Impacts of Lentils on Metabolism and Inflammation
Condition: Hypertriglyceridemia; Inflammation; Waist, Hypertriglyceridemic
MeSH terms: Hypertriglyceridemia; Hypertriglyceridemic Waist; Inflammation
Study type: Interventional

Summary: 
      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.
    

Detailed description: 
      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, 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.

      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.
    

Intervention name(s): Dietary Lentil; Control
Intervention type(s): Behavioral; Behavioral
Intervention description(s): Participants in the Lentil arm of the study will consume midday meals containing 0.66 cups of lentils seven days per week for 12 weeks.; Participants in the Control arm of the study will consume midday meals containing 0.00 cups of lentils seven days per week for 12 weeks.
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: This study will utilize a parallel intervention of meals with 0.66 cups of lentils each day versus macronutrient-matched (not including fiber) meals with 0 cups of lentils each day for 12 weeks in OW/OB adults with demonstrated risk for T2D and CVD.
Masking: None (Open Label)
Primary purpose: Prevention