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Unité INRA 914 de Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique Paris-Grignon, F75231 Paris Cedex 05, France and * Unité INRA AMIB, Institut National de la Recherche Agronomique, Centre de Recherche de Jouy-en-Josas, F-78352 Jouy-en-Josas, France
1To whom correspondence should be addressed. E-mail: gilles.fromentin{at}inapg.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • meal pattern • behavior satiety sequence • satiety • vagotomy • vagus nerve
Transition from a normal to a high protein diet induces a rapid depression in food intake on d 1 and a gradual but incomplete return to initial intake over subsequent days (1–5). This reduction in high protein diet intake may originate from nonvagal orosensory neuro-mediated signals, from vagal-mediated signals triggered by the presence of protein and amino acids in visceral tissues (6,7) and/or from the central detection of variations in plasma free amino acid or related metabolite concentrations. For the vagus-mediated mechanisms, an afferent signal may be generated by amino acids and protein while still in the digestive tract, and/or by an increase in the metabolic rate triggered by a greater amino acid flux into the liver (8–11). The aim of this study was to evaluate the relative involvement of these vagus-mediated pathways in the depression in food intake produced by a high protein diet. For this purpose, we measured the consequences of total subdiaphragmatic vagotomy on body weight gain, daily food intake, meal pattern and the behavioral satiety sequence (BSS) in rats fed a high protein diet.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Wistar rats (n = 21) (DEPRE, Saint Doulchard, France) weighing 290–310 g were housed in individual cylindrical cages in a room controlled for temperature (23 ± 1°C) under a 12-h light:dark cycle (lights off: 1700–0500 h). All experimental procedures used complied with the guidelines of the French National Animal Care Committee. Two diets were used. The control diet (P14) was an AIN-93M modified diet (5,12); instead of casein and cystine, this diet contained 140 g total milk protein/kg diet. The high protein diet (P50) was also an AIN-93M modified diet, containing 530 g total milk protein/kg diet. Total milk protein is a mixture of casein (85%) and other milk proteins (albumins and globulins). In the P50 diet, protein was added at the expense of equivalent amounts of sucrose and starch. The P14 and P50 diets were moistened (powdered diet to water; 1:1 and 1:2, respectively) to minimize spillage. Food containers were refilled daily with fresh food at 1700 h. Rats consumed water and food ad libitum throughout the experiments. Food intake was determined by the difference in the weight of the food cup before and after each experimental period, corrected for spillage and evaporation.
Surgical procedure.
Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (0.06 g/kg body) (Sanofi Santé Nutrition Animale, Libourne, France); they were separated into two experimental groups and underwent either a sham vagotomy (Sham group; n = 9) or a total subdiaphragmatic vagotomy (Vago group; n = 12). From the start of the surgery to reawakening, the body temperature was maintained with a heated pad. During total subdiaphragmatic vagotomy, connective tissue between the liver, stomach and esophagus was carefully removed and a 0.5-cm segment of the vagal dorsal and ventral subdiaphragmatic branches (including the hepatic, celiac, accessory celiac and ventral and dorsal gastric branches) were isolated from the esophagus and excised by cauterization. Surgery in the Sham group followed precisely the same procedure as in the vagotomized group, but the vagus nerve was left intact. After the laparotomy had been stitched and disinfected with Betadine, 0.05 mL of Gentaline (Schering-Plough) was injected intramuscularly to verify the success of the vagotomies (11); the gastric contents were removed after the rat had free access to a standard high fiber diet for 16 h (Harlan, Teklad Global 14%, N°2014), followed by 4 h of food deprivation. The contents of each stomach were freeze-dried, weighed and compared with the total amount ingested. The relative dry gastric contents were calculated for each rat as the weight of freeze-dried gastric content (g) divided by the total food intake. Vagotomy was considered to be total when the relative dry gastric contents of a vagotomized rat were at least 10 times greater than the mean relative dry gastric contents in sham rats.
Meal pattern analysis.
After surgery, rats consumed the P14 diet for 2 wk to acclimate them to the diet and experimental conditions. After 3 more days of P14 diet intake, both groups consumed the P50 diet for 16 d. Food intake was recorded by means of food cups placed on a strain-gauge (Entran SA, Les Clayes-sous-Bois, France, accuracy of 0.1 g) connected to a 34970A data acquisition/switch unit (Agilent Technology, Les Ulis, France) and to a personal computer programmed to record data every second via HP BenchLink Data Logger software (Agilent Technology, Les Ulis, France). The cumulative food intake was recorded every second during the last day of the P14 diet and during d 2 and 14 of the P50 diet. To be distinct, two meals had to be separated by an intermeal interval (IMI) 
10 min (13,14). Based on this criteria system, several parameters characterizing feeding patterns were obtained for each rat, such as the size (kJ) and duration (min) of meals, as well as the ingestion rate (kJ/min), calculated as the ratio of meal size to its duration, and meal frequency. The mean duration of all IMI and total daily food intake were also calculated. The quantity of ingested dry matter (g) was calculated as metabolizable energy ingested (kJ), using the conversion factor (14.6 kJ/g). All of the feeding pattern parameters listed above were studied during the first 16 h of presentation of a diet (i.e., 1700–0900 h). However, such an overall analysis of feeding patterns was of no value in identifying any discrete changes in feeding behavior. Consequently, we divided the entire period into hours, and studied the meal parameters, meal size and duration, ingestion rate and meal frequency for each hourly period. When a rat did not eat anything during an entire 1-h period, the data concerning this rat were taken into account only for the number of meals (expressed as n = 0).
Video-analysis (BSS).
Between 1400 and 1800 h, the rats were familiarized with their test chambers for 1 d before testing (d 15 of experimental period). The experimental chamber consisted of a circular Plexiglas tank (height: 320 mm; diameter: 300 mm), with a ring attached to the inside wall to hold the food cup, with a water bottle on the opposite side. The video cameras (JVC, Digital Still Camera GR-DVL557, Darty Fresnes 94 France) were placed outside the chamber, 
300 mm from the food cup ring. The video signal was recorded on conventional VHS tape (VHS-C, EC-60) at 50 frames/s using a recorder (Compact Super VHS, JVC: GR-SXM307, Darty Fresnes 94 France). Animal behavior was video-recorded during h 1 of food presentation. Videotapes were analyzed by a slow-motion playback (frame by frame to 1/6 of the normal playing speed) to count behavioral satiety or taste reactivity components. Food intake was measured at 1700 h and then at 0900 h the next morning. For analysis, the behavior of each rat was categorized as follows (5,15): 1) Activity: This included locomotion (e.g., walking and running), rearing (defined as rats standing up on its two hind limbs and jumping in a manner directed at escape from the experimental chamber, and sniffing. 2) Grooming: Scratching, licking or biting of coat. 3) Resting: Inactivity, standing, sitting or lying, sleeping with occasional changes of position. 4) Eating: Biting, gnawing, swallowing food.
Statistical analysis.
Results are expressed as means ± SEM. In most cases, the significance of differences between response variables was determined using one-way ANOVA. When appropriate, data were analyzed using ANOVA for repeated measures [PROC GLM: Statistical Analysis System (SAS) version 6.11, Cary, NC]. When the results of the ANOVA were significant, Tukey’s Honestly Significant Difference post-hoc test was used to compare between- or within-group means. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The relative dry gastric content of each vagotomized rat was more than ten times greater than the mean relative dry gastric contents of sham-operated rats (Sham: 3.5 ± 0.6 g and Vago: 0.1 ± 0.1 g, P < 0.05). Four vagotomized rats were not used because the vagotomy was incomplete.
Daily energy intake and body weight.
During the last 3 d of the prefeeding period (until d 0), the groups did not differ in daily intake of the P14 diet (Fig. 1) and body weight (d 0: Sham: 351 ± 4 and Vago: 352 ± 8 g). Transition from the P14 to the P50 diet induced an immediate depression in intake on d 1 and a gradual adaptation to the diet over subsequent days. Moreover, as early as d 1 of consuming the P50 diet, vagotomized rats ate less than sham rats (P < 0.05). During the 16 subsequent days, energy intake by the Vago group remained lower than that of the Sham group (P < 0.005), with daily energy intakes of 334.2 ± 10.9 and 388.8 ± 10.9 kJ, respectively (P < 0.005). At the end of the experiment, the body weights were 440 ± 10 and 452 ± 7 g in the Vago and the Sham groups, respectively.
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During the last day of consuming the P14 diet, the Vago and Sham groups did not differ in any total food intake meal parameter (Table 1). During the transition phase (P50 diet, d 2), meal size of the Vago group was less than that on the last day of consuming the P14 diet. After adaptation (P50 diet, d 14), meal size and rate of ingestion in both groups were not lower than those on the last day of P14. Other parameters (number of meals, intermeal intervals and meal duration) were not affected by the P50 diet. Because within- or between-group differences occurred only during h 1 of food availability, the results of h 1 meal parameters also are presented. Rats in the Vago group had a smaller meal size and tended to have a greater number of meals (P = 0.09) than those in the Sham group on all days tested. During the transition day (P50 diet, d 2), this was due mainly to a decrease in the rate of ingestion. After adaptation (d 14), it was mainly the consequence of reduced meal duration.
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After adaptation to the P50 diet, the four behaviors appeared in the Sham group in a well-defined sequence (Fig. 2). Most eating behavior occurred during the first 10 min after the P50 diet presentation. Resting became predominant after 15 min and its duration was the longest of the various parameters of the behavioral satiety sequence. In the Vago group, the profile during the first 10 min was slightly disturbed by a reduction in eating and an increase in activity.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Total subdiaphragmatic vagotomy neither modified the daily food intake of the control diet (18) nor suppressed the depression in food intake produced by transition to a high protein diet. These results indicated that nonvagus-mediated signals were involved in the central detection of a high protein diet by vagotomized rats. The principal mechanism could be a direct central recording of plasma components, including either free amino acid or amino acid catabolism–related components (6,19). These results did not exclude an orosensory nonvagus neuro-mediated poor palatability signal or a role for the sympathetic nervous system in monitoring peripheral status and nutrient information originating from the liver and gut.
Rats with total subdiaphragmatic vagotomization encountered some difficulties in eating the high protein diet because during the transition phase and even after adaptation, they ate significantly less than sham rats. The role of the vagus nerve in the peripheral regulation of stomach and gut kinetics to control the delivery of nutrients to the body from intraluminal nutrients has been documented. Indeed, a peripheral vago-vagal loop involving protein and amino acids (as well as the detection of other nutrients still in the intestinal lumen) is likely to regulate gastric kinetics (20). A defect in this vagus-mediated gastric control could be a reason why rats found it difficult to regulate and slow down the rates of amino acid release and absorption, thus reenforcing peripheral metabolic signal(s) and causing a depression of food intake triggered by a high protein intake.
The liver plays a central role in postprandial dietary amino acid metabolism because it modulates the inflow of amino acids from the intestine and controls amino acid supplies to peripheral tissues. However, although the modulatory role of liver anabolic and catabolic pathways is well established (4), less is known about the exact role of the hepatic vagus branch in dealing with an excess of dietary amino acids. Even after adaptation, vagotomized rats ate less of the high protein diet than control rats, and it is possible that vagotomy of the afferent branches influenced the hepatic processes involved in metabolic adaptation to high protein diets. Moreover, it has been shown that hepatic afferents alone apparently act during the adaptation to an amino acid–imbalanced diet (11).
In conclusion, the results of this study agreed with the idea that different and redundant vagus and nonvagus-mediated mechanisms are involved in the depression of food intake caused by a high protein diet in rats. As a consequence, the subdiaphragmatic vagus nerve is probably not an obligatory pathway for the transfer of information to the brain when a high protein diet is provided. In contrast, the results also indicated that a defect in the visceral and splanchnic regulating systems may reinforce other nonvagus metabolic food intake depression signals. This effect would limit the ability to regulate protein and energy intake both in the short term and after adaptation. It does not seem to generate major metabolic distress, however, because after the rats had eaten the high protein diet for 14 d, there was no difference in meal pattern parameters when either sham or vagotomized rat consumed normal or high protein diets.
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FOOTNOTES |
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Manuscript received 11 March 2003. Initial review completed 6 April 2003. Revision accepted 19 May 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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