The approach for Objective 1 is to enroll a population of overweight/obese individuals (n=24) in a two-armed (LENTIL and CONTROL), randomized, crossover trial in which each individual participant serves as his/her own control to compare postprandial serum bile acid responses between isocaloric meals with the same amount of fat with (LENTIL) and without (CONTROL) lentils. Isocaloric meals with the same total fat content in LENTIL and CONTROL study arms will provide the same stimulus to induce increased postprandial serum bile acids. The randomized crossover design with half of the participants starting in the LENTIL arm and the other half starting in the CONTROL arm will eliminate potential order effects. Block randomization with 3 blocks of two equally numbered levels (total of 8 per block) will be created. Having each person serve as their own control in a crossover trial will eliminate the influence of inter-individual variation. The MSU Nutrition Research team has extensive experience with postprandial testing protocols and dietary manipulation and will utilize this experience to perform the proposed protocol with appropriate pre-test standardization, uniformity of meal preparation, precise timing of blood collection, and established procedures for processing and analyzing blood samples. Bile acids, including all primary and secondary forms, will be analyzed from samples collected before the meal and at 30-minute intervals for 4 hours after the meal. Established methods in the MSU Proteomics, Metabolomics, and Mass Spectrometry Facility will be used to compare total and individual bile acids between LENTIL and CONTROL conditions.

The approach for Objective 2 will leverage banked samples from a highly synergistic ongoing clinical trial comparing 12 weeks of daily lentil consumption to control. This study utilizes a randomized, parallel research design to compare the impact of ingesting 140 g of lentils per day to 0 g per day for 12 weeks on serum triglyceride and inflammation responses to a high-fat meal. High fat meals induce the largest serum bile acid responses so the current proposal augments this study by adding assessment of the impact of prolonged lentil consumption on the appearance of bile acids in the systemic circulation under fasting and postprandial conditions. In contrast to Objective 1, which focuses on the acute impact of incorporating lentils into a meal, Objective 2 will measure the impact of adaptations to lentil ingestion that improve enterohepatic function. These changes may be facilitated by changes to the composition of the gut microbiome, enhanced bile acid excretion, or other mechanisms independent of having lentils in the challenge meal. Data from the ongoing trial will be analyzed to determine whether lentils induced changes in the composition of the gut microbiome. This proposal will analyze whether lentil consumption increases excretion of bile acids by analyzing bile acid composition of stool samples collected before and after lentil supplementation. In sum, adding analysis of serum and stool bile acids from samples collected and banked in an ongoing clinical trial, will allow the determination of whether lentil consumption over time improves enterohepatic health in a way that reduces appearance of bile acids in serum and or increases excretion of bile acids.