Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209
Performing Department
Chemistry
Non Technical Summary
Multiple risk factor syndrome, or metabolic syndrome (i.e., the coexistence of several risk factors for atherosclerosis, including hyperglycemia, dyslipidemia, and hypertension) is a growing medical problem in industrialized countries, with obesity as the central and causal component in this syndrome. But, the mechanistic role of obesity has not been fully elucidated, nor is it readily transferrable into clear instructions for effective parenting. Oxidative stress plays critical roles in the pathogenesis of various diseases. Increased oxidative stress also underlies the pathophysiology of hypertension and atherosclerosis by directly affecting vascular wall cells. Thus, to prevent some cellular damage caused by reactive oxygen species (ROS), the level of tissue antioxidant is critical. Dietary antioxidants, which complement the actions of enzymatic antioxidants, are now widely recognized. These include a-tocopherol (vitamin E), ascorbic acid (vitamin C), and b-carotene (8). Pharmacological doses of these vitamins are sometimes beneficial, but often times ineffective, making alternative natural antioxidants such as flavonoids more preferable.In the present study, we propose to further examine the following using the 3T3-L1 preadipocytes model for obesity: 1.) How quercetin, genistein and kaempferol enhances glutathione levels in cells to prevent oxidative damage. 2. How the above-mentioned flavonoids enhances cell differentiation in obese subjects.Results from this project can enhance the cell's protective mechanisms, thereby reducing the mutagenic burden of the cell to oxidative damage induced by obesity. Findings will then be transferred into short layman terms for use in poster presentations for use by child caregivers.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Goals of research: The proposed hypothesis is that certain plant flavonoids (namely, quercetin, genistein and kaempferol) can enhance (a.) GSH and its related enzymes through the induction of γ-glutamyl synthetase (γ-GCS) and glutathione synthetase (GS) activities; (b.) enhance the expression levels of PTEN in preadipocytes thereby resulting in decreased cell growth and differentiation. This hypothesis will be tested by two specific aims:Determine the relative efficiencies of the quercetin, genistein and kaempferol on the expression levels of γ-glutamylcysteine synthetase (γ-GGS) and glutathione synthetase (GS) activities in 3T3-L1 preadipocytes.Determine the relative efficiencies of the quercetin, genistein and kaempferol on the expression levels of PTEN (i.e., total and phospho-PTEN) in 3T3-L1 preadipocytes. There are very limited reports on investigations into the expression mechanisms underlying the effects that quercetin, genistein and kaempferol exert on γ-GCS, GS and PTEN activities. Thus, the purpose of this objective is to examine the structure-activity relationship to better understand how quercetin, genistein and kaempferol may modulate γ-GCS, GS and PTEN expression levels in 3T3-L1 preadipocytes. 3T3-L1 cell line was chosen because it is very easy to culture and many studies have been conducted using this cell line for investigative purposes on signaling with regards to obesity studies. Supporting objectives:Engage (6) more TSU students in flavonoids research and develop experience for minority students, and help familiarize source producers on the potential use and application(s) of flavonoids enriched products in prevention-based health marketing.Strengthen the prevention-based health advocacy and outreach within the disadvantaged communities near and around TSU's campus.Provide demonstrations/education to the general public on the health benefits gained from higher intake of flavonoids rich foods and beverages, with particular focus on customers and food stand sellers and Nashville's local farmers' market.
Project Methods
Treatment of 3T3-L1 Cells with the flavonoids:Treatment of 3T3-L1 for the analysis of γ-GCS, GS and PTEN will be carried out as described by Lee et al. (54) with the following modifications. Cells will be incubated (i.e., for 4, 8 and 24 hr) with or without flavonoid sample (0 -100 mM) following oxidation by the Fenton's pathway at 37°C, 5% CO2 and in Dulbecco's modified Eagle's medium (DMEM, Welgene Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) in cell culture plates. All test substances will be dissolved in DMSO; the solvent concentration in the incubation medium will not exceed 0.5%. Control incubations will contain the same concentration of DMSO. Cell number and viability (Tryphan blue exclusion) will be determined using a Haemocytometer, before and after the treatments.Principle of the assay:An assay of γ-GCS and GS activities in the cultured cells is based on the formation GSH from cysteine, glutamate and glycine in the presence of supernatant from the preadipocytes for the assay of γ-GCS, or from γ-glutamylcysteine and glycine for the assay of GS respectively.Expression levels of γ-GCS activity in 3T3-L1 preadipocytes following exposure to quercetin, genistein and kaempferol:The activity levels of γ-GCS following the treatments with the various concentrations of each of flavonoids for the different time periods as described under section 5a will be determined by the γ-GCS Elisa kit from Antibodies-Online Inc. (Cat. #ABIN364940, Atlanta, GA). The following precautions will be adhered to: Fresh samples will be used at all times for the assay to avoid protein degradation and denaturation which may lead to wrong results. γ-GCS activities for all the samples will be expressed/mg protein.Expression levels of GS activity in 3T3-L1 preadipocytes following exposure to quercetin, genistein and kaempferol:GS activities in the flavonoid treated samples for the different times (under section 5a) will be carried out by the GS Elisa kit from Antibodies-Online Inc. (Cat. #ABIN815651, Atlanta, GA). Care will be taken to predict the concentration of GS before the assay. If values are not within the range of the standard curve, the optimal sample dilutions for the particular experiments will be determined. GS activities for all the samples will be expressed/mg protein.. Analysis of total PTEN in Cell Lysates following exposure to quercetin, genistein and kaempferol: The lysates (from the various treatments including controls) will be diluted 1:1 in MILLIPLEX MAP Assay Buffer 2. 25 μl of diluted cell lysate will be added to each well and incubated overnight at 4°C (or 2 hours RT) on a plate shaker (600-800rpm) protected from light. Lysates will be removed by vacuum filtration and 100 μl/well of MILLIPLEXMAPAssay Buffer 2 added. Buffer will be removed by vacuum filtration and gently blot the bottom of the filter plate on a paper towel. 25 μl/well of 1X MILLIPLEX MAP Biotinylated Reporter will be added and incubated at room temperature for I hr. Following the incubation 25 μl of diluted (1:25) MILLIPLEX MAP Streptavidin-Phycoerythrin (SAPE) will be added and incubated at room temp for 15 min. 25 μl of MILLIPLEX MAP Amplification Buffer will be added to each well and incubated again at room temperature for 15 min. The MILLIPLEX MAP SAPE/Amplification buffer will then be removed vacuum filtration and the bottom gently blotted on a paper towel. Beads will be resuspended in 150 μl of MILLIPLEX MAP Assay Buffer 2. The samples will be analyzed and the Median Fluorescent Intensity (MFI) recorded using the Luminex® Instrument. The PTEN levels will be expressed as MFI/mg protein.Analysis of phospho-PTEN in Cell Lysates following exposure to quercetin, genistein and kaempferol:PC-3 cells will be treated with the flavonoids as described above and the levels of phospho-PTEN analyzed as described under Phospho-PTEN (Ser380) MAPmates™ Cat. # 46-679 by Millipore Corp. (St. Charles, MO).Determination of Protein Content in 3T3-L1 preadipocytes:The soluble protein content of all cell lysates will be determined by the Coomassie blue protein assay method (63) using bovine serum albumin (BSA) as standard. Absorbance of the samples together with standards will be read in a spectrophotometer at 595 nm (Synergy).Statistical Analysis: Statistical significance will be determined by two-way Analysis of Variance (2-Way ANOVA) followed by Duncan's test for multiple comparisons using SPSS 20. P<0.05 will be considered statistically significant. Each value in all figures will represent the mean for each dose level of flavonoid and time tested, which will be assayed in triplicates. Results will be expressed as means ± SD.