Associations Between Chemerin And Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) is defined as glucose intolerance that is first detected during pregnancy. The incidence of GDM is increasing year by year all over the world, especially in developing countries. It is a common perinatal complication which can threaten maternal and child health and even lead to energy metabolism disorders in pregnant women. It is associated with a variety of adverse pregnancy outcomes.such as macrosomia,polyhydramnios,spontaneous abortion,fetal malformation, fetal distress, stillbirth, neonatal hypoglycemia, hypocalcemia, hyperbilirubinemia and maternal ketoacidosis. Its pathogenesis is undefined. At present, it is widely accepted that GDM and type2diabetes (T2DM) share similar pathogenesis, namely there is insulin resistance (IR) and insulin secretion insufficiency on the basis of genetic defects, in which IR plays a major role.Human adipose tissue as one of the most important endocrine organ synthesize and secrete a variety of adipocytokines, including leptin, adiponectin, tumor necrosis factor-a (TNF-a), interleukin-6(IL-6) and newly discovered resistin, visfatin, apelin, fasting-induced adipose factor (FIAF), retinol-binding4(RBP4), Chemerin and so on. Chemerin are expressed in the body’s adipose tissue, adrenal gland, liver, lung, pancreas, placenta, ovary, skin and particularly in white adipose tissue, liver and lung. Chemerin can be involved in inflammation and regulate glucose and lipid metabolism. The expression of Chemerin is related to obesity. Chemerin has the biological effects of regulating adipocyte differentiation and lipolysis as well as promoting the insulin signal transduction pathway in adipose cells.As a newly recognized adipose factor, Chemerin is attracting more and more attention and in-depth research in obesity, insulin resistance, metabolic syndrome and its influence on the development of type2diabetes. However, the understanding of Chemerin is still incomprehensive and there are problems which remain to be clarified as it is a newly discovered area and there are racial differences. Topics such as the intrinsic relationship between the distribution and occurrence of Chemerin gene single nucleotide polymorphisms (SNP), the level of transcription and protein expression in adipose tissue and plasma concentration, and the correlation associated with obesity, insulin resistance and glucose metabolism abnormality have still not been revealed.Gestational diabetes mellitus is often combined with obesity, insulin resistance and abnormal glucose metabolism, but research related to Chemerin and gestational diabetes mellitus is rarely. This study intends to discuss the change of the gestational diabetes patients’peripheral blood and cord blood Chemerin,and its relationship with metabolism on the whole; discuss the Chemerin mRNA and protein expression of adipose cells and placenta in gestational diabetes mellitus at cell and molecular levels; analyze the change and distribution of Chemerin SNPs in gestational diabetes mellitus patients using direct sequencing method after gene amplification; and further clarify the relationship between Chemerin SNPs and the clinical parameters of gestational diabetes mellitus. In this way, we can further study the pathogenesis of gestational diabetes mellitus.provide evidence for the basis and therapeutic target to screen valuable markers of disease,and improve the prediction and diagnosis of gestational diabetes mellitus, thus exploring new ways to improve perinatal outcome of gestational diabetes mellitus.Part I. The Change of Chemerin in Gestational Diabetic Patients and Its Clinical SignificanceObjective:To explore the relationship between Chemerin and GDM, this study measured the change of peripheral blood and cord blood Chemerin respectively in patients with GDM and normal pregnant women, and its relationship with BMI, lipids and blood glucose.Methods:Select pregnant women who conducted regular prenatal examination and were hospitalized for delivery in Obstetrics Clinic of Women’s Hospital School of Medicine Zhejiang University from January2012to October2012. According to the2011ADA diagnostic criteria,46cases diagnosed as GDM in24to28weeks of pregnancy were selected as the GDM group.Simultaneously, select43cases of pregnant women with the age.gestational age and body mass index in progestation similar to that of the GDM group as the control group.All selected cases delivered in37to40weeks. After being admitted to hospital,detailed information of maternal age,height,body weight before pregnancy and pregnancy weight gain were recorded by a specially-assigned person. At the same time, determine the fasting blood glucose (Fast Plasm Glucose,FPG),fastinginsulin (Fast Insulins, Fins), glycosylated hemoglobin (HemoglobinAlc,HbAlc), total cholesterol (Total chole sterol, TC),triglyceride (Triglyceride,TG), high density lipoprotein cholesterol (HDL-C) and low densitylipoprotein cholesterol (LDL-C) of the two groups before delivery, and calculate the insulin resistance index (HOMA-IR),BMI in progestation and intrapartum BMI. After the delivery and umbilical cord clamp of the newborn, immediately extract10ml of venous cord blood from the placenta end with a disposable sterile syringe. Use enzyme linked immunosorbent assay (ELIS A) method to determine the peripheral blood and cord blood Chemerin value.Simultaneously, measure the weight (kg) and length (Ht) of the newborn, and calculate BMI.Results:1. Comparison of clinical data:there was no significant difference between two groups in maternal age, gestational age, parity, times of pregnancy,BMI in progestation. Weight gain of GDM group during pregnancy and intrapartum BMI was significantly higher than that of the normal control group.2. Comparison of2groups in glucose, lipid, inflammation, insulin and Chemerin:the level of Chemerin in peripheral blood of GDM group was significantly higher than that of normal control group;at the same time,its FPG, Fins,TG,HbAlc and HOMA-IR,CRP, WBC were higher than those in normal control group, but there is no statistical significance in two groups in terms of TC,HDL, LDL level difference (P>0.05).3. Correlation analysis of peripheral blood Chemerin and indexes:the correlation analysis shows that:Chemerin and FPG and HOMA-IR and TG of the normal control group were of negative correlation (r=-0.582,-0.376,-0.427,P=0.000,0.016,0.008).There is no correlation between the GDM group and the Chemerin index,while the weight gain during pregnancy and intrapartum BMI show positive correlation (r=0.336,0.340, P=0.026,0.022).4. Comparison of umbilical cord blood Chemerin level and neonatal body mass between two groups:Chemerin level of cord blood in GDM group was significantly higher than that of the control group, which were22.50±4.36pg/ml and19.85±6.06pg/ml respectively, showing significant difference; the differences between neonatal weight and BMI of GDM group and control group had statistical significance.5. Comparison of Chemerin levels in peripheral blood and cord blood of two groups:the levels of Chemerin in peripheral blood and umbilical cord blood of GDM groups were significantly higher than the corresponding measured values of the normal control group (P=0.001,0.026). Chemerin level in cord blood of both GDM group and normal control group was significantly higher than that in the peripheral blood of the same groups (P=0.005,0.029).Conclusion:(1) Chemerin in the peripheral blood of normal control group is related to blood glucose and blood lipid metabolism indexes, which suggests that Chemerin is involved in the regulation of energy metabolism, and help to maintain the blood glucose and blood lipids at normal level.(2) The level of Chemerin in peripheral blood of the GDM group was significantly higher than that of the normal control group, and was related with weight gain during pregnancy and intrapartum BMI, but had no correlation with metabolism. It is suggested that the Chemerin dysfunction caused by excessive weight gain of GDM group during pregnancy may be concerned with the abnormal energy metabolism of GDM.(3) In group GDM, neonatal birth weight and cord blood Chemerin levels were increased significantly compared with those of the control group. This suggests that Chemerin may be related to the complications of GDM offspring. Part Ⅱ. The Expression of Chemerin mRNA and Protein in Adipose and Placenta with Gestational Diabetic PatientsObjective:To detect Chemerin mRNA in adipose and placenta and protein level of the GDM group and the normal control group, and explore the expression of Chemerin in adipose and placenta.Methods:Select pregnant women who conducted regular prenatal examination and were hospitalized for delivery in Obstetrics Clinic of Women’s Hospital School of Medicine Zhejiang University from January2012to October2012. According to the2011ADA diagnostic criteria,46cases diagnosed as GDM in24to28weeks of pregnancy were selected as the GDM group. Simultaneously, select43cases of pregnant women with the age, gestational age and body mass index in progestation similar to that of the GDM group as the control group. All selected cases delivered in37to40weeks. In the caesarean operation, collect5g maternal subcutaneous adipose; after the delivery of placent, leave1cm*1cm*1cm of placenta tissue at the root of umbilical cord, and store it in refrigerator at-70℃. Detect Chemerin mRNA in adipose and placenta of GDM group and normal control group with fluorescence quantitative PCR, and detect the protein expression level in adipose and placenta of both groups by Western blotting.Results:1. The quality inspection of total RNA extraction:Inspect the integrity of total RNA through1.5%agarose gel electrophoresis. It can be seen under the UV lamp that28s,18s and5s of rRNA were visible, the intensity of28s was nearly2times higher than that of18s, and the intensity of18s was higher than that of5s, indicating that RNA is of good quality.2. Real-time..fluorescent..quantitative.PCR..amplification.(SYBRGreenI.fluorescence quantitative PCR relative quantitative method)2.1Amplification curve:amplification curve automatically generated by the PCR instrument shows that the amplification curve of the target gene Chemerin and the reference gene Actin were smooth with similar span, suggesting that the expression of gene amplification was in good condition.2.2Comparison of Chemerin mRNA expressions in adipose and placenta of the two groups:the average expressions level of Chemerin mRNA in adipose tissue and placental tissue of GDM group were higher than that of normal pregnant women. The difference was significant (P<0.01). 3. Comparison of Chemerin protein expressions in adipose and placenta of the two groups:through gray statistical analysis, it can be seen that the mean values of Chemerin/GAPDH in adipose and placenta of GDM group were significantly higher than those in the control group, and the difference was significant (P<0.01).Conclusion:(1) Chemerin has its expression in gestational adipose and placental tissues and participates in the normal physiological metabolism during pregnancy;(2) the expression quantity of Chemerin mRNA and protein in adipose and placenta of GDM group were higher than those in normal control group, indicating that Chemerin derived from adipose and placenta participates in the occurrence and development of GDM. Part Ⅲ. Single Nucleotide Polymorphism Analysis of Chemerin Gene in Gestational Diabetic PatientsObjective:Analyze the change and distribution of Chemerin SNPs in gestational diabetic patients by using direct sequencing method after gene amplification; and further clarify the relationship between Chemerin SNPs and clinical parameters of gestational diabetes mellitus. Methods:Select pregnant women who conducted regular prenatal examination and were hospitalized for delivery in Obstetrics Clinic of Women’s Hospital School of Medicine Zhejiang University from January2012to October2013. According to the2011ADA diagnostic criteria,330cases diagnosed as GDM in24to28weeks of pregnancy were selected as GDM group. Simultaneously, select329cases of pregnant women with the age, gestational age and body mass index in progestation similar to that of the GDM group as the control group. All selected cases delivered in37to40weeks. Inspect the fasting blood-glucose, triglyceride and other biochemical indexes of the subjects. Conduct screening on the basis of literature,NCBI and bioinformatics software to obtain10loci including rs3735170, rs6970196,rs10244748, rs17173608,rs1804764,rs3210475, rs10278590(rs4721), rs3735168, rs9942696and rsl7835937. Extract genomic DNA of all subjects, analyze the genotype of the above mentioned loci by direct sequencing method, and conduct correlation analysis.Results:1. The clinical feature of research objects:there was no significant difference in the pregnant week, progestational BMI, SBP, DBP、TC、HDL-C、LDL-C、Gr、BUN. ALT、GOT between the two groups. There are significant difference in FPG, TC, WBC、CRP、HOMA-IR and Fins between the two groups.2. Sequencing:The sequencing results of all SNPs loci were completely consistent with classification results by probe method.3. Analysis of genotype and allele frequency 3.1HWE test:Through Hardy-Weinberg test and analysis, P value of7loci was much larger than the critical value0.05, the observed values of7SNPs loci was consistent with the expected values, the gene frequency reached genetic equilibrium and the research data was group representative, proving that they meet the Hardy-Weinberg equilibrium.3.2A comparative study of each SNP loci caseThe C allele frequency of rs3735170locus was13.5%in GDM group and33.4%in normal control group. C allele was a protective factor for GDM (P=0.038), and OR was0.303(0.103-0.886); T allele frequency was86.5%in the GDM group and66.6%in the control group. The frequency distribution of genotype CC+CT vsTT carrying C in GDM group decreased and the P value was0.027, reaching statistical significance;The C allele frequency of rs17173608locus was1.7%in GDM group and13.3%in normal control group. C allele was a protective factor for GDM (P=0.039), and OR was0.132(0.015-1.177); T allele frequency was98.3%in the GDM group and86.7%in the control group. The frequency distribution of genotype CC+CA vs AA carrying C in GDM group decreased and the P value was0.039, reaching statistical significance;The T allele frequency of rs9942696locus was25.2%in GDM group and16.3%in normal control group. T allele was a high risk factor for GDM, and OR was3.354(1.062-10.590); C allele frequency was74.8%in the GDM group and83.7%in the control group. The frequency distribution of genotype TT+TC vs CC carrying T in GDM group increased and the P value was0.035, reaching statistical significance;The alleles of the rest SNP loci and the relevant genotypes involved showed no statistical difference between the two groups; 3.3LD test:there was no locus in the same LD block, and haplotype associated with the occurrence of GDM was no found;4. Comparison of clinical and biochemical indexes of genotypes:there was no statistical difference index between the genotypes in rs17173608and rs9942696loci. There was significant difference in weight gain during pregnancy, FPG, TC and LDL index between the genotypes in rs3735170locus.Conclusion:There is relationship between Chemerin rs3735170(T>C) of different genotypes and the occurrence of GDM.