ABCA6转运蛋白对脂质稳态失衡的调控作用

The incidence of disturbed lipid metabolism, which tends to occur more commonly and at younger ages，increases rapidly in our country in recent years, making it important and necessary to illustrate the regulatory mechanism of lipid homeostasis. It has been established that some members of the ATP-binding cassette (ABC) transporter family play important roles in this regulation. ABCA6 is a ABC A subfamily transporter with a cholesterol-responsive expression in macrophages, but its biological significance is unknown so far. We found in our studies that transcription factor FoxO (Forkhead box, class O), which is involved in insulin signaling pathway, regulates the expression of ABCA6. We further cloned the mouse ABCA6 cDNA and generated a ABCA6-V5 expression plasmid. With immunofluorescence we found that different from ABCA1, ABCA6 proteins co-localized with marker protein for the Golgi apparatus. Realtime PCR analysis of cells stably expressing ABCA6 demenstrated that ABCA1 was induced dramatically while Insig1 was repressed compared with control. Moreover, significant increase in hepatic ABCA6 mRNA levels was observed in fasted mice. In streptozotocin-induced diabetic mice, fast-induced expression of ABCA6 were more prominent. Our findings point to the potential involvement of ABCA6 in lipid transport processes and cellular cholesterol homeostasis. The current research program aims to investigate the detailed mechanism of ABCA6 in maintaining cellular lipid homeostasis and its function in disturbed lipid metabolism by studies both in vivo and in vitro. The results will help to understand the pathogenesis of disorders of lipid metabolism and contribute to the prevention and therapies of these disease.

近年来,我国脂质代谢紊乱的发病率增长迅速，呈大众化和低龄化的流行趋势，阐明脂质稳态失衡机制是当前重要而迫切的问题。一些ABC转运蛋白（ATP binding cassette transporter）在脂质稳态调节中起重要作用，是当前研究热点之一。ABCA6是ABCA亚家族转运蛋白，在巨噬细胞的表达受胆固醇调节，目前对其认识极为有限。我们发现ABCA6受胰岛素调控的转录因子FoxO调节。我们克隆了小鼠ABCA6全长cDNA，通过免疫组化发现，ABCA6主要分布于高尔基体；稳定表达ABCA6细胞中，ABCA1表达明显增加，Insig1表达减少；小鼠禁食后肝ABCA6表达明显升高，糖尿病小鼠禁食后升高更显著。上述结果提示，ABCA6可能在脂质稳态调节和失衡中发挥独特的作用。本项目旨在深入研究和阐明ABCA6的功能，揭示ABCA6与脂质稳态失衡的联系，为深刻认识和防治脂质代谢紊乱性疾病做出贡献。

ABC转运蛋白（ATP binding cassette transporter）是一组跨膜蛋白, 以 ATP 为能源介导脂类、氨基酸、多肽等多种物质的跨膜转运。ABCA6是ABCA亚家族成员，在组织中广泛表达，以肝脏中含量最高。ABCA6的生物学功能目前尚不清楚。我们课题组在以往的研究中发现，ABCA6基因的转录受转录因子FoxO的调控。本项目首次通过体外和整体实验，对ABCA6的功能进行了研究。我们发现，ABCA6蛋白在小鼠肝脏的表达最强，而心脏、肺、骨骼肌中仅有少量表达；免疫组化显示，内源性ABCA6分布于细胞内，可能定位于Golgi体；ABCA6的表达受能量代谢状态的调控，禁食或其他导致脂肪酸氧化增强的刺激能上调ABCA6的表达。正常饮食时，ABCA6敲除（KO）小鼠与对照小鼠的血浆葡萄糖、胆固醇、甘油三酯、酮体、游离脂肪酸、谷丙转氨酶（ALT）等含量没有明显差异，肝脏中胆固醇和甘油三酯的含量也没有明显差异，但肝糖原含量明显低于对照小鼠；代谢实验结果提示，ABCA6 KO小鼠与对照小鼠对胰岛素的敏感性无明显差异；代谢监测显示，ABCA6 敲除小鼠与对照小鼠的呼吸商在夜间无明显差别，但是在白天时段ABCA6 KO小鼠的呼吸商明显高于WT小鼠，且ABCA6 KO小鼠进食量大于对照小鼠；Realtime PCR 检测显示，ABCA6 KO 小鼠肝脏中参与脂肪酸转运及微粒体、过氧化物酶体脂肪酸氧化的基因表达增多；HPLC检测显示两组小鼠的肝脏脂肪酸组成无明显差别。在禁食早期（0—6h），ABCA6 KO小鼠的呼吸商明显高于对照小鼠，但随着禁食时间的延长，这种差异逐渐消失；ABCA6 KO小鼠血糖和酮体在禁食早期显著低于对照小鼠。在高脂或高糖高脂饮食喂养下，与对照小鼠相比，ABCA6 KO小鼠体重显著增加，对胰岛素的敏感性下降，血糖显著升高，高糖高脂饮食时ALT和AST也显著增加，而血清总胆固醇、甘油三酯、酮体等则无明显改变；ABCA6 KO小鼠肝脏出现显著的脂质沉积；高糖高脂饮食时肝脏炎症因子的表达明显增加。以上结果提示，ABCA6可能参与脂肪酸氧化中间产物的转运，在快速增强脂肪酸氧化的过程起重要作用。ABCA6对于高脂或高糖高脂食物诱导的肥胖、胰岛素抵抗以及非酒精性脂肪性肝病具有重要的保护作用。