Performing Department
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Non Technical Summary
Obesity is a chronic progressive disease that leads to the development of type 2 diabetes, heart disease, and stroke, all ofwhich are among the top 10 leading causes of death in the U.S. Currently, lifestyle modification, including diet, exercise, andbehavior therapy, has remained the first option to effectively and safely control the progression of obesity and obesity-relateddiseases. However, low adherence to lifestyle interventions underscores the urgency to develop other strategies to combatobesity. We are the first research group to investigate the nutraceutical values of dietary exosome-like nanoparticles (ELNs) inobesity. We found that chive-derived ELNs (C-ELNs) improve metabolic health in diet-induced obese mice. Continuing alongthis line of research, in this project we will investigate (1) the bioavailability and distribution of C-ELNs in vivo; (2) absorption ofC-ELNs in intestinal epithelial cells; and (3) the impact of C-ELNs on the gut microbiome in obesity. Successful completion ofthe proposed research will provide mechanistic insights into how C-ELNs distribute and function in vivo and pave the way forapplying C-ELNs to combat obesity.
Animal Health Component
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Research Effort Categories
Basic
100%
Applied
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Developmental
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Goals / Objectives
The long-term goal of our research is to explore the nutraceutical values and underlying mechanisms of dietary ELNs in combatting obesity. Our pilot studies demonstrated that chive-derived ELNs (C-ELNs) suppress chronic inflammation and improve metabolic health in diet-induced obese mice. In this project, we aim to study the bioavailability and absorption of C-ELNs and their interaction with the gut microbiome in obesity, which will be achieved through completing the following three supporting objectives.Objective 1: Assess the bioavailability and distribution of C-ELNs in vivo.Objective2: Investigate absorption of C-ELNs in intestinal epithelial cells.Objective3: Investigate the impact of C-ELN on the gut microbiome in obesity.
Project Methods
Objective 1: Assess the bioavailability and distribution of C-ELNs in vivo.Experimental Design:1) Bioavailability pattern of C-ELNs. Exoglow-RNA EV labeling kit (green fluorescence) and Exoglow-protein EV labeling kit (red fluorescence) will be used to label RNAs and proteins in C-ELNs, respectively. The fluorescence-labeled C-ELNs will be orally administered to obese C57BL/6J mice. After 6 h, mice will be euthanized, and a variety of tissues will be collected to assess the fluorescence intensity of C-ELNs using an iBox small animal image system. The results from membrane lipid, RNA or protein-labeled C-ELNs will corroborate each other to define the exact bioavailability of C-ELNs in animals.2) Integrity of C-ELNs in animal organs. C-ELNs will be labeled with lipophilic dye DiI (red fluorescence) and RNA dye (green fluorescence) from Exoglow-RNA EV labeling kit. The labeled C-ELNs will be orally administered to obese C57BL/6J mice. The target tissues of C-ELNs, such as liver and eWAT, will be sectioned into slices. The signal of double fluorescence-labeled C-ELNs in the tissue sections will be assessed using fluorescence confocal microscopy.3) Target cells of C-ELNs in tissues. After the target tissues of C-ELNs are defined, the target cells of C-ELNs in these tissues will be further determined. The fluorescence-labeled C-ELNs will be orally administered to obese C57BL/6J mice and the target tissues, such as liver and eWAT, will be harvested and sectioned. Antibodies recognizing specific markers of main cell types in each tissue will be used to immunofluorescence stain the sections. The localization of fluorescence-labeled C-ELNs in each cell type will be examined under fluorescence confocal microscopy.Data analysis: The null hypothesis for the C-ELN bioavailability studies is that administration of fluorescence labeled C-ELNs does not alter the fluorescence levels in liver in animals. Mice injected with the solvent PBS will serve as the control group. One-way ANOVA will be used to compare fluorescence intensity of a specific organ of C-ELN-injected mice with the same organ from the control group, and Dunnett's posthoc procedure will be used to make individual comparisons44. Differences will be considered significant if p<0.05. Our biostatistics consultant Dr. Kachman provided sample size calculations to detect differences between groups for a = 0.05 and 1-b = 0.8. Power calculations are based on our preliminary data, suggesting that a 1.3-fold increase of fluorescent intensity of lipid dye-labeled C-ELNs was detected in the liver following oral administration of 0.5 ´ 1010/g DiR-labeled C-ELNs in mice. Based on these calculations, 5 mice will be sufficient to detect a 1.3-fold fluorescence increase in the livers of animals orally administered with fluorescence-labeled C-ELNs.Objective2: Investigate absorption of C-ELNs in intestinal epithelial cells.Experimental Design:1) Use inhibitors to determine specific endocytic pathway. Endocytosis has been reported to be the major mechanism through which dietary ELNs4, 5, 45 and endogenous exosomes46, 47, 48 are internalized by recipient cells. Endocytosis is generally divided into two main subgroups: phagocytosis and pinocytosis49, 50. Pinocytosis is further classified into clathrin-dependent endocytosis (CDE), clathrin-independent endocytosis (CIE), and macropinocytosis. CIE includes caveolae-, Arf-6, flotillin-1-, CD42-, and RhoA-dependent endocytosis51. Specific inhibitors of these endocytic pathways will be used to define which endocytic pathway is responsible for C-ELN uptake in intestinal epithelial cell line Caco-2 cells.2) Use siRNA knockdown techniques to confirm specific endocytic pathway. Chemical inhibitors could have low specificity in some cell types52, and therefore they alone are not sufficient to fully establish the absorption mechanisms. To confirm the involvement of a specific endocytic pathway in C-ELN uptake, siRNA-mediated knockdown of key proteins in the specific pathway will be conducted. For example, if we find macropinocytosis is important, the key proteins in this pathway, such as Rac1 or PAK150, will be knocked down in Caco-2 cells, and the uptake of C-ELNs will be assessed in these cells.Data analysis: The null hypothesis is that specific inhibitor or knockdown of key players in the endocytic pathway does not affect the uptake of C-ELNs in Caco-2 cells. Statistical analyses and power calculations will be conducted at a = 0.05 and 1-b = 0.8 as described in Objective1. Power calculations are based on a previous study46 suggesting that EIPA, the inhibitor of macropinocytosis, suppressed 48% of exosome uptake in the human cervix epithelial cell line Hela. Based on these calculations, 3 biological repeats will be sufficient to detect a 30% decrease in the fluorescence signal of C-ELNs in Caco-2 cells.Objective3: Investigate the impact of C-ELN on the gut microbiome in obesity.Experimental Design:1) Identify members of the mouse microbiome that take up C-ELNs. Two-month old C57BL6/J mice will be fed a high-fat diet (HFD) for 15 weeks with C-ELNs orally delivered weekly at 1´1010/g. Mice will be then orally given with C-ELNs (1´1010/g) labeled with the lipophilic membrane dye PKH26. After 2 h, mice will be euthanized and the intact colon will be dissected in an anaerobic chamber to collect contents for fluorescence-activated cell sorting (FACS) and cultivation (Objective 3.2). For FACS analysis, colon contents will be resuspended in PBS and sorted on a BD FACSAria II to collect bacteria that have taken up C-ELNs. Metagenomic libraries will be prepared from each sample (n=16) and sequenced on an Illumina HiSeq at a depth of twenty million reads/sample.2) Determine the growth rates of isolated gut bacteria. Mouse colon contents from Objective 3.1 will be resuspended in anaerobic skim milk medium58 for cryopreservation at -80°C. Once taxa capable of C-ELN uptake are identified in Objective 3.1, phylogenetic information from metagenomic sequencing will be used for sequence-guided isolation59 using established protocols59, 60, 61, 62. Isolates will be tested for their ability to take up PKH26-labeled C-ELNs as described8 with media specific for each isolate's growth. Isolates capable of C-ELN uptake will be grown anaerobically in culture medium supplemented with C-ELNs; growth rates will be compared to isolates grown anaerobically in culture medium lacking C-ELNs.3) Assess gene expression of bacterial isolates. The effects of C-ELNs on bacterial gene expression will be tested with four isolates identified in Objective 3.2 as having the most significant changes in growth rate following cultivation with C-ELNs. Isolates will be cultured anaerobically in growth medium lacking C-ELNs to the mid-exponential phase, then will be diluted 1:10 in fresh anaerobic culture medium without or with C-ELNs. One hour later, cells will be collected and RNA will be extracted and prepared for RNA-seq sequencing.Data analysis: All sample size and power calculations were performed to detect differences between groups for a= .05 and 1-b=0.8. For identification of murine fecal bacteria capable of CELN-uptake in mice, the null hypothesis is that the distribution of bacterial taxa in the fluorescently sorted samples will not differ from the distribution of taxa in unsorted samples. Metagenomic data will be processed and analyzed using MetaWRAP63. Power calculations indicated that the minimum sample size capable of detecting differences in 75% of species based on our pilot study is 8/group. For growth rate study, the null hypothesis is that rates of growth in the presence of C-ELNs will not differ from growth rate in the absence of C-ELNs. Power calculations indicated that the minimum sample size capable of detecting a 20% difference in growth rate between treatment groups with 100% variance is 4/group.