代谢相关药物对3T3-L1脂肪细胞促酰化蛋白分泌的影响

Effect of metabolic drugs on the secretion of acylation stimulating protein in 3T3-L1 adipocytes

目的:建立使用3T3-L1脂肪细胞评价药物影响促酰化蛋白(ASP)分泌的细胞模型和测定ASP分泌的ELISA方法,观察代谢相关药物二甲双胍、罗格列酮钠及立莫那班对ASP分泌的影响及机制,以及与补体C3、非酯化脂肪酸(NE-FA)释放、甘油三酯总量(TG mass)、脂肪酸摄取量的关系。方法:以3T3-L1前脂肪细胞为实验对象,诱导细胞分化,分别加入乳糜微粒、二甲双胍、罗格列酮钠及立莫那班作用48 h。采用ELISA法测定ASP、C3含量,使用酶标比色法测定NEFA含量,脂肪细胞FA摄取速度在荧光酶标仪进行动力学方法测量,采用分光光度法测定TG含量,考马斯亮蓝染料结合法测定细胞总蛋白。结果:乳糜微粒刺激ASP分泌(达411%±133%P<0.05);罗格列酮钠和立莫那班抑制ASP产生(-53%～-85%,P<0.05),同时抑制前体蛋白C3分泌(-37%～-65%,P<0.05);二甲双胍抑制ASP分泌(-54%～-100%,P<0.05),对前体蛋白C3分泌无影响;二甲双胍降低TG含量(达-60%,P<0.05)和FA摄取速度(达-75%,P<0.05)。结论:成功建立了评价药物影响促酰化蛋白(ASP)分泌的3T3-L1前脂肪细胞模型,并建立了测定ASP分泌的ELISA方法。

AIM： To develop a 3T3-LI pre-adipocyte model for evaluating the secretion of acylation stimulating protein （ASP） and an ELISA assay for measuring ASP production and investigate the effects and related potential mechanisms of metabolic drugs on the secretion of ASP, and on the complement C3, triglyceride （TG） mass, nonesterified fatty acids （NEFA） release and fatty acid （FA） uptake into adipocytes. METHODS： After differentia- ted, 3T3-LI pre-adipocytes were treated with chylomicrons, metformin, rosiglitazone, rimonabant for 48 h. ASP and C3 were measured using a sandwich ELISA. NEFA levels were measured using enzymatic colorimetric kits. FA uptake was measured in a bottom-reading fluorescent microplate read- er. TG mass and protein levels were determined using enzymatic colorimetric assay and the Bradford assay, respectively. RESULTS. Chylornicrons increased ASP production （up to 411%±133%, P 〈 0. 05）. Rosiglitazone and rimonabant decreased ASP production （ -53% to -85%, P 〈 0.05 ）, associated with a decrease in the precursor protein C3 （ -37% to -65%, P〈0.0]）. By contrast, metformin also decreased ASP （ - 54% to - 100%, P 〈 0.05）, but with no change in precursor protein C3. In addition, metformin decreased TG mass （ maximum - 60%, P 〈 0.05 ） and real-time FA uptake （maximum - 75%, P 〈 0.05）.CONCLUSION： 3T3-Ll pre-adipocyte model is successfully developed, which can be used for evaluating the effects of metabolic drugs on the secretion of ASP. ELISA assay for measuring ASP production is also developed.