| BACKGROUNDDiabetes has become one serious chronic disease that damages the health of human. At present, the incidence of diabetes is going up by years, giving our community seriously economical burden. There is 90％Type 2 diabetes mellitus (T2DM) between all diabetic patients. The mechanism of T2DM has not clinically explanation yet. The present study believes that insulin resistance is an importance step in the incidence of T2DM that is document by much study. Most T2DM patients is existence insulin resistance. Banerji reports 60％males in African-American that BMI of are less than 30kg/m2 exist insulin resistance. Haffner reports 92％white people induced African origin Spanish origin and non Spanish origin exist insulin resistance. It is perceived that insulin resistance is universally existence. So to improve the insulin resistance is very importance to T2DM patients.The definition of insulin resistance is: normal measurement of insulin produces less than normal function. The classic insulin sensitive organ is adipose tissue liver and skeletal muscle. Present study in adipose tissue is a hotspot in mechanism of insulin resistance. Adipocyte defects express integrally first is adipose tissue anomally distribute, especially the cumulate of abdominal adipose tissue. Second is the body of adipocyte become multiplication. The reason of dysfunction in adipocyte has relations with adipocyte-secreted factors. The function of endocrine secretion of adipocyte has made a big progress at present studies; it has broken out the conventional idea that adipose tissue was only a store organ. Up to now, there is discovered so many different adipocyte-secreted factors. As if Adipsin RBP TNF-a PAI-1 and so on. They had something to do with insulin resistance.Retinol Binding Protein, RBP is a protein that transports vitamin A. At the period studies, RBP is mainly used to the detection in early diabetic nephropathy. In year 2004 a researcher name Yang and his group used Gene chip to compare the gene expression spectra in epididymis of mouse that GLUT4-Tg and GLUT4-/-, than they found a new adipocyte-secreted factors that is RBP4. There had document that, people before the diabetes but development IGT or IFG moreover the Glucose clamp indicated exist insulin resistance, the plasma of RBP4 is obviously higher than the normal people. RBP4 is seriously correlated with the level of insulin resistance. Elevated serum RBP4 was associated with components of the metabolic syndrome, including increased body-mass index, waist-to-hip ratio, serum triglyceride levels, and systolic blood pressure and decreased high-density lipoprotein cholesterol levels. Exercise training was associated with a reduction in serum RBP4 levels only in subjects in whom insulin resistance improved. Adipocyte GLUT4 protein and serum RBP4 levels were inversely correlated.Rosiglitazone as a new Thiazolidinediones euglycemic agent, has generally used to clinical treatment in T2DM. The mechanism of Rosiglitazone is mainly thought PPAR-γ, to raise the insulin sensitivity of skeleton muscle and adipose tissue. Investigate has prove that, Rosiglitazone had important function in depress glucose, to improve lipoids metabolism and hyperinsulinemia also in improve the sensitivity of insulin. In resent research rosiglitazone is documented that can decrease the level of plasma RBP4 in rat, than can improve the insulin sensitivity, the result impress that the mechanism of insulin resistance maybe have something to do with RBP4.To better observe the changes of RBP4 and GLUT4 in insulin resistance condition, and the effect of rosiglitazone interventions, we used vivo and vitro experiments. We choice spontaneous T2DM rat---OLETF rat as vitro experiment and the 3T3-L1 adipocyte that established insulin resistance condition as vivo experiment object. To observe the changes of RBP4 and GLUT4, at the same time to observe the effects that rosiglitazone contribute to RBP4 and GLUT4. We used RT-PCR to exam the mRNA expression of RBP4 and GLUT4 in adipocyte or adipose tissue, to approach the role of RBP4 and GLUT4 in the process of insulin resistance by the molecular biology.Chapter 1 Superficial Observed the Expression of Rebinding Protein 4 in 3T3-L1 adipocyteOBJECTIVEEstablished the insulin resistance model in vitro, utilized rosiglitazone to interfere in the adipocyte, observed the expression of RBP4 and GLUT4, at the same time observed the effect that rosiglitazone contribute to RBP4, analysis if the effect that rosiglitazone improve insulin resistance has something to do with the effect that it decreased the expression of RBP4.METHODS1. Chose the American Type Culture Collection, 3T3-L1 adipocyte as our experimental object than culture and derivation. Refer the differentiated method of Anil Kumar K. L, differentiated the adipocyte. Cultured 3T3-L1 adipocyte in the DMEM high glucose medium that contain 10％calf serum, and the condition of the evidence is 37℃, 5％CO2. when the cell was in good condition, put it to cultivated plates, when the cell conjugation after two days, culture the cells for 48 hours use the DMEM medium that contain 0.5 mmol/L IBMX 10mg/L insulin 1μmol/L dexamethasone 10％calf serum, and than change the DMEM medium that contain 10mg/L insulin 1μmol/L dexamethasone 10％calf serum, cultivate for 48 hours, after that culture with DMEM medium cantina 10％calf serum for 8-12 days. When there are 90％3T3cells become mature adipocyte, it can use in study.2. Use oil red O to improve 3T3 cells had yet induced to mature adipocyte. Remove the old medium; wash the cells by PBS for three times, use Ca-formalin fixation for 5-15min, removed the fixation fluid, wash the cells by PBS three times, open-air drying for 20min; ad move oil red O, dyeing for 1 hour, remove oil red O, avantin clean the rest oil red O, than campeachy dyeing the cell nucleus, observe the cell appearance and get the photos.3. 3T3-L1 adipocyte insulin resistance model established. After the cells differentiation, use DMEM medium that contain 1umol/L dexamethasone to induce insulin resistance model, change the medium every two days, and than use the old medium to exam the consumption of glucose, depend on the consumption we can judge the level of resistance. We use the GOD-POD kit to exam the level of glucose in the old medium. When the resistance model has established, set up the adipocyte as blank group dexamethasone model group rosiglitazone treatment group. After rosiglitazone has intervention for 48 hours, exam the level of glucose in the medium, than exam the mRNA expression of RBP4 and GLUT4.RESULT1. the absorbance of dexamethasone model group is significant higher than the blank group (P＜0.01). the absorbance level OD of rosiglitazone group is lower than dexamethasone model group, but it is not significant(P＞0.05).2. The expression of GLUT4mRNA in dexamethasone model group is significant lower than in the blank group(P＜0.01). The expression of GLUT4mRNA in rosiglitazone group is significant higher than the dexamethasone model group (P＜0.01). The expression of RBP4mRNA in dexamethasone model group is higher than the blank group, but the expression of RBP4mRNA in rosiglitazone group is lower than in the dexamethasone model group (P＜0.05).3. The expression of GLUT4mRNA and the expression of RBP4mRAN in dexamethasone model group exists nothing correlate relationship.CONCLUSION1. The expression of RBP4 in 3T3-L1adipocyte that used dexamethasone induced into insulin resistance model growth higher, but the expression of GLUT4 is lower.2. Rosiglitazone can decrease the expression of RBP4mRNA in 3T3-L1adipocyte that used dexamethasone induced into insulin resistance model, and can improve the expression of GLUT4mRNA.3. Rosiglitazone can increase the glucose intake in 3T3-L1 adipocyte.Chapter 2 Superficial Observed the Expression of Rebinding Protein 4 in OLETF ratsOBJECTIVECompare the level of the expression RBP4 between OLETF rats and LETO rats in adipose tissue; observe the function that rosiglitazone interfere in the expression of RBP4; observe the relationship between RBP4 and GLUT4, then approach the possible mechanism that RBP4 induce to insulin resistance.METHODOLETF rats random distribute to control group and rosiglitazone intervention group, with their lean nondiabetic counterparts, LETO rats as blank group. Use rosiglitazone 3mg/kg intragastric since 8 week of age; continue to 40 week of age. The blood glucose was determined by OGTT, to diagnoses diabetes. The RBP4 in adipose tissue was determined by RT-PCR.RESULTS1. The control group has appearance adiposities before it has not appearance diabetes. The body mass of control group is significant higher than the LETO group (P＜0.05), since 6 week of age. Abdominal adipose tissue of the control group is significant higher than the LETO group(P＜0.05) since 8 week of age.2. The AUC of control group and rosiglitazone is significant higher than the LETO group (P＜0.05), since 13 week of age. As the age growth, the AUC rising. The control group appearance the first diabetes, as the age growth, the incidence is rising, the incidence achieve 92.5％when it was 40 week of age.3. The expression of RBP4 mRNA in control group is significant higher than LETO group, since 8 week of age, and is rising as the age growth. The expression of GLUT4mRNA in control group is significant lower than LETO group, since 8 week of age, and is decreasing as the age growth.4. Between RBP4mRNA and GLUT4mRNA has nothing relationship.CONCLUSION1. RBP4 of adipose tissue in OLETF rats appearance higher expression when it has not exist diabetes. It has importance relationship with insulin resistance.2. There is nothing correlate relationship between RBP4 and GLUT4.3. Rosiglitazone can decrease the level of RBP4, improve insulin sensitivity, it may be one impossible mechanism of the rosiglitazone prevent T2DM.
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