Study On The Regulatory Mechanism Of VASPIN Expression And Its Functions

Background:Recently, increasing evidence have suggested that adipose tissue is not only an energy storage organ, but also a key endocrine organ which could secrete various adipokines such as leptin, resistin, adiponectin, visfatin, tumor necrosis factor, plasminogen activator inhibitor, and interleukin-6,etc. Epidemiological and animal studies have shown that visceral obesity is associated with increased prevalence of insulin resistance, type 2 diabetes and cardiovascular diseases. Adipokines secreted by visceral fat tissues play an important role in the occurrence and development of insulin resistance, type 2 diabetes and metabolism syndrome. Vaspin, a newly identified adipokine, is secreted by visceral adipose tissue with predominantly insulin-sensitizing effects,However, its physiological role is largely unknown. Although current studies have shown plasma vaspin level has direct effects on obesity, the result is still in debate. In the present study, we investigated 1) the relationship between serum vaspin level and macrovascular and retinopathy complications in diabetic patients,2) the regulatory mechanism of vaspin expression,3) the potential physiological function of vaspin. We first examined whether serum vaspin levels are associated with the presence of carotid plaque in early stage of type 2 diabetes. Next, we analysed the relationship between serum vaspin levels and diabetic retinopathy. Finally, we explored in vitro the effects of high glucose, insulin, dexamethasone, palmitate, TNF-a, pioglitazone, fenofibrate and lipoic acid on vaspin expression in 3T3-L1 cell and investigated the potential molecular mechanism involved. PartⅠSERUM VASPIN LEVELS IN TYPE 2 DIABETIC PATIENTS WITH CAROTID PLAQUEObjective:1) To explore whether serum vaspin levels are associated with the presence of carotid plaque in early stage of type 2 diabetes; 2) To explore the association between serum vaspin levels and FPG,2HPG, HbAlc, serum lipid and insulin resistance.Materials and Methods:Sixty-one type 2 diabetic patients within 1 years's diabetes duration were divided into two groups by the presence or absence of carotid plaque. Twenty-six age-matched apparently healthy controls were recruited as well. Serum vaspin was measured by enzyme-linked immunosorbent assay.Results:After controlling age and gender, fasting serum vaspin levels were significantly higher in the diabetic patients without carotid plaque than controls (0.65±0.24 vs.0.46±0.12 ng/ml, p<0.01). However, circulating vaspin level was much lower in the diabetic subjects with carotid plaque than those without (0.52±0.23 vs. 0.65±0.24 ng/ml, p<0.05).Partial correlations demonstrated that vaspin level was positively correlated with waist circumference (WC) (r=0.269, p=0.041), negatively correlated with HOMA-IR(r=-0.262, p=0.047) in all subjects after controlling age, gender and BMI. Logistic regression analysis showed that SBP and fasting serum vaspin levels were significantly associated with the presence of carotid plaque in type 2 diabetes.Conclusion:Serum vaspin levels were significantly elevated in type 2 diabetic patients while its level was negatively associated with the presence of carotid plaque in type 2 diabetic patients,indicating that vaspin might play a compensative role in the early course of diabetes while exert protective effects on atherosclerosis in early stage of diabetes. Part IIVASPIN SERUM CONCENTRATIONS IN TYPE 2 DIABETIC PATIENTS WITH DIABETIC RETINOPATHYObjective:1) To explore the relationship between serum vaspin level and type 2 diabetic retinopathy and its severity.2) To explore the association between vaspin level and age, duration, FPG,2HPG, HbAlc, serum lipid, urine albumin excretion rate (UAER) and other adipokinesMaterials and Methods:94 patients with type 2 diabetes were recruited through in-patients or clinic service from February to June 2009 at Qilu Hospital of Shandong University, Jinan, China. Based on ophthalmologic examination,94 patients with type 2 diabetes were divided into three subgroups:group 1 without diabetic retinopathy (NDR) (n=40), group 2 with simple diabetic retinopathy (SDR) (n=38) and group 3 with proliferative diabetic retinopathy (PDR) (n=16). Serum vaspin was determined by enzyme-linked immunosorbent assay.Results:In the diabetic patients, subjects with simple diabetic retinopathy had significantly lower vaspin levels than subjects without diabetic retinopathy (0.45+ 0.13 versus 0.53+0.22 ng/ml, p= 0.023).Moreover, subjects with proliferative diabetic retinopathy had much lower vaspin levels as compared with subjects with simple diabetic retinopathy. Age, disease duration, UAER and retinol binding protein-4 (RBP4) were negatively correlated with vaspin level in correlation analysis (r=-0.259, p= 0.012; r=-0.277, p= 0.007; r=-0.322, p=0.002; r=-0.234, p=0.023 respectively). Partial correlations demonstrated that UAER was still negatively correlated with vaspin levels (r=-0.281, p= 0.007) after controlling age and BMI, A stepwise linear multiple regression model revealed that age and UAER were significant determinants of serum vaspin levels. Conclusion:Tanken together, these results suggest that the reduction of vaspin level may be involved in the process of diabetic retinopathy and thereby provides a novel biomarker for evaluating its severity in diabetic patients. PartⅢSTUDY ON THE REGULATORY MECHANISM OF VASPIN EXPRESSION IN 3T3-L1 CELLObjective:To explore the effect of metabolic factors on vaspin expression in 3T3-L1 cell and the regulatory mechanism involved.Methods:1.3T3-L1 preadipocytes were cultured in DMEM medium supplementd with 10% FBS and differentiated into adipocytes. The total RNA was extracted in the preconfluent, confluent and 2-day,5-day,8-day,10-day confluent cells respectively. The expression of vaspin mRNA was determined by quantitative real-time polymerase chain reaction and vaspin level in cell supernatant was determined by ELISA.2. Mature 3T3-L1 adipocytes was treated with indicated concentrations of dexamethasone, insulin, glucose, palmitate, TNF-a, pioglitazone, fenofibrate and lipoic acid for 24 or 48 h.The cells were starved for 6 h in DMEM with 0.5% bovine serum albumin (BSA) before the study.3. TNF-a was added to the cell culture medium alone or with the indicated "concentrations of pioglitazone, fenofibrate or lipoic acid respectively for 48 h. During the treatment period, fresh TNF-a, pioglitazone, fenofibrate or lipoic acid was added every 24 h.Results:A marked increase in vaspin expression was observed during 3T3-L1 differentiation, with a 1.7-fold increase on 2-day confluent as compared with preconfluent. A further 3.8-fold increase was induced from day 0 to day 8 of differentiation (overall six fold). Overnight incubation with dexamethasone increased vaspin expression in adipocytes (100μM-1.6 fold). High glucose increased vaspin secretion by 6.6-fold at 25 mM, pioglitazone by 1.6-fold at 100μM, fenofibrate by 1.5-fold at 100μM, and lipoic acid by 1.6 fold at 500μM. While 10-100 nM insulin, 1mM palmitate and 10-20 ng/ml TNF-a decreased vaspin expression (.37-53%,48%, 15-40% respectively). Futhermore, pioglitazone and lipoic acid reversed the reduction of vaspin by TNF-α.Vaspin protein level in cell supernatant increased gradually during differentiation, rapidly in beginning 5 days after differentiation and slowly after 6 days. Dexamethasone and glucose promoted vaspin secretion in mature adipocytes while insulin and TNF-a inhibited vaspin level. Pioglitazone, fenofibrate and lipoic acid reversed the reduction of vaspin induced by TNF-a.Conclusion:Taken together, vaspin levels are associated with the extent of the differentiation and its level is regulated by multiple hormones and metabolic factors which could be associated with severity of metabolic disorders. The underlying mechanism of vaspin action remains to be elucidated futher.