The Study On The Mechanisms Of Increasing Insulin Resistance In Adipocytes By LRP16

LRP16 is a human gene cloned from the peripheral blood lymphocyte of the healthy adult and overexpressed in the malignant tumorous tissue related to estrogen. Previous studies showed that LRP16 promoted the MCF-7 breast cancer cell differentiation from Gl-phase to S-phase.Growth inhibiting is essential for the differentiation of adipocytes.To make cells maintain growth-inhibiting phase,it is essential to make cyclin stasis in Gl-phase.There is close relation between insulin resistance and malignant tumour.Therefore,we hypothesized that LRP16 may participate in regulating the differentiation of adipocytes and the mechanism of insulin resistance.This study investigated the changes of LRP16 protein expression during 3T3-L1 preadipocyte differentiation,the possible mechanisms of regulative effects of LRP16 on adipocyte differentiation and insulin resistance.The study can be divided into following three parts:一.Expression of LRP16 protein during 3T3-L1 preadipocyte differentiation and the regulative role of insulin on itObjective:To investigate the changes of expression levels of LRP16 protein during 3T3-L1 preadipocyte differentiation,and to explore the regulative role of insulin on LRP16 protein in adipocytes.Methods:1.3T3-L1 preadipocytes were cultured in vitro and differentiated into the matured adipocytes,then protein samples were collected everyday(0-8d).2.Various concentrations of insulin(0,1,10,100 nmol/L) were added into the culture medium of adipocytes for 24h,and then protein samples were collected.3.Expression levels of LRP16 protein in the 3T3-L1 cells were detected by means of Western-blot analysis.Results:1.Faint LRP16 protein was detected in preadipocytes.Expression level of LRP 16 protein increased from the d3 or d4 day rapidly and reached to the peak on d7(all P values＜0.01 except for from d6 to d8 P＞0.05).2.There was negative correlation between the concentration of insulin and expression level of LRP16 protein in adipocytes after stimulated by insulin for 24h(p＜0.01).Conclusions:1.LRP16 protein gradually increased during 3T3-L1 adipocyte differentiation.2.Insulin down-regulates expression of LRP16 protein in matured adipocytes.二.The mechanisms of LRP16 on regulation of adipocyte differentiationObjective:To investigate the effects and mechanisms of LRP16 on the regulation of adipocyte differentiation.Methods:1.3T3-L1 preadipocyte model with overexpression of LRP16 gene:LRP16 eucaryotic expression vector (pcDNA3.1-LRP16)was transfected into 3T3-L1 cells with the control vector (pcDNA3.1(+)),respectively,and then the cells were selected by G418 and cultured for further study.2.Overexpression of LRP16 protein in preadipocytes was determined by western-blot analysis.3.The morphologic changes during differentiation of 3T3-16 preadipocytes were observed by inverted microscope.4. Adipogenesis during differentiation of 3T3-16 and 3T3-3.1 adipocytes was observed by inverted microscope using staining by oil red-O.5.Expression levels of PPARγand C/EBPαmRNAs in 3T3-16 and 3T3-3.1 adipocytes were evaluated by Real-time PCR.Results:1.Expression levels of LRP16 protein were much higher in 3T3-16 adipocytes than 3T3-3.1 adipocytes(P＜0.01).2.Differentiation of 3T3-16 preadipocytes was more hysteretic than 3T3-3.1 preadipocytes.3.3T3-16 adipocytes had fewer lipid droplets than 3T3-3.1 adipocytes.4.Both PPARγand C/EBPαmRNA levels in 3T3-16 adipocytes were lower than that in 3T3-3.1 adipocytes(both P values＜0.01).Conclusions:LRP16 inhibits adipocyte differentiation possibly by down-regulating of PPARγand C/EBPαexpression in adipocytes.三.The mechanisms of LRP16 on regulation of insulin resistanceObjective:To investigate effects and mechanisms of LRP16 gene on regulation of insulin resistance.Methods:1.Glucose uptake rates were evaluated by 2-deoxy-[~3H]-D-glucose in 3T3-16 and 3T3-3.1 adipocytes.2.Expression levels of GLUT4 mRNA in 3T3-16 and 3T3-3.1 adipocytes were evaluated by Real-time PCR.Results:1.Basic and insulin-induced glucose uptake rates in 3T3-16 adipocytes both were lower than that in 3T3-3.1 adipocytes.(P＜0.01).2.Expression levels of GLUT4 mRNA in 3T3-16 adipocytes was lower than that in 3T3-3.1 adipocytes(P＜0.01). Conclusions:1.LRP16 may decrease glucose uptake in adipocytes through down-regulating of GLUT4 expression.Conclusions1.LRP16 inhibits adipocyte differentiation possibly by down-regulating of PPARγand C/EBPαexpression in adipocytes.2.LRP16 may decrease glucose uptake in adipocytes through down-regulating of GLUT4 expression.3.Insulin down-regulate expression of LRP 16 protein in adipocytes. These reulsts indicate that LRP16 may play an important effect on adipocyte differentiation and insulin resistance.