Source: PENNSYLVANIA STATE UNIVERSITY submitted to NRP
SEX-SPECIFIC DIFFERENCES IN THE MITIGATION OF GASTROINTESTINAL INFLAMMATION BY COCOA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032138
Grant No.
2024-67017-42459
Cumulative Award Amt.
$649,588.00
Proposal No.
2023-08247
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2027
Grant Year
2024
Program Code
[A1343]- Food and Human Health
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
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)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70222331010100%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
2233 - Cocoa;

Field Of Science
1010 - Nutrition and metabolism;
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.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The target audiences reached by this project over the last year include members of the cocoa and chocolate processing industries; researches in the field of plant science, food science, and human health; and the general public. Industry stakeholders were engaged as part of Penn State's Chocolate Short Course. Other scientists were engaged at the Plant Biology Symposium on Specialized Metabolites that was held at Penn State. The general public was engaged through press releases and a pod cast developed by the local NPR affiliate. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Threegraduate students were responsible for performing the experiments conducted so far on this protocol. They were primarily responsible for treating the mice, performing necropsies, and collecting and collating resultant data. As part of these studies, the students learned new laboratory techniques (RNASeq, western blot, and reverse transcriptase quantitative PCR) and statistical analysis programs (R program). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will continue to perform analysis on molecular markers of potential mechanisms of action for the anti-inflammatory effects of cocoa. We will also continue to examine molecular markers to understand the role of estogen in mediating the sex specificity in the anti-inflammatory effects of cocoa. In addition, we plan to initiate studies to examine differences in the effect of cocoa on the gut microbiota of male and female mice. We will conduct 16S rRNA sequencing and use bioinformatic pipelinesto determine the composition of fecal and cecal bacterial microbiome.

Impacts
What was accomplished under these goals? We initiated threeexperiments to accomplish objective 1. The first experiment examined differences in the response of male and female mice to cocoa treatment for the mitigation of obesity-related fatty liver disease.C57BL/6J mice were fed high-fat diet (HFD) for 8 wks to induce obesity and then randomized to HFD or HFD supplemented with 80 mg cocoa/g diet for an additional 8 wks. Hepatic responses were analyzed at the mRNA and protein levels. Cocoa-treated male mice had decreased liver weight and enhanced expression of mitochondrial oxidative and endoplasmic reticulum (ER) stress response markers (p < 0.05). In female mice, cocoa reduced ER stress response markers and increased expression of very low-density lipoprotein-triglycerides biogenesis compared to HFD-fed controls (p < 0.05). Two-way multivariate analysis of variance revealed significant main effects of sex, diet, and a significant interaction. Canonical variate analysis demonstrated the efficacy of fermented/unroasted and fermented/roasted cocoas in both sexes, and unfermented cocoas in females.Based on this, we conclude that cocoa mitigates obesity-related NAFLD by sexually dimorphic mechanisms. Further research is needed to develop personalized nutritional recommendations incorporating cocoa. The secondexperiment examined the efficacy of cocoa against obesity-related hepatic inflammation using the ob/ob genetic mouse model of obesity. Male and female ob/ob mice were treated with 20 or 80 mg/g dietary cocoa for 8 weeks. Body weight was measured weekly. 80 mg/g cocoa reduced the rate of body weight gain in female but not male ob/ob mice. Fasting blood glucose was measured every two weeks. Cocoa-treated ob/ob mice had significantly higher fasting blood glucose levels thanob/ob mice treated with control diet. We are currently investigating the effect of cocoa on molecular markers of hepatic and gastrointestinal inflammation. The thirdexperiment examined the role of estrogen in mediating differences in the efficacy of cocoa in male and female mice. Male and female mice were fed high fat diet for 8 weeks to induce obesity. Male mice were then randomized to receive high fat diet orhigh fat diet supplemented with 80 mg/g cocoa. Female mice received high fat diet, high fat diet supplemented with tamoxifen, high fat diet supplemented with 80 mg/g cocoa, or high fat diet supplemented with coco and tamoxifen. Control male and female mice were fed low fat diet. Body weight was recorded weekly and mice were euthanized after 10 weeks. Cocoa treatment reduced rate of body weight gain in both male and female mice. Using RNASeq approaches, we have identified several pathways in the liver that differ between obese male and female mice treated with cocoa. In cocoa-treated males, pathways related to mitochondrial energy metabolism and beta oxidation are enriched, whereas in cocoa-treated females, expression of Cyp3A16 and pathways related to hormone biosynthesis were enriched. On-ging studies using PCR and western blot are focused on confirming these results and examining the effect of tamoxifen.

Publications