- Email: [email protected]
Dexamethasone stimulation of glutaminase expression in mesenteric lymph nodes
Dexamethasone Stimulation of Glutaminase Expression in Mesenteric Lymph Nodes* Paul S. Dudrick, rat), Peter Sarantos, rap, Kimberly Ockert, as, Ratna Chakrabarti, PhD, Edward M. Copeland, MD, FACS, Wiley W. Souba, MD, SeD, FACS,Gainesville,Florida
Lymphocytes in the mesenterie lymph nodes (MLNs) play a key role in protecting the body from the translocation of bacteria through the bowel during catabolic states. Lymphocytes use glutamine for energy and for DNA synthesis and have high levels of the glutaminase (GA) enzyme that regulates intraeellular glutamine metabolism. This study tested the hypothesis that the increase in circulating glucocorticoid hormones that occurs in respouse to stress states regulates MLN lymphocyte GA expression at the molecular level. Adult male rats (n = 60) received a single dose of dexamethasone (DEX 0.5 mg/kg intraperitoneally) or saline vehicle (CONT). lleocolic lymph nodes were excised from anesthetized rats via laparotomy at 2, 4, 12, 24, 48, and 72 hours after injection, and GA activity was assayed in MLN homogenates. A second group of rats received repeated doses of DEX (0.5 mg/kg/d for 4 days). GA activity was assayed in MLN homogenates, and total RNA was extracted for quantitation by Northern hybridization using a phosphorus 32-labeled rat GA eDNA probe. GA activity was increased within 2 hours after a single dose of DEX, with a peak after 4 hours. Kinetic analysis at the 4-hour time point showed an increase in the maximum GA activity (maximal transport velocity [Vmax]:619 4- 107 nmol/mg protein/hr in DEX versus 380 4- 53 nmol/mg protein/ hr in CONT, p < 0 . 0 5 ) , with no change in GA affinity (Michaelis constant 0Kin]: 2.28 4- 0.39 mM in DEX versus 2.20 4- 0.36 mM in CONT, p = NS). Repeated doses of DEX resulted in a twofold increase in GA activity (550 4- 125 nmol/mg protein/hr versus 1,175 4- 40, p < 0 . 0 1 ) . Simultaneously, GA mRNA levels were increased by 70%. Glucocorticoids stimulate GA activity and gene expression in lymphocytes that reside in MLNs.
From the Department of Surgery, Universityof Florida Collegeof Medicine, Gainesville,Florida. Supported by National Institutes of Health (NIH) grantCA 45327 (Dr.Souba),a grantfromthe Veterans AdministrationMerit ReviewBoard (Dr. Souba), and NIH Training Grant T32-CA09605-03 (Drs. Dudrickand Sarantos). *Dr. Dudrickis the recipientof the Resident'sPrizeawardedannuallyby the Societyfor Surgeryof the AlimentaryTract. Requestsfor reprintsshouldbe addressedto WileyW. Souba,MD, Department of Surgery,JHMHSC Box 100286, Gainesville,Florida 32610-0286. Presentedat the 33rdAnnualMeetingofthe Societyfor Surgeryof the AlimentaryTract, San Francisco,California,May 11-13, 1992. 34
n critically ill patients, bacterial translocation may Imucosal 9occur as a consequence of the failure of the intestinal and immune barriers [1]. Recent studies have demonstrated the importance of glutamine metabolism in maintaining intestinal mucosal integrity [2] and improving healing of injured mucosa [3], processes that may diminish the translocation of intestinal bacteria. The function of the cellular components of the gut immune system is also related to the availability and utilization of glutamine [4,5]. In the intestinal tract, the gut-associated lymphoid tissue (GALT) and mesenteric lymph nodes (MLNs) contain the cellular elements of the intestinal immune system. Glutamine deficiency results in a decrease in populations of lymphocytes from the GALT [6] and is also associated with decreased levels of secretory immunoglobulin A (IgA) in the gastrointestinal tract [7]. Glutamine levels drop dramatically in blood and tissues in catabolic states despite a massive efflux from skeletal muscle [8,9] and the lungs [10,11]. The resulting relative glutamine deficiency may adversely the affect immune function of the intestinal tract. Glucocorticoid hormones are released in increased amounts after injury and infection [12,13] and are important mediators of the altered glutamine metabolism that occurs in stress states [10]. In experimental models of physiologic stress, dexamethasone mediates glutamine release from skeletal muscle [8,9] and the lungs [10,11]. The effect of dexamethasone is due, at least in part, to an increase in the activity of glutamine synthetase in these tissues [9,I1], which supports the subsequent efflux of glutamine. During critical illness, this efflux is accompanied by a decline in plasma glutamine concentrations [8,9], which suggests that other tissues are using glutamine at increased rates. Dexamethasone stimulates the uptake and utilization of glutamine by the kidneys [14] and intestine [14,15] in vivo and enhances uptake by hepatocytes in culture [16]. The increase in intestinal utilization of glutamine in response to dexamethasone has been associated with an increase in the specific activity of intestinal glutaminase [17]. The dexamethasonemediated alterations in glutamine metabolism thus represent a balance of cellular glutamine uptake and release, depending on the metabolic needs of the tissue in stress states. Lymphocytes consume relatively large amounts of glutamine, and the rate of utilization increases in response to an immune challenge [4]. Lymphocytes require glutamine for DNA synthesis and cell division in culture and for their proliferation in response to mitogenic stimuli [4]. Glutamine is essential for the transformation of B lymphocytes to immunoglobulin-secreting plasma cells [18]. Depletion of glutamine in vivo is considered specifi-
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eally immunosuppressive [19]. During stress states, glutamine requirements of lymphocytes in the GALT and in MLNs may increase in order to support the cellular response to translocating bowel bacteria. Induction of the glutaminase enzyme may then be necessary to increase the capacity for glutamine utilization in lymphocytes. We hypothesized that the elevated circulating concentration of the glucocorticoid hormones that occurs after surgical stress stimulates lymph node glutaminase activity. The purpose of this study was to examine the regulation of glutaminase expression in MLNs in response to exogenous dexamethasone administration.
MATERIALS AND METHODS All studies were approved by the Committee for the Use and Care of Laboratory Animals at the University of Florida. Adult male Sprague-Dawley rats weighing 200 to 250 g were obtained from the Animal Farm at the University of Florida College of Medicine. Rats were housed in standard rodent cages, exposed to 12-hour dark and light cycles, and allowed intake of water and standard rat chow ad libitum. Food intake in all rats was similar. After the rats were allowed at least 3 days to acclimate to the animal care facility, they were randomly divided into control and experimental groups. Dexamethasone (sodium phosphate salt) for injection was purchased from American Regent Laboratories, Inc. (Shirley, NY). Bovine glutamate dehydrogenase used for the glutaminase assay was purchased from Calbiochem Corp. (La Jolla, CA). All other chemicals used in assay reagents were purchased from Sigma Chemical Co. (St. Louis, MO). A rat complimentary DNA (eDNA) probe for the glutaminase enzyme was obtained as a gift from Dr. Norman P. Curthoys, Department of Biochemistry, Colorado State University, Fort Collins, CO. The aphosphorus 32 (32p) used to label the eDNA probe was purchased from DuPont Co. (Wilmington, DE). The/~actin gene was a gift from Dr. Laurence Kedes, Professor and Chairman, Department of Biochemistry, University of Southern California, Los Angeles, CA. Determination of glutaminase activity: Sixty rats were randomly divided into experimental and control groups. Treated rats received an intraperitoneal injection of dexamethasone (0.5 mg/kg), and control animals received an equal volume of saline vehicle. At various time points after treatment (2, 4, 12, 24, 48, and 72 hours), the animals were anesthetized with ketamine (10 mg/100 g body weight intraperitoneally) for tissue harvest. The ileocolic lymph nodes were excised through a midline laparotomy en bloc with a 2- X 7-mm segment of mesentery and dissected free from surrounding fat. A second group of rats were randomly assigned to receive repeated doses of dexamethasone (0.5 mg/kg/d for 4 days, intraperitoneal) or an equal volume of saline vehicle. MLNs were similarly excised from anesthetized animals 4 hours after the last injection. Animals were killed by cervical dislocation after harvest of the tissue. The lymph nodes from each rat were immediately homogenized in a chilled buffer composed of 50 mM Trizma (Sigma Chemical Co.), 330 mM sucrose, and 5 mM magnesium chloride.
Glutaminase activity of the lymph node homogenate was determined using a phosphate-dependent glutaminase assay described by Windmueller [20]. Protein content of the homogenate was determined using a commercial protein assay (Bio-Rad Laboratories, Richmond, VA). Samples of excised lymph nodes were also fLXedfor histologic examination to quantitate cellularity. Measurement of glutaminase mRNA: Sixteen rats were randomly assigned to receive repeated doses of dexamethasone (0.5 mg/kg/d for 4 days intraperitoneally) or an equal volume of saline vehicle. Four hours after the fourth and final injection, the animals were anesthetized with ketamine (10 mg/100 g body weight intraperitoneaUy), and the ileocolic lymph nodes were excised using a midline laparotomy. The tissue was immediately processed for total RNA extraction using the method described by Chomczynski and Sacchi [21]. Briefly, the lymph nodes were homogenized by dounce in 5 mL of 4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, and 0.1% mercaptoethanol, followed by the sequential addition of 500 #L 2 M of sodium acetate (pH 4.0), 5 mL of H20-saturated phenol (pH 4.0), and 2 mL chloroform:isoamyl alcohol solution (49:1). RNA was precipitated by cold incubation in 2-propanol. The RNA precipitate was centrifuged, and the pellet resuspended in 600 #L of homogenizing solution, followed by reprecipitation by cold incubation in 600 ~L 2-propanol. The RNA was washed again with 70% ethanol and re,suspended in diethylpyrocarbonate (DEPC)-treated water. The resuspended total RNA was reprecipitated by cold incubation in a solution containing 0.1 volume 3 M sodium acetate (pH 5.0) and 2.2 volume 100% ethanol. After the RNA was centrifuged, the final R N A pellet was washed in 70% ethanol and then resuspended in DEPCtreated water. A mRNA for the rat glutaminase gene was used as a probe to measure the concentration of glutaminase mRNA from the rat ileocolic lymph nodes by Northern blot analysis. Equal amounts of RNA (40 tzg), as determined both spectrophotometrically and through ethidium bromide staining, were fractionated by electrophoresis through denaturing agarose gels containing formaldehyde [22]. The RNA was transferred to nylon membranes and cross-linked to the membrane by ultraviolet irradiation. A 1.1-kilobase pair rat glutaminase eDNA [23] containing the N-terminal sequence of glutaminase was cloned in the Bluescript vector (Stratagene Cloning Systems, La Jolla, CA) in reverse orientation. A radiolabeled glutaminase mRNA was synthesized by in vitro transcription using a-32p-CTP (cytidine triphosphate) and a Riboprobe kit (Promega Corp., Madison, WI) according to the manufacturer's specifications. The labeled probe was hybridized overnight at 68~ using the method of Feinburg and Vogelstein [24]. Autoradiographic detection of the hybridization was accomplished by exposing the hybridized nylon membranes to Kodak XAR film for 48 hours at -700C. The glutaminase mRNA bands were quantitated by laser densitometry. Then the GA eDNA probe was stripped off of the membrane by boiling in 0.1% sodium dodecyl sulfate (SDS)
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Figure 1. Glutaminase activity after a single dexamethasone (DEX) injection. Glutaminase activity was significantly increased compared with control values at the 4-hour time point (627 4- 50 nmol/mg protein/hr in control rats versus 980 4- 70 in DEXtreated rats, *p <0.05). Control values obtained for each time point were unchanged and hence were pooled.
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Activity/[GLN] Figure 3. Eadie-Hofstea plot at the 4-hour time point demonstrates that the increase in glutaminase (GLN)activity at 4 hours is due to an increase in Vmax in the dexamethasone-treated rats compared with controls. Data from three separate experiments yielded a mean Vmaxof 619 4- 107 nmol/mg protein/hr in treated rats versus 380 4- 53 in controls (p <0,05). The Krn values were not significantly different between groups (2,28 4- 0.39 mM in treated rats versus 2.20 4- 0.36 mM in controls, p -- NS).
36
(aqueous). The blots were then rehybridized with a eDNA probe for fl-actin, a constitutively expressed gene used as a control for mRNA loading, fl-Actin eDNA was radiolabeled using cP2p-dCTP and a random primer labeling kit (Stratagene Cloning Systems) according to the manufacturer's protocol. The radioactivity of the/3-actin bands was also quantitated by laser densitometry. Calculations and statistical analysis: Glutaminase activity is expressed in nanomoles per milligram of lymph node protein per hour. The Michaelis-Menton kinetic parameters, Vmax (nmoles/mg protein/hr) and Km (raM), of the glutaminase enzyme were determined by measuring enzyme activity over a range of glutamine concentrations. Glutaminase mRNA is expressed in relative units with control values being 100%. The results from the experimental and control groups were compared using the Student's t-test, with a p value of less than 0.05 considered statistically significant. RESULTS Histologic examination of the ileocolic lymph nodes excised from the control and dexamethasone-treated rats demonstrated that the lymphocytes comprised 95% of the total cellularity. Treatment of the rats with a single dose of dexamethasone resulted in an increase in glutaminase activity in MLNs within 2 hours after injection reaching a maximum after 4 hours (Figure 1). The GA activity at 4 hours was significantly elevated in dexamethasone-treated rats compared with saline-treated controls. A plot of glutaminase activity versus glutamine concentration (Figure 2) gives a rectangular hyperbola for each group, with the asymptote representing the maximum rate of substrate conversion (Vm~x), or maximum enzyme activity. Linear transformation of these data with an Eadie-Hofstee (EH) plot (Act versus Act/[Gin], Figure 3) shows that the increase in glutaminase activity after dexamethasone injection was due to an increase in Vmax,represented by the y-intercept of the lines in the E-H plot. The affinity of the enzyme for glutamine (Km)represents the substrate concentration at which half of the available enzyme molecules are bound with substrate and is given by the negative slope of the line. Enzyme affinity was not different between dexamethasone-treated and control rats, as demonstrated by the parallel lines in Figure 3. The increase in Vmaxwith no change in Kmis consistent with an increase in the number of enzyme molecules expressed by the lymphocytes of the MLN, rather than a change in the enzyme affinity for glutamine. Repeated injections (0.5 mg/kg for 4 days) of dexamethasone produced an even greater increase in glutaminase activity assayed 4 hours after the fourth and final dose (Figure 4). The increase in glutaminase activity in the MLN homogenate from dexamethasone-treated rats was associated with an increase in glutaminase mRNA. Northern blot analysis of glutaminase mRNA content, normalized to the constitutively expressed fl-actin gene (Figure 5), demonstrated an increase in the amount of mRNA in the MLN homogenate of dexamethasonetreated rats compared with that of the controls. Quantita-
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tion of the glutaminase mRNA bands from three control and three dexamethasone-treated rats showed a 70% increase in the dexamethasone-treated group compared with the control group (Figure 6). COMMENTS The effects of dexamethasone on glutaminase expression in lymphocytes from MLNs were studied in order to learn about the regulation of glutamine metabolism in the cells of the gut immune system during stress states. The results suggest that the lymphocytes in the MLNs of the bowel mesentery account for a considerable portion of the augmented glutamine uptake by the portal-drained viscera that occurs after glucocorticoid treatment. The dose of dexamethasone used in this study was similar to that used in previous studies [10,14] and was chosen to approximate the threefold to fivefold elevation in circulating cortisol that occurs after a major operation or injury [13]. Glucocorticoid hormones are released by the adrenal gland in increased amounts in stress states, and these hormones have profound effects on amino acid metabolism in many organs. We chose to study the effects of dexamethasone on glutamine metabolism in lymphocytes because: (1) glutamine is the most abundant amino acid in the body and the amino acid most affected by critical illness, (2) lymphocytes are avid glutamine consumers, and (3) the requirement for glutamine by lymphocytes is increased during stress states, which may be an adaptive response designed to support cellular metabolism and function. Glutamine levels dropped dramatically in blood and tissues in catabolic states, producing a relative glutamine deficiency that is associated with adverse structural and functional alterations in tissues that characteristically utilize glutamine at high rates in both the normal and physiologic stress states [25]. The present study demonstrated that the administration of a single physiologic dose of dexamethasone resuited in a twofold increase in MLN glutaminase activity. The increase in activity was prompt and was relatively short-lived. Kinetic analysis indicated that the increase in activity was secondary to an increase in the number of enzyme molecules expressed intracellularly (Vmaxeffect), rather than to a change in the affinity of the enzyme for g l u t a m i n e (Kin effect). Repeated doses of dexamethasone produced an even greater increase in glutaminase activity, consistent with a persistent augmentation in lymphocyte glutamine utilization during chronic stress. This increase was associated with a 70% increase in levels of mRNA for the glutarninase enzyme detected in MLN lymphocytes after repeated dexamethasone injections. Collectively, these data suggest that, in response to glucocorticoids, glutaminase expression is regulated at the transcriptional level. The increase in glutaminase activity reflects an increase in the capacity of lymphocytes to utilize glutamine, which may be essential for the proliferation of these cells in response to an immune challenge, such as translocation of bacteria from the gut lumen. An important feature of the increases in both mRNA and specific activity in this study is the similarity in the magnitude of their increases. This is in contrast to the
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Figure 4. Effect of multiple doses of dexamethasone (DEX) on mesenteric lymph node glutaminase activity. Repeated treatment with DEX resulted in a twofold increase in glutamlnase activity (550 4- 125 nmol/mg protein/hr in control versus 1,175 4- 40 nmol/mg prctein/hr in DEX, "p <0.01).
Glutaminase mRNA
[5-actin mRNA
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Figure 5. Northern blot analysis of glutaminase mRNA after repeated doseS,of dexamethasone (DEX). Representative autoradiograms of glutaminase mRNA (left) and/3-actin mRNA (rlgM).
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Figure 6. Quantitative increase in glutaminase mRNA in dexamethaSone (DEX)-treated rats. Values are expressed In relative units as percent of control and represent quantitation of three separate autoradiograms normalized to/3-actin. *p <0.01.
disparity between glutaminase mRNA and activity seen in the gut mucosa after dexamethasone treatment. In general, levels of mRNA may be increased through several processes, which include an enhancement in mRNA stability, more efficient processing of the primary transcript, increased nucleocytoplasmic transport, or, more likely, an increase in the rate of transcription. Additional-
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ly, translational or post-translational modification may be taking place. Eventually, it will be possible to study the regulation of glutaminase in lymphocytes in more detail when the genomic clone for the enzyme is available. Such information will enhance our understanding of any differenceg between glutaminase regulation in the gut mucosa and gut immune cells that develop during critical illness. Dexamethasone accelerates glutamine efflux from skeletal muscle, which eventually leads to a profound depletion of intracellular glutamine in muscle [8]. Both the activity of glutamine synthetase [9,26] and the amount of m R N A [26] for the enzyme are elevated in skeletal muscle of dexamethas0ne-treated rats. This increase in the glutamine-synthesizing capacity of the muscle presumably supports the release of glutamine in response to glucocorticoids. The lungs have recently been shown to also release substantial amounts of glutamine in catabolic states [27]. Dexamethasone has an effect on the lungs that is similar to its effect on skeletal muscle, i.e., it accelerates glutamine release from the lungs [9,10] and produces an increase in glutamine synthetase activity [28]. Plasma levels of glutamine drop desPite this efflux of glutamine from skeletal muscle and the lungs, indicat, ing that other tissues are utilizing glutamine at an increased rate. Dexamethasone has been shown to enhance the uptake and utilization of glutamine by the intestine in rico [14,15] and to stimulate an increase in the activity of glutaminase in cultured human epithelial cells (Dudrick PS et al, unpublished data). In dexamethasone-treated rats, intestinal glutaminase activity is increased [17], and the increase in enzyme activity has recently been associated with elevated levels of glutaminase m R N A [29]. The data presented in this report suggest that lymphocytes represent another population of cells that demonstrate an increased utilization of glutamine in response to dexamethasone via a similar mechanism. The transport of glutamine across the plasma membrane of lymphocytes occurs via carrier-mediated systems that have a high affinity for glutamine [30,31]; correspondingly, intraceUular rates of utilization are high. Lymphocytes need glutamine as a substrate for energy production and as a precursor for de novo pyrimidine synthesis [4]. Mitogenic stimulation with concanavalin-A produces a 40% to 50% increase in the utilization of glutamine by lymphocytes [4] and a threefold increase in the rate of glutamine uptake secondary to the increased capacity of the transport systems [31]. Lymphocytes will not divide in vitro without the inclusion of glutamine in the culture mediUm [4]. A deficiency of circulating glutamine has been demonstrated to be specifically immunosuppressive [19], possibly interfering with the immune response to a challenge. Thus, glutamine is a critical nutrient for maintenance of metabolic homeostasis in lymphocytes. Glutaminase, which hydrolyzes glutamine to glutamate and ammonia, is the enzyme primarily responsible for the regulation of the intracellular glutamine metabolism in lymphocytes and other replicating cells. Although the transport of glutamine into cells is an important regulator of utilization [30], the activity of glutaminase in 38
tissue homogenates correlates with the capacity to utilize glutamine intracellularly [32]. Lymphocytes possess a high basal activity of phosphate-dependent glutaminase, which is increased during an immunologic challenge in vivo [4]. The high rate of glutamine breakdown in lymphocytes is considered an essential mechanism for directing the flow of intermediates to both the Krebs cycle for energy production and the pathways of nucleotide biosynthesis [4,33]. This presumably optimizes the utilization of glutamine to support key biosynthetic reactions and provides energy and intermediates to drive intracellular reactions. In conjtmction with an accelerated rate of membrane transport, a high rate of intracellular glutamine metabolism ensures adequate amounts of glutamine-derived by-products to support cellular biochemical pathways. Although glucocorticoids enhance the capacity for glutamine utilization in gut-associated lymphocytes, they have been shown to have detrimental effects on gut barri, er function in other models. Dexamethasone impairs rat intestinal mucosal resistance to bacterial translocation to MLNs and is associated with both a decrease in IgA levels in bile [34] and an increase in bacterial adherence to intestinal mucosal cells (unpublished data). Thus, the role of glucocorticoids in bacterial translocation across the gut lumen is complex; however, the effects of glucocorticoids on MLN glutaminase expression must be viewed as beneficial, supporting lymphocyte intracdlular metabolism in stress states. Glutamine-supplemented nutritional regimens have been shown to improve gut immune function by preserving mucosal cell integrity [2,3], maintaining levels of secretory IgA [7], and maintaining B-cell and T-cel!populations of the GALT [6]. Augmentation of T-cell function may be important for reducing the number of bacteria that survive in the MLNs, possibly by enhanced killing of bacteria within the MLNs [35]. This increased capacity for intracellular glutamine metabolism may be necessary to support intracellular metabolic pathways that are essential for lymphocyte proliferation and immunoglobulin synthesis. During stress states when glutamine depletion is characteristic, the elaboration of steroid hormones may help preserve the metabolic function of gut lymphoid tissue by inducing the glutaminase enzyme. This could optimize the effectiveness of the immune component of the intestinal barrier to translocation of bacteria. REFERENCES 1. Deitch EA. The role of intestinal barrier failure and bacterial translocationin the developmentof systemicinfectionand multiple organ failure. Arch Surg 1990; 125: 403-4. 2. Souba WW, Klimberg VS, Plumley DA, et al. The role of glutamine in maintaininga healthy gut and supporting the metabolicresponseto injuryand infection.J Surg Res 1990;48: 383-91. 3. Klimberg VS, Salloum RM, Kasper M, et al. Oral glutamine accelerates healing of the small intestine and improves outcome after whole abdominal radiation. Arch Surg 1990; 125: 1040-5. 4. NewsholmeEA, Crabtre~B, ArdawiMSM. Glutaminemetabolism in lymphocytes:its biochemical,physiologicaland clinicalimportance. Q J Exp Physiol 1985; 70: 473-89.
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5. Newsholme P, Gordon S, Newsholme EA. Rates of utilization and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages. Biochem J 1987; 242: 631-6. 6. Alverdy JA, Aoys E, Weiss-Carrington P, Burke DA. The effect of glutamine-enriched TPN on gut immune cellularity. J Surg Res 1992; 52: 34-8. 7. Burke DJ, Alverdy JA, Aoys E, Moss GS. Glutamine-supplemented total parenteral nutrition improves gut immune function. Arch Surg 1989; 124: 1396-9. 8. Muhlbacher F, Kapadia CR, Colpoys MF, Smith R J, Wilmore DW. Effects of glucocorticoids on glutamine metabolism in skeletal muscle. Am J Physiol 1984; 247: E75-83. 9. Ardawi MSM, Jamal YS. Glutamine metabolism in skeletal muscle of glucocorticoid-treated rats. Clin Sci 1990; 79: 136-47. 10. Souba WW, Plumley DA, Salloum RM, Copeland EM. Effects of glucoeorticoids on lung glutamine and alanine metabolism. Surgery 1990; 108: 213-9. 11. Ardawi MSM. Glutamine metabolism in the lungs of glucocorticoid-treated rats. Clin Sci 199i; 81: 37-42. 12. Kinney JM, Felig P. The metabolic response to injury and infection. In: DeGroot L J, editor. Endocrinology, vol 3. New York: Grune & Stratton, 1979: 1963-86. 13. Vaughn GM, Becker RA, Allen JP, et al. Cortisol and corticotropin in burned patients. J Trauma 1982; 22: 263-73. 14. Souba WW, Smith R J, Wilmore DW. Effects of glucocorticoids on glutamine metabolism in visceral organs. Metabolism 1985; 34: 450-6. 15. Ardawi MSM, Majzoub MF, Newsholme EA. Effect of glucocorticoid treatment on glucose and glutamine metabolism by the small intestine of the rat. Clin Sci 1988; 75: 93-100. 16. Gebhardt R, Kleeman E. Hormonal regulation of amino acid transport system N in primary cultures of rat hepatocytes. Eur J Biochem 1987; 166: 339-44. 17. Fox AD, Kripke SA, Berman JM, McGintey RM, Settle RG, Rombeau JL. Dexamethasone administration induces increased glutaminase specific activity in the jejunum and colon. J Surg Res 1988; 44: 391-6. 18. Crawford J, Cohen HJ. The essential role of L-glutamine in lymphocyte differentiation in vitro. J Cell Physiol 1985; 124: 275-82. 19. Kafkewitz D, Bendich A. Enzyme-induced asparagine and glutamine depletion and immune system function. Am J Clin Nutr 1983; 37: 1025-30. 20. Windmueller HG. Phosphate dependent glutaminase in the small intestine. Arch Biochem Biophys 1977; 182: 506-17. 21. Chomczynski P, Sacehi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-choloform extraction. Anal Biochem 1987; 162: 156-9. 22. Rave NR, Crkvenjakov R, Boedtker H. Identification of procollagen mRNAs transferred to diazobenzylmethyl paper from formaldehyde agarose gels. Nucleic Acids Res 1979; 6: 3559-67. 23. Banner C, Hwang J, Shapiro RA. Isolation of a cDNA for rat brain glutaminase. Mol Brain Res 1988; 3: 247-54. 24. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease to high specific activity. Ann Biothem 1983; 132: 6-10. 25. Souba WW, Herskowitz K, Austgen TR, Chen MK, Salloum RM. Glutamine nutrition: theoretical considerations and therapeutic impact. JPEN J Parenter Enteral Nutr 1990; 14: 237S-43S. 26. Max SR, Mill J, Mearow K, et al. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles. Am J Physiol 1988; 255: E397-403. 27. Plumley DA, Souba WW, Hautamaki RD, et al. Accelerated lung amino acid release in hyperdynamic septic surgical patients. Arch Surg 1990; 125: 57-61. 28. Ardawi MSM. Glutamine-synthesizing activity in lungs of fed,
starved, acidotic, diabetic, injured and septic rats. Biochem J 1990; 270: 829-32. 29. Sarantos P, Abouhamze A, Souba WW. Glucocorticoids regulate intestinal glutaminase expression. Surgery 1992; 112: 278-83. 30. Ardawi MSM, Newsholme EA. The transport of glutamine into rat mesenteric lymphocytes. Biochem Biophys Acta 1986; 856: 413-20. 31. Schroeder M, Schaefer G, Schauder P. Characterization of glutamine transport into resting and concanavalin A-stimulated peripheral human lymphocytes. J Cell Pbysiol 1990; 145: 155-61. 32. Ardawi MSM, Majzoub MF, Kateilah SM, Newsholme EA. Maximal activity of phosphate-dependent glutaminase and glutamine metabolism in septic rats. J Lab Clin Med 1991; 118: 26-32. 33. Newsholme EA, Newsholme P, Curi R, Challoner E, Ardawi MSM. A role for muscle in the immune system and its importance in surgery, trauma, sepsis and burns. Nutrition 1988; 4: 261-8. 34. Alverdy J, Aoys E. The effect of glucocorticoid administration on bacterial translocation. Ann Surg 1991; 214: 719-23. 35. Maddaus MA, Wells CL, Platt JL, Condie RM, Simmons RL. Effect ofT cell modulation on the translocation of bacteria from the gut and mesenteric lymph node. Ann Surg 1988; 387-98.
DISCUSSION
Christopher C. B a k e r (Chapel Hill, N C ) : Did you attempt to identify the cells that you were observing? M y knowledge about glutaminase production in macrophages is limited, but certainly the macrophages are the cells that carry bacteria across the cell wall in translocation. It seems to me that you did not use separation or identification techniques but that you simply homogenized the lymph nodes. Do you have any information about glutaminase production or changes in signal transduction in a model that might increase translocation? P a u l S. D u d r i e k : The mesenteric lymph nodes ( M L N s ) were histologically examined by a board-certified pathologist at our institution. Examination of the nodes from both normal and dexamethasone-treated animals revealed about 95% lymphocytes. Therefore, we believe that our measurements are quantifying glutaminase activity in lymphocytes. W e have also studied M L N glutaminase activity in septic rats. In results that were similar to those of the current study, we demonstrated that there was an increase in glutaminase activity in the lymph nodes of endotoxin-treated animals. It is very possible that the glucocorticoid hormones are an important regulator of this septic-induced increase. A. B a r b u l (Baltimore, MD): Could you explain an apparent dichotomy? Dexamethasone appears to decrease the activity of lymphocytes, but you observed enhanced metabolic activity, at least in utilizing the major metabolic fuel, which is glutamine. H o w do you explain these two seemingly disparate effects? Paul S. I)udriek: The dose of dexamethasone that we used was designed to mimic moderate to severe stress. Similar doses have been used by other investigators to simulate injury. The suppressive effects of steroids on lymphocytes are generally mediated at higher pharmacologic doses. Therefore, we purposely avoided using large nonphysiologic doses and attempted to choose a dose that was physiologic and not immunosuppressive.
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Lymphocytes in the mesenterie lymph nodes (MLNs) play a key role in protecting the body from the translocation of bacteria through the bowel during catabolic states. Lymphocytes use glutamine for energy and for DNA synthesis and have high levels of the glutaminase (GA) enzyme that regulates intraeellular glutamine metabolism. This study tested the hypothesis that the increase in circulating glucocorticoid hormones that occurs in respouse to stress states regulates MLN lymphocyte GA expression at the molecular level. Adult male rats (n = 60) received a single dose of dexamethasone (DEX 0.5 mg/kg intraperitoneally) or saline vehicle (CONT). lleocolic lymph nodes were excised from anesthetized rats via laparotomy at 2, 4, 12, 24, 48, and 72 hours after injection, and GA activity was assayed in MLN homogenates. A second group of rats received repeated doses of DEX (0.5 mg/kg/d for 4 days). GA activity was assayed in MLN homogenates, and total RNA was extracted for quantitation by Northern hybridization using a phosphorus 32-labeled rat GA eDNA probe. GA activity was increased within 2 hours after a single dose of DEX, with a peak after 4 hours. Kinetic analysis at the 4-hour time point showed an increase in the maximum GA activity (maximal transport velocity [Vmax]:619 4- 107 nmol/mg protein/hr in DEX versus 380 4- 53 nmol/mg protein/ hr in CONT, p < 0 . 0 5 ) , with no change in GA affinity (Michaelis constant 0Kin]: 2.28 4- 0.39 mM in DEX versus 2.20 4- 0.36 mM in CONT, p = NS). Repeated doses of DEX resulted in a twofold increase in GA activity (550 4- 125 nmol/mg protein/hr versus 1,175 4- 40, p < 0 . 0 1 ) . Simultaneously, GA mRNA levels were increased by 70%. Glucocorticoids stimulate GA activity and gene expression in lymphocytes that reside in MLNs.
From the Department of Surgery, Universityof Florida Collegeof Medicine, Gainesville,Florida. Supported by National Institutes of Health (NIH) grantCA 45327 (Dr.Souba),a grantfromthe Veterans AdministrationMerit ReviewBoard (Dr. Souba), and NIH Training Grant T32-CA09605-03 (Drs. Dudrickand Sarantos). *Dr. Dudrickis the recipientof the Resident'sPrizeawardedannuallyby the Societyfor Surgeryof the AlimentaryTract. Requestsfor reprintsshouldbe addressedto WileyW. Souba,MD, Department of Surgery,JHMHSC Box 100286, Gainesville,Florida 32610-0286. Presentedat the 33rdAnnualMeetingofthe Societyfor Surgeryof the AlimentaryTract, San Francisco,California,May 11-13, 1992. 34
n critically ill patients, bacterial translocation may Imucosal 9occur as a consequence of the failure of the intestinal and immune barriers [1]. Recent studies have demonstrated the importance of glutamine metabolism in maintaining intestinal mucosal integrity [2] and improving healing of injured mucosa [3], processes that may diminish the translocation of intestinal bacteria. The function of the cellular components of the gut immune system is also related to the availability and utilization of glutamine [4,5]. In the intestinal tract, the gut-associated lymphoid tissue (GALT) and mesenteric lymph nodes (MLNs) contain the cellular elements of the intestinal immune system. Glutamine deficiency results in a decrease in populations of lymphocytes from the GALT [6] and is also associated with decreased levels of secretory immunoglobulin A (IgA) in the gastrointestinal tract [7]. Glutamine levels drop dramatically in blood and tissues in catabolic states despite a massive efflux from skeletal muscle [8,9] and the lungs [10,11]. The resulting relative glutamine deficiency may adversely the affect immune function of the intestinal tract. Glucocorticoid hormones are released in increased amounts after injury and infection [12,13] and are important mediators of the altered glutamine metabolism that occurs in stress states [10]. In experimental models of physiologic stress, dexamethasone mediates glutamine release from skeletal muscle [8,9] and the lungs [10,11]. The effect of dexamethasone is due, at least in part, to an increase in the activity of glutamine synthetase in these tissues [9,I1], which supports the subsequent efflux of glutamine. During critical illness, this efflux is accompanied by a decline in plasma glutamine concentrations [8,9], which suggests that other tissues are using glutamine at increased rates. Dexamethasone stimulates the uptake and utilization of glutamine by the kidneys [14] and intestine [14,15] in vivo and enhances uptake by hepatocytes in culture [16]. The increase in intestinal utilization of glutamine in response to dexamethasone has been associated with an increase in the specific activity of intestinal glutaminase [17]. The dexamethasonemediated alterations in glutamine metabolism thus represent a balance of cellular glutamine uptake and release, depending on the metabolic needs of the tissue in stress states. Lymphocytes consume relatively large amounts of glutamine, and the rate of utilization increases in response to an immune challenge [4]. Lymphocytes require glutamine for DNA synthesis and cell division in culture and for their proliferation in response to mitogenic stimuli [4]. Glutamine is essential for the transformation of B lymphocytes to immunoglobulin-secreting plasma cells [18]. Depletion of glutamine in vivo is considered specifi-
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eally immunosuppressive [19]. During stress states, glutamine requirements of lymphocytes in the GALT and in MLNs may increase in order to support the cellular response to translocating bowel bacteria. Induction of the glutaminase enzyme may then be necessary to increase the capacity for glutamine utilization in lymphocytes. We hypothesized that the elevated circulating concentration of the glucocorticoid hormones that occurs after surgical stress stimulates lymph node glutaminase activity. The purpose of this study was to examine the regulation of glutaminase expression in MLNs in response to exogenous dexamethasone administration.
MATERIALS AND METHODS All studies were approved by the Committee for the Use and Care of Laboratory Animals at the University of Florida. Adult male Sprague-Dawley rats weighing 200 to 250 g were obtained from the Animal Farm at the University of Florida College of Medicine. Rats were housed in standard rodent cages, exposed to 12-hour dark and light cycles, and allowed intake of water and standard rat chow ad libitum. Food intake in all rats was similar. After the rats were allowed at least 3 days to acclimate to the animal care facility, they were randomly divided into control and experimental groups. Dexamethasone (sodium phosphate salt) for injection was purchased from American Regent Laboratories, Inc. (Shirley, NY). Bovine glutamate dehydrogenase used for the glutaminase assay was purchased from Calbiochem Corp. (La Jolla, CA). All other chemicals used in assay reagents were purchased from Sigma Chemical Co. (St. Louis, MO). A rat complimentary DNA (eDNA) probe for the glutaminase enzyme was obtained as a gift from Dr. Norman P. Curthoys, Department of Biochemistry, Colorado State University, Fort Collins, CO. The aphosphorus 32 (32p) used to label the eDNA probe was purchased from DuPont Co. (Wilmington, DE). The/~actin gene was a gift from Dr. Laurence Kedes, Professor and Chairman, Department of Biochemistry, University of Southern California, Los Angeles, CA. Determination of glutaminase activity: Sixty rats were randomly divided into experimental and control groups. Treated rats received an intraperitoneal injection of dexamethasone (0.5 mg/kg), and control animals received an equal volume of saline vehicle. At various time points after treatment (2, 4, 12, 24, 48, and 72 hours), the animals were anesthetized with ketamine (10 mg/100 g body weight intraperitoneally) for tissue harvest. The ileocolic lymph nodes were excised through a midline laparotomy en bloc with a 2- X 7-mm segment of mesentery and dissected free from surrounding fat. A second group of rats were randomly assigned to receive repeated doses of dexamethasone (0.5 mg/kg/d for 4 days, intraperitoneal) or an equal volume of saline vehicle. MLNs were similarly excised from anesthetized animals 4 hours after the last injection. Animals were killed by cervical dislocation after harvest of the tissue. The lymph nodes from each rat were immediately homogenized in a chilled buffer composed of 50 mM Trizma (Sigma Chemical Co.), 330 mM sucrose, and 5 mM magnesium chloride.
Glutaminase activity of the lymph node homogenate was determined using a phosphate-dependent glutaminase assay described by Windmueller [20]. Protein content of the homogenate was determined using a commercial protein assay (Bio-Rad Laboratories, Richmond, VA). Samples of excised lymph nodes were also fLXedfor histologic examination to quantitate cellularity. Measurement of glutaminase mRNA: Sixteen rats were randomly assigned to receive repeated doses of dexamethasone (0.5 mg/kg/d for 4 days intraperitoneally) or an equal volume of saline vehicle. Four hours after the fourth and final injection, the animals were anesthetized with ketamine (10 mg/100 g body weight intraperitoneaUy), and the ileocolic lymph nodes were excised using a midline laparotomy. The tissue was immediately processed for total RNA extraction using the method described by Chomczynski and Sacchi [21]. Briefly, the lymph nodes were homogenized by dounce in 5 mL of 4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, and 0.1% mercaptoethanol, followed by the sequential addition of 500 #L 2 M of sodium acetate (pH 4.0), 5 mL of H20-saturated phenol (pH 4.0), and 2 mL chloroform:isoamyl alcohol solution (49:1). RNA was precipitated by cold incubation in 2-propanol. The RNA precipitate was centrifuged, and the pellet resuspended in 600 #L of homogenizing solution, followed by reprecipitation by cold incubation in 600 ~L 2-propanol. The RNA was washed again with 70% ethanol and re,suspended in diethylpyrocarbonate (DEPC)-treated water. The resuspended total RNA was reprecipitated by cold incubation in a solution containing 0.1 volume 3 M sodium acetate (pH 5.0) and 2.2 volume 100% ethanol. After the RNA was centrifuged, the final R N A pellet was washed in 70% ethanol and then resuspended in DEPCtreated water. A mRNA for the rat glutaminase gene was used as a probe to measure the concentration of glutaminase mRNA from the rat ileocolic lymph nodes by Northern blot analysis. Equal amounts of RNA (40 tzg), as determined both spectrophotometrically and through ethidium bromide staining, were fractionated by electrophoresis through denaturing agarose gels containing formaldehyde [22]. The RNA was transferred to nylon membranes and cross-linked to the membrane by ultraviolet irradiation. A 1.1-kilobase pair rat glutaminase eDNA [23] containing the N-terminal sequence of glutaminase was cloned in the Bluescript vector (Stratagene Cloning Systems, La Jolla, CA) in reverse orientation. A radiolabeled glutaminase mRNA was synthesized by in vitro transcription using a-32p-CTP (cytidine triphosphate) and a Riboprobe kit (Promega Corp., Madison, WI) according to the manufacturer's specifications. The labeled probe was hybridized overnight at 68~ using the method of Feinburg and Vogelstein [24]. Autoradiographic detection of the hybridization was accomplished by exposing the hybridized nylon membranes to Kodak XAR film for 48 hours at -700C. The glutaminase mRNA bands were quantitated by laser densitometry. Then the GA eDNA probe was stripped off of the membrane by boiling in 0.1% sodium dodecyl sulfate (SDS)
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Figure 1. Glutaminase activity after a single dexamethasone (DEX) injection. Glutaminase activity was significantly increased compared with control values at the 4-hour time point (627 4- 50 nmol/mg protein/hr in control rats versus 980 4- 70 in DEXtreated rats, *p <0.05). Control values obtained for each time point were unchanged and hence were pooled.
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Activity/[GLN] Figure 3. Eadie-Hofstea plot at the 4-hour time point demonstrates that the increase in glutaminase (GLN)activity at 4 hours is due to an increase in Vmax in the dexamethasone-treated rats compared with controls. Data from three separate experiments yielded a mean Vmaxof 619 4- 107 nmol/mg protein/hr in treated rats versus 380 4- 53 in controls (p <0,05). The Krn values were not significantly different between groups (2,28 4- 0.39 mM in treated rats versus 2.20 4- 0.36 mM in controls, p -- NS).
36
(aqueous). The blots were then rehybridized with a eDNA probe for fl-actin, a constitutively expressed gene used as a control for mRNA loading, fl-Actin eDNA was radiolabeled using cP2p-dCTP and a random primer labeling kit (Stratagene Cloning Systems) according to the manufacturer's protocol. The radioactivity of the/3-actin bands was also quantitated by laser densitometry. Calculations and statistical analysis: Glutaminase activity is expressed in nanomoles per milligram of lymph node protein per hour. The Michaelis-Menton kinetic parameters, Vmax (nmoles/mg protein/hr) and Km (raM), of the glutaminase enzyme were determined by measuring enzyme activity over a range of glutamine concentrations. Glutaminase mRNA is expressed in relative units with control values being 100%. The results from the experimental and control groups were compared using the Student's t-test, with a p value of less than 0.05 considered statistically significant. RESULTS Histologic examination of the ileocolic lymph nodes excised from the control and dexamethasone-treated rats demonstrated that the lymphocytes comprised 95% of the total cellularity. Treatment of the rats with a single dose of dexamethasone resulted in an increase in glutaminase activity in MLNs within 2 hours after injection reaching a maximum after 4 hours (Figure 1). The GA activity at 4 hours was significantly elevated in dexamethasone-treated rats compared with saline-treated controls. A plot of glutaminase activity versus glutamine concentration (Figure 2) gives a rectangular hyperbola for each group, with the asymptote representing the maximum rate of substrate conversion (Vm~x), or maximum enzyme activity. Linear transformation of these data with an Eadie-Hofstee (EH) plot (Act versus Act/[Gin], Figure 3) shows that the increase in glutaminase activity after dexamethasone injection was due to an increase in Vmax,represented by the y-intercept of the lines in the E-H plot. The affinity of the enzyme for glutamine (Km)represents the substrate concentration at which half of the available enzyme molecules are bound with substrate and is given by the negative slope of the line. Enzyme affinity was not different between dexamethasone-treated and control rats, as demonstrated by the parallel lines in Figure 3. The increase in Vmaxwith no change in Kmis consistent with an increase in the number of enzyme molecules expressed by the lymphocytes of the MLN, rather than a change in the enzyme affinity for glutamine. Repeated injections (0.5 mg/kg for 4 days) of dexamethasone produced an even greater increase in glutaminase activity assayed 4 hours after the fourth and final dose (Figure 4). The increase in glutaminase activity in the MLN homogenate from dexamethasone-treated rats was associated with an increase in glutaminase mRNA. Northern blot analysis of glutaminase mRNA content, normalized to the constitutively expressed fl-actin gene (Figure 5), demonstrated an increase in the amount of mRNA in the MLN homogenate of dexamethasonetreated rats compared with that of the controls. Quantita-
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tion of the glutaminase mRNA bands from three control and three dexamethasone-treated rats showed a 70% increase in the dexamethasone-treated group compared with the control group (Figure 6). COMMENTS The effects of dexamethasone on glutaminase expression in lymphocytes from MLNs were studied in order to learn about the regulation of glutamine metabolism in the cells of the gut immune system during stress states. The results suggest that the lymphocytes in the MLNs of the bowel mesentery account for a considerable portion of the augmented glutamine uptake by the portal-drained viscera that occurs after glucocorticoid treatment. The dose of dexamethasone used in this study was similar to that used in previous studies [10,14] and was chosen to approximate the threefold to fivefold elevation in circulating cortisol that occurs after a major operation or injury [13]. Glucocorticoid hormones are released by the adrenal gland in increased amounts in stress states, and these hormones have profound effects on amino acid metabolism in many organs. We chose to study the effects of dexamethasone on glutamine metabolism in lymphocytes because: (1) glutamine is the most abundant amino acid in the body and the amino acid most affected by critical illness, (2) lymphocytes are avid glutamine consumers, and (3) the requirement for glutamine by lymphocytes is increased during stress states, which may be an adaptive response designed to support cellular metabolism and function. Glutamine levels dropped dramatically in blood and tissues in catabolic states, producing a relative glutamine deficiency that is associated with adverse structural and functional alterations in tissues that characteristically utilize glutamine at high rates in both the normal and physiologic stress states [25]. The present study demonstrated that the administration of a single physiologic dose of dexamethasone resuited in a twofold increase in MLN glutaminase activity. The increase in activity was prompt and was relatively short-lived. Kinetic analysis indicated that the increase in activity was secondary to an increase in the number of enzyme molecules expressed intracellularly (Vmaxeffect), rather than to a change in the affinity of the enzyme for g l u t a m i n e (Kin effect). Repeated doses of dexamethasone produced an even greater increase in glutaminase activity, consistent with a persistent augmentation in lymphocyte glutamine utilization during chronic stress. This increase was associated with a 70% increase in levels of mRNA for the glutarninase enzyme detected in MLN lymphocytes after repeated dexamethasone injections. Collectively, these data suggest that, in response to glucocorticoids, glutaminase expression is regulated at the transcriptional level. The increase in glutaminase activity reflects an increase in the capacity of lymphocytes to utilize glutamine, which may be essential for the proliferation of these cells in response to an immune challenge, such as translocation of bacteria from the gut lumen. An important feature of the increases in both mRNA and specific activity in this study is the similarity in the magnitude of their increases. This is in contrast to the
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Figure 4. Effect of multiple doses of dexamethasone (DEX) on mesenteric lymph node glutaminase activity. Repeated treatment with DEX resulted in a twofold increase in glutamlnase activity (550 4- 125 nmol/mg protein/hr in control versus 1,175 4- 40 nmol/mg prctein/hr in DEX, "p <0.01).
Glutaminase mRNA
[5-actin mRNA
Cont DEX DEX DEX
Cont DEX DEX DEX
Figure 5. Northern blot analysis of glutaminase mRNA after repeated doseS,of dexamethasone (DEX). Representative autoradiograms of glutaminase mRNA (left) and/3-actin mRNA (rlgM).
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Figure 6. Quantitative increase in glutaminase mRNA in dexamethaSone (DEX)-treated rats. Values are expressed In relative units as percent of control and represent quantitation of three separate autoradiograms normalized to/3-actin. *p <0.01.
disparity between glutaminase mRNA and activity seen in the gut mucosa after dexamethasone treatment. In general, levels of mRNA may be increased through several processes, which include an enhancement in mRNA stability, more efficient processing of the primary transcript, increased nucleocytoplasmic transport, or, more likely, an increase in the rate of transcription. Additional-
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ly, translational or post-translational modification may be taking place. Eventually, it will be possible to study the regulation of glutaminase in lymphocytes in more detail when the genomic clone for the enzyme is available. Such information will enhance our understanding of any differenceg between glutaminase regulation in the gut mucosa and gut immune cells that develop during critical illness. Dexamethasone accelerates glutamine efflux from skeletal muscle, which eventually leads to a profound depletion of intracellular glutamine in muscle [8]. Both the activity of glutamine synthetase [9,26] and the amount of m R N A [26] for the enzyme are elevated in skeletal muscle of dexamethas0ne-treated rats. This increase in the glutamine-synthesizing capacity of the muscle presumably supports the release of glutamine in response to glucocorticoids. The lungs have recently been shown to also release substantial amounts of glutamine in catabolic states [27]. Dexamethasone has an effect on the lungs that is similar to its effect on skeletal muscle, i.e., it accelerates glutamine release from the lungs [9,10] and produces an increase in glutamine synthetase activity [28]. Plasma levels of glutamine drop desPite this efflux of glutamine from skeletal muscle and the lungs, indicat, ing that other tissues are utilizing glutamine at an increased rate. Dexamethasone has been shown to enhance the uptake and utilization of glutamine by the intestine in rico [14,15] and to stimulate an increase in the activity of glutaminase in cultured human epithelial cells (Dudrick PS et al, unpublished data). In dexamethasone-treated rats, intestinal glutaminase activity is increased [17], and the increase in enzyme activity has recently been associated with elevated levels of glutaminase m R N A [29]. The data presented in this report suggest that lymphocytes represent another population of cells that demonstrate an increased utilization of glutamine in response to dexamethasone via a similar mechanism. The transport of glutamine across the plasma membrane of lymphocytes occurs via carrier-mediated systems that have a high affinity for glutamine [30,31]; correspondingly, intraceUular rates of utilization are high. Lymphocytes need glutamine as a substrate for energy production and as a precursor for de novo pyrimidine synthesis [4]. Mitogenic stimulation with concanavalin-A produces a 40% to 50% increase in the utilization of glutamine by lymphocytes [4] and a threefold increase in the rate of glutamine uptake secondary to the increased capacity of the transport systems [31]. Lymphocytes will not divide in vitro without the inclusion of glutamine in the culture mediUm [4]. A deficiency of circulating glutamine has been demonstrated to be specifically immunosuppressive [19], possibly interfering with the immune response to a challenge. Thus, glutamine is a critical nutrient for maintenance of metabolic homeostasis in lymphocytes. Glutaminase, which hydrolyzes glutamine to glutamate and ammonia, is the enzyme primarily responsible for the regulation of the intracellular glutamine metabolism in lymphocytes and other replicating cells. Although the transport of glutamine into cells is an important regulator of utilization [30], the activity of glutaminase in 38
tissue homogenates correlates with the capacity to utilize glutamine intracellularly [32]. Lymphocytes possess a high basal activity of phosphate-dependent glutaminase, which is increased during an immunologic challenge in vivo [4]. The high rate of glutamine breakdown in lymphocytes is considered an essential mechanism for directing the flow of intermediates to both the Krebs cycle for energy production and the pathways of nucleotide biosynthesis [4,33]. This presumably optimizes the utilization of glutamine to support key biosynthetic reactions and provides energy and intermediates to drive intracellular reactions. In conjtmction with an accelerated rate of membrane transport, a high rate of intracellular glutamine metabolism ensures adequate amounts of glutamine-derived by-products to support cellular biochemical pathways. Although glucocorticoids enhance the capacity for glutamine utilization in gut-associated lymphocytes, they have been shown to have detrimental effects on gut barri, er function in other models. Dexamethasone impairs rat intestinal mucosal resistance to bacterial translocation to MLNs and is associated with both a decrease in IgA levels in bile [34] and an increase in bacterial adherence to intestinal mucosal cells (unpublished data). Thus, the role of glucocorticoids in bacterial translocation across the gut lumen is complex; however, the effects of glucocorticoids on MLN glutaminase expression must be viewed as beneficial, supporting lymphocyte intracdlular metabolism in stress states. Glutamine-supplemented nutritional regimens have been shown to improve gut immune function by preserving mucosal cell integrity [2,3], maintaining levels of secretory IgA [7], and maintaining B-cell and T-cel!populations of the GALT [6]. Augmentation of T-cell function may be important for reducing the number of bacteria that survive in the MLNs, possibly by enhanced killing of bacteria within the MLNs [35]. This increased capacity for intracellular glutamine metabolism may be necessary to support intracellular metabolic pathways that are essential for lymphocyte proliferation and immunoglobulin synthesis. During stress states when glutamine depletion is characteristic, the elaboration of steroid hormones may help preserve the metabolic function of gut lymphoid tissue by inducing the glutaminase enzyme. This could optimize the effectiveness of the immune component of the intestinal barrier to translocation of bacteria. REFERENCES 1. Deitch EA. The role of intestinal barrier failure and bacterial translocationin the developmentof systemicinfectionand multiple organ failure. Arch Surg 1990; 125: 403-4. 2. Souba WW, Klimberg VS, Plumley DA, et al. The role of glutamine in maintaininga healthy gut and supporting the metabolicresponseto injuryand infection.J Surg Res 1990;48: 383-91. 3. Klimberg VS, Salloum RM, Kasper M, et al. Oral glutamine accelerates healing of the small intestine and improves outcome after whole abdominal radiation. Arch Surg 1990; 125: 1040-5. 4. NewsholmeEA, Crabtre~B, ArdawiMSM. Glutaminemetabolism in lymphocytes:its biochemical,physiologicaland clinicalimportance. Q J Exp Physiol 1985; 70: 473-89.
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5. Newsholme P, Gordon S, Newsholme EA. Rates of utilization and fates of glucose, glutamine, pyruvate, fatty acids and ketone bodies by mouse macrophages. Biochem J 1987; 242: 631-6. 6. Alverdy JA, Aoys E, Weiss-Carrington P, Burke DA. The effect of glutamine-enriched TPN on gut immune cellularity. J Surg Res 1992; 52: 34-8. 7. Burke DJ, Alverdy JA, Aoys E, Moss GS. Glutamine-supplemented total parenteral nutrition improves gut immune function. Arch Surg 1989; 124: 1396-9. 8. Muhlbacher F, Kapadia CR, Colpoys MF, Smith R J, Wilmore DW. Effects of glucocorticoids on glutamine metabolism in skeletal muscle. Am J Physiol 1984; 247: E75-83. 9. Ardawi MSM, Jamal YS. Glutamine metabolism in skeletal muscle of glucocorticoid-treated rats. Clin Sci 1990; 79: 136-47. 10. Souba WW, Plumley DA, Salloum RM, Copeland EM. Effects of glucoeorticoids on lung glutamine and alanine metabolism. Surgery 1990; 108: 213-9. 11. Ardawi MSM. Glutamine metabolism in the lungs of glucocorticoid-treated rats. Clin Sci 199i; 81: 37-42. 12. Kinney JM, Felig P. The metabolic response to injury and infection. In: DeGroot L J, editor. Endocrinology, vol 3. New York: Grune & Stratton, 1979: 1963-86. 13. Vaughn GM, Becker RA, Allen JP, et al. Cortisol and corticotropin in burned patients. J Trauma 1982; 22: 263-73. 14. Souba WW, Smith R J, Wilmore DW. Effects of glucocorticoids on glutamine metabolism in visceral organs. Metabolism 1985; 34: 450-6. 15. Ardawi MSM, Majzoub MF, Newsholme EA. Effect of glucocorticoid treatment on glucose and glutamine metabolism by the small intestine of the rat. Clin Sci 1988; 75: 93-100. 16. Gebhardt R, Kleeman E. Hormonal regulation of amino acid transport system N in primary cultures of rat hepatocytes. Eur J Biochem 1987; 166: 339-44. 17. Fox AD, Kripke SA, Berman JM, McGintey RM, Settle RG, Rombeau JL. Dexamethasone administration induces increased glutaminase specific activity in the jejunum and colon. J Surg Res 1988; 44: 391-6. 18. Crawford J, Cohen HJ. The essential role of L-glutamine in lymphocyte differentiation in vitro. J Cell Physiol 1985; 124: 275-82. 19. Kafkewitz D, Bendich A. Enzyme-induced asparagine and glutamine depletion and immune system function. Am J Clin Nutr 1983; 37: 1025-30. 20. Windmueller HG. Phosphate dependent glutaminase in the small intestine. Arch Biochem Biophys 1977; 182: 506-17. 21. Chomczynski P, Sacehi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-choloform extraction. Anal Biochem 1987; 162: 156-9. 22. Rave NR, Crkvenjakov R, Boedtker H. Identification of procollagen mRNAs transferred to diazobenzylmethyl paper from formaldehyde agarose gels. Nucleic Acids Res 1979; 6: 3559-67. 23. Banner C, Hwang J, Shapiro RA. Isolation of a cDNA for rat brain glutaminase. Mol Brain Res 1988; 3: 247-54. 24. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease to high specific activity. Ann Biothem 1983; 132: 6-10. 25. Souba WW, Herskowitz K, Austgen TR, Chen MK, Salloum RM. Glutamine nutrition: theoretical considerations and therapeutic impact. JPEN J Parenter Enteral Nutr 1990; 14: 237S-43S. 26. Max SR, Mill J, Mearow K, et al. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles. Am J Physiol 1988; 255: E397-403. 27. Plumley DA, Souba WW, Hautamaki RD, et al. Accelerated lung amino acid release in hyperdynamic septic surgical patients. Arch Surg 1990; 125: 57-61. 28. Ardawi MSM. Glutamine-synthesizing activity in lungs of fed,
starved, acidotic, diabetic, injured and septic rats. Biochem J 1990; 270: 829-32. 29. Sarantos P, Abouhamze A, Souba WW. Glucocorticoids regulate intestinal glutaminase expression. Surgery 1992; 112: 278-83. 30. Ardawi MSM, Newsholme EA. The transport of glutamine into rat mesenteric lymphocytes. Biochem Biophys Acta 1986; 856: 413-20. 31. Schroeder M, Schaefer G, Schauder P. Characterization of glutamine transport into resting and concanavalin A-stimulated peripheral human lymphocytes. J Cell Pbysiol 1990; 145: 155-61. 32. Ardawi MSM, Majzoub MF, Kateilah SM, Newsholme EA. Maximal activity of phosphate-dependent glutaminase and glutamine metabolism in septic rats. J Lab Clin Med 1991; 118: 26-32. 33. Newsholme EA, Newsholme P, Curi R, Challoner E, Ardawi MSM. A role for muscle in the immune system and its importance in surgery, trauma, sepsis and burns. Nutrition 1988; 4: 261-8. 34. Alverdy J, Aoys E. The effect of glucocorticoid administration on bacterial translocation. Ann Surg 1991; 214: 719-23. 35. Maddaus MA, Wells CL, Platt JL, Condie RM, Simmons RL. Effect ofT cell modulation on the translocation of bacteria from the gut and mesenteric lymph node. Ann Surg 1988; 387-98.
DISCUSSION
Christopher C. B a k e r (Chapel Hill, N C ) : Did you attempt to identify the cells that you were observing? M y knowledge about glutaminase production in macrophages is limited, but certainly the macrophages are the cells that carry bacteria across the cell wall in translocation. It seems to me that you did not use separation or identification techniques but that you simply homogenized the lymph nodes. Do you have any information about glutaminase production or changes in signal transduction in a model that might increase translocation? P a u l S. D u d r i e k : The mesenteric lymph nodes ( M L N s ) were histologically examined by a board-certified pathologist at our institution. Examination of the nodes from both normal and dexamethasone-treated animals revealed about 95% lymphocytes. Therefore, we believe that our measurements are quantifying glutaminase activity in lymphocytes. W e have also studied M L N glutaminase activity in septic rats. In results that were similar to those of the current study, we demonstrated that there was an increase in glutaminase activity in the lymph nodes of endotoxin-treated animals. It is very possible that the glucocorticoid hormones are an important regulator of this septic-induced increase. A. B a r b u l (Baltimore, MD): Could you explain an apparent dichotomy? Dexamethasone appears to decrease the activity of lymphocytes, but you observed enhanced metabolic activity, at least in utilizing the major metabolic fuel, which is glutamine. H o w do you explain these two seemingly disparate effects? Paul S. I)udriek: The dose of dexamethasone that we used was designed to mimic moderate to severe stress. Similar doses have been used by other investigators to simulate injury. The suppressive effects of steroids on lymphocytes are generally mediated at higher pharmacologic doses. Therefore, we purposely avoided using large nonphysiologic doses and attempted to choose a dose that was physiologic and not immunosuppressive.
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