Gastric bypass surgery efficiently and lastingly reduces excess body weight and reverses type 2 diabetes in obese patients. Although increased energy expenditure may also play a role, decreased energy intake is thought to be the main reason for weight loss, but the mechanisms involved are poorly understood. Therefore, the aim of this study was to characterize the changes in ingestive behavior in a rat model of Roux-en-Y gastric bypass surgery (RYGB). Obese (24% body fat compared with 18% in chow-fed controls), male Sprague-Dawley rats maintained for 15 wk before and 4 mo after RYGB or sham-surgery on a two-choice low-fat/high-fat diet, were subjected to a series of tests assessing energy intake, meal patterning, and food choice. Although sham-operated rats gained an additional 100 g body wt during the postoperative period, RYGB rats lost ∼100 g. Intake of a nutritionally complete and palatable liquid diet (Ensure) was significantly reduced by ∼50% during the first 2 wk after RYGB compared with sham surgery. Decreased intake was the result of greatly reduced meal size with only partial compensation by meal frequency, and a corresponding increase in the satiety ratio. Similar results were obtained with solid food (regular or high-fat chow) 6 wk after surgery. In 12- to 24-h two-choice liquid or solid diet paradigms with nutritionally complete low- and high-fat diets, RYGB rats preferred the low-fat choice (solid) or showed decreased acceptance for the high-fat choice (liquid), whereas sham-operated rats preferred the high-fat choices. A separate group of rats offered chow only before surgery completely avoided the solid high-fat diet in a choice paradigm. The results confirm anecdotal reports of “nibbling” behavior and fat avoidance in RYGB patients and provide a basis for more mechanistic studies in this rat model.
- bariatric surgery
- high-fat diet
- food preference
- meal patterns
- satiety ratio
the disappointing effectiveness and serious side effects of drugs have made surgical approaches very attractive alternatives for treatment or even prevention of obesity, type 2 diabetes, and other sequelae of the metabolic syndrome. Major progress in surgical methodology and experience with an increasing number of operations each year has resulted in a much-improved rate of serious complications and mortality during the last decade. Large prospective studies with up to 15 years follow-up have shown that an obese patient with a body mass index (BMI) of ≥35 has a longer life expectancy with gastric bypass surgery than without, and a >80% chance of resolving preexisting diabetes, cardiovascular disease, and sleep apneas (1, 5, 25, 33, 42, 43).
Given these impressive effects and the expected continued rise in the number of bariatric surgeries, it is vexing not to understand the mechanisms involved. Identification of these mechanisms should eventually lead to the development of pharmacological or behavioral tools without the need for surgery, or less invasive surgery. Roux-en-Y gastric bypass surgery (RYGB) involving anastomosis of the midjejunum to a small gastric pouch and completely bypassing the remaining stomach, duodenum, and upper jejunum has been found to be the safest and most efficient operation to produce sustained weight loss (26, 48), although gastric banding and sleeve gastrectomy, interventions that do not change intestinal continuity, continue to be popular. The major proposed candidate mechanisms for the superior effectiveness of RYGB are increased circulating levels of the lower-gut hormones glucagon-like protein (GLP)-1 and peptide YY (PYY), and to a lesser extent decreased circulating levels of the gastric hormone ghrelin (7, 16, 17, 19–21, 34). The effectiveness of gastric banding and sleeve gastrectomy appears not to depend on changes in gut hormone levels, but restriction of passage of food from the esophagus to the duodenum. Although there is considerable support for these candidate mechanisms, more mechanistic studies will be necessary to assess the specific contribution of each candidate, and such studies are difficult to carry out in human subjects.
A rat model of RYGB was originally applied to obese Zucker rats (38, 51) and subsequently to high-fat diet-induced obese male Sprague-Dawley rats (12, 27). These studies demonstrated that decreased calorie intake was a major factor in the weight loss and confirmed that circulating PYY levels were significantly increased in rats with successful surgeries. In addition, the most recent study of these investigators suggests that decreased endocannabinoid signaling via the CB1 receptor may be critically involved (12). In another RYGB rat model, male Sprague-Dawley rats made moderately obese by high-fat diet weighed 20% less than controls 3 mo after surgery, but, unexpectedly, serum total ghrelin levels were 38% lower than before surgery, suggesting that RYGB-induced weight loss is correlated with decreased circulating ghrelin levels (44).
A different surgical approach used vertical sleeve gastrectomy and a clip on the proximal duodenum with the cut jejunum anastomosed to the proximal stump of the duodenum (30). Compared with sham controls, these rats ate significantly less and lost significant body weight and fat tissue over 7 wk, with only an initial decrease in energy expenditure. Hypothalamic neuropeptide Y and agouti-related protein gene expression were significantly increased at the end of the study (30). Furthermore, jejunoileal bypass and biliopancreatic diversion surgery in male Wistar rats with no reduction in stomach size significantly decreased food intake and body weight and increased fasting plasma levels of both PYY and GLP-1 over a period of 28 days (4, 21). When contact of ingested nutrients with the duodenum and proximal jejunum was prevented by duodenojejunal bypass, glucose tolerance improved dramatically, and when gastroduodenal continuity was reestablished in a second operation, glucose tolerance worsened again, suggesting involvement of an anti-incretin factor originating from the duodenum (28, 39).
Observations in RYGB patients suggest that, at least initially, decreased food intake may result mainly from severely limited meal size and a lack of compensation by increased meal frequency (45). In addition, decreased acceptance for fatty foods and increased acceptance for fruits and vegetables was reported (14, 31, 33, 47). In the rat models mentioned above, these effects on meal patterns and food choice were not investigated. Therefore, the aim of our study was to develop a rat model of RYGB, to analyze meal patterns during the acute and chronic phase after surgery, and to determine food and macronutrient choice.
MATERIALS AND METHODS
Animals and Housing
Male Sprague-Dawley rats of 200 g body wt were purchased from Harlan Industries (Indianapolis, IN) and housed individually in wire-mesh cages at a constant temperature of 21–23° C with a 12:12-h light-dark cycle (lights on 0700, off at 1900). Food and water were provided ad libitum except before treatments or tests. For 16–20 wk, rats were on a three-choice diet consisting of normal laboratory chow containing 58% carbohydrates, 13.5% fat, and 28.5% protein by calories (no. 5001 LabDiet; Purina, Richmond, IN), high-fat diet containing 20% carbohydrates, 60% fat, and 20% protein (D12492, Research Diets, New Brunswick, NJ), and chocolate-flavored Ensure containing 64% carbohydrates, 21.6% fat, and 14.4% protein (Abbott Ross, Columbus, OH), with each of the diets containing sufficient minerals and vitamins. They were then randomly assigned to either RYGB or sham surgery, after which different diets were used as indicated below. An additional group of 6-mo-old rats was fed only chow before RYGB or sham surgery but, after surgery, were given the same diets as above. In addition, a group of rats that was never exposed to high-fat or Ensure diets was used as a control in some test paradigms.
All protocols involved in this study were approved by the Institutional Animal Care and Use Committee at the Pennington Biomedical Research Center in accordance with guidelines established by the National Institute of Health.
Our model consists of 1) a completely separated gastric pouch of roughly 20% of the total stomach volume created by a cutting stapler (Ethicon, Endo-surgery, cincinnati, OH) with two straight triple-staple lines between the lesser and greater curvature, 2) a jejunal transection 40 cm from the ileocecal valve, 3) an end-to-side gastrojejunostomy, and 4) an end-to-side jejunojejunostomy 25 cm from the ileocecal valve, creating a 15-cm-long Roux limb, a 25-cm-long common limb, and a roughly 40-cm-long biliopancreatic limb. All of the nerves crossing the gastric cut line are obviously transected, leading to partial denervation of the pyloric sphincter, proximal duodenum, and pancreas (2, 3). In addition, the continuity of the enteric nervous system is interrupted by the jejunal transection. However, the vagal and sympathetic supply to the gastric pouch (gastric branches), most of the intestines, pancreas, and liver (vagal celiac and hepatic branches), remain intact.
Sham surgery consisted of the same procedure, except that the transected jejunum was reanastomosed, one small incision in the jejunum 25 cm from ileocecal valve and one in the gastric fundus were sutured closed, and the cutting stapler was laid over the stomach without firing. Thus a similar amount of surgical trauma was inflicted, but the normal flow of nutrients was preserved in sham-operated rats. Because most of our endpoints were food intake and meal patterns, we did not run a control group pair-fed to the RYGB rats.
Body weight was monitored daily for the first 2 wk, and then was recorded weekly. Body composition was also measured before and 22 wk after surgery by using a Minispec LF 90 NMR Analyzer (Bruker, The Woodlands, TX). This method uses whole body magnetic resonance relaxometry in unanesthetized rodents with excellent linearity and reproducibility (18).
Measurement of Liquid Diet Intake
On the 2nd day after surgery, RYGB and sham-operated rats were given access to water and chocolate Ensure from separate drinking spouts in their home cage. Fresh Ensure was provided daily, and intake was measured during daily 21-h periods from ∼1200–0900. The health of each rat was checked, and body weight was measured during the daily 3-h maintenance period. After 10 days on Ensure only, rats were slowly accustomed to ingesting normal laboratory chow and high-fat chow by providing increasing amounts of preweighed pellets on the cage floor.
During the acute phase (weeks 2–3) and chronic phase (weeks 18–22) after surgery, patterns of Ensure intake were measured by means of monitoring lick behavior over periods of 24 h. When the rat's tongue made contact with the drinking spout, electrical conductance between electrodes connected to the cage floor and drinking spout was established, thus registering each lick in time on a computer (Vital View; Mini Mitter, Bend, OR). With the use of custom software, the number of licks was counted every second for 24 h. Initiation of a meal was defined as ≥3 licks with interlick intervals of <250 ms (8), and the end of a meal was defined by the start of a period of >5 min without licking (40). Meal duration was calculated by subtracting the time of the first lick from the time of the last lick in a meal. Meal size was calculated on the basis of the number of licks in a meal and the average calculated lick size (total amount of Ensure consumed divided by the total number of licks over 24 h). Because lick size was consistently lower in RYGB compared with sham-operated rats, we made sure that the spout size and geometry was identical for both treatment groups. In addition, to rule out differences in viscosity of Ensure after longer periods within the bottle, we also conducted short-term tests, confirming the lower lick size in RYGB rats. In addition, we trained rats to lick sucrose (0.06 M) in the brief access lick test and determined the average intraburst interlick intervals as a sensitive measure of motor performance (9). Only intervals in the range of >50 but <250 ms, typical for the 5–6 licks/s emitted within a burst, were considered.
The satiety ratio (min/g) was calculated by dividing the intermeal intervals by the amount (g) of food consumed in the preceding meal. Ingestion rate during a meal (g/min) was calculated by dividing meal size by meal duration.
Measurement of Solid Diet Intake
To assess solid food meal patterns, rats were exposed to powdered normal laboratory chow or high-fat diet as the sole source of calories for 1 wk each, ∼5 wk postsurgery. Food was presented in a continuous feeding monitoring system consisting of wire mesh cages with a small alcove leading to a food cup containing powdered diet continuously weighed by a balance and registered by a central computer. This custom-built system is spillage-proof and able to accurately analyze meal patterns over extended periods of time. Total intake was measured during the first 3 days, and meal pattern analysis was performed during days 4–6 in the feeding system on either diet. Meals were defined by at least 0.3 g eaten without interruption by a pause of >10 min.
Measurement of Food Choice
Nutritionally complete diets either low in fat (70% carbohydrates, 10% fat, and 20% protein; no. D124508-L) or high in fat (20% carbohydrate, 60% fat, and 20% protein; no. D12492-L; Research Diets) were reconstituted with water to yield 0.5 kcal/g and provided in separate bottles before dark onset. Twelve-hour dark intake was measured by weighing bottles before and after and subtracting spillage from tilting the bottles.
To measure solid food preferences, rats were presented with a choice of normal laboratory chow and high-fat chow presented fresh daily in two separate food hoppers attached to the cage during weeks 3–5 postsurgery. Intake of each diet was measured for 24-h periods subtracting carefully measured spillage.
Measurement of Energy Expenditure
Energy expenditure and respiratory exchange rate were measured after 2 days of adaptation during three consecutive days, 20 wk after surgery in a Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH).
Body weight, body composition, single-diet intake, water intake, solid food meal pattern, food preference data, mean energy expenditure, and respiratory exchange rate were analyzed with one-way or repeated-measures ANOVA, followed by Bonferroni's or Tukey's post hoc multiple-comparison tests. Ensure meal pattern data were analyzed by two-way ANOVA followed by Fisher's least-significant difference test. Solid food meal pattern and food preference data were analyzed by two-way repeated-measures ANOVA, followed by Bonferroni-adjusted multiple-comparison tests. Energy expenditure data were analyzed by repeated-measures ANOVA and ANCOVA to test if there is a common slope. Mean respiratory exchange rate was analyzed with one-way ANOVA.
Body Weight and Adiposity
Exposure to the high-fat diet for 14–16 wk increased body weight from 198 ± 3 to 484 ± 9 g. Sham surgery had only minor and transient effects on body weight (Fig. 1A). At the end of the 4- to 5-mo survival period, sham rats had gained another 140 ± 15 g body weight. In contrast, RYGB surgery resulted in rapid weight loss of 90 ± 6 g during the first 10 days and only minimal weight regain thereafter, so that 4–5 mo after surgery they weighed ∼200 g less than sham-operated rats (Fig. 1A).
Measurement of body composition revealed that, during the “fattening” period, fat mass increased from 38 ± 1.6 to 102 ± 2.3 g, and lean mass from 163 ± 4 to 321 ± 4 g, so that, at the time of surgery, adiposity was 23.2 ± 0.4% and relative lean mass was 72.8 ± 0.4%. Rats with sham surgery gained another 26 g of fat mass and 40 g of lean mass, whereas rats with RYGB lost 41 g of fat mass and 61 g of lean mass. After surgery (5 mo), relative fat mass (adiposity) was significantly lower (17.9 ± 0.7 vs. 25.0 ± 0.8%, P < 0.05), and relative lean mass was significantly higher in RYGB compared with sham-operated rats (77.7 ± 0.6 vs. 70.9 ± 1.0%, P < 0.05), but not significantly different from values before exposure to the palatable diet (Fig. 1, B and C).
Obviously the body weight curves of both RYGB and sham rats were influenced by the various dietary changes and the behavioral testing that occurred throughout the postoperative period.
Food and Water Intake
After an initially profound hypophagia typically lasting for a few days, Ensure intake in RYGB rats recovered quickly to ∼70% of sham controls 9 days after surgery (Fig. 2A). When exposed to a three-choice diet 10 days after surgery, intake initially dropped again in RYGB rats and then stabilized at ∼80% of sham rats (Fig. 2B). During this period, some RYGB rats ate large amounts of Ensure or later chow on a given day, only to eat very little the next day (yo-yo effect). Food intake later after surgery was clearly influenced by the various dietary manipulations and other behavioral tests. Nevertheless, average suppression of food intake in RYGB compared with sham-operated rats at 22–40, 41–80, and 81–150 days after surgery was 23.5+4.7% (P < 0.05), 24.4+9.7% (P < 0.05), and 27.2+19.6% [not significant (NS)], respectively. Starting at ∼2 wk after surgery, energy intake corrected for body weight did not differ between RYGB and sham-operated rats.
Water intake was measured during 5 days at ∼5 wk after surgery and compared with lean control rats. ANOVA showed no significant treatment effect (F = 2.92, P = 0.09), although RYGB rats tended to drink more compared with sham-operated rats. (Fig. 2C).
Meal Patterns with Liquid Food
The pattern of liquid Ensure intake was assessed 2–3 wk (acute phase) and 18–20 wk (chronic phase) after surgery. At the time of the acute phase measurements, RYGB rats weighed 88 g less than sham-operated controls (383 vs. 471 g), and at the time of chronic phase measurements they weighed 249 g less (373 vs. 622 g).
During the acute phase, meal size (−62%, P < 0.001), intermeal interval (−55%, P < 0.005), ingestion rate (−55%, P < 0.01), and total 24-h intake (−24%, P < 0.05) were all significantly reduced, whereas meal frequency (+107%, P < 0.0001) and satiety ratio (+63%, P < 0.005) were significantly increased in RYGB compared with sham-operated rats (Fig. 3, A–C). Lick size as calculated by dividing the total amount consumed by the total number of licks was reduced by 44% (P = 0.058). Meal duration was similar in RYGB compared with sham-operated rats, indicating that the reduction in meal size was due exclusively to a combination of slower ingestion rate and smaller lick volume. Analysis of intraburst interlick intervals when licking 0.06 M sucrose, a sensitive test for basic motor performance, revealed similar values for RYGB and sham-operated rats (RYGB: 176.4 ± 4.9 ms; sham: 182.5 ± 4.8 ms, NS).
RYGB attempted to compensate for the smaller meal size by increasing meal frequency, but fell short of full compensation as indicated by the significantly smaller total intake. Differential patterns of intake were similar for the dark and light periods (data not shown).
During the chronic phase, intermeal interval (−54%, P < 0.02) was still significantly reduced, and meal size was reduced (−34%, P = 0.08) but only at a marginal significance level. Meal frequency (+90%, P < 0.01) and satiety ratio (+70%, P < 0.01) were significantly increased in RYGB compared with sham-operated controls (Fig. 3C). Lick size (+18%, P = 0.69) and ingestion rate (−15%, P = 0.65, not shown) were no longer significantly reduced. Because the increase in frequency remained similar to the acute phase, it resulted in slightly higher (+15%, NS) total intake during the 24-h observation period in RYGB compared with sham-operated controls.
Meal Patterns on Solid Foods
At 5–7 wk after surgery, solid food meal patterns were determined, first during 1 wk on powdered normal laboratory chow (low fat) and 1 wk on powdered high-fat diet. When switched from the two-choice maintenance diet to powdered chow, sham-operated rats drastically lowered their intake to below the intake of RYGB rats and, when switched to the high-fat diet, they rebounded by almost doubling their calorie intake (Fig. 4A). Because this under- and overeating on the chow and high-fat diet moderated after 2–3 days, meal patterns were analyzed on days 4–6 after the respective switch. Furthermore, because the differential feeding pattern could be best seen during the dark period (Fig. 4B), meal pattern analysis was carried out for three consecutive 12-h dark periods.
On the chow diet, RYGB rats exhibited significantly increased meal size (+77%, P < 0.01) and meal duration (+112%, P < 0.01), significantly reduced meal frequency (−66%, P < 0.05), and similar intermeal interval, satiety ratio, and rate of eating compared with sham-operated controls (Fig. 5). Clearly, the paradoxically increased meal size in RYGB rats was because of the abnormally low intake of chow in sham rats.
On the high-fat diet, RYGB rats showed significantly decreased meal size (−45%, P < 0.01) and rate of eating (−50%, P < 0.01), significantly increased satiety ratio (+77%, P < 0.01), and similar meal frequency, meal duration, and intermeal intervals compared with sham-operated controls (Fig. 5).
Comparisons across diets further show that meal size (+170%, P < 0.01) and rate of eating (+160%, P < 0.01) expressed in calories dramatically increased in sham rats from chow to high fat, but did not significantly change in RYGB rats (Fig. 5).
The reduced acceptance of high-fat diet by RYGB rats was indicated by a lack of increase of energy intake when switched from chow to high-fat diet (Fig. 4A). Although sham-operated rats roughly doubled their energy intake, RYGB rats rather showed a trend for reduced energy intake when switched. Acceptance for low- and high-fat solid diets was assessed in direct choice tests at different time points after surgery, and in addition to RYGB and sham-operated rats, a third group of rats with no surgical intervention served as additional control. Both control groups highly preferred (96%) the high-fat diet throughout the 20-wk postoperative period (Fig. 6B). RYGB rats initially (1–7 wk postsurgery) showed only slightly reduced acceptance (86%) for the high-fat diet compared with the control groups, but acceptance decreased further (58%) during the later phase (8–20 wk postsurgery; Fig. 6B). During both periods, total calorie intake was lower (−26%, P < 0.05) in RYGB compared with sham-operated rats.
Measured at 3 wk after surgery, total intake of the two liquid diets in 12 h was only marginally smaller in RYGB rats (26 vs. 23 kcal, NS), but they ingested significantly fewer calories from the high-fat diet compared with the low-fat diet (4.0 vs. 20.0 kcal, P < 0.01), and their percent acceptance for fat was significantly lower than in sham-operated controls (20 vs. 55%, P < 0.01; Fig. 6C).
An additional group of rats was fed chow only and not exposed to high-fat diet before surgery. Similar to the obese rats, RYGB resulted in substantial weight loss and initial hypophagia compared with sham-operated controls (data not shown). Ensure meal patterns 2–3 wk after RYGB surgery revealed drastically reduced meal size and intermeal intervals and increased meal frequency and satiety ratio, similar to the effects in obese rats (data not shown).
In contrast to obese rats, lean rats after RYGB surgery completely avoided high-fat solid food, whereas sham-operated controls showed a very high acceptance (Fig. 6D). Total intake was significantly lower (−37%, P < 0.05) in RYGB rats compared with sham-operated controls.
Energy expenditure was measured during three consecutive days with the CLAMS system, 10 wk after surgery. Although there was a tendency for increased energy expenditure in RYGB rats compared with sham-operated rats if expressed on a per body weight basis [F(1,14) = 4.3, P = 0.058; Fig. 7A], this difference disappeared if expressed per body weight0.75 [F(1,14) = 0.45, P = 0.53] or per lean body mass [F(1,14) = 0.39, P = 0.54]. Furthermore, RYGB and sham-operated rats shared a common slope for the curve depicting energy expenditure as a function of lean body mass (Fig. 7B). The respiratory exchange rate was significantly higher in RYGB rats [F(1,14) = 7.64, P = 0.016], indicating increased carbohydrate oxidation (Fig. 7, C and D).
Our model demonstrates that RYGB in high-fat diet-induced obese rats almost fully reverses obesity. These findings confirm the usefulness of rat models for understanding the basic mechanisms leading to the impressively beneficial effects of RYGB surgeries performed in an increasing number of obese and diabetic patients. Here we focus on the effects of RYGB on food intake and food preference that have not been addressed in-depth in previous studies. Available data from animal models and human studies suggest decreased energy intake to be the major cause of the observed weight loss, at least during the early postsurgery period. This conclusion is supported by the following three lines of evidence: 1) direct measurement of decrease in food intake (11, 21), 2) weight loss similar to bypass in the pair-feeding paradigm (11, 38, 46, 49), and 3) minimal or absent effect on energy expenditure or energy loss in feces (10, 21, 37). We found no change in energy expenditure expressed per lean mass measured 7 wk after surgery. Furnes et al. (10) reported significantly increased energy expenditure 3 and 14 wk after gastric bypass surgery; however, if corrected for fat loss, the effect disappeared (10). In the only human study by Rodieux et al. (37), no significant change in energy expenditure was found. Together, the studies indicate that, although not increased, energy expenditure after RYGB does not show the expected weight loss-induced decrease observed in calorie restriction experiments (36). However, fecal energy loss through fat malabsorption cannot be excluded as an important contributor to the observed weight loss (12) and should be further investigated in future studies.
The significantly increased mean respiratory exchange rate in RYGB compared with sham-operated controls indicates increased carbohydrate oxidation. This suggests that RYGB rats are not in a chronically food-restricted state, which is typically associated with increased fat oxidation. Rather, RYGB rats are at a new steady-state body weight level, with increased carbohydrate oxidation reflecting preferential chow intake and increased insulin sensitivity compared with sham-operated rats.
Weight Loss and Body Composition
Our RYGB model was effective in reversing diet-induced obesity, confirming earlier studies using a similar approach (11, 12, 46). After surgery (5 mo), RYGB rats had lost ∼20% of their preoperative body weight by losing both fat mass and lean mass. In contrast, sham-operated rats gained ∼30% of body weight during this period by increasing both fat and fat-free mass. Both, percent fat mass (adiposity) and percent lean mass in RYGB rats were similar to the starting values before exposure to the high-fat diet, demonstrating full reversal of obesity.
Our surgical model was based on the pioneering studies by the group of Meguid and colleagues, the first to systematically refine the surgical procedure and study possible mechanisms of weight loss. Using control groups pair-fed to the food intake of RYGB rats, they demonstrated that ∼50–70% (depending on the time post surgery and individual study) of the weight loss can be attributed to decreased food intake, whereas the remaining 30–50% was due to mechanisms other than food intake (11, 12, 46). In another model, RYGB surgery in Wistar rats maintained on regular chow diet before and after surgery also resulted in significantly greater weight loss than pair-feeding over 3–4 wk postsurgery (4, 21). However, there was no indication of malabsorption as determined by measurement of the fecal energy content (21).
In humans, weight loss after RYGB varies widely. In the Swedish Obese Subjects Study (SOS; see Refs. 42 and 43), initial weight loss was ∼35% and sustained body weight loss after 10 years was ∼25%. This pattern is very similar to the one observed in rats in the present and other studies (11, 12, 46). However, obesity was not completely reversed in the SOS and most other human studies, as indicated by a body mass index of 32, 10 years postsurgery.
A limitation of our study is that we have not measured fat absorption, fecal energy loss, physical activity, and energy expenditure throughout the postoperative period, and we have not utilized pair-feeding, necessary to obtain a more complete characterization of mechanisms contributing to weight loss.
Total energy intake.
Energy intake was significantly lower in RYGB compared with sham-operated rats throughout the 150-day observation period, but hypophagia was most expressed during the first 2 wk, with significant improvement over time. Within the first 9 days after surgery, liquid Ensure intake of RYGB rats recovered from very little immediately after surgery to ∼65% of intake by sham-operated controls, and, after slowly adapting to regular and high-fat chow, RYGB rats ingested roughly 75% of the calories ingested by sham controls. This degree and pattern of food intake suppression is very similar to earlier studies in rats reporting decreases of food intake ranging from 10 to 50% (4, 11, 12, 21). It is also generally consistent with those few human studies reporting energy intake quantitatively. In the above-mentioned SOS study, energy intake of RYGB patients was decreased by ∼50% at 6 mo and by 12.6% at 10 years after surgery (42, 43).
Effect of switching diets.
RYGB rats generally showed less contrast-induced changes in caloric intake compared with sham-operated controls. Unlike sham-operated rats, which drastically reduced caloric intake when switched from the two-choice maintenance diet (normal and high-fat diet) to normal chow only, and increased intake when subsequently switched to high-fat only, RYGB rats showed relatively small changes. This resulted in paradoxically higher caloric intake during the first 3–4 days after exposure to normal chow in RYGB compared with sham-operated rats, and may be best explained by a strong negative contrast effect in sham rats that much prefer the high-fat diet. However, interpretation of this experiment is limited by the fact that rats were not switched from high fat to low fat (chow), in a complete crossover design. These results complement the two-choice acceptance tests and indicate that, although sham rats continue to prefer high fat, RYGB rats gradually shift preference from high fat to chow.
Two previous reports in rat models of RYGB produced variable and inconclusive outcomes regarding changes in meal patterns. One study using powdered regular chow reported strong trends for decreased meal size (−30%) and increased meal frequency (+32%), and satiety ratios (+60%), particularly during the light period (10). However, these changes did not reach the level of statistical significance. The inability to detect significant changes was likely because of the criteria used to separate meals (>10 s of feeding inactivity), which resulted in an unusually small meal size of ∼0.5 g in control rats. In the only other study reporting meal parameters, chow meal size was significantly reduced 5–10 days but not 11–19 days after RYGB in obese Zucker rats, without any changes in meal frequency (51).
Our analysis of liquid meal patterns generally demonstrated that RYGB results in decreased meal size with partial compensation by increased meal frequency/decreased intermeal intervals compared with sham-operated controls. As a consequence, satiety ratios drastically increased after RYGB and total intake decreased, at least during the first month after surgery. It has been noted early on that the typical RYGB patient learns to eat several small liquid meals a day for the first few weeks after surgery, gradually returning to a more normal pattern (45). In our RYGB rats, the smaller liquid meal size is achieved by reducing lick volume and rate of ingestion, with the meal duration remaining relatively unchanged. It does not appear that lick performance in the sense of motor capability was decreased, since RYGB rats emitted a far greater number of licks with low concentrations of sucrose and corn oil, and analysis of intraburst interlick intervals when licking sucrose was not reduced compared with sham-operated rats.
These profound changes in liquid meal patterning are likely the result of the negative impact of dumping. Under normal conditions, dumping is prevented by feedback control of gastric emptying, but, after RYGB, there is no mechanism to retain fluids or solids in the gastric pouch. Ingested food reaching the pouch is rapidly squeezed into the anastomosed midjejunum, limited only by the size of the stoma. Human RYGB patients show typical signs of the dumping syndrome by exhibiting severe nausea, light-headedness, flushing, and diarrhea after ingestion of 100 g oral glucose (45). Even though Ensure provides only ∼30% of energy from sugars, we noticed frequent reversals (yo-yo effect) of Ensure intake in some of our RYGB rats, with high Ensure intake (up to 100 ml) on one day followed by complete avoidance of Ensure the next day, during the first 10 days after RYGB. This is highly suggestive of learning through the negative effects of dumping. In a large human sample 1.5 to 4 yr after RYGB surgery, it was found that snacks accounted for 37% of their daily caloric intake (50).
It appears that, over time, slightly larger meals of Ensure are tolerated by our RYGB rats. This likely reflects adaptive changes in the Roux limb (30), minimizing the negative impact of large liquid meals.
Meal patterns on solid food (chow or high fat) revealed additional differences. Comparison between RYGB and sham-operated rats was complicated by the extreme acceptance of sham rats for the high-fat diet. This confound was particularly obvious when both groups were given regular chow as the sole diet. Paradoxically, obese sham rats ate smaller chow meals than RYGB rats (1.1 vs. 2.0 g). This meal size is very low for Sprague-Dawley rats, and typical average meal size measured with the same monitoring system and software in lean, chow diet-maintained control rats was around 2.3 g (52). This low meal size likely reflects the low preference and palatability of chow in obese sham-operated rats. The negative contrast of withdrawing the preferred diet in obese rats has been shown to induce hypophagia, anxiety, and increased locomotion (6). Surprisingly, chow meal size in RYGB rats was not substantially smaller than in chow-fed lean rats (52).
In contrast and as expected, meal size on the high-fat diet was significantly higher in sham-operated compared with RYGB rats (1.9 vs. 1.0 g). As a consequence, obese rats increased energy consumed in an average high-fat meal 2.5-fold over chow (9.5 vs. 3.8 kcal), whereas RYGB rats ate roughly the same number of calories in average high-fat and chow meals (5.6 vs. 6.0 kcal). By extending analysis of meal size in calories to liquid Ensure meals (∼1 kcal/g; see Fig. 3), it becomes quite clear that RYGB rats eat average meals of around 5–6 kcal, no matter which food. In contrast, obese sham-operated rats increase meal size to around 8–10 kcal when the food is palatable but reduce meal size drastically when the food is less preferred. It also suggests that meal size of RYGB rats is not exclusively controlled by volume but also by the macronutrient content and/or caloric density. Also, fat appears to be more satiating in RYGB rats than in obese sham-operated rats.
The mechanisms reducing meal size with solid diets are likely different than for liquid diets, since solids are more slowly squeezed through the stoma, producing less dumping. In addition, it is possible that intestinal peristalsis is compromised by the jejunal transection. Studies in RYGB patients suggest that, while emptying of liquids is accelerated, emptying of solid food is slower (13, 29, 32), and 1 mo after surgery, gastric emptying rate measured 4 h after the start of 2-h access to regular chow was ∼25% slower after RYGB compared with control obese Sprague-Dawley rats (46). The slower emptying of solids could bring mechanosensory mechanisms in the gastric pouch into play to explain RYGB-induced reduction of solid food meal size. Reaching critical distension of the gastric pouch earlier and activating gastric vagal afferent mechanosensors seems one plausible mechanism (35). However, the remarkably similar meal size in calories in the face of widely varying volumes suggests a major role for postgastric effects in limiting meal size after RYGB. This could be accomplished by exaggerated secretion of anorexigenic gut and pancreatic hormones such as PYY, GLP-1, and amylin, and/or enhanced meal-induced suppression of ghrelin secretion, in a calorie-dependent manner (15, 16, 21, 22). To identify the potential mechanisms underlying reduced meal size in our rat model, future studies will have to measure pouch emptying, intestinal transit time, gut hormone profiles, and changes in vagal activity with different diets. It will also be important to elucidate how these changes in hormonal and neural signals from the gut bring about changes in meal size and total food intake.
Our RYGB rats clearly demonstrated decreased acceptance for high-fat diet compared with sham rats, and this effect increased with time postsurgery. These results generally confirm observations in RYGB patients. In one study, preference for high-fat over low-fat potato chips before surgery was reversed after RYGB surgery (47). In another study, the number of high-fat food items selected from a food preference checklist before a test meal was significantly reduced after jejunoileal bypass surgery compared with before surgery (31). Many RYGB patients reported various combinations of nausea, vomiting, and abdominal pain after ingestion of milk products, especially ice cream, while some of them could tolerate skimmed or 1% fat milk (14). Finally, in a more recent study, laparoscopic RYGB but not vertical-banded gastroplasty resulted in avoidance of fatty foods in one-third of patients, as measured at 1 yr after surgery (33).
Together, the findings suggest that RYGB changes food choice, with lower acceptance of fatty foods. The mechanisms leading to decreased fat acceptance are not known. One potential mechanism is through learning to avoid unpleasant or painful consequences after ingestion, as indicated by the study of Kenler et al. (14). These effects could be mediated by vagal or spinal afferents. The other alternative involves increased “natural” satiety for fatty foods induced by changes in gut hormone release and their actions on the brain. However, this is unlikely to be mediated by cholecystokinin (CCK), the gut hormone preferentially released by dietary fat and protein (23). Because food no longer passes through the upper small intestine, it is less likely to stimulate CCK secretion, although it has been shown in dogs that CCK can be released by infusing fat in the distal small intestine (24). There are few and inconsistent reports on CCK in RYGB patients or animal models. One study in rats reported significantly increased plasma CCK levels 14 but not 28 days after RYGB compared with sham surgery (10).
In lieu of CCK, exaggerated PYY and GLP-1 responses after RYGB are the major candidates for inducing fat-specific satiety, although the neural mechanisms involved are unknown. It has been shown that GLP-1 released from taste receptor cells signaling in a paracrine fashion through GLP-1 receptors on afferent nerve endings may enhance sweet taste sensitivity, but it is unlikely that this pathway is modulated by changes in circulating GLP-1 (41).
Conclusions and Perspectives
RYGB is presently the only highly effective obesity treatment, but the mechanisms mediating hypophagia, weight loss, and resolution of diabetes are unknown. Changes in gut-brain communication involving both hormones and neural pathways are thought to be the major candidates for these beneficial effects. Although RYGB patients are increasingly used in controlled clinical studies, animal models will also be important for identification of the underlying neural mechanisms. Here we present a rat model of RYGB that produces similar weight loss and changes in eating behavior as has been described in RYGB patients. Therefore, this model should serve well for more invasive mechanistic studies.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-47348 (H.-R. Berthoud), and the Alberta Children's Hospital Professorship in Pediatric Surgical Research (D. L. Sigalet).
The technical support of Laurie Wallace and Elaine de Heuval is gratefully acknowledged.
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