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Previous studies have shown that administration of the fatty acids, linoleic and oleic acid, either by intragastric or intraintestinal infusion, suppresses food intake and body weight in rats. While still not fully understood, gut-mediated satiety mechanisms likely are potential effectors of this robust response to gastrointestinal fatty acid infusions. The objective of this study was to assess the effects of voluntary access to an oleic acid derivative, ethyl oleate (EO), on subsequent food intake and body weight in rats. Animals were randomized either to a 12.5% EO diet or a soybean oil diet as a “breakfast,” followed either by two one-hour or one five-hour access periods to standard rodent diet, and food intake and body weights were collected. Across 14 days access, rats consuming EO on both feeding schedules gained less weight and consumed less total kilocalories than rats consuming the SO diet. Further, plasma levels of glucose and insulin were comparable in both EO and SO diet groups. In summary, EO was found to increase weight loss in rats maintained on a 75% food-restriction regimen, and attenuate weight-gain upon resumption of an ad-libitum feeding regimen. These data indicate that voluntary access to EO promoted short-term satiety, compared to SO diet, and that these effects contributed to a important and novel attenuated weight gain in EO-fed animals.
Regulation of body weight involves a complex interaction of peripheral and central signals, and is governed in part by peripheral body fat stores, circulating factors such as insulin and leptin, and short-term meal-related cues (1, 19, 21). A large body of literature suggests that some of these meal-related signals originate in the gastrointestinal tract and serve to terminate ingestion (5, 11). Previously, studies have shown that intraintestinal infusions of long-chain fatty acids significantly reduced voluntary food intake (4, 16): the mechanisms that control this response are complex, but likely involve gut-initiated satiety signals (9). Numerous gastrointestinal peptide hormones are released during and immediately after food intake, including cholecystokinin, peptide YY, glucagon-like peptide 1, and oxyntomodulin, which act either distally, signaling satiety and suppressing food intake via receptors located in the central nervous system, or proximally, by slowing gastric emptying (22, 27). Importantly, numerous reports have demonstrated that infusions of nutrients directly into the small intestine can activate a satiety mechanism initiated and mediated by the gut (9, 16).
Cholecystokinin (CCK) is a well-studied gut peptide hormone, which plays a fundamental role in the gut-satiety cascade that terminates a meal (18). Specifically, CCK signals via vagal afferent neurons, which are responsible for the central transfer of peripheral sensations (2). Secreted by enterendocrine cells in the duodenal and jejunal mucosa, and released in response to the presence of fatty acids and protein in the intestinal lumen, CCK inhibits gastric emptying and stimulates intestinal motor activity (17). Together, these effects maximize nutrient absorption from the gut. Further, CCK signals satiety, reducing the number of meals, the duration of time spent eating and the total amount of food eaten (10).
Currently, the most efficacious long-term clinical interventions for morbid obesity require radical surgical procedures; therefore, the characterization of a gut-mediated, nutrient-activated satiety mechanism has important clinical implications. Numerous studies have described a reduction in food intake following infusion of fatty acids directly into the small intestine: French et al demonstrated that upper intestinal infusions of 20% oil emulsions enriched with either stearic, oleic, or linoleic acid significantly reduced food intake compared controls infused with saline, and that this reduction in food intake was accompanied with increased plasma CCK concentrations (9); Meyer et al described reductions in food intake following intraintestinal infusions of either fatty or amino acids (16); Cox et al have reported that jejunal infusions of linoleic or oleic acid elicit increased satiety, decreased food intake and decreased body weight in rats (6). Matzinger et al demonstrated that this effect is mediated by cholecystokinin, abolishing the effects of intraduodenal fat infusions on food intake with a specific CCK-A receptor antagonist (15).
Further, previous studies have reported a significant amelioration of the effects of fatty acid infusions by celiac vagal deafferentation (7, 8, 26). These data support the hypothesis that the small intestine generates a robust and important satiety signal capable of regulating meal size and, possibly, long-term food intake. Almost without exception, previous studies of gut-satiety mechanisms, have utilized only intragastric, or intraintestinal delivery of fatty acid infusions. In a study of potential therapies for chronic diarrhea, Lin et al observed decreased gastrointestinal transit times in human subjects orally administered oleic acid emulsions before meals (12). The activation of gut-mediated satiety mechanisms as a potential clinical intervention for obesity has not yet been fully addressed.
In previously published human studies, subjects orally administered oleic acid emulsions reported a slight, temporary burning sensation in the throat and mouth (12). In a recent study, Chalé-Rush et al suggest the acidic moiety of fatty acids is an irritant that serves as a physiologic cue for their presence, as detected by the chemosensory system (3). Additional studies have shown that intraintestinal infusions of oleic acid are responsible for significant and extensive mucosal damage, which is absent following infusions of an oleic acid derivate: ethyl oleate (24, 25, 30). Despite the absence of mucosal damage following ethyl oleate infusions, studies have demonstrated a long-lasting suppression of caloric intake following gastric EO infusions in rats (8).
The purpose of this study was to assess the effectiveness of an orally delivered ethyl-ester of oleic acid to reduce food intake and suppress body weight gain. To accomplish this, we placed rats on restricted food intake regimens throughout which they received access to regular chow for limited periods during the day. In addition, all rats received two-hours of fixed-volume access to an emulsion that contained either ethyl oleate (EO) or a soybean (SO) diet. Subsequent chow intakes, total kilocalorie intake, and body weights were determined. It is hypothesized that orally-administered EO enhances post-prandial satiety mechanisms, reflected by reductions in next-meal food intake and, in the long term, a decrease in body weight or a decrease in the rate of body-weight gain, in rats consuming EO, relative to rats receiving SO diet.
All procedures were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and were conducted in AAALAC-accredited facilities. Male Long-Evans rats (300–400 g; Harlan, Indianapolis, IN) were housed individually in Plexiglas tubs and maintained on a 12:12-h light-dark cycle at constant temperature and humidity (20 degrees C; humidity here). For all three experiments, lights were off from 10AM to 10PM and daily body weights, emulsion intake, and food intake of animals were measured from 9AM to 10AM in order to track changes in body weight and total caloric intake.
Diets contained, by volume, 12.5% ethyl oleate (Victorian Chemicals, Victoria, Australia) or soybean oil (Crisco®, J.M. Smucker Co, Orrville, OH), 28.1% vanilla-flavored Boost® Nutritional Energy Drink (Mead Johnson Nutritionals, Evansville, IN), and 59.4% water. Available caloric densities were calculated to be 1.08kcal/ml for ethyl oleate diet, and 1.26kcal/ml for soybean oil diet. Both diets were prepared by mixing, followed by brief sonication to promote emulsification, and were administered in 50ml sipper tubes placed on top of the home-cages.
For all experiments, animals were maintained on a pelleted rat diet (Harlan-Teklad, Indianapolis, IN) with a caloric density of 3.4kcal/gram.
Prior to the beginning of the experiment, animals acclimated for one week to a meal-feeding schedule, receiving chow from 10AM to 12PM (“breakfast”), 1PM to 2PM (“lunch”), and from 5PM to 6PM (“dinner”). At the end of one week, animals were weight-matched into two groups: ethyl oleate (EO, n = 15) and SO (n = 15). On Day 1 of the experiment, the “breakfast” chow access was replaced with 25ml of EO or SO emulsion. For eleven days, body weights were recorded before animals were given access to emulsions, and intakes of EO or SO emulsions and standard chow were recorded following administration of emulsion.
Upon arrival, animals were acclimated to meal-feeding schedules, and baseline body weights and food intakes were tracked for one week. Animals were then assigned to one of the following weight-matched groups: small-meal EO diet regimen; small-meal SO diet regimen; large-meal EO diet regimen; and large-meal SO diet regimen (n = 10–11 rats per group). For the duration of the study, animals in the “short access” groups followed a meal-feeding schedule identical to that of Experiment 1. Animals in the “long access” group were given EO or SO diet from 10AM to 12PM followed by one long period of access to chow from 1PM to 6PM daily. For the first 17 days of the study, animals received a 15ml does of experimental diet daily. For the remaining 14 days, the dose was increased to 25ml of diet. Body weights, experimental diet intakes, and food intake data were collected in a manner identical to that of Experiment 1.
Upon arrival, baseline body weights and food intakes of all animals were tracked for one week. Animals were then assigned to one of the following weight-matched groups: “restricted” EO diet (n = 21); restricted SO diet (n = 21); ad-libitum EO diet (n = 11); and ad-libitum SO diet (n = 10). On Day 1, all animals were placed on a meal-feeding schedule, and received 25ml of either EO diet or SO diet from 10AM to 12PM, followed by standard rat chow from 1PM to 6PM. For the first part of the experiment, animals in the “restricted” groups received food representing 75% of the calories consumed by meal-fed animals, or 11-grams of chow pre-weighed daily, while “ad-lib” animals received access to an unlimited amount of chow. After 17 days, it was determined that the body weights of rats in the “restricted” groups were significantly lower than those of their ad-lib counterparts. At that time, half the animals in each “restricted” group were sacrificed after a 21-hr fast (determined by weight-matching), and trunk blood was collected for plasma glucose and insulin analysis. In the second part of the experiment, all animals continued to follow the same meal-feeding schedule, including access to 25ml of EO or SO emulsions each day. However, the remaining animals in each “restricted” group received access to unlimited chow (1–6 PM) rather than a pre-weighed amount. Thus, the “restricted” groups were allowed to refeed ad-libitum and regain body weight lost during the restriction period. Body weights and total caloric intakes were monitored daily for 12 days.
All food and experimental diet intake data are reported in kilocalories. For all experiments, data were analyzed by one-way ANOVA and Tukey’s post-hoc tests. Significance level was set at (P < 0.05), two-tailed.
Figure 1 depicts mean body weight gain from first to last day of experimental diet meal-replacement: rats given access to SO diet gained significantly more weight during the meal replacement than rats that received EO diet (P < 0.05). Further, consumption of EO diet reduced subsequent chow intake and total daily kcal intake relative to SO diet (Figure 2). Specifically, relative to the SO diet, EO significantly reduced chow intake during the first meal following experimental diet access (P < 0.05), but had no effect on chow intake during the second meal. Further, mean total daily caloric intake was significantly less in rats receiving EO than SO diet (P < 0.05). The difference in daily total caloric intake was relatively constant across the 11-day study period.
The purposes of this experiment were twofold: first, to assess the effects of a lower dose of EO on food intake and body weight; and, second, to assess the effects of two one-hour periods of chow access versus one five-hour period of chow access following experimental diet meal-replacement. During the first 17-days of the experiment, the lower dose of EO (15ml of diet daily) reduced body weight gain and total energy intake relative to SO diet in the long access meal group. However, an increase in EO dose to that used in Experiment 1 (25ml of diet daily) was required to reduce these parameters in the short access meal group, and to maintain the reductions observed in the long access meal group. Figure 3 depicts mean body weight gain and total caloric intake during the first 17-days of the experiment using the low dose of EO. As shown, neither body weight gain nor total caloric intake of rats in the short access meal groups differed (P > 0.05). Conversely, these parameters were significantly different in rats assigned to the long access meal groups (P < 0.05). Furthermore, Figure 4 depicts data from the remaining 14-days of the experiment, after which the daily dose of EO or SO diet was increased to that used in Experiment 1. Across these 14-days, the larger dose of EO significantly reduced total daily caloric intake and body weight gain in animals assigned to both the short and long access meal groups (P < 0.05). Thus, a 25ml daily volume of 12.5% EO diet was effective to reduce food intake and body weight gain in both groups whereas the 15ml volume was effective to reduce intake and weight gain in only the long access meal group.
The purpose of this experiment was to assess the effectiveness of EO to attenuate the rebound weight gain observed after a period of food restriction. Following breakfast of either EO or SO diets, rats were given either: ad-libitum access to standard chow for five-hours; or were food-restricted, receiving 11-grams of chow during the same five-hour time period. Figure 5 depicts body weight gain in the “restriction” phase (top panel) and “refeeding” phase of Experiment 3. First, during the restriction period, rats receiving either EO or SO diet lost body weight relative to rats given five-hours of ad libitum access to chow, regardless of diet ingested (P < 0.05). Importantly, the rate of body weight loss in the restricted groups was dependent on diet received, with rats that received EO losing significantly more body weight than rats receiving SO (P < 0.05). Following a return to ad libitum feeding (from 1–6 PM), body weight was increased in both groups (EO and SO). However, rats receiving EO gained less weight than rats receiving SO diet (P < 0.05). Finally, Figure 6 depicts plasma glucose and insulin analysis from Experiment 3. As shown, there were no differences in fasting plasma glucose or insulin levels between rats receiving EO or SO diet.
Intragastric and intraintestinal infusions of either linoleic or oleic acid have been demonstrated to reduce food intake and body weight in humans, rats and other animal models (4, 6, 7, 8). Numerous studies have indicated a role for CCK, signaling via vagal afferent neurons in the gut and setting in motion a robust gut-satiety cascade. Importantly, these data support the hypothesis that specific fatty acids are able to engage distal gut satiety mechanisms, decreasing subsequent food intake and, when administered long term, suppressing body weight gain. Previous studies of the suppression of food intake and reduced body weight gain following fatty acid administration all have used intragastric or intestinal delivery systems. The data collected for the current study demonstrate that voluntary oral ingestion of fatty acids also brings about a marked and long-lasting reduction in body weight gain and caloric intake.
A cohort of meal-fed animals was given access either to EO diet, an ethyl-ester of oleic acid, or SO diet, in place of breakfast. In this study, it increased short-term satiety and reduced body weight gain when administered daily. Importantly, pilot experiments demonstrated that EO diet has no effect on food intake or body weight in 24-hr ad-lib fed rats, suggesting the short-term satiety that follows EO diet ingestion is compensated when rats are allowed to feed ad-libitum. These results were recapitulated by a second experiment, in which an effect on food intake or body weight gain was not observed in rats maintained on two one-hour meals of chow following a reduction to the dose of EO diet by 40%. This reduced dose of EO diet, however, was sufficient to significantly reduce food intake and suppress body weight gain in animals maintained on chow for longer time periods of five-hours. Taken together, these data indicate that the effect of EO diet to promote satiety and reduce food intake is dependent on both the dose of EO diet and the amount of food available during normal chow meals.
An important conclusion, suggested by these data, is that a lower dose of EO diet is ineffective in countering the regulatory physiological mechanisms activated by negative energy balance. Specifically, a low dose of EO diet activates short-term satiety, but is less effective in influencing food intake or changes in body weight during a period of food restriction. Clearly, this effect is dependent on the dose of EO diet, with larger doses of EO diet suppressing food intake and body weight gain in rats given access to chow for short periods of time. Overall, these results suggest that EO diet dose-dependently activates a gut-initiated satiety mechanism that interacts with the length of food availability.
A third experiment assessed the effect of EO diet on the characteristic weight gain observed in free-fed animals following a period of food restriction. Subsequent results indicate that consumption of EO diet can increase the weight loss caused by further food restriction. Rats were maintained at 75% of daily calories consumed by the meal-fed groups. As expected, restricted rats lost weight relative to meal-fed rats. Unexpectedly, however, the food-restricted rats consuming a “breakfast” of EO diet lost significantly more weight than food-restricted rats consuming an equicaloric control diet. Importantly, the additional body weight loss observed in EO-fed animals occurred despite an equal daily caloric intake by rats assigned to receive SO diet. These data support a tentative hypothesis that EO diet and related nutrients function to increase the thermic effects of food or activate energy expenditure systems, independent of total calories consumed; regardless of caloric value, orally consumed EO diet was fundamentally more satiating than the soybean oil diet in restricted animals. Finally, relative to the SO diet, EO diet ingestion significantly attenuated weight regain following a return to five-hours of daily ad-libitum access to food after a period of food restriction. These data further support the hypothesis that EO diet promotes short-term satiety and reduces subsequent food intake and body weight gain, compared to animals assigned to SO diet.
Consumption of fatty acids has been linked to impaired glucose tolerance and insulin sensitivity (13, 14, 23, 28); consequently, consumption of large doses of EO diet reasonably could be expected to impair insulin secretion or disrupt glycemic control. However, we observed no significant differences in fasting plasma glucose of insulin levels in rats receiving the EO or SO diets. While additional long-term confirmation is desirable, these data suggest that ingestion of an EO diet activates gut-initiated satiety cascades without promoting the development of overt impaired glucose regulation.
This study is not without limitations: clearly, the collection of metabolic data, such as locomotor activity and energy expenditure, would be valuable, as would body composition, and behavioral data. However, the suppression in body weight gain and the reduction in caloric intake were small and likely would have been significantly ameliorated or abrogated completely by the collection of such data. Importantly, repetitive blood sampling, and metabolic studies have the potential to activate the HPA axis and alter food intake and these effects were considered sufficiently detrimental that this study be designed and carried out in such a way as to avoid them. Future studies should be designed in such a way as to address these concerns and important considerations.
Furthermore, the present experiments do not address the precise mechanisms through which EO diet reduces food intake and suppresses body weight gain. Previous work on intragastric and intestinal infusion of fatty acids suggests several important potential routes through which EO diet ingestion might exert its effects. Numerous studies have demonstrated a role for CCK and other gut peptides in the reduction of food intake and the activation of satiety mechanisms following intraintetsinal infusions of fatty acids. These robust effects have been ameliorated by both vagotomy and CCK-A receptor antagonism (7, 8, 15, 32). Taken together, these novel findings both demonstrate the potential clinical relevance of gut-initiated satiety mechanisms in the treatment of obesity, and suggest important information on the means to best initiate them.
This study recapitulates previous reports demonstrating a reduction in food intake and body weight following intraintestinal infusions of fatty acids, using a paradigm of voluntary oral administration to deliver an oleic acid derivate proven not to injure the intestinal mucosa. Importantly, using this paradigm, EO diet increases weight loss disproportionate to its caloric value, relative to SO diet. The apparent ability of EO diet to promote satiety and reduce food intake is dependent on both the dose of EO diet given and the amount of food available. Further, EO diet attenuated weight rebound following a period of food restriction. In summary, this study presents ethyl oleate as a potential pharmacologic therapy for the manipulation of intestinal satiety mechanisms, with relevance to clinical interventions in obese individuals.
This work was supported by grants from the NIH (DK54890, DK056863) and funds from Procter and Gamble.
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