Mechanistic Study Of Adiponectin And Vaspin In Insulin Resistance And Clinical Expression Of The Metabolic Syndrome In Type 2 Diabetes Patients

Objective:Insulin resistance is a common pathophysiological condition of type 2 diabetes, metabolic syndrome and atherosclerosis. There are numerous mechanisms by which obesity can adversely affect the vasculature changes in diabetes and metabolic syndrome. White adipose tissue, the main energy store of the body, is also a source of factors that modulate the immune/inflammatory response and promote atherosclerosis, vascular dysfunction, insulin resistance, metabolic syndrome and type 2 diabetes. Therefore, investigations focused on the identification of adipose tissue derived molecules will provide further understanding of mechanisms for insulin resistance.Vaspin, a serine protease inhibitor derived from visceral adipose tissue, has been identified as an adipokine with insulin-sensitizing effects in a rat model of type 2 diabetes. The study by Hida et al. showed that the administration of recombinant human vaspin significantly improved insulin sensitivity and glucose tolerance, and reversed the expression of genes that may promote insulin resistance in diet-induced obese mice. However, the regulation of vaspin serum concentrations in human insulin resistance, and relationship between vaspin and insulin resistance related disorders is unclear. The present study focused on the role of vaspin in obesity-associated disorders.NF-κB is a major transcription factor in inflammatory responses, regulating a plethora of genes, activating endothelial cells, playing a vital role in the initiation, progression of type 2 diabetes, vessel complication and insulin resistance related disorders. Recent research has suggested that over-activation of NF-κB may be a mechanism of insulin resistance. It is important to investigate the relationship between NF-κB and diseases associated with insulin resistance.We postulated that adipokines, such as adiponectin and vaspin, can be included in the criteria used for the identification of subjects with metabolic syndrome. Therefore, our aim was to evaluate the determinants and associations of vaspin and adiponectin in relation with the number of clinical and metabolic abnormalities constituting the metabolic syndrome as defined by the International Diabetes Federal (IDF) diagnostic criteria. We also evaluated the performance characteristics of adiponectin, vaspin and the adiponectin/vaspin ratio (A/V) as markers for the identification of patients with the metabolic syndrome. For further exploring the risk of metabolic syndrome, the study was performed to evaluate the associations of adiponectin, vaspin, and their ratio (adiponectin/vaspin) and obesity, insulin resistance, and the metabolic syndrome in patients with type 2 diabetes.Adipocytes within adipose tissue directly augment systemic inflammation. Increased systemic inflammation is associated with higher prevalence of obesity, insulin resistance, type 2 diabetes, metabolic syndrome, and the risk of cardiovascular disease. With the aforementioned in mind, we sought to investigate whether Vaspin activates NF-κB inducing the intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and monocyte chemoattractant protein (MCP-1) in the vascular endothelium. Vaspin could affect the equilibration inflammatory state, which in turn may have a significant effect in the insulin resistance.Research Design and Methods:260 patients including 126 male and 134 female with newly diagnosed type 2 diabetes were recruited in this study. Patients were classified based on the degree of adiposity, insulin resistance (IR) (homeostasis model assessment of insulin resistance (HOMA-IR)) and the number of IDF criteria of the metabolic syndrome. The relationship between adiponectin, vaspin, A/V and the metabolic syndrome,degree of adiposity, IR, and the correlation with metabolic variables were investigated.Spearman's correlation coefficients (r) and multiple regression analysis were used to describe the association between serum vaspin, adiponectin or A/V and other continuous variables of interest. Multiple regression analysis was used to analyze the relationship between vaspin, adiponectin, A/V and age, gender, BMI (Body mass index), HOMA-R, WC (Waist circle), TG (Triglyceride), HDL (High-density lipoprotein-cholesterol). Logistic regression analysis was used to ascertain the association of vaspin and adiponectin with the metabolic syndrome as dependent variable. Potential confounding variables such as BMI, WC, WHR (Waist-to-hip ratio), TC, TG, HDL, LDL, FPG (Fasting plasma glucose), FIRI (Fasting plasma insulin level), HbA1c (GlycohemoglobinA1c), HOMA-IR and A/V ratio were included. To assess the diagnostic accuracy of the adiponectin, vaspin, A/V ratio in predicting the metabolic syndrome, receiver operating characteristic (ROC) curve was also constructed.To study NF-κB activation, HUVEC cells were transient transfected with a cis-reporter plasmid (Promega, E849A, #25236701). The pNF-κB-Luc plasmid was transfected together withβ-gal plasmid (Aldevron, #5002) into HUVEC cells using a QIAGEN transfection reagent, GFP plasmid was transfected simultaneously. Intra-reference transfection efficiency was measured byβ-gal activity, which may revise NF-κB chemistry luminous intensity in corresponding cell disruption. With the aforementioned in mind, a set of high efficiency and stable transfection method was established in HUVEC cells. The relative light unit (RLU) of NF-κB was measured using Promega GLOMAX Multi Detection System. Since most Hygromycin-based vectors used in gene transfer experiments harbor the E. coli Hygromycin B phosphotransferase (Hph) gene in mammals, cells were cultured in the presence of Hygromycin B (Catalog # ant-hg-5,InvovoGen) at a concentration of 200μg/ml and the medium was replaced every two to three days. Approximately three weeks after transfection, Hygromycin B-resistant clones were isolated using a cloning cylinder and analyzed individually for the expression of luciferase activity. We obtained the EA.hy926 cell clones with stable expression of pNF-кB-1uc. Multiple clones were also selected for analysis of NF-κB activation. Luciferase activity was measured using a luciferase assay kit (Luciferase Assay system, Promega, # E1501).Tumor necrosis factor (TNF-α), a strong inducer of NF-κB activity, activates cis-reporter plasmid containing luciferase reporter gene linked to five repeats of NF-κB binding sites. Addition of Bay11-7082, an irreversible inhibitor, results in the inactivation of NF-κB and NF-κB-dependent transcriptional expression.EA.hy926 cells or HUVEC cells transfected with pNF-κB-Luc were treated with various concentrations (0-3200 ng/ml) of Vaspin, After two hours, cells were harvested and lysed, and luciferase activity was measured based on NF-κB-dependent transcriptional activity as mentioned above. The effects of Vaspin on ICAM-1,VCAM-1 and MCP-1 activity was performed in EA.hy926 cells or HUVEC cells. In addition, cells pre-incubated with Vaspin were then treated with TNF-α(10 ng/ml), the effect of Vaspin on TNF-αinduced NF-κB activation was then evaluated using the same method. The expression of ICAM-1, VCAM-1 and MCP-1 were investigated using quantitative real time PCR (mRNA level), western blot analysis (protein level) following treatment with Vaspin and also the NF-κB inhibitor, Bay 11-7082.All data in the present study are expressed as mean±SD. Differences between two groups were assessed using the t-test. Comparisons among groups were made by ANOVA and LSD. A p<0.05 was considered statistically significant. All statistical analysis was performed using SPSS 16 and Sigma plot. Serum-starved HUVECs transfected with pNF-κB-Luciferase were treated with or without vaspin. Each experiment was carried out in triplicates. Results:Compared to healthy adult, the patients with type 2 diabetes had lower adiponectin (P<0.05) and A/V (P<0.05), but higher Vaspin level (P<0.05). Serum Vaspin was elevated in females (P<0.05). In general, patients who were metabolic syndrome positive had more atherogenic lipid profile, exhibited higher insulin resistant, lower adiponectin and A/V, and had higher Vaspin level than patients who were negative of metabolic syndrome.Adiponectin and A/V ratio showed stepwise decrease with increasing number of the criteria for diagnosis of the metabolic syndrome, while Vaspin showed the opposite trend. Vaspin was the highest while five constituents of the metabolic syndrome were present. Receiver operating characteristic analysis showed that vaspin and A/V ratio had significantly larger area under the curve (AUC) compared with adiponectin, TG and HDL for the detection of the metabolic syndrome, suggesting the importance of Vaspin and A/V in the diagnosis of this syndrome. Vaspin, A/V and adiponectin were shown to be protector factors of the metabolic syndrome by multiple logistic regression analysis. The odds ratios (OR) are 0.469(95%CI,0.252-0.873;P=0.017), 0.368(95%CI,0.171-0.791;P=0.010), and 0.279(95%CI,0.097-0.804;P=0018)respectively.Vaspin levels were significantly correlated with BMI, WC, WHR, TG, HDL, FIRI, and HOMA-IR (P<0.01). Adiponectin levels were significantly correlated with other parameters in an opposite manner except for SBP, DBP, and FBG (P<0.01) (P<0.05). On the other hand, A/V ratio was significantly correlated with these parameters (P<0.01) (P<0.05). Multiple regression analysis was used to analyze the relationship between vaspin, adiponectin, A/V and gender, BMI, HOMA-R, WC, TG, HDL. Overweight obese patients had significantly lower adiponectin, A/V ratio levels and higher vaspin comparing with non-obese patients. Insulin-sensitive patients had significantly higher adiponectin, A/V ratio levels and lower vaspin than those with IR when adjusted by BMI, WC and other variable.The association between A/V ratio or HOMA-R and objective variables were investigated by multiple regression analysis. Significant correlations between these parameters and A/V ratio were observed (P<0.05). Furthermore, multiple linear regression analysis with A/V ratio or HOMA-R as dependent variables and gender, BMI, WC, TG and HDL as independent variables revealed that the A/V ratio (R2=0.692) was higher than HOMA-R (R2=0.433). This model suggested that A/V ratio might be a more accurate surrogate index for determining insulin resistance in patients with type 2 diabetes.Vaspin (0-3200 ng/ml) treatment for 2 hours induced a significant dose dependent increase in NF-κB mediated transcriptional activity in pNF-κB-Luc transfected EA.hy926 cells (P<0.05). Similar results were obtained in transiently transfected HUVEC (P<0.05). When cells were pretreated with Vaspin (0-3200 ng/ml) for varies time points and then subjected to TNF-α(10ng/ml) treatment for 2 hours. A significant inhibition of TNF-αinduced NF-κB transcriptional activity in human ECs was observed (P<0.05), a greater inhibition was observed in cells with a longer period of incubation with Vaspin (24 hours).Vaspin significantly increased ICAM-1, VCAM-1 and MCP-1 mRNA expression and protein levels in HUVEC cells. Pretreatment of HUVEC cells with Bay 11-7082 (10μM), a NF-κB inhibitor, for 1 hour, the mRNA levels of ICAM-1, VCAM-1 and MCP-1 induced by Vaspin (3200 ng/ml, 4 hour) was significantly reduced (P<0.05). Similarly, in Vaspin/TNF-αtreated human ECs, ICAM-1 and VCAM-1 and MCP-1 protein levels were also significantly decreased by pre-incubation with Bay 11-7082 (10μM).Conclusions:The present study investigated the relationship between adipokines and insulin resistance by examining the Adiponectin, Vaspin, insulin resistance and clinical manifestations of the metabolic syndrome in patients with Type 2 diabetes, and the effects of Vaspin on NF-κB activation and its downstream gene expression in Human vascular endothelial cells. The results revealed that adiponectin, vaspin, and A/V ratio were associated with obesity, insulin resistance, and the clinical manifestations of the metabolic syndrome. Vaspin participated in the process of insulin resistance. Further investigation using human ECs endothelial cells transient or stable transfected with pNF-κB-Luc plasmid revealed that Vaspin increased NF-κB transcriptional activity, significantly enhanced ICAM-1, VCAM-1 and MCP-1 mRNA expression and protein levels. We reaffirmed the emerging role of adipokines as mediators of inflammatory responses. Vaspin activated NF-κB, which may play an important role in developing insulin resistance. At the same time we observed that Vaspin induced NF-κB activation that led to the suppression of cytokine-induced NF-κB activation in Human vascular endothelial cells. Therefore, the complexity of vaspin offers the advantage of further exploring insulin resistance, and in consequence might benefit the treatment specifically targeting insulin resistance.