JOURNAL OF CELLULAR PHYSIOLOGY 143:94-99 (1990) zy Comparison Between Transport and Degradation of Leucine and Glutamine by Peripheral Human Lymphocytes Exposed to Concanavalin A zyxwvuts zyxwvu zyxwvu BRIGITTE KOCH, MARIE-THERES SCHRODER, CERTRUD SCHAFER, AND PETER SCHAUDER* Department of Medicine, Division of Gastroenterologyand Endocrinology, University of Gottingen, 3400 Cottingen, Federal Republic of Germany Transport and pathways of leucine and glutamine degradation were evaluated in resting h u m a n peripheral lymphocytes and compared with the changes induced by concanavalin A (ConA).Cells were incubated with [l-’4C]leucine(0.15 mM), [U-’4C]leucine(0.15 mM), or [U-’4C]glutamine (0.4 mM) after culture with or without 2 , 5, 7, or 10 (*g/ml ConA for 2, 18, or 24 hours, respectively. Initial rates of transport of leucine and glutamine were augmented 2.7-fold and threefold by t h e mitogen. Leucine transamination, irreversible oxidation, and catabolism beyond isovaleryl-CoA were increased by 90%, 20%, and GO%, respectively. Glutamine utilization increased threefold; accumulation of glutamate, aspartate, and ammonia increased by 700%, SO%, and loo%, respectively, and 14C0, production by about 400% in response to ConA. The results indicate that ConA stimulates to about the same extent transport of leucine and glutamine into lymphocytes. Glutamine is mainly channeled into catabolic pathways, while leucine remains largely preserved. It is suggested that these metabolic changes provide more leucine for incorporation into protein and more N- and C-atoms required for the synthesis of macromolecules and energy from glutamine. In lymphocytes, mitogens activate a series of metabolic pathways, thereby finally enabling cells to proliferate. Early events associated with the interaction between mitogens and cell membranes include changes in metabolic flux (Hedeskov, 1968; van den Berg and Betel, 1971; Peters and Hansen, 1971; Ardawi and Newsholme, 1983). As a consequence, the intracellular processing of the respective metabolites becomes affected. It is important to characterize these adaptations because they provide the basis for the increase in the synthesis of protein, RNA, and DNA occurring during cell proliferation. Leucine is a n essential amino acid that is critical for protein synthesis (Adibi, 19841, and glutamine is essential for proliferating lymphocytes (Baechtel et al., 1976; Crawford and Cohen, 1985).We report on transport and degradation of leucine and glutamine in human peripheral lymphocytes exposed to concanavalin A (ConA). phate-buffered saline (PBS) containing 136.9 mmol/l NaC1,2.7 mmol/l KCl, 8.1 mmol/l Na,HPO,, 1.5 mmol/l KH,PO,, 0.5 mmol/l MgC1,6H2O, and 0.8 mmol/l CaC1, were obtained from Biochrom KG (West Berlin). Penicillirdstreptomycin was from Gibco Europe (Karlsruhe, FRG), human serum albumin (HSA) was from Paesel & Lorei (Frankfurt, FRG), and ConA was obtained from Sigma Chemie GmbH (Deisenhofen, FRG). [U-14C]glutamine was purchased from Amersham-Buchler (Brau:nschweig, FRG); [l-14Clleucine, IU-14Clleucine, Aquasol, and Liquifluor were from New England Nuclear (Dreieich, FRG). Unlabeled amino acids were obtained from Serva (Heidelberg, FRG), and toluene, ceric sulphate, silicone oil 550, dinonylphthalate, and sulphosalicylic acid were from Merck (Darmstadt, FRG). Enzymes, coenzymes, and other chemicals used for spectrophotometric measurements were products of Boehringer Mannheim (Mannheim, FRG). Coomassie blue reagent for protein assay was from Bio-Rad (Miinchen, FRG). Monoclonal antibodies for characterization of lymphocyte subsets were from Dakopatts ais (Glostrup, Denmark), i.e., Dako CD 22 (B lymphocytes), Dako-T, (pan T cells), Dako-T, (suppressodcytotoxic T cells), Dako-T, (helperhnducer T cells), or zyxwvutsr SUBJECTS, MATERIALS, AND METHODS Venous blood was obtained from healthy blood donors of both sexes. All had a normal body weight a s defined by a body mass index (kgim’) <24 and normal serum bilirubin, creatinine, electrophoresis, glutamate pyruvate transaminase, and alkaline phosphatase, as well as normal red and white blood cell counts. Ficoll, medium RPMI 1640, fetal calf serum (FCS), and phos0 1990 WILEY-LISS, INC. Received September 6, 1989; accepted December 15, 1989. *To whom reprint requests/correspondence should be addressed. z GLUTAMINE AND LEUCINE METABOLISM IN HUMAN LYMPHOCYTES from Becton-Dickinson (Mountain View, CAI, i.e., antiLeu-19 (natural killer cells). zyxwv 95 natants were neutralized with 1 M KOH, containing 10.5 mM triethanolamine-HC1, and stored at -20°C pending spectrophotometric measurements. Controls Lymphocyte preparation and culture without lymphocytes were carried through the same Venous blood samples were obtained from healthy procedure. Concentrations of glutamine and of glublood donors of both sexes. Mononuclear cells were pre- tamine breakdown products in neutralized extracts of pared according to the method of Boyum (1968). The cells plus medium were measured spectrophotometripercentage of monocytes/macrophages was reduced to cally on a Kontron Uvicon 960 spectophotometer by about 3% by allowing these cells to adhere to plastic using enzymic assays: glutamate and glutamine by the during a 15 min incubation in RPMI 1640 containing method of Beutler and Michal(19741, aspartate by the 10% (viv) heat-inactivated FCS. Cells were pelleted by method of Bergmeyer et al. (19741,and ammonia by the centrifugation for 10 rnin at 400g, suspended in 1ml of method of Gutmann and Bergmeyer (1974). Production RPMI 1640, and transferred to culture medium con- of 14C02from [U-'4C]glutamine was determined as detaining ConA (2, 5, 7, or 10 pg/ml). Controls were cul- scribed for incubations with [U-14C]leucine,except that tured in the absence of the mitogen. Culture medium a-ketoglutarate was omitted from the incubations. consisted of RPMI 1640 supplemented with 15% FCS Blank values were obtained from incubations without (v/v), penicillin (100 U/ml), streptomycin (100 pg/ml), the cells. and glutamine (2 mM). Lymphocytes (1.5-3.106 cells/ ml) were cultured for 2, 18, or 24 hours at 37°C in a 5% Characterization of lymphocytes C 0 2 incubator. Upon completion of the cultures, cells to incubations, lymphocytes were characterized Prior were harvested by centrifugation (10 min at 1,4OOg), for subsets using monoclonal antibodies (Cordell et al., washed with PBS, and incubated. 1984), for viability (eosin red exclusion), and for their Determination of amino acid transport rates content of protein (Bradford, 1976) as well as DNA Rates of leucine and glutamine transport were deter- (Burton, 1956). mined as described previously (Schauder et al., 1989). Briefly, 2.106 cells were incubated for 0.5 min at 37°C Expression of results in 0.5 ml PBS, pH 7.4, containing 0.5 g/dl HSA and Transport rates were calculated after subtraction of either leucine (0.15 mM) or glutamine (0.4 mM) with 0.5 pCi [l-14C]leucine or [U-14C]glutamine, respec- blank values from the radioactivity recovered in the tively. Cells were separated from incubation medium cell pellet and the specific radioactivity of substrate in by centrifugation through a silicone oil-dinonylphtha- the media, and expressed as pmol transported by 2-106 late layer. Radioactivity was measured in cell lysates cells per 0.5 min. Likewise, rates of leucine transamiadded to 6 ml of Aquasol. Blank values were obtained nation and oxidation, or rates of CO, production from glutamine, were determined from the radioactivity in from cells incubated on ice. the 14C02trapped and from the specific radioactivity of Leucine degradation substrates in the media. Values are expressed as pmol/ Rates of leucine transamination and oxidation were 2.106 cells/40 min. Glutamine, glutamate, aspartate, determined by incubating lymphocytes with 11-l4C1 and ammonia utilization or accumulation rates were leucine as previously described (Schauder and Schafer, calculated as net differences between incubations with 1987; Schafer and Schauder, 1988). Degradation of leu- and without cells. Results are expressed as nmol uticine beyond isovaleryl-CoA was measured by incubat- lized o r accumulated by 2.107 cells/l20 min. Statistical analysis was made by Student's t test ing 2.106lymphocytes for 40 min a t 37"C, pH 7.4, in 1.0 ml PBS buffer (150 mM), containing HSA (0.5 g/dl), based on paired comparisons. Results were considered pyridoxal phosphate (0.1 mM), a-ketoglutarate (0.1 statistically significant if the significance level was mM), unlabeled leucine (0.15 mM), and [l-'4C]leucine <0.05. (0.2 pCi) or [U-14Clleucine(0.45 pCi). Incubations were stopped with 2 N H2S04(0.5 ml) and 14C02collected for RESULTS 60 min in center wells containing 0.3 ml hydroxide of hyamine, as previously described (Schauder and SchaCharacterization of lymphocytes fer, 1987). Rates of leucine degradation beyond isovaleryl-CoA are obtained by subtracting the production of Lymphocytes were characterized after the 24 hour 14C02from [l-'4Clleucine from the respective rate gen- culture period for distribution of subsets, cell viability, and protein and DNA content. Distribution of pan T erated by cells incubated with [U-14C]leucine. cells, T,, T,, pan B cells, and natural killer cells was Glutamine degradation 75 k 1% (4),44 & 1%(4), 18 2 2% (41, 9 k 1%(41, and Glutamine utilization and accumulation of glu- 11 k 1%(41, respectively. tamine breakdown products were determined by incuCell viability was determined by eosin exclusion. bating %lo7lymphocytes for 120 min at 37°C in 1.5 ml The percentage of nonviable cells was 3.5 2 0.2% (n = PBS buffer (pH 7.4; 0.5 g/dl HSA, 0.4 mM glutamine). 55) in the absence and 9.4 k 0.5% (n = 55) in the Prior to incubation, reaction mixtures were gassed presence of 10 pg/ml ConA. Protein content in resting with 100% O2 for 10 sec. Reactions were stopped by and stimulated cells was 29.2 f 1.3 (n = 12) VS. 36.8 adding 0.3 ml of 30% (w/v) sulphosalicylic acid. Incu- 2.0 (n = 12) pg/cell ( P c 0.05), and DNA content was bation vessels were kept on ice for 10 min prior t o cen- 11.9 0.23 (n = 12) vs. 12.8 0.78 (n = 12) pgicell, trifuging the precipitates at 12,OOOg for 2 min. Super- respectively. zyxw zyxwvu zy zyx zyx * zyxwvuts zyx zyxwvutsrq zyxwvuts zyxwvutsrq zyxwvutsrqpo zyxwvutsrq zyxwvutsr 96 KOCH ET AL. TABLE 1. Leucine transport and degradation by incubated resting and stimulated human peripheral lymphocytes' TABLE 2. Glutamine transport and degradation by incubated resting and stimulated human DeriDheral Ivmohocvtes' Parameter Rate measured Increase Compound (%) ConA measured Controls N tested 267 18t3 48 2 12 [l-14Clleucine 9 Transport' 194 9 Tran~amination~370 f 30 716 ? 98 122 264 2 16 322 2 34 9 Oxidation" 182 271 2 18 493 2 54 8 Oxidation" [U-'4C]leucine- Parameter analvzed Glutamine transport' Glutamine utilization3 Glutamate accumulation" Aspartate accumulation3 Ammonia accumulation3 14C0., aroduction4 'Lymphocytes isolated from venous blood of healthy donors were cultured for 24 hours with or without ConA (10 Fg/ml)and subsequently incubated with ll-14Clor LU-i4Clleucine(0.15 mM). Values are means S E M . N, number o f donors. LpmoV30sec/2.10ficells. 3pmo1/40 min/2-10ficells. N 11 12 12 12 12 13 Rate measured Controls ConA 34f4 102 2 16 49 f 3 157 2 12 12 f 3 97 t 6 21 f 3 32 IT 3 71 t 8 146 f 19 984f 88 4.548 t 346 Increase (%c) 300 320 808 152 206 462 'Lymphocytes isolated from venous blood of healthy donors were cultured for 24 hours with or without ConA (10 pg/ml), and subsequently incubated with or without [U-L4Clglutamine(0.4 mM). Values are means 2 SEM. N, number of donors. 'pmo1/30 sec/2.10fi cells. 'nmol/120 mid2.i07 cells. 4pmo1/40 min/2.106 cells. Transport rates Initial rates of uptake were determined by incubating cells for 30 sec with [l-14Clleucine or [U-14Cl nmolil20 min/2.107 cells in resting and stimulated glutamine after a 24 hour culture period with or with- cells, respectively. out 10 kgiml ConA. In resting lymphocytes, transport End products of glutamine degradation also inrates of leucine and glutamine were 18 +- 3 and 34 4 creased in response to ConA. Ammonia accumulated pmoli30 ~ e c i 2 . 1 0cells, ~ respectively. In cells exposed to from 71 +- 8 to 146 19 nmol/l20 min/2.107 cells, while ConA, transport rates were augmented to 48 12 14C02 production from [U-14C]glutamine increased pmol/30 sec/2.106 cells for leucine (2.7-fold) and to 102 from 984 2 88 to 4,548 346 nmol/40 min/2.106 cells. 2 16 pmoli30 ~ e c i 2 . 1 0cells ~ for glutamine (threefold) (Tables 1, 2). Concanavalin A concentration, culture time, and 14C0, production rates Leucine degradation As shown in Figure 1, a linear relationship was obPathways of leucine catabolism are shown in Table served between the concentration of ConA ( 0 , 2, 5, 7, 1. The first step of leucine catabolism, i.e., leucine and 10 pg/ml) and 14C02 production from [U-l4C1 transamination, was determined from the sum of 14C02 glutamine. Augmentation was about fivefold. Leucine liberated enzymatically and chemically from [l- transamination increased linearly up to 5 p,g/ml ConA. 14]leucine (Schauder and Schafer, 1987). As shown in The increment was less than twofold (82 +- 16%). No Table 1, transamination was 370 ? 30 vs. 716 34 further increase occurred if ConA was augmented to 10 pmol/40 min/2.106 lymphocytes in resting and stimu- pg/ml. Leucine oxidation at 2, 5 , and 10 pg/ml ConA lated cells, respectively. was 16 2 16%, 52 5 lo%, and 22 10% higher than in The second step of leucine catabolism, i.e., leucine controls. As shown in Figure 2, the stirnulatory effect of oxidation via branched chain keto acid dehydrogenase, ConA (10 pg/ml) on 14C02production is time depenwas only slightly augmented by ConA (10 kg/ml) from dent. As indicated by the production rates a t 2, 18, and 265 & 16 to 322 k 34 pmol/40 mi1d2.10~lymphocytes. 24 hours, the relationship is not linear. Leucine oxidaLeucine catabolism beyond leucine oxidation was de- tion, transamination, and I4CO2 production from [lJtermined by measuring I4CO2 production from [U- 14Clglutamine was higher in lymphocytes exposed to 14C]leucine in relation to 14C02 production from [l- ConA for 18 hours instead of 2 hours. Subsequently, 14C]leucine. As seen in Table 1, production rates of i.e., when the exposure to ConA was prolonged to !24 14 CO, from [U-14C]- and [l-'4Clleucine were identical hours, l4CO, production from [U-14C]glutamine inin resting lymphocytes. In cells exposed to ConA the creased sharply, while leucine transamination aprespective 14C02production rates were 493 ? 54 vs. peared to level off and leucine oxidation became even 322 34 (P < 0.05). slightly diminished. * * zyxwv * * zyx * * * Glutamine degradation DISCUSSION The metabolic events t h a t were analyzed probably Pathways of glutamine degradation in resting and stimulated lymphocytes are shown in Table 2. Glu- reflect T-lymphocyte activity mainly, because the cell tamine utilization, reflecting the sum of anabolic and population investigated consisted of 75% T lymphocatabolic processes of lutamine, was 49 3 vs. 175 % cytes and because the T-cell activator ConA was stud12 nmol/l20 min/2-10? cells in resting and stimulated ied. lymphocytes, respectively. Glutamate, resulting from In human peripheral lymphocytes exposed for 24 deamination of glutamine by the phosphate-dependent hours to ConA, initial rates of leucine and glutamine glutaminase, accumulated in resting cells and in the transport were enhanced. The respective augmentation lymphocytes exposed to ConA during incubation in a was similar in magnitude (Tables 1, 2). Previous studmedium containing 0.4 mM glutamine. Glutamate ac- ies suggest preferential stimulation of Na +-dependent cumulation in resting and stimulated cells was 12 * 3 transport processes by the mitogen (van den Berg, vs. 97 k 6 nmol/l20 min/2.107 cells. 1974; Borghetti et al., 1979, 1981), possibly via changAspartate, generated from glutamine breakdown be- ing membrane potentials (Borghetti et al., 1979; Mit3 vs. 32 * 3 sumoto et al., 1988). yond glutamate, accumulated to 21 * * zy zyxw 97 GLUTAMINE AND LEUCINE METABOLISM IN HUMAN LYMPHOCYTES zyxwvuts zyxwvutsrq zyxwvutsr zyxwvutsrqponm zyxwvutsr a 0 [U-14C]glutamine ( 0 . 4 m M ) [ 1 - 14C] leucine (0.15 m M [U-'4C]glutamine ( 0 . 4 m M ) [l -14C]leucine(0.1S m M ) c + c - * ?- rl 5 E 4.0- - 3.2- .- 0 'LI g * R - - 2.4- P g 2 1.6- f u- 0 * 0.8- u) .m 0-L 0 2 5 Concanavalin A (,ug/rnl) 10 24 Culture time [hrs] * * . 9, c = * 18 Fig. 2. Degradation of glutamine and leucine by incubated peripheral human lymphocytes exposed to ConA for various periods of time. Lymphocytes isolated from venous blood of healthy donors were cultured for 2, 18, or 24 hours with or without ConA (10 Fgiml) and subsequently incubated with [U-'*Clglutamine (0.4 mM) or [1-14Cl leucine (0.15 mM). Values are means ? SEM obtained from blood of eight or more donors and are expressed as percentage from controls incubated without ConA. Asterisks indicate a significant difference from cells incubated without ConA (*P < 0.05; **P < 0.01; ***P < 0.001). 0, Leucine transamination; A, leucine oxidation. zyxw zyxwvuts Fig. 1. Degradation of glutamine and leucine by incubated peripheral human lymphocytes in response to various concentrations of ConA. Lymphocytes isolated from venous blood of healthy donors were cultured for 24 hours with or without ConA (2, 5, 7, or 10 kg/ml) and subsequently incubated with [U-'4Clglutamine (0.4 mM) or [l14C]leucine(0.15 mM). Values are means ? SEM obtained from blood of eight or more donors and are expressed as nmol I4CO, produced in 40 min by 2,106cells. Asterisks indicate a significant difference from cells incubated without ConA (*P < 0.05; **P < 0.01; ***P < 0.001). 0, Leucine transamination; A, leucine oxidation. Leucine transport into human lymphocytes is through the Na+-independent system L (Segel et al., 1985; Schauder et al., 1989). Glutamine transport has a broader specificity and occurs via Na+-dependent carriers similar to system N and system ASC (Schroder et al., submitted). From the results presented in Tables 1 and 2, it can be inferred that ConA affects the function of at least three carrier systems. Considering the heterogeneity of the carriers that are affected rather similarly by ConA, we suggest as underlying mechanism some nonspecific effect such as a steric change in the structure of the amino acid carriers. Following transport into lymphocytes, leucine can be either incorporated into protein or degraded. Table 1 shows effects of ConA on the first step of leucine catabolism (transamination), the second step (oxidation), and on leucine catabolism beyond the second step. Transamination is stimulated about twofold by ConA. This may result from increased leucine transport or from some direct effect on the transaminase. Leucine oxidation is only 22%higher in mitogen-stimulated lymphocytes than in the controls. This step is rate limiting for leucine catabolism in human lymphocytes (Schauder and Schafer, 1987) and irreversible, thereby submitting leucine definitely to catabolism (Paul and Adibi, 1984). Lymphocytes stimulated by ConA require protein synthesis in order to proliferate. It is, therefore, of considerable interest that ConA is greatly promoting transport of leucine into lymphocytes while barely stimulating its catabolism. As a con- sequence, more leucine becomes available for incorporation into protein (see Results). Effects of ConA on leucine degradation beyond the second step was tested by comparing 14C02production from lymphocytes incubated with [U-'4Clleucine or with [ l-14C]leucine. In resting lymphocytes, complete leucine oxidation barely seems to occur (Table 1). However, in stimulated lymphocytes, 14C02 production from [U-l4C1leucineis significantly greater than from [ l-'4C]leucine, indicating stimulation of some enzymatic step distal to isovaleryl-CoA. Leucine intermediates are precursors for lipid synthesis (Goodman et al., 19841, and it is conceivable that more complete breakdown of isovaleryl-CoA is providing more precursors for the lipid moiety of membranes in proliferating cells. Glutamine is essential for lymphocyte metabolism by generating a significant portion of their energy requirements and by providing N- and C-atoms for the synthesis of macromolecules (Newsholme et al., 1985). Glutamine utilization by lymphocytes, which reflects the sum of its anabolic and catabolic processes in these cells, was stimulated more than threefold by ConA (Table 2). The first step of glutamine degradation is deamination to glutamate by a phosphate-dependent glutaminase. ConA augmented glutamate accumulation eightfold (Table 2), indicating that glutaminase is not rate limiting for degradation. Glutamate is further degraded to a-ketoglutarate by oxidative deamination via glutamate dehydrogenase or by transamination via amino acid transferases. Degradation of a-ketoglutarate in the citric acid cycle generates C02 and oxaloacetate, which can be converted to aspartate. As shown in Table 2, ConA augments aspartate accumulation by about 50%. Aspartate fulfills critical functions for cell proliferation such as providing N- and 98 KOCH ET AL zyxwvut zyxwvu zyxwvut C-atoms for the synthesis of purine and pyrimidine. In resting lymphocytes at least 43% of glutamine metabolism beyond glutamate is via aspartate, as estimated from the ratio between glutamine utilization and aspartate accumulation and disregarding aspartate utilization. In stimulated lymphocytes, only 20% of glutamine metabolism is via aspartate, i.e., other glutamine-degrading pathways become activated preferentially. ConA stimulated 14C0, production rates from [U14C]glutamine about fivefold (Table 2). The metabolic significance of this effect is difficult to appreciate because many pathways are contributing, such as the phosphoenol pyruvate carboxykinase, the NAD(P) linked malate dehydrogenase reaction, and complete glutamine oxidation in the citric acid cycle (Ardawi and Newsholme, 1983; Curi et al., 1986). Since much of the carbon during glutamine degradation is lost as CO, rather than being incorporated into macromolecules, ConA-stimulated glutamine degradation may be supplying energy to drive macromolecular synthesis. Glutamine catabolism generates ammonia, and ConA augments ammonia accumulation about twofold (Table 2). However, the ratio between glutamine utilization and ammonia accumulation is significantly smaller in resting than in stimulated lymphocytes (P < 0.051, suggesting that ConA may promote nitrogen retention, possibly by increasing the incorporation of glutamine into macromolecules. Rates of glutamine utilization and accumulation of glutamine-derived metabolites have been reported previously from rat lymphocytes (Ardawi and Newsholme, 19831, rat thymocytes (Brand, 1985; Brand et al., 1987), and human lymphocytes (Ardawi, 1988). In these studies, nitrogen retention in response to ConA was not observed. This is probably due to at least three major differences in the experimental design. First, in our study glutamine was tested at physiological concentration (0.4 mM), compared with 2.0 mM in the other reports. Second, in the study of Brand e t al. (19871, there is a problem with the controls. Accumulation rates from cells incubated for 60 hours with ConA were compared with those from freshly isolated thymocytes, but not with those from cells incubated for 60 hours in the absence of the mitogen. Third, in the study of Ardawi (1988), cells were stimulated for 1 hour, while our cells were exposed to the mitogen for 24 hours. To further characterize the effects of ConA on leucine and glutamine degradation, lymphocytes were incubated with various concentrations of the mitogen or for different periods of time. As shown in Figures 1 and 2, the more lymphocytes become activated in response to ConA, the greater becomes the dissociation between the rates of glutamine and leucine degradation. It remains to be investigated whether similar findings would occur with other mitogens, and how the dissociation between rates of glutamine and leucine is affected in cells cultured for more than 24 hours. Furthermore, a n important way to appreciate the metabolic role of glutamine for lymphocyte proliferation is to compare its effects with those of other metabolites, such a s glucose or fatty acids, using physiological concentrations. Finally, there is no ready explanation of how ConA + should preferentially augment glutamine catabolism over leucine degradation. Irrespective of the proper explanation, this metabolic adaptation is providing the proliferating cells with more leucine for incorporation into protein, with more N- and C-atoms for synthesis of macromolecules, and with more energy. ACKNOWLEDGMENTS The careful technical assistance of Miss A. Struck is greatly appreciated. LITERATURE CITED Adibi, S.A. (1984) Nutritional, physiological and clinical significance of branched chain amino acids. In: Branched Chain Amino and Keto Acids in Health and Disease. S.A. Adibi, W. Fekl, U. Langenbeck, and P. Schauder, eds. Karger, Basel, pp. 1-14. Ardawi, M.S.M. 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