Impacts of Lentils on Metabolism and Inflammation

Investigators will utilize a parallel intervention of lentil versus macronutrient-matched (not including fiber) control treatment for 12 weeks in OW/OB adults with demonstrated risk for T2D and CVD. Experimental diets will be provided to participants in the form of pre-made midday meals to exploit the second meal effect and lower caloric intake at the evening meal. Pre- and post-intervention assessments will be made for the following variables: makeup of the gut microbiome (microbial species and relative abundance), gut metabolome, postprandial response of TG, inflammatory cytokines, and serum metabolome to a high-fat meal challenge (established inflammation stimulus), fasting serum glucose, lipid, insulin, inflammation markers and metabolome, blood pressure, and anthropometric measures including weight, body composition, waist circumference, and quantity of visceral adipose tissue. Physical activity, sedentary behavior, and habitual diet will be measured so that these variables can be used to aid in our characterization of participants and aid in analysis and interpretation of data.

Procedures:

Postprandial lipidemic and inflammation responses: High-fat meal challenges with 40 to 100 g of dietary fat are an established laboratory test to measure both postprandial triglyceridemic and inflammation responses. Investigators have used a 50 g dose of fat delivered in the form of butter on toast on > 50 individuals because this particular dose is effective at discriminating between low versus high TG and inflammation responders. In brief, participants will report to the laboratory after an overnight fast, an indwelling venous catheter will be placed in an antecubital vein, and blood samples will be collected before, and 1, 2, 4, and 6 hours following ingestion of the high-fat meal. Samples will be analyzed in real time for TG (and full lipid panel plus glucose) using a clinical chemistry analyzer (Piccolo xpress), while serum samples will be aliquoted and stored at -80 C until analysis for inflammatory cytokines, metabolomics, and insulin. Investigators will measure inflammatory cytokines (TNF-α, interleukin (IL)-1β, IL-6 IL-17, IL-23, and granulocyte macrophage colony stimulating factor (GM-CSF)) using high-sensitivity Luminex multiplexing technology (Bio-Rad Bio-Plex® 200 HTS) prepared by Millipore.

Dietary intervention: Using methods established for an ongoing CRT with lentils, investigators will prepare 7 meals per participant per week to deliver a dose of 4.6 or 0 cups of lentils per week for the experimental and control groups. Meals are matched for macronutrient content (except for fiber), and ground turkey replaces lentils in the control meals. Similar meals will be developed for black beans. Participants will be instructed to consume food provided for their midday meal, and then to proactively reduce portion sizes and to not eat beyond fullness at the evening meal. This strategy exploits the satiety effect of pulses at the midday meal and the 'second meal effect' in which volitional consumption is reduced at the next meal, the evening meal. Each participant will be surveyed once weekly to determine whether they consumed the experimental meal that day, their perceptions of hunger, fullness, satiety, and satisfaction with that day's meal (at 4:00 pm), and their gastrointestinal comfort (level of bloating, flatulence, cramping, and comfort) throughout the day (at 8:00 pm). This methodology has been successfully implemented in our ongoing study to demonstrate that lentil meals are equally pleasing, produce greater satiety, and are well-tolerated.

Gut microbiome analysis: Bulk DNA will be extracted from fecal samples using the Powersoil® DNA Isolation Kit (Mo Bio Laboratories Inc.). DNA will be shipped overnight to the University of Michigan, Center for Microbial Systems, for Illumina MiSeq amplicon sequencing of the 16S V4 variable region. Raw sequencing reads will be processed and curated using the mothur (v.1.39.5) software package, following the mothur MiSeq standard operating procedure, potentially chimeric sequences will be identified and removed using the Uchime (v4.2.40) algorithm, and taxonomic classifications will be assigned using the Bayesian classifier of the Ribosomal Database Project, and operational taxonomic units (OTUs) will be assigned in mothur using the VSEARCH distance-based clustering algorithm at the 97% sequence similarity threshold.

Metabolomic analysis: Samples will be analyzed by high resolution liquid chromatography mass spectrometry (LCMS). Hydrophilic interaction chromatography (HILIC) and reverse-phase (RP) columns will be used for deep coverage. Metabolite identification will use fragmentation pattern matching, authentic standards and database matching with METLIN and the Human Metabome Database (HDB). Novel features of significant interest will be characterized with liquid chromatography mass spectrometry solid phase extraction nuclear magnetic resonance (LCMS-SPE-NMR). Pathway analysis will use XCMS and mummichog.

Dietary analysis. Long-term dietary habits may create adaptations that influence the response to the short-term supplementation of Aronia and lentil. This study will use the most recent version (2018) of the web-based Diet History Questionnaire (DHQ III), a food frequency questionnaire designed for adults 19 and older, developed by staff at the Risk Factor Monitoring and Methods Branch (RFMMB) of the NIH National Cancer Institute. The outputs of the DHQ III include carbohydrate constituents, carotenoids and tocopherols, dietary constituents from supplements, fats, fatty acids and cholesterol, macronutrients and energy, minerals, protein constituents, and vitamins are dietary constituents and food groups available in the DHQ III output files.

Physical activity and sedentary behavior monitoring: Participants will wear an Actical omni-directional accelerometer during weeks 1 and 12 of the intervention to measure total physical activity, time in low intensity activity, moderate to vigorous activity, and vigorous activity, and sedentary activity in minutes.

Statistical analysis: H1 will be tested using two-sample t-tests to compare the difference between pre- and post-intervention assessments. Using preliminary data for TG responses and 50% effect sizes consistent with published findings in animal models, it was estimated that 18-24 participants will be required per group to achieve power of 0.76 to 0.86 at alpha = 0.05. To test H2, investigators will identify changes in the gut microbiome using the methods utilized in our preliminary research to identify characteristics of the gut microbiome that differentiate low versus high TG responders, and then use regression analysis to determine the level of variability in changes to the pulse and control treatments explained by changes in relative abundance of gut microbial species. To test H3, investigators will identify changes in the gut and serum metabolomes, and then determine the metabolic pathways associated with the metabolomic changes to identify potential mechanisms underlying health impacts of pulses.