Cytokine regulation of intestinal glutamine utilization

Cytokine Regulation of Intestinal Glutamine Utilization Thomas R. Austgen,

MD,

Mike K. Chen, MD, Paul S. Dudrick, MD, Edward M. Copeland, Wiley W. Souba, MD, ScD, FACS, Gainesville,Florida

The effects of cytokines on intestinal glutamine metabolism were studied to gain further insight into the regulation of altered glutamine metabolism that occurs during severe infection. One hundred thirteen adult rats were given a single dose of interleukin- 1 (IL- 1,50 &kg), tumor necrosis factor (TNF, 50 &kg or 150 &kg), or saline (controls) , and flux studies were performed 4 or 12 hours later. Intestinal blood flow was not different between control and cytokine-treated animals at either time point. At the &hour time point, arterial glutamine fell by 16% to 21% in the cytokinetreated animals (p <0.05); at the l%-hour time point, the arterial glutamine concentration had returned to normal. Intestinal glutamine extraction decreased iu the animals treated with IL-l at both time points (4 hours: 13% f 1.3% in IL-l versus 20% f 1.6% in controls, p <0.05; and 12 hours: 9% f 2% in IL-1 versus 17% f 2% in controls, p <0.05). Consequently, net intestinal glutamine uptake fell iu the animals treated with IL-I at both time points (p
From the Laboratories of Surgical Nutrition and Metabolism, Department of Sureerv. Universitv of Florida. Gainesville. Florida. Dr. Souba is the recipient of Grant CA 45327 from the National Institutes of Health and a grant from the Veterans Administration Merit Review Board. Requests for reprints should be addressed to Wiley W. Souba, MD, SeD, Box 5286, JHMHC, University of Florida, Gainesville, Florida 32610. Presented at the 32nd Annual Meeting of the Society for Surgery of the Alimentary Tract, New Orleans, Louisiana, May 20-22,199 1.

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MD, FACS,

epsis evokes profound changes in protein metabolism. Severe infection and endotoxemia result in muscle S proteolysis, increased nitrogen excretion, and prolonged negative nitrogen balance. During critical ilhress, the ability to maintain amino acid stores diminishes as does the efficiency of nitrogen utilization by individual tissues. Glutamine is the most abundant amino acid in the body and the amino acid most affected by catabolic disease. During endotoxemia, muscle glutamine efflux increases [I], liver glutamine uptake increases [2], renal glutamine uptake falls [3], and intestinal glutamine consumption falls [4]. The decrease in gut glutamine utilization associated with sepsis and endotoxemia may be detrimental to the hc&. Glutamine is the primary fuel source for the intestinal mucosa and a requisite substrate for de nmo deoxyribonucleic acid (DNA) biosynthesis necessary for cell repair and turnover [5,6J. Following operative stress, gut utilization of this amino acid is enhanced [7]. Presumably, this is an adaptive mechanism that supplies carbons for energy and nitrogen for DNA biosynthesis, thus supporting gut mucosal metabolism, function, and structure. During this period of operative stress, the gut can extract up to one third of circulating glutamine; however, during endotoxemia and sepsis, the extraction of circulating glutamine falls [4], brush border glutamine transport decreases [S], and the activity of the major enzyme of intracellular glutamine hydrolysis, glutaminase, decreases [4]. Cytokines are polypeptide signals that are produced by host cells in response to various stimuli including endotoxemia and bacteremia. Interleukin 1 (IL-l) and tumor necrosis factor (TNF) have received much attention as potential mediators of the septic response. These cytokines have been found to exert numerous biologic effects, some of which appear to be deleterious to the host, even at very low blood concentrations. Some of the characteristic physiologic changes seen with sepsis and endotoxemia can be reproduced with the administration of either TNF or IL-1 [9-141. In addition, soon after a bacterial or endotoxic insult, measurable amounts of IL-1 and TNF appear in the bloodstream [16,17]. Furthermore, pretreating animals with monoclonal antibodies directed against TNF can attenuate the deleterious effects of an endotoxic or bacterial challenge [ 17,181. We hypothesized that cytokines are important mediators of the altered intestinal glutamine metabolism that occurs during severe infection. Therefore, this study was designed to examine the effects of IL-1 and TNF on intestinal glutamine metabolism. The results suggest that IL- 1 induces changes in intestinal glutamine metabolism similar to those we previously observed in endotoxemic rats and septic patients [4], whereas TNF, in our model, did not cause significant perturbations.

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MATERIALS AND METHODS Model: Adult male Sprague-Dawley

rats (225 to 275 g) obtained from the Animal Farm at the University of Florida were used for this study. The animals were housed in the animal care facility and allowed at least 5 days to acclimate. They were housed in stainless steel cages, exposed to alternating 1Zhour dark and light cycles, and fed standard rat chow (Purina Rodent Chow, Purina Inc., St. Louis, MO) and water ad libitum.The evening prior to study, all chow was removed (water was allowed), and the rats were randomly divided into differing study groups. The control rats received an intraperitoneal injection of 0.9% saline (1 mL/ 100 g body weight [SW]). The IL-1 rats (IL-l) received an intraperitoneal injection of 50 pg/kg of IL-l dissolved in 0.9% saline (1 mL/ 100 g BW). The TNF rats received an intraperitoneal injection of either a standard TNF dose (50 pg/kg, TNFss) or a 3-fold larger dose (150 pg/kg, TNFi50) dissolved in 0.9% saline (1 mL/ 100 g BW). The rats were studied either 4 hours after injection (Chour study) or 12 hours after injection (12-hour study). To minimize diurnal variations, the intraperitoneal injections were administered such that the study procedure was performed at the same time in the morning (8:OOto 1O:OO AM) for both the 4-hour study and the 12-hour study. Recombinant IL-l-a was a gift from Peter Lomedico, PhD (HoffmannLa Roche, Nutley, NJ). The recombinant TNF-a was provided by Chiron Corporation (Emeryville, CA), as a gift of Patricia Olson, PhD. The cytokines were diluted in sterile 0.9% saline immediately prior to injection. Prior to dilution, the activity of the IL-l was 2 X lo* U/mL by D 10 assay, and the endotoxin content was 0.5 EU (endotoxin units) /mL by the limulus amebocyte assay test. Prior to dilution, the TNF had a specific activity of 2 to 4 X lo8 U/mg by the L929 cell cytolytic assay, and the endotoxin content was less than 5.0 EU/24 pg protein by the limulus amebocyte lysate test. Study procedure: The rats were studied in the morning in the postabsorptive state. All rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (0.75 mg/kg BW) and acepromazine maleate (0.075 mg/kg BW). The rats were placed on a heated surgical board. Body temperature was monitored with a rectal thermometer and maintained at 37OC to 38OC using a heating lamp. In the 12-hour studies, tail vein blood cultures were taken aseptically directly after anesthetizing the rats. The tail was prepared in sterile fashion using a povidone-iodine solution and alcohol. A 27-gauge needle was introduced into a lateral tail vein, and at least 0.1 mL of blood was obtained. This blood was plated on standard brain-heart infusion medium culture plates. Next, a midline neck incision was done. The trachea was identified, a small tracheotomy was made, and a 3-cm segment of PE240 polyethylene tubing (Intramedic, Clay Adams, Parsipanny, NJ) was inserted into the airway. The left carotid artery was identified and dissected free of surrounding structures and cannulated with a lo-cm segment of PE50 polyethylene tubing attached to a 1-mL syringe containing heparinized saline. This catheter was secured with 4-O silk sutures. THE AMERICAN

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A midline abdominal incision was then made, and the portal vein was identified. A 27-gauge needle was introduced into the portal vein and secured in place with cyanoacrylate adhesive. Next, a tertiary mesenteric vein in the mid-small bowel was identified and cannulated with a 30-gauge needle attached to a syringe containing C-14 para-amino hippurate (C-14 PAH, DuPont, New England Nuclear, Wilmington, DE). This was secured in place using cyanoacrylate adhesive. Intestinal blood flow was measured using the dye dilution technique as previously described [2,19]. Briefly, 1.5 to 2.5 &i of C-14 PAH was dissolved in 3 mL of 0.9% saline and infused into the tertiary mesenteric vein at a rate of 0.039 mL/min using a Braintree Scientific BS99 constant infusion pump (Braintree, MA). After a 30-minute infusion period, steady state was achieved, and blood samples (0.6 mL) were obtained from the carotid artery and portal vein simultaneously. The blood samples were processed for determination of C-14 PAH radioactivity and whole blood glutamine concentration. After completion of blood sampling, the ligament of Treitz was identified, and the proximal 20 cm of jejunum was removed and immediately processed for determination of the activity of glutaminase. During the infusion period, the abdominal and neck wounds were covered with warm saline-soaked gauze. The animals were killed by cervical dislocation. Processing of samples: Blood and tissue samples were processed immediately after obtaining them. Whole blood C-14 PAH radioactivity was determined as previously described [2] allowing for calculation of whole blood flow rates rather than plasma flow rates. A Beckman LS9800 Series Scintillation Counter (Beckman Industries, Fullerton, CA) was used to determine C- 14 PAH radioactivity of the blood samples. The blood samples were prepared as previously described [2]. A microfluorometric assay similar to that described by Bergmeyer [20] was used to determine whole blood glutamine levels. Blood glutamine concentrations were expressed in pmol/L. The proximal jejunal mucosa was obtained by incising the intestinal segment along the antimesenteric border and gently scraping the mucosa with a glass microscope slide. Gut mucosal glutaminase activity was determined using the method of Pinkus and Windmueller [21]. Protein determinations on these samples were determined using the Bio-Rad protein assay (Bio-Rad Chemical Division, Richmond, CA). Culture plates were inspected for 72 hours for the presence of growth. Colonies were Gram stained and examined under the light microscope. Only those plates with gram-negative growth were considered positive cultures. Calculations: Intestinal blood flow was determined using the following formula: Blood Flow (mL/lOO g BW/min)

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TABLE I Gut Glutamine Metabolism 4 Hours After Cytoklne InjectIon*

Art GLN

Gut Blood Flow (mL/lOO g BW/min)

Art-PV GLN

Group

(pmol/L)

Control (n = 15)

715 * 21

3.5 2 0.15

IL-1

598 f 14*

3.2 ” 0.23

78 -t a-55**

TNFw !“= 15)

567 * 12s

3.5 * 0.11

128 + 7.1**

TNFISI (n = 10)

a20+ 20t

3.4 2 0.8

139 r 20**

(n =

(pmol/L) 139 2 Q.S*

15)

Art GLN = arterial glutamine coffientration; Art-PVGLN = aitetiaCportalvein glutamine concentration; IL-1 = interleukin-1: TNFm = 50 pg/kg tumor necrosis factor; lNh~c = 150 &g/kg tumor necrosis factor. *Data = mean * SEM. +p CO.05 versus control group. *p CO.01 versus wntrol group. sp < 0.01 versus other groups. l*Signi%anUy different from 0.

TABLE II Gut Glutamine Metabollsm 12 Hours After Cytoklne InjectIon*

Group

(wmol/L)

Gut Blood Flow (mL/lOO g BW/min)

Control (n = 16)

661 2 33

3.6 + 0.2

IL-1 (n = 16)

626 f 22

3.5 f 0.3

56 k 13t

TNFw

684 -t 22

3.2 + 0.4

105 + 34*

633klQ

3.3 2 0.7

132 t la*

Art GLN

Art-PV GLN (wmol/L) 111 2

a.a*

(n = 16) TNFEQ (n = 10)

Abbreviations as in Table I. *Data = mean + SEM. +p < 0.05 versus other groups. *Significantly diierent from 0.

where CPM is the radioactive counts per minute in the C14 PAH infusate, 0.039 mL/min is the infusion rate of the C-14 PAH, CPM, and CPMiti are the radioactive counts per minute in the portal vein blood and arterial blood, respectively, and where BW is the body weight of the rats in grams. Fractional uptake of glutamine by the intestine was determined using the following formula: [(Art GLN) - (PV GLN)] + (Art GLN)

where Art GLN is the arterial glutamine concentration and PV GLN is the portal vein glutamine concentration. Intestinal glutamine uptake was determined by multi176

plying intestinal blood flow by the arterial portal venous glutamine concentration difference. All arteriovenous concentration differences were tested for significance from 0. A positive Art GLN to PV GLN concentration difference indicates net glutamine uptake. Data analysis was done using analysis of variance and Student’s unpaired t-test using a Macintosh Plus computer and Statview 512 software (Apple Computers, Inc, Cupertino, CA). The Fisher’s Exact test was used to compare blood culture results.

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RESULTS Intestinal blood flow was not significantly affected by the doses of IL-l or TNF given at either 4 or 12 hours after injection (Tables I and II). Four hours after the administration of IL-1 and TNF, the systemic arterial glutamine concentration decreased by approximately 20% (Table I), However, 12 hours after treatment, the arterial glutamine levels in the cytokine-treated animals were similar to the levels observed in the saline-treated control rats (Table II). Neither the small dose (50 pg/kg) nor the larger dose (150 pg/kg) of TNF affected the arterial-portal venous glutamine concentration difference 4 or 12 hours after injection (Tables I and 11). However, IL-1 produced a 44% fall in the arteriovenous glutamine concentration difference 4 hours postinjection, and, by 12 hours postinjection, the arteriovenous concentration difference decreased to 50% of the control value (Tables I and II). Intestinal glutamine extraction was diminished in the IL-1 treated animals at both the 4-hour (13% f 1.3% in IL-1 rats versus 20% f 1.6% in control rats, p <0.05) and the 1Zhour (9% f 2% in IL-1 rats versus 17% f 2% in control rats, p CO.05) time points. The intestinal glutamine extraction in the TNF-treated animals was not different from the saline-treated controls (Figures 1 and 2). Since the intestinal blood flow was not different between groups (Tables I and II) but the arteriovenous glutamine concentration difference fell in the IL-1 treated animals, the glutamine uptake by the IL-l animals was decreased by 46% at the 4-hour time point and by 41% at the 1Zhour time point (p <0.05 versus control rats, Figures 1 and 2). Once again, intestinal glutamine uptake was not different in the TNF-treated animals compared with saline-treated control rats (Figures 1 and 2). The activity of glutaminase fell by 50% 4 hours after IL- 1 injection and by 39% 12 hours after IL- 1 injection compared with saline-treated controls (Figures 1 and 2). The activity of glutaminase was not different in the TNF-treated animals compared with controls (Figures 1 and 2). The animals treated with IL- 1 after 12 hours were the only rats to demonstrate positive blood cultures (4 of 16, p = 0.05 versus controls, 0 of 16). All other animals had negative blood cultures. COMMENTS The effects of IL- 1 and TNF on intestinal glutamine metabolism were studied to gain further insight into the mechanisms responsible for the altered gut glutamine metabolism observed in critical illness. IL-1 and TNF were administered to rats since both of these cytokines

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F@re 1. Intestinalglutamineuptakeand from animals 4 helm after tr&ment. lp <0.05, l*p
GUT GLUTAMINE UPTAKE

GUT GLUTAMINASE ACTIVITY

Figuv2.int&id~ineuptakeand mucosalglumhseedMtyincontrol and cytokmmals. Data are from animals 12 bows after treabnent. lp <0.05, l*p
are important mediators of the catabolic response to inflammation and infection. Glutamine metabolism by the intestine was studied because of the fundamental role glutamine plays in the maintenance of intestinal metabolism, function, and structure. Glutamine is the primary fuel source for the gut and a necessary substrate for de nouo DNA biosynthesis [5,q. In this study, the dose of IL-1 used was similar to that utilized by other investigators [23,22]. In those studies, effects on both peripheral protein metabolism and central stress hormone production were observed. We have ob served this dose of IL- 1 to stimulate glutamine release by skeletal muscle (Austgen TA, Souba WW: unpublished observations). In the present study, the dose of IL-1 administered resulted in a fall in systemic arterial glutamine levels and effected a change in gut glutamine metabolism. TNF was administered initially at a dose of 50 pg/kg. Although this dose did produce a decrease in systemic blood glutamine levels, no change in gut glutamine metabolism was observed. Therefore a 3-fold increase in TNF dosage was administered to determine if a larger dose was required to cause an effect on gut glutamine metabolism. Previously we have observed this larger dose of TNF to produce changes in glutamine metabolism in other tissues. Hepatocyte plasma membrane vesicle glutamine transport was increased by 70% in rats 4 hours after treatment with an identical dose of TNF (Souba WW, Pacitti A: unpublished observations). In addition, this dose of TNF routinely produces visible physical changes in the rat consistent with systemic inflammation and similar to the physical changes noted with endotoxin administration-namely, decreased physical activity, piloerection, and chromodacryorrhea. The smaller dose of TNF and the dose of IL-l produced less pronounced signs of systemic inflammation. THE AMERICAN

The 4-hour and 1Zhour time points were chosen as study points for various reasons. Physical signs of illness became manifested as early as 3 hours after intraperitoneal cytokine injection, and other investigators have ob served effects as early as 1 hour after infusion [9]. The 12-hour time point was chosen in order to compare early and later effects. In general, cytokines regulate cellular metabolism within hours such that longer periods of time are not required to detect changes in substrate handling. The etiology of the fall in the systemic arterial glutamine concentration is not entirely clear. Both of these cytokines are known to induce an acute phase response with the induction of hepatic protein synthesis [ IO,11,23,24]. They also accelerate hepatic glutamine uptake (Scuba WW: unpublished observations) and endothelial cell transport [25]. TNF is a known mitogen of hepatocytes [I 11, and IL-1 is a mitogen of fibroblasts [23]. In addition, both of these cytokines activate the immune system [23,24]. Presumably glutamine is needed as a substrate for hepatic protein synthesis, as a substrate for DNA biosynthesis for replicating hepatocytes, fibroblasts, and immune cells, and as an energy source for activated immune cells [2q. In order to determine organ substrate fh.tx measurement, an accurate method of determining blood flow is necessary. The dye dilution technique was chosen because intravascular sampling catheters were already placed for obtaining blood samples for determination of blood glutamine concentration. This obviated the need for implanting multiple flow probes. After a 30minute infusion period, C-14 PAH steady state is obtained, and accurate blood flows can be determined [2]. The values we obtained for control rats are similar to the flows obtained by other investigators [27]. The single dose of cytokines we used resulted in no

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change in intestinal blood flow. This is in contrast to previous studies in which endotoxin [4] or TNF [28] administration resulted in a decrease in intestinal blood flow. In the aforementioned study, much larger doses of TNF were administered. Although intestinal blood flow was unchanged, the glutamine load (flow X arterial glutamine concentration) presented to the intestines was decreased by cytokine administration at the 4-hour time point. At the 12-hour time point, the delivered intestinal glutamine load was not different between groups since the arterial glutamine concentration had normalized. Even though the glutamine load was decreased at the 4hour time point, it only fell by 15%,whereas the fractional extraction of glutamine decreased nearly 50%. This suggests that the decrease in glutamine uptake noted in the IL-1 treated rats was, in part, secondary to a fall in membrane transport and/or in intracellular metabolism. Consistent with the fall in gut glutamine utilization in the IL-1 treated animals was a 40% to 50% fall in glutaminase activity in the intestinal mucosa. The administration of TNF had no such effect at either dose or either time point. This is similar to a previous study where no change in intestinal glutamine flux was observed in dogs treated with a large constant TNF infusion [28]. Glutaminase is the primary enzyme of intracellular glutamine hydrolysis in the gut mucosal cells, and therefore glutaminase activity may be an index of intestinal glutamine utilization [5]. In other studies where gut glutamine uptake falls, the activity of glutaminase has been observed to decrease. Such is the case in starvation [29], and sepsis and endotoxemia [4]. Conversely, when gut glutamine utilization is increased, the activity of glutaminase increases. This has been observed in gut mucosal hyperplasia after massive small bowel resection [ 301, glutamine feeding [31], and treatment with glucocorticoids [32]. The doses of TNF we administered had no effect on gut glutamine metabolism. The importance of intestinal glutamine metabolism for maintenance of gut structure and function has been demonstrated in several studies. Oral diets supplemented with glutamine have been observed to reduce bacterial translocation and bowel injury following whole abdominal radiation [33,34] and chemotherapy [35]. When compared with standard glutamine-free total parenteral diets, glutamine-enriched total parenteral nutrition increases gut nitrogen content [36], increases villous height [37], and decreases the incidence of bacterial translocation [38]. In the current study, 25% of the IL- 1 treated rats developed positive blood cultures. It should be emphasized, however, that a causal relationship between impaired intestinal glutamine metabolism and the breakdown of the gut mucosal barrier has not been demonstrated in this model. Whether or not the changes in gut glutamine metabolism observed with IL- 1 treatment are a primary effect of IL- 1 on the gut mucosa or a secondary consequence is not entirely clear. In in uiuo studies, it is often difficult to distinguish a direct effect of a specific cytokine from an effect that is caused by release of another mediator, particularly if specific receptor blockers are not available. 178

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The small intestine comprises multiple different cell populations, and IL-1 may affect glutamine metabolism by these cells differently. Furthermore, one cell population may affect the other cells of the gut in a paracrine fashion. Despite these potential difficulties, previous studies have shown that the enterocytes represent the principal glutamine-utilizing cell population of the intestinal tract [5]. Therefore, although the mechanism(s) by which IL1 exerts its effects have not been completely elucidated by this work, it is clear that the small intestinal mucosal cells are an important target population. Previous studies have demonstrated that IL-1 does have some direct effects on glutamine metabolism in other tissues. Pulmonary endothelial cells grown in cell culture and treated with IL-1 exhibit a 75% increase in membrane transport [25]. IL-1 is also recognized as a direct stimulant of the hypothalamic-pituitary-adrenal axis with subsequent elaboration of adrenocorticoids [39]. However, it has been demonstrated that glucocorticoids enhance uptake of circulating glutamine rather than reduce gut glutamine utilization [40]. To further investigate, more directly, the effects of IL-1 on the gut mucosa will require cell culture studies of the specific cell populations in which the hormonal/cytokine milieu can be controlled. The current study suggests that the alterations in gut glutamine metabolism and gut barrier function observed during sepsis and endotoxemia can be reproduced by a single dose of IL-l. Whether or not the changes in gut glutamine metabolism observed are direct effects of IL- 1 or are secondary to IL-1 induction of other mediators is not clear.

REFERENCES 1. Parry-BillingsM, LeightonB, DimitriadisG, de Vasconcelos PRL, NewsholmeEA. Skeletal muscle glutamine metabolism during sepsis in the rat. Int J Biochem 1989; 21: 419-23. 2. Austgen TR, Chen MK, Flynn TC, Souba WW. The effects of endotoxin on the splanchnic metabolism of glutamine and related substrates. J Trauma 1991; 31: 742-52. 3. Austgen TR, Chen MK, Moore W, Souba WW. The effects of endotoxin on renal glutamine metabolism. Arch Surg 1991; 126: 23-7. 4. Souba WW, Herskowitz K, Klimberg VS, et al. The effects of sepsis and endotoxemia on gut glutamine metabolism. Ann Surg 1990; 211: 543-50. 5. Windmueller H. Glutamine utilization by the small intestine. Adv Enzymol 1982; 53: 201-37. 6. Frisell WR. Synthesis and catabolism of nucleotides. In: Frisell WR, editor. Human Biochemistry. New York: MacMillan Co, 1982: 292-304. 7. Souba WW, Wilmore DW. Postoperative alteration of arteriovenous exchange of amino acids across the gastrointestinal tract. Surgery 1983; 94: 342-50. 8. Salloum RS, Copeland EM, Souba WW. Brush border transport of glutamine and other substrates during sepsis and endotoxemia. Ann Surg 1991; 213: 401-10. 9. Michie HR, Spriggs DR, Manogue KR, et al. Tumor necrosis factor and endotoxin induce similar metabolic responses in human beings. Surgery 1988; 104: 281-6. 10. Bibby DC, Grimble RF. Temperature and metabolic changes

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in rata after various doses of tumour necrosis factor. Am J Physiol 1989; 410: 367-80. 11. Fong Y, Moldawer LL, Marano M, et al. Cachectin/TNF or IL-la induces cachexia with redistribution of body proteins. Am J Physiol 1989; 256: R659-65. 12. Tracey KJ, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986; 234: 470-4. 13. Zamir 0, Hasselgren PO, von Allmen D, Fischer JE. In oivo administration of interleukin-la induces muscle proteolysis in normal and adrenalectomized rata. (In review). 14. Flares EA, Bistrian BR, Pomposelli JJ, Dinarello CA, Blackburn GL, Istfan NW. Infusion of tumor necrosis factor/cache&in promotes muscle catabolism in the rat. A synergistic effect with interleukin 1. J Clin Invest 1989; 83: 1614-22. 15. Hease DG, Tracey KJ, Fong Y, et al. Cytokine appearance in human endotoxemia and primate bacteremia. Surg Gynecol Obstet 1988; 166: 147-53. 16. Michie HR, Manogue KR, Spriggs DR, et al. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 1988; 318: 1481-6. 17. Tracey KJ, Fong Y, Hesse DG, et al. Anticachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 1987; 330: 662-4. 18. Beutler B, Milsark IW, Cerami A. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science 1985; 229: 869-71. 19. Katz ML, Bergman EN. Simultaneous measurements of hepatic and portal venous blood flow in the sheep and dog. Am J Physiol 1969; 216: 946-52. 20. Bergmeyer HU, editor. Methods of enzymatic analysis, vol. 4. New York: John Wiley and Sons; 1974: 1704-8. 21. Pinkus LM, Windmueller HG. Phosphate dependent glutaminase of small intestine: localization and role in glutamine metabolism. Arch Biochem Biophys 1977; 182: 506-17. 22. Sapolsky RC, Rivier G, Yamamoto P, el al. Interleukin-1 stimulates the secretion of hypothalamic corticotropin-releasing factor. Science 1987; 238: 522-4. 23. Le J, Vilcek J. Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Lab Invest 1987; 56: 234-48. 24. Dinarello C. Interleukin-1 and its biologically related cytokines. Adv Immunol 1989; 44: 153-95. 25. Souba WW, Salloum RM, Bode BP, Herskowitz K. Cytokine modulation of glutamine transport by pulmonary artery endothelial cell. Surgery 1991; 110: 295-302. 26. Brand K, Fekl W, von Hintzenstem J, Langer K, Luppa P, Schoemer C. Metabolism of glutamine in lymphocytes. Metabolism 1989; 8(1 Suppl): 29-33. 27. Welbourne TC. Interorgan glutamine flow in acidosis. Am J Physiol 1987; 253: F1069-76. 28. VanLanschot JB, Mealy K, Wilmore DW. The effects of tumor necrosis factor on intestinal structure and metabolism. Ann Surg 1990; 212: 663-70. 29. Windmueller HG, Spaeth AE. Respiratory fuels and nitrogen metabolism in vivo in small intestine of fed rats. J Biol Chem 1980; 255: 107-13. 30. Klimberg VS, Souba WW, Salloum RM, et al. Intestinal glutamine metabolism after massive small bowel resection. Am J Surg 1990; 159: 27-32. 31. Salloum RM, Souba WW, Klimberg VS, et al. Glutamine is superior to glutamate in supporting gut metabolism, stimulating intestinal glutaminase activity, and preventing bacterial translccation. Surg Forum 1989; 40: 6-8. 32. FOXAD, Kripke SA, Berman JM, et al. Dexamethasone administration induces increased glutaminase specific activity in the THE AMERICAN

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jejunum and colon. J Surg Res 1988; 44: 391-6. 33. Souba WW, Klimberg VS, Hautamaki RD, et al. Oral glutamine reduces bacterial translocation following abdominal radiation. J Surg Res 1990; 48: 1-5. 34. 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. 35. Fox AD, Kripke SA, DePaula J, et al. Effect of a glutaminesupplemented enteral diet on methotrexate-induced enterocolitis. JPEN J Parenter Enteral Nutr 1988; 12: 325-3 1. 36. Hwang TL, O’Dwyer St, Smith RJ, ef al. Preservation of small bowel mucosa using glutamine-enriched parenteral nutrition. Surg Forum 1986; 38: 56-8. 37. Grant J. Use of L-glutamine in total parenteral nutrition. J Surg Res 1988; 44: 506-13. 38. Burke D, Alverdy JC, Aoys E, et al. Glutamine supplemented total parenteral nutrition improves gut immune function. Arch Surg 1989; 124: 1396-9. 39. Gwosdow AR, Kumar MSA, Bode JJ. Interleukin 1 stimulation of the hypothalamic-pituitary-adrenal axis. Am J Physiol1990; 258: E65-70. 40. Souba WW, Smith RJ, Wilmore DW. Effect of glucocorticoids on glutamine metabolism in visceral organs. Metabolism 1985; 34: 450-6.

DISCUSSION Marcel Messer (Boston, MA): Any hints of how in-

terleukin-I (IL-l) would interfere in the glutamine metabolism pathway? Any hints why you couldn’t demonstrate IL-l with the endotoxin or with tumor necrosis factor (TNF)? Emmanuel Opara (Durham, NC): Have you thought of blocking IL- 1 and then seeing what that does to the glutamine? Donald L. Kaminski (St. LOUIS, MO): I know YOU didn’t measure these factors when you introduced the cytokines intraperitoneally, but can you tell us what would happen to these rats with regard to their body temperature and blood pressure utihzing these doses of cytokines? Thomas R. Austgen (closing): Dr. Messer, the current study does not allow us to consider all of the mechanisms by which intestinal glutamine metabolism is altered by IL-l. Certainly treatment with IL-l results in a decrease in glutaminase activity. However, it is unclear whether this is due to a direct effect of IL-1 on the enterocytes or some secondary effect that is mediated by endothelial cells or possibly changes in the microcirculation. We have not, to date, studied the effects of IL-1 on basolateral membrane transport. We do have preliminary work in our laboratory that demonstrates that IL- 1 does exert direct effects on glutamine transport by cultured rat crypt cells and human enterocytic cells. In regard to your second question, we did not measure blood levels of endotoxin or cytokines in our study. However, others have shown that, following a single dose of endotoxin, there are relatively prompt increases in TNF and IL- 1 levels in the blood stream. Dr. Opara, we have not done studies using monoclonal antibodies to block the effects of IL-l. Using such an

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inhibitor would help us gain further insight into the role of IL-1 in mediating some of the events that we have observed. Although we did not measure cardiac output in these rats, Dr. Kaminski, others have shown that IL-l increases cardiac output. We would predict a simultaneous in-

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crease in hepatic blood flow. In general, these animals develop signs of systemic inflammation including piloere&ion, red tear secretion, and decreased physical activity. Rats receiving TNF also developed signs and symptoms of illness, but we did not observe any changes in intestinal glutamine metabolism.

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