Physiology &Behavior,Vol. 21, pp. 957-965. PergamonPress and BrainResearch Publ., 1978. Printedin the U.S.A.
Long Term Inhibition of Intake by Mannitol B A R B A R A J. C O L L I N S A N D J O H N D. D A V I S
Department of Psychology, University of Illinois at Chicago Circle Chicago, IL 60680 ( R e c e i v e d 8 M a y 1978) COLLINS, B. J. AND J. D. DAVIS. Long term inhibition of intake by mannitol. PHYSIOL. BEHAV. 21(6) 957-%5, 1978.--The addition of mannitol, a slightly sweet carbohydrate which retards absorption, to a palatable glucose-saccharin solution results in the retention of fluid in the intestine for up to 4 hr after intake of the fluid in a 15 min drinking bout. When given access for 4 hr to glucose-saccharin solutions to which mannitol is added, intake is reduced in proportion to the concentration of mannitol (0, 0.05, 0.1, 0.2 M) present. Since rats with access to a 0.2 M mannitol solution consume most of their total intakes in the first 15 or 30 min exposure, and this is sufficient to cause prolonged retention of fluid in the intestine, it was hypothesized that initial exposure to mannitol would suppress intake of a glucose saccharin solution subsequently offered. Intake of a glucose-saccharin solution was diminished for only 1.5 hr when rats were switched from a mannitol-glucose-saccharin solution, indicating that the effect of mannitol was mitigated but not abolished. The suppression of intake by mannitol does not appear to involve a conditioned taste aversion, but may activate a mechanism which is normally involved in limitation of meal size. Fluid intake
IT IS well established that daily food intake in the rat consists of a number of discrete meals in each 24 hr period, separated by intervals in which eating does not occur [7, 8, 11, 12]. The physiological determinants of meal size and meal frequency have been sought by many investigators with the hope that such knowledge will result in a better understanding of the mechanism by which food intake is controlled. The present paper offers further evidence that an intestinal factor is involved in the termination of a meal and in the maintenance of post-meal satiety. Davis, Collins and Levine  have investigated the possibility that gastrointestinal filling is a factor in inducing satiety in rats. The experiments described in that paper involved the addition of mannitol, a slightly sweet carbohydrate which retards absorption, to a palatable glucosesaccharin solution, and studied the effects of various concentrations of mannitol on intake and absorption of the solutions. They reported that the addition of mannitol to a solution containing 0.1 M glucose and 0.006 M Na saccharin reduced 30 min intake in proportion to the amount of mannitol present (0-0.5 M). They also reported that the amount of fluid which was retained in the gastrointestinal tract as a whole was directly proportional to the concentration of mannitol ingested in a 14 min test period. At concentrations above 0.072 M mannitol (by calculation from least squares fit) fluid was actually drawn into the intestine. Retention of fluid in the stomach was not apparent, nor did the rate of gastric clearance appear to be affected. These results suggested that distention of the small or possibly large intestine was an important factor in limiting meal size. The present experiments were designed to explore this
hypothesis in more detail, investigating whether mannitolinduced satiety showed characteristics similar to the rat's normal eating patterns and whether the disposition of fluid in the gut was correlated with the intake of the rat over a longer period of time than 14 rain, as previously observed.
Animals In all of the experiments reported the animals were male albino rats bred in our own colonies from Charles River stock. Animals were maintained on a 12:12 LD cycle with lights on at 0600 and off at 1800 hr. Since many of the experiments were terminal, rats were often used which had been in other experiments but whose experience would not interfere with the determinations made in the present experiments. Except where deprivation conditions are stated, rats were given ad lib Wayne Lab-BIox small animal chow and tap water. EXPERIMENT 1 This experiment observed the change in weight of gut contents at 0, 30, 60, 120, and 240 min after 15 min of ingestion of either a 0.1 M glucose + 0.006 M Na saccharin (G-S) solution or this same solution to which 0.2 M mannitol (M-G-S) was added.
Method One hundred five male albino rats weighing an average of 497 g were used throughout this experiment. The rats had been adapted for several weeks to drinking a solution of 0.1 M glucose + 0.006 M Na saccharin (G-S) on their home cage.
1This work was supported in part by NSF grant BMS 75-17091. Portions of this paper were presented at the 6th International Conference on the Physiology of Food and Fluid Intake, July, 1977. Paris-Jouy en Josas (France).
C o p y r i g h t © 1978 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/78/120957-09502.00/0
C()LLINS AND DAVIS
On the day of testing beginning at approximately 900 the animals were deprived of food. one animal every 20 rain in order that sufficient time intervene for subsequent sacrifice and weighing of tissue. No more than 8 animals were tested and sacrificed on any single day. After 4 hr of food deprivation the water bottle was removed from the home cage and replaced by a calibrated drinking tube with a stainless steel spout, filled with a solution of either 0.1 M glucose + 0.006 M Na saccharin (G-S) or this same solution + 0.2 M mannitol (M-G-S). The rat was given access to this solution for 15 rain at which time the tube was removed. Animals were sacrificed by etherization at either 0, 30, 60, 120, or 240 rain after the 15 rain drinking period. There were 7 animals for each condition of time and solution. As soon as the animal was judged to be dead it was quickly removed from the ether jar and placed on a dissecting table. A transverse cut was made in the abdominal cavity with scissors followed by a midline cut so that the entire abdominal viscera were easily accessible. Clamps were quickly placed at the junction of the esophagus and stomach, two at the pylorus and two at the ileo-caecal junction. If any of the sections to be removed were distended with fluid (this was particularly true of the stomach at the early time points) sutures were tied just inside the clamps in order to prevent escape of fluid when the clamps were removed. The stomach was removed first by cutting above the esophageal clamp and between the two pyloric clamps, holding the stomach by the two clamps at either end and with a small scissors freeing it from adhering tissue, being careful to trim away fat. The excised stomach was placed on pre-weighed weighing paper. The small intestine was removed by grasping the duodenal end by its pyloric clamp and gently pulling it free from adhering mesenteries, using a blunt forceps if necessary. Preweighed weighing paper received the intestine as it was pulled free of the abdominal cavity. The caecum-colon was removed by grasping the clamps at the caecal end, lifting the organ and with scissors freeing the caecum and colon from the omentum, which is more firmly attached here than to the small intestine. Care must be taken not to incise the caecum or colon. After cutting away the omentum the colon was freed from the body by cutting as close to the rectum as possible. Usually there were no loose stools in the colon so that clamping of this end of the colon was not necessary. Each of the 3 sections was weighed on a double pan balance to within 0.01 g and then transferred to a drying oven. Gut sections and their contents were dried at 100°C for 24 hr after which time they were reweighed. The difference between the wet weight and dry weight was calculated and represented the water present in that compartment. Seven no-drink controls for each time point were run. These animals were not given access to the drinking solution but were sacrificed at the specified times. The amount of fluid recovered (wet weight minus dry weight) from the gut compartments of these no-drink controls represented the cellular water and residual gut fluid which presumably would be present in any of the animals. A figure arrived at by averaging the results from the seven no-drink animals at each time point was subtracted from the appropriate group mean for each of the drinking groups. This corrected for tissue water and residual fluid in all animals, giving a final result which more accurately represents percent of ingested fluid. Results and Discussion
Figure 1 shows the distribution of fluid in the three gut
4(7 ~ ,
\~ 20 - ~ %
: GLUCOSE-SACCHARIN GLUCOSE-SACCHARIN +MANNITOL
I'~L=~-- ~ . . . . . . . . . . . O u.I Io9 uJ O _z p. Z
<~ u. O
Z uJ n,. uJ o.
8C7C 6C- jt
SMALL I N T E S T I N E
TIME ( m i n )
FIG. 1. Recovery of fluid in gut compartments over 4 hr after drinking either glucose-saccharin or mannitol-glucose, saccharin SolUtions for 15 rain. Each point represents data from 7 rats. (Exp. 1)
compartments at each time point (which represents time measured from the end of the 15 rain drinking period) for the two G-S and M-G-S groups of rats. The data are presented as percent of amount ingested, representing a ratio of amount of fluid recovered to amount ingested, with no-drink corrections made as denoted above. The raw data from which the graph in Fig. 1 was made are presented in Table 1. There is no correction made for residual water in Table 1. However. the average values for the no-drink condition are given. These combine the values from all 5 time points, since there was no significant difference between them. The reader should recognize that this residual water representing tissue water and residual fluid in the gut has not been subtracted. Therefore, for example, in Group G-S at time 0, there is virtually no ingested fluid in the large intestine (10.10_+0,67 ml) since 9.60_+0.28 ml can be accounted for as residual fluid. As expected at 0 time, much of the fluid recovered is in the stomach for both the G-S and M-G-S groups. Fluid clears rapidly from the stomach and intestine of the G-S groups; 60 min there is scarcely any fluid left in any of the gut compartments of this group. Clearance from the stomach by the M-G-S group, though somewhat slower initially than the G-S group, is no different from that group after 30 min. However. considering that the rats in the 30 min G-S group drank 25_+2 ml of fluid and those in the M-G-S group drank only 13-+3 ml,
M A N N I T O L A N D I N H I B I T I O N OF I N T A K E
TABLE 1 AMOUNTOF FLUID RECOVEREDIN EXPERIMENT I No Drink Condition (n=35) 2.49 ___ .11 ml Stomach 9.35 +- .26 Small Intestine Large Intestine 9.60 --- .28 Time Compartment (min)
Group G-S Amount Amount Drunk Recovered ml ml
11.71 +_ 1.05 14.35 +_ 0.90 10.10 -+ 0.67 4.34 _+ 0.21 ! 1.87 +- 0.66 11.52 _+ 0.44 22---2
18-+3 Stomach Small Intestine Large Intestine
0.1618 4.0552 0.8823
n.s. <.01 n.s.
5.40 +- 1.70 19.00 _+ 1.74 10.92 ___0.52
0.6122 n.s. 5.0310 <.001 0.8759 n.s.
3.22 - 0.46 16.92 +- 1.64 12.62 -+ 0.82
0.1098 n.s. 4.2234 <.002 2.5262 <.05
3.38 -+ 0.58 15.31 +- 1.67 15.51 _+ 0.90
0.5681 n.s. 3.7592 <.01 5.6801 <.001
2.50 ___0.16 10.08 _+ 0.28 15.11 _+ 1.39
2.0878 n.s. 5.4988 <.001 4.3892 <.002
18-+2 2.93 +- 0.54 8.97 +- 0.25 8.58 +- 0.82
23-+ 1 Stomach Small Intestine Large Intestine
11.43 -+ 1.21 19.29 -+ 0.82 9.34 +- 0.54
14-+2 3.29 +- 0.52 9.83 _-. 0.34 10.12 +- 0.55
Stomach Small Intestine Large Intestine
25-+2 Stomach Small Intestine Large Intestine
22---2 Stomach Small Intestine Large Intestine 30
Group M-G-S Amount Amount Drunk Recovered ml ml
20_+ 2 2.05 -+ 0.15 7.63 +- 0.34 8.64 -+ 0.50
*d.f.= 12 for all tests.
the fact that there is no difference in the amount recovered at 30 rain necessitates that clearance in the G-S group be much more rapid in order to have cleared twice as much fluid in this time. There was already significantly more fluid in the small intestine of the M-G-S group at 0 time, which increased still more at 30 min after which clearance proceeded gradually. The clearance of fluid from the small intestine in the M-G-S group is mirrored in the large intestine which initially was empty but began to accumulate fluid after 30 min, the point at which the small intestine was losing fluid. This fluid retention is even more striking when one considers that the average amount of fluid ingested is greater in the G-S group (22.0---0.9 ml) than in the M-G-S group (16.5+-1.1 ml). After 60 min the curve of the large intestine is essentially flat at about the 25% mark suggesting that the fluid being received from the small intestine was in equilibrium with the fluid being absorbed from the large intestine. The course of gut contents was not followed beyond the 240 min time point. However, it was observed that in only 2 of the M-G-S rats were there any watery stools in the colon, but almost all of the M-G-S rats, particularly at the later time points, did show extreme distention of the ileum and caecum. Note that for the G-S group the maximum total recovery of fluid was about 60% at the zero time point, whereas in the
M-G-S group approximately 100% recovery was found up to 60 min post ingestion. This indicates that for the G-S group some of the glucose-saccharin solution was being absorbed from the gut as it was being drunk. It has been shown previously  that glucose solutions are rapidly absorbed. However, the addition of mannitol greatly retards absorption and this effect is seen primarily in the small and large intestine. The results of this experiment show that the addition of mannitol to a palatable glucose-saccharin solution retards absorption of the ingested fluid from both the small and large intestines for up to 4 hr after ingestion. Stomach clearance is not markedly changed by the addition of mannitol. The addition of mannitol provides an experimental method of gut distention which can be observed for up to 4 hr after ingestion. EXPERIMENT 2 In the first experiment, rats had access to either the G-S or M-G-S solution for only 15 min. However, the consumption during this short exposure produced large differences in gut fluid absorption and thereby distended the intestine. Experiment 2 investigates whether these differences in intestinal absorption will produce a change in intake over a longer period of time.
COI,LINS AND I)AVIS
0 M MANN 70 M MANN
tinues virtually unabated for the duration of the test period while the addition of mannitol suppresses drinking suggests that this fluid retention after drinking mannitol may activate some gut signal which limits meal size and whose duration may determine the post prandial intermeal interval. Whether such a signal is from distention of the intestinal wall or is an osmotic effect or some other consequence is not known. However, it is clear that when mannitol is present in the glucose-saccharin solution, suppression of intake is seen as early as the second 15 rain interval. Since there is virtually no drinking after the first 30 min of exposure, except in the case of the 0.05 M mannitol solution, it appears thai the physiological action of mannitol affects the animal within 30 rain after beginning to drink. Whatever this action, it is effective in inhibiting further intake of the mannitol solution.
.1 M M A N N
.2 M M A N N
FIG. 2. Four-hour cumulative intakes of solutions of mannitol in varying concentrations (0, 0.05, 0.1, 0.2 M) + glucose-saccharin. N=6 for each group. (Exp. 2)
As shown in Fig. 2, rats with continuous access to the M-G-S solution (0.2 M mannitol + 0.1 M glucose + 0.006 M Na saccharin) do almost all of their drinking in the first 30 min and drink virtually nothing for the rest of the 4 hr test period. Since only a 15 min drinking period of this M-G-S solution is also sufficient to cause a prolonged retention of fluid in the intestine, as seen in Experiment 1, it would be expected that initial ingestion of the M-G-S solution should suffice to suppress further intake of any solution. The present experiment investigated whether a short (30 min) period of exposure to mannitol could affect intake of a glucosesaccharin solution without mannitol offered subsequently. Method
Four groups of 6 male albino rats were used. They had been adapted to drinking the 0.1 M Glucose + 0.006 M Na saccharin (G-S) solution on their home cages. On the day of testing they were deprived of food for 4 hr after which time the water bottles were removed from their cages and replaced by drinking tubes fitted with stainless steel spouts. Intake was measured at 15 rain intervals for the next 4 hr. Test solutions were the G-S solution to which mannitol in the concentrations 0, 0.05, 0.1 and 0.2 M were added. Results and Discussion
The 4 hr cumulative intakes of each of the 4 groups are presented in Fig. 2. It can be seen that as mannitol concentration is increased, intake is suppressed. Both the magnitude and duration of the suppression increases with increasing mannitol concentration. A two-way A N O V A reveals a significant time effect, F(3,80)=56.67, p<0.01, and a significant group effect, F(3,80)=9.72, p<0.01. The suppression of intake of the 0.2 M mannitol solution is effective throughout the entire 4 hr period. As demonstrated in Experiment 1 (Fig. 1) at this time point, the large intestine contains significantly more fluid than after G-S alone, and the small intestinal fluid is still slightly elevated above the level for G-S alone. Although data are not available for gut fluid retention after the ingestion of solutions containing 0,05 M or 0.1 M mannitol, it is assumed that they would demonstrate a pattern that is intermediate to the two extremes since Davis et al.  have shown a linear relation between mannitol concentration of the drinking fluid and gut fluid retention in rats sacrificed immediately after drinking. The fact that in the absence of mannitol, drinking con-
Male albino rats were adapted for one week or more to the 0.1 M glucose + 0.006 M Na saccharin (G-S) solution on their home cages. On the day of testing they were deprived of food for 4 hr prior to testing. At the end of the deprivation period the water bottles were removed from their home cages and replaced by drinking tubes containing either the G-S or M-G-S solution. The first of three groups received the G-S solution throughout the entire 4 hr test period (Group G - , G , n= 18). The second group received the M-G-S solution for the first 30 min and was then switched to the G-S solution (Group M--,G, n=18). The third group ( M ~ M , n--18) received the M-G-S solution throughout the 4 hr test period. Readings of intakes were taken every 15 rain. Results and Discussion
The results are displayed in Fig. 3, a graph of the cumula, tive intakes for the 4 hr test period. There is no significant difference between the three groups in the first 15 rain of drinking, F(2,51)= 1.97, n.s. However, in the second 15 rain period the intakes of the rats on the G-S solution were sig, nificantly higher, F(2,51)=9.25, p<0.001 than those in Groups M---,G and M--~M which were drinking the M-G-S solution. At the end of this first 30 min the M--*G group was switched to the G-S solution. This group showed a modest but insignificant increase in drinking when first presented with the G-S solution. However, gradually increasingintakes of this solution produced a significant, F(i,34)=8.55, p<0.01, difference by the 2 hr 15 rain time point. (Although a difference yielding F(1,34)=5.30, p<0.05, was found as early as the 1 hr 30 rain time point, because of the number of tests a more stringent criterion of 0.01 was set for significance.) Intake of Group M--~M remained suppressed throughout the
M A N N I T O L AND INHIBITION OF I N T A K E
5C IZ t~ >
TIME (hr) FIG.
3. F o u r h o u r c u m u l a t i v e
intakes of t h r e e g r o u p s o f rats: G r o u p
G--~G, glucose-saccharin for entire test period; Group M--*G,0.2 M mannitol + glucose-saccharin for 30 rain, then switched to glucosesaccharin alone; Group M---~M,0.2 M mannitol + glucose-saccharin through the 4-hr test period. N= 18 for each group. (Exp. 3) TABLE 2 INCREMENTALINTAKES(ML) IN EXPERIMENT 3 Hr 0
Time Min 15 30 45 0 15 30 45 0 15 30 45 0 15 30 45 0
13.78 _ 1.47 8.17 - 1.60 3.94 _+ 1.04 3.22 - 1.01 3.17 - 1.05 2.61 +- 0.96 0.78 _ 0.41 1.72 _+ 0.97 0.72 _+ 0.43 1.44 _+ 0.93 3.11 -+ 1.12 2.56 -+ 1.00 3.50 +- 1.07 2.50 _ 1.09 1.72 _+ 0.90 3.67 +_ 1.41
10.94 _ 1.16 0.78 _ 0.32 4.67 _ 1.49 0.22 -+ 0.17 1.11 _+ 0.69 1.72 - 0.85 1.22 _ 0.51 1.33 _ 0.69 1.00 _ 0.52 1.50 ___1.12 0.39 +- 0.39 0.61 +- 0.40 4.06 _+ 1.34 1.94 + 0.87 3.67 _ 1.03 3.56 _+ 0.86
10.22_ 1.37 2.78 -+ 1.10 0.89 _ 0.42 0.28 +--0.18 0.17 +- 0.09 0.41 _+ 0.29 0.33 - 0.33 0- 0 0.06 _+ 0.06 0.11 +- 0.11 0.43 +_ 0.31 0.39 + 0.20 0.61 + 0.41 0.44 + 0.20 0.67 -+ 0.46 0 -+ 0
4 hr test period. However, the rats of Group M--~G continued to increase their intakes. A burst of drinking in the fourth hour appears to match the intake of the G---~G rats in this fourth hour. Therefore, it can be said that the initial exposure to mannitol in the M--~G group suppressed intake for no more than 2
hr, although within the 4 hr test period the M - , G rats approached but did not match the intake of the G-~G rats. These data are also presented as the incremental intakes in Table 2, broken down into 15 rain intakes. Two-way ANOVA (Multivariate General Linear Hypothesis) yielded a significant time effect, F(15,37)=19.537, p<0.001, which shows up in the univariate test as a significant difference between the first and second 15 min time periods and between the twelfth and thirteenth 15 rain time periods. An inspection of Fig. 3 indicates that this shows up as the suppression of drinking in the second 15 rain period by the two groups on the M-G-S solution. The other point is due to the increase of drinking of the M--~G group seen in the interval from 3 hr to 3 hr 15 min. There is an overall group difference, F(2,51)-- 16.789, p<0.001, but a univariate interaction effect is significant only in the comparison between the second and third 15 rain periods. This is due to the increased intake of the M--~G group at this time combined with a decrease in drinking rate of the Cr--,G rats. The fact that initial ingestion of the mannitol solution in the M-*G group suppresses drinking for less than 2 hr after those animals are switched to a glucose-saccharin solution is not easily explained in accordance with the results from the previous 2 experiments. It is possible that drinking the G-S solution dilutes the M-G-S solution and thereby enhances the rate of absorption. Another possibility is that these rats which have never before experienced mannitol do not pay attention to the feedback which arises from its ingestion. However, the M--,M rats are also naive with respect to mannitol and their intakes do remain suppressed. Furthermore, during the second 15 min period, the M-*G rats are suppressed as much as the M--~M rats with respect to the G---~G group, which continues drinking avidly. A third possibility is that, despite the apparent evidence to the contrary reported by Davis et al.  rats can detect the presence of mannitol in the glucose-saccharin solution by taste, and avoid it either because of its novelty or because it is associated with a sensation of fullness or illness. The subsequent experiments attempt to investigate some of these possibilities. EXPERIMENT4 This experiment looked at the differences in gut clearance in rats representative of the three groups in Experiment 3, in order to see if switching from the M-G-S solution to G-S after 30 min affected the rate of fluid absorption. Method
Three groups of 5 rats each drawn from animals used in Experiment 3 were used in this experiment. All of the rats had experience drinking a glucose-saccharin-mannitol solution (0.2 M mannitol) prior to this experiment, but had only glucose-saccharin solutions for at least 1 week before this test. The first group (G--~G) of 5 rats had access to the 0.1 M glucose + 0.006 M Na saccharin (G-S) solution throughout the 2 hr test period. The second group of 5 (M-*G) began on the 0.2 M mannitol + G-S (M-G-S) solution and were switched to G-S after 30 min. The third group (M--~M) had the M-G-S solution throughout. At the end of 2 hr the animals were sacrificed by etherization and their gut contents measured in the manner described in Experiment 1. The 2 hr time point was chosen from the previous experiment because it represented an intermediate period at which point the intake of the M--~G rats differed from the intake of the M--,M
COlA.INS ANI) DAVIS
o--.o GLUCOSE-SACCHARIN +MANNITOL
v < I
SMALL INTESTINE G~G M~G
TIME (hr) FIG. 4. Two-hour cumulative intakes of three groups of rats: Group G-,G, glucose-saccharin for entire 2-hr test period; Group M-oG, 0.2 M mannitol + glucose-saccharin for 30 min, then switched to glucose-saccharin alone; Group M-+M, 0.2 M mannitol + glucosesaccharin throughout the 2-hr test period. N=5 for each group. (Exp. 4)
rats but before the recovery of drinking rate seen in the fourth hour.
/o- ~ " -
Results and Discussion
FIG. 5. Recovery of fluid in gut compartments of rats sacrificed after 2 hr of drinking (see Fig. 4). Points representing percentage of ingested fluid recovered are superimposed on data from Fig. 1. (Exp. 4)
Figure 4 is a graph of the cumulative intakes of these rats in the 2 hr test period. As can be seen, the drinking of the M----G rats in this experiment remains suppressed throughout the 2 hr period, at no point differing from the M--*M group, though in the last half hour intake appears to be increasing. Drinking rates in the G-+G group, on the other hand, are significantly higher than the M ~ G group from the second 15 min onward, F(2,12)=7.11 or greater, p<0.01. The results of this and the previous experiment indicate that there is some difference between the intakes of those rats in the M---,M group and those in the M--~G group which are switched to the G-S solution after the first 30 rain. Although this experiment does not demonstrate a significant difference at the 2 hr time point at which they were sacrificed, the intake of the M ~ G group is beginning to increase and one might expect on the basis of the results of Experiment 3 that this difference would increase if the rats were allowed to drink for another 2 hr. An examination of gut contents supports the possibility of a difference in clearance rates between these two groups. Figure 5 superimposes on the graphs from Fig. 1 the data compiled from the examination of the contents of the
stomach, small and large intestines of the rats sacrificed after 2 hr of drinking. As seen in the previous Fig. 4, the G--,G rats had consumed an average of 4 1 . 8+- 5.3 ml of the G-S solution at this time. Unlike the rats on which the data from Fig. I are based, these rats were still drinking up to the time of sacrifice. Yet it can be seen that the amount of fluid in all three gut compartments is essentially the same as that from the rats on G-S, shown in Fig. 1. which stopped drinking 2 hr prior to the measurement. This indicates that clearance of the G-S solution is almost immediate. At the other extreme. rats in the M---,M group, which were given the M-G-S solution throughout, had consumed an average of only 15.4_+3.9 ml in the 2 hr period, 13.2 of which was drunk in the first 30 rain. The fact that very little was consumed just prior to sacrifice is reflected in the low value for stomach fluid, which indicates that what had been drunk had already passed to the intestines. The value for fluid recovery in the small intestine is nearly as high as that seen in Experiment 1. demonstrating retention of fluid in this compartment. Although statistics cannot be done on percentages. Student t-tests on
MANNITOL AND INHIBITION OF INTAKE
TABLE 3 AMOUNT OF FLUID (ML) RECOVERED IN EXPERIMENT 4 Group G---~G Amt. Drunk Stomach Small Intestine Large Intestine
42 +_ 5
22 -+ 3.5
15 - 4
5.43 +_ 1.27 9.82 -+ 0.63 9.10 +_ 1.24
6.61 -+ 1.15 13.02 -+ 0.93* 13.41 -+ 0.89*
4.71 _+ 1.35 14.86 _+ 2.69 13.75 +-_ 1.58"
*Significantly different from G---~G,p<0.05. the untransformed data (Table 3) show no differences between the G - * G group and the M---~M group for either the stomach (t=0.39, n.s.) or for the small intestine (t=1.83, n.s.). The lack of effect in the small intestine is due to the large standard error for 'this group. This is somewhat misleading however, since the rats in the G--*G group consumed almost 3 times the fluid that those in Group M---~M did. As in the results of Experiment 1, a lack of effect therefore means a great difference in clearance rates, and therefore considerable retention in the M---,M group. Likewise in the large intestine the value for the M---~M group is quite high, approaching that for the first experiment. A t-test on the raw data gives a value of 2.56, p<0.05. If one examines the position of fluid retention for the M---~Ggroup, it can be seen first that the stomach fluid contents are greater than both other groups, this no doubt due to the recent burst of drinking of the G-S solution prior to sacrifice. The fact that the G-S solution did not clear from the stomach of these rats as fast as it does from those in the G--,G group which did not have mannitol suggests that the presence of the mannitol solution in the intestines retards stomach emptying. On the other hand the figures for small and large intestines are both intermediate between the G---~G and M---,M groups, giving support to the hypothesis that ingestion of G-S solution after initial exposure to M-G-S may act to speed up fluid absorption. Statistics on untransformed data (Table 3) show significant effects in both small (t=2.84, p<0.05) and large intestine (t=2.82, p<0.05). However, since the M---~G group drank an average of only 11.4-+ 1.9 ml of M-G-S as opposed to 15.4+_3.9 ml in the M---~M group, the difference may be accounted for by the differential intakes of mannitoi. It is strikingly apparent in the data of Experiment 3 and suggested in the data of this experiment that when switched from a glucose-saccharin solution containing 0.2 M mannitoi to the same solution without mannitol, rats are much more likely to continue drinking than are rats which are given the M-G-S only. Although the data of Fig. 5 indicate that there may be a difference in gut clearance due to subsequent ingestion of G-S, it is not clear that these rats are not detecting a difference in taste. The next experiment examines the possibility of taste-associated cues. EXPERIMENT 5 Throughout the course of all of our experiments with mannitol, there existed the possibility that rats were suppressing their intakes because of illness due to mannitoi. Although we never observed diarrhea in any of the rats in the time periods used, such effects have been reported in humans. Administration of mannitol solutions in humans has also been found to cause cramps (J. Rodin, personal corn-
munication). Since gastric distress from lithium chloride and other agents has been shown by several investigators  to become associated with the taste of ingested substances, it was decided to perform an experiment in which mannitol was paired with a distinctive flavor. If the suppression of intake after ingestion of mannitol could be conditioned to a distinctive flavor, this would suggest that the animals were experiencing gastric distress, which, like that produced by LiCI, becomes associated with taste. Method Four groups o f 5 male albino rats were used. The rats, adapted to the 0.1 M glucose + 0.006 M Na saccharin (G-S) solution, were 4 hr food deprived on the days of testing. The test period was two hr. On Day 1, all groups received unflavored G-S solution, and 2 hr intakes were recorded. On Day 2 all 4 groups received the M-G-S solution (G-S+0.2 M mannitol) flavored with 2.5% chocolate extract (McCormick). On Day 3, Group 1 received chocolate flavored M-G-S solution, and Group 2 received the M-G-S solution without flavoring. Group 3 received the G-S solution flavored with chocolate, and Group 4 received unflavored G-S, the solution to which the rats were adapted. Results and Discussion Figure 6 is a graph of the 2 hr intakes for each of the 4 groups on the three successive days of testing. On Day 1, all four groups received the unflavored G-S solution, which gave uniformly high intakes for all the groups. On Day 2, all 4 groups received chocolate flavored M-G-S solution, which reduced intake in all groups to about the same level. On Day 3, each group received a different solution as designated in the Methods section. Groups 1 and 2 both received the M-G-S solution, the former with chocolate flavoring, the latter without. Nevertheless, both groups were equally suppressed. Groups 3 and 4 both received the G-S solution, Group 3 with chocolate flavoring, Group 4 without. The intakes of both these groups were at that level seen on Day 1, regardless of flavor. A 2-way A N O V A reflects these results with no significant group effects over the 3 test days, F(3,48)=2.4145, n.s., but with a significant day effect, F(2,48)=15.6323, p<0.01. A significant interaction effect, F(6,48) = 3.6772, p <0.01, reflects the results of the third day, These results are not consistent with those seen in a conditioned aversion paradigm in which flavor associated with gastric distress suffices to inhibit intake in the absence of the poison. Under such circumstances we would have expected Group 3 of Day 3 to have shown suppressed intake. To the contrary, this group showed the highest intake. Therefore, flavor does not provide an associable cue to the effects of
COLI,INS AND I}AVIS Mannitol, on the other hand, may produce cramps in fals ~,~ it has been reported to do in humans, and this form of dis. tress may not become associated with taste. We would pFefer to believe that, in the absence of a demonstrable conditioned taste aversion, it is likely that mannitol acts through a normal feedback mechanism related to intestinal distention which serves to limit intake in a manner comparable to that which limits normal food intake. GENERAL DISCUSSION
v, i--z -r
NO FLAVOR FLAVOR 1 2 3 4 G-S M-G--S FIG. 6. Two-hour intakes (ml) of solutions on 3 consecutive test days. Day 1: All groups drank unflavored 0.1 M glucose + 0.006 M Na saccharin (G-S). Day 2: All groups drank the G-S solution to which 0.2 M mannitol was added (M-G-S), flavored with 2.5% chocolate extract. Day 3: Group 1, Flavored M-G-S; Group 2, Unflavored M-G-S; Group 3, Flavored G-S; Group 4, Unflavored G-S. (Exp. 5)
mannitol, even when there is no doubt that the flavor can be detected. It is then not likely that a possible slight difference in flavor between the basic G-S and M-G-S solutions can account for the difference in intakes seen between these solutions. A similar result has recently been reported by Bernstein and Vitiello [I]. In their experimental procedure, rats with no previous experience with a saccharin solution were given a 0.1% saccharin solution to drink on the first test day. Shortly after 1 hr exposure to the saccharin solution, the animals were intubated gastrically with a solution of either 0.15 M saline, 0.15 M LiCI, 0.4 M mannitol or 0.8 M mannitol. The procedure was repeated every 3 days for 3 more sessions. Only the LiCI group showed significant suppression of intake. Neither mannitol group (both on concentrations of mannitol higher than that used in our experiments) showed a difference in intake from the saline control. The results of both Bernstein and Vitiello's experiment and our own suggest that mannitol does not make the animal sick, at least in the same way as do LiCI and other poisons which become associated with taste. It may be, however, that these other agents cause illness which produces nausea and that only this symptom becomes associated with taste.
The five experiments presented here indicate that intestinal filling is a factor in the limitation of intake. This finding corroborates and extends the conclusions arrived at in the previous paper by us [31 which established intestinal filling as one of several possible negative feedback loops on the ingestion mechanism. The present experiments also confirmed the conclusion from the previous work that the stomach is excluded from any apparent role in this inhibitory mechanism since not only was fluid retention not apparent in the stomach when intake was inhibited, but the point at which the stomach clearly did have appreciable fluids was a point at which there was no reduction of drinking. These experiments measured fluid retention in the small and large intestines as a whole, without giving information on their subdivisions. However, it is our opinion based on subjective observation that the regions which presented the most visible increases were the ileum and the caecum. This is not surprising in view of the fact that these are normally the most active regions of water absorption, and one would therefore expect an inhibition of absorption to be the most noticeable at these points. Mannitol causes a retention of water in the intestine because of an osmotic effect. The absorption of water is essentially passive, depending on an osmotic gradient. Mannitol in the intestinal lumen retains water as long as it is present. Since it is absorbed only very slowly, fluid absorption is retarded for some time. As seen in Experiment 1, even after" 4 hr there is an elevation of fluid content in the large intestine. Experiments 2, 3, and 4 indicate that ingestion is retarded as long as there is intestinal distention. Presumably the bulk of fluid which accumulates after intake of a mannitol solution exerts an effect which inhibits the ingestion mechanism, but it is not known what this effect is. It may be activation of mechanoreceptors by distention of the gut wall, or stimulation of osmotic receptors. Distention of the gut may also stimulate production of the hormone cholecystokinin (CCK) which has been shown to inhibit food intake by Gibbs, Young, and Smith . The work of Liebling and coworkers 191 and Meyer and Grossman  suggests that this hormone is liberated when liquid food is infused into the duodenum. Although more than one factor limiting intake is no doubt involved in the course of normal ingestion the experiments presented here seem to implicate a mechanism which is involved further along in the gastrointestinal tract than the duodenum, since the inhibition of ingestion endures much longer than the point of maximal fluid accumulation in the small intestine (see Experiment 1). This is, of course, not incompatible with the action of a hormone which is liberated at the time of passage of the fluid through the duodenum and acts for some time thereafter. Glick and Modan 16] have performed experiments using duodenal and ileal glucose infusions in which they suggest that intestinal satiety' mechanisms may exist in other portions of the intestine than the duodenum. The possibility that the caecum, which has a
MANNITOL AND INHIBITION OF INTAKE
great capacity for distention, may be involved in this inhibition is strongly suggested, as mentioned above. As yet, little work has been done on the rodent caecum that would lead one to postulate a role for it in the control of food intake; however, it may be worthy of further investigation. The possibility that mannitol may inhibit intake because it produces gastrointestinal distress was examined in Experiment 5. The results show that the effects of mannitol cannot be used as an unconditioned stimulus to produce a conditioned taste aversion,-and it is therefore suggested that illness is not produced. Although this experiment does not rule out an unpleasant effect of mannitol, it does weaken the probability. Additional evidence against illness is seen in Experiments 3 and 4 in which rats pretreated with mannitol solutions for 30 min readily accepted plain glucose-saccharin solutions subsequent to the initial mannitol ingestion. If mannitol did in fact make the animals sick, they presumably would not be likely to ingest anything. This is also seen in rats which were given G-S solution after 4 hr of M-G-S exposure and began drinking the G-S solution copiously at that time (unpublished results). The inhibition of intake seems to be specific to mannitol solutions. The fact that these experiments indicate that the satiety produced by a mannitol solution is fully capable of inhibiting further ingestion of a mannitol solution, but only partially capable of reducing intake of a glucose-saccharin solution may at first seem to make the results of these experiments inapplicable to feeding behavior in general. However, when one realizes that mannitol exerts its effect through an osmotic action which is diminished by subsequent ingestion of the dilute glucose-saccharin solution, and that solid food exerts, if anything, a stronger osmotic effect than mannitol until all of its components are digested and absorbed, it is not so unrealistic to extrapolate from these results to normal feeding behavior. In order to test whether our previous results  were
generalizable to solid food intake, Bernstein and Vitiello  have examined the effect of mannitol in solid (or semi-solid) food. By combining ground Purina chow with a solution of 0, 0.2, 0.4, or 0.8 M mannitol (2:3, W:V) a mash was prepared for feeding rats after 5 hr deprivation. These investigators found that addition of mannitol did not significantly change the size of the mash meal, but that the post-meal interval was significantly prolonged in rats consuming the mash made with 0.4 M or 0.8 M mannitol. The fact that in their study mannitol did not reduce meal size whereas in ours it did suggest that the controls operating to determine meal size of liquid diets are different from those operating to control meal size of solid food diets. This possibility is also supported by the fact that meals in the Bernstein and Vitiello study lasted only 10 min whereas in ours rats drinking the glucosesaccharin solution drank almost continuously for as long as an hour. Although our results differ from theirs with respect to meal size they are similar with respect to post prandial feeding. In their study mannitol increased the intermeal interval and in ours it significantly suppressed feeding following the first meal. In summary, we believe that our results are consistent with the idea that ingestion of the mannitol solution activates a normal feedback mechanism, and that the rat learns to respond to this signal. In some cases, inhibition of drinking can be seen within 30 rain of the first exposure to mannitol, in other cases the effect is not strongly seen until the second exposure. This is no doubt due to each individual rat's own capacity, the amount of food and fluid present in the gut at the time of exposure, and the rat's disposition to heed the signal. The latter factor involves many component,, among which are thresholds for a putative mechanoreceptor in the gut and the sensitivity of the hypothesized neural network in the central nervous system which is involved in the control of feeding and drinking.
REFERENCES 1. Bernstein, I. L. and M. V. Vitiello. The small intestine and the control of meal patterns of the rat. Physiol. Behav. 20: 417-422, 1978. 2. Crane, R. K. Absorption of sugars. In: Handbook of Physiology, Section 6: Vol. 1117,Intestinal Absorption, edited by C. F. Code. Washington, D.C.: American Physiological Society, 1968, pp. 1323-1352. 3. Davis, J. D., B. J. Collins and M. W. Levine. Peripheral control of drinking: Gastrointestinal f'dling as a negative feedback signal, a theoretical and experimental analysis. J. comp. physiol. Psychol. 89: 985-1002, 1975. 4. Garcia, J., W. G. Hankins and K. Rusiniak. Behavioral regulation of the milieu interne in man and rat. Science 185: 824--831, 1974. 5. Gibbs, J., R. C. Young and G. P. Smith. Cholecystokinin decreases food intake in rats. J. comp. physiol. Psychol. 84: 488-495, 1973. 6. Glick, Z. and M. Modan. Behavioral compensatory responses to continuous duodenal and upper ileal infusions in rats. Physiol. Behav. 19: 703-705, 1977.
7. Kissileff, H. R. Free feeding in normal and "recovered lateral" rats monitored by a pellet-detecting eatometer. Physiol. Behav. 5: 163-173, 1970. 8. LeMagnen, J. and S. Tallon. La periodicit6 spontanre de la prise d'aliments ad libitum du rat blanc. J. Physiol. (Parrs) 58: 323-349, 1966. 9. Liebling, D. S., J. O. Eisner, J. Gibbs and G. P. Smith. Intestinal satiety in rats, J. comp. physiol. Psychol. 89: 955-965, 1975. 10. Meyer, J. H. and M. I. Grossman. Release of secretin and cholecystokinin. In: Gastrointestinal Hormones, edited by L, Demling. Stuttgart: Springer-Vedag, 1972. 11, Panksepp, J. Hypothalamic regulation of energy balance and feeding behavior. Fedn Proc. 33:1150-1165, 1974. 12, Panksepp, J. On the nature of feeding patterns--Primarily in rats. In: Hunger: Basic Mechanism and Clinical Implications, edited by D. Novin, W. Wyrwicka and G. Bray. New York: Raven Press, 1976, pp. 369-382.