Body fat reserves attenuate gastric ulcers induced by restricted feeding in rats

Body fat reserves attenuate gastric ulcers induced by restricted feeding in rats

Physiology & Behavior, Vol. 59, Nos. 4 / 5 , pp. 931-936, 1996 Copyright @ 1996 ELsevier Science Inc. Printed in the USA. All rights reserved 0031-938...

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Physiology & Behavior, Vol. 59, Nos. 4 / 5 , pp. 931-936, 1996 Copyright @ 1996 ELsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/96 $15.00 + .00

ELSEVIER

SSDI 0031-9384(95)02169-8

Body Fat Reserves Attenuate Gastric Ulcers Induced by Restricted Feeding in Rats I M G A P YI A N D F R I E D R I C H K. S T E P H A N 1

Neuroscience Program, Department of Psychology, Florida State University, Tallahassee, FL 32306-1051 USA Received 31 May 1995 YI, I. AND F. K. STEPHAN. Body fat reserves attenuate gastric ulcers induced by restricted feeding in rats. PHYSIOL BEHAV 59(4/5) 931-936, 1996.--The purpose of this experiment was to explicitly test the hypothesis that the depletion of fat reserves is a critical factor in the generation of ulcers in the glandular stomach. Fat reserves were systematically manipulated by using chow vs. high-fat diets and by using rats of different ages (3, 7, and 17 months). The ulcerogenic procedure consisted of limiting food intake to 6 g of chow per day. For chow-fed rats, 20 days were required to induce ulcers in 3-month-old rats whereas more than 40 days were required in 7- and 17-month-old rats. In 7-month-old rats fed a high-fat diet, the ulcerogenic phase lasted 63 days. Virtually no ulcers were found in control groups with larger body weights that were yoked to the duration of the ulcerogenic phase. Measurement of retroperitoneal and epididymal fat pads indicated that these were nearly depleted in rats with ulcers whereas some fat remained in ulcer-free rats, regardless of age, diet, or the duration of the ulcerogenic phase. These results suggest that the depletion of fat reserves to a critical level triggers a metabolic stress response that then produces gastric ulcers. Restricted feeding High-fat diet Stress ulcers Retroperitoneal fat pads Epididymal fat pads

Glandular stomach

Body fat reserves

being exposed to an A-S ulcer procedure, rats show a similar high running activity with a large increase in diurnal activity, which is accompanied by severe gastric ulcerations (19). This suggests that the depletion of fat reserves could play an important role in the formation of gastric ulceration. Furthermore, the depletion of fat reserves due to the low energy input during restricted feeding and the high energy expenditure of wheel running can be expected to aggravate ulcerations. Although rats exposed to restricted feeding without activity wheel access spend less energy compared to A-S rats, if energy input is low enough to result in fat depletion these rats may also develop gastric ulcers. Previous experiments in our laboratory, show that rats housed in hanging cages, as well as those housed in activity wheels, developed gastric ulcers in the glandular stomach if the daily amount of food was restricted to 6 g per day and in both conditions terminal body weight had a high negative correlation with the severity of ulceration (20,21), Furthermore, total food deprivation or access to nonnutritive cellulose also induced ulcers in the glandular portion of the stomach, regardless of whether the animals had access to activity wheels or were housed in hanging cages (22). These results strongly suggest that reduced food intake and the subsequent depletion of body fat reserves play an

IN the activity-stress (A-S) ulcer model developed by Pare and Houser (12), it is assumed that the limited food access time of 1 h / d a y creates a stress that induces high levels of wheel running, which in turn leads to ulcerations of gastric mucosa in a few days. However, the short food access time necessarily results in a severe reduction in food intake and a loss of body weight. These factors themselves could be sufficient to induce ulcers. During fasting, after initial usage of glycogen and protein for 1 or 2 days, animals mainly rely on fat reserves (3). Therefore, body weight loss reflects mainly the depletion of fat reserves. The length of fasting tolerance depends on the accumulation of fat prior to the fasting. An 8-week-old rat, with an adiposity of about 10%, tolerates only 4 - 5 days of fasting, but a rat of the same age, with an adiposity of 20% due to a high-fat diet, survives twice as long (4). With prolonged fasting, whether in activity wheels or in hanging cages, rats use body fat reserves through three distinct phases in terms of protein utilization. In phase I, protein utilization is minimized after initial increase. In phase II, protein utilization remains at a very low level, whereas lipid utilization is high. Finally, in phase III, lipid reserves are depleted and protein utilization rises rapidly while at the same time locomotor activity increases sharply with high diurnal activity (1). A few days after

To whom requests for reprints should be addressed.

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important role in ulcerogenesis, whereas the high energy expenditure incurred by wheel running contributes to the severity of resulting ulcers. The purpose of the present study was to examine the potential influence of body fat reserves on the susceptibility of rats to gastric ulceration induced by restricted feeding. In the present study only one housing condition (hanging cage) was used because our previous results indicate that decreased food intake is a sufficient ulcerogenic factor (20-22). Body weights were manipulated by using rats of different ages and by providing different diets to reveal the relationship between body fat reserves and susceptibility to ulceration caused by food restriction. In addition, remaining adipose tissue from two most distinct fat depots (retroperitoneal and epididymal) was measured at the conclusion of the experiment. Based on the adipose depletion hypothesis, the expected outcome was that the ulcerogenic phase would be significantly longer in obese and older rats than in lean or young rats and that rats with higher body weights yoked to the duration of the ulcerogenic phase would not have gastric ulcers. METHOD

Animals and Housing Male Sprague-Dawley rats were purchased from Charles River Laboratory at three different time points of the experiment. They were housed in a colony maintained at a controlled ambient temperature (22 + 2°C) on a standard LD 12:12 cycle (lights on 0700 h). Food (Purina Rat Chow, No. 5001) and water were available ad lib. Fresh water was provided at 2- or 3-day intervals. After about 10 days of habituation, rats were moved to the laboratory and housed individually in standard hanging cages. Food was provided in a glass jar secured to the inside of the cage. Some rats continued to receive chow (powdered) whereas others were fed a high-fat diet consisting of chow, vegetable oil (Crisco®), and chocolate (Quick®/Nestle®) in the ratio of 2, 1, and 1 in volume, respectively.

Procedures Rats were fed chow or the high-fat diet ad lib until they reached a certain age or body weight criterion (see below) before

the ulcerogenic phase started. To induce ulcers, all groups received 6 g chow / d a y , given at 0900 h. Access time was not limited. The amount of food was selected because it proved effective in causing ulcers in about 50% of rats housed in hanging cages (20) and approximates average food consumption when access time is limited to 1 h [e.g., (19)]. By holding food amount, rather than access time, constant, both within- and between-group variability in food intake was avoided. In the experimental groups, the duration of the ulcerogenic phase was determined for individual rats by weight loss to a certain body weight criterion, which based on previous experiments (20,21) was at or slightly below the body weight at which rats developed ulcers and close to the estimated depletion of fat reserves. In a few cases, rats were sacrificed prior to the criterion based on behavioral assessment of signs of weakness, reduced movement or alertness, or failure to consume the 6 g of food. These symptoms are good predictors of ulcers in the A-S paradigm (8). Yoked control groups were sacrificed on the day when the last of the rats in the experimental group was sacrificed. Consequently, the average duration of the ulcerogenic phase for these controls was slightly longer than for the experimental groups (see Table 1). Seven groups were formed, representing combinations of age and diet as follows: 1. Group 3MC (3-month-old/chow; n = 8). This experimental group was fed ad lib chow until rats were 76 days ( < 3 months) old at the start of the ulcerogenic phase. A second group of young rats received a high-fat diet but failed to gain weight significantly faster than the chow fed group and was discarded from the experiment. Consequently, a group of 7-month-old chow-fed rats (below) was yoked to the duration of the ulcerogenic phase. 2. Group 7MCY (7-month-old/chow/yoked control; n = 7). This control group received ad lib chow until they were 224 days ( > 7 months) old at the start of the ulcerogenic phase. The length of the ulcerogenic phase was " y o k e d " to that of group 3MC. 3. Group 7MC (7-month-old/chow; n = 8). This experimental group received ad lib chow until they were 224 days ( > 7 months) old at the start of the ulcerogenic phase. 4. Group 7MFY (7-month-old/fat diet/yoked control; n = 6). This group received a high-fat diet ad lib for 138 days until they were 224 days ( > 7 months) old at the start of the

TABLE 1 SUMMARY OF ULCER INCIDENCE, INITIAL AND TERMINAL BODY WEIGHTS, BODY WEIGHT LOSS, WET WEIGHTS OF RETROPERITONEALAND EPIDIDYMAL FAT PADS, AND DAYS ON FEEDING SCHEDULES

Group

N

Days on Sched.

3MC 7MCY 7MC 7MF 7MFY 17ML 17MHY

8 7 8 8 6 6 6

20.5 23.0 41.8 62.6 48.0 43.7 48.0

Rats With Ulcers

Area of Ulcers (mm 2)

Initial Body Weight /g)

Terminal Body Weight (g)

Body Weight Loss (g)

Retroperitoneal Fat Pads (g)

Epididymal Fat Pads (g)

7 ! 4 5 0 5 1

2.97 0.01 1.28 2.51 0.00 18.98 0.08

371.6 623.6 636.5 793.8 767.8 651.5 743.5

196.9 425.3 310.4 318.8 408.2 312.3 374.3

174.8 198.3 326.1 475.0 359.6 339.2 369.2

0.00 4.76 0.31 0.80 5.91 0.00 0.53

0. l 0 4.59 0.53 1.34 4.96 0.32 1.18

3MC: 3-month-old/chow-fed rats. 7MCY; 7-month-old/chow-fed rats, yoked to 3MC rats. 7MC: 7-rnonth-old/chow-fed rats. 7MF: 7-momh-old/high-fat diet-fed rats. 7MFY: 7-month-old/high-fat diet-fed rats, yoked to 7MC rats. 17ML: l%month-old/chow-fed/low body weight rats. 17MHY: 17-month-old/chow-fed/heavy body weight rats, yoked to 17ML rats.

FAT RESERVES AND STRESS ULCERS

ulcerogenic phase. The length of the ulcerogenic phase was "yoked: to that of group 7MC. 5. Group 7MF (7-month-old/fat diet; n = 8). This experimental group received a high-fat diet ad lib for 138 days until they were 224 days ( > 7 months) old at the start of the ulcerogenic phase. 6. Group 17ML (17-month-old/lean; n = 6). This experimental group was fed ad lib chow until rats were 522 days ( > 17 months) old at the start of the ulcerogenic phase. They were selected from a group of 12 rats, based on the lowest body weights. 7. Group 17MHY (17-month-old/high body weight/yoked control; n ~ 6). This yoked control group consisted of the six heaviest of a group of 12 rats at 522 ( > 17 months) days of age at the start of the ulcerogenic phase. The length of the ulcerogenic phase was yoked to group 17ML.

Assessment of Ulcer Incidence Rats were euthanized with an overdose of pentobarbital. After ligating the esophagus and duodenum, the stomach was inflated by injection of 8 ml 10% formalin solution to flatten out the major folds. The stomach was then removed, cut along the greater curvature, rinsed with 10% formalin, pinned flat on a paraffin bed, and preserved in a glass container filled with a 10% formalin solution. All stomachs were inspected under a low power ( 1 0 × ) light microscope and loose blood clots were removed by spraying a 10% formalin solution over the tissue. An image processing system (Macintosh II Quick Capture Board with S / W Image 1.22) was then used to outline and quantify the total area of ulceration in each stomach. Shallow erosion that did not appear to penetrate the mucosal layer was noted but not included in this analysis.

Quantification of Body Fat The retroperitoneal fat pads were removed by incising the sac membrane covering the pads and dissecting and pulling them out. Epididymal fat pads were also removed by dissecting them away from the major blood vessels at the base of the pad. These pads were separately stored for about 10 days in small glass container filled with 10% formalin. Prior to weighing, they were blotted with soft tissue paper and left exposed to room air for 2 h. Wet weights of the retroperitoneal and epididymal pads were measured separately.

Statistics Differences in ulcer incidence, number of days on the ulcerogenic feeding schedule, terminal body weight, body weight loss, and remaining fat pads weight among the seven groups were assessed using a one-way ANOVA. If this analysis showed a significant main effect ( p < 0.05), subsequent comparisons of groups were done with a post hoc Duncan test. Pearson productmoment correlation coefficients were calculated for some of the variables of interest.

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Ulcer Incidence and Days on Restricted Feeding Twenty-one of 30 rats in the four experimental groups had ulcers in the glandular portion of the stomach whereas only 2 of 19 rats in the yoked control groups had ulcers. The latter were very small, almost punctate lesions. Because body weights were manipulated by age and diet, overall analysis of variance (ANOVA) showed highly significant weight differences in initial body weight [ANOVA: F(6, 48)= 28.30, p < 0.0001] and in terminal body weight [ANOVA: F(6, 48) = 22.38, p < 0.0001]. As expected, differences in the duration of the ulcerogenic phase among experimental groups were also highly significant [ANOVA: F(3, 29) = 82.62, p < 0.0001]. Post hoc comparisons using the Duncan test showed that 7-month-old rats on a high-fat diet (7MF) required significantly more days to develop ulcers than the other experimental groups ( p < 0.05) and that 7- and 17-month-old experimental rats (7MC and 17ML) took longer to develop ulcers than the 3-month-old (3MC) rats ( p < 0.05). An ANOVA revealed significant between-group differences in the accumulated area of ulcers, F(6, 48) = 7.65, p < 0.0001, which was mostly attributable to differences between the yoked and nonyoked groups. A selective comparison among the four experimental groups also showed a significant difference in ulcer severity, F(3, 29) = 7.48, p < 0.001, and post hoc comparisons revealed that this difference was solely attributable to the larger ulcers in old, lean rats (i.e., these rats had substantially larger ulcers than the younger experimental rats) (Duncan test: 17ML vs. 7MF, 7MC, 3MC; all p < 0.05), but no significant differences were found among the latter three groups (all p > 0.05).

Body Weight and Body Fat Figure 1 shows the group mean initial and terminal body weights, body weight loss, and the number of days of restricted feeding each group was exposed to. Although there were significant between-group differences in absolute body weight loss, F(6, 48) = 40.37, p < 0.0001, and in percent body weight loss, F(6, 48)= 40.97, p < 0.0001, mean daily body weight losses were almost same, ranging between 7.0 and 7.6 g per day (after about a 30 g loss on the first day), regardless of age or initial 1000 B 0 D Y

800

W E

68O

I G

H T

400

200

3MC

7MC

7MF

17ML

7MCY

7MFY

17MHY

GROUP

RESULTS

Table 1 contains a summary of the data for each group: the number of days on the ulcerogenic feeding schedule, the number of ulcerated rats, the accumulated area of ulcers, initial body weights (last day prior to the ulcerogenic procedure), terminal body weights (prior to sacrifice), weight loss during the ulcerogenic procedure, and wet weights of the retroperitoneal and epididymal fat pads.

FIG. 1. Mean initial (full height) and terminal (dark area) body weight, body weight loss (light area), and number of days of restricted feeding (numbers on top of each bar). 3M, 7M, and 17M designate approximate ages in months. C = chow and F = high-fat diet prior to the ulcerogenic phase, L and H designate low and high body weights. Y = control group yoked for the duration of the ulcerogenic phase. The four experimental groups are on the left and the three yoked control groups are on the right side of the figure.

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body weight in each group. Consequently, body weight loss mainly reflected the number of days on restricted feeding each group had been exposed to (Pearson correlation coefficient: r = 0.95, p < 0.001). There were significant differences in terminal body weights among groups, F(6, 48) = 22.38, p < 0.0001, which consisted of differences between each pair of yoked and experimental groups (3M vs. 7MCY: t = 13.21, p < 0.001; 7MC vs. 7MFY: t = 2.77, p < 0.05; 17ML vs. 17MHY: t = 7.94, p < 0.001) and among experimental groups, F(3, 29) = 27.7614, p < 0.0001. As shown in Table 1, the experimental rats were characterized by an almost complete depletion of fat reserves. In particular, retroperitoneal fat pads were absent in 3MC and 17 ML rats, the two groups with the highest incidence of ulcers. The experimental groups with the lowest ulcer incidence (7MC and 7MF) had an average of 0.3 and 0.8 g, respectively, of retroperitoneal fat remaining. Epididymal fat pads showed the same trend. Wet weights of retroperitoneal, as well as epididymal fat pads, were significantly different among groups [F(6, 48) = 4.52, p < 0.01; F(6, 48) = 6.08, p < 0.1301]. The yoked 7-month-old group, which had the highest terminal body weight, had significantly heavier retroperitoneal and epididymal fat pads than the other groups (Duncan test: p < 0.05). Figure 1 shows clearly that the terminal body weights for three of the experimental groups (7MC, 7MF, 17ML) were very similar and less than that of the three yoked control groups, which were virtually ulcer free. On the other hand, for young rats (3MC), the terminal body weight at which ulcers occurred was considerably lower. Of the eight 3MC rats, seven rats with ulcers had a terminal body weight below 205 g and had little retroperitoneal or epididymal fat reserves, whereas the ulcer-free rat had a terminal body weight of 227 g and 0.81 g of epididymal fat (no retroperitoneal fat). Out of eight 7MC rats, four rats with ulcers and two rats with signs of mucosal erosions (but not with ulcers) had terminal body weights below 298 g and little fat reserves, whereas one of the remaining rats had ulcers despite 391 g of terminal body weight and 4.58 g of fat pads. Another rat with 333 g body weight and 0.12 g of fat pads had no ulcers. Among eight of the 7MF rats, five rats with ulcers and one rat with erosions had terminal body weights below 304 g and epididymal fat pads less than 0.71 g (no retroperitoneal fat), whereas two other rats without ulcers had terminal body weights of 397 g and 382 g, and 8.61 g and 6.34 g of fat pads, respectively. All 17ML rats had body weights below 330 g and no retroperitoneal fat pads with

A R E A

50 40, 30

O F

20:

U

1o

C, E R

o

~IO GIIo 0o ( D o

o oo

O

h (ram')

Wet

weight of epididymal fat pads (g)

FIG. 2. Area of ulcers in mm 2 as a function of wet weight of epididymal fat pads: ulcerated rats ( 0 ) , nonulcerated rats (C)). Note that most of ulcerated rats are clustered around 0.5 g and that all rats above 3 g were

ulcer free.

small remnants of epididymal fat of less than 0.48 g. Only one rat did not have ulcers. Most of the 19 yoked rats still had substantial fat pads at sacrifice and only two rats had very small punctuate ulcers (0.01 and 0.08 mm 2, respectively). Thus, with the few exceptions noted above, terminal body weights and depletion of fat pads were good predictors of ulcers within and between groups. In general, for 3-month-old rats there was a high probability of ulcers when their terminal body weights decreased to around 200 g. Older rats started to have ulcers when their terminal body weights decreased to the range of 330-300 g, despite big differences in initial body weights or body weight loss. Figure 2 shows the area of ulcers as a function of wet weight of epididymal fat pads for individual rats in all experimental groups. Regardless of age, obesity, or terminal body weights, ulcers developed when epididymal fat pads decreased below a certain level (of about 0.5 g) and none of the rats with fat pads of 3 g or more had ulcers. DISCUSSION

Twenty-one (70%) of the 30 experimental rats that were exposed to restricted feeding of 6 g lab chow per day had ulcers in the glandular portion of the stomach. No ulcers or erosions were observed in the nonglandular portion of the stomach (rumen). Thus, unlike total food deprivation, which causes ulcers in the rumen (13,15) or in both portions of the stomach (22), severely curtailed and prolonged food restriction causes only glandular ulcers. As expected, control groups yoked to the duration of restricted feeding but with higher initial and terminal body weights than their paired groups, were ulcer free except for 2 of 19 rats with small punctate lesions. This result again supports our findings in previous studies that curtailed daily food intake (and subsequent body fat depletion) is a sufficient condition for generating glandular ulcers (20-22). In these previous studies, it was also the case that ulcer severity was highly correlated with terminal body weight and that ulcers developed mostly in animals in which terminal body weights had decreased below a certain level, depending on the initial body weight, that is, from 300 g to about 210 g, from 310 g to about 220 g (20), and from 270 g to about 170 g (22). These critical weight levels seemed to differ depending on the ages of the animals used in each experiment, because all were exposed to the same ulcerogenic procedure (i.e., ad lib chow feeding prior to each experiment and then restricted feeding of 6 g/day lab chow). In the present study, the critical levels of terminal body weights for ulceration fell into two groups (i.e., about 205 g for 3-month-old rats and between 300 and 330 g for 7- and 17month-old rats). It is interesting that in the 7-month-old and older groups the critical terminal body weights needed to induce ulcerations were very similar, despite enormous differences in their initial body weights and ages. This suggests that body weight gain after a certain age depends mostly on increases in body fat reserves with relatively small increases in bone and lean body mass. A recent study of growth in male Wistar rats shows a steep increase in body weight, hindlimb length, and weight of the plantaris muscle between 2 and 10 weeks of age (16). Thereafter, hindlimb length stayed nearly constant whereas body and muscle weight increased at a much slower rate. An earlier study indicates that male Sprague-Dawley rats housed in colonies gained little body weight and showed only minor changes in body composition between 11 and 18 months (5). Consequently, it seems reasonable to assume that the 3-month-old rats in the present study were still in the rapid growth phase whereas the older rats

FAT RESERVES AND STRESS ULCERS

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were in a relatively slow growth phase and that this accounts for the difference in terminal body weights needed to generate ulcers. In earlier A-S studies, as well as our previous experiments, ulcers were observed between 5 and 14 days of restricted feeding (8,19). In contrast, the experimental groups in the present study were on restricted feeding for 20 to 63 days before ulcers occurred, with rats having the highest initial body weights having the longest ulcerogenic phase. Although this result was in the expected direction, the number of days required to produce ulcers was somewhat surprising. The rate of weight loss was very similar in all groups (experimental and yoked controls), averaging about 7 g per day. This low rate of weight loss is probably the result of metabolic adaptations that take place when food supply is curtailed [e.g., (6)]. The length of the ulcerogenic phase was, of course, dependent on the initial body weight, which in turn is highly indicative of fat reserves. Most importantly, this result indicates that a reduced food supply itself is not ulcerogenic unless it persists until fat reserves are nearly depleted. Yoked control rats were also maintained on restricted feeding for 23 to 48 days but were virtually ulcer free, presumably because some fat reserves remained. Measurements of wet weights of the retroperitoneal and epididymal fat pads confirmed this interpretation (see below). Interestingly, it appeared that retroperitoneal fat reserves were depleted earlier than epididymal fat reserves. Some rats without ulcers or erosions were depleted of the former, but not of the latter. Most rats with ulcers had little or no retroperitoneal fat and very small reserves of epididymal fat. Therefore, it is likely that prolonged restricted feeding results in depletion of body fat reserves, with some priority for the utilization of the retroperitoneal fat. Although no ad lib-fed rats were included in this study, previous results indicate that epididymal fat pads in male Fischer rats increased from about 5 to 8 g between 6 to 18 months of age (2). By comparison, in the present study, the remaining epididymal fat pads in the three yoked control groups ranged from 1.2 to 5.0 g. Figure 2 clearly shows that body fat depletion is the most critical factor in ulceration by restricted feeding, because ulcers appeared almost exclusively in animals that had utilized most epididymal fat reserves, regardless of age or adiposity at the beginning of the ulcerogenic restricted feeding. Again, terminal body weight levels critical for ulceration were considerably different for 3-month-old rats than for older rats. Furthermore, percent body weight loss was not a good predictor of ulceration because it depended on the adiposity of the animals prior to the ulcerogenic phase. For example, in this study, none of the 7-month-old rats on the high-fat diet (7MFY) had ulcers after 48 days of 6 g chow per day, whereas all but one of the 3-month-old chow-fed rats (3MC) had ulcers although the former lost far more body weight (in both percent and absolute measurements). In addition, 7MFY rats had been on restricted feeding more than twice as long as 3MC rats (see Table 1). Unfortunately, the attempt to include a 3-month-old dietary obese group failed because weight gain was not sufficiently faster

for young rats on the high-fat diet. A similar result has been reported recently, that is, postweanling rats fed a high-fat diet for 4 weeks were not significantly heavier than those fed a low-fat diet (18). The present results show that the group averages of ulcerated area were not reliably different in 3- and 7-month-old rats. However, the l%month-old rats had ulcers six times larger than the 3MC rats and 15 times larger than the 7MC rats. Compared to the 7MC rats, the 17ML rats had similar initial and terminal body weights and were exposed to about the same number of days of restricted feeding. One possibility is that older rats are more susceptible to ulcers induced by food restriction. However, another possibility is that the number and size of ulcers increases rapidly once the ulcerogenic process is triggered, that is, if these rats had been sacrificed 1 or 2 days earlier, the ulcer incidence and size might have been comparable to those in the other groups. Age effects have not been observed consistently using waterrestraint stress to induce ulcers (11) and may be strain dependent (9,10). In the A-S ulcer paradigm, 100-g rats began to die after 5 days, 200-g rats after 12-13 days, and 300-g rats after 17-18 days (8). However, younger rats had higher activity levels than older rats and this undoubtedly contributed to these results. Nevertheless, it is of interest that this is one of very few studies to find ulcers in food yoked controls housed in hanging cages. Ulcers were observed in 8 of 15 rats in the 100-g body weight group but not in the heavier animals (8). It seems reasonable to assume that depletion of adipose reserves plays an important role in the A-S ulcer paradigm. Procedures that attenuate these ulcers [e.g., preadaptation to limited feeding time in hanging cages (7,14) or multiple short access times (17)] result in higher daily food intakes during the ulcerogenic phase, which may reduce ulcer incidence and size. On the other hand, when food intake in hanging cage housed controls is yoked to that of rats in activity wheels, the high levels of activity result in greater weight loss [e.g., (8)]. Because body weight, age, weight loss, food intake, and activity levels are interrelated, it is difficulty to ascribe causality to any particular variable. The design used here and in previous studies (20-22) eliminates activity levels and differences in food intake as variables. The results provide strong evidence that depletion of adipose tissue to a critical level is sufficient, and perhaps necessary, to induce ulcers in the gastric mucosa. At this point, the mechanisms by which depletion of fat reserves leads to ulcers in the gastric mucosa are not known. Possibly, a decline in available free fatty acids, elevated ketone bodies, or the beginning of protein catabolism induces a metabolic stress response involving the autonomic nervous system that then leads to ulcerogenesis. The procedure used to induce such ulcers could be regarded as a "metabolic stress ulcer" model. ACKNOWLEDGEMENTS The authors would like to thank Dr. Karen Glendenning for the use of an image processing system for ulcer assessment. This work was partially supported by the FSU Foundation.

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