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Toll-like Receptor 4 Negatively Mediates Islet Function and Insulin Resistance in Diet-Induced Obesity

Author: LiJuan
Tutor: ZouDaJin
School: Second Military Medical University
Course: Endocrine and metabolic diseases
Keywords: Obesity Islet function Toll-like receptor 4 Glucose-stimulated insulin secretion function Cytokines Inflammation
CLC: R589.2
Type: PhD thesis
Year: 2009
Downloads: 612
Quote: 2
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Abstract


TOLL-like receptor 4 with a high-fat diet-induced obese mice islet function relationship studies obesity is an important predisposing factor of type 2 diabetes, is also an important component of the metabolic syndrome, can cause the body dyslipidemia and high free fatty acidemia. However, the exact mechanism of sustained high-fat diet-induced obesity damage islet beta cells is still not very clear. We assume that the cytological mechanisms of long-term high-fat diet-induced obesity impairs islet beta cell function may be beta cells by upregulating surface of TLR4 and activation of the TLR4 signaling pathway, and enhance the level of expression of the downstream inflammatory factors, and thus damage the beta cell function. In order to verify the above assumptions, we have designed the following three-part test. The model of the first part of the high-fat diet-induced obese mice Objective: To establish a high-fat diet-induced obesity mouse model. Test methods: 1 male 8-10 weeks old C57BJ/L6 mice 40, were randomly divided into normal diet group and the high fat diet group (n = 20). Ordinary feed energy components: 10% fat, 20% protein, carbohydrates and 70%. The high-fat feed energy components: 35% fat, 20% protein, carbohydrates and 45%. Every two weeks, said the body weight, food intake and the amount of water diverted calculated weekly, specimens from serum of mice in each group at the end, said take epididymal fat weight, liver weight, calculate visceral fat mass (epididymal fat / body weight), liver content (liver / body weight). 3 blood glucose meter blood glucose, blood glucose, blood lipids (TG, TC), enzyme-linked immunosorbent assay (ELISA) serum insulin biochemical methods. 4 by the glucose tolerance test, the insulin release test, insulin tolerance test, to determine the insulin sensitivity and β-cell function. 5 high-fat group and the control group of mice, respectively, at 4 weeks, 12 weeks were sacrificed 24 weeks (n = 5); take the six groups of liver pathology HE staining, light microscope steatosis and TUNEL assay liver apoptosis changes. Results: 1. Compared to the control group, high-fat mice body weight, visceral fat content (epididymal fat / body weight), the liver content (liver / body weight) was significantly higher, especially in the high fat diet for 12 weeks significantly different and differences persist to the end point of 24 weeks. Fat mice fasting plasma glucose, fasting serum insulin levels, and blood lipids (TC, TG) are significantly higher, higher than that of the control group; triglyceride content of the liver, muscle and pancreas tissue of high-fat group was also significantly high in the control group. Compared to the control group, diminish fat mice insulin tolerance, insulin sensitivity, reduce acute glucose-stimulated insulin secretion capacity. With the time extension of the high-fat diet, the liver gradually diffuse steatosis fat 12-week group weight in fat 4 weeks (HF4,), fat 24 weeks (HF24) heavier than fat 12 weeks (HF12), HF24 livers large number of lipid droplets in the most obvious around the centrilobular vein, liver cell volume increases, there is a large number of lipid droplets in vacuoles in the cytoplasm, nuclear Habitat edge, and inflammatory cell infiltration in the lobules. The control group liver cells arranged rules, no lipid droplets, no inflammatory cell infiltration. TUNEL assay found the fat mice hepatocyte apoptosis obvious control group does not appear hepatocyte apoptosis. Conclusion: The success of high-fat diet-induced mouse model of obesity, insulin resistance, its weight, the liver content (liver / body weight), visceral fat content (epididymal fat / body weight), blood lipids (TC, TG), fasting blood glucose, plasma insulin levels are significantly higher, impaired glucose-stimulated insulin secretion, impaired insulin; liver steatosis was significantly more evident with the feeding time steatosis and liver cell apoptosis. Due to the high-fat diet to simulate the characteristics of the human diet, create the conditions for the study of high-fat islet function. Therefore, the high-fat-induced obesity, insulin resistance model is ideal test animal model for in-depth study of beta cell function. The second part of the expression of TLR4 in high fat-induced obese mice islets Objective: To observe the TLR4 differentially expressed in the high-fat diet-induced obese mice islets, free fatty acid (palmitate) stimulate mouse insulinoma cell line MIN6 TLR4 expression explicitly obesity, high free fatty acid damage islet beta cell function and the relationship between the beta cell TLR4 expression. Method: 1. 40 C57BJ/L2 mice were randomly divided into two groups, 20 in each group (the same as the first part). Immunohistochemical method to detect high-fat 24 weeks (HF24) and control (ND24) 24 weeks group pancreatic tissue islet insulin TLR4 expression. 3. Cultured mouse pancreatic tumor cell lines in MIN6 free fatty acids (Palmitate) stimulation for 24 hours and BSA observed insulin secretion changes. Mouse pancreatic tumor cell lines in MIN6, respectively, with the free fatty acid (Palmitate) and BSA before stimulation 2,4,5,6-hour observation TLR4 mRNA expression IL-6mRNA in, TNFamRNA, and MCP-1 mRNA expression changes. Results: 1. Immune staining confirmed that TLR4 expression in islet insulin positive staining of cells, and TLR4 expression levels in mouse islets of high-fat group was significantly higher than that of the control group. 2 cultured insulinoma cell line MIN6 stimulation give free fatty acid (palmitate), compared to BSA group, sustained after 24 hours of free fatty acid (palmitate) stimulate a significant reduction in the secretion of insulin. 3 compared to BSA group, mouse pancreatic tumor cell lines of MIN6, TLR4 mRNA levels 2,4,5 hours after stimulation with the stimulation time increases with the free fatty acids (Palmitate), respectively, after 6 hours TLR4 mRNA levels reduced proinflammatory cytokines IL-6mRNA TNFamRNA MCP-1 mRNA levels of free fatty acids (Palmitate) 2,4,5 hours after the stimulus also significantly increased, decreased after 6 hours. Conclusion: The sustained high-fat diet can induce islet beta cells in TLR4 upregulation. Continuous stimulation for 24 hours of free fatty acids (palmitate), insulin secretion dysfunction found in the MIN6 the culture of the pancreatic tumor cell lines in vitro, and as extend the time for stimulation of free fatty acids, TLR4 and proinflammatory cytokines IL-6 mRNA TNFamRNA, MCP -1mRNA expression levels were elevated, indicating that the high-fat diet and high continuous stimulation of free fatty acids upregulated TLR4 expression in islet beta cells and promote TLR4 signaling pathway middle and lower reaches of the proinflammatory cytokines IL-6, TNFa, MCP-1 increased expression. Prompted the free fatty acid (palmitate) stimulate insulin secretion dysfunction may be raised with the beta cell surface expression of TLR4 related. The third part TRL4 knockout mechanism to protect the high-fat diet-induced obese mice islet function Objective: To study the TLR4 knockout turn against obesity, genes in mice after the high-fat diet to protect islet function mechanism and TLR4 signaling pathway with the free fatty acids (palmitate clarify obesity) to stimulate insulin secretion in the relationship between the initial, chronic free fatty damage pancreatic beta cells function may be through a raised surface of the beta cells of TLR4, and activation of the TLR4 signaling pathway, enhanced Preparation inflammatory cytokine expression levels achieved. Methods: 1. High fat feed ingredients with the first portion, the same high fat diet (HFD) were reared in the wild-type (SNF) mice and TLR4 knockout (NJ) mice, and each of the normal diet (ND) as the control group continuous feeding for 24 weeks. . Fortnightly weighed, calculated weekly food intake and the amount of water diverted serum specimens when at the end, weighing epididymal fat weight and liver weight, calculate visceral fat mass (epididymal fat / body weight), the content of the liver (liver / body weight). 3 fasting glucose, fasting insulin, blood lipids measured insulin sensitivity test, glucose tolerance test and insulin release test and evaluation TLR4 knockout mouse islet beta cell function (NJ). 4 The end point of 24 weeks, four groups of mice (SNJ HF group were sacrificed SNJ ND group the, NJ HF group NJ ND group) to take liver histopathology HE staining the liver steatosis differences observed between the different groups. Observe the changes the groups oil red staining of the liver and pancreas, as well as group islet ultrastructure. 5 from fat induced TLR4 knockout (TLR4-/ -) mice and wild-type mice isolated primary islet cells give static cultivate observed changes in islet function and the extracted RNA line realtime PCR detection of IL-6, TNFa and so the expression of inflammatory factors. Results: 1. Hyperlipidemia SNJ (SNJ HF) mice fatty liver changes similar to the first part of the SNJ HF mice fasting hyperglycemia, hyperinsulinemia, impaired glucose tolerance, decreased insulin sensitivity, and significantly reduced acute glucose-stimulated insulin secretion, and face the same high-fat diet fed for 24 weeks continuous, high-fat TLR4-/ - (NJ HF) group of mice that are resistant to obesity, insulin sensitivity enhanced insulin secretion by protection acute glucose-stimulated insulin secretion function protected. Face the same high-fat diet continued fed for 24 weeks, high-fat TLR4-/ - (NJ HF) group mouse liver, muscle and pancreas tissue triglyceride content was significantly lower than the SNJ HF group. Liver volume of 3.24 weekends NJ HF group was significantly greater than the NJ ND group, the liver surface dark brown, the blunt edge section greasy, NJ HF group liver reddish color, with sharp edges. Light microscope, SNJ HF group liver cells diffuse steatosis, lobular inflammatory cell infiltration. NJHF of liver cells arranged in basic normal lipid droplets and no hepatic steatosis. The 4 red staining of the liver and pancreas oil mice fat SNJ severe hepatic steatosis, liver red stained lipid droplets area increased significantly, there are a large number of lipid droplets red dye area, NJ HF group showed significant lipid droplets. The red dye District, the background liver cells arranged basic rules. SNJ HF group pancreatic tissue surface area of ??a large number of red dye showed red oil droplets like background pancreatic tissue arranged in the basic rules, NJ HF group no significant pancreatic tissue lipid droplets red dye, 5.SNJ HF mice islet ultrastructure fat TLR4 knockout NJ mice beta cells in the absence of change, its shape is close to normal diet lipid droplets within the beta cell vacuoles increased significantly reduce the number of insulin secretory vesicles, and accompanied by mitochondrial swelling; group of beta cells. Primary islet isolation static cultivate display continued to reduce high-fat fed for 24 weeks in wild-type mice the SNJ HF group primary islet glucose-stimulated insulin secretion (GSIS), NJ HF group was significantly lower than TLR4 knockout mice. NJ HF group islet inflammatory cytokines IL-6, TNFa, MCP-1mRNA level significantly lower than that in wild-type mice. Conclusion: This study TOLL-like receptor 4 in the status and role of insulin secretion from the live animal level and cell level study found that high-fat diet can upregulate the expression of TLR4 in islets, activated TLR4 signaling pathway, enhanced downstream inflammatory factors expression levels; found that activation of TLR4 signaling pathway and secretory function of the same islet beta cells damage are closely related. TLR4 knock divided mice that are resistant to high-fat diet-induced obesity, and protect islet function. Continued free fatty acids (palmitate) stimulate damage insulin secretion, and enhanced the expression of TLR4 activation of TLR4 signaling pathway, prompted the beta cell insulin secretory function is closely related with TLR4, TLR4 knockout can protect the islet beta cell function. Animal level and cell level results: Cellular mechanisms of chronic free fatty acids stimulate damage islet beta cell function by up-regulating beta cell TLR4 expression levels, and thus activated TLR4 signaling pathway.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Metabolic diseases > Lipodystrophy
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