| Objective:The aim of this study is to disclose the PAI-1 genotype, alleles distribution features in obese type 2 diabetic patients of northern China Han nationality and their relationships between the body composition, insulin sensitivity and plasma fibrinolysis function, and from view of the blood fibrinolysis mechanism, we want to explore the relationships between PAI-1 gene polymorphism and vascular complications in obese type 2 diabetic patients.Subjects and methods:1.Subjects: 187 patients with no consanguinity were selected from Han nationality in Shandong district.(1)T2DM group: 147 patients, 76 men 71 women . All the patients were over 50 years old old. The mean age was (59.98 ±10.52) years old .The mean body mass index (BMI) was (25.58±3.90)kg/m~2.Based on the BMI, the patients were divided into two groups: ①Obese and overweight(BMI ≥ 23kg/m~2) group (OBDM): 105 patients, there are 57 male and 48 female patients among them, the mean BMI was (26.99 ± 3.28)kg/m~2. ② Normal weight(23kg/m~2>BMI≥18.5kg/m~2)T2DM (NWDM) group:42 patients, there are 19 male and 23 female patients among them, the mean BMI was(20.89 ± 1.37)kg/m~2.Based on the vascular complication conditions, the patients were divided into 4 groups:①DM with no complications group(DMNC group):49 patients, 22 men, 27 women. There were 16 normal weight (NWDMNC group) and 33 overweight or obese (OBDMNC group) patients among them.②Diabeticnephropathy group (DN group): DN was defined as 24 hours urine albumin excretion rate(UAER)>20mg/min, UAER between 20 and 200mg /min was diagnosed as micro-albumin urine, UAER over 200mg/min was diagnosed as macro- albumin urine.59 patients in all, there were 28 men and 31 women in them, the mean age was(59.20 ± 11.08)years old ,the mean BMI was(26.02 ± 4.25)kg/m2. Among them, 44 were in DNIII phase ,12 in IV phase and 3 in V phase .Based on the BMI, the patients were divided into two groups: normal weight DN group(NWDN),it involved 19 patients ; overweight and obese DN group(OBDN), it involved 40 patients;(3)Diabetic retinopathy(DR)group:69 patients in all, 60 patients were of background type, 9 patients were of proliferative type. Among them , 38 men,31 women, the mean age was (61.87±9.75)years old, mean BMI was (24.46±3.70)kg/m2.Based on the BMI the patients were divided into two groups: normal weight DR group(NWDR) involved 27 patients, and overweight and obese DR group(OBDR) involved 42 patients; ?T2DM complicated with coronary heart disease(DM-CHD)group: involved 75 patients,35 men ,40 women, the mean age was(61.88 ± 9.65)years old ,the mean BMI was (25.89 ± 4.19)kg/m2.Based on BMI the patients were divided into two groups: normal weight DMCHD (NWDMCHD) group involved 13 patients, and overweight and obese DMCHD (OBDMCHD) group involved 62 patients. (2)Normal control(NC) group:38 subjects, 17 men ,21 women, the mean age was (61.55±7.23)years old, the mean BMI was (23.78±2.27)kg/m2. Based on BMI the subjects were divided into normal weight control(NWNC) group involved 17 subjects; obese control(OBNC group) involved 21 subjects.2.Research content and methods: (l)History collection; (2) Anthropometric measurements: including height > weighu waist circumference> hip circumference and blood pressure. BMI and WHR were calculated from above measurements; (3) Body composition analysis: the whole body and local composition were measured by bioelectrical impedance assay (BIA) and dual energy x-ray absorptiometry (DEXA); (4) Samples collection: Over night fasting venous blood was collected .After centrifugation, we extracted the plasma or serum for measurement of:? Metabolic variables: glucose-, blood lipid% HbA^ insulin. HOMA-IR.% HOMA-IS and ISI were calculated respectively; ?Plasma fibrinolysis system variables: the activity of tissue- plasminogen activator (t-PA) and concentrations of plasminogen activator inhibitor l(PAI-l), plasmin-antiplasmin complex (PAP) and D-dimer (D-D) were determined. (5) 24 hours urine albumin excretion rate (UAER): urine sample of 24 hours were used for measurement of albumin content and UAER calculation. (6) Gene analysis.PAI-1 gene promotor 4G/5G polymorphism was analysed by PCR technology using the DNA extracted from peripheral blood white cells. We made sure that it was consist with the Hardy-Weinberg balance. 3.Statistical analysis: Statistical analysis was performed using SPSS statistical software (version 11.0). Quantitative data were subject to normal test. Natural logarithm were taken for FINS, HOMA-IR, HOMA-IS and ISI to achieve normal distribution. All the numerical data are expressed as mean±SD. According to the different aims and data types, we used t-test, analysis of variance (ANOVA), multiple factors correlation analysis, Logistic multiple stepwise regression analysis. The significance level was defined as a= 0.05.Results:1 .Relationship between plasma fibrinolysis and type 2 diabetes mellitus: (1) Comparison of the clinical data: BML waist circumference, WHR, blood pressure, body fat mass (general, trunk and waist),PAI-l and D-D levels in T2DM group were significant greater than those in NC group (P value<0.05, respectively);(2)Comparison of genotype:?Genotype and alleles frequencies: There was no statistical difference in 4G/4G genotype frequency between T2DM group and NC group (53.1% vs. 36.8%, P=0.21). 4G allele frequency in T2DM group was significantly higher than that in NC group (61.9% vs. 54.0%, P=0.042). ? Comparison between different genotype T2DM patients: BMI, WHR, body fatmass (general, trunk and waist), FINS, HOMA-IR, TG, HDL-C and PAI-1 levels were significantly increased in 4G homozygous patients compared with 5G homozygous patients (P value<0.05, respectively),but ISI in 4G homozygote patients was less than that in 5G homozygote patients;(3) Multiple factors correlation analysis: genotype in T2DM group was inversely correlated with waist circumference, WHR, blood pressure, TC, LDL-C and body fat mass (general,'trunk and waist), all the P value<0.05 respectively; BMI, WHR and Waist were positively correlated with FINS, HOMAIR, HOMAIS, PAI-1, TG and LDL-C (P value<0.05, respectively), and they were negatively correlated with ISI and HDL-C (all the P value<0.05, respectively). Body fat mass (general, trunk and waist)was positively correlated with blood pressure, FINS, HOMAIR, HOMAIS, HbAlc and UAER (P value <0.05 , respectively) and negatively correlated with ISI(P value<0.05, respectively). Fat mass in waist was positively correlated with PAI-1 level (P value < 0.05, respectively), it suggested that body fat accumulation in northern China Han nationality T2DM patients are mainly in the trunk especially in the waist. The conditions of insulin resistance, glucose and lipid metabolism disturbances and plasma fibrinolysis activity reduction are aggravated with the increase of the obesity degree; HOMAIR, HOMAIS and FINS levels were positively correlated with TG and PAI-1 concentrations (P<0.05), and negatively correlated with HDL-C level (P<0.05, respectively). ISI was negatively correlated with TG and PAI-1 concentrations (PO.05) and positively correlated with HDL-C level (PO.05), suggestting that insulin resistance is closely associated with lipid metabolism disturbance and increased PAI-1 level. TG level was not only positively correlated with BMI and waist circumference (P<0.05), but also positively correlated with HOMAIR and PAI-1 (PO.05, respectively), negatively correlated with ISI, t-PA activity, PAP and DD levels (PO.05, respectively); TC level was negatively correlated with PAP and DD levels (PO.05, respectively); LDL-C level was negatively correlated with t-PA activity; HDL-C level was positively correlated with t-PA activity andnegatively correlated with PAI-l(P<0.05, respectively),suggesting that lipid metabolism disturbances is.closed associated with fibrinolysis dysfunction.2. Relationship between plasma fibrinolysis and obese type 2 diabetes mellitus(OBDM):(1) Comparison of clinical data: body fat mass (general, trunk, limbs and waist), HbAic, FINS, HOMA-IR and TG levels in OBDM group were greater than those in NWDM, NWNC and OBNC group respectively. PAI-1 levels and UAER in OBDM group and NWDM group were greater than those in the 2 control groups. PAI-1 levels in OBDM group were greater than those in NWDM group (P<0.05~P<0.01, respectively), demonstrating that in DM patients especially OBDM patients there are not only obvious glucose and lipid metabolism disturbances, but also obvious fibrinolysis mechanism dysfunction;(2) Comparison of genotype: (DGenotype and alleles frequencies: there was no statistical difference between OBDM group and NWDM group. Allele 4G frequency in OBDM group (71.4%) was greater than that in NWDM group (53.6%, P<0.05). No statistical difference was observed when compared with NC group, ?comparison between different genotype T2DM patients: DP, BMI, Waist, WHR, body fat mass (general, trunk and waist), TG, LDL-C, FINS, HOMA-IR, PAI-1, DD and UAER in 4G homozygote OBDM patients were greater than those in 5G homozygote patients, ISI in the former was less than that in the later (P<0.05, respectively).(3) Correlation analysis: The genotypes of OBDM patients were negatively correlated with DP, trunk fat mass and PAI-1 level (r = -0.233, -0.236 and -0.369, respectively, P value<0.05); BMI and WHR were significantly positively correlated with TG, PAI-1 and PAP levels, body fat mass (general, trunk and waist) were all significantly positively correlated with PAI-1 level (P value<0.05, respectively). HOMA-IR was significantly positively correlated with PAI-1 level. HDL-C, LDL-C was significantly positively and negatively correlated with plasma t-PA activity respectively (both P value<0.05), suggesting that increase orinappropriate distribution of general and local fat mass in OBDM patients are closely associated with the degree of glucose and lipid metabolism disturbances and insulin resistance, plasma fibrinolysis mechanism dysfunction, PAI-1 genotype may have some impact on body fat mass and distribution, other body composition such as lean body mass, lipid metabolism is closely associated with plasma fibrinolysis mechanism. 3.RelatiotiShrp between plasma fibrinolysis and diabetic nephropathy(1) Comparison of clinical data: PAI-1 levels of NWDNl group, NWDN2+3 group, OBDN2+3 group were greater than that in NWDMNC group respectively (PO.05, respectively); PAP levels of NWDMNC group, OBDMNC group, NWDNl group and NWDN2+3 group were lower than that in OBDN2+3 group (P value<0.05, respectively). PAP levels in OBDMNC group, NWDNl group were lower than that in OBDN1 group (PO.05, respectively); DD levels in 0BDN1 group, OBDN2+3 group were higher than that in NWDNl group (PO.05, respectively).(2) Comparison of genotype:?Genotype and alleles frequencies: There was no statistical difference of genotype distribution between DN group and DMNC group. Allele 4G frequency in DN group was greater than that in DMNC group (Allele 4G freqnencies in DN group and OBDN group were greater than those in NC group and its subgroup respectively). ?Comparison between DN patients with different genotype : DP, BMI, Waist, Fat%, TG, HDL-C, UAER, HOMA-IR and PAI-1 levels in 4G homozygote DN patients were greater than those in 5G homozygote patients while ISI in the former was less than that in the later (PO.05, respectively).(3) Multiple factors correlation analysis: The genotype of DN patients was significantly negatively correlated with BMI, Waist, DP, HOMA-IS, fat% and PAI-1 levels respectively, and significantly positively correlated with lean body mass. Fat% was significantly positively correlated with FINS, HOMA-IR, PAI-1 and TC respectively, and significantly negatively correlated with ISI (PO.05,respectively); HOMA-IR was significantly correlated with TG and HDL-C respectively. There was no statistical correlation with plasma fibrinolysis variables; TG was significantly positively correlated with t-PA, PAI-1 and UAER. TC, LDL-C were significantly correlated with PAI-1 respectively. UAER was significantly positively correlated with BMI, DP and PAI-1 (PO.05, respectively). The result suggests that body fat distribution, insulin sensitivity, glucose and lipid metabolism, plasma fibrinolysis function and UAER in DN patients interact in the whole DN pathophysiological process and they are closely associated with PAI-1 genotype and other heredity factors. (4) Logistic stepwise regression analysis: Considering DN as the dependent variable and other variables as independent variables, we performed logistic multiple stepwise regression analysis .The results demonstrated that duration, systolic blood pressure, BMI, Fat%, HbAic, HOMA-IR, TC, TG, genotype, PAI-1 and DD all entered the regression equation. They are risk factors of DN. 4. Relationship between plasma fibrinolysis and diabetic retinopathy (DR)(1) Comparison of clinical data: FINS, HOMA-IR in OBDR group were greater than those in NWDR group and normal control group respectively. PAI-1 level in OBDR group was significantly greater than that in non DR group but was not statistically different from that in NWDR group; DD level in OBDR group was significantly greater than that in NWDMNC group and NWDR group.(2) Comparison of genotype:?Genotype and alleles frequencies: There was statistical difference of genotype and alleles frequencies when compared with DMNC group. 4G/4G genotype frequency in DR group (59.4%) and allele 4G frequency (71.0%) were greater than those in DMNC group respectively (33.3% and 51.5%, %2=6.l2, P=0.043 and %2=7A3, P=0.007, respectively). There was no statistical difference in genotype frequency between DR subgroup and NC subgroup. Alleles frequencies in OBDR group (75.0%) were greater than those in NWNC group (52.9%) and OBNC group (54.8%), all the P value<0.05.? Comparison between DR patients with different genotype: diastolic bloodpressure, Fat%, FINS, HOMA-IR, TG, UAER, PAI-1 and DD levels in 4G homozygote DR patients were less than those in 5G homozygote patients. Duration and HDL-.C in the former were less than those in the later (P value<0.05, respectively).(3) Multiple factors correlation analysis: Genotype of DR patients was significantly negative correlated with FINS, HOMA-IR, PAI-1, DD and LDL-C respectively but was significantly positively correlated with lean body mass (P value<0.05, respectively). BMI and Fat% were significantly positively correlated with PAI-1 level (P value<0.05, respectively). WHR and Waist were significantly negatively correlated with DD (P value<0.05, respectively). FINS and HOMA-IR were significantly correlated with PAP and DD. HOMA-IR was significantly negatively correlated with t-PA. TG and LDL-C were significantly positively correlated with PAI-1 respectively. TG and TC were significantly negatively correlated with PAP respectively (P value<0.05, respectively),.The result suggests that body fat distribution, insulin sensitivity, glucose and lipid metabolism, plasma fibrinolysis system function and kidney function ect. in DR especially OBDR patients interact in the DR whole pathophysiological process and closely associated with PAI-1 genotype and other heredity factors.(4) Logistic stepwise regression analysis: Considering DR as the dependent variable and other variables as independent variables, we performed logistic multiple stepwise regression analysis .The results demonstrated that duration, smoking index, HbAlc, HOMA-IR, TC, DD, PAI-1 and genotype all entered the regression equation. They are risk factors of DR.5. Relationship between plasma fibrinolysis and DM-CHD(1) Comparison of clinical data: Smoking index, SP, DP, BMI, WHR and Waist, body fat mass (general, trunk and waist), TG, LDL-C, FINS, HOMA-IR, HOMA-IS, PAI-1 and DD levels in OBDM-CHD group were greater than those in DMNC group but duration and ISI in the former were less than those in the later, suggestting that OBDM-CHD patients are characterized by shorter duration,more smoking, high blood pressure, obesity, inappropriate body fat distribution, high blood lipid, obvious insulin resistance, decreased plasma fibrinolysis activity, which are consist with metabolic syndrome features. (2) Comparison of genotype:?genotype and alleles frequencies: There were statistical difference of genotype and alleles frequencies when compared with DMNC group. 4G/4G genotype frequency in DM-CHD group (58.7%) were greater than those in DMNC group (34.7%, X2=6.83, P=0.033). Allele 4G frequency (70%) was greater than that in DMNC group (52%,^2 =8.19, P=0.004). There was no statistical difference in genotype frequency between when compared with NC group. There was statistical difference in alleles freqnencies. 4G Allele frequency in DM-CHD group (70%) was greater than that in NC group (54%, x2 = 5.68, P = 0.018). ?comparison between DM-CHD patients with different genotype: diastolic blood pressure, BMI, Fat%, waist fat mass, LDL-C, HOMA-IR and PAI-1 levels in 4G homozygote were greater than those in 5G homozygote patients, but ISI in the former was less than that in the later (P value<0.05, respectively).(3) Multiple factors correlation analysis: Genotype of DM-CHD patients was significantly negatively correlated with PAI-1 and fat mass in trunk and waist. BMI, general and waist fat mass were significantly positively correlated with PAI-1. WHR and Waist were significantly positively correlated with UAER.FINS and HOMA-IR were significantly negatively correlated with HDL-C. ISI was significantly positively and negatively correlated with HDL-C and LDL-C respectively. TG and TC were significantly negatively correlated with PAP and DD (P value < 0.05, respectively). HDL-C was significantly positive and negatively correlated with t-PA and PAI-1 (P value<0.05, respectively), suggestting that obesity especially abdominal obesity, glucose and lipid metabolism, insulin resistance, decreased plasma fibrinolysis system function in DM-CHD especially OBDM-CHD interact in the pathophysiological process and closely associated with PAI-1 genotype and other heredity factors.(4) Logistic stepwise regression analysis: Considering DM-CHD as the dependent variable and other variables as independent variables, we performed logistic multiple stepwise regression analysis .The results demonstrated that systolic blood pressure, BMI, lumbar fat content, HbAlc, ISI, TC and PAI-1 are risk factors of DM-CHD.Conclusion:1. There is apparent plasma fibrinolysis system function disturbance in T2DM especially OBDM patients, which is mainly presented by increased PAI-1 levels and is closely associated with obesity, inappropriate body fat distribution, glucose and lipid metabolism disturbance, insulin resistance and genotype and other heredity factors. 4G homozygote OBDM patients are characterized by high blood pressure, abdominal obesity, insulin resistance, glucose and lipid metabolism disturbance and decreased plasma fibrinolysis activity.2. PAI-1 genotype and alleles distribution features in T2DM patients of northern China Han nationality: There is no statistical difference in genotype frequency between T2DM patients and normal population, but 4G allele frequency in the former was greater than that in the later; similar condition existed between OBDM patients and NWDM patients;3. Comparison between DM patients with different vascular complications and DMNC patients: 4G allele frequency in DN, DR and DM-CHD patients was greater than those in the later. 4G/4G genotype frequency in DR and DM-CHD patients was greater than that in DMNC patients respectively.4. Body fat distribution, insulin sensitivity, glucose and lipid metabolism, plasma fibrinolysis system function and UAER in DN patients especially in OBDN patients interact in the whole DN pathophysiological process and they were closely associated with PAI-1 genotype and other heredity factors. Duration, systolic blood pressure, BMI, Fat%, HbAlc, HOMA-IR, TC, TG, genotype, PAI-1 and DD are risk factors of DN. |
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