Effects Of Green Tea Polyphenols On Metabolic Disorders And Endothelial Dysfunction Induced By High Fat Diet

Part1The effect of GTPs on high-fat diet induced metabolic disordersObjective:The metabolic abnormalities associated with metabolic syndrome include obesity, hyperglycemia, dyslipidemia, elevated blood pressure and decreased insulin sensitivity. Liver is the biggest and most important organ that regulates metabolism, and fatty liver is considered to be a performance of the metabolic syndrome in liver. This part of the study investigated the effect of high-fat diet on metabolic markers in rats, liver fat deposition and expression of genes related to glucose and lipid metabolism in the liver, the effects of GTPs on high-fat diet-induced metabolic changes was focused.Methods:Male SPF Wistar rats after weaning and weight between40-60g were used. After acclimation for a week, the rats were randomly divided into the following five groups:the control group, fed on standard chow and drank deionized water; the high fat group, fed on high fat diet, drank deionized water; three GTPs treated groups, fed on high fat diet, and the deionized drinking water was replaced with different concentrations of tea polyphenols solution (0.8g/L,1.6g/L and3.2g/L) when the rats weights over180g. The oral glucose tolerance test (OGTT) was taken at the26th week, and then the rats were sacrificed. The serum was separated for measurement of serum insulin levels by ELISA and determination of blood sugar levels and blood lipid levels by commencial kit. HOMA-IR index was calculated according to the formula. The liver, epididymal fat and perirenal fat weight, were accurately weighed and the visceral fat coefficient and liver coefficient were calculated. The fat deposition in liver was measured by oil red O staining on frozen sections. Real-time quantitative PCR was used to detect the mRNA expression of fatty acid synthase (FAS),3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), peroxisome proliferator activated receptor a,(PPARa), sterol regulatory element binding protein-lc (SREBP-lc), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK) and glucose transporter2(GLUT2) in the liver..Results:The food intake of the control group is higher than that of the other groups; however, the energy intake is equal in all groups due to the different energy density of the standard chow and the high fat chow. From the9th week on, the weight of high fat fed rats was significantly higher than that of the control group. At the end of the experiment, body weight and visceral fat coefficients of high fat fed rats were significantly higher than that of the control group. Compared with the high-fat group, the body weight and visceral fat coefficients were significantly reduced in GTPs treated groups. High fat fed rats exhibited significantly higher blood glucose levels than the control group; the blood glucose of GTPs treated groups was significantly lower than that of the high-fat group. The glucose tolerance in high-fat rats was lower and the insulin HOMA-IR index was higher than that in the control group, and GTPs intervention improved the glucose tolerance and down-regulated the HOMA-IR index in high-fat fed rats. The serum insulin level in high-fat group is significantly higher-than that in the control group, GTPs treatment didn’t change the insulin level in high fat fed rats. High fat diet induced dyslipidemia in rats. Compare to the control group, the serum triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) level was significantly increased. The serum high-density lipoprotein cholesterol (HDL-C) was significantly decreased, and the LDL-C/HDL-C ration increased significantly, all these alterations except for HDL-C levels were alleviated by GTPs treatment to different extents. In the liver, high-fat diet induced fat deposition, increased mRNA expression of genes related to fat synthesis (the FAS HMG-CoAR and SREBP-1), and reduced mRNA expression of fatty acid oxidation gene, PPARa. The alterations in fat deposition and expression of genes were all alleviated by GTPs treatment. High-fat diet also significantly raised the mRNA expression of the key and rate-limiting enzymes of gluconeogenesis (PEPCK and G6Pase), and reduced the mRNA expression of the major glucose transporter in the liver (GLUT2), these alterations in hepatic glucose metabolism gene expressions induced by high fat diet were also mitigated by GTPs. Conclusion:The high-fat diet induced metabolic syndrome, fat deposition in the liver and dysregulated the hepatic mRNA expression of genes related to fat and glucose metabolism in rats, these changes are irrelative to total energy intake. GTPs alleviated the high-fat diet-induced metabolic syndrome, fat deposition in the liver and abnormal hepatic mRNA expressions of glucose and lipid metabolism related genes.Part2The Effect and cell signaling mechanism of GTPs on adiponectin productionObjective:The mechanism underlying the metabolism regulating effects of GTPs remains unclear. Hypoadiponectinemia plays an important role in the development of obesity, metabolic syndrome and related diseases, and adiponectin had been considered to be a new therapeutic target for treatment of obesity and insulin resistance. In this section we observed the effects of GTPs on adiponectin levels in high-fat fed rats and explored the possible mechanism.Methods:Animal management was stated in part1. The serum adiponectin levels were determined by ELISA, The mRNA expression of adiponectin and PPARy in visceral fat of the rats were determined by real-time quantitative PCR. The protein expression of PPARy, the phosphorylation of PPARy, and protein expression extracellular signal-regulated kinase extracellular signal regulated kinase (ERK)1/2and phosphorylated ERK1/2levels were detected by Western blot. Rat visceral adipose tissue were cultured in vitro with high glucose DMEM medium, and treated with GTPs or pretreated with PD98059, a specific inhibitor of ERK1/2pathway. After24hours intervention, the culture medium was collected and the adiponectin levels in it were determined by ELISA, the cultured visceral adipose tissue were collected and the mRNA expression of adiponectin and PPAR Y and protein expressions PPAR Y phosphorylated PPAR Y, ERK1/2and phosphorylated ERK1/2were determined by real-time quantitative PCR and western blot respectively.Results:The adiponectin mRNA levels and serum adiponectin level was significantly lower in the high fat group than those in the control group, and GTPs treatment significantly mitigated the decrease in adiponectin in high fat fed rats. Compared with the control group, the ERK1/2activation and PPARy phosphorylation in the visceral fat were increased significantly. Meanwhile, the PPARy mRNA levels and protein levels were decreased significantly. Compared with the high-fat group, the activation of ERK1/2and PPARy phosphorylation were significantly lower, and PPARy expression levels were significantly increased. High glucose cultured visceral adipose tissue also exhibited significantly reduced mRNA and secretion levels of adiponectin, up-regulated ERK1/2and PPARy phosphorylation levels and decreased PPAR Y expression levels. GTPs intervention and ERK1/2inhibitor pretreatment both inhibited the ERK1/2activation, PPAR Y phosphorylation, and raised the PPARγ expression and adiponectin expression and secretion in high glucose cultured rat visceral adipose tissue.Conclusion:The regulation of adiponectin levels is an important mechanism underlying the preventive and mitigative effects of GTPs on high-fat diet-induced metabolic disorders. GTPs increase the adiponectin level by inhibition of ERK1/2activation, decrease the PPARy phosphorylation and increase the level of PPARy expression.Part3The effects and mechanism of GTPs on vascular endothelial hyperpermeabilityObjective:Atherosclerotic cardiovascular diseases, which make biggest contribution to morbidity and mortality of cardiovascular diseases, are metabolic syndrome related diseases. Endothelial hyperpermeability, referring to increased transport of large molecules including AGE and lipoproteins to the subendothelial space, is the primary reaction of endothelium to damage factors like hyperglycemia or hyperlipidemia, an early marker and one of the major performances of endothelial dysfunction, and the primary change in the development of atherosclerosis. The objective of this part is to observe the effect of high-fat diet on endothelial permeability in rat aorta with the focus on effects of GTPs intervention, and to explore the mechanisms underneath both in vivo and in vitro.Methods:Animal management and intervention were same with part1, bovine aortic endothelial cells (BAECs) were cultured with high glucose culture medium, and treated with GTPs (0.4, or4μg/mL.24h), or pretreated with DPI (10μM) or SU5416(10μM) pre-incubated for30min or incubated with rhVEGF (8ng/mL), beta cyclodextran intervention (8μM) for30min before the test of permeability in monolayer bovine aortic endothelial cells. The endothelial permeability in rat aortic was measured using Evans blue injection, and the permeability in monolayer bovine aortic endothelial cells was measured by the fluorescein isothiocyanate (FITC) glucan assay. Dihydro-ethidium (DHE) fluorescent probe was used to detect the reactive oxygen species (ROS) level in rat aortic, the2’,7’-dichloro-fluorescein diacetate (DCFH-DA) fluorescent probe to detect ROS levels in cultured BAECs. The real-time quantitative PCR was used to detect the mRNA levels of vascular endothelial growth factor (VEGF) and caveolin-1. VEGF levels in serum and cell culture medium were measured by ELISA.Western blot assay was used to detect the protein levels of cav-1, p22phox and p67phox in cultured BAECs.Results:Compared with the control group, the high fat diet increased the endothelial permeability in rat aorta, comparing with the high-fat group, the permeability in aorta of GTPs treated groups were significantly lower. Meanwhile, high-fat diet significantly increased the ROS level and the mRNA level of vascular endothelial growth factor (VEGF), a strong permeability factor, in the aorta and upregulated the serum level of VEGF, GTPs treatment significantly reduced the high fat diet induced increase in VEGF and ROS. In high glucose cultured BAECs, the endothelial permeability, VEGF expression and secretion, ROS level were all increased. High glucose culture also increase the expression of p22phox and p67phox subunits of NADPH oxidase and the expression of caveolin-1, GTPs mitigated the elevated VEGF expression and secretion, ROS increase, up-regulated expression of p22phox, p67phox and caveolin-1induced by high glucose incubation. Similar to high glucose culture, rhVEGF treatment increased the endothelial permeability in monolayer BAECs cultured in normal DMEM. Preincubation with SU5416(VEGF receptor inhibitor), DPI (NADPH oxidase inhibitor) or treatment with0cyclodextran (structural inhibitor of caveolae) all down-regulated the endothelial permeability in high glucose cultured monolayer BAECs. SU5416also down-regulated the cavelin-1expression in BAECs cultured in high glucose DMEM.Conclusion:High fat diet could induce endothelial hyperpermeability in rat aorta, and this variation could be mitigated by GTPs treatment. GTPs regulated the endothelial permeability in high fat rats by down-regulating NADPH oxidase, ROS production, thus decreased the VEGF levels and the expression of cav-1, which induced down-regulation of caveolae mediated large molecule transport and alleviate the endothelial hyperpermeability.