Assessing Gut Microbiota Mediated Health Outcomes of Whole Wheat and Its Major Bioactive Components

This study will investigate the gut microbiota-mediated effects of whole wheat consumption on human health in adults with pre-diabetes. Participants will complete two phases of intervention in random order in which they will consume either whole wheat bread (4 servings) or white bread a day for two weeks prior to collecting specimens (stool, urine, and plasma/serum).

Accumulating clinical evidence suggests positive effects of whole grain on cardiometabolic risk. However, outcomes of controlled trials indicate that substantial interpersonal variation occurs in these studies with regard to glucose homeostasis, with some persons being unaffected and others experiencing glucose-lowering effects due to whole wheat bread consumption. Whole grain (whole wheat) contains bioactive phytochemicals in addition to its well-recognized fiber content, and these constituents have not received adequate study to inform dietary recommendations. The objective of this study is to investigate the glucose-lowering effects of whole wheat bread in persons with prediabetes using multi-omics platforms that can provide an understanding of the complex interactions among the gut microbiome, gut metabolome, host metabolome, and gut barrier function. The hypothesis is that gut microbial metabolism of whole wheat and its major bioactive components is a determining factor of human health benefits. This will be tested by conducting a randomized, controlled crossover trial in persons with pre-diabetes who follow a controlled diet containing whole wheat bread or white bread for 2-weeks. Outcomes are expected to significantly advance an understanding of personalized, gut microbiome-mediated approaches in individuals with pre-diabetes to help guide dietary recommendations of whole wheat intake. In addition, novel evidence that maps out the differential functions of diverse genus/species of microbiota to biotransform whole wheat nutrients into more bioactive metabolites are expected.

Gut microbiota, Microbiome, Metabolomics, Prediabetes, Glycemic response, Intestinal permeability, Gut dysbiosis, Microbiome metabolism, Whole Wheat, Phytochemicals

Participants will be randomized to receive 4 servings per day of whole wheat bread (WWB) or white bread (WB) for 2 weeks. During each study arm, participants will be provided eucaloric standardized diets that are devoid of whole wheat products, probiotic-containing foods, and fermented foods. Before and after each study arm, a fasting blood sample and fecal sample will be obtained for metabolomic and clinical measures. Anthropometric measurements and blood pressure will be assessed at days 0, 7, and 14 of each study arm. On day 14, participants will complete an oral glucose tolerance test (OGTT) with collection of blood samples every 30 minutes for 3 hours and a simultaneous gut permeability test that entails ingesting non-digestible sugar probes and subsequent 24-h collection of urine. Upon completion of these procedures, participants will undergo a ~1 month washout before repeating the study identically, with the crossover to the alternate bread diet.

Fasting plasma glucose concentrations (mg/dL) will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Plasma concentration (μIU/mL) of insulin will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Evaluated through an oral glucose tolerance test and quantified using the area under the curve (AUC) of the temporal changes in blood glucose and insulin conducted at the end of each intervention arm to assess between-treatment effects.

Lactulose/mannitol ratio will be measured in urine collected 0-5 h following the digestion of non-digestible sugar probes to assess small intestinal permeability. Excretion ratios will be measured at the end of each intervention arm to assess between-treatment effects.

Sucralose/erythritol ratio will be measured in urine collected 6-24 h following digestion of non-digestible sugar probes to access colonic permeability. Excretion ratios will be measured at the end of each intervention arm to assess between-treatment effects.

Serum endotoxin concentration (EU/mL) will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Serum concentration (ng/mL) of myeloperoxidase will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Expression of pro-inflammatory toll-like receptor 4 gene from peripheral blood mononuclear cells will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Expression of pro-inflammatory myeloid differentiation factor 88 gene from peripheral blood mononuclear cells will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Expression of pro-inflammatory tumor necrosis factor alpha gene from peripheral blood mononuclear cells will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Expression of pro-inflammatory p65 subunit of nuclear factor kappa B gene from peripheral blood mononuclear cells will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Expression of pro-inflammatory interleukin-6 gene from peripheral blood mononuclear cells will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Expression of pro-inflammatory interleukin-8 gene from peripheral blood mononuclear cells will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Expression of pro-inflammatory myeloperoxidase gene from peripheral blood mononuclear cells will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Expression of pro-inflammatory monocyte chemoattractant protein-1 gene from peripheral blood mononuclear cells will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentration (μg/g) of calprotectin will be measured in samples collected and the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentration (ng/g) of myeloperoxidase will be measured in samples collected at the beginning and end of each study arm to assess within-treatment and between-treatment effects.

Fecal concentrations (mmol/kg) of butyrate will be measured in samples collected at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentrations (mmol/kg) of acetate will be measured in samples collected at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentrations (mmol/kg) of propionate will be measured individually in samples collected at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentrations (mmol/kg) of isobutyric acid will be measured in samples collected at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentrations (mmol/kg) of isovaleric acid will be measured in samples collected at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Serum concentrations of (nmol/L) alkylresorcinol and alkylresorcinol derivatives will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Serum concentrations of (nmol/L) benoxazinoids and benoxazinoids derivatives will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Serum concentrations of (nmol/L) phenolic compounds and phenolic derivatives will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentrations of (μmol/L) alkylresorcinols and alkylresorcinol derivatives will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentrations of (μmol/L) benoxazinoids and benoxazinoids derivatives will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Fecal concentrations of (μmol/L) phenolic compounds and phenolic derivatives will be measured at the beginning and end of each intervention arm to assess within-treatment and between treatment effects.

Plasma triglyceride concentrations (mg/dL) will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Plasma cholesterol concentrations (mg/dL) will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Plasma HDL-cholesterol concentrations (mg/dL) will be measured at the beginning and end of each intervention arm to assess within-treatment and between-treatment effects.

Serum concentrations (U/L) of alanine transaminase will be measured at the beginning and end of each intervention arm to assess within-treatment and between treatment effects.

Serum concentration (mg/L) of C-reactive protein will be measured at the end of each intervention arm to assess between-treatment effects.

Serum interleukin-6 concentrations (pg/mL) of interleukin-6 will be measured at the end of each intervention arm to assess between-treatment effects.

Serum interleukin-8 concentrations (pg/mL) will be measured at the end of each intervention arm to assess between-treatment effects.

Serum tumor necrosis factor alpha concentrations (pg/mL) will be measured at the end of each intervention arm to assess between-treatment effects.

Within-treatment and between-treatment effects regarding alpha-diversity will be determined based on the Shannon-Wiener diversity index. Fecal samples collected at the beginning and end of each intervention period will be used to perform microbiota assessments and the subsequent determinations of alpha-diversity.

Within-treatment and between-treatment effects regarding beta-diversity will be determined based on the Bray-Curtis diversity index. Fecal samples collected at the beginning and end of each intervention period will be used to perform microbiota assessments and the subsequent determinations of beta-diversity.

Gut microbiota relative abundance (percent order, genus, and species level) will be measured in fecal samples collected at the beginning and end of each intervention arm to assess within-treatment and between treatment effects.

Inclusion Criteria: Fasting blood glucose between 100-125 mg/dL BMI of 30-35 kg/m2 Exclusion Criteria: History of liver disease, cardiovascular disease, overt diabetes, or cancer Prescribed medications for hyperglycemia or dyslipidemia Use of dietary supplements, prebiotics, or probiotics Usage of antibiotics or anti-fungals within 3 months prior to enrollment Smoker Alcohol consumption greater than 2 drinks per day Aerobic exercise greater than 5 hours per week Pregnancy or fertility treatments History of chronically active inflammatory or neoplastic disease in 3 years prior to enrollment History of chronic gastrointestinal disorder including diarrhea, inflammatory bowel disease, celiac disease; coagulation disorders, chronic immunosuppressive medication usage History of myocardial infarction or cerebrovascular accident within 6 months prior to participation