Performing Department
(N/A)
Non Technical Summary
Increasing evidence from various studies suggests that consuming fruits and vegetables containing polyphenols, including berries, especially blueberries in particular may influence carbohydrate metabolism by controlling postparandial gylcemic responses and fasting hyperglycemia, as well as improving insulin response and sensitivity.However, the hypoglycemic activities of polyphenol fractions other than anthocyanins have not been properly understood. Therefore, the objective of this research is to investigate the possible roles of blueberry polyphenols in preventing metabolic syndrome-induced diabetes.Whole blueberries will be processed into freeze-dried powders and assayed for total polyphenolic content, in vitro antioxidant and hypoglycemic properties and then formulated into feed diets according to standard methods. Fifty 5 week old male C57/B16 mice will be housed 10 per cage, acclimatized for a week and then placed on very high fat diets (VHFD) for 12 weeks to induce obesity, insulin resistance and hyperglycemia. The mice will be placed on intervention diets made up of VHFD containing either 0, 5, 10, 15 and 20g whole freeze-dried blueberry for additional 13 weeks.Feed and water intake will be measured daily while body weight will be measured weekly. Urine, blood samples, oral glucose tolerance and fasting blood glucose tests will be collected at baseline, midpoint and at termination. After the 13 weeks period, the rats will be anesthetized and blood, heart, liver, kidney and adipose tissue collected for in vivo assays for biomarkers of oxidative stress, obesity and diabetes using biochemical, molecular and metabolomic analyses. This research will advance knowledge in diabetic health effects of blueberries.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The long term goal of this research is to investigate the possible roles of blueberry polyphenols in the prevention of metabolic syndrome induced diabetes. Specifically, the study aims to quantify and characterize blueberry polyphenols, determine in vitro and in vivo antioxidant, hypoglycemic and hypolipidemic activities of blueberry polyphenols using biochemical, molecular and metabolomic approaches
Project Methods
Experimental material: The highbush blueberry (Vaccinium corymbosum) variety will be used for this study. Fresh samples ofblueberry fruits will be obtained directly from a known producer in Tuskegee (AL, United States). The harvested fruits will be sorted for maturity and stored immediately under refrigeration prior to processing. All analytical grade reagents and materials will be purchased from Fisher Scientific (Waltham, MA, United States). The samples will be processed into blueberry powder accordingto the methods of Ozga et al [35].HPLC analysis of blueberry polyphenols: The freeze-dried blueberry powder will be purified in a Seph-Pak C18 cartridge, eluted, vacuum concentrated and evaporated to dryness. The dried sample will be redissolved in 200 µl for C18-HPLC of 50% aqueous methanol (v/v) and a 20 µl aliquot chromatographed on C18-HPLC according to the method of Ozga et al [35] with modifications according to Jiang et al [36].LC-MS analysis of blueberry polyphenols: The most abundant polyphenolic peaks will be identified using a liquid chromatography - mass spectrometry (LC-MS) system according to the method of Ozga et al [35] with modifications according to Jiang et al [36].In vitro assays antioxidant and antidiabetic assayTotal Phenols Assay by Folin-Ciocalteu Reagent: Total phenols analysis will be determined by the method described by Grussu et al [18] with 1000 µM solution of catechin as the standard. An aliquot of standard, blank, or the extract (up to 100 µl) will be added to 1000 µl of the Folin-Ciocalteu reagent (Sigma Chemical Company, St. Louis, MO), which has been diluted previously 1:9 with water. The solution will be allowed to stand at 20-25 ° for 20 min before colorimetric measurement at 750 nm.Determination of α-amylase activity: The α-amylase inhibitory activities will be determined by the method of McDougall et al [21] using porcine pancreatic α-amylase.Determination of α- glucosidase activity: The α-glucosidase inhibitory activities will be determined according to the method of Zhao et al [37] with minor modifications according to Jiang et al [36] using α-glucosidase from S. cerevisiae.In vivo studies using C57/B16 diabetic mouse modelsAnimal study conditions: Samples will be formulated into feed diets to meet the American Institute of Nutrition (AIN-93) recommended purified diets for laboratory rodents [38]. Fifty 5 week old male C57/B16 mice will be housed 8 per cage and acclimatized for a week. At 6 weeks of age, the mice will be placed on very high fat diets (VHFD) for 12 weeks to induce obesity, insulin resistance and hyperglycemia. They will afterwards be divided into 5 groups (10 rats per group) after baseline data collection. The mice will be placed on the intervention diets made up of VHFD containing 0, 5, 10, 15 and 20g freeze-dried blueberry with the VHFD to serve as a control for additional 13 weeks. Feed and water intake will be measured daily throughout the experiment while urine and blood samples will be collected at baseline, 6 weeks and at termination. Oral glucose tolerance test and fasting blood glucose test will also be done at baseline, midpoint, and termination. Body weight (to assess responses of the animals to the diets) will be measured weekly. After the 13 weeks period, the rats will be anesthetized by isoflurane administration and exsanguinations by cardiac puncture and blood, heart, liver, kidney, and adipose tissue will be collected, weighed, flash frozen in liquid nitrogen and stored under appropriate conditions (- 80oC) for biochemical tests. The above animal care procedure will be subjected to the Tuskegee University Institutional Animal Care and Use Committee (IACUC). Serum will be obtained from the collected blood samples by centrifuging at 5000 rpm for 10 mins and stored at - 80oC for analyses.Analysis of blood, urine, and tissue samples: Blood, urine and tissue samples will be assayed for glucose, lipids (triglycerides and total cholesterol), insulin, insulin resistance, β cell function, adiponectin, RBP4 and inflammatory cytokines.Determination of oral glucose tolerance test: Oral glucose tolerance test (OGTT) will be measured vial tail vein after an overnight fast according to the method described by Kim et al [3].Fasting blood glucose test: Fasting blood glucose test will be measured during the day after 6 and 9 hour fast using a glucometer (AlphaTRAK 32004-02, Abbot Animal Health) according to the method of Roopchand et al [12].Serum lipid analysis: Blood lipids (triglycerides, total cholesterols, LDL- and HDL-cholesterols) will be assayed by oxidase method using a commercial kit (Yeongdong Pharm. Corp., Korea) according to Kim et al [3].Serum insulin analysis and insulin resistance: Serum insulin will be assayed with a commercial rat/mouse insulin ELISA kit (EZRMI -13K) from Linco Research, Inc. (St. Charles, MO). The degree of insulin resistance will be estimated by a homeostasis assessment model (HOMA-IR) as described by Prior et al [39]. HOMA-IR = {plasma glucose (mmol/l) x serum insulin (mU/l)}/22.5.Assessment of β cell function: The β-cell function will also be assessed by a homeostasis assessment score (HOMA-BCF) as described by Prior et al [39] using the formula: HOMA-BCF = {20 x serum insulin (mU/l)}/{plasma glucose (mmol/l) - 3.5}.Cytokines analysis: Serum RBP4 will be measured by ELISA kits while adiponectin will be assayed using Milliplex MAP single plex adiponectin kit (Millipore). The concentration of inflammatory cytokines IL-6, IL-1β, and TNFα will be quantified according to Roopchand et al [12] using the Milliplex MAP mouse cytokine/chemokine kit (Millipore).Metabolomic analyses of blood, tissue, and urine samplesLC-QTOF-MS metabolomics analysis will be performed on a 1290 Infinity Agilent Rapid Resolution HPLC system coupled to a 6538 UHD Accurate LC-QTOF MS (Agilent Technologies, Santa Clara, CA, USA) equipped with dual electro-spray ionization (ESI) source according to Onuh et al [40].Statistical analysis: Results of all determinations will be expressed as means of triplicate values. Data will be subjected to 2-way Analysis of Variance (ANOVA) and significance will be detected using Turkey test. An IBM SPSS Statistical package (version 20) will be used for all statistical analyses.