Study On The Mechanism Of Insulin Resistance Induced By Tumor Necrosis Factor-α And Soluble Receptor And Gene Polymorphism In Gestational Diabetes Mellitus

PrefaceDiabetes is a major disease endangering human health. Its continuing increased epidemiological tendency poses a serious public health problem. Gesta-tional diabetes mellitus (GDM)is defined as glucose intolerance of variable severity with onset or first recognition during pregnancy. GDM may not exclude those with pre - existing glucose intolerance before pregnancy. GDM has short term and long term adverse effect on mothers and infants.The goal of GDM control is to maintain the level of the blood glucose and glycosylated hemoglobin to normal level as possible. At present it is not clear the best blood glucose control level for GDM. The monitoring of blood glucose for consecutive three days by dynamic glucose monitoring system (DGMS) can find the damage of glucose intolerance during pregnancy at an early age and direct the blood glucose control program during pregnancy.The etiology of GDM is unclear. Insulin resistance (IR) plays an important role in the initiation and development of GDM. Tumor necrosis factor system ( TNF) including TNFα and soluble receptors have a close relationship with IR and GDM. The TNFa gene polymorphism is related to the onset of type 2 diabetes. But it is unclear whether it is related to the occurrence of GDM at present time. The purpose of this research is to monitor the dynamic blood glucose level during pregnancy and provide theoretical foundation for the blood glucose control program based on the blood glucose variation pattern during pregnancy. The aim of the research also include the understanding of GDM pregnant woman serum TNF system and the distribution frequency of genetic polymorphism, its relation-ship with IR to explore its role in the mechanism of GDM onset.Object and method120 cases of pregnant woman at 24 -28 weeks'gestation and 60 cases of non - pregnant health woman in health examination were selected. Among them, 62 cases of pregnant woman underwent DGMS monitoring.The two - step diagnosis method of GDM recommended by the American Diabetes Association (ADA,2001)was referred . The uniform questionnaire was designed. Regular body check - up and obstetric examination were done. Four ml fasting blood from ulnar vein were taken. The serum TNFa level was measured by ELISA method. Genomic DNA was extracted by salt fractionation. TNF - a gene - 308 and - 238 allel and genotypic distribution frequency were tested by polymerase chain reaction - restriction fragment length polymorphism (PCR - RFLP). The blood glucose for pregnant woman was monitored by DGMS.Reagents and equipments;Blood glucose was measured by plasma glucose oxidase method (Japan OLYMPUS - AU1000 automatic biochemistry analyzer). DGMS (American Sat Meditech 1.0 edition). Insulin was measured by electro-chemiluminescence method (Beyer Acs 180SE). Serum TNFa and soluble receptor I , II test kit ( Shenzhen Jingmei biotech company). PCR amplification instrument ( 2400 American PerkinElmerGetus company). Gel imaging system (Lamwork3.0 imaging system software , American gene company). Taq DNA polymerase , Ncol restriction enzyme(Takara company).The major observation index;1. The average blood glucose level for pregnant woman, the maximum and nadir blood glucose level in a day, fasting and after - dinner average blood glucose level ,the proportion of hyperglycemia and hypoglycemia time in 24 hours.2. The comparison of FPGNFINSAHOMA - IR^TNF - a.sTNFR I fP sTN-FR1I level.3. TNF - a gene -308 and 238 locus allel and genotype and its correlation withlR.Results(1) Glucose level variation within 24 hours by DGMS monitoring1. Blood glucose is low at midnight and before three meals. It increased after three meals. The average blood glucose level in GDM group (6. 7 ±1. 1 mmol/L) is significantly higher than normal pregnant woman(5.7 ±0.6 mmol/ L).2. The percentage of hyperglycemia and hypoglycemia variation in 24 hours.The percentage of blood glucose after meals >7. 8 mmol/L is 60.4% (32/ 53 ), > 11.1 mmol/L is 5.7% ( 3/53 ). The percentage of blood glucose fast or before meals < 7. 8 mmol/L is 28. 3% (15/53 ). The percentage of blood glucose variation between 3.9-7.8 mmol/L is 26.4% (14/53 ).3. The occurrence of fetal macrosomia has a significant correlation with the blood glucose level after breakfast, after lunch and after dinner and the average level of three meals by dynamic blood glucose monitoring.The occurrence of fetal macrosomia is 43. 3% (23/53). The pregnant women with abnormal 75g OCTT glucose metabolism delivered 5 cases(9.4% ). The pregnant women for whom the time of blood glucose exceed 7.8 mmol/L is over 10% in 24 hours DGMS monitoring delivered 21 cases (39.6% ).4. In ROC curve, when the time of blood glucose level variation exceed 7. 8 mmol/L is over 10% in 24 hours DGMS monitoring and Youden index is the maximum 0. 55, the ideal cutting point for diagnosis of fetal macrosomia infant is over 10%.5. The occurrence of asymptomatic hypoglycemia is 28. 3% ( 15/53). The majority happened at midnight 3;34 ± 122 minute and the time length is 296. 1± 152.3 minntes. Among them, seven people had blood glucose <2. 8mmol/L. The time length is 40.1 ±28mmol/L. The nadir blood glucose is 2.24mmol/L. (2)The TNF - c^sTNF I fP sTNF II level in GDM group and normal pregnancy group are higher than healthy non - pregnancy group. The GDM group is higher than normal pregnancy group( P < 0.01). TNF - a, sTNF II have signifi-? 8 ?cant positive correlation with FPG^FTNS^BMI and IR. sTNF I has no correlation with the above indexes.(3)11ie -238 polymorphism locus A allel gene frequency and GA + AA genotype frequency in GDM group are higher than normal pregnant control group, but the difference is not statistically significant. The -308 polymorphism locus A allel gene frequency and GA + AA genotype frequency in GDM group are higher than normal pregnant control group, and the difference is statistically significant.DiscussionDynamic glucose monitoring system is consist of three parts: signal used glucose transmitter, data recorder and computer workstation. DGMS can monitor the daily blood glucose profile variation of pregnant women and reveal hypergly-cemia and hypoglycemia at an early time, especially the asymptomatic hypogly-cemia at night. These important information are neglected by the individual blood glucose monitoring technology. It is convenient for clinicians to identify blood glucose pattern and set a better program for blood glucose control. The treatment rectification is not merely based on blood glucose meter and venous blood glucose data.When pregnancy associated with diabetes and carbohydrate tolerance happened , the incidence rate of fetal macrosomia increased. At present, fetal mac-rosomia usually born to the pregnant women with normal glycometabolism. Only 3-5% newborn body weight can be explained by 75g carbohydrate tolerance variation. The prediction of infant body weight by blood glucose fast and after dinner is poor. By the consecutive three days, 24 hours monitoring of pregnant women blood glucose through DGMS, the occurrence of fetal macrosomia has a significant correlation with blood glucose level after breakfast, after lunch , after dinner and the average blood glucose level of three meals. It is important to guide a balanced, scientific and rational nutrition for pregnant women.Hypoglycemia has adverse effects on foetus, mainly on his nervous system, especially the trisplanchnic nerve and brain. Hypoglycemia has adverse damageon the brain similar to that of brain ischemic impairment, but it is not totally the same. The research by DGMS demonstrates that the hypoglycemia occurrence rate is 10.1% in children with impuberal type 1 diabetes. It is more common at midnight, lliis illustrates that DGMS can provide an effective way to find asymptomatic hypoglycemia in pregnant women and children. DGMS can make early diagnosis of hypoglycemia and guide the food rectification and treatment program for the less happening of hypoglycemia.There is a physiological insulin resistance during pregnancy. The onset of GDM is related to IR and insulin secretion defect. The elevated TNF - a in body can cause body insulin resistance. The increased TNF - a in body initiate IR, cutting down the insulin sensitivity. The incidence rate of GDM in obese pregnant women before pregnancy is evidently higher than the non - obese pregnant women. Moreover, pre -pregnancy BMI in GDM pregnant women is significantly higher than the normal pregnant women. Serum TNF - a>sTNFR H have a significant correlation with BMI,HOMA - IR,BMI and HOMA - IR.TNF - a performs its biological activity through the specific bonding to the two different receptors TNFR I andTNFR II in target cell membrane. TNFR is classified into two category according to their presence;one kind of receptor which mainly binds to cell membrane is called membrane receptor;another kind of receptor in body fluid such as serum is called soluble receptor. TNF system is consist of TNF - a, sTNFR I and sTNFR ]I. sTNFR is probably formed by the different enzyme modification of mTNFR extracellular region shredded from cell membrane surface which enters the blood circulation and filtered, reabsorbed in kidney. So, sTNFR has biphasic effect on TNF - a. The elevated sTNFR probably means the decrease of mTNFR. sTNFR has competitive binding to TNF - a with mTNFR, neutralizing TNF - a and preventing its adverse effect on body. Low concentration of sTNFR has promotion effect on the induction of cell growth by TNF - a, attenuating the retrogradation of TNF - a. It plays an role as a " TNF — a slow releasing reservoir " or stabilizing and maintaining the TNF - a trimer activity.TNF belongs to HLA - M family gene. TNF - a gene has several polymorphic site. More recent researches focus on the two gene polymorphic sites rela-? 10-tionship with diseases. One polymorphic site guanine (C) to adenine (A) locates in the TNF - a gene promoter region - 308 locus. Another polymorphic site guanine ( G) to adenine (A) locates in the Y box 238 locus. For non - mutation allel, restriction enzyme Ncol and Mspl could cut it. There are three genotypes for the two polymorphic sites :GC(non - mutant) ,GA( mutant heterozy-gote) and-AA(mutant homozygote).Hie gene polymorphism has effect on its products. Different genotypes can produce different proteins and has influence on the disease sensitivity and severity. Hie relationship between the TNF - a gene polymorphism and insulin resistance attract more and more attentions. Hie conclusion about the relationship between TNF - a gene polymorphism and insulin resistance is different. A study shows that insulin sensitivity index decreases for gene carriers with 308 locus A allel. Hie mutant increases the TNF - a transcriptional efficiency by 6 to 7 times in Raji cell. One of the mechanisms for TNF - a induced insulin resistance is the fortification of lipoclasis and the increase of FFA. the difference between the lipoclasis by genetic mutation probably is the mechanism of insulin resistance in population. It is then deduced that TNF - a gene polymorphism has a certain relationship with insulin resistance. Hie contradictory researches believe that the genetic mutation in 308 locus has nothing to do with insulin resistance. TNF - a gene expression decreases for TNF - a genotype AA at locus 238. HOMA insulin resistance index decreases for A allel gene carrier, implying that the TNF -a gene locus 238 G—?A mutation could improve the insulin resistance. Our stud-y discover that the - 308 polymorphism locus A allel gene frequency and GA + AA genotype frequency in northeast of China Han ethnic GDM group are higher than normal pregnant control group. Hie serum TNF - a level in mutant group is higher than non - mutant group. Our results imply that TNF - a - 308 locus gene mutation probably increase the gene expression to a great extent, and the latter has a close relationship with the onset and development of gestational IR and GDM. Although the - 238 TNF - a polymorphism locus A allel gene frequency and GA + AA genotype frequency in GDM group are higher than normal pregnant control group, the difference is not statistically significant. Hierefore, TNF - a - 238 locus gene mutation may not participate in the onset and develop-ment of IR. Meanwhile, there is no linkage relationship among the TNF - a polymorphic sites.Conclusion1. DGMS could diagnose the after - meal hyperglycemia and asymptomatic hypoglycemia at night for pregnant women which could not identified by the discontinuous blood glucose monitoring. DGMS is a useful tool for the management of blood glucose monitoring for pregnant women.2. TNF - a and sTNF II in women with gestational diabetes mellitus is correlated to IR. In human, TNF system has effect on IR probably and mainly through sTNF II.3. In Han ethnic pregnant group of northeast China, TNF - a gene 308 G—? A mutation can increase the risk of gestational diabetes mellitus for pregnant women. A allel could increase the release of TNF - a participating insulin resistance, leading to the onset of GDM.