Internal signals cause large changes in food intake in one-way crossed intestines rats

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Vol. 14, pp. S-603,

198.5. ’ Ankho

International

0361-9230185 $3.00 + .OO

Inc. Printed in the U.S.A.

Internal Signals Cause Large Changes in Food Intake in One-Way Crossed Intestines Rats HENRY S. KOOPMANS Deportment

c?fMedical

Physiology,

University

of Cdgrrry,

Cnlgrrry, Alberta

T2N 4NI,

Canuda

KOOPMANS, H. S. Intc~mul sign& CLIUSClarge changrs in food intukr in one-nxy c~rosscvf intestines rats. BRAIN RES BULL 14(6) 595-603, 1985.-Fifteen pairs of parabiotic rats had either a surgical operation in which a 15 or 30 cm segment of upper small intestine was disconnected from the digestive tract of one rat in the pair and reconnected to the transected duodenum of its partner or had control surgery. Food eaten by one rat in the pair went to the rat’s own stomach, traveled through 5 cm of its upper duodenum and then crossed into the isolated segment of the partner’s small intestine. After traversing the 15 or 30 cm isolated segment, the remaining unabsorbed food crossed back into the lower duodenum of the rat that fed. Food eaten by the partner went through its own digestive tract, but bypassed the isolated segment of its own upper small intestine. The operation produced a large and sustained change in food intake of both rats in a pair. For the rats with 30 cm crossed segments, the rat that lost intestinal thyme into its partner ate 3.6 times as much food as did its partner for a period of many months. At sacrifice. the rats that ate more, weighed less and had less body fat. These large changes in food intake may be caused by internal changes associated with changes in the amount of food absorbed into each rat or by differential stimulation of the lower digestive tracts. The results clearly show that there is a major internal control mechanism for the amount of food eaten. Food intake

Body

weight

Small intestine

Absorbed

THE study of the internal control of food intake began with the early experimental physiologists. Such well-known figures as Magendie, Bernard and Pavlov did studies trying to locate the anatomical site responsible for the control of feeding behavior [62]. Since that time, many investigators have done experiments that have provided new information about the possible internal mechanisms for regulating food intake, but no generally accepted explanation has emerged. In fact, one prominent investigator believes that there is no internal control mechanism and explains human efforts to regulate body weight as a cognitive response to changes in the tightness of the belt [21]. Because the ingestion of food leads to large changes in metabolism and to the stimulation of many nerves and the release of several hormones, there are many possible signals that could affect food intake. Since feeding is a behavior, psychological factors can also change intake. The taste, type of food and time of feeding have all been shown to alter feeding behavior [ 10. 49, 61, 651. If one looks at the control of intake during a single meal, there are several regions of the digestive tract that have been implicated. Studies with esophageal and gastric fistulas show that signals from the mouth and throat have only a minor role in terminating a single meal [28,30,45,54], but they are important in motivating an animal to ingest food [ 181. In recent years, a number of investigators have shown that the stomach is involved in terminating a single meal [Il. 13, 16, 37, 39, 531, but the nature of this signal is still in dispute. Some investigators believe that the signal is neural [ 131and others believe that it is hormonal 1391. The results of studies that have looked for

food

Ileum

Stomach

Body fat

satiety signals that arise in the small intestines are quite contradictory [5, 6, 17, 691. It is important in evaluating these studies to determine whether the delivery of food to the intestine is physiological and which segment of the small intestine is being stimulated by the food infusion. Other investigators have chosen to investigate peptides that have been isolated from the gastrointestinal tract. There is a great deal of recent evidence that the hormone, CCK, may play some role in producing satiety [2, 9, 23, 27, 47, 67, 681. Other researchers have shown that the infusion of bombesin [22], glucagon [46,51], insulin [72] and somatostatin [48] reduce food intake. With all peptide infusions, there is a continuing concern about whether these hormones inhibit food intake at physiological doses or simply make the animals feel sick at pharmacological doses [ 14, 15, 381. Still other investigators believe that the food must be absorbed from the digestive tract and alter liver metabolism in order to terminate a single meal [57,64]. This view has recently been challenged [41]. When we turn to the long-term control of food intake, a new set of theories emerge. Some investigators believe that metabolic signals from absorbed or infused food are the major controls of food intake [ 12, 55, 701. Recent work with diabetic rats that lose glucose through the urine has led to the hypothesis that the body senses the amount of utilizable fuel and adjusts intake accordingly [20]. Other researchers have attributed the long-term regulation of food intake to signals arising in the lower small intestine [4, 40, 43, 661. These researchers point to the effectiveness of intestinal surgery in causing long-term weight loss in both humans and animals [3, 4, 43, 591. Still other investigators believe that there are un-

595

596

KOOPMANS

known signals from body fat that are involved in controlling food intake [19, 24, 25, 33, 52, 56, 581. Force-fed obese animals tend to reduce their food intake and starved animals increase intake [26]. Enzymes associated with adipose tissue, like lipoprotein lipase, may be involved in driving food intake [71]. Other researchers believe that the main control of body weight is through energy output and they point to the role of brown fat in burning off excess energy intake [63]. This profusion of experimental explanations for the regulation of food intake and energy balance suggests that either the control of food intake is extraordinarily complicated or that we have not yet found a good explanation. It points out the need for experiments that evaluate the relative importance of the various types of signals that have been suggested. The purpose of the present experiment is to show that there definitely is an internal control mechanism for the regulation of food intake and to begin to sort out the relative importance of the various types of signals that have been proposed. SURGERY

Thirty inbred male Lewis rats weighing 300-325 g were divided into pairs of rats matched for body weight. In the first operation, which produced parabiosis, the rats were anesthetized with ether and shaved along the left or the right flank of the body. An oval incision about 10 cm in length and 3 cm in width was made in the shaved skin of each rat and, then, the lower skin flap of each rat was sewn to that of its partner with 3-O silk. A round 3 cm incision was made in the abdominal wall of each rat and it was closed by continuous suture with 2-O absorbable cat gut to create a common abdominal cavity. Finally, the upper skin flap was closed with 3-O silk and two stay sutures were placed before and after the parabiotic union. A few weeks after the surgery, the capillaries in the skin and muscle of the two rats is known to make connections with each other and about 1% of each rat’s blood mixes every minute leading to a complete mixing of the blood every 3 hours [25]. In twelve of the fifteen pairs of rats, a second operation was done under ether anesthetic to change the way in which food moved through the digestive tracts of the two rats. In six of these pairs, one of the rats had its small intestine transected midway through the duodenum about 1 cm below the common bile and pancreatic duct. It also had the small intestine transected 30 cm further down the intestine in midjejunum. This 30 cm isolated segment of upper small intestine was connected to the ends of a single transection of the partner’s duodenum (see Fig. 1). The upper half of the partner’s duodenum was connected to the upper portion of the isolated 30 cm segment and the lower part of this 30 cm segment was connected to the lower duodenum of the partner. The upper duodenum of the rat that had the 30 cm segment disconnected was reconnected with 7-O continuous Connell sutures to its own lower jejunum so that the food eaten by this rat bypassed the disconnected 30 cm segment. In the other six experimental rat pairs, the isolated segment of the intestine was only 15 cm but the surgery was the same. During the transections of the small intestine, no major nerves or blood vessels were cut. Any food absorbed from or any signals arising in a particular segment of the small intestine went into the bloodstream or through the nerves to the brain of the rat in which that segment of the intestine originated. The 3 other pairs of rats underwent sham intestinal surgery in which

1 or 2 transections

of the intestine

were

made in

FIG. 1. Diagram of the one-way crossed intestines preparation. The stipled digestive tract belongs to the rat on the left. Either 15 or 30 cm of one rat’s upper small intestine was disconnected from its own digestive tract and reconnected to the mid-duodenum of its partner. There are no transections of major nerves or blood vessels during the surgery.

each rat but the small intestines of these rats were reconnected in the normal way. As a result of the surgery, food eaten by one rat of each pair traveled to its stomach, traversed 5 cm of its own upper duodenum and then passed into the 15 or 30 cm segment of its partner’s upper small intestine. After moving through the partner’s intestinal segment, the thyme that was not absorbed into the partner’s bloodstream returned to the lower duodenum of the rat that fed. It then travelled down the rest of this rat’s digestive tract and was excreted in the usual way. Food eaten by the partner rat stayed in its own digestive tract, but bypassed the 15 or 30 cm segment that was connected to the other rat’s digestive tract. The normal length of the rat’s small intestine is about 110 cm. The end

INTERNAL

CONTROL

597

OF FOOD INTAKE

result of the surgery was that food leaving the stomach of the rat that lost food to its partner travelled through 125 or 140 cm of small intestine before reaching the cecum. Food leaving the stomach of the partner rat traversed 95 or 70 cm of small intestine before entering its own cecum.

130‘ 120 -

too-

y

vo-

a

z

METHOD

after the initial parabiotic surgery, the rats were put on a 17 hour feeding schedule (4 p.m. to 9 a.m. with lights off at 5 p.m. and on at 9 a.m.) and fed the complete liquid diet, Nutrament (Mead Johnson Nutritionals). Nutrament has 53% of its calories as carbohydrate, 21% as fat and 26% as protein and contains adequate minerals and vitamins. Its caloric value is about 1 kcal/ml. During the nighttime feeding the rats were placed in modified Bollman restraining cages that prevented the rats from eating each other’s food. During the day, the rats were allowed free movement in their home cages. Water was available at all times. The rat pairs were weighed daily during the hour before feeding. After being placed in the restraining cages. the rats were offered food and the amount ingested was recorded 30 min later for measurement of short-term intake and at 9 a.m. the next morning for total daily intake. On a randomized and balanced schedule, one or the other rat in each pair was fed 30 min before its partner throughout the study. The objective of this time delay was to determine whether the presence of food in the crossed 15 or 30 cm segment would have any effect on the short-term food intake of the rats. The rats were maintained on this feeding schedule until their daily food intake was stable for at least 8 days. On the afternoon after the eighth stable day, the rats underwent the second surgery (described above) to alter the movement of food through the digestive tract. They were not fed for the next 24 hours to allow the suture lines of the intestine to heal and then they were returned to their normal feeding schedule. Their food intake and body weight were followed for the next 29 days. After completion of this study, the rats were allowed to eat lab chow ad lib in their home cages for 2 to 3 months, were returned to the liquid diet schedule as described above and then meal patterns were recorded for 5 days. Meal patterns were recorded with an apparatus that consisted of a movable sensor that followed changes in the level of the liquid diet in a 100 ml buret. The sensor had a light source on one side of the buret and a photocell to detect light levels on the other side. As the rat ate, the level of the liquid diet declined exposing the photocell to the beam of light. This activated a relay that turned on a 1 rpm motor that slowly lowered the sensor along the buret until the new level of liquid diet was reached and the light beam was interrupted. An Esterline-Angus recorder kept a record of the times when the motor was on. This information could be converted to the size of the meals eaten by the rats because the motor ran at a fixed speed. The criterion for a meal was the ingestion of a minimum of 0.5 ml preceded and followed by at least IO min without feeding. The animals were sacrificed with ether the day after the meal pattern measurements were complete. At sacrifice, the rats were weighed as a pair and then separated and weighed individually. Several internal organs-liver, kidneys, heart, small intestine and testes-were dissected free of other tissue, blotted and weighed for each rat. Five definable fat pads-retroperitoneal, epididymal, mesenteric, inguinal and subcutaneous-were carefully delineated, dissected and weighed. In addition, the skin and carcass (defined as the remaining body tissue) were weighed. Two

weeks

HO-

= E -

80. 70.

8

60.

e

50-

d

40

E

3o 20L

-

IO 3

5

7

9

(I

13

15

17

IV

21

23

25

27

29

31

33

35

37

DAYS

FIG. 2. The total daily food intake (ml) for the four groups of oneway crossed intestines rats and the controls. Baseline food intake was measured for 8 days and the surgery (see Fig. 1) was done on day 9. The post-surgical changes in food intake were followed for 29 days. The food intake is reported separately for the rats that lost food into the partner’s I5 cm (A) and the 30 cm (0) segment and for the rats that received food into the I5 cm (A) and 30 cm (m) segment. The controls (0) simply had sham intestinal surgery. The liquid diet, Nutrament, has a caloric value of about I kcaliml.

Statistics

All statistical tests were done with one-way ANOVAs or where relevant with more complex repeated measures ANOVAs. Individual tests were done by specific comparisons. RESULTS

The average food intake for all of the rats during the 8 days of baseline measurements was 61.6k2.0 ml/day. The rats were divided into 5 groups of six rats each. In both the 15 cm and the 30 cm intestinal loss pairs, the 6 rats that lost food to the intestine of their partners were defined as one group. In both types of pairs, the 6 rats who gained food from the intestine of other rats were also defined as a group. The final group of 6 rats came from the 3 pairs of control parabiotic rats. The results of the total daily food intake measurements are presented in Fig. 2. Although there were small differences in the average intakes of the five groups of rats during baseline, there was no statistically significant difference among these groups (range 58.6 to 66.0, p>O.6). After surgery, the control rats showed a reduction of food intake that lasted one day. Thereafter, their food intake returned to previous baseline levels and averaged 72.5e6.9 kcal/day during the last eight days of the experiment. The rats that lost food into the 30 cm segment of their partner’s small intestine showed a gradual increase that was significantly greater than their baseline intake on the 5th day after surgery (p
59X

food intake were not as large in the groups in which food was lost into the 15 cm upper intestinal segment. The rats that lost food to their partner’s intestines increased their food intake more gradually and showed a significant difference only on day 10. Their food intake stabilized at 90.9 f 4.1 kcahday during the last 8 days. Their partner’s intake decreased rapidly and stabilized at a post-surgical average of 41.7k3.8 kcaI/day. The changes in food intake were highly significant across all groups (F=48.3, p>0.05). There was no significant interaction between time of feeding and before or after surgery, indicating that the presence of food in the crossed intestinal segment had no effect on subsequent short-term food intake. For the rats with 30 cm segments of crossed intestine, their food intake during the baseline period was 5.2 ml when fed first and 6.1 ml when fed 30 min after their partners. During the last 8 days of the study, these rats had reduced the size of their initial meal to approximately half of what they previously ate (p>0.05). Apparently, the control of short-term food intake was not affected by the intestinal surgery. Toward the end of the study, the meal patterns of 4 rats in each group were measured on the apparatus described

KOOPMANS

710 700 690 660. ;

670

E

.Y :

653.

B

640.

3

630. 6ZO. 610 600.

DAYS

FIG. 3. The daily body weight for the pairs of 15 cm (A) and 30 cm (D) crossed intestines rats and for the control parabiotic rats (0). For further details, see caption in Fig. 2.

above. The rats continued to show the large differences in food intake (see Table 2) that were noted at the end of the first behavioral experiment. The 4 rats that lost food into the 30 cm intestinal segment of their partners ate 128 ml/day over the 5 day period. The rats that lost food into the 15 cm segment ate 102 ml/day while the two pairs of control rats ate 76 ml/day. The 4 rats that received food into the 15 cm segment ate 65 ml/day and those that received food into the 30 cm segment ate 52 ml/day. These changes in the total amount of food eaten were not reflected in a consistent change in meal patterns (Table 2). The rats that showed the largest increase in total food intake did so by significantly increasing their average meal size from 3.7 to 6.5 ml. The rats that most greatly reduced their intake showed a significant reduction in meal number from 20.5 to 14.3 but no change in meal size. At sacrifice 3 to 4 months after surgery, the rats that lost food to their partners always weighed less than their partners. The rats with 30 cm crossed intestinal segments weighed an average of 332 g compared to their partner’s 413 g (see Table 3). The rats with 15 cm segments weighed 352 g and 416 g, respectively, while the control rats weighed the most, 441 g. Because of these differences in total body weight, the various internal organs are expressed as weight per 100 g body weight. The two major changes in the weight of internal organs occurred in the percentage weight of dissectable body fat and of the liver. The amount of body fat was inversely related to the amount of food that the animals ate. Those rats that ate the most had the smallest percentage of body fat @
INTERNAL

CONTROL

OF FOOD

599

INTAKE

DISCUSSION

The major finding from these experiments is that rats that lose a substantial amount of food from their intestine show a large and sustained increase in total daily food intake. Their partners that receive food directly into their upper small intestine show a substantial and persistent reduction in intake. When 30 cm of one rat’s intestine was connected to the intestine of its partner, the two rats of a pair showed an average 3.6 fold difference in total daily food intake. The rats with 15 cm of crossed intestinal segment had an average 2.2 fold difference in food intake. These large changes in daily food intake must be caused by internal changes resulting from the amount of food absorbed or by changes in the amount of food stimulating the lower portion of the digestive tract. They can not be attributed to the psychological characteristics of the feeding cages, the type of feeding schedule, the food eaten or to social factors related to delays in feeding or the parabiotic attachment to other rats. The control rats had the same experience of surgery, diet, schedule and the presence of other rats, but did not show a significant change in food intake. Thus, the normal internal control of food intake goes beyond the human psychological process of noting changes in the fastening of a belt and making appropriate cognitive adjustments in total amount of food eaten [2l]. The most obvious consequence of the surgery on these parabiotic rats is that the amount of food eaten by the rats differs from the amount of food absorbed into their bloodstreams. The rats that show a large increase in their food intake lose some of that food into the intestine and subsequently into the bloodstream of their partners. In contrast, the partners that receive nutrient-rich thyme into their intestine, absorb some of this thyme and reduce their food intake. The size of these changes in intake depend upon the length of the intestinal segment that was disconnected from the digestive tract of one rat and reconnected to the small intestine of its partner. Rats with 30 cm crossed intestinal segments change their food intake by 38-43 ml while rats with I5 cm segments change their intake by IS26 ml. It should be noted that the method ofdelivering nutrients to the bloodstream of the partner rat was highly physiological. The food that was eaten by one rat was mixed with saliva, gastric and pancreatic secretions, and bile before it entered the partner’s crossed segment. The rate of entry was controlled by the normal motility of the stomach and intestine. Absorption into the partner’s bloodstream followed the usual physiological pathways and nutrients were delivered to the portal vein and to the liver before entering the systemic blood circulation. These large and consistent changes in food intake can be compared to changes in intake that result from caloric dilution or concentration of the diet. Previous investigators have shown that animals adjust rapidly and reasonably accurately to dilution of their diet with water, kaolin or cellulose [I, 26, 29, 50, 601.They adjust back to previous levels of intake when the diluent is removed. The present experiments are different in one critical dimension. There was no change in the diet that the rats were fed and, thus, no changes in the taste of the diet or in the psychological experience of it. The rats were changing the amount of food eaten only because of the internal physiological changes that they were experiencing. The changes in food intake in these experiments can also be compared to the changes noted when nutrients are infused directly into the bloodstream of animals [12, 36, 55, 731.

TABLE

1

THIRTY MINUTE FOOD INTAKE OF RATS RECEIVING NUTRIENTS FROM THEIR PARTNER’s INTESTINE (ml r S.E.M.)

15 cm Cross Fed First* Fed Second

Pre-op 1-8

Post-op 32-39

5.1 -c 3.2 5.3 f 4.0

5.0 -t 2.8 5.4 -t 0.4

F interaction=O.Olt

30 cm Cross Fed First Fed Second

p=O.94

2.5 + 1.4 2.8 2 1.9

5.2 + 2.1 6.1 + 2.9 F interaction=O.

19

p=O.67

*Rats fed first do not receive nutrients from their partners intestine while those fed 30 min later (second) have already received intestinal nutrients before feeding during postoperative days 32-39. tTwo-way repeated measured ANOVA on both factors.

Those animals that remain healthy during the infusion generally reduce their intake to partially compensate for the additional calories that they are receiving, but the adjustment is usually incomplete. In human subjects receiving total parenteral nutrition because of GI problems, the patients report that they remain hungry despite the infusion of adequate calories, but they do not eat much when offered food, possibly because of their gut problems [31,70]. An alternate way to explain the large changes in intake that occur in this crossed intestine experiment is by noting the changes in the stimulation of the lower small intestine. The rats that lose food into the 30 cm segment of their partner’s upper small intestine have some of that food absorbed into the partner, less food reaches their own lower duodenum and less food travels down through the rest of the intestine to reach the ileum. It may be necessary for these rats to increase their food intake to get a constant level of ileal stimulation by ingested nutrients. In contrast, the rats that receive food into the 30 cm segment, eat food that bypasses 30 cm of upper small intestine and may reach the ileum more quickly. They may have to eat less to reduce the stimulation of their lower ileum. There are a number of experiments in the literature that are consistent with the role of the ileum in the long-term control of food intake [42, 43, 661. in both ‘humans and rats that have a jejunoileal bypass, food travels through a shortened segment of upper small intestine and arrives in the ileum more quickly. Jejunoileal bypass has been an effective way to cause a reduction of food intake and a loss of body weight [4,59,66]. Furthermore, several experiments on the transposition of a short ileal segment to the duodenum. show a substantial reduction of food intake and a loss of body weight similar to the jejunoileal bypass preparation [3, 42-441. In these experiments, the ileal segment is stimulated by a large amount of unabsorbed food, but the length of the functional small intestine is not reduced and the rats do not have discomfort associated with malabsorption following jejunoileal bypass. Another possible way to explain these results is that changes in the amount of body fat or in the nutritional state of the liver cause the observed changes in food intake. At

KOOPMANS TABLE 2 MEAL PATTERNS FOR ONEWAY CROSSED INTESTINES (FOOD INTAKE MEASURED IN ml rt s.e.m.) Total Intake

30 cm 15 cm Control 15 cm 30 cm

127.9 101.6 76.2 64.6 51.6

Loss Loss Gain Gain

+ 2 ” -t t

Meal Number 21.6 19.3 20.5 17.9 14.3

13.7* 17.4” 16.6 7.2’ 22.6*

*Signi~cantly different from controls %After 7 hour deprivation.

c t + It rt

RATS

Meal Size

6.9 4.3 1.5 3.4 s.3*

6.5 5.5 3.7 3.8 3.9

First Meal

+ 2.1* i 1.4 + 0.6 t 0.7 ‘-’ 1.6

15.3 9.4 7.7 5.1 5.3

r rt r +t

4.5* 2.6 3.0 1.6 2.1

p
TABLE 3 SACRIFICE DATA FOR ONEWAY CROSSED INTESTINES 15 cm Loss

30 cm Loss

Weight

332

+- 30

352

Weight Carcass Skin Fat Liver Heart Kidneys Intestine Testes

64.1 15.4 5.05 3.35 0.30 0.88 1.79 0.48

c -t 2 2 t r -t 2

0.9 0.8 1.20 0.55 0.03 0.04 0.28 0.23

62.7 16.5 6.00 3.31 0.28 0.84 1.75 0.57

t f * + 2 rt 5 2

1.6 0.9 0.79 0.45 0.04 0.09 0.43 0.15

441

30 cm Gain

15 cm Gain

Contr

It 33

RATS (g i: s.e.m.1

rt 64

416

t 48

413

F-test P

r 47

10.01

t z!z -+ tf -t +

co.01 n.s.
per 100 g Body Weight 60.3 17.0 9.25 3.59 0.26 0.75 1.37 0.36

sacrifice, it was found that the percentage of dissectable body fat and the total body weight varied inversely with the daily food intake. The wet weight of the liver also varied systematically with food intake. It is not clear from the sacrifice data, which was gathered 3-4 months after surgery, at what time the changes in the weight of both fat and liver occurred. They may have occurred soon after surgery and triggered the changes in food intake or they may be the longterm consequence of changes in food intake that were not complete. That is, the rats that overate may not have been able to push their food intake sufficiently high to prevent a loss of body fat. By estimating the caloric value of the changes in body fat and body protein in the two partners with 30 cm crossed segments and dividing by the product of the average daily caloric intake and the number of days after surgery, the changes in body composition can be explained by a small 1 to 2% error in total daily food intake. It is interesting that the average amount of body fat in the 30 cm rats was 8.27 g per 100 g while the average amount of fat in the 15 cm rats was 8.26 g. These averages suggest that the total amount of fat in the pair may be regulated [24, 25, 56,581. The average for the controls was 9.25 g per 100 g, but the weight of the control rats was also greater than that of either group of crossed intestines rats. Many investigators have thought that the body fat stores

+z?z t r rt t + I

3.0 1.7 2.9 0.45 0.01 0.07 0.30 0.10

59.0 16.3 10.52 3.87 0.28 0.74 1.43 0.46

f 2 2 ? + f + f

2.3 0.6 1.6 0.54 0.02 0.05 0.13 0.16

58.2 16.0 11.49 4.06 0.27 0.75 1.54

0.37 i:

4.0 0.4 3.0 0.40 0.02 0.05 0.20 0.10

may play a role in the long-term control of food intake 119, 33, 521. In an unpublished experiment that I did with Irving Faust and Jules Hirsch (1980), we transplanted large retroperitoneal or epididymal pads that were depleted of fat by starvation ( 1.40 g per pad at time of transplantation) from five inbred Lewis rats to five recipients. The blood vessels to these fat pads were connected to the abdominal aorta and ascending vena-cava within the abdominal cavity. We thought that the depleted fat cells would withdraw nutrients from the plasma in the early post-surgical period and that the lowered plasma levels of these nutrients might activate the feeding mechanism [713. We followed the food intake of the 3 rats that had the most successful transplants (10.84 g average at sacrifice) for 30 days and found that the rats with transplants ate 66.2r3.6 kcal/day during this time while the surgical controls ate 69.1 rt 1.6 k&/day (F=0.58, p =0.49). These results suggest that there was no change in food intake as a consequence of the transplantation of fat depleted cells, but we were not sure that the large increase in fat pad size occurred during the first month. At sacrifice 8 months later, the five transplanted fat pads were greatly enlarged (8.53 g/pad) and averaged 19.042 million cells/pad. The rats own equivalent fat pad averaged 16.459 million cells. The fat celI size in the transplanted tissue (0.421t0.091 fig of lipid per cell) was nearly the same as the cell size in the equivalent pad of the

INTERNAL

CONTROL

OF FOOD INTAKE

recipient (0.41920.076 pg) (F=0.0003, p=O.98). Thus, the transplant of fat-depleted fat pads did not cause an increase of food intake in the recipient during the 30 post-surgical days but the denervated fat pads grew and eventually had the same cell size as the recipient’s own equivalent fat pads. In summary, the most likely explanation for the large changes of food intake that we have observed is the change in the amount of nutrient that was absorbed into the body of each of the rats. This change in the amount of absorbed nutrient may act directly on the brain or through other tissues such as the liver and body fat. A less likely, but consistent explanation of the changes in feeding behavior is the altered stimulation of the lower small intestine by nutrientrich thyme. The rats may have eaten the amount of food that they did to maintain a constant level of undigested food in the ileum, Of course, a combination of all of the factors that we have discussed-the amount of food absorbed, the stimulation of the ileum with thyme and the changes in the amount of body fat-may be necessary to explain the large and sustained changes in food intake that were observed. Further experiments are needed to sort out the relative importance of these different possible explanations. Stomach signals have received a lot of attention in recent years because they are thought to control short-term feeding behavior [ll, 13, 39, 531. The large and persistent changes in food intake seen in the present experiment can not be readily explained by signals arising in the stomachs of these rats. Food eaten by either one of these one-way crossed intestines rats goes directly to the stomach. Since one rat ate 3 to 4 times the amount of food as its partner, the stomach of this rat will have had to hold and process 3 to 4 times the amount of food. If the stomach were able to assess the quantity of food that is eaten by the rat and if the stomach alone controlled food intake, then it should have sent back signals that would bring the intake of these rats back to normal levels. Apparently, short-term stomach signals are overridden by long-term signals such as the amount of food absorbed or lower gut signals f43]. A further study is needed to determine whether there are changes in the spontaneous stomach emptying rate of these rats. The studies should be done after the rats had fasted for a period that was sufficiently long that the contents of the stomach and upper intestines had emptied out. In this case, the measured rate of stomach emptying would not be affected by the recent nutritional state of the animal and the presence of food in other parts of the upper digestive tract. For the rat of the one-way crossed intestines experiment that overate, the food eaten would probably have moved more rapidly out of the stomach than for its partner, but these changes in emptying rate may have been due to the pressure of increased food intake and increased gastric fill and not to an increased spontaneous rate of stomach emptying. The results of this experiment are not in conflict with the previous work on the short-term control of food intake in two-way crossed intestines rats. In these previous studies, a 30 cm segment of upper small intestine was disconnected from the digestive tracts of both rats in a parabiotic pair [34, 35, 371. These 30 cm segments were then connected to the digestive tract of their partners in the same location where the partner’s 30 cm segment had been detached. Food eaten by either rat in the pair passed into its own stomach, travelled through 5 cm of its own upper duodenum and then crossed into the 30 cm upper intestinal segment of its partner. The intestinal thyme was partly absorbed by the partner’s 30 cm intestinal segment and the remaining thyme returned to the

601

lower jejunum of the rat that fed. In this experiment, one rat in each pair was fed at a fixed time each day and its partner was fed with a time delay of either 30 or 60 min. The objective of the experiment was to see whether food present in the 30 cm segment of the upper small intestine would affect the short-term food intake of either rat. The rat fed first would lack signals arising in its own crossed segment because that segment was connected to the intestine of the unfed partner. If signals arising in the intestinal segment were impo~ant for the short-term control of food intake, this rat should overeat. The rat fed 30 or 60 min later would already have food present in its own 30 cm segment (from the feeding of its partner) and should reduce its own intake. The results of the experiment showed that 30 cm segment was not important for the short-term control of food intake 135,371. Both rats in the crossed intestines pair ate the same amount of food as control parabiotic rats that did not have their intestines altered. The delay in the feeding of the second rat had no differential effect on the food intake of the crossed intestines rats and of control parabiotic rats. These results on short-term feeding behavior have been repeated in the present experiment. The effects of the presence of food in the 15 or 30 cm crossed segment can only be seen in the one rat of each pair because undigested food passes from only one rat to the other (see Fig. 1). As shown above, feeding the partner 30 min before feeding the test rat had no significant effect on the rat’s subsequent 30 min food intake. Thus, the short-term food intake of this rat was not affected by the presence of food in the 1.5or 30 cm crossed intestinal segment. These results are particularly interesting because the two rats of a pair show large changes in their total daily intake, but still fail to show any effect of stimulating the crossed intestinal segment on their short-term feeding behavior. Since the changes in the long-term control of total daily food intake must be attributed to the effects of the crossing of the same 30 cm segment, this study provides a pa~icul~ly clear case for differentiating between short- and long-term controls of food intake. The results of this experiment on the long-term control of food intake provide an interesting and informative contrast to the results of previous long-term studies on two-way crossed intestines rats [7,34]. In previous studies, we found that depriving one of the two rats in a symmetrical two-way crossed intestines pair (see description two paragraphs above) for a period of 5 to 15 days led to a very gradual increase in food intake in the feeding rat. That increase did not compensate for the amount of food that the partner would normally have eaten and the pair as a whole continued to lose weight. When the partner was again refed, the continuously feeding rats failed to show a substantial decrease in their food intake 171. This failure to decrease food intake was unusual because the food eaten by the other rat was being absorbed into the bloodstream of the continuously feeding rat and the pair as a whole was gaining weight rapidly. These results are in apparent conflict with the results of the present experiment because the one-way crossed intestines rats in this study show major changes in food intake that seem to occur more rapidly and that allow the rat pair to continue to gain weight. There are two major differences in the two long-term studies. In the two-way crossed intestines experiments, the rats were fed for either 11 or 14 hours per day. In the present experiment, the rats were fed for 17 hrlday. It is possible that in the present experiment. the longer feeding period allowed for greater adjustments in food intake because the capacity of the digestive tract was

602

KOOPMANS

not as seriously challenged. The difference in feeding schedule can not explain why the two-way crossed intestines rats failed to significantly decrease their food intake when lheir partners were fed again. The second major difference between the two preparations follows from the consequences of the surgery. In the two-way crossed intestines rats, each rat can lose food into the intestine of its partner. If one rat is deprived of food intake, then it can no longer lose food into the partner but it can lose digestive enzymes and shed cells into the crossed segment. These internal secretions can themselves be digested and absorbed in the partner rat. It is possible that these substances which are in contact with the crossed segment or are absorbed into the bloodstream can exert a restraint on the food intake of the singly feeding rat. Further studies are needed to make direct comparisons of these two types of rat pairs under similar conditions of schedule and diet. The changes in meal patterns that were observed in this experiment were not consistent for the rats that showed the largest increases and the largest decreases in total intake. Those rats that increased their food intake showed an increase in meal size but no change in meal number while those that reduced their intake showed a decrease in meal number but no change in meal size. Normally, these different outcomes would be interpreted as being caused by different mechanisms. In a recent study on the slow infusion of nutrients into the stomach, duodenum or jejunum of rats, Canbeyli and I found that the rats showed a consistent reduction of meal size but no change in meal number [6] and similar results have been reported by others in the literature [.5,69].We interpreted this consistent change in meal size for

the different infusions as evidence for the same type of underlying satiety mechanism. On the other hand. Collier and his students have shown that both meal size and meal number can be radically changed by simply changing the work requirements placed on rats or other animals in order to obtain food 18,321. In the present experiment, it is difficult to see how changes in one rat of the one-way crossed intestines pair would not be the inverse of changes for the partner. Perhaps, the changes in intake patterns are a consequence of the psychological strategies that the rats adopt to greatly increase or decrease their total food intake f8] and they have no major significance for underlying internal satiety mechanisms. In summary. the rats with one-way crossed intestines surgery show a substantial and sustained change in the total daily food intake. The rats in the 30 cm one-way crossed intestines pairs ate approximately 3.4 times the amount eaten by their partners over periods of months. The size of the change in food intake depends upon the length of the crossed segment of the upper small intestine and is probably related to the amount of nutrient absorbed into each rat of the pair. Signals from the lower small intestine and from the body fat may also play a role in the long-term change in feeding behavior. The study clearly shows that there are strong internal signals that control food intake. Further studies are needed to pinpoint the exact nature of the internal control system. ACKNOWLEDGEMENTS The author

thanks Mr. Barry Mahier for his fine technical assistance. The research was supported by NIH grant AM I?%!6 and by tl grant from the AHFMR.

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