摘要
在整个生物体能量平衡中肝脏代谢调控系统起着关键作用,因为肝脏是糖代谢(糖酵解和肝糖原合成)和甘油三脂合成(脂肪生成)的主要部位。脂肪生成会受到关键酶的调控,这些酶的活性依靠变构和共价修饰产生。大部分糖酵解和脂肪生成过程中的酶的合成会受到进食状况的调控,其中葡萄糖是起到至关重要作用的营养物质。葡萄糖主要作用于编码这些酶的基因的转录调控过程。
糖应答元件结合蛋白(ChREBP)和乙酰辅酶A羧化酶(ACCs)是糖／脂代谢过程中两个重要因子。ChREBP是碱性／环-螺旋-环／亮氨酸拉链(bHLH/LZ)转录因子家族成员,在肝脏内大量表达的。它在进食高碳水化合物食物后会高效表达,而在进食高脂类食物后其表达会受到抑制。它对很多脂酶有调控作用。同时,ChREBP的活性又会被胰岛素、葡萄糖和抗糖尿病类药物的调节。因此,研究ChREBP的作用机理有着重要的意义。
ACCs家族目前发现的有两个成员：ACC1和ACC2。ACC1主要是在肝脏和脂肪组织的细胞质中表达；ACC2主要是在心脏和肌肉组织的线粒体中表达。ACCs是ChREBP的靶基因之一,会受到食物、激素和其他一些生理因子的调控。
本研究采用real-time PCR和细胞转染等技术,对猪ChREBP基因的生物学特性,ChREBP的功能和转录水平的表达调控,ACC1转录水平的表达调控等进行了研究,获得如下结果：
1.通过同源分析比较,以小鼠和人的ChREBP基因序列为参照,运用RT-PCR方法,获得了猪ChREBP基因；以猪的心、肝、脾、和肺等11个组织样品作为研究材料,通过半定量RT-PCR分析获得了ChREBP的组织表达谱；通过辐射杂种板分析进行了ChREBP的染色体定位,并与人的定位结果进行了比较。
2.在无血清无糖培养条件下,对HepG2人肝癌细胞系进行葡萄糖和胰岛素的时间和梯度刺激。采用real-time PCR方法,检测了ChREBP、胆固醇应答元件结合蛋白(SREBP-1c).ACC1和脂肪酸合成酶(FAS)mRNA的表达量。结果表明,葡萄糖在单独作用时也可引起ChREBP.SREBP-1c.ACC1和FAS表达量的升高。
3.以RT-PCR的方式,克隆得到ChREBP和SREBP-1c的编码序列,分别构建真核表达载体。通过转染和共转染,研究ChREBP和SREBP-1c对靶基因(ACC1)的调控作用。结果表明,ChREBP和SREBP-1c并无协同作用,相反它们有可能对靶基因启动子区的调控位点有竞争作用。
4.在游离脂肪酸的作用下,细胞中ChREBP和SREBP-1c mRNA的表达量下降,而ACC1的表达量却在花生四稀酸存在的情况下大幅度升高。相关因子肝脏X蛋白受体(LXR)和过氧化酶增值体激活受体(PPARa)在花生四稀酸的作用下表达量无明显变化,但cAMP应答元件结合蛋白1 (CREB1)的表达量有所增高。这表明,花生四稀酸不是通过已知两条糖／脂代谢途径来诱导ACC1表达的。
5.检测花生四稀酸诱导或转染CREB1后的细胞培养液中的葡萄糖含量和细胞中甘油三酯的含量,同时通过real-time PCR,检测了细胞中葡萄糖吸收和脂肪酸合成的标志性基因——葡萄糖转运蛋白2 (GLUT2),葡萄糖-6-磷酸酶(G6Pase)和FAS的表达量。综合以上结果,表明花生四稀酸对ACC1的诱导会引起细胞中甘油三酯的沉积,但对葡萄糖的摄取没有明显影响。
6.构建CREB1的真核表达载体及ACC1的启动子荧光素酶载体,进行转染和共转染。通过real-time PCR检测转染CREB1的细胞中ChREBP, SREBP-1c和ACC1的表达量。在共转染后,检测荧光素酶的活性,观察ACC1启动子活性的变化。结果表明,CREB1可结合到ACC1的PⅡ启动子上。这与花生四稀酸的诱导实验的结果相同,进一步证实花生四稀酸是通过CREB1来调控ACC1的表达的。这一作用可能与前列腺素(PG)途径相关。
Liver tissue is a major organ of glycometabolism (glycolysis and hepatic glycogen synthesis) and lipometabolism (lipid synthesis). The metabolic system plays an important action during the maintain energy balance. Lipid synthesis are regulated by some pivotal enzymes which activated by allosterism and contravalence modification. Most of the enzymes linked to glycometabolism and lipometabolism are regulated by the diet. And glucose is one of the significant nutrients. Glucose could act on the transcription of the genes.
Carbohydrate response element-binding protein (ChREBP) and Acetyl-CoA carboxylase (ACCs) are both important factors of glycometabolism and lipometabolism in liver. ChREBP is a member of the bHLH/ZIP transcription factor family. It is expressed in the liver significantly. The high carbohydrate diets could up regulate the expression of ChREBP, as the high lipogene diets would suppress its expression. It could bind to the ChoRE region of many lipogenic enzymes to actived their gene transcription. Meanwhile, its activity would be regulated by insulin, glucose and antidiabetiecs, that why we need to study the mechanism of ChREBP.
ACC has two isoforms:ACC1 and ACC2. ACC1 is mainly expressed in the cytoplasm of liver and adipose tissue, and proposed to be involved in fatty acid synthesis, whereas ACC2 is predominantly expressed in skeletal muscle and heart, which is believed to be responsible for fatty acidβ-oxidation. ACC is one of the factors-induced by carbohydrate response element-binding protein (ChREBP). It is regulated by diet, hormone and other physiological factors else.
In this study, by choosing pig and mouse ChREBP and ACC1 and using real-time PCR, transfection and western blotting, we studied the gene characteristics, transcriptional regulation and function of ChREBP, and transcriptional regulationg of ACC1 and got the following results:
1. By the comparative analysis, according mouse and human ChREBP sequences, pig ChREBP gene was cloned. By semi-quantitative RT-PCR, the tissue expression profile of ChREBP in eleven tissue samples was determined. By radiation hybrid (RH) mapping analysis, pig ChREBP was located and compared with the result of human ChREBP.
2. With different time and different dose treatment of glucose and insulin on HepG2 hepatoma cell line, by real-time PCR, the mRNA expression of ChREBP, sterol regulatory element binding protein-1c (SREBP-1c), ACC1 and fatty acid synthetase (FAS). The results indicated that glucose could increased the expression of ChREBP, SREBP-1c, ACC1 and FAS separately.
3. By RT-PCR, CDS of ChREBP and SREBP were cloned. Subsequently, their expression vectors were constructed, respectively. By transfection or co-transfection, their effects on transcription of ACC1 and FAS were investigated. The results indicated that there is no cooperation but competition between ChREBP and SREBP-1c.
4. By using arachidonic acid (AA), ACC1 was obviously activated while as the expression of ChREBP and SREBP-1c was reduced. The expression of liver X receptor (LXR) and peroxisome proliferators-activated receptorα(PPARα) which bind to the promoter of ACC1 were reduced while as cAMP-response element binding protein 1 (CREB1) was activated. It suggests that the stimulation of AA was not via the glycometabolism approaches which have been studied.
5. By real-time PCR, the expression of the symbol genes in cells linked to glucose absoption and fatty acid synthetic, such as glucose transport 2 (GLUT2), glucose-6-phosphatase (G6Pase) and FAS was checked after dealing with AA. Measured the wastage of glucose in culture media and the creation of triacylglycerol (TG) in cells. The results domanstrated that the inducement of AA on ACC1 can promote fatty acid synthesis but has no relation to glycometabolism.
6. Constructing the expression vector of CREB1, by transfecting and co-transfecting, its effect on transcription of ChREBP, SREBP-1c and ACC1 were investigated. Subsequently, the promoter vector of ACC1 was constructed. By co-transfecting with the expression vector of CREB1, the activity of firefly luciferase was checked. The datas showed that CREB1 could bind to the PⅡpromoter of ACC1. It is match the result of AA stimulation. It confirmed the effect of AA on ACC1 expression via activation of CREB1.
引文
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