Expression Of Adiponectin And Retinol Binding Protein 4 In Patient With Metabolic Syndrome And Mechanism Involved In Gender Difference

Background and Objective:Metabolic syndrome (MS) is a group of metabolic risk factors characterized by central obesity, insulin resistance, hypertension, dyslipidemia, and impaired glucose tolerance or type 2 diabetes mellitus. Insulin resistance is thought to be the main pathophysiologic basis of metabolic syndrome, in which underlying pathogenesis involved remains unclear. Recently, a body of reports demonstrates that central obesity and endocrine dysfunction of adipose tissue might pay an important role in the pathogenesis of metabolic syndrome, and related issues have become the focus of research in this field. Many kinds of adipocytokines secreted by adipocytes are implicated in homeostasis of the whole body, including regulation of many pathophysiologic processes, such as insulin sensitivity, blood pressure, endothelial function, fibrinolytic system, and anti-inflammatory. Adiponectin (ANP) is a kind of protein hormone secreted specifically by adipocyte, which has been reported can improve insulin resistance, against metabolic syndrome, anti-atherosclerosis, and anti-inflammatory. ANP is forward to be the predictor and target for diagnosis and treatment of the obesity related disturbance characterized by insulin resistance, such as metabolic syndrome, type 2 diabetes mellitus and related cardiovascular disease. Retinol binding protein 4 (RBP4) is a new adipocytokine identified recently, which is reported to be involved in the glucoregulation, and would induce insulin resistance. However, the role of RBP4 in the pathogenesis of MS and its regulation factors are still in suspense. The present study aims to identify the role of ANP and RBP4 in metabolic syndrome and the relationship with its components through measuring plasma level of ANP and RBP4. Previous study indicates that there has gender difference in plasma level of ANP and RBP4. The underlying mechanism is partly thought to be related to sex hormones, but is still unknown, thus needs further investigation. In order to explore the underlying mechanism and identify our hypothesis, the present study has evaluated plasma level and expression of ANP and RBP4 in dissected adipose tissue and cultured adipocytes. Project has been divided into 3 parts: (1) Detecting plasma level of APN, RBP4, sex hormones, and sex hormone binding globulin (SHBG) in the patient with different pathophysiologic state of metabolic syndrome, to explore the relationship between these indicators and clinical feature; (2) Examining the expression of APN, Adiponectin receptor 1 (Adipo R1) and RBP4 in intra-abdominal (omental) adipose tissue and subcutaneous tissue, to identify the character of expression for these indicators in central obesity; (3) Effect of sex hormones on differentiation and adipogenesis of 3T3-L1 preadipocyte, and the expression of APN, Adipo R1 and RBP4 in the course of preadipocyte differentiation, to explore the underlying mechanism in gender difference of plasma level of APN and RBP4, and the role of sex hormones.Material and Methods:The project is composed of clinical observation and experimental investigation. General information and plasma characters of patients were detected in the part of clinical observation. Expression of interested proteins was measured in intra-abdominal and subcutaneous adipose tissue, and in the course of 3T3-L1 preadipocyte differentiation.1. Patients were assigned into MS group, non-MS group, and healthy control group. Parameter of body fat (BMI, WC, and WHR) and blood pressure were measured, general blood characters, plasma level of glucose, lipid, inflammatory indicator, APN and RBP4 were detected, and cigarette smoking and alcohol intake were surveyed with subjective questionnaire, in order to identify plasma level of APN and RBP4 in diverse disorders and its regulatory factors.2. Patients were divided into (central) obesity group and non-obesity group. General blood characters, plasma level of APN, RBP4, sex hormones, and SHBG were detected for the investigation of molecular mechanism, especially the role of sex hormones in the gender difference of APN and RBP4 level.3. The presence of APN, Adipo R1 and RBP4 in intra-abdominal and subcutaneous adipose tissue was detected by immunofluorescence. Expression of APN, Adipo R1 and RBP4 was measured with western blotting, in order to clarify the relationship among plasma level, protein expression in adipose tissue, and the plasma level of sex hormones. 4. 3T3-L1 preadipocytes were cultured with estradiol of 10-10M (low level), 10-8M (physiological level), and 10-6M (high level), and testosterone of 3×10-10M (low level), 3×10-8M (physiological level), and 3×10-6M (high level), then the adipogenesis was evaluated with oil red staining.5. 3T3-L1 preadipocytes in state of differentiation inducing, non-differentiation inducing, and maturation were cultured with estradiol of 10-10M (low level), 10-8M (physiological level), and 10-6M (high level), and testosterone of 3×10-10M (low level), 3×10-8M (physiological level), and 3×10-6M (high level), or without sex hormones. Expression of APN, Adipo R1 and RBP4 was measured with western blotting, in order to investigate does-dependent effect on APN, Adipo R1 and RBP4 expression in 3T3-L1 preadipocyte and mature adipocyte. In addition, molecular mechanism for the gender difference of APN and RBP4 will be identified in this sectionResults:1. Results of clinical observation: (1) Plasma level of RBP4 in MS group is significant higher than control group, while APN in MS group is decreased significantly. (2) Plasma level of RBP4 in male is significant higher, and APN is lower than that in female. (3) Plasma level of RBP4 is relation to gender and BMI independently, while plasma level of APN is relation to WC and level of RBP4.2. Results of clinical observation: (1) Plasma level of RBP4 in obesity group (BMI≥25) is significant higher than control group, while APN in obesity group (BMI≥25) group is decreased significantly. (2) Subgroup analysis for male and female, the results (1) also establish. (3) Plasma level of SHBG in obesity group (BMI≥25) decreases significantly. (4) In male, plasma level of RBP4 is independently relation to BMI, WHR, and level of APN; plasma level of APN is independently relation to WC, HDL-C, age and level of RBP4. In female, plasma level of RBP4 is independently relation to BMI and level of LH; plasma level of APN is independently relation to WC and level of hsCRP.3. The presence of APN, Adipo R1 and RBP4 in intra-abdominal and subcutaneous adipose tissue was determined by immunofluorescence.4. Results of western blotting: (1) Expression of APN is decreasing significantly in intra-abdominal adipose tissue of MS patients. Expression of APN in intra-abdominal adipose tissue of male MS subjects is significant lower than female, while in subcutaneous adipose tissue of female MS subjects is significant higher than male. In non-MS group, expression of APN in both kind of adipose tissue is similar. (2) Expression of Adipo R1 in intra-abdominal adipose tissue of female MS subjects is significant lower than control group, but is higher than that in male. Expression of Adipo R1 in intra-abdominal and subcutaneous adipose tissue of female MS subjects is significant higher than that in male. Expression of Adipo R1 in subcutaneous adipose tissue of male MS subjects is significant higher than that in intra-abdominal adipose tissue. (3) Expression of RBP4 in intra-abdominal adipose tissue from male MS patients is decreasing significantly than control group or female patients. Expression of RBP4 in subcutaneous adipose tissue of male subjects is significant lower than female subjects. Expression of RBP4 in intra-abdominal adipose tissue of female subjects is increasing significantly. However, this difference was not been observed in male subjects.5. Comparison of lipogenesis of 3T3-L1 preadipocyte with oil red staining: (1) in non-differentiation inducing group, lipogenesis after intervention with estradiol only in the concentration of 10-8 M for 72 hours is increasing significantly than control group. (2) In differentiation inducing group, lipogenesis after intervention with sex hormone is similar with control group. (3) In the mature adipocyte, lipogenesis after intervention with estradiol in the concentration of 10-10 M or 10-8 M, and testosterone in the concentration of 3×10-8 M is increasing significantly than control group, while that with testosterone in the concentration of 3×10-10 M is decreasing significantly.6. Culture of 3T3-L1 preadipocyte: (1) in non-differentiation inducing group, expression of APN after intervention with estradiol only in the concentration of 10-10 M, and in any concentration of testosterone is increasing significantly than control group;Expression of Adipo R1 after intervention with testosterone in the concentration of 3×10-10 M and 3×10-6 M is increasing significantly than control group; Expression of RBP4 after intervention with testosterone in the concentration of 3×10-10 M is significant lower than control group. (2) In differentiation inducing group, expression of APN after intervention with estradiol testosterone in the concentration of 3×10-8 M is increasing significantly than control group; Expression of Adipo R1 after intervention with testosterone in the concentration of 3×10-8 M and 3×10-6 M is increasing significantly, while that with 10-6 M estradiol is significant lower; Expression of RBP4 after intervention with testosterone in the concentration of 3×10-8 M and 3×10-6 M is increasing significantly, while that with 10-6 M estradiol is significant lower. (3) In the mature adipocyte, expression of APN after intervention with estradiol in the concentration of 10-8 M and testosterone in the concentration of 3×10-8 M is increasing significantly; Expression of Adipo R1 after intervention with any level of testosterone is significant higher; Expression of RBP4 after intervention with estradiol in the concentration of 10-10 M and10-8 M, or any level of testosterone is significant higher than control group.Conclusion:1. Plasma level of RBP4 in MS patient is significant increasing, while APN is significnat lower than control group. Plasma level of RBP4 and APN is closed to components of MS, moreover plasma level of RBP4 and APN is related independently. In additon, RBP4 and APN would become the indicators of obesity and obesity related disorders.2. Disorders of sex hormone level in MS patients are change to reverse trands with gender difference, and level of SHBG is significant decreasing. Plasma level of RBP4 and APN changes significantly to the gender difference, and is closely related to the level of sex hormones and SHBG.3. Expression of APN, Adipo R1 and RBP4 is present in human intra-abdominal and subcutaneous adipose tissue. (1) Expression characters of APN in intra-abdominal adipose tissue is similar to plasma level of APN, which indicates that lower level of APN in patients with central obesity or MS is attributed to the significant lower secration of APN from intra-abdominal adipose tissue. (2) Expression characters of Adipo R1 in intra-abdominal adipose tissue is similar to plasma level of APN, which indicates that level of APN can enhance the biological effect of APN through positive regulation on its expression level. (3) Expression characters of RBP4 in intra-abdominal adipose tissue is different to plasma level, which indicates that increasing plasma level in central obesity, MS patients, and male patients is not from intra-abdominal adipose tissue, but related to the metabolic and functional state of metabolic organs.4. Lipogenesis of preadipocyte is regulated by estradiol and testosterone. Physiologic or lower level of estradiol and physiologic level of testosterone can induce, but lower level of testosterone will suppress lypogenesis in mature adipocyte. 5. Expression of APN, Adipo R1, and RBP4 in preadipocyte and adipocyte is regulated by sex hormones. Plasma level of SHBG is decreasing in patients with MS, and testosterone level in female MS patients is higher than non-MS group. In mature adipocyte, expression of APN is induced by physiologic level of estradiol and testosterone. Expression of Adipo R1 is induced by testosterone, especially in physiologic level. Expression of RBP4 in patients with central obesity and MS is not anly regulated by sex hormones, but also by other factors, such as metabolic state. Higher RBP4 and lower ANP level maybe a protective mechanism in MS.