Influence of Prostaglandins

Interleukin-1 -induced Anorexia in the Rat Influence of Prostaglandins Marc K. Hellerstein, Simin Nikbin Meydani, Mohsen Meydani, Ken Wu, and Charles A. Dinarello U. S. Department ofAgriculture Human Nutrition Research Center on Aging at Tufts University, Department of Medicine, Division of Geographic Medicine and Infectious Diseases, Tufts University School of Medicine and New England Medical Center, Boston, Massachusetts 02111; Department of Nutritional Sciences, University of California at Berkeley, Berkeley, California 94720; and Department ofMedicine, Division of Endocrinology and Metabolism, San Francisco General Hospital, University ofCalifornia at San Francisco, San Francisco, California 94110 Abstract The anorexia associated with acute and chronic inflammatory or infectious conditions is poorly understood. Our objectives were to explore the anorexigenic effects of interleukin-1 (IL-1) in the rat. Recombinant human (rh) IL-1,6, murine (rm) IL-la and to a lesser extent rhIL-la significantly reduced food intake at 24.0 gg/kg i.p. but not at lower doses, in young (200-250 g) meal-fed rats on chow diets. The anorexic effect appears to be mediated by prostaglandins since pretreatment with ibuprofen completely blocked it, and a fish oil based diet abolished it, in comparison to corn oil or chow diets. Fish oil feeding also decreased basal and IL-1 stimulated prostaglandin E2 production by tissues in vitro (liver, brain, peritoneal macrophages) and in the whole body. Constant intravenous infusions of lower doses of IL-1 also diminished food intake, though intravenous boluses did not (reflecting rapid renal clearance). Chronic daily administration of IL-1 caused persistent inhibition of food intake for 7-17 d in chow and corn oil fed rats, but had no effect in fish oil fed rats. There was an attenuation of the effect (tachyphylaxis) after 7 d in corn oil and chow fed rats, but slowed weight gain and lower final weights were observed after 17-32 d of daily IL-1. Old (18-20 mo Fisher 344) rats showed less sensitivity to IL-1 induced anorexia. In conclusion, IL-1 is anorexigenic in the rat, but this is influenced by the structural form of IL-1, the route and chronicity of administration, the source of dietary fat, and the age of the animal. The ability of prior fat intake to influence the anorexic response to IL-1 represents a novel nutrient-nutrient interaction with potential therapeutic implications. Introduction Both acute and chronic inflammatory conditions as well as infectious diseases are associated with alterations in nutrition and metabolism (1, 2). One of the most important of these is decreased food intake (anorexia), which can progress to caPortions of this work have appeared previously in abstract form. 1988. FASEB (Fed. Am. Soc. Exp. Biol.) J. 2:Al 198. (Abstr.) Address reprint requests to Dr. Hellerstein, Room 4101, Koret Center for Human Nutrition, San Francisco General Hospital, 1001 Potrero Avenue, University of California, San Francisco, CA 941 10. Receivedfor publication 2 June 1988 and in revisedform 7 March 1989. J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/89/07/0228/08 $2.00 Volume 84, July 1989, 228-235 228 chexia and death by starvation (3, 4). With its high prevalence in human immunodeficiency virus-infected patients (5-7), cachexia is becoming an increasingly important problem, yet it remains poorly understood. The availability of recombinant cytokines (8, 9) allows experimental testing of new hypotheses about the mediators and mechanisms of the anorexia of inflammation. Tumor necrosis factor/cachectin (TNF)' has received attention in this regard (9-12). Administration of a solution containing macrophage secretory products including TNF suppresses food intake in mice (10) but these preparations contain other cytokines such as IL- I13 (13) in addition to TNF. Tumor cells secreting TNF can induce cachexia in mice (1 1) but again the presence of other mediators cannot be excluded. When given in sublethal doses to mice (12, 13), recombinant TNF results in decreased appetite and weight loss. However, the effect appears to be short lived ( 12) in addition to requiring escalating doses (13). The effect of IL-l on food intake in rats is largely unknown (14). McCarthy et al. (15) reported decreased intake of a liquid diet in rats given recombinant murine IL-la, but the effect was relatively minor, lasting only for the first hour of refeeding. More recently (16), rhIL-lB administered to ad lib. fed mice at 6 ,g/kg per d in two divided doses was reported to decrease food intake by 10- 15%, a similar quantitative effect as for rhIL- 1 a but significantly less than recombinant murine IL-la given at similar doses (40-50% reduction in intake). The effect of IL- 1(3 on food intake is relevant to interpretation of TNF effects, since TNF stimulates the release of IL- 1f (16, 17). Our objectives in these studies were, first, to develop a quantitative, reproducible and physiologically relevant animal model for the anorexia of inflammatory illnesses, using IL-l. Dose-response relationships, potential mediation by eicosanoid pathways, involvement of central or peripheral appetite regulatory mechanisms, effects of normal aging, differences between the two structural forms of IL-I and persistence of any effects during chronic IL- 1 administration were then examined. Methods Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were trained to a single daily meal (meal-fed) after reaching 150 g body wt. Meal-feeding has advantages over ad lib. feeding for studies of drug or hormone effects on appetite (18, 19), in that the effects of anorexigens with a short biological duration of action 1. Abbreviations used in this paper: C.O., corn oil; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; F.O., fish oil; LA, linoleic acid; TNF, tumor necrosis factor. M. K Hellerstein, S. N. Meydani, M. Meydani, K. Wu, and C. A. Dinarello are not lost over a 24-h period, since all intake occurs over 3 h. Mealfed animals grow well and eat amounts comparable to ad lib.-fed rats (20), and the metabolic and enzymatic consequences of meal-feeding have been well characterized (21, 22). Studies were performed at 200-250 g body wt. Aged Fisher 344 rats (18-20 mo old, National Institute of Aging) were used in some studies. All animals were housed in individual cages in a temperature controlled room (220C) with a 12-h dark/light cycle daily. Animals were fed nonpurified diets consisting of ground Purina Rat Chow (Ralston Purina Co., St. Louis, MO), or purified nutritionally adequate diets containing 10% by weight of corn oil (C.O.) (Mazola; Best Foods, Englewood Cliffs, NJ) or 1.2% C.O. plus 8.8% fish oil (F.O.) (MaxEPA, a gift from R. P. Scherer Co., Troy, MI) for 6 wk. The details of diet preparation and precautions to minimize oxidation of oils have been previously published (22). As previously reported (23), C.O. contained 65.6±0.6% linoleic acid (LA) while MaxEPA contained 2.0±1.4% LA, 16.7±1.8% eicosapentaenoic acid (EPA), and 12.2±1.1% docosahexanoic acid (DHA). The F.O. diet, therefore, provided about 1% by weight of LA. Food was provided daily in spill-proof glass containers placed inside cages. Water was provided ad lib. RhIL- I( was produced, purified and characterized as described (8) and consisted of the carboxy-terminal 157 amino acids. This and subsequent lots were active in the rabbit pyrogen test at 100 mg/kg and the mouse lymphocyte activating factor (LAF) assay. The endotoxin concentration in the present preparation was < 60 pg/mg. The specific activity as measured by half-maximal units (LAF) on C3H/He5 mouse thymocytes was 5.5 X 107 U/mg. The rhIL-lI3 was diluted in 0.1% serum which had been dialyzed overnight at 4VC then heated to 560C for 20 min. The diluted rhIL-1If was administered either intravenously or intraperitoneally. RhIL-la and recombinant murine (rm) IL-la were generously provided by Dr. Peter Lomedico (Hoffmann-La Roche, Nutley, NJ). The activity of rmIL-la is 1.3 X 108 U/mg (D 1O.G4. 1 cells, where I LAF unit = 20-50 DI 0 units) and the endotoxin concentration is 0.55 ng/mg protein (LAL). The activity of rhIL-la is 2.5 X I09 U/mg (D0 assay) and the endotoxin concentration is 65 pg/mg protein (LAL). Meal-fed rats (Chow, C.O., or F.O.) given 0.1% serum i.p. or heat inactivated rhIL- IB i.p. 1 h premeal had no change in food intake from baseline. The technique for placement and maintenance of indwelling intrajugular silastic catheters has been described elsewhere (25). Catheterization may depress food intake for 24-48 h, therefore no studies were performed for at least 72-96 h after catheter placement, until food intake had stabilized at precatheterization values. A parenteral preparation of ibuprofen (50 mg/ml) was the gift of Upjohn Co. (Kalamazoo, MI) and was administered intravenously or intraperitoneally in sterile 0.45% saline. 48-h urine was collected on dry ice from rats before and after intraperitoneal injection of rhIL-l. The urinary metabolite of PGF2a [ 13,14-dihydro- l 5ketoPGF2a (MetF2a)], an indicator of total body PG production, was extracted using a Sep-pak C18 cartridge (Waters Associates, Milford, MA) as described by Powell (26). MetF2a in the extracts was measured by RIA as described previously (27), and urinary creatinine was measured by Roche Cobas Fara Centrifugal Analyzer (Nutley, NJ) using Roche Diagnostic Systems Reagent and procedure number 44905, a modification of the method of Larson (28). Rats were anesthetized with metaphane and peritoneal leukocytes were obtained by lavaging the peritoneal cavity with 50 ml of Ca2l and Mg2e free PBS solution. The cell pellet was obtained by centrifugation at 1,200 rpm for 10 min. Cells were washed two times in RPMI 1640 (Gibco Laboratories, Grand Island, NY) and resuspended in RPMI 1640 at a concentration of 5 X 106 cells/ml. 100 gl of cell suspension (5 X 105 cells) was cultured in 96-well culture plates (Becton-Dickinson Co., Oxnard, CA) in the presence of 0.5% fetal bovine serum (Gibco Laboratories) and 50 gl of 0.1% BSA (Sigma Chemical Co., St. Louis, MO) or 50 ,l of 60 ng/ml rhIL-lIfl solution in 0.1% BSA (final rhIL-113 concentration 15 ng/ml) for 24 h in a 37°C, 5% CO2 humidified incubator. Cell-free supernatant was saved at -70'C for PGE2 analysis. PGE2 was analyzed by RIA (27). PGE2 antibody had a - cross-reactivity of 5.6% with PGE3 standard (Cayman Chemical Co., Ann Arbor, MI). Blood was collected from the vena cava and brain dissected on an ice-cold platform and immersed with ice-cold buffer. The mid-brain (including hypothalamus, striatum, and hippocampus) and brain stem (including medulla oblongata and pons) were dissected with precooled tools. 10% homogenates from brain regions and liver were incubated for 30 and 10 min, respectively, in a shaking water bath at 370C. PGE2 was measured in the supernatant of the incubation media. Student's t test or one-way ANOVA (when significant, followed up by Tukey-HSD procedure with a procedure-wise error rate of 0.05) was used to determine significance of differences between the means of the dietary groups. When unequal variances were present, logarithmic transformation of the data was performed. If this resolved the unequal variances, statistics were performed on the transformed data. Results First, the anorexigenic dose of intraperitoneal rhIL- 13 in meal-fed rats was established. Administration of 0.1% serum, alone or with 80 ng (400 ng/kg) or 240-400 ng (1.2-2.0 ug/kg) of rhIL- 13 i.p. 1 h before the daily meal had no effect on food intake in young (200 g) chow fed rats (Fig. 1). Administration of 800 ng (4 gg/kg) rhIL- 13 had a marked anorexic effect (Fig. 1), decreasing food intake by 37.5±6.6% (P < 0.005) in this set of rats. Food intake increased to 97.0±3.3% of baseline values by the next day (NS vs. baseline). Boiled rhIL- 13 given at 4-6 ug/kg i.p. had no anorexic effect. Because many, but not all of the diverse effects of IL-lI3 are mediated by PGE2 (29), we administered the cyclooxygenase inhibitor ibuprofen 10 mg/kg i.v. 10 min before injection of 800 ng rhIL-lB i.p. This completely blocked the acute anorexic effect of rhIL-l1( (Fig. 1). Acetaminophen, which preferentially inhibits brain cyclooxygenase relative to that in peripheral tissues (30, 31), did not block rhIL- 13 anorexia (not shown). § Day of IL- o: 80 no 240-400mg (6) (6) m00ng (10) Administration DayFoliowinglL-1 Administration 800 g * IBU (8) Figure 1. Effect of intraperitoneal rhIL-l1( alone and with intravenous ibuprofen on food intake in meal-fed rats. Rats weighing 175-225 g were studied. Housing was in individual cages in a temperature controlled room (220C) with a 12-h dark/light cycle daily. Catheterization may depress food intake for 24-48 h, therefore no studies were performed for several days after catheter placement, until food intake had stabilized at precatheterization values. RhIL-lfI was administered intraperitoneally 1 h before the daily meal. Baseline food intake represents the mean±SEM daily intake of the previous 7-10 d. P values are in comparison to baseline food intake, using a two-tailed I test. Ibuprofen (IBU) was administered at a dose of 10 mg/kg i.v., 10 min before administration of rhIL-Ifl. *P < 0.005. Interleukin-I Anorexia in the Rat 229 Different structural forms and species sources of recombinant IL- I were also tested. RhIL- 1 a was less anorexigenic than rhIL-1I3 in chow-fed rats (although this did not reach statistical significance), while the effect of rmIL- la was similar to that of rhIL-1 I3 (Table I). Anorexia from intraperitoneal rmIL-la, as from intraperitoneal rhIL-1I, was completely blocked by ibuprofen 10 mg/kg i.v. (not shown). To exclude any possible effects due to the intravenous route of ibuprofen administration or due to catheterization itself, ibuprofen 10 mg/kg or 0.9% saline were administered to noncatheterized rats by the intraperitoneal route 1 h before rmIL-la 6 jm/kg i.p., (Fig. 2). IP ibuprofen completely abolished rmIL-la anorexia (food intake 99.4% with ibuprofen/IL-l, 56.3% with saline/IL-l). Next, the effect of dietary fat source on IL- 1 anorexia was examined (Table I). Exogenous N-3 fatty acids, which are present at high levels in cold water F.O. (24) are known to reduce PGE2 synthesis in rats (32) as well humans (33). Groups of rats were chronically fed chow or purified diets based on AIN diet recommendations for rats (34) containing 10% by weight of either F.O. or C.O. Animals were begun on purified diets at 75-100 g body wt and continued for up to 6-10 wk. Composition of tissue fat in growing rats has been shown to be altered by dietary fat source (35). Serum tocopherol levels in these F.O., C.O. and chow fed rats were comparable (not shown). The effect of rhIL-1I3 4 jg/kg or rmIL-la 6 ,jg/kg injections on Table I. Effect of Dietary Fat Source and Recombinant IL-I Structural Form and Species Source on rIL-J Induced Anorexia Dietary group Chow IL- I form Effect on food intake n % Decrease Mean±SE rhIL-11, (12) rmIL-la (41) rhIL- I a (16) Corn oil rhIL-113 (12) rmIL-1a (12) rhIL- I a 18.9±3.2%$ 23.6±3.2%$ 12.7±3.6% 32.1±5.3%$ 44.8±5.5%* 8.5±4.6% 211 * 8sseline I Figure 2. Effect of pre- Postl-treatment with intraper- 7 _S S. itoneal ibuprofen (10 mg/kg) or 0.9% saline 1 h before administration 1 of rmILla 6jg/gkg on Intake food intake in chow fed rats. Ibuprofen or 0.9% 5 saline were given in 1 ml volume. Baseline values were calculated the mean intake for 0.3SI /Ltas 0.12 Saillne/1L.I lbuprafanflt.- I (N=6) (N1-I) the previous 9 d for each animal. Values shown as mean±SE for each group. P values are calculated by twotailed t test. **P < 0.01; NS, not significant. 15 food intake was significantly different between the F.O. and C.O. groups with chow-fed rats in between (Table I). Administration of 0.9% saline i.p. had no effect on food intake in any group. The effect of dietary fat source was similar for rmIL- 1 a as for rhIL- 1, (Table I). To test the hypothesis that this nutrient-nutrient interaction (prior dietary fat source influencing food intake response to IL- 1 administration) is mediated by eicosanoid pathways, PGE2 synthesis was determined in vitro and in vivo in basal and IL- I stimulated states. Consistent with the model, in vivo production of PGs of the 2 series, represented by the urinary excretion of the PGE2a metabolite 13,14-dihydro- 1 Sketo-PGF2a (MetF2a) was higher in C.O. fed rats after rhIL- 1, injection than in F.O. fed rats (Table II). Moreover, peritoneal leukocytes from C.O. fed rats synthesized more PGE2 than F.O. fed rats in vitro, both in the presence and absence of rhIL- 113 (Table III). Brain stem and liver homogenates from F.O. fed rats also produced significantly less PGE2 than C.O. and chow fed rats (Table III), although no such difference was observed in mid-brain. When rhIL- 113 was given intravenously as a 800-ng (4 jig/kg) bolus i h before the daily meal, there was no effect on food intake (Table IV). The same dose administered by constant intravenous infusion at 200 ng/h over 4 h beginning I h before the meal reduced food intake by 34.8±15.9% (P < 0.005), which was not significantly different from equal doses given intraperitoneally. When rhIL- 11 was infused overnight at 80 ng/h for the 20 h before and during the daily (5) Fish oil rhIL-1# 6.4±6.5% (1 1) rmIL-la (12) 4.9±4.6% rhIL-la -1.1±5.3% (6) Rats were fed fish oil, corn oil, or chow diets as described in the text. RhIL-1,B 4 ug/kg, rmIL- la 6 ug/kg, or rhIL- la 6 jg/kg i.p. were administered 1 h before the daily meal. Changes in food intake were calculated by comparison to the animal's mean daily intake over the preceding 7 d. P values are calculated using one-way ANOVA followed up by Tukey-HSD procedure when significant. P value for overall effect of dietary fat or IL- I source on food intake was 0.0001 by one-way ANOVA. * P < 0.05 vs. all other groups. t P < 0.05 vs. fish oil fed (all IL- I sources). 230 M. K Table I. Urinary MetF2a, Concentration in Rats Fed Different Diets before and after Intraperitoneal rhIL-I1 Injection (Mean±SEM, n = 6) MetF2, (ng/ng creatinine) Diet Before injection After injection Fish oil Corn oil Chow 0.33±0.02 0.35±0.05 0.35±0.06 0.22±0.04* 0.52±0.16 0.40±0.15 * P < 0.09 vs. corn oil fed rats. 48 h urine was collected on dry ice from rats before and after intraperitoneal injection of rhIL- I#. Logarithmic transformation of data was used when there were unequal variances, and one-way ANOVA was then applied with Tukey's followup. Hellerstein, S. N. Meydani, M. Meydani, K Wu, and C. A. Dinarello Table III. PGE2 Synthesis by Peritoneal Leukocytes and Liver and Brain Stem Homogenates of Rats Fed Different Diets in the Presence or Absence of rhIL-If (Mean±SEM) Peritoneal macrophages Control +rhILlj3 Liver (n) (n) (n) Diet pg/S X 106 cells Fish oil Corn oil Brainstem (n) ng/g 220±52 328±73* 42±7* (4) (5) (6) (5) 941±440 1327±493 329±72 18±2 (5) (5) Chow 9±1§ (6) (5) 304±63 19±3 (5) (5) Tissues were isolated as described in the text. PGE2 was measured by RIA (26) in the supernatant of the incubation media. P values are calculated using two-sample Student's t test for peritoneal macrophage data (two groups) and one-way ANOVA for liver and brainstem data (three groups). Statistics were performed on logarithmically transformed data to resolve unequal variances. * P < 0.09 vs. corn oil fed. P < 0.0001 for overall diet effect by ANOVA. Fish oil significantly different from corn oil and chow (P < 0.05) by Tukey's followup. § P < 0.0012 for overall diet effect by ANOVA. Fish oil significantly different from corn oil and chow (P < 0.05) by Tukey's followup. meal (1,600 ng total, Table IV), food intake was markedly decreased, and remained decreased the next day in comparison to preceding and succeeding intakes while catheterized. Inclusion of ibuprofen at 2.5 mg/kg per h to the overnight Table IV. Effect of Intravenous rhIL-I1f Alone and with Intravenous Ibuprofen on Food Intake (g/d) in Meal-fed Rats Group (n) Baseline food intake Day I intake (% Baseline) Day 2 intake (% Baseline) 11.0±1.0 12.4±1.8 (113.0±16%) II Constant infusion at 13.2± 1.8 8.6±2.1t 13.6±0.7 200 ng/h (65.2±15.9%) (103.0±5.0%) X 4 h (7) III Constant infusion at 11.1±0.6 1.3±1.2* 6.1±1.9* 80 ng/h (11.7±10.8%) (55.0±17.1%) X 20 h (6) IV Constant infusion at 12.7±1.0 6.2±1.6t§ 8.6±1.9* 80 ng/h X 20 h (48.8±12.6%) (67.7±14.9%) + i.v. ibuprofen 2.5 ng/kg per h X 20 h (5) I Bolus 800 ng i.v. (4) intravenous rhIL-13 infusion partially restored food intake toward baseline values (Table IV), but not completely. RmIL- la administered at 80 ng/h (400 ng/kg per h) for 20 h also decreased food intake (to 66% baseline) and this was blocked by co-administration of ibuprofen 2.5 mg/kg per h (intake 108% baseline). The question whether normal aging alters the anorexic effect of rhIL- 1 was addressed, because many normal homeostatic responses are diminished with aging (36) and because there exists some evidence (Hellerstein, M. K., unpublished observations) that elderly humans are less able to mount other features of the acute-phase response to inflammation (e.g., fever, leukocytosis, etc.). NIA aged, 18-20-mo old chow-fed Fisher 344 rats had diminished sensitivity to IL-113 anorexia (8.3±3.9% inhibition of food intake, P < 0.05 vs. young chowfed rats). Finally, we examined the effect ofchronic IL-l administration on food intake and body weight in rats fed different fat sources. These experiments consume large amounts of recombinant protein. Accordingly, only a relatively small number of animals could be studied (5 chow rats, 3 F.O., 3 C.O.). To minimize the possible confounding effects of antibodies to a foreign protein developing with chronic administration in this rodent model, rmIL-la was given. Rats were given daily rmIL- I a 6 gg/kg or 0. 1% serum i.p. Average food intake over the first 7 d of IL-1 administration was significantly diminished in chow-fed rats (16.6± 1.0 to 13.5±0.9 g/d, a decrease of 18.7%) and C.O.-fed rats (19.3±2.6 to 13.4±2.3 g/d, a decrease of 30.6%) but not in F.O.-fed rats (14.2±1.9 to 13.7±2.6 g/d, a decrease of 3.5%) (Fig. 3). IL-la administration was continued in the C.O. and chow-fed groups beyond the initial 7 d. The anorexic effect in both groups was attenuated during days 8-17 of IL-l administration (Fig. 3), and there was no apparent anorexic effect days 18-25 of IL- 1 administration (done in chow group only). After discontinuation of IL-1 treatment (days 18-25 for C.O., 26-32 for chow), food intake returned to baseline levels in both groups (chow 99.4%, C.O. 97.9%). The chow group was rechallenged with IL- 1 on days 33-40 and 120 110 U Baseline Intake 100 S Baseline0PesFood Intake 0 OtffIL-lIalpha 80 70 60 rhIL-lI was administered intravenously to chronically catheterized rats. Bolus refers to a single injection 1 h before the daily meal. Constant infusions were performed as detailed in the text, using a Harvard infusion pump. Baseline, day one and day two food intakes and P-values for comparison were calculated as described in Fig. 1. * P < 0.025 vs. baseline. * P < 0.005 vs. baseline. I P < 0.025 vs. group III, day 1 intake. hRechallange with IL-I alpha 90 Fish Oil (N-3) Corn Ol (N-3) Chow (N=S) Figure 3. Effect of chronic rmIL- Ia administration on food intake in fish oil, corn oil, and chow-fed rats. A dose of 6 ug/kg i.p. was given daily. P values calculated by one-way ANOVA, which revealed an interaction between diet and time on effect of IL- 1 (e.g., diet alters pattern over time, P < 0.03). *P < 0.05 vs. baseline intake. Each bar represents the mean±SE from 21-50 data points (three to five animals X 7-10 d of rmIL-lIa). Interleukin-J Anorexia in the Rat 231 there was no anorexic effect observed (intake 1011.8% with or without IL- 1). Body weights for chow fed rats giveen IL-la are shown in Fig. 4. Initially, weight gain is entirely pirevented but after 7-10 d weight gain is again observed, though the slope of gain is less than it was before rmIL- la administration and is less than in paired rats (average weight gain duriing IL- 1 administration was 1.5±0.2 g/d, P < 0.05 vs. pre--IL- 1 weight gain [3.6±0.2 g/d] and P < 0.05 vs. control group weight gain [2.7±0.2 g/d]). Similar results were observed in 1the C.O.-fed group (not shown). Discussion These results demonstrate that the meal-fed ralt is a useful animal model for quantifying the effects of purificad mediators on food intake and that rhIL-l(3 and rmIL-1a reduce food intake in the rat at doses 2 4 ug/kg i.p. PGE2 pr()ductiOn appears to be required for the IL- 1 effect, based on 1the ability of intravenous or intraperitoneal ibuprofen to blo(ck the effect completely, the ability of chronic F.O. feeding 1to markedly decrease it, and the observation that F.O. fed rats have signifi cantly lower in vivo and in vitro PGE2 producti on The anorexic effect of rhIL-lI3 and rmIL- la were similarl modulated by F.O. feeding and ibuprofen treatment. The interaction between IL- 1 secretion and P4GiE2 production is complex. PGE2 may inhibit further IL- 1 secretion by endothelial cells and macrophages (37), represeniting a short negative feedback loop. Since not all the actionss of IL- are mediated by PGE2 (e.g., certain immunostimulastory effects, reference 29), inhibition of macrophage and other tissue PGE2 production (Table III) may increase IL- 1 secreti(on and nonPGE2 mediated IL- 1 effects, even as it decrease-s those mediated by PGE2. Proper classification of IL- 1 ano4rexia a presumably undesirable effect in the clinical setting,;as PGE2 mediated is therefore important. It is worth inoting that cyclooxygenase inhibition also prevents a numbeir of effects of TNF (38, 39) and endotoxin (39) without prevernting the endotoxin-induced rise in circulating TNF (39). iy 300 r OW* ,"le Begin IL- ,e-a* or 0. 1%Serum l4 Injections Cr -0-C -- - Control Rats - -- IL-I Rats Average Body Weight (grams) 200 [ *ff4184Alao aos3°° a0 . , 10 5 20 25 30 35 Days Figure 4. Effect of chronic rmIL- I a administration on in chow-fed rats. A dose of 6 tog/kg i.p. was given daily.bAverage weight gain of IL-I group was 1.5±0.2 g/d during IL-I administration, P < 0.05 vs. weight gain pre-IL- 1 (3.6±0.2 g/d) anid P < 0.05 vs. control group (2.7±0.2 g/d), by two-tailed t test. 232 M. K Hellerstein, S. Comparisons with previous studies of the effect of IL- I on food intake in the rat are complicated by methodologic considerations. The report of McCarthy et al. (40) that intracerebroventricularly administered endotoxin and "IL-I" induced fever but did not suppress intake of a liquid diet in previously fasted rats must be interpreted with caution, since human monocyte supernatant containing IL- 1 was administered, not recombinant IL- 1. The brief and relatively minor effect of rmIL- la on intake of a liquid diet reported in the rat (14) in comparison to the more robust effects on total daily intake shown here with rhIL-l( and rmIL-la has several possible explanations. The liquid diet is not a physiologic form of food and may modify any effects of IL- 1 on appetite that are mediated by suppression of gastric motility (41). Due to rapid clearance of administered IL- 1f (37), the meal-feeding model may be superior for revealing IL-1,8 anorexia. The increased hepatic lipogenesis of meal-fed rats (19-21) may also increase sensitivity to IL- 1 anorexia, if the mechanism is a peripheral one and involves alterations in hepatic carbohydrate utilization or triose metabolism, as appears to underlie several other metabolic models of anorexia (17, 18, 42-46). The ability of cytokines to stimulate hepatic lipogenesis (47) in the rat is suggestive in this regard. The difference is unlikely to be due to the form of IL- 1 used, since we (Table I) and Moldawer et al. (16) have found rmIL- 1 a to be an effective anorexigen in rodents. Our observation of differences between rhIL-l1B and rhIL-1a (Table I) is consistent with other reports of different activities of IL-la and in some systems (e.g., 48) and may reflect different affinities of IL-la and IL-113 for the IL-1 receptor, and different abilities to generate PGE2. The difference between rhIL- 1 a and rmIL- 1 a emphasizes the importance of species source. The molecular basis of these variations in actions between IL- 1 from different species is unknown, although the profound effect of dietary fat source on both rhIL-lI3 and rmIL-la anorexia suggests a shared mechanism for their anorexigenic actions. The question whether IL- 1 anorexia operates by a central or peripheral mechanism can not be definitively answered yet. The failure of intravenous boluses of rhIL-l(I (800 ng) to cause any decrease in food intake (Table IV) is probably due to rapid renal clearance of IL- 1( (37), in view of the ability of intravenous rhIL- 1(3 or rmIL- 1 a continuously infused at lower doses to suppress food intake. The inability of acetaminophen, which preferentially inhibits brain cyclooxygenase relative to that in peripheral tissues (30, 31), to block rhIL-lB anorexia supports a peripheral mechanism. The inability of intravenous ibuprofen to completely block the anorexic effect of prolonged, low dose rhIL- 1( infusion also suggests that other mechanisms besides PGE2 production may be involved in this setting, although other explanations are possible since PG production was not measured. These questions require further study. The finding that normal aging decreases sensitivity to the anorexic effect of rhIL-1( is of interest. One might have expected a greater effect on food intake in these old, presumably less resilient animals. The fact that the opposite was observed indirectly supports the IL- 1(3 effect as being part of a "designed" physiologic response, which is attenuated with aging, in contrast to a model wherein anorexia is a "nonspecific" effect of illness. N. Meydani, M. Meydani, K Wu, and C. A. Dinarello By analogy to TNF, the possibility needs to be considered that IL- 1 induced suppression of food intake is a toxic effect rather than a primary anorexigenic effect. Daily administration of recombinant human TNF-a to rodents (12) initially caused acute gastrointestinal inflammation, with edema and hemorrhage, followed by rapid recovery within 24-48 h (tolerance). Food intake and weight loss followed a similar time course, with an acute effect followed by rapid tolerance (12). However, IL- 1 anorexia is unlikely to be due to acute gastrointestinal toxicity, for several reasons. First, extensive toxicity studies in mice and rats using 100-fold higher IL-l doses than were used here have failed to reveal any organ toxicity (Dinarello, C. A., unpublished observations). In fact it is quite difficult to kill a nonadrenalectomized rodent with massive doses of IL- 1, in contrast to TNF. Second, one would expect gastrointestinal toxicity to be worsened by prostaglandin inhibition, rather than ameliorated, since PGE2 is cytoprotective in the gut (49, 50). Finally, the anorexia and slowing of body weight gain that we observed were not restricted to the first 24-48 h of IL-l administration, but persisted for at least 7 d (Figs. 3 and 4), and withholding of IL- 1 for a day during chronic administration immediately restored food intake to normal (not shown). The effects of chronic rmIL- 1 a administration are notable for several reasons. The issue of tachyphylaxis to cytokine anorexia is central if a physiologic role for cytokines in the anorexia of chronic disease is to be considered tenable. Our observations indicate that tachyphylaxis to IL- 1 anorexia (attenuation of anorexigenic effects and return of some weight gain) does occur, but equally importantly show at least a 7-d effect for the anorexia at a constant rmIL- 1 a dose (Fig. 3) and a net lower weight in the IL- 1 treated rats compared to controls after several weeks (Fig. 4). Thus, IL- 1 by itself has long-term nutritional consequences when administered chronically, in this model. Also, the dietary fat effects observed in acute food intake experiments were reproduced for at least the first 7 d of treatment, supporting the physiologic relevance of the acute data. The mechanism for tachyphylaxis was not established by these studies (e.g., development of antibodies to the recombinant IL- 1, a well-documented event even in the same species, receptor down-regulation, metabolic adaptations, etc.). The question whether synergy with other cytokines (51, 52) would overcome this attenuation is currently under investigation. Placed in the larger picture, these findings add support to the concept that nutrients and inflammation are intimately intertwined, in a complex bidirectional fashion mediated by cytokines (Fig. 5). Thus, dietary protein and source of fatty acids may alter IL-1 release (53, 54), dietary fats may alter SUBSTRATE AVAILABILITY ( NUTRITION ) PROTEIN CYTOKINE RELEASE FAT CYTOKINE ACTIONS CHO L - ANOREXIA ALTERED METABOLISM: - NITROGEN WASTING - PYRUVATE OXIDATION - FAILURE OF KETOSIS - HYPERLIPIDEMIA Figure 5. Cytokine-mediated interactions between nutrients and inflammation. See text for details. ml cytokine actions (see above), carbohydrates may in theory be involved as signals in their anorexic effects (18, 42-46), and inflammatory mediators not only alter metabolism of all classes of substrates (47, 55-57) but reduce their overall intake (cause anorexia). Inclusion of IL-1 anorexia in this scheme is now necessary. From a clinical perspective, perhaps most interesting is the ability of a dietary supplement to diminish IL- 1 anorexia. This would be a simple and safe intervention in patients experiencing (or expected to experience) anorexia and weight loss in association with an inflammatory illness or injury. If IL-l is involved in the human syndrome and acts through PGE2 generation, as is the case in the rat, therapeutic trials of F.O. supplementation may be rewarding. By analogy with F.O. supplementation used for other clinical purposes (58) it is unlikely that humans would need to ingest a diet with 80% of the fat as F.O. to test the hypothesis. Human volunteers adding 3 g/d of EPA in F.O. capsules to their otherwise normal western diets demonstrated a 50% reduction in IL- 1 production when their blood leukocytes were stimulated ex vivo (59). 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