Recipient Organization
WEILL MEDICAL COLLEGE OF CORNELL UNIVERSITY
407 E 61ST ST RM 106 1ST FL
NEW YORK,NY 10065
Performing Department
Office of Sponsored Research
Non Technical Summary
Type 2 diabetes is a pressing problem in the United States. The burden of the disease presents both a health and financial challenge due to the chronic effects and long term health care. Identifying food components that aid in diabetes prevention is one method for addressing this problem. Type 2 diabetes is diagnosed as elevated blood sugar, resulting from a combination of decreased blood sugar absorbance by tissues and decreased insulin secretion from the pancreas. Elevated cholesterol within the insulin-secreting portion of the pancreas prevents insulin secretion. Phytosterols are similar to cholesterol, but are found in plant food stuffs and studies show that phytosterols can help to decrease blood and tissue cholesterol. Thus, increased phytosterol ingestion may help to prevent type 2 diabetes.This project will examine the link between phytosterol consumption and diabetes prevention. In order to assess the ability of phytosterols to prevent diabetes, this project will use both cell and animal mouse models to measure the function of insulin secreting cells. Insulin secreting cells will be incubated with phytosterol components in the presence of elevated sugar and fatty acids, conditions that mimic diabetes. Increased insulin secretion in response to sugar will serve as an indicator of diabetes prevention. The underlying cause behind phytosterols' actions will also be examined using a combination of molecular biology and fluorescent microscopy. Because phytosterols have been shown to decrease cholesterol, cholesterol will be measured in insulin secreting cells after phytosterol treatment. Inflammation has been documented as an underlying cause of type 2 diabetes and will be measured as well. It is expected that the result of this project will establish the benefits of phytosterols consumption, including increasing insulin secretion, decreasing cholesterol, and decreasing inflammation. The results from this project will be written as a manuscript for scientific publication. The knowledge gained from this study will be useful to dieticians, and the pharmaceutical industry. This postdoctoral fellowship will also provide training and aid in the project director's transition to an independent academic career.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Due to number of Americans with type 2 diabetes, it is necessary to identify therapeutic candidates that aid in diabetes prevention. Phytosterols are cholesterol analogs found in plant products and may promote cholesterol depletion in tissues. The major goal of this project is to determine if phytosterols from foods can prevent diabetes progression by measuring their effects at the pancreas level. The training from this project will be vital to gaining independence in an academic career. The following are the objectives for this project:Experimental Objectives1. Determine whether phytosterols can promote glucose stimulated insulin secretion from pancreatic beta cellsDiabetes occurs when blood glucose is elevated due to an inability of tissues to take in glucose. A major contributor to type 2 diabetes is the lack of insulin production from pancreatic beta cells. For this objective, both in vitro and ex vivo beta cells will be incubated with phytosterol components and the ability of the cells to secrete insulin will be measured. Campesterol, beta-sitosterol, and stigmasterol will be tested, as these are the most abundant phytosterols in food.2. Determine the mechanism by which phytosterols act on beta cellsThis objective will examine the mechanism of action for phytosterols. Excess cholesterol in beta cells is proposed to inhibit beta cell secretion of insulin. Phytosterols are plant analogs of mammalian cholesterol, and studies show that some phytosterols can effect parameters of intracellular cholesterol. For this objective, the effect of phytosterols will be measured in terms of their effect on beta cell cholesterol. Following treatment with phytosterols, cholesterol abundance and cholesterol efflux and influx will be measured.Inflammation is also implicated in beta cell dysfunction. As such, markers of beta cell inflammation will be measured in beta cells exposed to phytosterols.3. Determine if phytosterol consumption protects pancreatic islets from dysfunction.Mice will be fed phytosterols and islets will be isolated from these mice. The islets will be assessed for morphology and function. This objective will determine if increased phytosterol consumption is a viable therapy for improving insulin secretion.Career objectives1. Gain laboratory experience that will be useful for a career in academics.Previous experience was in adipose tissue biology. Training in diabetes research will help guide future projects as an independent scientist.2. Publish articles in scientific journals.3. Obtain an academic position.
Project Methods
Cell culture The primary cell culture model for this study is the INS-1 cell, which is often used for beta-cell studies. In order to mimic the diabetic environment, cells will be incubated in the presence of elevated glucose (25-30 mM) and palmitate (0.5 mM) levels. Phytosterol components will be added to the treatments in an attempt to rescue the diabetic phenotype. The project will test the phytosterols campesterol, beta-sitosterol, and stigmasterol, as these are the most abundant phytosterols in foods. The ability of beta-cells to secrete insulin will be measured through static glucose stimulated insulin secretion assays.Ex vivo Interesting results found in INS-1 cells will be repeated in islets isolated from mouse pancreas.Animal studies Mice will be fed modified diets enriched with phytosterols. High fat diets will be used to promote obesity, a condition strongly correlated with type 2 diabetes. Male C57BL/6 mice (n=12) will be placed in dietary treatment groups for 12 weeks:Low-fat diet--10% kcal fat ad libitum High-fat diet--42% kcal fat ad libitum High-fat diet + phytosterols--High fat diet supplemented with 2% phytosterols ad libitumIntraperitoneal glucose tolerance tests will be performed every week to monitor diabetic progression. Serum will be collected for analysis of circulating glucose and insulin. Serum will also be analyzed for fatty acid profile and cholesterol through commercially available kits. In addition, immunohistochemistry of the pancreas will be performed at sacrifice to assess the morphology of the islets. Macrophage infiltration is associated with type 2 diabetes. To measure macrophage infiltration, pancreatic sections will be stained with the macrophage markers CD68 and CD11b.We will isolate islets after dietary treatment and perform GSIS to assess islet health. Intra-islet cholesterol and phytosterols will be measured by gas chromatography-mass spectrometry.Molecular Biology To measure lipid within cells, the fluorescent probes filipin and lipidtox will be utilized to measure free cholesterol and esterified lipid respectively. Molecular biology techniques such as RT-PCR and western blot will be used to measure markers of inflammation and cholesterol trafficking.Analysis: Fluorescent images will be analyzed using Metamorph software from Molecular Probes. Statistics will be performed using SAS software.Efforts: The results of this project will be disseminated through research articles and presentations at scientific conferences. Laboratory techniques such as islet isolation and glucose stimulated insulin secretion will be learned and passed on to mentees (graduate student, postdocs) in future career positions.Evaluation: The success of this project will be measured by reaching key milestones.Key milestones:1. Completion and analysis of experiments examining the effect of phytosterols on insulin secretion.2. Preparation and submission of manuscripts for scientific publication.2. Attendance and presentation of results at scientific conferences.