent development of cancer cachexia (Chen et al. 1993, LeBricon et al. 1995). Compared with glutamine alone, providing glucose in the incubation media reduced the oxidation of glutamine by MH 7777 cells. For other cell types, it has been reported that glucose decreases glutamine metabolism via decreasing intracellular phosphate concentrations which result in a decrease in glutaminase activity (Sri-Pathmanathan et al. 1990). Glutamine also serves as an important nitrogen precursor for the biosynthetic processes associated with cellular proliferation (Souba 1993). Consistent with reports of preferred glutamine oxidation by tumors (Sauer et al. 1983), including the MH 7777 (Mares-Perlman and Shrago 1988), when a mixture of amino acids were provided to MH 7777 cells, large amounts of glutamine were utilized and glutamate produced (Fig. 2). It is unclear why poorly differentiated tumor cells consume and incompletely oxidize such large amounts of glucose and glutamine, but it is hypothesized that high rates of glycolysis and glutaminolysis are necessary to enable sensitive and precise metabolic control of pathways that synthesize macromolecules (Souba 1993). In addition, it was reported recently that hepatoma cells synthesize lipids from glutamine via a reductive carboxylation of -ketoglutarate (Holleran et al. 1995).
). A tumor could disrupt this balance by changing host protein intake, intestinal absorption, hepatic synthesis, tissue oxidation and protein turnover or its own rates of utilization and production. Some studies have suggested a relationship between tumor size and extent of aberration in amino acid kinetic parameters (reviewed by Pisters and Pearlstone 1993). Changes in specific amino acids have been related to tumor growth. Tumors require a supply of ornithine or its precursors to maintain growth (Marquez et al. 1989). Feeding the unsupplemented diet was associated with a lower plasma ornithine level, suggesting that ornithine was utilized to a greater extent by the larger tumor mass in these rats. Tryptophan depletion alters tumor growth in vitro (Marten et al. 1994). In vitro, the MH 7777 was observed to consume tryptophan, methionine and the branched-chained amino acids (valine, isoleucine and leucine). This is consistent with arterio-venous difference in amino acid concentrations measured across the MH 7777 (Sauer et al. 1982). Thus, the smaller tumor burden in glutamine-supplemented rats might account for their higher plasma concentration of tryptophan and branched-chained amino acids. Large amounts of ammonia were produced by tumor cells when provided with glutamine in vitro. This is consistent with elevated plasma ammonia concentration in tumor-bearing rats (Chance et al. 1991). Given the high utilization of amino acids by the MH 7777 cells (Le Bricon et al. 1995), this would result in a considerable ammonia burden to the host. Glutamine occupies a unique role in interorgan ammonia metabolism (Souba 1993). Hyperammonemia is proposed to be related to changes in brain neurotransmitters and anorexia (Chance et al. 1991).
). With other experimental tumors, glutamine supplementation was reported to increase (Fahr et al. 1994) or not change (Jadeski and Hoffman-Goetz 1996) NK activity in tumor-bearing animals. In the present study, glutamine supplementation resulted in a higher proportion of NK cells in spleens of tumor-bearing rats but did not alter the ability of splenocytes to lyse YAC-1 cells. In vivo, NK cells contribute to limiting both the development of transplanted primary tumors and metastasis from established tumors (Hanna 1985). Although the cytotoxic activity of splenocytes against YAC-1 cells correlates well with NK cell activity in the host early in the establishment of other tumors (Hanna 1985), it is possible that the higher proportion of NK cells seen in glutamine-supplemented rats may reflect more NK cells with specific reactivity against the MH 7777 but which are not cytotoxic against YAC-1 cells. Further studies, using the Morris Hepatoma as the target, are required to establish NK activity as the mechanism for a smaller tumor burden in glutamine-supplemented rats.
). Dietary glutamine supplementation resulted in a higher 48-h response to Con A by both the exercising and sedentary groups and at 72 h by the sedentary group. This suggests that providing glutamine may improve the early cell-mediated response to an immune challenge. Investigations with rodents have suggested that exogenous nucleotides provided in diets may be essential to ensure an adequate pool of nucleotides for lymphocytes (Van Buren et al. 1994). Restricting dietary nucleotides (by feeding purified casein diets) has been demonstrated to decrease immune function (Van Buren et al. 1994). Although casein is the basis for many of the current enteral formulas available to feed cancer patients, it is possible that all of our rats demonstrated some immunosuppression due to the lack of nucleotides in both diets.
). Surgical, drug and radiation therapies have immunosuppressive effects on cancer patients (Longo and Hubbard 1991). Regular exercise, by augmenting cell-mediated immune function may help in patient tolerance and recovery from treatment. Exercise did not significantly affect the activity of the oxidative enzyme succinate dehydrogenase in the soleus muscle, an indicator of training. We have previous demonstrated in nontumor-bearing rats that swimming increases succinate dehydrogenase activity by 170% (Shewchuk et al. 1996a). The activity of this enzyme found in the present study was less than one half of that previously reported in sedentary nontumor-bearing rats (Shewchuk et al. 1996a). Surprisingly, feeding the glutamine-supplemented diet resulted in lower succinate dehydrogenase activity. The reason for this is not clear at this time. Similar to nontumor-bearing rats (Shewchuk et al. 1996a), rats following the swimming protocol used in this study did not have altered plasma or muscle glutamine concentrations. The lower plasma concentrations of histidine and threonine in exercise-trained rats are consistent with lower concentrations of these amino acids in soleus muscle.
FOOTNOTES
gln, glycine-supplemented diet; Iono, ionomycin; MH 7777, Morris Hepatoma 7777; NK, natural killer; PMA, phorbol myristate acetate.
Manuscript received 28 May 1996. Initial reviews completed 25 June 1996. Revision accepted 4 September 1996.
ACKNOWLEDGMENT
The authors would like to acknowledge the excellent technical assistance of S. Goruk.
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