Performing Department
(N/A)
Non Technical Summary
Cocoa is a popular food ingredient. Research from our laboratory and others have shown that dietary cocoa can mitigate obesity-related inflammation and non-alcoholic fatty liver disease (NAFLD). The effects are related to improved gut health. Our research team recently published two papers showing significant differences in the response of obese mice to dietary cocoa based on sex. Prior studies have reported that sex and sex hormones can impact development of NAFLD and other co-morbidities of obesity in gutmicrobiota-dependent and independent ways. Based on this, we hypothesize that dietary cocoa mitigates inflammation and NAFLD in obese mice in a sex-specific manner, and that the influence of sex hormones on the composition of the gut microbiome and mammalian signaling pathways drive these differences in biological response. We willcharacterize the sex-specific beneficial effects of cocoa against obesity-related enterohepatic inflammation and fatty liver disease. Additionally, we willcharacterize the role of the gutmicrobiome in mediating the sex-specific effects of cocoa against obesity-related inflammation and liver disease.These studies will provide greater insight into the anti-inflammatory and enterohepatic protective activity of cocoa, and the impact of sex as moderator. These data will support the development of personalized nutritional approaches and novel products containing cocoa to improve humanhealth.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of the project is to characterize the efficacy of dietary cocoa to mitigateenterohepatic inflammation and non-alcoholic fatty liver disease (NAFLD) in obese mice, and to determine the role of biological sex as an influence on the underlyingmechanisms of action, including modification of the mammalian signaling and the gastrointestinal (GI) microbiome. We plan to accomplish this goal through two specific objectives. In objective 1, we willcharacterize the sex-specific beneficial effects of cocoa against obesity-related enterohepatic inflammation and fatty liver disease. We propose to conduct dose-response studies in a diet-induced mouse model of obesity and a genetic mouse model of obesity and NAFLD. We will examine changes in signaling pathways related to GI barrier function, enterohepatic inflammation, and hepatic oxidative stress, lipid trafficking, and endoplasmic reticulumstress. We will use surgical and pharmacological approaches to directly examine the role of estrogen and testosterone. In objective 2, we willcharacterize the role of the GI microbiome in mediating the sex-specific effects of cocoa against obesity-related enterohepatic inflammation and fatty liver disease. We propose to examine changes in the composition of the cecal microbiome and metabolome. We will examine the role of the microbiome using cecal transplants in germ-free mice, and we will study the interactions between the microbiome and sex hormones using surgical and/or pharmacological approaches. We will extend our mouse studies to humans using in vitro fecal fermentation approaches and in vitro models of GI barrier function and inflammation.
Project Methods
To accomplish objective 1, we propose to conduct dose-response studies in a diet-induced mouse model of obesity (high fat diet induced obesity)and a genetic mouse model of obesity (leptin-deficient Ob/ob mice). We will examine changes in signaling pathways related to gastrointestinal (GI) barrier function by looking at permeability to FITC-conjugated dextran and expression of barrier related genes and proteins. Markers of enterohepatic inflammation, hepatic oxidative stress, lipid trafficking, and endoplasmic reticulum (ER) stress will be examined using quantitative reverse transcriptase (qRT)-PCR, western blot, and ELISA. We will use surgical castration/ovariectomy and pharmacological approaches (tamoxifen treatment) to directly examine the role of estrogen and testosterone.To accomplishobjective 2, we will examine changes in the composition of the cecal microbiome and metabolome using 16s rRNA sequencing and LC-MS-based metabolomics approaches, respectively. We will directly test the role of the microbiome using cecal transplants from obese control and cocoa treated mice into germ-free mice. We will extend our mouse studies to humans using in vitro fecal fermentation approaches and in vitro models of GI barrier function and inflammation. We will determine how in vitro cocoa treatment affects the microbiome of human fecal samples from male versus female subjects. We will also examine how cocoa components are metabolized by the fecal microbiome and determine the potential bioactivity of these metabolites using established in vitro models of GI barrier function.For in vivo studies, body weight, and food and fluid intake data will be analyzed by repeated measures ANOVA with time, treatment group, and sex as co-variables. Intermediate and endpoint markers of NAFLD, GI barrier function, inflammation, oxidative stress/antioxidantresponse, and ER stress will be analyzed by two-way ANOVA with sex and treatment group being co-variables or by regression analysis, as appropriate. Canonical variate analysis (CVA) will be performed using the R package candisc with sex and treatment as fixed variables to provide an overall picture of the impact of cocoa on GI barrier function, enterohepatic inflammation, and NAFLD. Colonic and hepatic gene expression, rate of body weight gain, molecular markers of oxidative stress, antioxidant response, endoplasmic reticulumstress, and inflammation will be incorporated into the model.