Human Nutrition and Metabolism

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1 Human Nutrition and Metabolism Protein Source, Quantity, and Time of Consumption Determine the Effect of Proteins on Short-Term Food Intake in Young Men 1,2 G. Harvey Anderson, 3 Sandy N. Tecimer, Deepa Shah, and Tasleem A. Zafar Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 ABSTRACT The objective of these 4 studies was to describe the effects of protein source, time of consumption, quantity, and composition of protein preloads on food intake in young men. Young men were fed isolates of whey, soy protein, or egg albumen in sweet and flavored beverages (400 ml) and provided a pizza meal 1 2 h later. Compared with the water control, preloads (45 50 g) of whey and soy protein, but not egg albumen, suppressed food intake at a pizza meal consumed 1 h later. Meal energy intake after egg albumen and soy, but not after control or whey treatments, was greater when the treatments were given in the late morning (1100 h) compared with earlier ( h). Suppression of food intake after whey protein, consumed as either the intact protein or as peptides, extended to 2 h. Altering the composition of the soy preload (50 g) by reducing the soy protein content to 25 g and by adding 25 g of either glucose or amylose led to a loss in suppression of food intake by the preload. Egg albumen, in contrast to whey and soy preloads, increased cumulative energy intake (sum of the energy content of the preload plus that in the test meal) relative to the control. We conclude that protein source, time of consumption, quantity, and composition are all factors determining the effect of protein preloads on short-term food intake in young men. J. Nutr. 134: , KEY WORDS: proteins food intake men Dietary protein contributes more strongly than carbohydrate or fat to short-term satiety in humans as indicated by both quantitative and subjective measures (1). Subjects consuming a fixed energy content lunch composed of foods or meals high in protein, compared with those low in protein, decrease their food intake at either a later meal (2 4) or from food consumed immediately after (5). In addition, less total energy is consumed during a meal composed of only a highprotein food, compared with one of a low-protein food (5). Subjective measurements of satiety after meals or foods also showed that protein is more satiating than fat or carbohydrate (3,5 7) and that it delays the return of hunger (8). The protein source, in addition to protein quantity or concentration in food, may be a determinant of the satiating efficacy of protein, but very limited data from studies in humans exist on this topic. Greater subjective satiety was found over a 3-h period when young men were fed a 50-g meal of lean fish compared with an equivalent amount of either beef or chicken (9). However, only 1 report in the literature (10) suggests that response in short-term food intake of humans to protein differs among protein sources. Whey (48 g) increased subjective satiety and decreased food intake more than casein 1 Presented in part at the Proceedings of the 44th Annual Conference of the Canadian Federation of Biological Sciences, June 2001, Ottawa, Canada [Tecimer, S. N. & Anderson, G. H. (2001) Effect of Protein Source on Suppression of Food Intake in Young Men (abs. # T0571, p. 63)]. 2 Supported by the Natural Sciences and Engineering Research Council of Canada and an Unrestricted Discovery Grant from the Bristol-Myers Squibb Foundation and Mead Johnson Nutritionals. 3 To whom correspondence should be addressed. harvey.anderson@utoronto.ca. (48 g) at a buffet meal consumed 90 min later by healthy volunteers. Because no control treatment was provided and the preloads contained an additional kj from fat and carbohydrate, the effects on food intake of these 2 proteins alone or compared with an energy-free drink were not described. In contrast, the protein source was not found to be a factor affecting later food intake when fed in a meal (11). When 6 dietary protein sources (egg albumen, casein, gelatin, soy protein, pea protein, and wheat gluten) were fed in lunches containing 5192 kj, no difference in energy intake was found at a dinner 8 h later. However, both the low concentration of the test proteins in relation to the total energy content (22% of energy as protein) in the lunch meal and the 8 h duration to dinner may have contributed to the negative conclusion. In a later study (12), a gelatin lunch was found to be satiating for longer than the casein lunch, but there was a return of hunger occurring 3 to 7 h after both lunches, suggesting that measuring food intake at a meal 8 h later would not detect differential effects of protein source when given in a mixed macronutrient meal. Thus, the source, quantity, and composition of the protein treatment and time to the next eating occasion are factors to be considered in the evaluation of the effect of protein on satiety and food intake, as they are with carbohydrate (13). The primary objective of these studies was to compare the effect of whey, egg, and soy proteins when consumed in the form of isolates by young men on their food intake 1 2 h later. Additionally, we investigated the effect on food intake of reducing the protein content of the preload and replacing it by /04 $ American Society for Nutritional Sciences. Manuscript received 2 February Initial review completed 3 March Revision accepted 25 August

2 3012 ANDERSON ET AL. either low- or high-glycemic carbohydrate. The proteins tested in these studies (e.g., egg albumen, whey, and soy) were chosen because they were available in almost pure forms in commercially sold products and they included both animal and plant proteins that are commonly consumed, both naturally and in supplemental form. SUBJECTS AND METHODS Subjects. Young men (18 35 y old) with a BMI between 20 and 25 kg/m 2 were recruited. Diabetics, breakfast skippers, those dieting or taking medicine, and restrained eaters who scored 11 or more on the Eating Habits Questionnaire (14) were excluded. The study was approved by the Human Subjects Review Committee, Ethics Review Office, University of Toronto, Canada. Four experiments were conducted. Preload treatments were administered in a repeated measure design. In Expt. 1 the effect of protein source on food intake was determined 1 h later. Four isoenergetic treatments administered to the subjects were sucrose, egg albumen, whey, soy protein in addition to the water control. The sucrose and protein treatments provided the same subject with 0.65 g/kg body weight. Men (n 13) aged y (mean SEM: y) completed the experiment. On test days, 6 men (BMI, kg/m 2 ; body weight, kg) started treatments at 0910 h and 7 (BMI, kg/m 2 ; body weight, kg) at 1100 h. Because egg albumen did not suppress food intake and the time of morning at which the subjects began their treatments in Expt. 1 affected the results, Expt. 2 was conducted with a larger sample size to examine these results more fully. The test treatments were water control, 50 g whey protein, or egg albumen. Food intake was measured 1 h after the treatments were given at 2 times in the morning (0830 and 1100 h). Men (n 22) aged y ( y) completed the experiment; 12 men (BMI, kg/m 2 ; body weight, kg) started the test session at 0830 h and 10 men (BMI, kg/m 2 ; body weight, kg) at 1100 h. To determine whether the duration of effect of whey protein on food intake lasted 1 h, Expt. 3 measured food intake at 2 h after the preloads. The treatments were 50 g of intact whey protein or its enzymic hydrolysate, and an energy-free water control. A total of 10 men with an age range of y and BMI range of kg/m 2 completed the experiment. Experiment 4 aimed to determine the effect of adding high- or low-glycemic carbohydrate to replace 50% of the protein on food intake after a protein load. The experiment consisted of 4 treatments: control, soy protein alone (50 g) or soy protein (25 g) with either amylose (25 g) or glucose (25 g). Experiment 4 was completed by 13 men (19 24 y) with BMI of kg/m 2 and body weight of kg. Glycemic response to the treatments was also measured by taking blood samples every 15 min over the course of 1 h, and food intake was measured 1 h after consumption of the preloads. The treatment sources were as follows: soy protein (Swiss Herbal Remedies), whey (Expts. 1 and 2, Ultimate Balance; Expt. 3, Just Whey, Sport Pharma USA), whey hydrolysate (Ultimate Whey Designer Protein, Next Nutrition), egg albumen (Egg D Lite, Optimum Nutrition), sucrose (Redpath Industries), glucose monohydrate (Grain Process Enterprises) and amylose (Hylon VII, National Starch and Chemical). The protein sources contained negligible amounts of carbohydrate or fat. The protein content claim of the sources was verified by Kjeldahl analyses to ensure that target protein intakes were equivalent among treatments. Treatments were matched for sweetness and flavor. Sucralose (Tate and Lyle Specialty Sweeteners) was chosen as the no-energy sweetener because it is not metabolized in the body and has no effect on blood glucose or insulin secretion (15). In Expts. 1 and 2, aspartame-sweetened, strawberry-flavored crystals (Kool-Aid, Kraft) were added to standardize flavor among treatments. Because a small amount of maltodextrin was found in the commercial egg protein mix, a proportional amount was added to all drinks. Thus, the mean energy content of the test preload drinks was 67 kj for the water control and 833 kj for the whey, soy, egg, and sucrose treatments. In Expts. 3 and 4, lemon concentrate was added to provide the flavor and reduce the sweetness of the drinks. All treatments were adjusted to a total volume of 400 ml. After preparation, the drinks were stored in the refrigerator and served chilled to subjects the following morning. Subjects consumed their treatments, followed by 50 ml of water to minimize aftertaste, in 5 min. Experimental procedure. Subjects were administered the treatments after an overnight fast of h in a covered, opaque cup. Pizza was served with 1.5 L bottled spring water (Danone Crystal Springs). Four varieties of pizza (McCain Deep N Delicious; 5 diameter: Deluxe, Pepperoni, Three Cheese, and Deli Lovers; Mc- Cain Foods) purchased from local retailers, were provided as choices. Subjects were instructed to eat until they were comfortably full and were made aware that they would be presented with identical trays of hot pizzas at 5- to 10-min intervals. Each variety of pizza was weighed separately and the energy consumed was calculated by converting the net weight consumed to kj using information provided by the manufacturer (McCain Foods). Total energy contents for the Pepperoni, Deluxe, Three Cheese, and Deli Lovers pizzas were 213, 192, 229, and 225 kj, respectively. Their protein, fat, and total carbohydrate contents were similar, averaging 10.0, 7.6, and 26.6 g, respectively. An advantage of using these pizzas was the lack of outer crust, which results in a pizza with more uniform energy content and eliminated the possibility of the subjects eating the more energy dense filling and leaving the outside crust of the pizza. Each pizza was cut into 4 slices before serving. Blood samples (Expt. 4) obtained using a Monojector Lancet Device (Kendall, Tyco Healthcare), were placed on glucometer test strips of a portable glucose monitoring system (Fast Take One Touch, Life Scan Canada) and blood glucose concentration recorded. Data analysis. Statistical analyses were conducted using SAS version 7.1 (Statistical Analysis Systems, SAS Institute) and SPSS version (Statistical Package for Social Sciences). One-way repeated-measures ANOVA was performed to test for the effect of treatment on outcome variables, energy intake, and blood glucose. A 2-way repeated-measures ANOVA in 1 factor (treatment) was conducted to determine the effect of treatment and time in Expts. 1 and 2. Students unpaired t test was used to compare the effect of time on the response to the preloads. The General Linear Models (GLM) procedure was used for the ANOVAs. Duncan s post-hoc tests were performed for mean comparisons. All values are presented as means SEM. Differences with P-values 0.05 were considered significant. Correlation analysis was conducted using the Partial Pearson Correlation coefficient. RESULTS Food intake Expt. 1: Protein source and food intake. Treatment was the main factor affecting energy consumption at the test meal (P 0.001) and cumulative energy intake (P 0.001) (Table 1). Whey and soy protein decreased food intake compared with the control (P 0.05), whereas egg protein and sucrose did not. There was no difference in energy intake from the test meals between sucrose and soy protein, or between whey and soy protein. Cumulative (meal plus preload) intake after egg albumen and sucrose, but not after whey or soy treatments, was greater than for the control (P 0.001). Compensation for energy consumed in the drinks was different from control for whey and soy (P 0.05), but not for the egg and sucrose treatments. The pattern of effect of treatment was affected by arrival time, as shown by an interaction (P 0.05) between the main factors of treatment (P 0.03) and time (P 0.23) on food intake (Table 2). Within the 0910 h group (n 6), sucrose, whey, and soy protein suppressed food intake compared with the control, whereas whey and soy suppressed food intake more than sucrose (P 0.05). The egg protein preload resulted in food intake similar to the control and sucrose preloads. For the 1100 h group (n 7), pizza energy intake after egg albumen was greater than after the control, whey, or soy (P 0.01) but not different from sucrose. Cumulative energy intake by the men consuming the treat-

3 PROTEIN SOURCE AND FOOD INTAKE 3013 TABLE 1 Effect of protein source in preload treatments on food intake 1 h later in adult men1 Preload treatments Control Egg Whey Soy Sucrose Experiment 1 Meal,2 kj a a c bc ab Meal Preload,3 kj c a c bc ab Compensation,4 % 33 30c 96 24a 59 35ab 12 25c Experiment 2 Meal,2 kj a a b Meal Preload,3 kj b a b Compensation,4 % 27 30b a 1 Values are means SEM, n 13 (Expt. 1) and n 22 (Expt. 2). Means in a row without a common letter differ, P Cumulative energy intakes from pizza meal and the preload treatment. 4 Compensation (%) (control meal (kj) treatment meal (kj)/preload (kj) 100. ments at 0910 h was greater after egg and sucrose than after whey and soy (P 0.01) (Table 2). However, none were different from the control. At 1100 h, all treatments except whey resulted in cumulative intakes greater than the control (P 0.01). The largest cumulative energy intake occurred when egg protein was the preload. Expt. 2: Protein source, time of arrival and food intake. Treatment, (P 0.01) but not time of arrival (P 0.25) affected food intake and in contrast to Expt. 1, there was no interaction (P 0.11) between the factors (Table 2). Whey suppressed food intake compared with the control and egg treatments, which did not differ from each other (Table 1). As in Expt. 1, egg treatment resulted in a net increase in cumulative energy consumption. Energy compensation at the pizza meal was greater after the whey than for the egg treatment (Table 1). Again, the pattern of effect of treatment was slightly different at the 2 times (Table 2). For the 0830 h arrival time, both egg and whey protein suppressed food intake compared with the control with whey having the greatest effect (P 0.01). At 1100 h, there was no difference between the egg and control treatments, but whey protein suppressed food intake more than the egg treatment (P 0.05). Treatment at 0830 h resulted in cumulative energy intakes that were similar to the control after whey and egg, but higher after egg than after whey (P 0.01). At 1100 h, cumulative energy intake after egg albumen was greater than after either control or whey (P 0.01). In both experiments, more pizza was consumed when the egg preload was given at the later time (P 0.05). Expt. 3: Whey and food intake at 2 h. Energy intake 2 h later was less after hydrolyzed whey ( kj) or intact whey ( kj) than after the control ( kj) (P 0.01). The sums of the intakes of the preload plus the test meal were not different (P 0.7). For the control, whey hydrolysate, and intact whey, cumulative intakes were , , and kj, respectively. TABLE 2 Effect of protein source in preload treatments on food intake by time of treatment in adult men1 Preload treatments n Control Egg Whey Soy Sucrose Experiment 1 Meal h a ab c c b 1100 h bc a* c bc* ab Meal Preload h ab a b b a 1100 h d a* cd bc* ab Experiment 2 Meal h a b c 1100 h ab a* b Meal Preload h ab a b 1100 h b a* b 1 Values are means SEM. Means in a row without a common letter differ, P * Different from 0910 or 0830 h (Student s unpaired t test, P 0.05). 3 Cumulative intakes from pizza meal and the preload treatment. kj

4 3014 ANDERSON ET AL. Expt. 4: Soy protein, glycemic carbohydrate and food intake. There was an overall treatment effect on pizza intake (P 0.01) (Table 3). The soy treatment reduced meal intake compared with the control and the soy/amylose mixture. Neither the soy/glucose nor the soy/amylose treatment decreased food intake. The treatment effect was not significant for the percentage compensation (P 0.11). Cumulative energy intake after the soy/amylose and soy/glucose treatments was greater than that after either the control or soy treatments (P 0.05), which were similar. Blood glucose. In Expt. 4, blood glucose concentration was affected by treatment (P 0.001) and time (P 0.001), and there was a time treatment interaction (P 0.001). Post-hoc analysis showed differences among treatments at 15, 30, and 45 min (P 0.05) (Table 4). The net incremental area under the curve (AUC) 4 for blood glucose to 60 min was highest after the soy/glucose treatment [ (mmol min)/l; P 0.05]. The AUC for the soy/amylose treatment [ (mmol min)/l] was significantly greater than the AUC for the soy treatment [ (mmol min)/l], and both were greater than the control [ (mmol min)/l]. No correlations were found between food intake and blood glucose at 60 min (r 0.05, P 0.71) or area under the blood glucose curve to 60 min (r 0.12, P 0.40). DISCUSSION The results of these studies show that protein source, quantity, and composition of protein preloads and time of food consumption in the morning are factors determining the effect of protein ingestion on short-term food intake in humans. The importance of protein source for its effect on food intake is clearly illustrated. Whey consistently resulted in the greatest food intake suppression, decreasing food intake relative to control and sucrose, as well as in comparison with egg albumen and soy protein. Whey (45 50 g) reduced food intake when consumed either 60 min (Expts. 1 and 2) or 2 h later (Expt. 3). An unexpected observation was that egg albumen preloads did not suppress food intake and resulted in cumulative energy intakes that were higher than after the control treatments, an effect that was accentuated when the subjects arrived late compared with earlier in the morning (Table 2). Because egg albumen, whey, and soy are high-quality proteins for meeting the amino acid requirements of humans (16), it is highly unlikely that suppression of food intake after whey and soy protein can be attributed to a single amino acid imbalance and depletion in the central nervous system, as occurs with amino acid imbalanced diets or diets deficient in an amino acid (17). Rather differences in the effect of these protein sources on food intake are more likely due to their effects on pre- and postmetabolic responses. The failure of egg protein to suppress food intake in these young men was surprising and is in contrast to its effect on food intake in rats (18). Digestibility is an unlikely explanation. Spray-dried egg white powder as used in the present studies, was estimated to have a digestibility of 92 97% in nitrogen balance studies (19), and cooked egg white was estimated to be 94% digestible by stable isotope techniques (20,21). A more likely explanation might reside in the effect of egg albumen on satiety hormones. In rats, egg albumen suppresses 4 Abbreviations used: AUC, area under the curve; CCK A, cholecystokinin-a; CMP, caseinomacropeptide; GLP-1, glucagon-like peptide-1. TABLE 3 Experiment 4: Effect of carbohydrate addition to soy protein preloads on food intake 1 h later in adult men1 Treatment Meal2 Meal Preload3 Compensation4 kj % Control a b Soy b b Soy/glucose ab a Soy/amylose a a Values are means SEM, n 13. Means in a column without a common letter differ, P Cumulative intake from pizza meal and the preload treatment. 4 Compensation (%) (control meal (kj) treatment meal (kj)/ preload (kj) 100. food intake and leads to activation of cholecystokinin-a (CCK A ) receptors (18), but in humans, egg whites, in contrast to whole egg or egg yolk, do not raise the blood concentration of CCK (22). Other satiety hormones also appear to be stimulated less by egg albumen than by other proteins. Because insulin is a satiety hormone (23), the low plasma insulin concentrations observed after egg, compared with ham consumed at breakfast (24), may also explain its failure to affect food intake in the present study. The effect of whey on short-term food is consistent with several aspects of its metabolism. First, whey compared with casein, digests quickly, resulting in a rapid increase in plasma amino acids that is sustained for 2 h (10,25), perhaps contributing to its suppression of food intake through increased brain amino acid concentrations (17). Second, whey ingestion results in the release of several gut peptides involved in satiety including CCK, glucagon-like peptide-1 (GLP-1) and glucosedependent insulinotropic polypeptide (10). Third, commercial whey products prepared by ultrafiltration, like the one used here, contain up to 15 20% of the bioactive macropeptide, caseinomacropeptide (CMP), which is a water-soluble compound of casein that is released during the cheese-making process. CMP is a strong stimulant of CCK release in rats (26). Soy protein also stimulates mechanisms that regulate food intake. Soy protein digestion releases biologically active peptides that stimulate peripheral opioid and CCK A receptors in rats (27). In humans, soy protein in a test meal of 46 kj/kg was reported to increase blood glucose, insulin, and glucagon concentrations for up to 8 h (28). The greater satiety value of protein compared with carbohydrate, as noted in Expt. 1, is emphasized by the results of substituting glycemic carbohydrate for 50% of the soy protein in the preload treatment (Table 4). The rationale for adding glycemic carbohydrate to soy protein was based on the hypothesis that the combined satiety signals arising from protein and carbohydrate would be stronger than for each alone. For example, protein ingestion results in an increase in plasma amino acids concentration (10) and stimulates the satiety hormones glucagon (29), CCK (10,18), and GLP-1 (10,30), whereas carbohydrate ingestion leads to increased plasma concentrations of glucose and insulin (13,31). The replacement of 25 g of soy protein in the preload with glucose and amylose raised blood glucose above that found after soy protein alone but only to 30 min (Table 4). As a result, no association was found between blood glucose immediately before the meal and food intake at 60 min as was reported after 75-g carbohydrate

5 PROTEIN SOURCE AND FOOD INTAKE 3015 TABLE 4 Experiment 4: Effect of soy preloads with glycemic carbohydrate on blood glucose concentration in adult men1 Time Control Soy min mmol/l Soy/ Glucose preloads (32). Perhaps a larger amount of glucose, resulting in elevated blood glucose immediately premeal would enhance the effect of protein on food intake suppression. However, it is clear that a reduction from 50 to 25 g of soy protein in the preload, and its replacement with a carbohydrate source of equal energy value, did not duplicate the effect of soy protein on food intake, suggesting too that 25 g is below the threshold dose of soy protein required to affect food intake 1 h later. Arrival time, that is, the time at which the pizza was consumed in the morning, was a factor affecting the results in Expts. 1 and 2. Although starting time did not prove to be an overriding factor affecting the results, in both experiments, more energy was consumed after the egg treatment was given in the later morning compared with earlier in the morning (Table 2). A weakness of the design of the present studies was that different subjects were administered the treatments at each of the 2 times of the morning. Thus, the effect of time is not known and can be determined only by a repeated-measures design in which the same subject is administered the same treatment compared with the control at each time. The relevance to body weight regulation of these differences among protein sources in their effect on short-term food intake remains to be determined. However, evaluation of the effect of amount and protein source, and the interaction between these 2 variables over a period of several days or weeks on energy intake regulation and body weight, is required to provide guidance for diets aimed at body weight maintenance or body weight loss. Milk proteins might be of specific interest and perhaps provide an explanation for the association noted between increased dairy product consumption and lower BMI (1). In conclusion, protein source, time of consumption, quantity, and composition are factors determining the effect of protein on short-term satiety and food intake in young men. LITERATURE CITED Soy/ Amylose F P c bc a b c c a b b ab a a Values are means SEM, n 13. Means in a row with superscripts without a common letter differ, P Anderson, G. H. & Moore, S. E. (2004) Dietary proteins in the regulation of food intake and body weight in humans. J. Nutr. 134: 974S 979S. 2. Booth, D. A., Chase, A. & Campbell, A. T. 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