articles
nature publishing group
Integrative Physiology
Dietary Whey Protein Decreases Food Intake and Body Fat in Rats June Zhou1−3, Michael J. Keenan2, Jack N. Losso2, Anne M. Raggio1,2, Li Shen1,2, Kathleen L. McCutcheon2, Richard T. Tulley2, Marc R. Blackman3 and Roy J. Martin1,2 We investigated the effects of dietary whey protein on food intake, body fat, and body weight gain in rats. Adult (11–12 week) male Sprague–Dawley rats were divided into three dietary treatment groups for a 10-week study: control. Whey protein (HP-W), or high-protein content control (HP-S). Albumin was used as the basic protein source for all three diets. HP-W and HP-S diets contained an additional 24% (wt/wt) whey or isoflavone-free soy protein, respectively. Food intake, body weight, body fat, respiratory quotient (RQ), plasma cholecystokinin (CCK), glucagon like peptide-1 (GLP-1), peptide YY (PYY), and leptin were measured during and/or at the end of the study. The results showed that body fat and body weight gain were lower (P < 0.05) at the end of study in rats fed HP-W or HP-S vs. control diet. The cumulative food intake measured over the 10-week study period was lower in the HP-W vs. control and HP-S groups (P < 0.01). Further, HP-W fed rats exhibited lower N2 free RQ values than did control and HP-S groups (P < 0.01). Plasma concentrations of total GLP-1 were higher in HP-W and HP-S vs. control group (P < 0.05), whereas plasma CCK, PYY, and leptin did not differ among the three groups. In conclusion, although dietary HP-W and HP-S each decrease body fat accumulation and body weight gain, the mechanism(s) involved appear to be different. HP-S fed rats exhibit increased fat oxidation, whereas HP-W fed rats show decreased food intake and increased fat oxidation, which may contribute to the effects of whey protein on body fat. Obesity (2011) doi:10.1038/oby.2011.14
Introduction
Whey protein is an abundant byproduct in cheese production (10 liter of milk produces about 9 liter of whey during cheese manufacture (1). Because it contains all essential amino acids and can rapidly increase plasma amino acid concentrations, whey protein is considered to be a high quality protein (2,3), and is thus commonly used for maintaining healthy body weight in humans (4–6). Although, in general, a high-protein diet decreases food intake (7–10), whey protein shows greater suppression of appetite in the next hour of feeding than do egg-albumin, soy protein, and casein (5,11,12). Whey protein is also more effective than red meat in reducing body weight gain and increasing insulin sensitivity (6). These studies indicate that HP-W may have unique properties in maintaining healthy body weight. However, most studies on whey protein suppression of appetite in humans have been shortterm, with food intake or satiation measured several hours after consumption of a single whey protein meal. The results are also contradictory because the duration to the next meal and composition of the test meals were varied in these studies (5,11–13). Thus, longer term studies and use of carefully matched dietary components for test and control diets are
needed to clarify the role of whey protein on food intake and body weight. In this study, we used a rat model and carefully designed control diets to examine the longer term effects of dietary whey protein on body fat and food intake. During a 10 week study period, food intake, body fat, body weight gain, and metabolic rate (respiratory quotient (RQ)) were measured in rats fed whey protein or two different control diets. There is considerable interest in whether whey protein affects satiety and food intake via alterations in the secretion or action(s) of the gut secreted anorectic hormones, glucagon like peptide-1 (GLP-1), peptide YY (PYY), and cholecystokinin (CCK) (14–16). Thus, we measured plasma concentrations of GLP-1, PYY, and CCK. Leptin, another important hormone in regulation of food intake and body fat, was also measured. Two control diets were used in this study: one had the protein content of the AIN-93M based rodent diet (14%), whereas the other had the same higher protein content (38%) as the whey protein diet, although another source of protein was used. This study design allowed us to examine the effects of dietary whey protein vs. high dietary protein in general, on food intake and body fat.
Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana, USA; 2Louisiana State University Agricultural Center, Baton Rouge, Louisiana, USA; 3Research Service, Veterans Affairs Medical Center, Washington, DC, USA. Correspondence: June Zhou (
[email protected]) 1
Received 1 September 2010; accepted 8 January 2011; advance online publication 17 February 2011. doi:10.1038/oby.2011.14 obesity
1
articles Integrative Physiology Methods and Procedures Animals and housing conditions Thirty healthy adult male Sprague–Dawley rats (11–12 week old) were purchased from Harlan Industries (Indianapolis, IN). After quarantine, the rats were single housed in suspended wire-bottom cages in a humidity and temperature controlled room (22 ± 2 °C, 65–67% humidity) on a 12:12 h light:dark cycle with free access to food and water. Wire-bottom cages were used to measure food spillage. All rats were fed semipurified powder diets prepared in our laboratory. Rats were fed the AIN-93M based rodent control diet and acclimated to experimental conditions until their body weights were stable (about 2 weeks). The protocol was approved by the Pennington Biomedical Research Center’s Animal Care and Use Committee. Diets The regular control (control) diet was prepared based on the AIN-93M formula for laboratory rodents (17) and egg albumin was used as the protein source. The components of the whey protein (HP-W) diet were the same as those of the control diet, except that half the starch in the diet was replaced by whey protein. The high-protein content control (HP-S) diet had the same protein content as the HP-W diet, although the whey protein was replaced by isoflavone-free soy protein. This HP-S diet was designed to compare whey protein with a non-milk protein source. Isoflavone-free soy protein was used to avoid the reported bioactivities of isoflavones that are naturally associated with soy protein. The total protein contents were 14% for Control diet, 38% for HP-W diet (14% egg albumin plus 24% HP-W), and 38% for HP-S diet (14% egg albumin plus 24% isoflavone-free soy protein). The detailed compositions of the three experimental diets are listed in Table 1. Table 1 Experimental diets components Control diet
HP-W diet
HP-S diet
140
140
140
Whey protein concentrate 80
0
300
0
Soy protein
0
0
353.74
Ingredients (g) Albumin (egg white)
Starch
612.5
312.5
258.8
Sucrose
100
100
100
Soybean oil
40
40
40
Cellulose
50
50
50
Mineral mix for egg white protein
35
35
35
Vitamin mix
10
10
10
Choline chloride
2.5
2.5
2.5
Biotin/Sucrose Premix
10
10
10
Total (g)
1,000
1,000
1,000
BHT (in oil)
0.008
0.008
0.008
14
38
38
Total protein content (%) Total fat content (%)
4
4
4
Total carbohydrate content (%)
82
58
58
The unit for all ingredients is gram. • Whey protein (whey protein concentrate 80, a gift from the Dairy Council) contains 79.7% whey protein. • Isoflavone-free soy protein (PROCON 2000, a gift from The Solae Company, St Louis, MO) has 67.6% soy protein. • Starch (100% amylopectin) was a gift from National Starch Food Innovation (Bridgewater, NJ 08807) • All other ingredients were purchased from Dyets (Bethlehem, PA). • The energy content of the three experimental diets were similar (3.6–3.7 kcal/ kg diet). HP-S, high-protein content control; HP-W, whey protein; BHT, butylated hydroxytoluene. 2
Experimental protocol After acclimatization to the experimental conditions (powder diet and wire cages), rats were divided into three dietary treatment groups for a 10 week study. Each group had a similar average body weight and body fat at the beginning of the study. Fresh food was provided to each rat three times per week by changing the food jar of each rat. Food intake was recorded by weighing the food jar before it was placed into the cage with fresh diet (full jar weight) and when it was taken out of the cage (empty jar weight) 2 or 3 days later. Food spillage was also recorded by carefully weighing the spillage under the wire cage for each rat. Because all three diets were powder diets, there were no differences in diet consistency that would have made the spillage of one diet harder to measure than that of the other diets. The food intake was calculated by subtracting the weights of the empty food jars and spillage from the weights of the full food jars for each rat. Body weight was also measured three times per week when food intake was measured. During the first 6 weeks of the study period, rats were undisturbed except for food intake and body weight measurements. At the 6th week of the study, body composition was measured by nuclear magnetic resonance (NMR) in all rats. On the 7th and 8th weeks of the study, rats were placed in metabolic chambers for measurement of respiratory exchange ratios (RQ’s). Body weight gain was calculated as the body weight on the day of measurement minus the body weight recorded at the beginning of study (daymeasurement−day0). All rats were killed by decapitation at the end of the study. Trunk blood was collected from each rat for measurements of GLP-1, PYY, CCK, and leptin. Body fat and lean body mass measurements during the study Body fat and lean body mass were measured during the study by NMR (Bruker minispec Live Rats Analyzer model mq7.5, LF90; Bruker Optics, Woodlands, TX) (18). Each rat was weighed before it was placed in the tube for NMR measurement and the recorded body weight was used for body composition calculation. For each rat, the measurements were repeated twice and the difference of % fat for each rat was
