The overall goal of this investigation is to determine gut microbiome dependent and independent impacts of pulse consumption on metabolic resilience and metabolic risk profiles for type 2 diabetes (T2D) and cardiovascular disease (CVD) risk. Specifically, pulse crop consumption has unrealized potential to fundamentally alter how the body responds to disease promoting metabolic stresses of postprandial triglyceride and inflammation responses. The specific objectives are to (1) Determine the impact of green lentil consumption on postprandial triglyceride (TG) and inflammation responses to a high-fat meal challenge. (2) Determine the extent to which the gut microbiome and changes in the gut microbiome induced by pulse consumption influence health impacts (3) Measure metabolomic profiles to elucidate underlying mechanisms linking pulse consumption to improved health. To achieve these objectives investigators will determine the effects of green lentil on high impact risk factors of large postprandial triglyceride excursions and inflammation, composition of and changes in the gut microbiomes, and both gut and serum metabolomes in overweight/obese (OW/OB) individuals with elevated risk. The 12-week intervention will consist of consumption of 4.6 or 0 cups of pulses per week across 7 pre-made meals (matched for macronutrient content (except fiber) provided to experimental and control groups. The following hypotheses will be tested in the proposed investigation:

H1: Lentil consumption lower postprandial TG and inflammation responses and improve overall metabolic health.

H2: Characteristics of the gut microbiome and changes in the gut microbiome induced by lentil consumption substantially influence health impacts of pulse consumption.

H3: Features of the fecal and serum metabolomes distinguishing lentil and control treatments correspond to metabolic pathways elucidating potential gut microbiome dependent and independent mechanisms linking pulse consumption to improved health.

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. 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, 3, 4, and 5 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, IFN-y, interleukin (IL)-1β, IL-6 IL-17, IL-23, IL-10, 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 or chicken replaces lentils in the control meals. Dietary intervention will be 12 weeks in length with a possible extension to 13 weeks if participants either 1) miss 3 or more meals in a row at any point during the intervention or 2) miss any meals at all in the last week of the intervention prior to their final visit. To keep the meal intervention period between 12 and 13 weeks, participants will be dropped from the study if meal adherence failures result in more than a one week intervention extension.

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.

Inclusion Criteria:

• Waist circumference > 35 inches for women and > 40 inches for men
• Non-fasting serum triglyceride concentration > 175 mg/dl

Exclusion Criteria:

• Allergy to wheat
• Taking medication that will influence cholesterol, lipids, or inflammation
• Pregnant or use of hormonal contraceptive method
• Have diabetes, a pacemaker, or other health conditions that may interfere with the study outcomes
• Planning to undergo a weight loss intervention or change in exercise regimen

Adverse events were monitored for an average of 12-13 weeks for all participants. This time frame is equivalent to the study length from the first/baseline visit.