| Objective:To determine the expression of Chemerin level in serum and subcutaneous adipose tissue both for gestational diabetes mellitus(GDM) and normal glucose tolerance(NGT) pregnant women at term of before delivery, and to detect serum insulin, blood glucose, and TNF- α leves in GDM and normal pregnant women. To observe the changes of Chemerin levels in patients with GDM. To analyze its relationship with inflammation, insulin resistance, obesity etc in GDM fat. To Study the effect of Chemerin on the fat cells and its mechanism through cultured 3T3-L1 preadipocytes, in order to further explore the mechanism of Chemerin in gestational diabetes.Methods: 1. A total of 90 individuals collected from Tianjin gynecology and obstetrics Central Hospita. The experiments were divided into NGT group and GDM group, according to BMI before pregnancy, each group was subdivided into normal weight, overweight, obese group. We completed the following work, inquire history, record progestation and current weight, measure height, blood pressure; detect serum Chemerin, TNF α, insulin and blood glucose, blood lipid, liver and kidney function; observe changes in Chemerin; analyse its relationship with insulin resistance, lipid metabolism, obesity, weight gain, inflammation etc. 2. Chemerin and its receptor chem R23(CMKLR1) m RNA were detected in 30 cases of subcutaneous adipose tissue from pregnant women at cesarean section. 3. Chemerin overexpression lentiviral vector Chemerin-lenti and Chemerin RNA interference lentiviral vector shchemerin-lenti infect in 3T3-L1 adipocytes. And then glucose consumption test was performed to observe adipocyte insulin resistance; MTT assay was used to observe the effects of Chemerin on 3T3-L1 preadipocyte proliferation; Hoechst33258 staining to observe the effects of Chemerin on apoptosis of mature fat cells; Quantitative real-time PCR to detect the key factor related insulin resistance and inflammation in insulin metabolism pathway; Western-blot to detect the expression of AKT1 protein expression in insulin metabolism pathway.Results:1. Obese pregnant women have higher serum Chemerin, HOMA-IR, TNF-α, and lower weight gain than normal weight women in NGT group. GDM patients have higher serum Chemerin, HOMA-IR and TNF-α than normal weight NGT women. 2. The serum Chemerin is positively correlated with TG, Cholesterl, HOMA-IR, weight gain, SBP, TNF-α assessed by univariate correlation analysis. The serum Chemerin is positively correlated with TG and HOMA-IR assessed by Multiple linear regression analysis. 3.The level of Chemerin, weight gain, TG, HOMA-IR,TNF-α and BUN were significantly lower for diet control women with GDM han no diet Control women with GDM. 4. Obese NGT pregnant women have higher serum Chemerin, lower Chemerin and CMKLR1 m RNA expression in subcutaneous adipose tissue than normal weight NGT women; GDM patients have higher serum Chemerin, lower Chemerin and no different CMKLR1 m RNA expression in subcutaneous adipose tissue than normal weight NGT pregnant women. 5. 3T3-L1 preadipocyte differentiate into mature adipocytes induced by the mixture of BMX, dexamethasone, insulin for 8-12 days after 3T3-L1 preadipocyte cell contact inhibition. Oil red O staining confirmed that more than 90% of 3T3-L1 preadipocyte differentiate into mature fat cells. 6. Chemerin Lentivirus Expression Vector was successfully constructed. The Chemerin expression was up-regulated 3 fold compared with contral group after overexpression Vector transfect 3T3-L1 cell. 7. Shchemerin lentiviral vecto was successfully constructed. The Chemerin expression was down-regulated 30% account for contral group after overexpression Vector transfect 3T3-L1 cell. 8. 3T3-Ll cell glucose consumption decline after it was transfected by overexpression of Chemerin lentiviral vector, and 3T3-Ll cell glucose consumption increase after knockdown of Chemerin gene. 9. MTT results showed that OD value was increased in chemerin overexpression group compared with overexpression control group. OD value decreased in Chemerin silencing group compared with silencing control group. 10. Hoechst staining showed that Chemerin had no effect on the apoptosis of 3T3-L1 cells. 11. Real-time quantitative PCR results showed that Chemerin had no effect on the expression of IRS1, IRS2; Overexpression of Chemerin may lead to down-regulation of Akt1 expression, up-regulated expression of Fox O1, Erk1, Erk2; Knockdown of Chemerin gene could cause up-regulation of Fox O1, Erk1, Erk2 expression, down-regulation of Akt1 expression.Conclusions:1. Obesity and GDM affect of pregnancy serum Chemerin, HOMA-IR, TNF- α concentration, weight gain as independent factor. 2. Maternal serum Chemerin positively related to TG and HOMA-IR assessed both by univariate correlations and multivariate analysis. 3. The reduction of the chemoattractant protein Chemerin and inflammatory molecules TNF-α causing by diet control results in decline of inflammation in adipose tissue, and as a consequence decrease HOMA-IR and improve insulin sensitivity. 4. Obese NGT pregnant women have higher serum Chemerin, lower Chemerin and CMKLR1 m RNA expression in subcutaneous adipose tissue; GDM patients have higher serum Chemerin, lower Chemerin and no different CMKLR1 m RNA expression in subcutaneous adipose tissue. 5. Increased Chemerin expression in 3T3-L1 adipocytes can lead to decreased glucose uptake, cause insulin resistance. The possible mechanism maybe realizes through Down-regulation of Akt1 expression, reduction of GLUT4 membrane translocation, and then the descrease the absorption of glucose. 6. Increased expression of Chemerin can promote the proliferation of 3T3-L1 preadipocytes. The mechanism is realized probably by up-regulating the expression of Erk1, Erk2 7. Chemerin had no effect on the apoptosis of 3T3-L1 cells. 8. Chemerin may affect lipid metabolism through the up-regulation of Fox O1 expression... |
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