Ethics Statement. The protocol was approved by the Institutional Review Board at Montana State University. Written informed consent was obtained from all participants prior to their participation. This study was retrospectively registered October 2019 at ClinicalTrials.gov (NCT04128839).
Study Population. Potential participants were recruited via advertisement and were excluded if they had taken oral antibiotics within 90 days of study enrollment, regular use of anti-inflammatory medications, use of estrogen-only contraceptives, wheat and/or dairy allergies, were pregnant, or had any musculoskeletal, cardiovascular, gastrointestinal, or immunological condition that could interfere with the study (Additional File 1). Forty overweight and obese men and women participated in testing of cardiovascular, anthropometric, and metabolic markers and ingestion of a 50 g high-fat meal challenge. Participants were 18–55 years old within a body mass index (BMI) 27–36 kg/m2.
Research Design. The study followed a 1-group pretest-posttest quasi-experimental design. Participants were asked to attend two visits. The initial visit involved questionnaires and analysis of body composition and cardiorespiratory fitness. The second visit occurred within two weeks after the first visit and involved blood collection before and after a high-fat meal challenge. Fasting and postprandial lipids, glucose, and insulin were measured for four hours postprandially. Blood pressure, visceral adipose tissue, physical activity frequency, and substrate utilization crossover during a submaximal exercise test were measured.
Physical Activity Frequency. Participants were asked to complete a written 3-question questionnaire on their physical activity in the past week (24). Questions asked “On how many of the past 7 days did you -” perform 30–60 minutes of aerobic exercise, strengthening activities, and stretching exercise with written examples of each provided for reference.
Anthropometrics. Measurements were collected from participants using the validated segmental multifrequency bioelectrical impedance analysis (SECA mBCA 515, Hamburg, Germany) (25). Participants were instructed to refrain from eating, drinking, or exercising in the three hours prior to testing. Fat mass percentage and estimated visceral adipose in liters were used for analysis.
Blood Pressure. Systolic and diastolic blood pressure measurements were performed on seated participants after 5–10 minutes of rest. Two automated measurements were taken with the average of the two measurements used for analysis.
Cardiorespiratory Fitness. Participants were asked to complete a modified Bruce protocol on a treadmill for determination of calculated absolute oxygen consumption (VO2) max at their age-predicted heart rate max. Speed and grade of the treadmill (Woodway GmbH D-79576, Weil am Rhein, Denmark) were manually changed by the researcher with each progressive three-minute stage until the participant reached 85% of their age-predicted maximal heart rate. Expired gases were collected for analysis through a metabolic cart system (ParvoMedics, TrueMax 2400 Metabolic System, Sandy, Utah, USA). Heart rate (bpm) and VO2 (ml/kg/min) data from each participant were input into a simple linear regression model to predict the absolute VO2 at the age-predicted maximal heart rate based on the equation presented by Tanaka, Monahan, and Seals (26).
Exercise requires metabolically flexibility, the ability to switch between glucose and fat use in response to metabolic demand (27). The switch or “crossover” point of substrate utilization during exercise reflects the point at which kilocalories per minute of carbohydrate expended exceeded that from fat. If the participant had intermittent periods early in the test where carbohydrate exceeded fat expenditure, the crossover point was determined as the point at which carbohydrate and fat expenditure most rapidly differentiated from the other. Using the VO2 at the crossover point, we derived the crossover percentage of the calculated absolute VO2 max.
High-Fat Meal Challenge. The high-fat meal challenge was performed after an overnight fast during the morning hours. Total energy content of the high-fat meal challenge was 714 kcal, with 43.1% from fat, with a macronutrient breakdown of 50 g fat, 54 g carbohydrate, and 12 g protein. Water was provided with the meal. Caffeinated black tea was provided instead for participants who identified as habitual coffee consumers. Participants had 15 minutes to consume the meal, and the postprandial period timing began when participants started the meal.
Blood Sampling. Participants were instructed to avoid alcohol consumption and strenuous physical activity in the 24 hours before blood collection and to complete an overnight fast (10–12 hours) before blood collection. Venous blood samples were collected through a cannula inserted into the antecubital vein after a 3-mL waste withdrawal, then followed by a sterile saline flush performed by a physician or nurse on the research team. The fasting sample was drawn 30 minutes after catheter insertion. After meal ingestion, blood was drawn every hour for four hours in the postprandial period, totaling five time points including fasting. Blood was collected into 8.5 mL endotoxin-free serum separating and 4.0 mL heparinized vacutainer tubes (BD Vacutainer, Franklin Lakes, New Jersey, USA). The serum tube was allowed to clot for 15 minutes at room temperature before centrifugation (3000 rpm, 15 min). Serum aliquots were frozen at -80ºC until analysis.
Biochemical Analyses. Blood triglycerides, glucose, and high-density lipoprotein were determined using the Picollo Xpress Chemistry Analyzer lipid panels (Abaxis, Union City, USA). Insulin was determined through ELISA (MP Biomedicals, USA) performed according to manufacturer instructions, with the average used for analysis. Mean inter-assay coefficient of variation for samples run in duplicate was 13.3%.
Insulin Resistance. Fasting blood glucose and insulin were used to determine the homeostatic model of insulin resistance (HOMA-IR) in the original HOMA-IR formula (28):
$$\frac{Glucose \left(\frac{mmol}{L}\right)*Insulin\left(\frac{mU}{L}\right)}{22.5}$$
Postprandial Lipemic Response. The postprandial lipemic response to the high-fat meal was summarized as iAUC, a calculation method that accurately represents the postprandial TG response to a high-fat meal (16). The magnitude of the postprandial lipemic response was also calculated by subtracting the fasting TG value from the maximum TG value during the 4-hr postprandial period after the high-fat meal.
Statistical Analysis. Analysis was conducted in RStudio (1.3.1073) running R 4.0.2 (29), and data was visualized using ggplot2 (30).
To assess which variables most influence TG iAUC and the TG magnitude, initial saturated multivariate linear regression models were created with the following predictor variables: age, sex, relative exercise intensity of substrate utilization crossover, visceral adipose tissue in liters, HOMA-IR, systolic and diastolic blood pressure in mmHg, and aerobic exercise frequency. Model refinement was performed by stepping down one main effect at a time from the initial mode. Model reduction was determined through strength of evidence against the null hypothesis using Type III F-tests, in which every test is conditional on every variable in the model. Model refinement stopped when the majority of predictor variables reached their smallest p-value. Final linear models were screened for shared information among predictor variables using variance inflation factors from the car package (31), with values > 5 set as the threshold for predictor removal. Further validity conditions were confirmed through residual visualizations. Power was calculated a posteriori from the final TG iAUC regression model using the power.f2.test in the pwr package. Computed power was 95% at a Type I probability of 0.05.