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APPETITE, OBESITY, DIGESTION, AND METABOLISM
University of Adelaide Discipline of Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia
Submitted 18 August 2007 ; accepted in final form 13 November 2007
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15,400 kJ per day), containing either 44% (high-fat, HF) or 9% (low-fat, LF) fat, for 21 days in single-blind, randomized, cross-over fashion. Immediately following each diet (i.e., on day 22), subjects received a 45-min intravenous infusion of CCK-8 (2 ng·kg–1·min–1), and effects on antropyloroduodenal motility, plasma CCK, PYY, ghrelin concentrations, hunger, and fullness were determined. Thirty minutes after commencement of the infusion, subjects were offered a buffet-style meal, from which energy intake (in kilojoules) was quantified. Body weight was unaffected by the diets. Fasting CCK (P < 0.05), but not PYY and ghrelin, concentrations were greater following the HF, compared with the LF, diet. Infusion of CCK-8 stimulated pyloric pressures (P < 0.01) and suppressed antral and duodenal pressures (P < 0.05), with no difference between the diets. Energy intake also did not differ between the diets. Short-term consumption of a HF diet increases fasting plasma CCK concentrations but does not affect upper gut motility, PYY and ghrelin, or energy intake during CCK-8 infusion, in a dose of 2 ng·kg–1·min–1, in healthy males. antropyloroduodenal motility; appetite; gut hormones; healthy subjects
Only two studies have hitherto investigated the effects of a HF diet on plasma CCK concentrations in humans (3, 14). In one of these, the plasma CCK response to a standardized breakfast was greater following exposure to a HF diet, when compared with an unstandardized "prediet" condition (14). In contrast, the plasma CCK response to intraduodenal infusion of lipid (Intralipid at 2.8 kcal/min) did not differ following exposure to a HF (44% energy as fat), when compared with a LF (10% energy as fat), diet for 2 wk (3), suggesting that the increased postprandial plasma CCK concentrations observed following consumption of a HF diet (14) are likely to reflect more rapid gastric emptying (11). Interestingly, Boyd et al. (3) reported in healthy humans that consumption of a HF diet attenuated the stimulatory effects of intraduodenal lipid on pyloric pressures, despite a similar increase in plasma CCK concentrations. Because pyloric motility is modulated, at least in part, by the activation of CCK1 receptors located on the pylorus (33), it is conceivable that, despite an unchanged plasma CCK response, the sensitivity of the antropyloroduodenal region to CCK is reduced following exposure to a HF diet. This hypothesis has, hitherto, not been investigated in humans.
CCK also modulates the secretion of ghrelin and PYY (5, 12), both of which are likely to be important in the regulation of energy intake. In response to both exogenous (5) and endogenous (12) CCK, plasma ghrelin concentrations are suppressed and plasma PYY concentrations are increased. Postprandial plasma ghrelin concentrations have been reported to be decreased in healthy male subjects following exposure to a high-fat diet (24). If CCK were less effective at suppressing ghrelin and stimulating PYY secretion in response to a HF diet, this may be of relevance to the hyperphagia observed in response to HF diets (18, 27). To our knowledge, no studies have hitherto investigated the effects of a HF diet on PYY secretion in humans.
The aims of this study were, therefore, to evaluate in healthy lean male subjects the hypothesis that exposure to a HF diet for a period of 3 wk would attenuate the effects of an intravenous infusion of CCK-8 on antropyloroduodenal motility, plasma PYY and ghrelin concentrations, appetite perceptions, and energy intake.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ten healthy male subjects [age: 22.4 ± 1.0 (range 18–28) years] with normal body weight for their height (body mass index: 22.1 ± 0.8 kg/m2) were studied. The number of subjects was based on previous studies demonstrating the effects of HF diets for periods of 14 days on antropyloroduodenal pressure responses to intraduodenal lipid (3) and postprandial plasma CCK concentrations (14). We calculated that 10 subjects would allow detection of a 10% difference in antropyloroduodenal pressures and gut hormone concentrations with an 
of 0.05 and a power of 0.8. Subjects were unrestrained eaters [scoring <12 on the eating restraint section (Factor 1) of the Three-Factor Eating Questionnaire (28)], did not have any gastrointestinal disease or symptoms or any other illnesses, take any medication known to affect gastrointestinal motility or appetite, habitually smoke >10 cigarettes, or consume >20 g of alcohol per day. Before inclusion in the study, subjects completed a 5-day (3 week days and a weekend) diet diary to ascertain that their habitual energy and fat intake were within the range of 25–35% energy from fat, as recommended in the National Health and Medical Research Council Dietary guidelines for adult Australians. The study was conducted in accordance with the institutional ethical guidelines, and the protocol was approved by the Royal Adelaide Hospital Research Ethics Committee. All subjects provided written, informed, consent before their enrollment in the study.
Protocol
Subjects completed two 21-day diet periods, during which they consumed, in single-blind, randomized, cross-over fashion, isocaloric diets (15,400 kJ per day) that were either high in fat, where fat contributed 44% of the total daily energy or low in fat, where fat contributed 9% of the total daily energy intake. The two diet periods were separated by a 14-day "wash-out" period, during which subjects were instructed to consume their habitual diet, that is, to return to the diet reported during the 5-day baseline period. Immediately after each diet (day 22), the effects of a 45-min intravenous infusion of CCK-8 (CCK-8 sulfated, Clinalfa; Merck Biosciences AG, Läufelfingen, Switzerland) at 2 ng·kg–1·min–1 on antropyloroduodenal (APD) pressures, plasma gut hormone concentrations, appetite perceptions, and energy intake were evaluated. The dose of CCK-8 was selected on the basis of previous studies, which indicated that it would have submaximal effects on energy intake (4, 19) and APD pressures (23), while resulting in plasma concentrations comparable to those observed postprandially (22, 29). Blood samples were collected at 10-min intervals for determination of plasma concentrations of CCK, PYY, and ghrelin.
Diets
The difference in fat intake between the two diets was 150 g/day, and the protein content of the diets was matched; however, carbohydrate content differed to make the diets isocaloric. The planned total daily energy and macronutrient intakes are displayed in Table 1. Six daily meal plans were designed for each diet to increase variety and, thus, ensure compliance with the prescribed diets; an example of a daily menu for each diet is shown in Table 2. These menus were rotated over the 21-day periods. The macronutrient distribution of the foods provided was achieved by supplying the subjects with similar HF or LF varieties of foods (cheeses, yogurts, biscuits, butter, margarine, etc.), respectively (3). In addition, subjects received HF or LF snacks (prepackaged in the study kitchen), such as potato crisps or biscuits, to be eaten between main meals (3). Foods were repackaged to remove nutritional information, ensuring that subjects were unaware of the macronutrient composition of the foods that they were consuming during the respective diets, and supplied to subjects at 3- to 4-day intervals. A detailed plan outlining what and when to eat was provided with the food, and subjects were required to document, in an accompanying diary, that they had consumed all of the food provided. These diaries were reviewed regularly during each diet period to determine compliance of the subjects to the prescribed diet.
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Antropyloroduodenal Motility, Hormone, Appetite, and Energy Intake Responses During Intravenous CCK-8 Infusion
On the day immediately following each of the diet periods (day 22), subjects attended the Department of Medicine at 0830 following an overnight fast from both solids and liquids from 2200. Subjects were intubated with a 16-channel manometric catheter (Dentsleeve, Adelaide, Australia) via an anesthetized nostril. The catheter was allowed to pass through the stomach and into the small intestine by peristalsis. Six side-holes (channels 1–6) were positioned in the antrum, a 4.5-cm sleeve sensor, with two channels present on the back of the sleeve (channels 8 and 9), was positioned across the pylorus, and 7 channels were positioned in the duodenum (channels 10–16). The correct positioning of the assembly, with the sleeve sensor straddling the pylorus, was determined by continuous measurement of the transmucosal potential difference (TMPD) between the most distal antral (channel 6) (–40 mV) and the most proximal duodenal channel (channel 10) (0 mV) (16). For this purpose, a 20-gauge saline-filled cannula was placed subcutaneously in the left forearm as a reference electrode. All manometric channels were perfused at a rate of 0.15 ml/min with degassed, distilled water, except for the channels measuring TMPD, which were perfused with degassed 0.9% saline. Once the catheter was in the correct position, fasting motility was monitored until the occurrence of phase III of the migrating motor complex (1). Two intravenous cannulas were inserted, one into each forearm, for intravenous infusion and blood sampling, respectively. A baseline blood sample was taken immediately, and the subject rated perceptions of appetite on a visual analog scale questionnaire (VAS) (t = –10 min). At t = 0 min, an intravenous infusion of CCK-8 at 2 ng·kg–1·min–1 was commenced and maintained for 45 min. APD pressures were recorded continuously from t = 0–30 min. Blood samples were taken and VAS completed throughout the infusion at 10-min intervals. At t = 30 min, the subject was extubated and immediately offered a cold, buffet-style, meal consisting of white and brown bread, margarine, mayonnaise, ham, chicken, cheese, lettuce, tomato, cucumber, yogurt, custard, fruit salad, iced coffee, orange juice, water, an apple, and a banana, as described in detail previously (13). This meal provided food in excess of what the subject was expected to consume. Each subject was allowed 30 min to eat (t = 30–60 min) and instructed to eat until comfortably full. The CCK-8 infusion was terminated 15 min after the commencement of the meal (i.e., at t = 45 min). At t = 60 min and t = 90 min, further blood samples were taken and a VAS was completed. The intravenous cannulas were then removed, and the subject was free to leave the laboratory.
Measurements
Antropyloroduodenal motility. Manometric pressures were digitized and recorded on a computer-based system (PowerMac 7100/75; Apple Computers, Cupertino, CA) running commercially available software (HAD, Professor GS Hebbard, Melbourne, Australia), written in LabView 3.1.1. (National Instruments, Austin, TX), and stored for later analysis. APD pressures were analyzed for 1) the number and amplitude of pressure waves (PWs) in the antrum and duodenum and 2) number and amplitude of isolated pyloric pressure waves (IPPWs), as described previously (13) using custom-written software [Gastrointestinal Motility Unit, University Hospital Utrecht, The Netherlands (25)], tailored to our requirements.
Appetite perceptions and energy intake. Ratings of appetite, that is, hunger and fullness, were measured using validated VAS (20). Nausea and bloating were also assessed. Each VAS evaluated a sensation on a 100-mm horizontal line, where 0 mm represented "sensation not felt at all" and 100 mm meant "sensation is felt the greatest." Subjects were asked to indicate how they were feeling at a particular time by placing a vertical stroke on the 100-mm line.
Energy intake (in kilojoules), amount eaten (in grams), and macronutrient distribution (% energy) of food consumed at the buffet meal were analyzed using commercially available software (Foodworks 3.01; Xyris Software, Highgate Hill, Queensland, Australia) (13).
Plasma CCK, PYY, and ghrelin concentrations. Venous blood samples (10 ml) were collected into ice-chilled EDTA-treated tubes containing 400 kIU aprotinin/ml blood (Trasylol; Bayer Australia, Pymble, Australia). Plasma was separated by centrifugation at 3,200 rpm for 15 min at 4°C within 30 min of collection, and stored at –70°C until assayed.
Plasma CCK concentrations (pmol/l) were determined following ethanol extraction using an established radioimmunoassay (19). A commercially available antibody raised in rabbits against synthetic sulfated CCK-8 was employed (C258, Lot 105H4852, Sigma Chemical, St. Louis, MO, USA). This antibody binds to all CCK analogs with the sulfated tyrosine residue in position 7, has a cross-reactivity of 26% with unsulfated CCK-8, less than 2% cross-reactivity with human gastrin (the cross-reactivity with gastrin I was 0.2% and with big gastrin 1%) and does not bind to structurally unrelated peptides. The intra-assay coefficient of variation (CV) was 9% and the interassay CV was 27%, with a sensitivity of 2.5 pmol/l.
PYY immunoreactivity (pmol/1) was measured with a specific and sensitive radioimmunoassay (22). The assay measured both the hormone fragment (PYY3–36) and the full-length hormone (PYY1–36), both of which are biologically active. This antibody cross-reacts fully with the biologically active circulating forms of PYY but not with pancreatic polypeptide, neuropeptide Y or other known gastrointestinal hormones. The intra-assay CV was 12.3% and the interassay CV was 16.6%, with a sensitivity of 1.5 pmol/l.
Total ghrelin (in nanograms per liter) was measured by an established radioimmunoassay using a commercial antiserum (RAST-4745, Bachem, CA) (21) that does not cross-react with human secretin, orexin, motilin, galanin, or vasoactive intestinal peptide. The intra-assay CV was 17% and the interassay CV was 23%, with a sensitivity of 40 ng/l.
Data and Statistical Analysis
Baseline values were calculated as the means of values obtained between t = –10 and 0 min for VAS scores, number of antral and duodenal PWs, number of IPPWs, basal pyloric pressure and plasma CCK, PYY, and ghrelin concentrations. The total number and mean amplitude of antral and duodenal pressure waves were expressed as means over the 30-min recording period. The number and amplitude of IPPWs and basal pyloric pressures were expressed as means over 10-min periods during the 30-min recording period. All data were expressed as changes from baseline. Areas under the curve (AUC) for plasma CCK, PYY, and ghrelin concentrations were calculated using the trapezoidal rule.
The number and amplitude of isolated pyloric PWs, basal pyloric pressure, and plasma CCK, PYY, and ghrelin concentrations were analyzed using repeated-measures ANOVA with treatment and time as factors. Antral and duodenal PWs, AUCs for plasma CCK, ghrelin and PYY concentrations, and energy intake were analyzed using one-way ANOVA. Post hoc paired comparisons, corrected for multiple comparisons by Bonferroni's correction, were performed if ANOVAs revealed significant effects. Data are presented as means ± SE. Statistical significance was accepted at P < 0.05.
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antropyloroduodenal Pressures
There was no effect of diet on the number, or amplitude, of antral, pyloric, or duodenal pressure waves (Fig. 1), or basal pyloric pressure (data not shown). Following both the HF and LF diets, CCK-8 decreased the number and amplitude of antral and duodenal PWs (time effect: P < 0.05) and stimulated basal pyloric pressure and isolated pyloric pressure waves (time effect: P < 0.01), when compared with baseline.
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CCK. Baseline concentrations of CCK were greater following the HF, when compared with the LF, diet (HF: 4.3 ± 0.4 pmol/l, LF: 3.1 ± 0.3 pmol/l; P < 0.05). Plasma concentrations of CCK increased during infusion of CCK-8 between t = 0–30 min (time effect: P < 0.001), with no difference in the magnitude of the response between the HF and LF diet (Fig. 2A). After the meal, plasma CCK fell and was less at t = 60 min, when compared with t = 30 min (P < 0.001). There was no effect of diet on the AUC for plasma CCK concentrations (Table 3).
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Plasma ghrelin. There was no effect of treatment on baseline ghrelin concentrations (HF: 1,017 ± 102 ng/l, LF: 969 ± 105 ng/l), and there was no significant effect of treatment, or time, on plasma ghrelin concentrations during CCK-8 infusion (Fig. 2C). There was no effect of diet on the AUC for plasma ghrelin concentrations (Table 3), although there was large variability between subjects.
Appetite and energy intake. There was no effect of diet on ratings of hunger or fullness. Following both diets, infusion of CCK-8 increased fullness between t = 10–30 min when compared with baseline (time effect: P < 0.05) (data not shown).
There was no difference in energy intake (kilojoules), amount (grams), or the macronutrient distribution of food consumed at the buffet meal following the HF, when compared with the LF, diet (Table 4).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our hypothesis that the sensitivity to exogenous CCK-8 would be reduced by a HF diet in humans, was based primarily on the observation that in healthy lean men, exposure to a hypercaloric HF diet (40% energy from fat, 20,123 kJ/day) for a period of 14 days attenuated the stimulatory effects of an intraduodenal lipid infusion on tonic and phasic pyloric pressures, when compared with a low-fat diet (11% energy from fat, 11,191 kJ/day), despite comparable elevations in plasma CCK (3). However, we observed no differences in the effects of an intravenous infusion of CCK-8 on antropyloroduodenal motility or energy intake, suggesting that the sensitivity to exogenous CCK-8, at least in the dose used, was unaltered. This contrasts with data from rodent studies reporting that the inhibitory effects of intraperitoneal CCK on both gastric emptying (8) and food intake (7, 9) were attenuated following exposure to HF diets (containing 34 or 54% energy as fat), when compared with exposure to an isocaloric low-fat diet (containing 5% energy as fat), for 2 wk, in the absence of any change in body weight or adiposity. It should, however, be recognized that the literature is inconsistent. In particular, Torregrossa and Smith (31) reported divergent effects of the same diet on the response to CCK-8 in rats. In their first study, the inhibitory effect of intraperitoneal CCK-8 on energy intake was attenuated following consumption of a 34% fat diet for 2 wk, supporting the observations of Covasa et al. (9); however, in a subsequent study, the suppressive effect of intraperitoneal CCK-8 on energy intake was maintained (31). Furthermore, in rats consuming a 60% fat diet for 2 wk, the sensitivity to the satiating effects of CCK-8 has been reported to be enhanced, rather than diminished, when compared with rats maintained on a LF diet (31). The reason(s) underlying these discrepant observations are unclear; however, it should be noted that rats consuming the 60% fat diet gained significantly more weight and adipose mass when compared with rats on the 5 or 34% diets, raising the possibility that the effects of CCK on energy intake may be dependent upon body weight. Furthermore, it is unclear whether observations from studies in animals are applicable to humans. In animal studies, the magnitude of the fat supplementation has been far greater (8) than in human studies, simply because the diet can be enriched to almost 100% with a particular macronutrient, whereas in human studies, the degree of fat supplementation is limited by the need to provide "normal" foods.
Exposure to a HF diet has been shown to modulate gastrointestinal function and energy intake in humans (3, 6, 11, 14). For example, in healthy lean male subjects, consumption of a high-energy, HF diet (2,340 kJ of fat, 19.3 MJ energy daily) for a period of 14 days accelerated gastric emptying and mouth-to-cecum transit of a HF test meal, when compared with a LF diet (105 kJ fat, 9.1 MJ energy daily) (11). Exposure to a HF diet has also been reported to increase daily energy intake, as measured from food diaries (HF: 10.3 ± 0.5 MJ/day, prediet: 9.6 ± 0.6 MJ/day; P < 0.05) (14). Furthermore, increased postprandial CCK concentrations have been reported following exposure to a HF diet when compared with a prediet condition (integrated plasma CCK: HF: 1,285 ± 153 pM/min, prediet: 897 ± 78 pM/min; P < 0.01) (14). However, during direct intraduodenal infusion of lipid (2.8 kcal/min), which "bypasses" the influence of gastric emptying, the CCK response did not differ following exposure to a HF, when compared with a LF, diet (3). It is, therefore, likely that the increased postprandial plasma CCK concentrations observed following consumption of a HF diet (14) are reflective of more rapid gastric emptying (11). Given the important role of CCK in the regulation of gastric emptying (15) and energy intake (2), it is intuitively likely that exposure to a HF diet may modulate the actions of CCK; however, most likely because of methodological limitations, studies in humans have been unable to directly relate the changes in gastrointestinal function and energy intake observed following a HF diet to a change in the sensitivity to CCK. Our observation of increased fasting concentrations of CCK following exposure to the HF diet does, however, indicate that the CCK response to fat is modulated, suggesting that changes in the mechanisms that mediate CCK secretion, metabolism, and sensitivity may occur.
It has recently been demonstrated by ourselves (5) and others (12) that CCK modulates the secretion of ghrelin and PYY. Both exogenous (5) and endogenous (12) CCK suppress plasma ghrelin and increase plasma PYY concentrations. In the current study, we observed no changes in the fasting concentrations of either ghrelin or PYY following exposure to the high-fat diet. While CCK-8 stimulated PYY following both diets, with no differences between them, there was, somewhat surprisingly, no effect on plasma ghrelin. It is, accordingly, possible that hypercaloric diets (which our diets were), regardless of their fat content, may attenuate the suppression of ghrelin by CCK-8, and this issue warrants formal evaluation.
The methodological approach taken in the current study warrants discussion. In contrast to previous studies in humans, which have evaluated the effects of HF diets that were also high in energy, and compared them to low-energy, LF diets (3, 11, 14), we employed isocaloric HF and LF diets and observed no changes in gastrointestinal function or energy intake in response to intravenous CCK. It is, accordingly, possible that the increased energy content of the HF diet, rather than the fat content per se, mediated the changes in gastric emptying (11), antropyloroduodenal motility (3), CCK secretion (14), and appetite (3, 14) observed in previous studies. Furthermore, while we matched the protein content of the diets, in attempting to increase the fat content of the diet, the carbohydrate content decreased, and vice versa; thus, we cannot discriminate between an effect of fat, as opposed to carbohydrate. It is also important to recognize that the duration of dietary modification (3 wk) may have been insufficient to change the gastrointestinal and energy intake responses to exogenous CCK-8, although previous studies have demonstrated significant changes in the gastric emptying and antropyloroduodenal motility responses to fat following exposure to HF diets for 2 wk in healthy humans (3, 11, 14). The interpretation of our current observations is also limited by the administration of only one dose of CCK. The dose of CCK-8 used was based primarily on the outcome of previous studies by our group, establishing that 2 ng·kg–1·min–1 results in a substantial, but probably submaximal, suppression of energy intake (4, 19), as well as stimulation of pyloric pressures (4), suppression of ghrelin, and stimulation of PYY (5). While it could be argued that the resulting plasma CCK concentrations were moderately supraphysiological, they are comparable with those observed following ingestion of a 750-kcal mixed-nutrient meal (29) and during intraduodenal infusion of the fatty acid, lauric acid (13), or the triacylglyderide emulsion, Intralipid (22), in our previous studies, in which CCK-8 was measured with the same assay. It should also be noted that because of the lack of a control (i.e., intravenous infusion of saline following each diet), for logistical reasons, we cannot determine whether the magnitude of the response to CCK was attenuated, when compared with baseline, following both diets, given that they were both hypercaloric compared with the subjects' habitual diet.
Perspectives and Significance
Studies in animals have characterized numerous changes in gastrointestinal function, particularly relating to the secretion of gastrointestinal hormones in response to increased fat intake that may predispose animals to an increase in energy intake, and consequently, weight gain and obesity. The influence of HF diets on gastrointestinal physiology and appetite in humans is less clear, largely related to methodological issues. In view of our observations, evaluation of the effects of a HF diet on the dose-response to exogenous CCK-8 would be of interest, albeit logistically difficult in human subjects. Interestingly, intravenous infusions of CCK-8 in doses of 1 and 4 ng·kg–1·min–1 for 30 min have been reported to have comparable effects on the gallbladder (30). Arguably of greater interest would be to evaluate the effects of endogenous CCK (by blocking the CCK1 receptor with a specific antagonist, such as dexloxiglumide) (12) to determine whether CCK is less potent at modulating appetite and gastrointestinal function following exposure to a HF diet in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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