Resistant starch and protein intake enhances fat oxidation and feelings of fullness in lean and overweight/obese women

A total of 70 women from the Saratoga Springs, NY area were recruited through newspaper advertisements and flyers and initially screened for participation, of whom 24 were eligible for participation. Participants were non-smoking, healthy women with no known cardiovascular or metabolic diseases as assessed by a medical history and examination by their personal physicians. Participants were middle-aged (45.8 ± 2.5 years), overweight/obese (BMI = 31.9 ± 1.4 kg/m²; % body fat = 41.2 ± 2.3) or lean (BMI = 21.0 ± 0.5 kg/m²; % body fat = 23.9 ± 1.4), and weight stable (±2 kg) for at least 6 months prior to beginning the study. Each participant provided informed written consent in adherence with the Skidmore College Human Subjects review board prior to participation, and the study was approved by the Human Subjects Institutional Review Board of Skidmore College. All experimental procedures were performed in accordance with the Federal Wide Assurance and related New York State regulations, which are consistent with the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research and in agreement with the Helsinki Declaration as revised in 1983. This trial was registered at clinicaltrials.gov as NCT02418429.

On four separate visits to the Human Nutrition and Metabolism Laboratory, all subjects consumed one of four pancake test meals in a single-blind, randomized repeated measures crossover design: 1) waxy maize (control) starch (WMS, n = 16); 2) waxy maize starch and whey protein (WMS+WP, n = 9); 3) resistant starch (RS, n = 16); or 4) RS and whey protein (RS+WP, n = 16). Due to subject availability and time constraints, only 9 subjects (lean, n = 5; obese, n = 4) completed the WMS+WP test meal condition. Each pancake test meal was consumed together with water (180 ml) only. Pancakes were prepared following institutional guidelines using pre-gelatinized test starch, sugar, maltodextrin, vegetable oil, baking powder, egg, non-fat dry milk powder and water. All dry and wet ingredients were mixed together separately and then combined. Each meal consisted of three pancakes that were cooked on a non-stick griddle until golden brown. The nutritional composition of the four test meals are shown in Table 1.

The day before each of the four test days, participants were required to prepare their own meals and follow a standardized daily menu plan consisting of 25 % protein, 50 % carbohydrate, and 25 % fat, based on their estimated caloric needs. The final evening dinner meal the night prior to each test condition was consumed between 1800 and 2000 h and was identical for all four test conditions. All laboratory testing was conducted between 0600 and 0700 following a 12-hour fast (water was allowed) and at least a 24-hour restriction of physical activity, caffeine, and alcohol intake. Details regarding the test-day timeline are shown in Fig. 1. Briefly, upon arrival to the laboratory, body weight was measured (Befour Inc., model number FS0900) with participants wearing only shorts and a t-shirt. Following ~15 min of resting supine in a quiet, dimly lit room, resting metabolic rate (RMR) was measured for 30 min followed by a fasted blood draw (for plasma insulin, glucose, GIP, GLP-1, PYY, ghrelin, and leptin), and completion of visual analog scales (VAS) of hunger, desire to eat, and satiety (see testing procedures below). Subjects then consumed one of the four test meals (WMS; WMS+WP; RS; RS+WP) within 12 min. For 3 h following the completion of the test meals, subjects remained in a sedentary and supine position while serial blood samples were taken and VAS were completed (minutes 60, 120, 180). Indirect calorimetry was used to determine the thermic effect of the meal (TEM) (min 45–60, 105–120, 165–180). Following completion of the first test meal, participants were measured for body composition using the Life Measurements BODPod Body Composition Tracking System (Concord, CA).

RMR (kilocalories per minute) was measured using the ventilated hood technique [17] with a computerized open-circuit indirect calorimeter (Parvomedics, Truemax 2400, Salt Lake City, UT). Participants were not allowed to sleep and all measurements were obtained in the supine position following at least 15 min of quiet resting in a thermo-neutral (22–24 °C), semi-dark room. Following the RMR, a thermic effect of a meal (TEM) challenge was administered and postprandial thermogenesis was measured every 45 min for 180 min (TEM 45–60; 105–120; 165–180 min). Steady state was achieved for all participants during the final 10 min of each 15 min measurement period, and used in the calculation of TEM (minutes 0–5 were discarded). The total 180-minute TEM was calculated by taking an average of each 10-min TEM measurement and multiplying it by 60 min (0–60; 61–120; 121–180 min). Each of the three 60-min TEM periods was then summed for the 180-min TEM value.

The RQ and substrate utilization were also calculated from the gas exchange data using computer software from the calorimeter (Parvomedics, Truemax 2400) to provide fat and carbohydrate oxidation rates based on standardized caloric equivalents. The total 180-minute oxidation rates (fat and carbohydrate) were calculated using the exact same method described above for TEM. RMR (kcal/d) was calculated as the average of the test period. A 180 min TEM was chosen to capture the majority of the postprandial response. The total kilocalories eaten for each of the 4 test meals were isocaloric and only differed in the type of starch and protein content (Table 1). This study design allowed for the direct comparison of differences in starch composition and the macronutrient distribution of protein content on the thermogenic response. Test-retest intraclass correlation (r) and coefficient of variation (CV) in n = 14 is: RMR (Kcal/min) r = 0.92, 4.2 %, respectively.

Venous blood samples (~20 ml) were obtained following the RMR and every hour after meal ingestion (TEM, minutes 60, 120, 180). Due to participant and resource constraints, no blood was obtained for WMS+WP. Blood was collected into EDTA-coated vacutainer tubes and centrifuged (Hettich Rotina 46R5) for 15 min at 2500 rpm at 4 °C. Plasma was then separated and stored at −70 °C in small aliquots until analyzed. Insulin, ghrelin, PYY, and GIP were determined using commercially available ELISA kits (Millipore, Inc. and DSL, Inc.). Plasma glucose concentrations were determined with a glucose analyzer using the glucose oxidase technique (GM7 Analyser, Analox Instruments, Lunenberg, MA). GLP-1 was analyzed using a radioimmunoassay with antiserum (no. 89390).

Visual analogue scales (VAS) were used to evaluate hunger, satiation, quantity of food that could be eaten, and desire to eat scores. Each VAS was 100 mm in length and anchored at each end. Participants were instructed to place a mark on the 100 mm line to indicate their levels of hunger, satiety, food quantity, and desire to eat. For hunger, a mark at 0 mm indicated no hunger, while a mark at 100 mm indicated extreme hunger. For satiety, a mark at 0 mm indicated no feeling of fullness, while a mark at 100 mm indicated an extreme feeling of fullness. For quantity of food that could be eaten, a mark at 0 mm indicated no food could be eaten, and a mark at 100 mm indicated a large quantity of food could be eaten. For desire to eat, a mark at 0 mm indicated no desire to eat, and a mark at 100 mm indicated extreme desire to eat. For each of the four measures (hunger, satiety, quantity of food eaten, and desire to eat), the degree to which each sensation was felt was quantified by measuring how far the mark was from the 0 mm point. For this measurement, a standard millimeter ruler was used and all scores were computed by the same investigator. VAS scales were completed during each of the four test meal conditions at baseline (RMR) and every hour during the TEM meal challenge (60, 120, and 180 min).

Resting heart rate and blood pressure were obtained manually in the supine position as previously described [18]. Heart rate and blood pressure were obtained by a trained investigator on each of the four test meal days following the RMR measurement.

Statistical analyses were performed using SPSS software (Ver. 21; IBM-SPSS Inc.). Significance was set at p <0.05. All values are reported as means ± SEM unless noted otherwise. Prior to the start of the study, subject number was determined from a power analysis based on the major outcome variables (TEM, fat oxidation, gastro-entero-pancreatic hormones, VAS) as reported by a previous study [10] with an alpha level set to 0.05 and a power of 0.8. This analysis determined we would require n = 12 participants to detect significant differences. Biomarker responses (glucose, insulin, GIP, GLP-1, ghrelin) were assessed by calculating the incremental area under the curve (AUC) using the trapezoid method. A 2 × 4 × 4 factor repeated measures ANOVA (2 groups; lean, obese: 4 test meals; WMS, WMS+WP, RS, RS+WP: 4 time points; 0, 60, 120, 180 min) was initially performed to determine differences among groups, test meals, and time points (time and group/test meal x time interactions). Thereafter, because no differences existed between lean and overweight/obese women for all variables, a 4 × 4 RMANOVA (meal × time) was run for all outcome variables. Absolute changes were calculated as the average baseline value (3 or 4 test meals) subtracted from each post-meal value (Figs. 2, 3, 5 and 6). Percent changes were calculated as the delta between baseline and each post meal time point divided by the baseline value (Fig. 4). Where significant main effects were identified, post hoc comparisons (paired sample t-test [time effects] and Tukey’s test [group differences]) were performed to locate differences. We utilized one-tailed t-tests for investigation of time effects for each test meal condition.