Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Biochemistry
Non Technical Summary
Orange carrots are one of the most widely consumed vegetables, and they are a significant source of vitamin A in the form ofß-carotene. Breeding efforts to improve food crops through biofortification have increased the value of this root vegetable in which other bioactive compounds such as lycopene and lutein introduced. We believe that these horticultural approaches, i.e., different colors of carrots,will lead toimproving the nutritional quality and visual appeal of our foods. Colored carrots have several pigments that have various biological benefits when consumed, such as the prevention of chronic diseases. Because carrots are so widely accepted and consumed in the US diet, increasing the nutrient content could bring so many health benefits to the public.Yellow carrots contain mainly the xanthophyll lutein, which is essential for a healthy vision, and this pigment aids in the fight against age-related macular degeneration. The rich red pigment in red carrots is lycopene, which acts as antioxidants as it is associated with a reduced risk of serum lipid oxidation.White carrots lack pigment, and they will be used as a control. Several human studies reported lycopene in red carrot was nearly 50% as bioavailable as that from tomato paste. Lutein bioavailability from yellow carrots was 65% as bioavailable as the lutein supplement. These studies indicate that carotenoids in these carrots displayed high bioavailability. It is not knownwhether these carotenoids are biologically active phytochemicals at the expense of colon epithelial cells. Another question is whether carotenoids in the colon could be fermented by our gut microbiota. Our project meets the goals of the program area priority as we plan to investigate the nutrients in food and their impact on the gut microbiota to improve our health. We will also use a whole food approach and plan to justify the relationship of the bioactive component(s) being studied to human health outcomes and the health of the human gut microbiome.This research could lead to more varieties of carrots on grocery shelves and into the hands of consumers. In taste evaluation, the carrots of various colors were accepted by consumers, especially when they were not blindfolded. Consuming these whole foods has an equal or more significant reduction in the prevention of chronic diseases.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Goals / Objectives
Our long-term goal is to developfunctional biomarkers related to the metabolic effects of circulation carotenoidsassociated withlow-grade inflammation.The overarching goal of this proposed study is to determine the impact ofdietary carotenoidsongut microbiota, intestinal barrier function, and colonic inflammation.Our central hypothesis is that carotenoids escaping the absorption in the small intestine can be transformed by the gut microbiota in the colon, and these undigested carotenoids and their microbial metabolites are available to the gut epithelium. They protect against obesity-associated gut dysfunction (e.g., microbial dysbiosis, impaired and low-grade inflammation).To test our hypothesis and achieve our overall objective, we propose the following objectives.Objective 1: Elucidate molecular mechanisms of action of major circulating carotenoids on inflammatory transcription factors in vitro using human colonic epithelial cells.Hypotheses:1-) SR-B1 mediates the uptake of dietary carotenoids reaching the colon and plays a role in carotenoid metabolism in colonic epithelial cells.2-) Carotenoids modulate nuclear translocation of Nf-kB and PPARg transcription factors, thereby playing a role in colonic inflammation.3-) Carotenoids blunt colonic inflammation and loss of barrier integrityObjective 2: Illustrate the impact of carotenoids on obesity-associated metabolic endotoxemia, gut dysfunction, and gut barrier using a murine model.Hypotheses: 1-) Dietary intake of carotenoids-rich foods reduces obesity-associated gut inflammation2-) Carotenoids reduce losses of tight junction proteins and subsequent gut barrier integrity3-) Carotenoids reduce circulating endotoxin levelsObjective 3: Characterize diet (rich in carotenoids)-induced changes in the gut microbiota utilizing a shotgun sequencing and multi-omic approaches, including metabolomics and metagenomics.Hypotheses:1-) Carotenoids alter the diversity and species composition of the gut microbiota.2-) Systems biology approaches provide us the answers to the questions in which bacteria are present, what functional capacities they exert, and what functional capacities they possess.
Project Methods
Objective 1Nuclear Translocation NF-kB and PPARg Profiles.Cells will be stimulated with TNF-aand treated with various doses of ß-carotene. We will also use extracts from orange carrots and white carrots. The transcription factors will be measured using a commercial TransAM DNA-binding ELISA kit according to the manufacturer's instructions.Cytokine Profiles.TNFa, IL-1b, IL-10, IFN-a, IL-8, and TGFb will be analyzed using ELISA kits from Cayman Chemicals according to manufacturer directions.Western blots for TLR2, Peptidoglycan Recognition Receptors (PGlyRP), occludin, claudin-1, and junctional adhesion molecule-2.Cell lysates will be prepared by using RIPA buffer with protease inhibitors. Following lyses, supernatants will be collected, and proteins will be quantified using BCA Protein Assay (Pierce, Rockford, IL). Cell lysates (amounts equalized by protein concentration) will be mixed with a 4 × SDS-PAGE sample buffers and boiled for 7 min. Lysates will be loaded on a 10% SDS polyacrylamide gel, and electrophoresis will be carried out according to standard protocols. Proteins will be transferred to a PVDF membrane by using an electroblotting mini transfer apparatus. Membranes will be blocked at room temperature for one h in Tris-buffered saline plus 0.05% Tween 20 (TBST) and 5% BSA, and then incubated overnight in the primary antibody at 4°C. After washings in TBST, membranes will be incubated with a horseradish peroxidase-conjugated secondary antibody and developed for visualization of protein with a luminol enhancer solution.Objective 2Nuclear Translocation NFκB and PPARγ Profiles. Colonic mucosa will be collected, and snap-frozen in liquid nitrogen for nuclear translocation DNA binding assays for NFκB, and PPARγ will be completed as described in Aim 1.Mucosal and Blood Cytokine Profiles.TNFa, IL-1b, IL-10, IFN-g, IL-8, and TGFb will be analyzed using ELISA kits from Cayman Chemicals companies according to manufacturer directions.Mucosal TLR2 and PGlyRP Western blots. Tissues will be thawed and lysed by homogenization in 10 vol/wt of chilled protein extraction buffer with protease inhibitors. Followinglysis, sampleswill be centrifuged at 6,000 x g for 10 minutes at 4°C to remove insoluble material. Supernatants will be collected, and protein will be quantified using BCA Protein Assay. Tissue extracts (amounts equalized by protein concentration) will be mixed with an equal volume of 4 × SDS-PAGE sample buffers and boiled for 7 min. Lysates will be loaded on a 10% SDS polyacrylamide gel, and electrophoresis will be carried out according to standard protocols. Proteins will be transferred to a PVDF membrane by using an electroblotting mini transfer apparatus. Membranes will be blocked at room temperature for one h in Tris-buffered saline plus 0.05% TBST and 5% BSA, and then incubated overnight in the primary antibody at 4°C. After washings in TBST, membranes will be incubated with a horseradish peroxidase-conjugated secondary antibody and developed for visualization of protein with luminol enhancer solution.Histology. H&E Staining. Intestinal sections will be assessed histologically for gross morphology, cuff depth, and cuff width measurements will be measured in the colon. Formalin-fixed segments will be embedded in paraffin, and five µm-thick sections will be cut and stained with H&E for examination by light microscopy. Computer-assisted morphometric measurements will be conducted with a video-imaging system (Nikon- FXA). All samples will be measured twice by two observers masked to the treatment groups. Six cuffs will be measured to determine themean cuff depth and cuff width.Immunofluorescence and Confocal Analysis. Colon sections will be stained with the primary antibody, as indicated by the manufacturer's instructions. Images will be obtained with a 3-Laser Nikon Confocal Laser Scanning Instrument (Nikon Instruments). Images will be obtained using EZC1 Nikon software. Threshold values will be determined using the appropriate isotype-matched controls. A channel series approach will be used to ensure no spectral overlap between fluorescent signals. Primary antibodies include occludin (mouse-anti-occludin), 1:1000. Secondary antibodies used include Fab-antibodies, Jackson Labs Inc (donkey-anti-mouse FITC), 1:1000. Nuclei will be visualized with To-Pro-3 iodide 642/661 1:1000. In addition to tight junction protein occludin, we will stain for TLR2 (mouse-anti-TLR2) and PGlyRP3 (mouse-anti-PGlyRP3).Inflammatory markers: Collect feces every week, and measure fecal calprotectin. Other biomarkers will include neutrophil myeloperoxidase, fecal neopterin, and citrulline in serum.Endotoxin Analysis.Blood levels of endotoxin will be quantified using Lonza rFC or LAL assay kits.Body Composition. Body composition (% fat, fluid, and lean mass) will be measured at the beginning and end of the study using the Bruker NMR analyzer in CLASObjective 3Sequencing and bioinformatics analysis will be performed at the David H. Murdoch Research Institute genomics core.METABOLOMICSSmall molecule metabolites will be extracted from samples and analyzed using the DHMRI Metabolomics Discovery PanelDISCOVERY METABOLOMICSThe spreadsheet containing semiquantitative measurements of metabolites measured inbiospecimens, including serum, feces, and urine.Data upload and access to MarkerLab, a metabolomics data visualization, and pathway analysis tool.TARGETED METABOLOMICSThe spreadsheet containing quantitative measurements of 8 short-chain fatty acids in 56 feces samplesThe spreadsheet containing quantitative measurements of 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) in 56 serum samples9-HODE and 13-HODE are stable oxidized derivatives of linoleic acid, which provides an insight in oxidative stress occurring in a biological systemThe spreadsheet containing quantitative measurements of cortisol in 56 serum samplesCortisol is a hormone released in response to physiological stress and low blood-glucose concentrationMETAGENOMICSBacterial DNA extraction from mice stool samples, sample QC, library construction, QC and sequencing (125 bp paired-end)Average of 2 Gb of data per sampleMetagenomics Analysis Deliverables:Raw Reads and QC ReportAssembled and Annotated GenomesFunctional Profiling Abundance TableSpecies Abundance Tables including Bar and Sunburst Charts (for visualization)Phylogenetic TreesAlpha Diversity and Beta Diversity at species level curvesDifferential species abundance analysisHeat maps of abundance tables and differential abundance analysis