Depot-specific Expression Of Caveolin-1in Human Subcutaneous And Visceral Adipose Tissue And Effects Of Insulin And Exenatide Intervention In Vitro

Introduction:With the social and economic development, obesity has become a serious threatto people's health and a major public health problem. Obesity-related insulinresistance is a significant risk factor for many serious diseases, such as type2diabetes,dyslipidemia and other cardiovascular diseases. Obesity not only means increased fataccumulation, but also means fat distribution abnormalities. Excessive accumulationof body fat, especially in the visceral fat depot, is a major risk factor to develop avariety of metabolic diseases such as type2diabetes. A growing set of studies haveshown the importance of adipose tissue distribution and type in the development ofobesity-related complications. Visceral adipose tissue (VAT) was not onlymorphologically but also functionally different from subcutaneous adipose tissue(SAT). Meanwhile, adipose tissue is a huge endocrine organ by secreting serials ofadipocytokines and inflammatory factors which involved in regulation of insulinsensitivity and insulin signaling pathways. The ectopic fat hypothesis proposes that:Since the subcutaneous fat tissue differentiation and secretion abnormal, excessive fatdeposition in visceral adipose tissue, liver, skeletal muscle, pancreatic β-cells,resulting in "acquired lipodystrophy", leading to the development of insulin resistance.Therefore, it has great significance to deeply explore the differentiational andsecretional function of visceral and subcutaneous adipose tissue and may provide theoretical and experimental basis for obesity-related diseases.Caveolae, a subset of membrane (lipid) rafts, are flask-like invaginations of theplasma membrane that contain caveolin proteins (caveolin-1,-2, and-3), which serveas organizing centers for cellular signal transduction. Many signaling proteins ofinsulin signal transduction pathway focus or localize in caveolae, such as: Src familytyrosine kinase, Ras/MAPK cascade members, etc. Their most important proteinmarker, caveolin-1, has been shown to play a key role in the regulation of severalcellular signaling pathways and in the regulation of plasma lipoprotein metabolism. Itplays a "switch" role in the cell proliferation, differentiation, migration, apoptosis andangiogenesis. We have found that caveolin-1is strongly linked to type2diabetes.Many researches suggest that insulin resistance means caveolins abnormal.Caveolin-1-knockout mice show insulin resistance no matter whatever diet. But thepart of the research focus on the animal experiment, the researches of human are stillrare.Our group have found that early intensive insulin therapy can improve insulinsensitivity, but the mechanism is not completely clear. So we choose insulin as theintervention drug to research its influence on caveolin-1. And we also chooseexenatide as the research object, which can reverse insulin resistance and reduce bodyweight.ObjectiveThe main purposes of this study are as following:①To observe the mRNA andprotein expression of caveolin-1genes in human subcutaneous and omental adiposetissue of different BMI subjects, compare their expression differences and analysistheir relationship with clinical variables.②Primary cultured human subcutaneousand omental preadipocytes, then observe the effects of different concentrations ofinsulin and exenatie on preadipocytes differentiation.③To investigate the role ofcaveolin-1in human preadipocytes differentiation and preliminary discussion itsmechanism of improving insulin sensitivity. ContentThe whole project includes three parts.Part Ⅰ Depot-specific expression of Caveolin-1in humansubcutaneous and visceral adipose tissue and their relationship withobesity and insulin resistance1Objective:This study aimed to explore the depot-specific mRNA and protein expression ofCaveolin-1(CAV-1) in human subcutaneous and omental adipose tissue, and theninvestigate their relationship with obesity and insulin resistance.2Subjects and Methods2.1SubjectsWe selected41cases of non-menopause women according to certain inclusioncriteria and exclusion criteria who were undergoing an elective abdominalgynecological surgery.2.2Methods2.2.1Anthropometric indices data collectionWe inquired patients' detailed medical history, and orally informed patients thepurpose and significance of the study to obtain the cooperation of patients beforesurgery. Anthropometric indices data were collected including height, weight, waistcircumference, hip circumference and other anthropometric indicators.2.2.2Metabolic measurementsAll patients underwent oral glucose tolerance test (OGTT) after more than10hours of fasting.Fasting venous blood samples were obtained for measurement ofglucose, HbA1c, lipid levels, insulin and adipokines concentration. Homeostasismodel assessment of insulin resistance index (HOMA-IR). Serum concentrations ofleptin, adiponectin, RBP4were detected by ELISA method. 2.2.3Adipose tissue samplingSubcutaneous and omental adipose tissue samples were paired collected duringthe surgery. Real-time quantitative PCR and Western Blot methods were used todertermine adipogenic and metabolic genes (Caveolin-1, PPAR-γ, C/EBP-α, Pref-1,,RBP4, adiponectin genes) mRNA expression and protein levels respectively.2.2.4Statistical analysesStudent's unpaired t test was used to compare the clinical and laboratorycharacteristics between the two groups. ANOVA analysis was used to compare therelative gene expression between subjects of different groups and between omentaland sc adipose tissue, respectively. Pearson's coefficient was used for analysis therelationships between gene expressions and clinic parameters and serum adipokinesconcentration.3Results3.1FINS and HOMA-IR were significantly higher in overweight and obesitygroup than those in the normal BMI group [FINS:(8.82±3.79)mU/L vs(6.43±4.38)mU/L, P<0.05; HOMA-1R:1.9l±0.85vs1.364±0.72, P<0.05].3.2The normal BMI group patients had the higher expression levels of caveolin-1mRNA in omental adipose tissue than overweight (1.33±0.09vs0.48±0.16, P<0.05).But the difference in subcutaneous adipose tissue was not significant (P>0.05).3.3The caveolin-1protein expression in omental adipose tissue of the normal BMIgroup was higher than overweight patients (1.68±0.67vs0.48±0.16, P<0.05). And thedifference between two groups was not significant (P>0.05).3.4The expressions of caveolin-1mRNA in the omental adipose tissue werenegatively correlated with BMI, waist circumstance, triglyceride andHOMA-IR(r=-0.441,-0.615,-0.539,-0.688, p<0.05). No correlations were found between the expressions of caveolin-1mRNA in subcutaneous adipose tissue withBMI,waist circumstance, and HOMA-IR (P>0.05).Conclusions4.1There is depot-specific expression of caveolin-1in human subcutaneous andomental adipose tissues．4.2A low expression of caveolin-1in omental adipose tissue may contribute to thepathogenesis of obesity and IR.4.3There are depot-specific differences in adipogenic and metabolic genetranscriptions in women.4.4These differences in gene expression may be involved obesity-relateddyslipidemia. Part Ⅱ Human Preadipocytes Primary Cultured and Inducedto Differentiation with Different Dose of Insulin1Objective1.1To establish cell models of primary cultured preadipocytes from human omentaladipose tissue.1.2To compare the morphological and functional variations in the course of thedifferentiation of human preadipocytes.1.3To observe the effects of different dose of insulin on the differentiation of humanpreadipocytes. 2Methods2.1Human preadipocytes primary cultureHuman preadipocytes were separation from adipose tissue by collagenase I andcultured in DEME/F12medium, and then they were induced to differentiation intoadipocytes, identified their surface antigens antigens by flow cytometry.2.2Compare the morphological and functional variations of humanpreadipocytesWe used inverted phase contrast microscope to observe the morphological featureof human preadipocytes, used MTT proliferation test to detect their proliferationalability, oil red O staining to measure their differentiational proficiency, RT-PCR andWestern Blot analysis to detect of differentiation-related genes and proteinexpression.2.3Preadipocytes were induced to differentiation with different dose of insulinHuman preadipocytes were induced to differentiation in serum-free medium withdifferent dose of insulin; insulin glargine concentrations were from0nm,0.1nm,1nm,10nm to100nm. Then we observed the effects of different insulin concentrations onadipogenic genes expression and adipokines secretion.3Results:3.1We successfully cultured human primary preadipocytes from omental adiposetissue respectively; and amplificated them in vitro.These researches laied thefoundation for further study. Human preadipocytes were fusiform, similar tofibroblasts, the surface antigen expression is also similar to that of fibroblasts, andthey can be induced to differentiate into adipocytes in vitro.3.2The result of MTT proliferation test is that with the increase of the concentrationof insulin intervention, proliferational ability human preadipocytes is decreased.3.3The result of oil red O staining is that the degree of human preadipocytesdifferentiation is proportional to the concentration of insulin intervention.3.4Caveolin-1gene expression did not change significantly during differentiation ofhuman preadipocytes (P<0.05).3.5With the increase of the concentration of insulin intervention, Caveolin-1expression is also increased. 3.6The content of intracellular triglyceride accumulation and the production ofglycerol release in the culture media during preadipocytes differentiation areincreased with the insulin intervention.4Conclusions4.1Human preadipocytes can be obtained from adipose tissue and can be amplificatedsuccessfully in vitro.4.2Insulin inhibits human preadipocytes proliferation, and promotes theirdifferentiation.4.3Insulin can raise caveolin-1expression of human preadipocytes. Part ⅢInduced Human Preadipocytes to Differentiation withDifferent Dose of exenatide1ObjectiveTo observe the effects of different dose of exenatide on the differentiation ofhuman preadipocytes.2Methods2.1Human preadipocytes were separated from adipose tissue and cultured.2.2Human preadipocytes were induced to differentiation in serum-free medium withdifferent dose of exenatide; insulin exenatide concentrations were from0nm,0.1nm,1nm,10nm to100nm.2.3We observed the effects of different exenatide concentrations on adipogenic genesexpression and adipokines secretion.3Results:3.1The result of MTT proliferation test is that with the increase of the concentration of exenatide intervention, proliferational ability human preadipocytes is alsoincreased.3.3The result of oil red O staining is that the degree of human preadipocytesdifferentiation is in contrast with the concentration of exenatide intervention.3.4With the increase of the concentration of exenatide intervention, Caveolin-1expression is also increased.3.5The production of glycerol release in the culture media during preadipocytesdifferentiation are increased with the exenatide intervention.4Conclusions4.1Exenatide promotes human preadipocytes proliferation, and inhibits theirdifferentiation.4.2Exenatide can raise caveolin-1expression of human preadipocytes.