Study On Serum Adiponectin Level And Its Gene Polymorphism In Different Glucose Tolerance Humans

Objective: With the increasing of obese subjects, the incidence strongly associated with it such as type 2 diabetes mellitus, cardiovascular, cerebrovascular disease, hyperlipidemia, hypertension and fatty liver is increasing too. Some studies showed that the incidence of type 2 diabetes mellitus in mid-obese subjects was two times and in severely obese subjects was 5~10 times higher than that in normal people. More than 80% type 2 diabetes mellitus company with overweight or obesity. Insulin resistance (IR) is the important pathophysiologic basis of the development of type 2 diabetes mellitus, and exists in the whole process of it. Insulin sensitivity in peripheral tissues often decrease in obese subjects which induce hyperinsulinemia and IR. Obesity, especially visceral adipose deposit takes part in the development of IR. Obesity is the risk factor in the development of type 2 diabetes mellitus. At present, it is unclear that how obesity causes IR. Some studies showed that the factors such as dyslipidemia,tumor necrosis factor-alpha (TNF-a),leptin contribute to the development of IR in obese subjects. Adiponectin is another adipose-secreted protein which has attracted a lot of attention recently. The serum level of adiponectin is abundant in normal subjects. Adiponectin has important metabolic and anti-inflammatory actions that suggest a protective role in diabetes development. Some studies showed that adiponectin levels decreased in subjects of obesity, insulin resistance and type 2 diabetes, and that the low levels of adiponectin were paralleled with the development of insulin resistance. It was said that adiponectin gene contributed to the susceptibility to type 2 diabetes. Several novel polymorphisms in the adiponectin gene have recently been identified. Of particular interest is a +45T/G polymorphism of the adiponectin gene, which has been found to regulate the serum level of adiponectin and is a functional mutation. Therefore, in this study serum adiponectin in different glucose tolerance humans was measured and adiponectin genotype was determined in order to elucidate the association of adiponectin and its +45T/G polymorphism with obesity, insulin resistance and type 2 diabetes, which would be helpful to deeply understand the pathophysiological mechanism of type 2 diabetes. Methods 1. Subjects: According to the standard of DM (WHO) in 1999, 104 type 2 diabetes mellitus patients (DM group) and 46 impaired glucose tolerance patients (IGT group) were chosen at the time of diagnosis, and 90 subjects with normal glucose tolerance from regular physical examination (NGT group) were chosen as the control group. Each group was divided into two subgroups based on BMI. All the subjects were excluded (1)Cardiomyopathy, valvular heart disease, cardiac function in Ⅱdegree or above; (2) Out of compensation period of renal dysfunction (Ccr ≥178mmol/l), hepatic dysfunction or the history of chronic hepatitis; (3) Pregnancy, lactation or taking the anticonceptive for a long period. The gender and age are matched in different groups, and there are no significant differences in statistics. 2. Physical measure: All subjects were measured blood pressure (SBP/DBP), height, body weight, waist and hip circumference,calculated body mass index (BMI), waist-hip ratio (WHR). After a over night fast(without calorie taken in at least 10 hours), 5ml blood samples were taken from the elbow vein. For human genomic DNA extraction, 2%Na-EDTA was added to 1ml whole blood, then frozen and stored at -40℃until use. Serum was separated from 4ml blood and conserved in the temperature of -40℃to be prepared for the test of fasting serum adiponectin level,fasting blood glucose (FBG), fasting insulin (FINS), blood lipid(including TG, TC, LDL-C, HDL-C), hepatic function and renal function; Oral glucose tolerance test (OGTT) were performed for 2 hour glucose (PBG). 3. Determination of the adiponectin polymorphism: Human genomic DNA was extracted from peripheral blood leukocytes of each individual. The required fragment was amplified by polymerase chain reaction (PCR) method. The primers were forward: 5 '-GCAGCTCCTAGAAGTAGACTCTGCTG-3', and reverse: 5'-GCAGGTCTGTGATGAAAGAGGCC-3'. Each ampliation was performed using 150ng of DNA in a volume of 25μl containing 12pmol of each oligonuleotide, 0.8 μl deoxyribonucleotide -5'-triphosphates (dNTP), 3.5μl 10×PCR Buffer, 2μl ingrain dyestuff and 1.25U of Taq polymerase. DNA templates were denatured at 95℃for 5 minutes, and then each PCR reaction was subjected to 32 cycles with a temperature cycle consisting of 95℃for 30 seconds, and 59℃for 30 seconds and 72℃for 30 seconds, and finally an extension at 72℃for 8 minutes. The PCR products were subjected to restriction enzyme analysis by digestion with 12 unit of the restriction endonuclease BsmⅠfor 5μl of PCR sample at 30℃for 5 hours in the buffer and the fragments separated by ployacrylamide gel electrophoresis. After electrophoresis, the gel was treated with ethidium bromide for 10 minutes and DNA fragments were scored according to the patterns of DNA bands. The resulting fragments were 372bp for the uncut T allele and 209bp and 163bp for the G allele. 4. The methods of laboratory: Blood glucose,hepatic function ,renal function and blood lipid were tested in auto-biochemical analyzer. Fasting serum insulin (FINS) was measured by radioimmunoassay. Fasting serum adiponectin concentration was examined by enzyme immunoassay, reagent boxes were obtained from Peking Wanfang Medicine company; Insulin resistance index (IRI) was calculated according to the HOMA model, IRI= (FINS×FBG)/22.5, because it is not normaldistribution, Ln was taken to analysis. The effect of adiponectin is discussed in the IR and development of type 2 diabetes mellitus according to the adiponectin level in different groups and the factors related to it; the relationship between adiponectin, IR and obesity is investigated. 5. Statistical analyses: All calculations were performed using the SPSS 10.0 version for Windows. Chi-square analysis was used to test whether adiponectin genotype distribution followed the Hardy-Weinberg equilibrium. All normally distributed variables were presented as means±SD( x ±s). One-way analysis of variance (ANOVA) was used to compare continuous variables among groups. A Chi-square analysis was performed to compare noncontinuous variables among groups. Multiple regression analysis was used to estimate the relationship between adiponectin and other clinical characteristics. Result 1. These was no significant difference in age and sex among NGT, IGT and DM groups. 2. The serum adiponectin in IGT patients is 5.29 ±1.13mg/L,which is lower than that in healthy controls(8.08±0.45mg/L)( P<0.01). And the patients with type 2 diabetes have more lower serum adiponectin level(4.15±0.76mg/L) than those with IGT( P<0.05). 3. The levels of BMI, WHR, FBG, TG in IGT group were significantly higher than those in controls, while the levels ofHDL-C were greatly decreased(P<0.05 or 0.01). Compared with IGT and NGT groups, the patients in type 2 diabetes had more higher BMI, WHR, SBP, FBG, IRI and TG and more lower HDL-C level (P<0.05 or 0.01). 4. Serum adiponectin was correlated negatively with WHR, IRI, SBP, FBG and TG and positively with HDL-C. There was no significant correlation between serum adiponectin and other clinical parameters. 5. The genotype GG distribution in IGT and DM groups were 8.7% and 11.6% respectively, which were significantly higher than those in NGT group. There was no significant difference in the distribution of G allele frequency between the 3 groups. Conclusion 1. Fasting serum adiponectin concentration in IGT and DM groups was lower than that in NGT group, which indicated that the adiponectin probably plays a role and is one of the risk factors in the development of type 2 DM. 2. IRI was markedly higher in obese subgroups, which showed that IR exists in obese subjects; adiponectin level was significantly lower in obese subgroups than in non-obese subgroups, which indicated that adiponectin maybe a binder linking obesity, IR and type 2 DM. 3. The level of serum adiponectin was closely related to +45T/G polymorphism. Subjects with G/G at SNP45 had lower serum adiponectin level.