Role Of Vaspin In The Pathogenesis Of Gestational Diabetes Mellitus

Gestational diabetes mellitus（GDM）, defined as the newly onset of glucose intolerance during pregnancy, occurring in 2⁵% of pregnant women, is a major cause of maternal and perinatal serious complications. GDM includes maternal hypertensive disorders, increased rates of caesarean delivery, macrosomia, neonatal hypoglycemia, and RIDS.Several mechanisms have been suggested for the pathogenesis of GDM, including altered adipose tissue endocrine function, placental and hormonal changes, inflammation, and oxidative stress, all contributing to insulin resistance. Visceral adipose tissue-derived serpin A12（vaspin）, is a novel adipokine with insulin-sensitizing effects, which is involved in the Type 2 diabetes. But its role in GDM is currently unknown.The first aim of the study is to investigate the serum levels of vaspin in GDM at 2^nd and 3^nd trimester, evaluating the role of vaspin in the pathogenesis of GDM and its correlation with insulin resistance. The second aim of the study is to investigate the vaspin m RNA and protein expression in the placental tissue from women with GDM and normal pregnant women, so as to explore its role in the pathogenesis of GDM. The third aim is to explore the effect of metablic factors on vaspin expression in Be Wo cell and the underlying mechanisms. Part 1 Serum vaspin levels in the second trimester of pregnancy ingestational diabetes melitusObjective: To investigate the role of vaspin in the pathogenesis of gestational diabetes mellitus（GDM） in the second trimester and its correlation with insulin resistance.Methods: The study recruited 58 pregnant women of 24 to 28 gestational weeks in the Hebei General Hospital Obstetrics and Gynecology from January to June 2013. Among them, 30 were patients with GDM（GDM group）, 28 had normal oral glucose tolerance test and was referred as healthy pregnancy group（NGT group）, and 27 were healthy nonpregnant women with normal oral glucose tolerance test referred as normal controls（NC）. The fasting plasma glucose（FPG）, 1 hour and 2 hours postprandial glucose levels were measured by glucose oxidase method. The fasting insulin（FIN） levels were measured by radioimmunoassay and the homeostatic model assessment-insulin resistance index（HOMA-IR） was calculated. The levels of total cholesterol（TC）, triglycerdes（TG）, high density lipoprotein cholesterol（HDL） and low density lipoprotein cholesterol（LDL） were determined. The vaspin and IL-6 levels were measured by ELISA.Results:1 The levels of TC, TG, HDL and LDL levels in GDM group were（5.52 ± 0.87）,（2.79±0.77）,（1.75±0.44） and（3.34±0.77） mmol/L, and were（5.85±0.78）,（2.52±0.71）,（1.94±0.39） and（3.4±0.62） mmol/L in NGT group. The levels of lipid in the two groups were significantly higher than those in NC group, respectively（P<0.05）.2 The levels of FPG were significantly higher in GDM and NC group [（5.48±0.69）mmol/L,（5.16±0.42）mmol/L] than those in NGT group [（4.54±0.28）mmol/L], respectively（P<0.05）.3 The levels of INS [（14.03±5.57）m U/L], HOMA-IR（3.45±1.46）, 1-hour [（10.87±1.84） mmol/L] and 2-hour [（8.55±1.76）mmol/L] postprandial glucose levels of GDM group were significantly higher than those in NGT group [（11.49±4.18）m U/L, 2.32±0.86,（7.42±1.33） and（6.2±0.92） mmol/L] and in NC group[（8.88±3.75）m U/L;2.03±0.87,（7.58±0.83、6.75±0.68）mmol/L], respectively（P<0.05）.4 The levels of vaspin in GDM group and NGT group [（5.10± 2.36）,（ 5.43±1.88） ng/m L] were significantly higher than those in NC group [（2.03±2.34）ng/m L ], respectively（P<0.05）, whereas the vaspin levels in GDM group were slightly lower than those in NGT group, and the difference was not statistically significant（P>0.05）.5 The levels of IL-6 in GDM group and NGT group [（296.96±122.38）,（286.80±97.66） ng/m L] were significantly higher than those in NC group [（234.38±90.86）ng/m L], respectively（P<0.05）; whereas the IL-6 levels in GDM group were slightly higher than those in NGT group, but the difference was not statistically significant（P>0.05）.6 The vaspin levels in GDM group and in NC group were positively correlated to the levels of TC（r:0.42, P:0.02; r:0.71, P<0.01） and TG respectively（r:0.51, P<0.01; r:0.38, P:0.046）. However, the vaspin levels were not correlated with the levels of FPG, FIN and HOMA-IR in the GDM and NGT group（P>0.05）. While in NC group, the levels of vaspin were negatively correlated with INS and HOMA-IR（r:-0.42,-0.41, respectively, P<0.05）.The vaspin levels were positively correlated to the levels of IL-6 in GDM group（r:-0.47, P:0.01）,7 The IL-6 levels were positively correlated to the levels of FPG in NC group（r:0.52, P:0.01）, While in GDM group, the levels of IL-6 were positively correlated with FPG（r:0.54, P:<0.01）, pre-pregnancy BMI（r:0.44, P:0.02） and current BMI（r:0.45, P:0.01）Conclusion:1 The levels of vaspin were significantly increased in pregnant women, irrespective to the status of their glucose tolerance. This elevation may be derived from the placenta.2 Vaspin maybe an important adipokine that involved in the carbohydrate and lipid metabolism in GDM, and is related to the pathogensis of GDM and insulin resistance.3 Vaspin may be a marker of lipid metabolism in pregnancy and affected by IL-6. Part Ⅱ Serum vaspin levels in the third trimester of pregnancy in gesta-tional diabetes melitusObjective: To investigate vaspin concentrations and its relationship with insulin resistance and lipid parameters in women with gestational diabetes mellitus（GDM）.Methods: A total of 30 GDM subjects and 27 age-matched pregnant women at term with normal glucose tolerance（NGT） were included. The fasting plasma glucose（FPG） was measured by glucose oxidase method, the fasting insulin（FIN） was determined by radioimmunoassay, the total cholesterol（TC）, triglyceride（TG）, high density lipoprotein（HDL） and low density lipoprotein（LDL） were determined, The HOMA-insulin resistance index（HOMA-IR） was caleulated based on FPG and FINS with the minimal model. The serum vaspin and IL-6 levels before and three days after delivery as well as fetal serum vaspin levels were measured by Enzyme-linked immunosorbent assay（ELISA）.Results:1 The levels of TC, TG, HDL and LDL levels in GDM group were（4.67 ± 0.89）,（3.26±1.03）,（1.32±0.23） and（2.73±0.75） mmol/L, and were（4.93 ±0.79）,（2.80±0.93）,（1.40±0.26） and（2.89±0.69） mmol/L in NGT group, there were no significant differences between the two groups（P>0.05）.2 Women with GDM showed significantly higher FPG（4.61±0.58 vs 4.23±0.51 P=0.01.） and glycated hemoglobin（5.88±0.38 vs 5.48±0 38 P<0.01.） than those in normal pregnant control.3 There were no significant difference of insulin levels and insulin resistance index between the GDM group and the NGT group（12.68±4.74 vs 11.63±4.91 P=0.41; 2.38±1.48 vs 2.24±1.08 P=0.17）.4 Serum vaspin levels were significantly lower in women with GDM compared to healthy controls [（0.49±0.24）,（0.83±0.27）] ng/m L,（P<0.01）. Three days after delivery, serum vaspin levels decreased significantly in GDM women [（0.36±0.13）,（0.49±0.24）] ng/ml（P<0.01）. However, the changes in NGT were not significant. The cord vaspin levels in the two groups were [（0.34±0.12）（0.44±0.25）] ng/m L, there were no significant differences between the two groups（P>0.05）.5 The serum IL-6 levels in GDM group [（358.70±169.39） pg/ml] were significantly increased than those in NGT group [（271.21±148.91）] pg/ml（P=0.04）. Three days after delivery, serum IL-6 levels were unchanged in the two groups. The cord blood IL-6 levels in GDM group [（383.92±213.25） pg/ml] were slightly higher than those in NGT group [（336.95±140.70） pg/ml], but the difference was not statistically significant（P>0.05）.6 The vaspin levels in NGT group were positively correlated to the levels of TG and LDL respectively（r=0.45,0.42, P<0.05）, while the IL-6 levels in NGT group were negatively correlated to the levels of VLDL（r:-0.42, P:0.03）, and were positively correlated to the levels of FPG. In the GDM group, there were positively correlation between the serum levels of IL-6 and prepregnancy and third trimester of pregnancy body mass index（r:0.50, P:0.01; r:0.40, P:0.03）. and there were negatively correlation between the serum levels of IL-6 and vaspin.Conclusion:1 The serum vaspin levels decreased in the GDM group in late pregnancy, and further decreased three days after delivery, indicating that vaspin may be a protective factor for GDM and secreted mainly by the placenta.2 In the NGT group, there were positively correlation between serum vaspin levels and TG, VLDL, suggesting that vaspin may be involved in the regulation of lipid metabolism.3 The serum IL-6 levels were increased in late pregnancy in the GDM group, supporting the theory that oxidative stress and inflammatory responses were involved in the pathogenesis of GDM.4 There were negative correlation between the vaspin serum levels and IL-6 levels in the GDM groups, suggesting there were correlation between the two factors. IL-6 may inhibit the expression of vaspin. Part Ⅲ Vaspin expression in term Placenta with GDMObjective: To investigate the vaspin m RNA and protein expression in the placental tissue from women with GDM and normal pregnant women, and in order to explore its role in the pathogenesis of GDM.Methods: A total of 20 GDM subjects and 20 age-matched pregnant women at term with normal glucose tolerance（NGT） were included. Placental tissue were collected during operation, the m RNA expression of vaspin and IL-6 in placenta were detected by fluorescence quantitative PCR（real-time FQ RT-PCR）, the protein expression of vaspin and IL-6 were investigated using immunoblotting（Western blotting）.Results:1 The expression of vaspin m RNA and protein in placenta were slightly decresed in GDM group than that in NGT group（0.60±0.32 vs 0.68±0.32 P=0.46; 0.30±0.08 vs 0.39±0.26 P=0.33）; but there were no significant differences between the two groups（P>0.05）.2 The expression of IL-6 m RNA and protein in placenta were significantly higher in GDM group than that in NGT group（1.35±0.67 vs 0.8±0.47 P=0.01; 1.23±0.25 vs 0.54±0.20 P<0.01）.3 There were no significant correlation between the expression of vaspin in placental tissue and serum vaspin（P>0.05）; In the GDM group, vaspin m RNA levels were negatively correlated to glycated hemoglobin（r:-0.71, P: 0.01）, but it was negatively correlated to the newborn body mass(（r=-0.48, P=0.03; r=-0.88, P<0.01） and IL-6 m RNA expression（r=-0.57, P=0.01）.Conclusion:1 The expression of vaspin m RNA and protein in placenta were negatively correlated with IL-6 in the GDM group, which showed that IL-6 was involved in the regulation of vaspin.2 The vaspin may act locally in the placenta as a paracrine/autocrine agent and thereby play a major role in fetal development. Part Ⅳ Study on the regulatory mechanism of vaspin expression inBe Wo cellObjective: Vaspin is mainly secreted by visceral adipose tissue, which acts as an insulin-sensitizing adipokine. It is expressed in the placenta during pregnancy, but the regulatory mechanism is unclear. The aim of the study is to explore the effecte of metablic factors on vaspin expression in Be Wo cell and the regulatory mechanisms involved.Methods: Be Wo cells were cultured with different concentrations of IL-6（1, 5, 10 ng/ml）, glucose（5.5, 10,30 mmol/L） and pioglitazone（10, 100 ummol/L）, respectively for 12 h, 24 h, 48 h and 72 h. After collecting cells, the transcription and protein levels of vaspin were detected by RT-PCR and westernblot respectively.Results:1 Different concentrations of IL-6（1, 5, 10 ng/ml） can decrease the vaspin m RNA and protein expression after Be Wo cell were cultrured for 12 h, 24 h, 48 h, 72 h.The expression of PPAR-γ m RNA and protein in the Be Wo cells were unchanged.2 Different concentrations of glucose（5.5, 10, 30mmol/L） decreased the expression of m RNA and protein of vaspin and PPAR-γ after cultured with the Be Wo cells for 48 hours respectively.3 In cultured Be Wo cells with different concentrations of pioglitazone（10, 100umol/L） after 12 h, 24 h, 48 and 72 hours respectively, it showed that 10umol/L of pioglitazone significantly increased the expression of vaspin m RNA in Be Wo cells after 12 h and 24h（*P<0.05）, while 100umol/L of pioglitazone slightly inhibited the expression of vaspin m RNA after 48h（#P<0.05）; 10umol/L and 100umol/L of pioglitazone inhibited the expression of PPAR-γ m RNA of Be Wo cells after 12 h and 24h（*P<0.05）, while the inhibitions effective disappeared after 48h（**P<0.01, *P<0.05）.Conclusion: IL-6, glucose and pioglitazone are involved in the regulation of trophoblast vaspin expression in a concentration-dependent manner. IL-6, glucose can suppress the expression of vaspin, while pioglitazone upregulates the expression of vaspin. IL-6 has no effect on the expression of PPAR-γ of the trophoblast cells, but it can reduce the expression of vaspin, showing that the vaspin is involved in the occurrence and development of GDM by the regulation of inflammatory cytokines. However, the effect on vaspin is not through PPAR-γ pathway. The secretion of vaspin in the trophoblast cells is regulated by many factors and may relate to oxidative stress and inflammation. The exact mechanism of its regulation needs further study.