胰岛素对小鼠原代肝细胞甘油三酯合成分解代谢的影响

The Effect of Insulin on Triglycerides Synthesis and Lipolysis of Mice Primary Hepatocytes

目的:建立小鼠肝细胞体外培养的方法,研究不同浓度胰岛素对肝细胞甘油三酯合成代谢、分解代谢及甘油三酯含量的影响。方法:通过肝脏灌注和胶原酶消化分离小鼠肝细胞,密度梯度离心纯化后,进行体外培养。在0 nmol/L,50 nmol/L,100 nmol/L,200 nmol/L胰岛素存在的情况下培养,通过3H标记的甘油测定细胞内甘油三酯合成速率,使用3H标记的油酸预孵育,加入triacsin C抑制脂肪酸重酯化,追踪掺入3H的甘油三酯分解的速率。采用甘油三酯检测试剂盒,测定不同浓度胰岛素对细胞内甘油三酯含量的影响。结果:成功分离了小鼠原代肝细胞,存活率达90%。50 nmol/L胰岛素对细胞甘油三酯含量及甘油三酯合成分解速率影响较小。100 nmol/L胰岛素可显著增加甘油三酯合成速率,减低分解速率,使细胞内甘油三酯含量增加。200 nmo/L胰岛素反而降低甘油三酯合成速率,细胞内甘油三酯含量少于对照组(0 nmol/L)。结论:本研究成功建立了小鼠原代肝细胞分离培养的方法,使用3H标记物敏感的检测肝细胞内甘油三酯合成分解速率。研究发现,过高浓度的胰岛素反而抑制肝细胞甘油三酯的储积。

Objective： To isolate the mice primary hepatocytes, and to investigate the effect of insulin on triglycerides accumulation, triglycerides synthesis and lipolysis. Methods： Mouse hepatocytes were isolated by using a two-step in situ collagenase perfusion procedure and purified by density-gradient centrifugation. Hepatocytes in vitro were incubated in experimental medium containing 0 nmol/L, 50 nmol/L, 100 nmol/L, 200 umol/L insulin respectively for 24 h. The amounts of [2-^3H] glycerol incorporated into the triglycerides was monitored to determine the rate of lipolysis, AML12 cells lacking LSDP5 were preloaded with [^3H]-oleate to incorporate into triglycerides and the efflux of [^3H]-oleate was monitored for 4 h and reflects the rate oflipolysis in cells to determine the rate of lipolysis. Triacsin C was administrated to block the re-esterification in this process. The triglyceride content was assayed using a TG test kit. Results： The primary hepatocytes were isolated and cultured successfully, 50 nmol/L insulin had little influence on triglyceride metabolism. 100 nmol/L insulin could increase the triglyceride content and the speed of triglycerides synthesis, but decrease the speed of lipolysis. 200 nmo/L insulin decreased the triglyceride content and the speed of triglycerides synthesis. Conclusion： The mouse primary hepatocytes in vitro was isolated and cultured and the speed of triglycerides synthesis and lipolysis was detected with radiolabeled tracers sensitively. The influence of insulin in different concentrations on triglyceride metabolism was determined.