| Objective:Metabolic syndrome (MS) is a cluster of metabolic and clinical disturbances featuring of central obesity and consists of type 2 diabetes mellitus, hypertension, hyperglycemia, dyslipidemia and so on. The pathogenesis of metabolic syndrome is extremely complicated and has been considered to be due to the interaction between the multiple genetic and environmental factors. Furthermore, insulin resistance is central and pathophysiology base of metabolic syndrome. Increasing evidence indicates that obesity leaded to the abnormal expression of adipose-derived signaling molecules of visfatin and chemerin. Visfatin not only increased adipogenesis, associated with obesity induces inflammation in adipose tissue or immune response, but decreased blood glucose by binding insulin receptor as well. Chemerin is a new adipokine that characterized the chemokines. It can activated macrophage, increased phosphorylation of tyrosine of insulin receptor substrate-1 and altered insulin sensitivity. In this case we hypothesize that visfatin and chemerin play important rules in the development of metabolic syndrome. Based on very high incidence of obesity and type 2 diabetes mellitus in Uighur population, we investigated the visfatin and chemerin mRNA expressions in abdominal subcutaneous and omental adipose tissues and the serum concentration of Uighur metabolic syndrome subjects, studied the visfatin and chemerin mRNA and protein expressions in different adipose tissues of metabolic syndrome rats, and measured the visfatin and chemerin mRNA and protein expressions in 3T3-L1 pre-adipocytes at different differentiation phases and visfatin, MCP-1 expression level in different times after adding different doses of recombinant mouse chemerin by using real time RT-PCR, Western blot, ELISA, and immunohisto-chemistry. According to discussions the role of visfatin and chemerin in glucose and lipid metabolism and the relation of visfatin and chemerin expression to the adipogenose, we provided experimental evidence of visfatin and chemerin rules in the development mechanism of metabolic syndrome.Method:1. The measurement of clinical and biochemical parameters in Uighur population: according to the diagnostic criteria for MS recommended by 2005 IDF, research subjects were enrolled.81 selective subjects were divided into non-MS control group (42; male-18, female-24) and MS group (39; male-16, female-23). Height, body weight, waist circumference (WC), hip circumference (HC) of all research subjects were measured and body mass index (BMI) and waist to hip ratio (WHR) were calculated. After fasting 8 hours, systolic blood pressure (SBP), diastolic blood pressure (DBP) were measured and 5ml blood samples were obtained to determine fasting plasma glucose, fasting insulin, triglyceride, total cholesterol, high density lipoprotein cholesterol (HDL-C) and low density lipoprotein (LDL).2. Measurement of mRNA expression level for visfatin and chemerin in abdominal subcutaneous and omental adipose tissues from human:extract total RNA by using subcutaneous and omental adipose tissues from surgery patients and then obtain cDNA after reverse transcription. Visfatin and chemerin mRNA expression level were determined by semi-quantitative RT-PCR and quantitative real time RT-PCR respectively.3.90 health, male SD rats aged 4-6 weeks and weight 100-120 gram were randomly divided into the normal control group (NC), MS model with normal-salt group (NS), and MS model with high-salt group (HS), and 30 animals in each group. The rats were fed with routine diet, high glucose-high fat diet, and high glucose-high fat-high salt diet in NC, NS and HS group respectively. After 10 weeks feeding, the rats in two model groups which waistline achieves the standard were injected streptozotocin (STZ). The weight, body length, waist circumference, blood pressure of rats were measured, and blood of rats were collected after 8 hours fasting to measure fasting blood glucose (FBG) and blood lipid including triglyeride, HDL-C and LDL every 2-4weeks. Semi-quantita-tive reverse transcription polymerase chain reaction (RT-PCR) was applied to detect visfatin and chemerin mRNA expression level in different adipose tissue.4. Plasma visfatin and chemerin of human and rats were measured by enzyme linked immunosorbent assay (ELISA). 5.3T3-L1 pre-adipocytes were cultured in vitro and differentiation were induced 2 days after the cultures reached 95% confluence by incubating the cells with 3-isobutyl-1-methylxanthine, insulin and dexamethasone. Oil red O dye was used to observe the process of differentiation. The adipocytes were collected on the 0th,2th,4th, 6th,8th,10th,12thdays.The mRNA expression levels of visfatin and chemerin in adipocytes was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) respectively and theirs protein expression levels was detected by Western blot during differentiation. After 3T3-L1 pre-adipocytes was induced to mature adipocytes, the different doses of the recombinant mouse chemerin(0,25ng/ml、50ng/ml、100ng/ml、200ng/ml) were added to cultured cells. And thent the adipocytes were collected after 12h,24h and 48h, and the mRNA expression levels of visfatin and monocyte chemotactin protein(MCP-1) were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) respectively.Result:1.Research data in Uighur population:1) Comparison of clinical and biochemical parameters in MS and non MS groups:MS subjects showed the higher levels of BMI, WC, HC, WHR, TG, FPG, HOMA-I and SBP (P<0.01～<0.05) and lower HDL-C (P=0.016) compared with non-MS controls. However, there were no significant difference of age, LDL-C, DBP and FINS.2) mRNA expression level of visfatin and chemerin in abdominal subcutaneous and omental adipose tissues:①there were no significant differences of visfatin mRNA expression between omental and abdominal subcutaneous adipose tissues between MS group and non-MS control and the expression level in abdominal subcutaneous is higher than in omental adipose tissues in two groups, however the difference is no significant.②There were no significantly differences of chemerin mRNA expression between omental and abdominal subcutaneous adipose tissues between MS group and non-MS control. There were also no significantly differences between omental and abdominal subcutaneous adipose tissues in two groups dividing by gender respectively.3) Both of the plasma level of visfatin and chemerin were significantly higher in MS subjects than in non-MS controls (P<0.01-P<0.05). The plasma level of chemerin was also significantly increased in MS female group dividing by gender (P=0.001).4) The correlation analysis:①The mRNA expression of visfatin in omental adipose tissue was positively correlated with HOMA-IR, and the plasma visfatin level was positively correlated with TC, TG and LDL-C.②The mRNA expression of chemerin in abdominal subcutaneous adipose tissues was positively correlated with that in omental adipose tissues and in plasma. The level of plasma chemerin was positively correlated with age, TG, WHR and HOMA-IR. After further multiple linear regression analysis by using plasma chemerin for dependent variable, age, TG and WHR entered into regression equation.2.Construction of MS rat model:1) The weight and waist circumference of 2 MS model groups were significant increased compare with control group at the end of 4th,8th, 14th week (P<0.05).2) The fasting blood glucose of 2 MS model groups were signifi-cant increased compare with control group at the end of 14th week (P<0.05), and the increase of FBG in HS group has statistical significance compare with that in NS group (P<0.05). 3) The increase of plasma triglyeride (TG), LDL-C and decrease of HDL-C in 2 MS model groups had statistical significance compare with that in control group at the end of 14th week (P<0.01). The concentration of TG and LDL-C of HS rats was significantly increased, and HDL-C was decreased compare with that of NS rats (P<0.05).4) Both systolic blood pressure (SBP) and diastolic blood pressure (DBP) were increased compare with that of control and NS rats, but the differences among three groups had not statistical significance at the end of 14th week (P>0.05).3.The visfatin and chemerin expression level of MS rats:1) visfatin and chemerin mRNA expression level in different adipose tissue:the mRNA expression level of visfatin in adipose tissue of omental, around kidney, and parastata, were decreased in MS rats compare with that in normal control rats, however the differences were no statistically significant, but the difference was significantly higher in adipose tissue of shoulder.The mRNA expression level of chemerin in adipose tissue around kidney, parastata, and shoulder were significantly higher in MS rats than that in normal control rats (PO.05). There were no significantly differences of visfatin and chemerin mRNA expression in different position adipose tissues between MS and control rats.2) The plasma level of visfatin and chemerin:the concentration of visfatin in MS rat was decreased compare with normal control and the expression level of chemerin was increased in MS rat, however the difference was no statistically significant.4.Ectopic lipidosis:there were formation of adipose vacuolization and a lot of lipid droplets in hepatocyte of MS rats. The diffused adipose degeneration was found in part of hepatic tissue. There were deposition of lipids in the stroma of somatic muscles and partial muscle fiber squirm of MS rats was found.5.During the differentiation phase of 3T3-L1 pre-adipocyte, the expression of visfatin mRNA is increased, and the mRNA expression of visfatin gene was kept higher level and remained unchanged in the telophase. Expression level of visfatin mRNA from the 6th day to 10th day is higher than 0 day (P<0.05), the expression level of visfatin is higher from the 10th to 12th day than from the second day to 6th day (P<0.01), the 12th day expression of visfatin is higher than the from 6th day to 8th day. The expression level of chemerin mRNA is increased also with the differentiation phase of 3T3-L1 pre-adipocyte, and the mRNA expression of chemerin was kept higher level and remained unchanged on the 6th day. During differentiation phase of 3T3-L1 pre-adipocyte, the expression level of chemerin mRNA from second day to 12th day is higher than 0 day (P<0.01), and starting from day 2, every going through 6 days, chemerin mRNA expression level increased significantly than foregoing phase (P<0.01). At same time, the expression of visfatin and chemerin protein were increased while the pre-adipocyte goes to mature.6. After 24 hours by adding recombinant mouse chemerin to the mature 3T3-L1cells, the visfatin mRNA expression level in the group of l00ng/ml doses was increased significantly than control group(P<0.05); and the MCP-1 mRNA expression levels were significantly higher in the groups of 50ng/ml、100ng/ml、200ng/ml doses respectively than that in normal control group(P<0.05). After 48 hours of the effect of recombinant mouse chemerin, the expression levels of visfatin and MCP-1 were significantly increased in the groups of 50ng/ml、100ng/ml、200ng/ml doses respectively than that in normal control group(.P<0.05).Conclusion:1.Metabolic syndrome patients of Uighur characterized by central obesity, impaired glucose tolerant and dyslipidemia. Both of the plasma level of visfatin and chemerin were significantly increased in MS Uighur subjects. The mRNA expression levels were increased both in omental and abdominal subcutaneous adipose tissues, however, there were no significantly differences between omental and abdominal subcutaneous adipose tissues in two groups dividing by gender respectively. The result of correlation analysis suggests that visfatin may regulate the formation of lipochondria in lipocyte and make function in lipid metabolism. The results of significantly increased plasma chemerin and its association with MS key marks seem to be partly responsible for the central obesity, high triglyceride level and IR in Uygur population.2.The mRNA expression high level of chemerin in adipose tissue around kidney, and parastata in MS rats suggest the function of chemerin in central obesity, and high glucose-high fat diet maybe one reason of the increased chemerin gene expression. The mRNA expression level of visfatin in adipose tissue was no significantly different in SD rats.The adipose degeneration was found in part of hepatic tissue of MS rats. There were deposition of lipids in the stroma of somatic muscles and ectopic lipidosis was found in MS rats.3.The mRNA and protein expression level of visfatin and chemerin were increased during the differentiation phase of 3T3-L1 pre-adipocyte, and its association with visfatin and chemerin seem to be partly responsible for the lipid accumulation in adipocytes. And the mRNA expression levels of visfatin and MCP-1 were up-regulated by the recombinant mouse chemerin in 3T3-L1 adipocytes. These two adipose cytokines may have the important role in formation of white adipose tissue of obesity. |
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