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Pharmacokinetics and feeding responses to muramyl dipeptide in rats
Physiology & Behavior 79 (2003) 173 – 182
Pharmacokinetics and feeding responses to muramyl dipeptide in rats Sophie Fosset a,b, Gilles Fromentina, Olivier Rampinc, Vincent Langb, Florence Mathieub, Daniel Tome´a,* a
Unite´ INRA/INAPG de Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique de Paris-Grignon, 16 rue Claude Bernard, F-75231, Paris, France b Danone Vitapole, 15 avenue Galile´e, F-92350 Le Plessis Robinson, France c AMIB, INRA, F-78352 Jouy en Josas, France Received 17 April 2002; received in revised form 13 December 2002; accepted 3 March 2003
Abstract N-acetyl-muramyl-L-alanine-D-isoglutamine or muramyl dipeptide (MDP) is the minimally active subunit of bacterial peptidoglycan. During a systemic infection, the involvement of MDP has been demonstrated in food intake depression by the macrophage hydrolysis of Gram-positive bacteria. Under normal conditions, mammals are constantly exposed to the release of endogenous MDP from degraded gut flora and that of exogenous MDP from the diet. However, MDP digestion and absorption in the gastrointestinal tract are not fully understood, and their physiological significance needs to be clarified. After gavage (1.5 mg/kg), very low levels of MDP were found in the systemic circulation of rats and feeding patterns were not altered. In contrast, after the intraperitoneal injection of a similar dose, a depression in food intake was observed. The rats reduced their meal frequency and constant feeding rate, showing signs of satiety. The behavioral satiety sequence (BSS) was modified by behavioral changes, similar to those which appear during sickness, such as an increase in resting and a reduction in grooming. Our data suggest that the hypophagic effect of MDP may result from satiety and sickness behavior. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Muramyl dipeptide; Pharmacokinetics; Meal pattern; Food intake; Anorexia; Conditioned food aversion; Behavioral satiety sequence
1. Introduction Muramyl dipeptide or N-acetyl-muramyl-L-alanine-D-isoglutamine (MDP) is a component of the muramyl peptides, which constitute the unit building blocks of bacterial cell walls. They are indispensable components of the outer membrane of Gram-positive bacteria, where they account for half of the cell wall. During Gram-positive bacterial infection, MDP is released at significant levels into the systemic circulation in the course of bacterial hydrolysis by macrophages [1 – 4]. MDP is involved in the food intake depression observed during bacterial infections [5– 7]. The origins and mechanisms underlying this anorexia are not clearly elucidated. Conditioned taste aversion or other sickness behaviors may play a role in mediating anorexia due to bacterial products
* Corresponding author. Tel.: +33-1-44-08-17-18; fax: +33-1-44-0818-25. E-mail address: [email protected] (D. Tome´).
[8 –11]. It may arise either from a direct effect of MDP or from an indirect effect through the release of anorexigenic cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a) [12 – 15] from lymphocytes and macrophages [16 – 18]. Under normal conditions, the intestinal lumen is probably continuously exposed to muramyl peptides since they arise naturally from the hydrolyzed cell walls of bacteria in the gut flora and from fermented food. However, no results are available concerning either the presence of MDP in the systemic circulation after its absorption by the intestinal mucosa or any associated effect on food intake. Our goal was to determine whether MDP could be detected under normal conditions in the circulation following oral administration and to investigate the capacity of orally administered MDP to decrease appetite in a rat model by comparison with systemic administration. A pharmacokinetic study was performed to compare the MDP plasma levels found after administration by gavage or systemic injection. A further study analyzed the meal pattern of rats receiving MDP by either gavage or intra-
0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0031-9384(03)00065-9
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peritoneal administration. To complete our findings on meal patterns, we examine the behavioral satiety sequence (BSS) in rats receiving an intraperitoneal injection of MDP [19,20].
2. Materials and methods 2.1. Chemicals MDP was purchased from Bachem (G-1055.0025, batch 116617, France). Analytical reagents were HPLCgrade acetonitrile (Carlo Erba 412391), HPLC-grade acetone (Prolabo 20066.296), formic acid (Merck 1.00264. 1000), 6 N hydrochloric acid (HCl) (Carlo Erba 502008), sodium chloride (NaCl) (Merck 1.06404.1000) and sodium iodide cesium iodide (Naics) (Waters 700000889, lot 904892 H). 2.2. Animals Animal experiments were carried out in accordance with the recommendations of the French Committee for Animal Care. Male Sprague –Dawley rats (De´pre´, SaintDoulchard, France) weighing 220 –240 g at the start of the experiment were housed in individual cylindrical cages in a room with controlled temperature (22 ± 1 C) and lighting (lights off from 10:00 to 22:00, 12:12 h cycle). During the entire experimental period, the rats had free access to water and food in the form of an AIN-93M modified diet (P14 diet) [21]. Instead of casein and cystine, this diet contained (g/kg of diet): total milk protein (Armor Proteins, Saint Brieuc, France) 140.0, corn starch (carbohydrate) (Cerestar, Haubourdin, France) 622.4, sucrose (carbohydrate) (Eurosucre, Paris, France) 100.3, soy bean oil (fat) (Medias Filtrants Durieux, ZI Torcy, France) 40.0, vitamin mixture AIN-93M (ICN Biomedicals, OH, USA) 10.0, salt mixture AIN-93M (ICN Biomedicals) 35.0; cellulose (Bailly, Aulnay sous Bois, France) 50.0 and choline chloride (ICN Biomedicals) 2.3. The diet had a metabolizable caloric density of 14.6 kJ/g and was moistened (water/powdered diet, 1:1 w/ w) to minimize spillage. All dietary components were purchased from the sources listed above or prepared by the Atelier de Pre´paration des Aliments Expe´rimentaux (APAE) (Experimental Food Preparation Unit, French National Institute of Agronomic Research, INRA, Jouy en Josas, France). Each day, at 09:00, the food intake was determined by the difference in the weight of the food cup before and after each experimental period, corrected for spillage and dehydration. The body weight of each rat was measured daily at 09:00. Before all the experiments, the rats had been adapted to the diet and housing conditions for 10 days. Whatever the experiment, during the prefeeding periods, the rats ate (27 ± 2.0 g/day) and grew similarly (4.6 ± 0.6 g/day).
2.3. Experiment 1: study of MDP plasma levels following the administration of MDP by gavage or intravenous injection To enable blood sampling, the rats (n = 32) had previously been implanted with a jugular catheter under anesthesia (sodium pentobarbital Sanofi, 0.07 ml/100 g of rat) [22,23]. They were allowed to recover from surgery for 1 week before the start of the experiment. Rats were habituated to handling for remote blood sampling during daily inspections for the patency of the cardiac catheter. During blood collection, the rats were continuously connected to a syringe filled with heparinized saline solution (10 U/ml) via polyethylene tubing. Venous blood samples were withdrawn 30 min before the intravenous (iv) injection of MDP or gavage (1.5 mg/kg b.wt.) and at different time points from 0 to 4 h after the administration of MDP (n = 8 rats by group). Control groups (n = 8) were treated identically but received a solution of NaCl 0.9% (saline) (intravenous injection) or water (gavage). Blood samples were centrifuged and plasma was frozen at 80 C for subsequent analysis. Before the analysis of MDP using Reverse Phase-High Performance Liquid Chromatography coupled with Mass Spectrometry (RP-HPLC/MS), plasma samples were deproteinized using five volumes of a mixture of acetone/0.1 N HCl (80:20 v/v) (double protein precipitation). Followed precipitation, the samples were centrifuged at 22,860 g for 15 min at + 4 C. The solvent mixture was brought to dryness in a speed-vac under vacuum. Each residue was redissolved in a volume of water, which allowed the concentration of plasma samples (concentration factor from 2 to 8) prior to RP-HPLC/MS analysis. HPLC equipment (Alliance system) comprised a Waters 2690 automated gradient controller and a Waters 996 UV detector, linked to a data acquisition and processing system (Waters Millenium32). A reverse-phase column symmetry C18 (2.1 150 ˚ ) (Waters WAT106172) was used with a C18 mm I.D., 300 A cartridge (Waters 186000198) as a guard column. Solvent A was 0.1% formic acid in water and solvent B was acetonitrile in 0.1% formic acid. Starting with 1% of solvent B (equilibration buffer), a gradient elution was generated by increasing this proportion to 15% in 10 –15 min, with a further rise to 100% in 5 min and then a pause for 3 min at 100% before returning to the starting conditions in 10 min. The column temperature was fixed at + 25 C and the flow rate was 0.2 ml/min. Eluted peaks were detected by absorbance at 210 nm. The mass spectrometer was a single quadripole detector with atmospheric pressure ionization (Micromass ZMD 4000 Waters). MS analysis was performed with an electrospray (ES) ion source set in the positive ionization mode. The m/z scale of the instrument was calibrated with Naics. The Selected Ion Monitoring function was used to detect and quantify MDP. The ES parameters were optimized: capillary voltage 3 kV, cone voltage 60 V for [M + H] + 493 (m/z) ion and 20 V for [M + Na] + 515 (m/z) ion, source block temperature 120 C,
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desolvation temperature 220 C and desolvation gas flow 420 l/h. RP-HPLC/MS (ESI + ) experiments were performed while scanning the m/z range 100 – 975 amu/s. Specific masses (m/z) were monitored to quantify MDP with a dwell time of 1 s, a span time of 1 s and an interchannel delay time of 0.02 s. The specificity of the analysis was verified in plasma samples by comparing a control sample (without MDP) with a plasma sample containing MDP. Data were processed using the MassLynx NT software package (version 3.4). MDP concentrations were calculated from the area of the peak compared with a nine concentration standard curve generated using standard solutions containing (4 – 20) 103 ng/ml MDP. Under these conditions, the limit of quantification was 13.3 ng/ml and the limit of detection was 4 ng/ml. 2.4. Experiment 2: effect of MDP on food intake The rats (n = 16) were housed in individual cylindrical cages designed to permit the automatic recording of food intake and thus analyze the meal pattern. After 10 days of habituation to laboratory conditions, one group of eight rats received at 3 day intervals an intraperitoneal injection of NaCl 0.9% solution (saline) or MDP solution (1.5 mg/kg b.wt.). The drug solution was prepared extemporaneously. Injections were performed at the onset of darkness and in counterbalanced day order so as to minimize any order effects. Under the same experimental conditions, another group of eight rats received MDP (1.5 mg/kg b.wt.) or water by gavage. The influence of the treatment order was studied in both groups, and no difference was detected. The food intake recording device consisted of an individual Plexiglas cylindrical cage equipped with an electronic balance (Ohaus, CT600, accuracy ± 0.1 g, Florham Park, USA) located outside the cage and connected to an IBM-compatible personal computer via a multiport controller (Baytech 528 from Baytech, Bay Saint Louis, USA) using a RS-232 output. Inside the cage, a food cup was supported by a bar fixed to the balance. The data acquisition
Fig. 2. Variations in mean concentrations of MDP in the plasma at various time points after different methods of administration (a) after gavage with MDP (1.5 mg/kg b.wt.) ( – – ) and (b) after an intravenous injection of MDP (1.5 mg/kg b.wt.) ( – & – ). The values are the mean concentrations in rats presented with their S.E.M. (expressed in ng/ml).
.
program scanned each on-line channel representing the output of each balance, recorded data and read the real time clock and saved the data with their corresponding time of recording. The time interval of recording was set up at 13 s. The cumulative food intake was continuously recorded on the last P14 day or at ‘‘baseline’’ and on subsequent experimental days. Food ingestion was measured during
Fig. 1. TIC profiles as measured by RP-HPLC/MS (ESI + ) of (a) plasma sample obtained at 60 min after gavage with MDP (1.5 mg/kg b.wt.) and (b) plasma sample obtained at 60 min after water administration. Peaks 1 and 2 represent the two anomeric forms of MDP.
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the 24 h period beginning at the time of injection. The food intake was filtered as follows: the minimum meal size was set at >0.1 g and the duration at >13 s and the intermeal interval (IMI) at at least 10 min [24]. Water evaporation was seen in the food intake record as a gradual uninterrupted decrease in food cup weight in the cumulative food intake signal. A number of parameters characterizing the rat’s feeding pattern are given for each rat, such as the size (g) and duration (min) of meals, meal frequency and ingestion rate (g/min), calculated by the ratio of meal size over its duration. The average duration of all IMI was also calculated. All eating parameters were measured over either a 24 h cycle or 3 h periods during the dark phase. The cumulative food intake (g) over 24 h is also presented. A microstructural study of ingestive behavior was achieved by determining the food intake but applying no hypothesis as to the minimum interval between feeding episodes. Following the application of this new filter, eating
parameters such as the size, frequency, duration, IMI and ingestion rate of eating bouts were calculated over a 24 h cycle and a 3 h cycle. 2.5. Experiment 3: effect of an intraperitoneal injection of MDP on the BSS During a third experiment identical to Experiment 2, six rats received at the onset of the dark cycle at 3 day intervals an intraperitoneal injection of NaCl 0.9% (saline) or MDP (1.5 mg/kg) so as to analyze the BSS. The experimental chamber consisted of a circular Plexiglass tank (height 320 mm, diameter 300 mm), containing a ring attached to the wall, where the food cup was fixed. A water bottle was placed on the opposite side of the chamber. The rats were habituated to using their test chambers for 3 days prior to testing. Animal reactions emitted by the rats were videorecorded while they ate during the first and third hours after
Fig. 3. Cumulative food intake of rats: (a) after gavage of MDP (1.5 mg/kg b.wt.) or water (n = 8) and (b) after intraperitoneal injection of MDP (1.5 mg/kg b.wt.) or saline (n = 8). Each value is presented as the mean ± S.E.M. (n = 8). * MDP, saline and baseline values are different ( P < .05).
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Table 1 Feeding patterns of rats during the baseline situation and thereafter an intraperitoneal injection of MDP (1.5 mg/kg b.wt.) or saline
Meal frequency
Meal duration (min)
Meal size (g)
Feeding rate (g/min)
Baseline MDP Saline Baseline MDP Saline Baseline MDP Saline Baseline MDP Saline
0–3 h
3–6 h
6–9 h
9 – 12 h
24 h
3.9 ± 0.4 3.8 ± 0.4 4.0 ± 0.7 4.1 ± 0.9 4.2 ± 1.0 4.4 ± 0.6 2.4 ± 0.4 1.9 ± 0.3 2.5 ± 0.4 0.8 ± 0.1 0.6 ± 0.1 0.8 ± 0.1
3.1 ± 0.5 1.5 ± 0.2 * 3.3 ± 0.4 5.4 ± 1.0 4.7 ± 0.8 3.9 ± 0.7 3.0 ± 0.4 3.4 ± 0.4 2.6 ± 0.3 0.8 ± 0.2 0.9 ± 0.1 0.8 ± 0.1
2.6 ± 0.4 1.9 ± 0.2 1.9 ± 0.4 5.5 ± 1.7 5.7 ± 1.1 6.0 ± 1.9 2.3 ± 0.4 3.8 ± 0.4 3.0 ± 0.5 0.7 ± 0.2 0.8 ± 0.1 0.7 ± 0.1
2.8 ± 0.5 2.6 ± 0.3 2.8 ± 0.3 7.0 ± 1.2 4.8 ± 0.7 4.4 ± 0.6 2.2 ± 0.4 2.6 ± 0.3 2.2 ± 0.2 0.5 ± 0.1 0.6 ± 0.1 0.6 ± 0.1
13.4 ± 1.2 10.8 ± 0.7 13.1 ± 1.1 5.3 ± 0.6 4.9 ± 0.6 4.7 ± 0.4 2.4 ± 0.2 2.7 ± 0.1 2.5 ± 0.2 0.6 ± 0.1 0.7 ± 0.1 0.7 ± 0.1
Values are expressed as means ± S.E.M. (n = 8) over a 24 h period and by 3 h periods during the first 12 h (nighttime). * MDP, saline and baseline values are different ( P < .05).
food presentation. The last P14 day was videorecorded and corresponded to the baseline period. During the experimental days, the video recording was started just after the intraperitoneal injection and meal presentation and lasted for 1 h (H1). The video recording was then started again 3 h after meal presentation (H3). Data were continuously recorded and not sampled. The choice of this schedule was based on the results of the meal pattern analysis made during Experiment 2. Food intake was also measured at the beginning and the end of each video analysis period. The video cameras (JVC, Digital Still Camera GR-DVL557) were placed outside the chamber, at a distance of approximately 300 mm from the food cup holder. The signal was recorded on conventional VHS tape (VHS-C, EC-60) at 50 frames per second using a video recorder (Compact Super VHS, JVC, GR-SXM307). Animal behavior was recorded on the last P14 day corresponding to the baseline situation and on subsequent experimental days. Videotapes were analyzed by a slow-motion playback (frame by frame at 1/ 6th of the normal playing speed) to count behavioral satiety. The behavior of each animal was categorized as follows for analysis [25 – 27]: (i) activity: this included locomotion (i.e.,
walking and running), rearing, defined as the rat standing on its two hind limbs, and jumping; (ii) grooming: scratching, licking or biting of the coat; (iii) resting: inactivity, standing, sitting or lying, sleeping with occasional changes of position; (iv) eating: the biting, gnawing or swallowing of food and (v) drinking. The analysis was made by dividing the 60 min of continuous record into 5 min periods. The frequency and duration of each behavioral phase were calculated for each period. 2.6. Statistics Results are expressed as means ± S.E.M. The statistical significance of differences between response variables was determined using one-way ANOVA. When appropriate, data were analyzed using ANOVA repeated measures to highlight a Treatment Time interaction (Proc GLM, SAS version 6.11, Cary, NC, USA) following a general linear model procedure. A P value lower than .05 was considered to be significant. When differences were detected, differences between individual means were determined using a post hoc test (Tukey, P < .05).
Table 2 Microstructural analysis data of rats during the baseline situation and after an intraperitoneal injection of MDP (1.5 mg/kg b.wt.) or saline
Eating bout frequency
Eating bout duration (min)
Eating bout size (g)
Feeding bout rate (g/min)
Baseline MDP Saline Baseline MDP Saline Baseline MDP Saline Baseline MDP Saline
0–3 h
3–6 h
6–9 h
9 – 12 h
24 h
6.5 ± 1.1 5.3 ± 0.5 6.1 ± 1.2 1.8 ± 0.4 2.0 ± 0.2 1.9 ± 0.3 1.6 ± 0.4 1.4 ± 0.2 1.8 ± 0.5 0.9 ± 0.1 0.7 ± 0.1 0.9 ± 0.1
5.8 ± 1.5 2.0 ± 0.4 * 5.0 ± 0.6 2.0 ± 0.3 3.6 ± 0.6 * 1.9 ± 0.3 2.0 ± 0.6 2.9 ± 0.5 1.9 ± 0.3 1.1 ± 0.2 0.9 ± 0.1 1.0 ± 0.1
4.6 ± 0.7 3.3 ± 0.6 3.4 ± 0.8 1.8 ± 0.2 2.6 ± 0.4 1.9 ± 0.3 1.4 ± 0.2 2.3 ± 0.4 1.8 ± 0.3 0.9 ± 0.2 0.9 ± 0.1 0.9 ± 0.1
6.6 ± 1.2 5.6 ± 0.7 5.1 ± 0.8 2.0 ± 0.5 2.0 ± 0.4 2.0 ± 0.4 1.0 ± 0.2 1.5 ± 0.2 1.3 ± 0.1 0.7 ± 0.2 0.8 ± 0.1 0.7 ± 0.1
25.3 ± 2.7 18.6 ± 0.8 22.1 ± 2.0 3.1 ± 0.9 2.6 ± 0.2 2.4 ± 0.3 1.7 ± 0.3 1.7 ± 0.1 1.5 ± 0.2 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.1
Values are expressed as means ± S.E.M. (n = 8) over a 24 h period and by 3 h periods during the first 12 h (nighttime). * MDP, saline and baseline values are different ( P < .05).
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3. Results 3.1. Experiment 1: study of MDP plasma levels following the administration of MDP by gavage or intravenous Injection The RP-HPLC (UV) profile of MDP in an aqueous solution displays two peaks, one for each anomeric form. a/b anomerization at the C-1 of N-acetylmuramic acid results in a splitting of the HPLC signal for each peptide. The TIC profiles of plasma samples taken from rats receiving MDP by gavage displayed peaks within the retention times for anomeric forms (Fig. 1a). The RP-HPLC elution profile for control plasma samples did not exhibit these peaks (Fig. 1b). Prior to the analysis of each unknown sample kinetic, standard curves were generated using spiked plasma samples following sample pretreatment procedures
similar to those applied to actual samples. Fig. 2a shows the mean plasma concentrations versus time profiles after gavage with MDP (1.5 mg/kg b.wt.). Plasma MDP concentrations peaked at 18.4 ng/ml at 60 min (less than 0.05% of the ingested dose) and then gradually declined. After 90 min, plasma MDP concentrations were near the limit of quantification (13.8 ng/ml). At 120 min, the plasma MDP concentrations were between the limit of quantification and the limit of detection (7.8 ng/ml). After the systemic administration of MDP, it was readily detected in plasma. The mean plasma concentration– time profile of MDP after a single intravenous injection is represented in Fig. 2b. Immediately after the intravenous injection, plasma concentrations rapidly declined; at 2 min, less than 35% of the injected dose was quantified. MDP was then slowly eliminated from the plasma, reaching a low level after 2 h (less than 0.3% of the injected dose).
Fig. 4. Temporal profiles of the four main categories of behavior (eating, grooming, activity and resting) observed: during the first hour (a) and the third hour (b) of recording after meal presentation during the baseline situation, (c) during the third hour of recording after meal presentation and intraperitoneal injection of saline and (d) during the third hour of recording after meal presentation and intraperitoneal injection of MDP (1.5 mg/kg b.wt.). The data are plotted as the percentage time occupied in each behavior during each 5 min period over the course of 1 and 3 h tests.
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3.2. Experiment 2: effect of MDP on food intake after the administration of MDP by gavage or intraperitoneal Injection After gavage, the cumulative food intake on baseline or after MDP or water treatments is shown in Fig. 3a. There was no decrease in the daily food intake by comparison with baseline or water gavage treatment nor was there any significant Time Treatment interaction on the cumulative food intake. After the intraperitoneal injection of MDP, the cumulative food intake for baseline, MDP and saline are shown in Fig. 3b. Although a reduction in food intake was seen after the intraperitoneal injection of MDP, the 24 h cumulative food intake did not differ significantly between treatments. A Time Treatment interaction was seen on the cumulative food intake [ F(2,97) = 2.2, P < 10 3]. After the intraperitoneal injection of MDP, the reduction in food intake became significant at 180 min by comparison with the baseline or saline treatment (Fig. 3b). A difference was no longer seen after 600 min. Overall, averaged feeding parameters during the 12 h of the nighttime period are presented in Table 1. Whatever the feeding parameters, the overall average between treatments did not differ. The IMI values obtained for baseline, MDP and saline were 75.9 ± 10.2, 87.7 ± 5.6 and 77.7 ± 5.4 min, respectively. When we examined the data concerning 3 h periods, the hypophagia induced by the intraperitoneal injection of MDP was characterized by a reduction in meal frequency within the 3– 6 h period [ F(2,21) = 7.9, P=.003]. Other meal parameters, such as meal duration, meal size and feeding rate were not affected by the MDP injection (Table 1). Microstructural analysis showed that after the intraperitoneal injection of MDP, differences were nonetheless observed in both eating bout frequency and eating bout duration during the 3 – 6 h period. The average eating parameters for 24 h and grouped by 3 h cycles are presented in Table 2. The overall average values of IMI for baseline, MDP and saline treatments were 43.1 ± 6.8, 66.5 ± 5.0 and 57.7 ± 4.4 min, respectively. There was a reduction in eating bout frequency [ F(2,21) = 4.8, P=.019] and an increase in eating bout duration [ F(2,21) = 4.7, P=.021]. 3.3. Experiment 3: effect of an intraperitoneal injection of MDP on the BSS The temporal profiles of the four major categories of behavior (eating, grooming, activity and resting) involved in the development of satiation are plotted in Fig. 4. The behavioral parameters during the first and third hour test sessions are summarized in Table 3. 3.3.1. Baseline During the first hour of video recording, the four behaviors occurred in a well-defined sequence: eating behavior followed by grooming and/or activity behaviors superseded by resting (see Fig. 4a). Most eating behavior occurred
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Table 3 Food intake (g) (1 h) and behavioral parameters of rats measured during the first hour of meal presentation (H1) and at 3 h after meal presentation (H3) during the baseline situation and after an intraperitoneal injection of MDP (1.5 mg/kg b.wt.) or saline
Eating Grooming Resting Activity 1 h food intake (g)
Baseline
MDP
Saline
H1 H3 H1 H3 H1 H3 H1 H3 H1
14.0 ± 0.7 6.5 ± 1.3 8.2 ± 3.2 13.1 ± 1.6 47.3 ± 7.4 54.2 ± 8.9 28.9 ± 5.2 25.9 ± 7.8 6.1 ± 0.5
9.6 ± 2 4.1 ± 0.9 7.0 ± 2.9 4.7 ± 1.5 * 54.1 ± 4.2 81.7 ± 3.0 * 27.9 ± 5.0 9.4 ± 1.7 6.3 ± 1.0
10.3 ± 0.8 6.6 ± 0.7 9.8 ± 3.3 13.5 ± 2.7 52.1 ± 4.2 58.4 ± 8.9 27.0 ± 2.3 21.3 ± 6.4 5.5 ± 1.2
H3
3.1 ± 0.4
2.2 ± 0.6
3.3 ± 0.3
These parameters are presented as the percentage of time spent on the different monitored behaviors. Each value is presented as the mean ± S.E.M. (n = 6). * MDP, saline and baseline values are differed ( P < .05).
during the first 15 min following presentation of the P14 diet. Resting predominated during the last 40 min and its duration was the longest in the BSS. During the third hour of video recording, the satiety sequence was shifted to the left. Feeding was terminated earlier, grooming occurred earlier, appearance of peak activity was shifted to the left and time course of resting was reduced from 20 to 10 min (Fig. 4b). A comparison of the first and third hours of video recording revealed a reduction of eating over time. The other compartments remained unchanged. 3.3.2. Saline There was no difference in behavioral profiles and parameters after the saline and the baseline treatments for the H1 and H3 (Table 3 and Fig. 4c) periods. 3.3.3. MDP During the first hour of video recording, there was no difference in behavioral profiles and parameters between baseline, saline and MDP treatments (Table 3) nor was the BSS any more disturbed during the third hour of video recording (Fig. 4d). The injection of MDP increased resting behavior by comparison with the baseline and saline treatments [ F(2,15) = 5.73, P < .05] (Fig. 4d and Table 3). In rats treated with MDP, resting was mainly preponderant while grooming was significantly decreased [ F(2,15) = 8.21, P < .01]. However, because of considerable variabilities within the group, the reduction in activity was not statistically significant. Eating behavior was not affected by treatment with MDP.
4. General discussion These results demonstrate that MDP administered orally could be identified in the blood but was not effective as an
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appetite suppressant. In contrast, after an intraperitoneal injection of MDP, a depression in food intake and a reduction in meal frequency were observed. This suppression of appetite does not seem to have resulted from mechanisms such as satiation or conditioned taste aversion but may be representative of an increase in satiety along with the sickness behavior, which can be induced by bacterial infection, such as a reduction in grooming and an increase in resting. During our experiments, a highly sensitive and selective RP-HPLC method coupled to MS detection was used to quantify MDP in plasma samples. Our findings indicate that MDP appeared in the systemic circulation after gavage, suggesting a partial resistance during the intestinal digestion and absorption processes. The intestinal absorption pathway for this peptide remains unclear. Specific transport systems, such as the di- and tripeptide transport systems, may be involved [28,29]. The MDP plasma peak was obtained at 60 min with plasma levels representing less than 0.05% of the ingested dose. However, MDP was also rapidly eliminated from the systemic circulation. At 120 min, MDP was detected but not quantified according to the current limit of sensitivity for the analytical method. These low levels of MDP found in the systemic circulation after oral administration could be explained by a marked retention in the stomach and intestinal tract, as is observed with the peptidoglycan monomer [30]. MDP may be also degraded by various enzymes and principally by a mammalian enzyme (N-acetyl-muramyl-L-alanine amidase) found in biological fluids such as serum, which inactivates muramyl peptides by hydrolyzing the lactylamide bond between N-acetylmuramic acid and the L-alanine residue [31 –33]. In relation to these plasma kinetics, MDP after oral administration cannot attain sufficient peripheral or central tissue levels to achieve an anorectic effect [34]. In contrast, following intravenous injection, MDP was readily detected in its intact form and at high levels in the circulation. Plasma levels of MDP rapidly decreased to less than 35% of the injected dose at 2 min. Our findings are consistent with previous reports by Ambler and Hudson [35] who studied the metabolic fate of an intravenous injection of 3 H-MDP in mice. They observed that MDP was rapidly eliminated from the circulation and excreted in its intact form in the urine, which is consistent with its water solubility [35,36]. However, the plasma levels seemed to be sufficient to reduce food intake by decreasing the number of meals without affecting their size. This finding is consistent with the results obtained by Langhans et al. [37,38] who studied similar doses and time courses after the intraperitoneal injection of MDP. The reduction in food intake was not immediate and could be related to an indirect effect induced by cytokine production [9,14,15,39 – 43]. The effect of an MDP injection on food intake would be more relevant to satiety and/or sickness behavior than to conditioned taste aversion. Analysis of the spontaneous meal pattern and the BSS have made it possible to clarify
whether experimental handling affects ingestion by inducing satiety or malaise [20,27,44 –49]. Our results showed that the depression in food intake induced by the injection of MDP resulted from a reduction in meal numbers, although no decreases in meal size, meal duration or feeding rate were observed. These results were also confirmed by microstructural analysis. The effect should not be interpreted as satiation because, in such a case, we would have observed a reduction in meal duration without any effect on feeding rate [46,50,51]. In contrast, the reduction of meal frequency, together with a constant feeding rate, could be interpreted as a sign of enhanced satiety. However, analysis of the BSS during the third hour of presentation makes this interpretation more puzzling. During the first hour of food intake, whatever the experimental day, the four principal behaviors always occurred in a welldefined sequence: eating followed by grooming and/or activity followed by resting. During the baseline and saline situations, video analysis of the third hour of the feeding period showed that the four principal behaviors always occurred in the same, well-defined sequence, with a reduction in the eating period and a shift to the left for all other behavioral profiles. This preserved BSS could be interpreted in favor of a reduction in food intake by a postingestive mechanism of satiety [46,20,27]. In contrast, after the intraperitoneal injection of MDP, video analysis of the third hour showed that resting was dramatically increased while the grooming period was reduced. At this dose of MDP, this profile could be related to the behavioral modifications observed during bacterial infection, i.e., a reduction in grooming and locomotor activity and increased sleepiness [11]. The latter may also be associated with other biological effects of MDP, which include the induction of drowsiness [4]. A characteristic feature of such a behavior might be suggested by the results of the microstructural analysis, which showed that rats visited the food cup less frequently but stayed longer, as demonstrated by the increase in eating bout duration. In addition to the nature of food intake depression, MDP administration did not act as an unconditioned stimulus for the conditioned aversion which might arise during bacterial infection. The lack of difference in meal size and feeding rate showed that the intraperitoneal injection of MDP did not act like lithium chloride (LiCl), a known aversive toxic [45,52,53]. Analysis of the BSS test reinforced this hypothesis, because it was preserved. A conditioned taste aversion induces a loss of the BSS associated with an increase in activity [54]. Moreover, Blundell et al. [46] showed that a cessation of eating was followed by resting before grooming appeared, which is in favor of an abnormal inhibition of eating, associated with the malaise caused by intestinal discomfort. In conclusion, the food intake depression caused by an intraperitoneal injection was not due to a conditioned taste aversion mechanism but probably an increase in satiety
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along with sickness behavior. After oral administration, MDP was poorly absorbed and did not act on food intake. We suggest that under normal conditions the release of MDP from gut flora or food bacteria is not able to exert any hypophagic effect. However, other peptidoglycan fragments may also be released from bacterial hydrolysis and should be investigated in the future with respect to their anorectic activity.
[20]
[21]
[22]
Acknowledgements
[23]
The assistance provided by Nathalie Jerome is gratefully acknowledged.
[24] [25]
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Pharmacokinetics and feeding responses to muramyl dipeptide in rats Sophie Fosset a,b, Gilles Fromentina, Olivier Rampinc, Vincent Langb, Florence Mathieub, Daniel Tome´a,* a
Unite´ INRA/INAPG de Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique de Paris-Grignon, 16 rue Claude Bernard, F-75231, Paris, France b Danone Vitapole, 15 avenue Galile´e, F-92350 Le Plessis Robinson, France c AMIB, INRA, F-78352 Jouy en Josas, France Received 17 April 2002; received in revised form 13 December 2002; accepted 3 March 2003
Abstract N-acetyl-muramyl-L-alanine-D-isoglutamine or muramyl dipeptide (MDP) is the minimally active subunit of bacterial peptidoglycan. During a systemic infection, the involvement of MDP has been demonstrated in food intake depression by the macrophage hydrolysis of Gram-positive bacteria. Under normal conditions, mammals are constantly exposed to the release of endogenous MDP from degraded gut flora and that of exogenous MDP from the diet. However, MDP digestion and absorption in the gastrointestinal tract are not fully understood, and their physiological significance needs to be clarified. After gavage (1.5 mg/kg), very low levels of MDP were found in the systemic circulation of rats and feeding patterns were not altered. In contrast, after the intraperitoneal injection of a similar dose, a depression in food intake was observed. The rats reduced their meal frequency and constant feeding rate, showing signs of satiety. The behavioral satiety sequence (BSS) was modified by behavioral changes, similar to those which appear during sickness, such as an increase in resting and a reduction in grooming. Our data suggest that the hypophagic effect of MDP may result from satiety and sickness behavior. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Muramyl dipeptide; Pharmacokinetics; Meal pattern; Food intake; Anorexia; Conditioned food aversion; Behavioral satiety sequence
1. Introduction Muramyl dipeptide or N-acetyl-muramyl-L-alanine-D-isoglutamine (MDP) is a component of the muramyl peptides, which constitute the unit building blocks of bacterial cell walls. They are indispensable components of the outer membrane of Gram-positive bacteria, where they account for half of the cell wall. During Gram-positive bacterial infection, MDP is released at significant levels into the systemic circulation in the course of bacterial hydrolysis by macrophages [1 – 4]. MDP is involved in the food intake depression observed during bacterial infections [5– 7]. The origins and mechanisms underlying this anorexia are not clearly elucidated. Conditioned taste aversion or other sickness behaviors may play a role in mediating anorexia due to bacterial products
* Corresponding author. Tel.: +33-1-44-08-17-18; fax: +33-1-44-0818-25. E-mail address: [email protected] (D. Tome´).
[8 –11]. It may arise either from a direct effect of MDP or from an indirect effect through the release of anorexigenic cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a) [12 – 15] from lymphocytes and macrophages [16 – 18]. Under normal conditions, the intestinal lumen is probably continuously exposed to muramyl peptides since they arise naturally from the hydrolyzed cell walls of bacteria in the gut flora and from fermented food. However, no results are available concerning either the presence of MDP in the systemic circulation after its absorption by the intestinal mucosa or any associated effect on food intake. Our goal was to determine whether MDP could be detected under normal conditions in the circulation following oral administration and to investigate the capacity of orally administered MDP to decrease appetite in a rat model by comparison with systemic administration. A pharmacokinetic study was performed to compare the MDP plasma levels found after administration by gavage or systemic injection. A further study analyzed the meal pattern of rats receiving MDP by either gavage or intra-
0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0031-9384(03)00065-9
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peritoneal administration. To complete our findings on meal patterns, we examine the behavioral satiety sequence (BSS) in rats receiving an intraperitoneal injection of MDP [19,20].
2. Materials and methods 2.1. Chemicals MDP was purchased from Bachem (G-1055.0025, batch 116617, France). Analytical reagents were HPLCgrade acetonitrile (Carlo Erba 412391), HPLC-grade acetone (Prolabo 20066.296), formic acid (Merck 1.00264. 1000), 6 N hydrochloric acid (HCl) (Carlo Erba 502008), sodium chloride (NaCl) (Merck 1.06404.1000) and sodium iodide cesium iodide (Naics) (Waters 700000889, lot 904892 H). 2.2. Animals Animal experiments were carried out in accordance with the recommendations of the French Committee for Animal Care. Male Sprague –Dawley rats (De´pre´, SaintDoulchard, France) weighing 220 –240 g at the start of the experiment were housed in individual cylindrical cages in a room with controlled temperature (22 ± 1 C) and lighting (lights off from 10:00 to 22:00, 12:12 h cycle). During the entire experimental period, the rats had free access to water and food in the form of an AIN-93M modified diet (P14 diet) [21]. Instead of casein and cystine, this diet contained (g/kg of diet): total milk protein (Armor Proteins, Saint Brieuc, France) 140.0, corn starch (carbohydrate) (Cerestar, Haubourdin, France) 622.4, sucrose (carbohydrate) (Eurosucre, Paris, France) 100.3, soy bean oil (fat) (Medias Filtrants Durieux, ZI Torcy, France) 40.0, vitamin mixture AIN-93M (ICN Biomedicals, OH, USA) 10.0, salt mixture AIN-93M (ICN Biomedicals) 35.0; cellulose (Bailly, Aulnay sous Bois, France) 50.0 and choline chloride (ICN Biomedicals) 2.3. The diet had a metabolizable caloric density of 14.6 kJ/g and was moistened (water/powdered diet, 1:1 w/ w) to minimize spillage. All dietary components were purchased from the sources listed above or prepared by the Atelier de Pre´paration des Aliments Expe´rimentaux (APAE) (Experimental Food Preparation Unit, French National Institute of Agronomic Research, INRA, Jouy en Josas, France). Each day, at 09:00, the food intake was determined by the difference in the weight of the food cup before and after each experimental period, corrected for spillage and dehydration. The body weight of each rat was measured daily at 09:00. Before all the experiments, the rats had been adapted to the diet and housing conditions for 10 days. Whatever the experiment, during the prefeeding periods, the rats ate (27 ± 2.0 g/day) and grew similarly (4.6 ± 0.6 g/day).
2.3. Experiment 1: study of MDP plasma levels following the administration of MDP by gavage or intravenous injection To enable blood sampling, the rats (n = 32) had previously been implanted with a jugular catheter under anesthesia (sodium pentobarbital Sanofi, 0.07 ml/100 g of rat) [22,23]. They were allowed to recover from surgery for 1 week before the start of the experiment. Rats were habituated to handling for remote blood sampling during daily inspections for the patency of the cardiac catheter. During blood collection, the rats were continuously connected to a syringe filled with heparinized saline solution (10 U/ml) via polyethylene tubing. Venous blood samples were withdrawn 30 min before the intravenous (iv) injection of MDP or gavage (1.5 mg/kg b.wt.) and at different time points from 0 to 4 h after the administration of MDP (n = 8 rats by group). Control groups (n = 8) were treated identically but received a solution of NaCl 0.9% (saline) (intravenous injection) or water (gavage). Blood samples were centrifuged and plasma was frozen at 80 C for subsequent analysis. Before the analysis of MDP using Reverse Phase-High Performance Liquid Chromatography coupled with Mass Spectrometry (RP-HPLC/MS), plasma samples were deproteinized using five volumes of a mixture of acetone/0.1 N HCl (80:20 v/v) (double protein precipitation). Followed precipitation, the samples were centrifuged at 22,860 g for 15 min at + 4 C. The solvent mixture was brought to dryness in a speed-vac under vacuum. Each residue was redissolved in a volume of water, which allowed the concentration of plasma samples (concentration factor from 2 to 8) prior to RP-HPLC/MS analysis. HPLC equipment (Alliance system) comprised a Waters 2690 automated gradient controller and a Waters 996 UV detector, linked to a data acquisition and processing system (Waters Millenium32). A reverse-phase column symmetry C18 (2.1 150 ˚ ) (Waters WAT106172) was used with a C18 mm I.D., 300 A cartridge (Waters 186000198) as a guard column. Solvent A was 0.1% formic acid in water and solvent B was acetonitrile in 0.1% formic acid. Starting with 1% of solvent B (equilibration buffer), a gradient elution was generated by increasing this proportion to 15% in 10 –15 min, with a further rise to 100% in 5 min and then a pause for 3 min at 100% before returning to the starting conditions in 10 min. The column temperature was fixed at + 25 C and the flow rate was 0.2 ml/min. Eluted peaks were detected by absorbance at 210 nm. The mass spectrometer was a single quadripole detector with atmospheric pressure ionization (Micromass ZMD 4000 Waters). MS analysis was performed with an electrospray (ES) ion source set in the positive ionization mode. The m/z scale of the instrument was calibrated with Naics. The Selected Ion Monitoring function was used to detect and quantify MDP. The ES parameters were optimized: capillary voltage 3 kV, cone voltage 60 V for [M + H] + 493 (m/z) ion and 20 V for [M + Na] + 515 (m/z) ion, source block temperature 120 C,
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desolvation temperature 220 C and desolvation gas flow 420 l/h. RP-HPLC/MS (ESI + ) experiments were performed while scanning the m/z range 100 – 975 amu/s. Specific masses (m/z) were monitored to quantify MDP with a dwell time of 1 s, a span time of 1 s and an interchannel delay time of 0.02 s. The specificity of the analysis was verified in plasma samples by comparing a control sample (without MDP) with a plasma sample containing MDP. Data were processed using the MassLynx NT software package (version 3.4). MDP concentrations were calculated from the area of the peak compared with a nine concentration standard curve generated using standard solutions containing (4 – 20) 103 ng/ml MDP. Under these conditions, the limit of quantification was 13.3 ng/ml and the limit of detection was 4 ng/ml. 2.4. Experiment 2: effect of MDP on food intake The rats (n = 16) were housed in individual cylindrical cages designed to permit the automatic recording of food intake and thus analyze the meal pattern. After 10 days of habituation to laboratory conditions, one group of eight rats received at 3 day intervals an intraperitoneal injection of NaCl 0.9% solution (saline) or MDP solution (1.5 mg/kg b.wt.). The drug solution was prepared extemporaneously. Injections were performed at the onset of darkness and in counterbalanced day order so as to minimize any order effects. Under the same experimental conditions, another group of eight rats received MDP (1.5 mg/kg b.wt.) or water by gavage. The influence of the treatment order was studied in both groups, and no difference was detected. The food intake recording device consisted of an individual Plexiglas cylindrical cage equipped with an electronic balance (Ohaus, CT600, accuracy ± 0.1 g, Florham Park, USA) located outside the cage and connected to an IBM-compatible personal computer via a multiport controller (Baytech 528 from Baytech, Bay Saint Louis, USA) using a RS-232 output. Inside the cage, a food cup was supported by a bar fixed to the balance. The data acquisition
Fig. 2. Variations in mean concentrations of MDP in the plasma at various time points after different methods of administration (a) after gavage with MDP (1.5 mg/kg b.wt.) ( – – ) and (b) after an intravenous injection of MDP (1.5 mg/kg b.wt.) ( – & – ). The values are the mean concentrations in rats presented with their S.E.M. (expressed in ng/ml).
.
program scanned each on-line channel representing the output of each balance, recorded data and read the real time clock and saved the data with their corresponding time of recording. The time interval of recording was set up at 13 s. The cumulative food intake was continuously recorded on the last P14 day or at ‘‘baseline’’ and on subsequent experimental days. Food ingestion was measured during
Fig. 1. TIC profiles as measured by RP-HPLC/MS (ESI + ) of (a) plasma sample obtained at 60 min after gavage with MDP (1.5 mg/kg b.wt.) and (b) plasma sample obtained at 60 min after water administration. Peaks 1 and 2 represent the two anomeric forms of MDP.
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the 24 h period beginning at the time of injection. The food intake was filtered as follows: the minimum meal size was set at >0.1 g and the duration at >13 s and the intermeal interval (IMI) at at least 10 min [24]. Water evaporation was seen in the food intake record as a gradual uninterrupted decrease in food cup weight in the cumulative food intake signal. A number of parameters characterizing the rat’s feeding pattern are given for each rat, such as the size (g) and duration (min) of meals, meal frequency and ingestion rate (g/min), calculated by the ratio of meal size over its duration. The average duration of all IMI was also calculated. All eating parameters were measured over either a 24 h cycle or 3 h periods during the dark phase. The cumulative food intake (g) over 24 h is also presented. A microstructural study of ingestive behavior was achieved by determining the food intake but applying no hypothesis as to the minimum interval between feeding episodes. Following the application of this new filter, eating
parameters such as the size, frequency, duration, IMI and ingestion rate of eating bouts were calculated over a 24 h cycle and a 3 h cycle. 2.5. Experiment 3: effect of an intraperitoneal injection of MDP on the BSS During a third experiment identical to Experiment 2, six rats received at the onset of the dark cycle at 3 day intervals an intraperitoneal injection of NaCl 0.9% (saline) or MDP (1.5 mg/kg) so as to analyze the BSS. The experimental chamber consisted of a circular Plexiglass tank (height 320 mm, diameter 300 mm), containing a ring attached to the wall, where the food cup was fixed. A water bottle was placed on the opposite side of the chamber. The rats were habituated to using their test chambers for 3 days prior to testing. Animal reactions emitted by the rats were videorecorded while they ate during the first and third hours after
Fig. 3. Cumulative food intake of rats: (a) after gavage of MDP (1.5 mg/kg b.wt.) or water (n = 8) and (b) after intraperitoneal injection of MDP (1.5 mg/kg b.wt.) or saline (n = 8). Each value is presented as the mean ± S.E.M. (n = 8). * MDP, saline and baseline values are different ( P < .05).
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Table 1 Feeding patterns of rats during the baseline situation and thereafter an intraperitoneal injection of MDP (1.5 mg/kg b.wt.) or saline
Meal frequency
Meal duration (min)
Meal size (g)
Feeding rate (g/min)
Baseline MDP Saline Baseline MDP Saline Baseline MDP Saline Baseline MDP Saline
0–3 h
3–6 h
6–9 h
9 – 12 h
24 h
3.9 ± 0.4 3.8 ± 0.4 4.0 ± 0.7 4.1 ± 0.9 4.2 ± 1.0 4.4 ± 0.6 2.4 ± 0.4 1.9 ± 0.3 2.5 ± 0.4 0.8 ± 0.1 0.6 ± 0.1 0.8 ± 0.1
3.1 ± 0.5 1.5 ± 0.2 * 3.3 ± 0.4 5.4 ± 1.0 4.7 ± 0.8 3.9 ± 0.7 3.0 ± 0.4 3.4 ± 0.4 2.6 ± 0.3 0.8 ± 0.2 0.9 ± 0.1 0.8 ± 0.1
2.6 ± 0.4 1.9 ± 0.2 1.9 ± 0.4 5.5 ± 1.7 5.7 ± 1.1 6.0 ± 1.9 2.3 ± 0.4 3.8 ± 0.4 3.0 ± 0.5 0.7 ± 0.2 0.8 ± 0.1 0.7 ± 0.1
2.8 ± 0.5 2.6 ± 0.3 2.8 ± 0.3 7.0 ± 1.2 4.8 ± 0.7 4.4 ± 0.6 2.2 ± 0.4 2.6 ± 0.3 2.2 ± 0.2 0.5 ± 0.1 0.6 ± 0.1 0.6 ± 0.1
13.4 ± 1.2 10.8 ± 0.7 13.1 ± 1.1 5.3 ± 0.6 4.9 ± 0.6 4.7 ± 0.4 2.4 ± 0.2 2.7 ± 0.1 2.5 ± 0.2 0.6 ± 0.1 0.7 ± 0.1 0.7 ± 0.1
Values are expressed as means ± S.E.M. (n = 8) over a 24 h period and by 3 h periods during the first 12 h (nighttime). * MDP, saline and baseline values are different ( P < .05).
food presentation. The last P14 day was videorecorded and corresponded to the baseline period. During the experimental days, the video recording was started just after the intraperitoneal injection and meal presentation and lasted for 1 h (H1). The video recording was then started again 3 h after meal presentation (H3). Data were continuously recorded and not sampled. The choice of this schedule was based on the results of the meal pattern analysis made during Experiment 2. Food intake was also measured at the beginning and the end of each video analysis period. The video cameras (JVC, Digital Still Camera GR-DVL557) were placed outside the chamber, at a distance of approximately 300 mm from the food cup holder. The signal was recorded on conventional VHS tape (VHS-C, EC-60) at 50 frames per second using a video recorder (Compact Super VHS, JVC, GR-SXM307). Animal behavior was recorded on the last P14 day corresponding to the baseline situation and on subsequent experimental days. Videotapes were analyzed by a slow-motion playback (frame by frame at 1/ 6th of the normal playing speed) to count behavioral satiety. The behavior of each animal was categorized as follows for analysis [25 – 27]: (i) activity: this included locomotion (i.e.,
walking and running), rearing, defined as the rat standing on its two hind limbs, and jumping; (ii) grooming: scratching, licking or biting of the coat; (iii) resting: inactivity, standing, sitting or lying, sleeping with occasional changes of position; (iv) eating: the biting, gnawing or swallowing of food and (v) drinking. The analysis was made by dividing the 60 min of continuous record into 5 min periods. The frequency and duration of each behavioral phase were calculated for each period. 2.6. Statistics Results are expressed as means ± S.E.M. The statistical significance of differences between response variables was determined using one-way ANOVA. When appropriate, data were analyzed using ANOVA repeated measures to highlight a Treatment Time interaction (Proc GLM, SAS version 6.11, Cary, NC, USA) following a general linear model procedure. A P value lower than .05 was considered to be significant. When differences were detected, differences between individual means were determined using a post hoc test (Tukey, P < .05).
Table 2 Microstructural analysis data of rats during the baseline situation and after an intraperitoneal injection of MDP (1.5 mg/kg b.wt.) or saline
Eating bout frequency
Eating bout duration (min)
Eating bout size (g)
Feeding bout rate (g/min)
Baseline MDP Saline Baseline MDP Saline Baseline MDP Saline Baseline MDP Saline
0–3 h
3–6 h
6–9 h
9 – 12 h
24 h
6.5 ± 1.1 5.3 ± 0.5 6.1 ± 1.2 1.8 ± 0.4 2.0 ± 0.2 1.9 ± 0.3 1.6 ± 0.4 1.4 ± 0.2 1.8 ± 0.5 0.9 ± 0.1 0.7 ± 0.1 0.9 ± 0.1
5.8 ± 1.5 2.0 ± 0.4 * 5.0 ± 0.6 2.0 ± 0.3 3.6 ± 0.6 * 1.9 ± 0.3 2.0 ± 0.6 2.9 ± 0.5 1.9 ± 0.3 1.1 ± 0.2 0.9 ± 0.1 1.0 ± 0.1
4.6 ± 0.7 3.3 ± 0.6 3.4 ± 0.8 1.8 ± 0.2 2.6 ± 0.4 1.9 ± 0.3 1.4 ± 0.2 2.3 ± 0.4 1.8 ± 0.3 0.9 ± 0.2 0.9 ± 0.1 0.9 ± 0.1
6.6 ± 1.2 5.6 ± 0.7 5.1 ± 0.8 2.0 ± 0.5 2.0 ± 0.4 2.0 ± 0.4 1.0 ± 0.2 1.5 ± 0.2 1.3 ± 0.1 0.7 ± 0.2 0.8 ± 0.1 0.7 ± 0.1
25.3 ± 2.7 18.6 ± 0.8 22.1 ± 2.0 3.1 ± 0.9 2.6 ± 0.2 2.4 ± 0.3 1.7 ± 0.3 1.7 ± 0.1 1.5 ± 0.2 0.8 ± 0.1 0.8 ± 0.1 0.8 ± 0.1
Values are expressed as means ± S.E.M. (n = 8) over a 24 h period and by 3 h periods during the first 12 h (nighttime). * MDP, saline and baseline values are different ( P < .05).
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3. Results 3.1. Experiment 1: study of MDP plasma levels following the administration of MDP by gavage or intravenous Injection The RP-HPLC (UV) profile of MDP in an aqueous solution displays two peaks, one for each anomeric form. a/b anomerization at the C-1 of N-acetylmuramic acid results in a splitting of the HPLC signal for each peptide. The TIC profiles of plasma samples taken from rats receiving MDP by gavage displayed peaks within the retention times for anomeric forms (Fig. 1a). The RP-HPLC elution profile for control plasma samples did not exhibit these peaks (Fig. 1b). Prior to the analysis of each unknown sample kinetic, standard curves were generated using spiked plasma samples following sample pretreatment procedures
similar to those applied to actual samples. Fig. 2a shows the mean plasma concentrations versus time profiles after gavage with MDP (1.5 mg/kg b.wt.). Plasma MDP concentrations peaked at 18.4 ng/ml at 60 min (less than 0.05% of the ingested dose) and then gradually declined. After 90 min, plasma MDP concentrations were near the limit of quantification (13.8 ng/ml). At 120 min, the plasma MDP concentrations were between the limit of quantification and the limit of detection (7.8 ng/ml). After the systemic administration of MDP, it was readily detected in plasma. The mean plasma concentration– time profile of MDP after a single intravenous injection is represented in Fig. 2b. Immediately after the intravenous injection, plasma concentrations rapidly declined; at 2 min, less than 35% of the injected dose was quantified. MDP was then slowly eliminated from the plasma, reaching a low level after 2 h (less than 0.3% of the injected dose).
Fig. 4. Temporal profiles of the four main categories of behavior (eating, grooming, activity and resting) observed: during the first hour (a) and the third hour (b) of recording after meal presentation during the baseline situation, (c) during the third hour of recording after meal presentation and intraperitoneal injection of saline and (d) during the third hour of recording after meal presentation and intraperitoneal injection of MDP (1.5 mg/kg b.wt.). The data are plotted as the percentage time occupied in each behavior during each 5 min period over the course of 1 and 3 h tests.
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3.2. Experiment 2: effect of MDP on food intake after the administration of MDP by gavage or intraperitoneal Injection After gavage, the cumulative food intake on baseline or after MDP or water treatments is shown in Fig. 3a. There was no decrease in the daily food intake by comparison with baseline or water gavage treatment nor was there any significant Time Treatment interaction on the cumulative food intake. After the intraperitoneal injection of MDP, the cumulative food intake for baseline, MDP and saline are shown in Fig. 3b. Although a reduction in food intake was seen after the intraperitoneal injection of MDP, the 24 h cumulative food intake did not differ significantly between treatments. A Time Treatment interaction was seen on the cumulative food intake [ F(2,97) = 2.2, P < 10 3]. After the intraperitoneal injection of MDP, the reduction in food intake became significant at 180 min by comparison with the baseline or saline treatment (Fig. 3b). A difference was no longer seen after 600 min. Overall, averaged feeding parameters during the 12 h of the nighttime period are presented in Table 1. Whatever the feeding parameters, the overall average between treatments did not differ. The IMI values obtained for baseline, MDP and saline were 75.9 ± 10.2, 87.7 ± 5.6 and 77.7 ± 5.4 min, respectively. When we examined the data concerning 3 h periods, the hypophagia induced by the intraperitoneal injection of MDP was characterized by a reduction in meal frequency within the 3– 6 h period [ F(2,21) = 7.9, P=.003]. Other meal parameters, such as meal duration, meal size and feeding rate were not affected by the MDP injection (Table 1). Microstructural analysis showed that after the intraperitoneal injection of MDP, differences were nonetheless observed in both eating bout frequency and eating bout duration during the 3 – 6 h period. The average eating parameters for 24 h and grouped by 3 h cycles are presented in Table 2. The overall average values of IMI for baseline, MDP and saline treatments were 43.1 ± 6.8, 66.5 ± 5.0 and 57.7 ± 4.4 min, respectively. There was a reduction in eating bout frequency [ F(2,21) = 4.8, P=.019] and an increase in eating bout duration [ F(2,21) = 4.7, P=.021]. 3.3. Experiment 3: effect of an intraperitoneal injection of MDP on the BSS The temporal profiles of the four major categories of behavior (eating, grooming, activity and resting) involved in the development of satiation are plotted in Fig. 4. The behavioral parameters during the first and third hour test sessions are summarized in Table 3. 3.3.1. Baseline During the first hour of video recording, the four behaviors occurred in a well-defined sequence: eating behavior followed by grooming and/or activity behaviors superseded by resting (see Fig. 4a). Most eating behavior occurred
179
Table 3 Food intake (g) (1 h) and behavioral parameters of rats measured during the first hour of meal presentation (H1) and at 3 h after meal presentation (H3) during the baseline situation and after an intraperitoneal injection of MDP (1.5 mg/kg b.wt.) or saline
Eating Grooming Resting Activity 1 h food intake (g)
Baseline
MDP
Saline
H1 H3 H1 H3 H1 H3 H1 H3 H1
14.0 ± 0.7 6.5 ± 1.3 8.2 ± 3.2 13.1 ± 1.6 47.3 ± 7.4 54.2 ± 8.9 28.9 ± 5.2 25.9 ± 7.8 6.1 ± 0.5
9.6 ± 2 4.1 ± 0.9 7.0 ± 2.9 4.7 ± 1.5 * 54.1 ± 4.2 81.7 ± 3.0 * 27.9 ± 5.0 9.4 ± 1.7 6.3 ± 1.0
10.3 ± 0.8 6.6 ± 0.7 9.8 ± 3.3 13.5 ± 2.7 52.1 ± 4.2 58.4 ± 8.9 27.0 ± 2.3 21.3 ± 6.4 5.5 ± 1.2
H3
3.1 ± 0.4
2.2 ± 0.6
3.3 ± 0.3
These parameters are presented as the percentage of time spent on the different monitored behaviors. Each value is presented as the mean ± S.E.M. (n = 6). * MDP, saline and baseline values are differed ( P < .05).
during the first 15 min following presentation of the P14 diet. Resting predominated during the last 40 min and its duration was the longest in the BSS. During the third hour of video recording, the satiety sequence was shifted to the left. Feeding was terminated earlier, grooming occurred earlier, appearance of peak activity was shifted to the left and time course of resting was reduced from 20 to 10 min (Fig. 4b). A comparison of the first and third hours of video recording revealed a reduction of eating over time. The other compartments remained unchanged. 3.3.2. Saline There was no difference in behavioral profiles and parameters after the saline and the baseline treatments for the H1 and H3 (Table 3 and Fig. 4c) periods. 3.3.3. MDP During the first hour of video recording, there was no difference in behavioral profiles and parameters between baseline, saline and MDP treatments (Table 3) nor was the BSS any more disturbed during the third hour of video recording (Fig. 4d). The injection of MDP increased resting behavior by comparison with the baseline and saline treatments [ F(2,15) = 5.73, P < .05] (Fig. 4d and Table 3). In rats treated with MDP, resting was mainly preponderant while grooming was significantly decreased [ F(2,15) = 8.21, P < .01]. However, because of considerable variabilities within the group, the reduction in activity was not statistically significant. Eating behavior was not affected by treatment with MDP.
4. General discussion These results demonstrate that MDP administered orally could be identified in the blood but was not effective as an
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appetite suppressant. In contrast, after an intraperitoneal injection of MDP, a depression in food intake and a reduction in meal frequency were observed. This suppression of appetite does not seem to have resulted from mechanisms such as satiation or conditioned taste aversion but may be representative of an increase in satiety along with the sickness behavior, which can be induced by bacterial infection, such as a reduction in grooming and an increase in resting. During our experiments, a highly sensitive and selective RP-HPLC method coupled to MS detection was used to quantify MDP in plasma samples. Our findings indicate that MDP appeared in the systemic circulation after gavage, suggesting a partial resistance during the intestinal digestion and absorption processes. The intestinal absorption pathway for this peptide remains unclear. Specific transport systems, such as the di- and tripeptide transport systems, may be involved [28,29]. The MDP plasma peak was obtained at 60 min with plasma levels representing less than 0.05% of the ingested dose. However, MDP was also rapidly eliminated from the systemic circulation. At 120 min, MDP was detected but not quantified according to the current limit of sensitivity for the analytical method. These low levels of MDP found in the systemic circulation after oral administration could be explained by a marked retention in the stomach and intestinal tract, as is observed with the peptidoglycan monomer [30]. MDP may be also degraded by various enzymes and principally by a mammalian enzyme (N-acetyl-muramyl-L-alanine amidase) found in biological fluids such as serum, which inactivates muramyl peptides by hydrolyzing the lactylamide bond between N-acetylmuramic acid and the L-alanine residue [31 –33]. In relation to these plasma kinetics, MDP after oral administration cannot attain sufficient peripheral or central tissue levels to achieve an anorectic effect [34]. In contrast, following intravenous injection, MDP was readily detected in its intact form and at high levels in the circulation. Plasma levels of MDP rapidly decreased to less than 35% of the injected dose at 2 min. Our findings are consistent with previous reports by Ambler and Hudson [35] who studied the metabolic fate of an intravenous injection of 3 H-MDP in mice. They observed that MDP was rapidly eliminated from the circulation and excreted in its intact form in the urine, which is consistent with its water solubility [35,36]. However, the plasma levels seemed to be sufficient to reduce food intake by decreasing the number of meals without affecting their size. This finding is consistent with the results obtained by Langhans et al. [37,38] who studied similar doses and time courses after the intraperitoneal injection of MDP. The reduction in food intake was not immediate and could be related to an indirect effect induced by cytokine production [9,14,15,39 – 43]. The effect of an MDP injection on food intake would be more relevant to satiety and/or sickness behavior than to conditioned taste aversion. Analysis of the spontaneous meal pattern and the BSS have made it possible to clarify
whether experimental handling affects ingestion by inducing satiety or malaise [20,27,44 –49]. Our results showed that the depression in food intake induced by the injection of MDP resulted from a reduction in meal numbers, although no decreases in meal size, meal duration or feeding rate were observed. These results were also confirmed by microstructural analysis. The effect should not be interpreted as satiation because, in such a case, we would have observed a reduction in meal duration without any effect on feeding rate [46,50,51]. In contrast, the reduction of meal frequency, together with a constant feeding rate, could be interpreted as a sign of enhanced satiety. However, analysis of the BSS during the third hour of presentation makes this interpretation more puzzling. During the first hour of food intake, whatever the experimental day, the four principal behaviors always occurred in a welldefined sequence: eating followed by grooming and/or activity followed by resting. During the baseline and saline situations, video analysis of the third hour of the feeding period showed that the four principal behaviors always occurred in the same, well-defined sequence, with a reduction in the eating period and a shift to the left for all other behavioral profiles. This preserved BSS could be interpreted in favor of a reduction in food intake by a postingestive mechanism of satiety [46,20,27]. In contrast, after the intraperitoneal injection of MDP, video analysis of the third hour showed that resting was dramatically increased while the grooming period was reduced. At this dose of MDP, this profile could be related to the behavioral modifications observed during bacterial infection, i.e., a reduction in grooming and locomotor activity and increased sleepiness [11]. The latter may also be associated with other biological effects of MDP, which include the induction of drowsiness [4]. A characteristic feature of such a behavior might be suggested by the results of the microstructural analysis, which showed that rats visited the food cup less frequently but stayed longer, as demonstrated by the increase in eating bout duration. In addition to the nature of food intake depression, MDP administration did not act as an unconditioned stimulus for the conditioned aversion which might arise during bacterial infection. The lack of difference in meal size and feeding rate showed that the intraperitoneal injection of MDP did not act like lithium chloride (LiCl), a known aversive toxic [45,52,53]. Analysis of the BSS test reinforced this hypothesis, because it was preserved. A conditioned taste aversion induces a loss of the BSS associated with an increase in activity [54]. Moreover, Blundell et al. [46] showed that a cessation of eating was followed by resting before grooming appeared, which is in favor of an abnormal inhibition of eating, associated with the malaise caused by intestinal discomfort. In conclusion, the food intake depression caused by an intraperitoneal injection was not due to a conditioned taste aversion mechanism but probably an increase in satiety
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along with sickness behavior. After oral administration, MDP was poorly absorbed and did not act on food intake. We suggest that under normal conditions the release of MDP from gut flora or food bacteria is not able to exert any hypophagic effect. However, other peptidoglycan fragments may also be released from bacterial hydrolysis and should be investigated in the future with respect to their anorectic activity.
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Acknowledgements
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The assistance provided by Nathalie Jerome is gratefully acknowledged.
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