| Part 1 Expression and regulation of RBP4 mRNA in human adipose tissue in obese and type 2 diabetic patientsObjective:To study the RBP4 mRNA expression between subcutaneous and visceral adipose tissue in obese and type 2 diabetic patients and to investigate the factors that influence RBP4 mRNA expression in Human adipose tissue.Methods:9 subjects with normal weight normal glucose tolerance, 9 obesity subjects and 9 type 2 diabetes subjects were enrolled. All of the subjects were prepared to undergone an operation because of nondiabetes disease. Subcutaneous and visceral adipose tissue were taken out as soon as the operation begin and treated the visceral adipose tissue of normal weight normal glucose tolerance subjects with INS, DEX, TZD, PA, TNF-α.RT-PCR and real-time PCR were used to assay the relative expression of RBP4 mRNA. Results:RBP4 mRNA level in visceral adipose tissue of obesity and type 2 diabetes groups were(2.10±1.84)and (1.54±0.46), respectively. Both were significantly higher than that of normal weight normal glucose group (0.75±0.28,P<0.01 or P<0.05). RBP4 mRNA level in subcutaneous adipose tissue among the three groups have no difference. INS, DEX, TZD, PA have an increase of 2.13 times, 0.84 times, 2.04 times, 4.88 times compared with the control group, respectively. Tumor necrosis factor-αdecreased RBP4 mRNA level by 38%, compared with the control group.Part 1 Conclusion:1. Both of the subcutaneous and visceral adipose tissue have RBP4 expression, and there were no difference between the two kinds of adipose tissue in subjects of normal weight normal glucose.2. RBP4 mRNA level in visceral adipose tissue were significantly higher in obesity and type 2 diabetes subjects.3. RBP4 gene expression was regulated by INS, DEX, TZD, PA and TNF-α, such factors were also participated in the pathophysiological process of insulin resistance and type 2 diabetes.4. The elevated RBP4 expression in visceral adipose tissue may be the main reason of increased serum RBP4.Part 2 The infulence and mechanism of PPAR-γagonists on the expression of RBP4 in SD rats fed high-fat diet.Objective:To investigate the mechanism of PPAR-γagonists on the regulation of RBP4 gene expression by observing the impact of PPAR-γagonist pioglitazone on RBP4 levels in SD rats fed high-fat diet.Methods:24 Male SD rats were randomly divided into 2 groups, normal diet group (NC, n = 8) and high fat diet froup (F, n = 16), after 8 weeks fed by different diets, F group were randomly divided into 2 groups: high fat diet control group (FC, n = 8), high fat diet treatd by pioglitazone group (F + Pio, n = 8), NC and FC groups were gavaged by normal saline, and the treatment group was gavaged by pioglitazone, 20mg?kg-1?day-1 for 4 weeks. Before and after the treatment, all of the Rats were carried out oral glucose tolerance (OGTT )and insulin tolerance (ITT ) to assay islet function, and the fasting serum were collected to measure blood glucose, lipid profiles, insulin as well as RBP4 levels and so on. At the end of the experiment, epididymal fat, adipose around kidney, liver were taked and weight their weight, calculating fat to body weight ratio. We used the method of RT-PCR and Western-blot to relatively quantify RBP4 and PPAR-γmRNA and protein levels in epididymal adipose and liver.Results:After 8 weeks fed, Rats in F groups had impaired glucose and insulin tolerance, lipid disorders. They also had higher fasting glucose and insulin levels, higher fat to body weight ratio and serum RBP4 levels than NC group(58.3±12.7 vs 26.8±7.3,p<0.01); after treated by pioglitazone, The fat to body weight ratio, liver to body weight ratio and blood glucose, lipid profiles, liver function as well as Serum RBP4 were decreased(P<0.01). RBP4 mRNA and protein in epididymal adipose tissue were significantly increased in FC group than NC group (P<0.01). after treated by pioglitazone, their expression were significantly decreased (P<0.01). There were no significantly difference among the three groups of the RBP4 levels in rat livers. Simple correlation analyses showed serum RBP4 were positively correlated with the RBP4 mRNA and protein levels of visceral adipose. and the three indices were positively associated with the blood glucose, serum lipids and insulin levels as well as the weight of adipose tissues and fat to body weight ratio.Part 2 Conclusion:5. The rat models with insulin resistance induced by high fat diet was successfully founded.6. High fat diets could lead to insulin resistance, and increased the levels of RBP4 both in the adipose tissue and serum, and there was a significant positive correlation between the two index.7. PPAR-γagonist pioglitazone can significantly reduce the levels of blood glucose and improve insulin resistance as well as decrease the level of adipocytokine RBP4 through the selective activation of adipose PPAR-γgene expression.Conclusion1. Both of the subcutaneous and visceral adipose tissue have RBP4 expression, and there were no difference between the two kinds of adipose tissue in subjects of normal weight normal glucose.2. RBP4 mRNA level in visceral adipose tissue were significantly higher in obesity and type 2 diabetes subjects.3. RBP4 gene expression was regulated by INS, DEX, TZD, PA and TNF-α, such factors were also participated in the pathophysiological process of insulin resistance and type 2 diabetes.4. The elevated RBP4 expression in visceral adipose tissue may be the main reason of increased serum RBP4.5. High fat diets could lead to insulin resistance, and increased the levels of RBP4 both in the adipose tissue and serum, and there was a significant positive correlation between the two index.6. PPAR-γagonist pioglitazone can significantly reduce the levels of blood glucose and improve insulin resistance as well as decrease the level of adipocytokine RBP4 through the selective activation of adipose PPAR-γgene expression.7. According to the first and second part of the experiment, the mechanism of PPAR-γagonist pioglitazone reduced RBP4 levels in vivo is indirectly by reducing the glucose and lipid toxicity and improving insulin resistance.
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