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Mechanism of the Treatment Effect of Resveratrol on the High-fat Induced Insulin Resistance and Hepatic Lipotoxicity

Author: ShangJing
Tutor: ChenZuoZuo
School: Huazhong University of Science and Technology
Course: Internal Medicine
Keywords: Insulin sensitivity Hyperinsulinemic - euglycemic clamp Tail artery Tail vein. Insulin resistance Glucose infusion rate Resveratrol Non - alcoholic fatty liver AMPK SREBP-1c FAS HepG2 cells Steatosis Oxidative stress Reactive oxygen species Malondialdehyde Superoxide dismutase Hydroxyl radical TNF - alpha
CLC: R285.5
Type: PhD thesis
Year: 2008
Downloads: 582
Quote: 1
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Abstract


The first part of the rat euglycemic - hyperinsulinemic clamp technique improvement purposes: to improve classic euglycemic - hyperinsulinemic clamp technique based on intubation, the end of the use of vascular catheter technology implementation in rats with insulin in the awake state - euglycemic clamp experiments and evaluate the technology. Methods: normal feeding and high-fat fed Wistar rats 5, after 4 weeks of feeding, rat root of the tail local anesthesia in the awake state, respectively tail vascular catheter from the tail vein input of insulin (speed 0.25u/kg.h) and the glucose, tail artery blood hyperinsulinemic - euglycemic clamp experiments. Results: Last action after venous catheter blood glucose in the normal range, no significant fluctuations in blood sugar after; fat diet rats glucose infusion rate was significantly lower than the control group (P <0.01), indicating that the high fat diet rats with insulin resistance. Conclusion: Last action the venous catheter technology, based on the high insulin - euglycemic clamp experiment is feasible, more traditional intubation techniques with simple to shorten experimental period, the rats with low mortality advantages. The second part of resveratrol to improve high-fat-fed rats with insulin resistance, fatty liver and its mechanism research purposes: To observe the resveratrol can improve high-fat feeding caused insulin resistance in rats and non-alcoholic fatty liver ( NAFLD) and its mechanism. Methods: Acute: 10 Wistar rats were divided into two groups (n = 5), the control group and resveratrol treatment group the treatment group resveratrol (100mg/kg.day) orally, the control group saline gavage, were sacrificed 4 hours after liver AMPK phosphorylation level. Chronic experiments: 30 Wistar rats were divided equally into 3 groups (n = 10): normal control group (standard diet), high-fat group (high fat diet), and resveratrol group, resveratrol alcohol group throughout the high fat diet, the start resveratrol intervention (100mg/kg.day), seven weeks were fed for 16 weeks. Euglycemic - hyperinsulinemic clamp technique in rats IR liver paraffin HE staining pathological changes, liver frozen sections of oil red O staining lipid deposition conditions in the liver cells, Western Blot Western blot detection of liver AMPK phosphorylation levels RT-PCR detection of liver sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) mRNA gene expression. Results: The the acute experimental results: Resveratrol intervention group liver tissue AMPK phosphorylation levels increased to 164.2% of the control group; chronic experimental results: the high fat diet group rats compared with the control group, visceral fat index, increased liver index (P <0.05), fasting insulin levels, glucose infusion rate decreased (P <0.01), the light microscopic liver obvious steatosis, liver tissue AMPK phosphorylation levels down to the 45.8% of the control group (P <0.01) lipogenic gene SREBP-1c and FAS mRNA expression was significantly increased; rats after treatment with resveratrol, visceral fat content decreased significantly (P <0.01), lower fasting insulin levels, glucose infusion rate increased significantly (P <0.01), hepatic steatosis light microscope has been significantly improved, the level of AMPK phosphorylation in the liver tissue to the control group 70.2% (P <0.05), and at the same time lipogenic genes SREBP-1c and FAS reduce the amount of mRNA expression. Conclusion: Resveratrol can promote liver AMPK activity in the body; resveratrol treatment can improve the high-fat fed rats the IR while reducing lipid accumulation in the liver thereby improving fatty liver, the mechanism is suppressed by activation of AMPK downstream lipid synthesis pathway. The third part of the resveratrol purpose of reducing lipid deposition in the liver cells through activation of AMPK pathway: To observe the resveratrol is able to directly act on the liver cells and reduce lipid deposition in the liver cells, and in-depth to explore its mechanism. METHODS: HepG2 cells were grown in 6 empty plate, when the cell fusion to 80% of the single-layer adherent cells were serum-starved overnight, were added containing 0μM, 10μM, 25 mM, 50μM resveratrol low glucose DMEM medium and incubated for 6 and 24 hours after the extraction of cells the AMPK protein detection intracellular levels; HepG2 medium containing 25mmol / L glucose and 10 nm / L of insulin medium were incubated 24 hrs induction of steatosis model, then a concentration of 50μM resveratrol intervention steatosis model cell for 24 hours, extract protein, WesternBlot that Western blot detection of intracellular levels of AMPK phosphorylation; Trizol extraction of intracellular mRNA detection of sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) gene expression levels, while oil red O staining. lipid deposition and extraction of intracellular lipid detection of intracellular TG content. Results: Resveratrol dose-and time-dependent manner to promote AMPKα phosphorylation in cells. HepG2 cells steatosis model group red fat droplet in (oil red O staining), intracellular TG content was significantly higher (P <0.01), the level of AMPK phosphorylation is reduced to the 33.5% of the control group, SREBP- 1C, FAS gene expression were increased to 182% and 167% of the control group; resveratrol treated HepG2 oil red O staining and intracellular TG quantitative steatosis has been significantly improved, the level of AMPK phosphorylation returned to control 72.6%, while the amount of gene expression of SREBP-1c, FAS was also significantly decreased. Conclusion: Resveratrol can increase AMPK activity in the liver cells in vitro; resveratrol can act directly on the liver cells to reduce TG deposition in the liver cells, the mechanism is inhibition of downstream activation of AMPK SREBP-1c, FAS of gene expression thereby inhibiting lipid synthesis. The fourth part of the resveratrol fat induced insulin resistance oxidative stress in rats purpose: To observe whether resveratrol can reduce the fat IR model rats induced by oxidative stress, and to explore the relationship between oxidative stress and IR. Methods: The animal model grouped with the second part of this article, hyperinsulinemic - euglycemic clamp test insulin sensitivity kits were used to detect serum samples hydroxyl radical, MDA level and SOD activity were detected by ELISA in serum TNF-alpha concentration. Results: The model rats serum hydroxyl radicals, MDA, TNF-alpha compared with the control group were significantly increased, respectively, 1.2 times that of the control group, 2.1 times the level of 1.69 times (P <0.05 or 0.01), SOD activity decreased to control group 86% (P <0.01), and is associated with insulin resistance, prompted the model group rats were in severe oxidative stress and IR state; After resveratrol treatment, the hydroxyl radical, MDA, TNF-alpha concentration reduces SOD activity recovery, IR also been significantly improved. Conclusion: IR and oxidative stress are closely related, oxidative stress is one of the important reasons for the IR, the effect of resveratrol against oxidative stress is one of the mechanisms of resveratrol to improve IR.

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