The Mechanisms Of Apolipoprotein A5Internalized In Adipocytes

ObjectivesThe aims of this study were to build matured3T3-L1adipocytes hypertrophy model and insulin resistance model and to observe the internalization of apolipoprotein A5(apoA5); The low density lipoprotein receptor-related protein1(LRP1) small interfering RNA (siRNA) was used to investigate whether apoA5could be internalized by3T3-L1adipocytes. The molecular mechanism of apoA5internalized by3T3-L1adipocytes was studied.MethodsMouse3T3-L1fibroblasts were differentiated into mature3T3-L1adipocytes as experimental cells. The following experiments were performed:(1) Establish3T3-L1adipocytes hypertrophy models:mature3T3-L1adipocytes after induction of differentiation medium was changed to21days;(2) Establish3T3-L1adipocytes insulin resistance model:3ng/ml murine TNF-a by incubation of mature3T3-L1adipocytes3days, medium was changed every day;(3) Recombinant human apoA5was prepared and labeled with125I,3T3-L1adipocyte hypertrophy and insulin resistance were incubated with125I-apoA5, to test apoA5can be internalized by3T3-L1adipocytes performing to pulse-chase experiments and Western Blot analysis;(4)3T3-L1adipocytes were treated with different concentration gradients1nM,5nM,20nM,50nM siRNA silencing LRP1, the optimum concentration of silencing efficiency siRNA was received by Western Blot analysis;(5) After using the optimal concentration of siRNA silencing LRP1intervened mature3T3-L1adipocytes, the3T3-L1adipocytes was incubated with apoA5, to test whether apoA5can be internalized by3T3-L1adipocytes by pulse-chase experiments and Western Blot analysis.Results1. Differentiation and maturation of3T3-L1adipocytes was changed to21days, oil red O staining bright red large lipid droplets integration, establish adipocytes hypertrophy model.2. Insulin resistance cell were obtained by incubating the differentiated3T3-L1adipocytes for3days in the presence of3ng/ml TNF-a with fresh media changes each day as described.3T3-L1adipocytes treated in parallel but without TNF-a were considered N adipocytes. In both cases, after overnight incubation in serum-free medium supplemented with0.2%bovine serum albumin, set up insulin resistance model.3. Pulse-chase experiments confirmed that I-apoA5was internalized into3T3-L1adipocytes hypertrophy and insulin resistance significantly decreased66%和36%(P<0.05)compared with normal3T3-L1adipocytes, respectively; Likewise, compared with normal3T3-L1adipocytes, the uptake of apoA5significantly reduced85%和52 %(P<0.05) respectively by3T3-L1adipocytes hypertrophy and insulin resistance was further evidenced by Western Blot analysis which showed the presence of apoA5in adipocytes after treatment with apoA5for4hours.4.3T3-L1adipocytes were treated with different concentration gradients1nM,5nM,20nM,50nM siRNA silencing LRP1, the best siRNA concentration detected by Western Blot analysis is5nM (silencing efficiency is87%(P<0.05).5. Pulse-chase experiments revealed that:after a treatment of5nM siRNA with mature3T3-L1adipocytes,125I-apoA5was significantly reduced72%(P<0.05) internalized into3T3-L1adipocytes compared with control during the pulse. Similarly, Western Blot testing found that the intervention of5nM siRNA markedly reduced70%(P<0.05) uptake of content apoA5by3T3-L1adipocytes compared with control.Conclusions1.We successfully established mature3T3-L1adipocyte hypertrophy and insulin resistance model.2. ApoA5can be internalized by3T3-L1adipocyte hypertrophy and insulin resistance obviously decreased.3. The uptake of apoA5by3T3-L1adipocytes mainly through LRP1...