胰岛素对肝细胞铁稳态的影响及其分子机制研究

It has been considered an indisputable fact that hepatic iron overload can lead to insulin resistance and cell damage, or even necrosis. However, the reason remains unclear. It's proposed that there may exist some signaling pathways between cellular glucose and iron metabolism. Our preliminary experiments found that the alternation of the insulin level and the activity of its signaling pathway can cause significant change of the expression of iron metabolism regulatory factors in hepatocytes. Therefore, the project will observe the expression and function characteristic of the hepatic iron homeostasis regulatory factors under different insulin levels and signaling pathway activity and discuss the mechanisms through experiments both in vivo and in vitro, which contains variant kinds of methods including molecular biology, immunohistochemistry, metabolic kinetics, mass spectrometry, confocal microscopy and so on. In addition, by comparing the effect of different iron nutritional status on hepatic iron metabolism in the body of insulin dysfunction, what will be also under research is that, whether or not, the dietary iron content has some affect on the onset or development of the hepatic iron overload among people with abnormal insulin sensitivity. On the basis of promoting the study in the relationship between glucose and iron metabolism, the results of the above research will provide new ideas for the prevention and treatment of hepatic iron overload in the crowd of insulin dysfunction, and provide scientific evidence for the formulation of Reference Dietary iron Intakes which is especially suitable for the population suffering from dysmetabolic iron overload syndrome(DIOS).

肝铁过负荷可导致肝细胞胰岛素抵抗和细胞损伤甚至坏死已是不争的事实。然而造成肝脏铁过负荷的原因还不清楚。有学者提出细胞糖代谢与铁代谢之间可能存在信号通路，我们的前期实验发现胰岛素及其信号通路活性变化可引起肝铁过负荷和肝细胞铁稳态调控因子表达发生明显变化。因此，本项目拟采用细胞实验和动物实验相结合的方法，以分子生物学、免疫组化、代谢动力学、质谱分析和共聚焦显微镜等多种技术手段，观察胰岛素及其信号通路活性变化条件下肝细胞铁稳态调控分子的变化特点及其机制，并通过比较不同铁营养状况对胰岛素功能异常机体肝铁代谢变化的影响，分析膳食铁含量对胰岛素功能异常人群肝细胞铁过负荷的发生、发展有无调节作用。研究结果将在推进糖代谢与铁代谢相互关系研究的基础上，为防治胰岛素功能异常人群的肝铁过负荷提供新的思路，并为制定患有"代谢紊乱型铁过负荷综合征"的人群膳食铁参考摄入量提供科学依据。

为阐明代谢性铁过负荷综合症发生原因，在项目资金资助下的研究发现：（1）胰岛素功能障碍大鼠小肠铁吸收增加，肝脏Hepcidin和STAT3表达降低，小肠Fpn表达升高，血清和肝脏铁过负荷，血清IL-6和IL-1β无明显变化。外源性给予胰岛素可逆转上述变化；（2）胰岛素可上调HepG2细胞Hepcidin和STAT3蛋白表达，对其磷酸化及SMAD1和SMAD4表达无影响。胰岛素对 STAT3/Hepcidin 轴的活化部分依赖于ERK 通路，而不是PI-3K 通路；（3）持续高胰岛素状态可引起大鼠轻中度肝铁沉积，伴有肝脏TfR-1、IRP-1、IRP-2表达增高；（4）胰岛素上调TfR-1促使HL-7702细胞铁负荷增加，而TfR-1高表达主要是由于胰岛素增加了TfR-1 mRNA稳定性。胰岛素还可通过IRP-2促进TfR-1表达增加；（5）胰岛素信号通路活性变化可影响HNF4α表达进而影响肝细胞Hepcidin的表达, 此过程不依赖于HNF4α反应元件，而SMAD4反应元件是HNF4α诱导Ｈepcidin表达的必要环节, HNF4α通过抑制BMPR1A通路影响hepcidin表达；（6）铁过负荷可加重胰岛素功能障碍。.本研究结果初步阐明，胰岛素信号通路功能障碍可导致机体铁代谢紊乱。一方面，胰岛素缺乏可通过STAT3信号通路和MEK信号通路抑制肝脏Hepcidin表达，另一方面，胰岛素信号通路还可以通过影响转录调节因子HNF-4α抑制肝细胞的hepcidin 的表达，进而引起肠铁吸收增加，血清铁升高和肝铁沉积。这部分研究结果 可以部分解释2型糖尿病患者铁过负荷的原因。另外，过量胰岛素可通过PI-3K/IRP-2通路上调TfR-1表达使得肝细胞摄铁增多，最终导致肝铁过负荷，这部分研究结果可能可以部分解释伴有高胰岛素血症的非酒精性脂肪肝患者出现肝铁沉积的原因。研究结果形成论文4篇。