1. Introduction
The increasing obesity epidemic has prompted researchers to seek novel nutritional strategies to manage a healthy body weight and composition. While dietary approaches such as macronutrient composition, caloric restriction, and meal frequency have been popular areas of focus, the use of specific food products and dietary components are increasingly showing promise in the treatment of obesity [
1,
2]. Of these, dairy protein fractions, including whey protein (WP), and indigestible carbohydrates, like resistant starch (RS), are both increasingly being viewed as strategies to control body weight and enhance body composition when incorporated into a nutritional intervention [
3,
4,
5,
6].
As a category, RS is starch that moves through the small intestine to the colon undigested/unabsorbed, where it can be metabolized through microbial fermentation processes and ultimately produce short-chain fatty acids (SCFAs) as well as small microbes with health-promoting properties [
7]. Multiple studies in animals [
8,
9,
10,
11], and a growing body of evidence in humans [
12,
13,
14], including from our lab [
6], report that dietary RS (obtained from RS4 hydroxypropyl distarch phosphate) may support healthy body weight management. There are several proposed mechanisms of action, including (1) the ability of RS to reduce the number of metabolizable calories because of its resistance to digestion and absorption [
9]; (2) the production of SCFAs, which may increase the absolute number of calories burned and lipid oxidation [
15]; and (3) reduction in calories consumed by promoting release of appetite and hunger signals, including gastro-entero-hepatic hormones peptide YY (PYY) and glucagon-like polypeptide-1 (GLP-1), both of which bind receptors in the brain and decrease appetite and serve as anorexigenics [
16]. In addition, clinical research has reported reductions in fasting and postprandial glucose levels and enhanced insulin sensitivity and gut-derived satiety peptides [
17].
There are four types of RS: RS found in seeds, germs, and whole grains that is resistant to digestive enzymes (RS1); high-amylose starch from maize that contains α-1,4 glycosidic bonds (RS2); starch from cooked and cooled grains such as pasta and rice that has been retrograded (RS3); and starch that has been chemically modified (RS4, hereinafter RS; [
18]). Some of these forms can be found naturally in the human diet, including RS1, provided by whole grains (rice, pasta). RS2 is derived from raw potatoes and unripe bananas and resists carbohydrate digestive enzymes. RS3 is retrograded starch that occurs after grains, such as rice and pasta, have been cooked then cooled [
3]. In contrast, RS4 is starch that is chemically altered and more resistant to retrogradation, with increased viscosity, but it is more stable in response to acid and increased temperature compared to naturally occurring starch [
3]. While less studied than other forms, RS4 consumption increases resting energy expenditure and lipid utilization in lean men [
19] and improves post-meal glucose and insulin levels in healthy adults [
20]. Therefore, RS4 may offer an attractive candidate as a potential functional ingredient in various food applications replacing other forms of starch with lower overall nutritional value.
Increasing the intake of dietary protein over the current recommendations is another nutrition-focused strategy proposed for weight maintenance and optimal body composition [
21,
22]. As with RS, increased dietary protein is associated with increased resting metabolism and satiety [
23,
24]. Previously, our lab showed higher quantity and frequency of dietary protein (termed “protein pacing”) optimizes fat loss and muscle mass retention along with elevating caloric expenditure in obese adults [
4]. Thus, combining RS, obtained from RS4 type hydroxypropyl distarch phosphate, and dietary protein, specifically WP, may offer a supra-additive thermogenic and metabolic advantage beyond each functional food alone. Indeed, our previous study was the first, and only one, to demonstrate an acute meal challenge combining dietary RS and WP increases fat oxidation, PYY, satiety and fullness compared to waxy maize starch control (WMS), WMS + WP, and RS alone in women [
6]. While the results of this research were notable, males were not assessed. Therefore, the current study examined breakfast meal challenges containing waxy maize (non-RS) ingredients or RS with and without added WP on fuel utilization, postprandial thermogenesis, subjective measures of hunger and appetite, and circulating signals of satiety hormones in healthy men. For this purpose, and similar to our previous study, we conducted a randomized, single-blind, crossover design with four meal challenges consisting of pancake meals provided for breakfast with (1) WMS (control); (2) WMS + WP; (3) RS; or (4) RS + WP.
4. Discussion
The major aim of the current study was to quantify the effects of pancake meals containing non-RS constituents or RS4 meals with and without greater protein contents on postprandial thermogenesis (TEM), fuel utilization, satiety, and gastro-entero-pancreatic hormones in healthy middle-aged men. The main findings reveal that RS + WP, compared to other test meals, elicited (1) a larger thermogenic effect; (2) greater rate of fat oxidation and lower CHO oxidation; and (3) reduced postprandial circulating glucose and insulin levels. Subjective feelings of hunger, satiation, and desire to eat were not different among the four meals. Taken together, these findings show acute ingestion of a solid food meal containing RS + WP elicits a significantly greater thermogenic response (total calorie burn), fat oxidation rates, and lower glucose/insulin responses compared to whole food meals of equal caloric and macronutrient composition. These results may have important implications regarding the incorporation of functional ingredients including resistant starches and isolated dairy proteins into various food matrices for weight control and mitigating metabolic-related health conditions (e.g., diabetes, obesity, inflammation).
The major finding of the current study is the significant magnitude of increase (>76%) in TEM with RS + WP compared to the other isocaloric test meal conditions. This clearly reflects a metabolic advantage of combined resistant starch with whey protein meals. The likely mechanism for this heightened thermic response is the different digestion and absorption and subsequent short-chain fatty acid (SCFA) production of RS. Specifically, the total macronutrient amount and proportion of calories entering circulation may be responsible for the drastic increase in thermogenesis with RS compared with isocaloric non-RS containing meals. Thus, further research needs to elucidate whether the greater TEM response with RS meals is due to altered gut metabolism or simply a reflection of altered amounts of calories/macronutrients being metabolized, or a combination of both. Further, given the addition of WP was necessary for this greater TEM response, there seems to be a supra-additive effect of both together compared to each alone. Our lab has previously demonstrated the “metabolic advantage” of increased TEM from consuming protein pacing meals/snacks compared to comparable isocaloric non-protein pacing meals [
4]. In addition, data from our laboratory have previously reported increased fat oxidation, PYY, and increased satiety and fullness following consumption of combined dietary resistant starch and whey protein in lean and overweight/obese females [
6]. Using identical study methodology as the current investigation, in females we did not find the same effect of significantly increased post-prandial energy expenditure nor the reductions in postprandial glucose and insulin concentrations. Though in contrast to the current study, RS + WP did increase the feelings of fullness, while decreasing the feeling of hunger versus non-whey conditions. The divergent findings between males and females highlight potential sex differences that may warrant further investigation. Interestingly, no previous rationale for sex differences in these responses following consumption of identical test meals exists. Thus, conflicting findings remain unclear at this time. In sum, the combination of RS and WP may elicit enhanced gut (SCFA production) and macronutrient (calorie) metabolism that results in greater postprandial thermogenic response compared to isocaloric meal challenges and thus may serve as a highly effective, long-term nutrition strategy to aid with weight control, body composition, and cardiometabolic health outcomes.
Our results align with previous research utilizing RS in a solid food matrix meal challenge, particularly in reducing postprandial elevations in blood glucose and/or plasma insulin compared to matched carbohydrate controls [
19,
20,
27,
28,
29,
30]. Similar in research design to our study, Shimotoyodome et al. (2011) provided participants with a mixed meal pancake breakfast challenge containing a comparable amount of RS (38 g) or WMS (38 g) [
19]. Not only did the RS meal elicit significantly lower postprandial glucose and insulin concentrations, but postprandial thermogenesis and fat utilization were also significantly elevated in healthy male participants. More recently in a double-blind, randomized crossover study, Mah et al. (2018) reported RS4, as a constituent of a breakfast bar, reduced glucose and insulin AUC [
28]. Findings from these studies, as well as the present study, have important clinical relevance due to the strong relationship between poorly controlled postprandial blood glucose control and development of diabetes. Indeed, more recent data point to a strong correlation between poor postprandial blood glucose control and the presence of coronary heart disease [
31].
The current findings support the replacement of a certain amount of available carbohydrate with RS4 to help lower postprandial glucose, as this may reduce the amount of carbohydrate contributing to blood glucose. This assertion is supported by animal studies that have shown decreased gastrointestinal transit time of RS-containing carbohydrates [
32], which in turn likely allows portions of the available carbohydrate to escape digestion and absorption in the small intestine. Once in the lower portions of the gastrointestinal tract, RS may be used as substrate for microbial fermentation and production of SCFAs, as well as other metabolites with beneficial metabolic properties. Indeed, supplementing with RS4 over a 12-week period in participants with signs of metabolic symptoms significantly altered the composition of the gut microbiome, including the enrichment of bacteria with starch-degrading enzymes and increased fecal SCFAs (such as butyrate, propionate, valerate, isovalerate, and hexanoate) [
33]. In addition, cholesterol, glycemic (fasting glucose, glycosylated hemoglobin) and proinflammatory markers, along with as anthropometric measures (waist circumference, percent body fat) were also reduced in the RS4 group versus the control group post intervention. These positive outcomes have been hypothesized to be, in part, mediated by SCFAs via microbial production from RS fermentation in the gut [
34]. These changes may also promote anti-inflammatory and signaling activity, both locally in the gut and systemically, which warrants further investigation in future studies.
Shimotoyodome et al. (2011) reported reduced postprandial GIP levels in the RS group, which may mediate some of the beneficial effects of RS [
19], and this is supported by others showing dietary carbohydrates and fats stimulate postprandial increases in GIP levels [
35,
36,
37]. GIP has been proposed to stimulate efficient fat deposition [
38,
39], whereas inhibition of GIP signaling increases fat oxidation and energy expenditure, as well as reduces high-fat diet-induced obesity in mice [
40,
41]. Daousi et al. (2009) demonstrated elevated blood GIP levels are associated with lower resting energy expenditure (REE) in healthy humans [
42]. Interestingly, our data did not show a difference between meal conditions on GIP. However, we did note RS + WP had a significantly lower GLP-1 compared to RS. As an anorexigenic gastrointestinal peptide, the lower GLP-1 levels in response to the RS + WP meal is surprising given whey protein has been well characterized to stimulate GLP-1 release in acute meal challenges over carbohydrate controls [
43,
44,
45]. However, in our previous work using the same research methodology and test meals we did not find a significant difference between conditions in females [
6].
In contrast to our test meals, the test meals provided by Shimotoyodome et al. (2011) and Mah et al. (2018) contained lower comparative amounts of protein [
19,
28]. As a functional food component used in meal replacement products and other food applications for the promotion of weight loss/maintenance, whey protein has been well-described to have positive effects on both body composition and cardiometabolic outcomes [
4,
5,
22,
46]. Currently, there is a paucity of research that has examined acute metabolic outcomes from the combination of both RS and isolated dairy proteins as functional ingredients in various processed food applications.
Several limitations in the present study should be noted. First, the number of participants was
n = 8, and only middle-aged men were included, which limits the applicability of our findings to women and other age ranges. We noted several outliers (i.e., z-scores ≥ 2.0) whose removal improved the trend toward significance for the interaction effects of postprandial change in TEM. While important to note, these outliers where included in our formal analysis. Such findings are not uncommon in small sample sizes and highlight the interindividual variability in metabolic response to these meal challenges and importance of displaying these data visually (i.e., individual response curves in
Figure 2,
Figure 4,
Figure 5,
Figure 6 and
Figure 7). Second, both the resistant starch and whey protein were supplemental powders; therefore, the physiological effects, including energy expenditure, hormone levels, and satiety, of powder may differ from whole food sources. Third, the glycemic load and index of the test meals were not calculated; thus, the contribution of these factors to the major outcomes of TEM, fuel utilization, hormone, and hunger responses is missing. Fourth, as mentioned above in the methods, plasma variables for WMS + WP were not obtained; therefore, the independent effects of whey protein on satiety/hunger hormones were not established. Lastly, subjective feelings of hunger/satiation did not differ significantly among conditions, which may limit the long-term efficacy of WMS + WP to sufficiently support a caloric deficit and promote weight loss.