【作者】 田利民；

【导师】 赵家军；

【作者基本信息】 山东大学 ， 内分泌与代谢病， 2010， 博士

【摘要】 研究背景：甲状腺疾病时机体常伴有血脂异常,如甲状腺功能减退时常合并有高胆固醇血症,而甲状腺功能亢进时则常伴有血胆固醇水平降低。以往认为甲状腺功能减退患者血胆固醇水平升高的主要机制是：甲状腺激素(TH)水平降低,导致肝脏低密度脂蛋白受体(LDL-R)表达减少、活性降低,血浆低密度脂蛋白(LDL)清除减少,因而引起血浆中的总胆固醇(TC)水平升高。然而,临床上出现的一些特点往往无法用这种传统理论去解释。例如,亚临床甲状腺功能减退时,促甲状腺素(TSH)水平升高,TH水平正常,但血清TC水平升高。另外在继发性甲减时,TSH、TH均降低,TC也随之降低。从以上现象我们可以看到在甲状腺功能减退时血清TC的变化除与TH有关外,也与TSH有某种不曾被人们认识的关系。而近几年,不断出现的一些大型流调研究也发现在亚临床甲减时TSH与TC相关。例如,最近Turha报道在亚临床甲状腺功能减退时血清TSH与TC有明显相关性(r=0.52；p=0.001)。另一项研究发现亚临床甲减患者经过L-T4治疗后血清TSH水平下降至0.2-2mU／L,TC也明显下降。亦有报道在妇女中TSH每升高1mU／L则伴有0.09mmol／L血胆固醇的升高。而在老年人群中,当TSH>5.5mU／L时血胆固醇较正常时有0.23mmol／l(9mg/dl)的升高,进一步的计算得出TSH水平每升高1mU／L,就伴随有0.09mmol／l(3.5mg／dl)血浆胆固醇浓度的提高。这些临床现象均无法通过传统理论中关于TH对胆固醇代谢调节的理论得到完全解释。在这之前,有研究发现另外一个垂体激素.促性腺释放激素(FSH)对骨吸收和骨形成有直接的调节作用。这一研究结果表明垂体激素可能参与周围组织器官生理调节作用。因此我们有信心也有必要对TSH在胆固醇合成过程中的作用及机制进行研究。一般情况下内源性合成是机体胆固醇最主要的来源。其中肝脏是机体胆固醇合成最旺盛的器官。3-羟基-3-甲基戊二酰辅酶A (3-hydroxy-3-methylglutaryl coenzyme A, HMG-CoA)还原酶(HMGCR)位于细胞内质网内,由它催化的由HMG-CoA还原成甲羟戊酸(Mevalonate, MVA)的反应是整个胆固醇合成途径中的限速反应,HMGCR则是合成反应的限速酶。通过调节该酶可维持体内胆固醇的稳定。现今,人们已公认肝脏在调节机体胆固醇代谢方面发挥中心作用。HMGCR也被发现存在于机体其它器官,但在肝脏却有更高水平的表达,在一些动物,肝脏具有最高的HMGCR活性和胆固醇生物合成能力。促甲状腺素受体(thyrotropin receptor, TSHR)是人体内介导TSH甲状腺调控功能的重要的蛋白分子。大量研究表明,TSHR不仅表达于甲状腺细胞表面,而且广泛表达于如脂肪细胞、成纤维细胞、淋巴细胞、骨细胞及神经细胞等在内的非甲状腺组织细胞内,且在这些组织细胞内发挥重要的病理生理作用。肝脏作为机体内最重要的组织器官之一,在肝细胞表面是否亦有TSHR的表达,进而介导TSH起调控作用的作用呢?我们前期的研究已证明人体正常肝组织、体外培养的人正常肝细胞株L-02细胞以及正常Wistar与Sprague Dawley (SD)大鼠肝组织有TSHR的表达,并且TSH可剂量依赖性地增加L-02细胞内cAMP的含量,说明肝细胞表面表达有功能性的TSH受体。本研究拟通过体外和体内实验研究TSH对肝细胞HMGCR表达的影响及相关机制,以探讨TSH在胆固醇合成中可能存在的调节作用。研究目的：1.前期研究已证明肝细胞膜上表达功能性TSH受体(TSHR),本课题拟通过研究TSHR介导的TSH作用,上调肝细胞HMGCR表达,增加肝细胞内总胆固醇(TC)含量,引起高胆固醇血症,从而揭示TSH在胆固醇合成中的可能机理。2、通过TSH刺激肝细胞cAMP/PKA/CREB信号途径的实验研究,揭示TSH增加肝细胞HMGCR的表达的作用机制。研究方法：1、为了研究肝细胞表达功能性TSHR,实验用牛TSH、胰高血糖素或福斯考林刺激L-02、培养的人原代肝细胞和小鼠胚胎肝细胞系BNL细胞,检测cAMP释放。福斯考林作为阳性对照。2、在体外实验,用牛TSH处理肝细胞,检测细胞HMGCR表达。在刺激实验中,用无血清培养基培养人正常肝细胞系L-02、人原代肝细胞和BNL细胞,用不同浓度的TSH作用一定时间,检测TSH对HMGCR剂效性和时效性关系。3、为表明TSH影响肝细胞HMGCR的表达是通过TSHR作用的,实验用人单克隆抗体CS-17与TSH竞争结合TSHR。L-02细胞和人原代肝细胞用CS-17预培养60分钟,然后用TSH继续作用60分钟,检测cAMP释放。另外,我们采用慢病毒介导的RNA干扰病毒感染培养的肝细胞,敲除肝细胞TSHR表达,检测TSH刺激后cAMP的释放和HMGCR的表达的变化。4、为了探讨TSH诱导上调肝细胞HMGCR表达是通过cAMP信号系统,用腺苷酸环化酶抑制剂SQ22536预孵肝细胞,再用TSH刺激肝细胞,检测cAMP和HMGCR变化。5、为了证实TSH刺激增加肝细胞HMGCR mRNA水平是通过增加HMGCR启动子的活性作用实现的,本研究设计相应HMGCR启动子的一个DNA片段,与荧光素酶基因融合,构建荧光素酶报告基因,然后转染到L-02细胞。在不同处理条件下,测定荧光素酶活性。实验选择福斯克林为阳性对照,用0.4和4μMTSH刺激L-02细胞8小时。6、通过ChIP实验定量分析TSH刺激L-02细胞激活HMGCR转录活性。将L-02细胞分为四组：福斯克林(0.5μM)组,TSH(4μM)组,H89(PKA阻断剂,20μM)组,RNA干扰组。7、为进一步明确TSH是否对CREB/DNA结合活性有影响,从L-02细胞提取核蛋白进行了EMSA实验研究。从未加处理的细胞,加了4μMTSH和20μMH89的细胞中提取等量的核蛋白,探针用生物素标记。8、为了进一步研究TSH增加肝细胞HMGCR表达,增加胆固醇水平,我们通过手术切除大鼠甲状腺建立了甲减大鼠模型。切除了大鼠甲状腺,就失去了对TSH的刺激反应,血中甲状腺素水平降低甚至消失,这样就可以通过注射外源性T4或TSH人为的控制大鼠体内TSH水平,达到实验目的。术后30天检测,血清TT4≤1.41ng/ml认为甲状腺已完全摘除,为实验组大鼠(48只),未达到要求的切除甲状腺大鼠排除实验外。同时设假手术组(30只)。研究结果：1、肝细胞膜表达具有功能活性的TSH受体与对照组比较,福斯考林显著增加L-02细胞内cAMP水平(约2.8倍,p<0.001),同样,bTSH刺激也明显增加了细胞内cAMP水平(约为福斯考林组的三分之二,p<0.05)。实验中以CHO细胞为阴性对照,因为在其细胞表面不存在TSH受体,本实验发现福斯考林显著增加CHO细胞内cAMP水平(p<0.05),但是bTSH刺激CHO细胞后细胞内cAMP水平无明显变化(p>0.05)。同样,我们用bTSH、福斯考林和胰高血糖素刺激原代培养的人肝细胞和BNL细胞均显著增加了细胞内cAMP水平。并且,与L-02细胞比较,人原代肝细胞对bTSH反应更明显(p<0.05)。表明新鲜分离的人肝细胞具有更好的肝细胞活性。2、TSH增加肝细胞HMG-CoA还原酶(HMGCR)的表达和活性(1)不同浓度bTSH对HMGCR蛋白的影响与对照组相比,0.1μM、1μM、4μM bTSH处理后L-02细胞内HMGCR蛋白均有明显增加,分别增加了30％、58％与110％,差异有统计学意义p<0.05或p<0.01)。说明bTSH可剂量依赖性地上调HMGCR蛋白水平。(2)不同浓度bTSH刺激对HMGCR mRNA水平的影响与正常对照组相比,bTSH刺激组HMGCR mRNA表达增强,2μM (p<0.05)与4μM (p<0.01)bTSH干预组与对照组相比分别提高了92％与150％,差异均有统计学意义,且4μM bTSH刺激作用强于0.2μM作用(p<0.01)。说明bTSH可剂量依赖性地上调HMGCR mRNA水平。(3)不同浓度bTSH对人原代肝细胞和小鼠肝细胞系HMGCR蛋白的影响：与L-02细胞相似,0.1μM、1μM、4μM bTSH刺激人原代肝细胞和NBL细胞48小时后,Wester blot示细胞内HMGCR蛋白水平均呈剂量依赖性增加。(4)不同浓度bTSH对HMGCR活性的影响：分别用0.1μM、1μM、4μM bTSH处理L-02细胞48h后发现,随bTSH剂量的增加L-02细胞HMGCR活性水平也呈增加趋势。与对照组比较,0.1μM bTSH和4μM bTSH增加HMGCR活性分别为2和4倍(p=0.013和p<0.001)。3、TSH刺激增加L-02细胞内总胆固醇(TC)含量随着bTSH浓度的增加,细胞内TC含量逐渐增加,与对照组比较,1μM和4μM bTSH刺激增加TC含量分别为2.2和3倍(p=0.032和p=0.004)。同时,刺激48h后TC含量明显高于24h(p<0.01)。TSH对肝细胞内TC水平影响的这种剂效和时效关系与对HMGCR刺激效应一致,表明TSH作用肝细胞后通过上调HMGCR表达,增加细胞内总胆固醇含量。4.TSH诱导肝细胞HMGCR表达依赖于TSHR(1)L-02细胞和人原代肝细胞在bTSH刺激下,细胞内cAMP水平分别增加了约2.8和3.5倍(p<0.001),在CS-17预处理后,bTSH刺激下细胞内cAMP水平均无明显增加(p>0.05)。表明CS-17抑制了bTSH诱导的细胞内cAMP水平的增加。同样,与对照组比较,TSH刺激可显著增加HMGCR蛋白表达,但是在CS-17处理L-02细胞后,TSH作用后细胞HMGCR蛋白表达无明显变化。表明CS-17抑制了bTSH诱导的肝细胞HMGCR蛋白的增加。(2)慢病毒介导的TSH受体RNA干扰：TSHR-RNAi-lentivirus显著降低TSH诱导的L-02细胞cAMP释放和HMGCR表达,以及TC含量的增加。(3)RNAi干扰TSHR基因表达后TSH对BNL CL.2细胞HMG-CoA还原酶含量的影响：siRNA干扰处理细胞HMG-CoA还原酶含量与正常对照组比较无明显变化,TSH明显提高未加siRNA干扰的细胞HMG-CoA还原酶含量,TSHR-siRNA干扰TSHR基因表达后,TSH不能引起细胞HMG-CoA还原酶含量的明显升高,而错配的non-targeting siRNA处理后TSH仍可明显增高细胞HMG-CoA还原酶含量,增高程度与未加siRNA干扰的正常细胞TSH处理后基本相同。5、TSH通过cAMP/PKA/CREB信号通路上调肝细胞HMGCR表达(1)用SQ22536预孵L-02细胞和人原代肝细胞1小时,继续TSH刺激细胞1小时。我们发现,无论是L-02细胞,还是人原代肝细胞,SQ22536均能显著降低TSH诱导的细胞内cAMP水平(P<0.01)。无SQ22536预处理的L-02细胞,在TSH刺激48小时后HMGCR蛋白表达明显增加,但是SQ22536处理后的细胞,在TSH刺激后,HMGCR蛋白表达无明显增加。(2)实验选择福斯克林为阳性对照,用0.4和4μM TSH刺激L-02细胞8小时。结果表明,福斯克林增加荧光素酶活性2.4倍,0.4和4μM TSH刺激L-02细胞增加荧光素酶活性分别为1.5和2.2倍。进而将HMGCR启动子上的CRE结合位点突变,突变序列为：TGACGTAG to TAAAAGGG。我们发现无论是TSH,还是福斯克林刺激均不能增加荧光素酶活性。这一实验表明TSH调控HMGCR基因转录是通过CRE结合位点实现的。(3)与对照组比较,福斯克林刺激L-02细胞显著增加磷酸化CREB(Ser133 pCREB)的结合能力(P<0.001),而TSH刺激L-02细胞增加pCREB结合能力约2.3倍(P<0.001)。H89可明显抑制TSH诱导的pCREB结合能力(P=0.019与对照组比较)。同样,RNA干扰也显著抑制TSH诱导的pCREB结合能力(P=0.002与TSH组比较)。(4) ESMA实验结果表明TSH可以增加CREB/DNA结合活性,在凝胶电泳图上可看到较强的CREB/DNA混合物条带。当加入H89后这个条带明显减弱,表明TSH增加CREB/DNA结合活性是通过PKA通路实现的。6、TSH增加甲状腺切除(Tx)鼠肝脏HMGCR表达,增加血清TC水平(1)Tx和(假手术)Sh组大鼠血清TT4分别为0.55±0.32(ng／ml)和7.60±2.15(ng／ml),血清TSH分别为41.9～6.20(uIU／mL)和0.63±0.23(uIU／mL)。T4和TSH的差异均具有统计学意义。Tx和Sh组大鼠血清总胆固醇水平分别为2.33±0.34mmol／l和1.46±0.0.32mmol／l,Tx大鼠血清胆固醇水平明显升高,差异具有统计学意义。Western blot分析HMGCR和LDLR蛋白表达。目的条带在90KD和160KD左右,以β-actin为参照。结果发现,与Sh组大鼠比较,Tx大鼠肝组织HMGCR蛋白明显升高(P<0.05),而LDLR明显降低(P<0.05)。(2)TSH对血浆TC水平的影响：将Tx大鼠分成4组,每组12只大鼠：①Tx；②Tx+T4；③Tx+T4+小剂量TSH；④Tx+T4+中剂量TSH；⑤Tx+T4+大剂量TSH。结果发现①组Tx大鼠TC水平较假手术组显著升高,当给予恒定量T4后,血浆中T4水平升高,而TSH水平下降,与假手术组大鼠相当,也就是说T4处理后恢复了甲减大鼠血浆T4和TSH水平。同样,TC水平也明显下降。当血中T4值达到恒定水平后,给外源性TSH注射,我们发现,与未注射TSH组的大鼠(即②)比较,注射TSH组大鼠(即③④⑤组)血浆TC水平剂量依赖性升高,但差异无统计学意义。进一步分析发现,在同一只大鼠,注射TSH前后血清胆固醇水平有变化,注射TSH后TC水平较注射前升高,且有统计学意义。(3)TSH对肝组织HMGCR和LDLR表达的影响：同时,上述前4组大鼠肝组织做HMGCR和LDLR检测,结果显示,与Tx大鼠比较,注射T4后HMGCR蛋白表达降低,而LDLR表达增加。与Tx+T4组比较,注射TSH后HMGCR升高,且大剂量TSH升高更明显,而LDLR无明显变化。表明TSH注射增加肝脏HMGCR的表达,但对LDLR无明显影响。结论：1、本研究通过体外细胞实验和建立甲减模型的体内实验研究,证明了TSH可以增加肝脏胆固醇合成的关键酶HMGCR的表达,通过体外研究发现TSH的这种作用主要是通过cAMP/PKA/CREB信号通路实现的,且依赖于肝细胞膜上的TSHR。与体外实验研究一致,目前的研究发现切除了甲状腺的甲减大鼠肝脏HMGCR表达升高,血清胆固醇水平相应升高,通过体内实验研究发现这种升高是由于血清TSH水平升高造成的,也就是说TSH促进肝脏HMGCR表达,增加了肝脏胆固醇的合成。2、我们的研究揭示了高胆固醇血症一个新的发病机理,尤其是甲减相关的高胆固醇血症。因此,本研究对于理解和处理甲减相关的高胆固醇血症有重要的病理生理意义和临床应用价值。研究背景：在人类,饮酒与血糖升高、葡萄糖耐受不良和和葡萄糖体内稳态相关。已有报告短期饮酒可引起大鼠糖耐量异常。而对于周围组织来说,长期饮酒可降低肝脏和骨骼肌葡萄糖的利用。而且,长期饮酒损伤胰岛素受体信号。前期的研究通过葡萄糖钳夹实验已证明饮酒可降低心脏和骨骼肌胰岛素刺激的葡萄糖吸收。总之,饮酒不仅导致肝脏和骨骼肌的葡萄糖耐受,而且可引起酒精性心肌病。然而,心肌胰岛素信号的损伤和酒精性心肌病之间的关系至今不明确。尽管已有研究报道酒精损伤胰岛素信号和葡萄糖转运是由于降低了葡萄糖的氧化和利用,但是潜在的分子机制仍然不清楚。胰岛素在心肌代谢功能方面起着重要作用。胰岛素通过受体发挥各种生理作用,例如增加葡萄糖吸收,诱导基因表达和细胞增殖。胰岛素结合到受体,可激活胰岛素B受体酪氨酸激酶,进而激活胰岛素信号通路的下游分子,包括胰岛素受体底物家族。然而,有关酒精对胰岛素信号损伤的研究主要集中宅骨骼肌和脂肪组织,在心肌上几乎没有研究。组织对葡萄糖的摄取主要依赖于质膜上的葡萄糖转运体,心脏主要表达葡萄糖转运体1和4,而胰岛素依赖的葡萄糖吸收主要依赖于葡萄糖转运体4。本研究拟建立饮酒大鼠模型,观察长期饮酒对大鼠心肌胰岛素受体B亚基,胰岛素受体底物和葡萄糖转运体4表达的影响。研究目的：拟通过建立饮酒大鼠模型,观察长期饮酒对大鼠心肌胰岛素受体β亚基,胰岛素受体底物和葡萄糖转运体4表达的影响。研究方法：1、动物分组及喂养：雄性Wistar大鼠72只,按体重随机分为6组,每组12只,即对照组(蒸馏水：5g·kg-1·d-1)；大剂量饮酒组(酒精量5g·kg-1·d-1)；中剂量饮酒组(酒精量2.5g·kg-1·d-1)；小剂量饮酒组：(酒精量0.5g·kg-1·d-1)；酒精由胃管灌入,每天早9点定时灌胃,喂养时间22周。2、机体水平胰岛素敏感性评价：喂养期间监测大鼠体重和血糖水平；大鼠处死前,测空腹血糖、空腹血胰岛素和血脂水平,计算胰岛素抵抗指数(HOMA-IR),同时进行口服葡萄糖耐量试验。3、长期饮酒对经典的胰岛素信号传导通路的影响：RT-PCR检测心肌IR、IRS1、IRS2及GLUT4的mRNA水平；Western blot检测IR、IRS1、IRS2及其磷酸化和GLUT4的蛋白水平。研究结果：1、机体水平胰岛素敏感性评价：大鼠喂养22周后,各组大鼠口服葡萄糖耐量试验0、30、60、120分钟血糖无显著性差异,糖耐量曲线下面积亦无明显差别。2、与对照组比较,中等剂量和高剂量饮酒组大鼠心肌胰岛素受体B亚基,胰岛素受体底物1和葡萄糖转运蛋白体1的mRNA水平明显降低；相应地,心肌胰岛素受体B亚基,胰岛素受体底物1和葡萄糖转运蛋白体4的蛋白水平也明显降低,尤其是高剂量饮酒组大鼠。3、长期饮酒大鼠心肌磷酸化胰岛素受体β亚基和磷酸化胰岛素受体底物1的水平降低,这与心肌胰岛素受体β亚基、胰岛素受体底物1和葡萄糖转运体4蛋白水平的变化一致。结论：本研究表明长期饮酒可下调大鼠心肌胰岛素受体β亚基,胰岛素受体底物1和葡萄糖转运蛋白体1的表达。更多还原

【Abstract】 Background:It is well known that serum total cholesterol (TC) level is decreased in hyperthyroidism, or conversely increased in hypothyroidism. It is also well established that these changes are attributed to the thyroid hormone (TH) and the TH-deprived state can result in hypercholesterolemia. However, elevation in serum TC has also been noted in subclinically hypothyroid (SCH) patients with the elevated thyroid-stimulating hormone (TSH) but normal TH levels. Obviously, only TH effect fails to explain the hypercholesterolemia which occurred in SCH.In recent years, the relationship between serum TC and TSH has been a concern. For example, in a cross-sectional study of 2799 white and black participants, aged 70-79 years, a positive association between TSH and TC was found in both racial groups. In another study investigating the prevalence of SCH in 1191 middle-aged men and women, a significant increase of 0.09 mmol/L (3.5mg/dL) in TC has been estimated in men and of 0.16 mmol/L (6.2mg/dL) in women for every 1 mU/L elevation in serum TSH. Recently, Turhan et al. also reported that serum TSH was significantly correlated with plasma TC (r=0.52; P=.001). Moreover, the fifth Tromso study, an intervention trial with LT4-treated patients with SCH, resulted in a significant reduction of TC only in those whose TSH was reduced to the range 0.2-2mU/L.Although the important roles of TH in regulating cholesterol metabolism are widely accepted, all the above data cannot be explained based only upon the role of TH. In view of all the above stated findings, the possibility that the increase in the level of TSH might be responsible for the increase in cholesterol was suspected.So we hypothesize that, TSH, in addition to regulating the synthesis and secretion of TH, also involves cholesterol levels. This hypothesis is developed based upon three premises. First, besides its expression in thyroid tissue, the presence of the TSH receptor (TSHR) in nonthyroid tissues has also been studied. Meanwhile, there is increasing evidence that TSH plays a physiological function in extra-thyroidal tissues. For example, TSHR on preadipocytes are thought to induce lipolysis and a thermogenic response. Likewise, another study defines a role for TSH as a single molecular switch in the independent control of both bone formation and resorption. Apart from TSH, Sun et al established that FSH directly regulates osteoclastic bone resorption and bone mass. Both studies demonstrate that the pituitary-derived hormones may play a role on the peripheral organs, besides the endocrine glands. Secondly, our previous study demonstrated that TSHR is present in the liver, and observes that TSH stimulation of cultured liver cells results in an increase of cAMP levels. It suggests TSH may play, at least to a certain extent, a physio-pathological role in the liver in addition to its role in regulating the thyroid via the TSHR. Finally, it is well known that HMG-CoA reductase (HMGCR) is essential for cholesterol synthesis, although HMGCR is found in virtually all tissues, and the liver expresses the highest levels of this enzyme. A previous study showed TSH could induce HMGCR gene expression and increase cholesterol levels in FRTL-5 rat thyroid cells.To test our hypothesis, we attempted in the present study, to examine unknown direct effects of TSH in the regulation of HMG-CoA reductase in liver cells leading to changes in cholesterol levels.Objective:1. With the functionality of TSHR in liver cells established, the main focus of this study was to test whether TSH has a novel and independent role in up-regulation of hepatic HMGCR expression and in the pathogenesis of hypothyroidism-associated hypercholesterolemia that is mediated through the TSHR in hepatocytes.2. To investigate whether the cAMP/PKA/CREB signaling pathway was required for TSH-induced up-regulation of HMGCR.Methods: 1. To examine and demonstrate a functional coupling of the TSHR to the cAMP system in these cells. L-02 cells, a human hepatocyte-derived cell line, cultured human primary hepatocytes and murine embryonic liver cell line BNL were treated with bovine TSH (bTSH), glucagons or with forskolin, an adenylate cyclase (AC) activator to induce cAMP production as a positive control.2. In vitro experiments, we test the effect of bovine TSH (bTSH) on the expression of HMGCR, For stimulation experiments, human normal liver cell line L-02, cultured human primary hepatocytes and murine embryonic liver cell line BNL were used and cultured in serum free medium and stimulated with bTSH for the indicated time and concentrations.3. To demonstrate that TSHR was indeed necessary for the effect of TSH on the HMGCR, we used a murine monoclonal antibody (mAb), CS-17 compete for TSH binding, L-02 cells and cultured human primary hepatocytes were pre-incubated with or without CS-17 for 60 minutes, followed by bTSH (0.1μM) for an additional 60 minutes, respectively. Next, we used a lentivirus-based RNAi delivery system to infect cultured hepatocytes with short interfering RNA targeting human TSHR (TSHR-RNAi-lentivirus).4. To investigate whether the cAMP signaling pathway coupled to the TSHR was required for TSH-induced up-regulation of HMGCR, liver cells were pre-incubated with the adenylyl cyclase inhibitor SQ22536 before subsequent stimulation with bTSH.5. To test whether the elevated HMGCR mRNA level upon TSH treatment reflects elevated HMGCR promoter activity, a DNA fragment corresponding to the HMGCR promoter was fused to a luciferase gene and the resultant luciferase reporter construct was transfected into L-02 cells.6. The PKA signaling pathway is thought to play a major role in CREB activation in many types of cells. ChIP assays were performed and immunoprecipitated DNA was analyzed using qPCR to measure the induction of transcription factor binding to the HMGR promoter in response to TSH.7. CREB DNA-binding activity in L-02 cells. In addition, to assess whether TSH has any effect on CREB DNA-binding activity, EMS A was performed. Equal amounts of nuclear protein extract were prepared from untreated cells and from cells treated with 4μM TSH or with 20μM H89. Biotin-labeled CRE oligonucleotide was used as a probe.8. To further investigate the role of TSH in the regulation of HMGCR and cholesterol levels, we pursued in vivo studies in rats. To remove the effect of TSH on the production of endogenous thyroid hormone in the thyroid gland, we performed total surgical thyroidectomies (Tx) and included a control group of animals receiving sham procedures without removing the thyroid gland. We only chose those rats with a complete Tx as demonstrated by the disappearance of thyroid hormone (undetectable T4) for further experimental manipulations with administration of exogenous T4 and TSH. With this strategy, we were able to keep thyroid hormone at a constant level and the endogenous TSH at suppressed levels in the body of the animal while administering exogenous bTSH.Results:1. TSHRs are functional in liver cells.As expected, forskolin increased the liver cells cAMP level. Treatment with bTSH also significantly stimulated cAMP production over the control (p<0.001), which was approximately two-thirds of that induced by forskolin. We found that the effect of TSH on cAMP was similar to that of glucagons in liver cells. Moreover, CHO cells that did not express TSHRs showed enhanced cAMP production in response to forskolin (p＜0.001) but not to TSH.2. TSH increased the expression and enzymatic activities of HMGCR in liver cells(1) A dose-dependent increase in the level of HMGCR protein in L-02 cells was observed following bTSH stimulation for 48 h.(2) Compared with the control, the HMGCR mRNA levels increased by 92%(p= 0.015) and 150%(p= 0.004) at 1μM and 4μM bTSH, respectively. The HMGCR mRNA level induced by bTSH at 4μM was nearly twice that induced by bTSH at 0.1 μM (p= 0.004), demonstrating a dose-dependent effect of TSH on the expression of the HMGCR gene.(3) We also examined the effects of TSH on HMGCR expression in the mouse embryonic liver cell line BNL and human primary hepatocytes, and we found a significant increase in the protein levels of HMGCR upon stimulation by TSH in a concentration-dependent manner in both cell types. These results were similar to those obtained in L-02 cells.(4) HMGCR activity increased following a 48-h treatment with bTSH in a dose-dependent manner, the activity nearly doubled at 0.1μM bTSH (p=0.013) and was four-fold at 4μM bTSH (p＜0.001) in comparison with the control.3. TSH increased the production of total cholesterol in liver cellsTreatment of L-02 cells with increasing concentrations of bTSH resulted in a concentration-dependent increase in intracellular TC. The bTSH-stimulated TC levels at the 48-h treatment were higher than those at the 24-h treatment with either 1.μM or 4μM bTSH (p=0.032 and 0.004).4. TSH-stimulated responses in liver cells were dependent on the presence of TSHR(1) TSH-stimulated production of cAMP both in L-02 cells and primary human hepatocytes cultured in the presence of CS-17 was significantly lower than that in cells cultured without CS-17 (p＜0.001). Similarly, both basal and bTSH-stimulated HMGCR protein levels were substantially reduced by CS-17.(2) TSH-stimulated cAMP production was greatly diminished in cells transfected with TSHR-RNAi-lentivirus to knock down TSHR. Correspondingly, TSH-stimulated HMGCR protein expression was also greatly diminished by TSHR knockdown using the TSHR-siRNA approach. In contrast, in cells transfected with control NS lentivirus, TSH could still enhance the expression of HMGCR. Similarly, like HMGCR, knockdown of TSHR by TSHR-RNAi inhibited TSH-induced TC production in L-02 cells (p＜0.001).(3) In separate experiments, we used siRNA to knock down TSHR expression in the mouse liver cell line BNL and achieved similar results to those in the L-02 cells with the TSHR-RNAi-lentivirus transfection approach. Specifically, two different TSHR siRNAs (1# and 2#) strongly inhibited TSHR expression and effectively suppressed bTSH-stimulated HMGCR expression.5. The cAMP/PKA/CREB signaling pathway was involved in TSH-induced up-regulation of HMGCR(1) bTSH-stimulated cAMP production in both L-02 cells and primary hepatocytes was significantly inhibited by treatment with SQ22536 (0.5 mM) (p< 0.001). Similarly, the expression of HMGCR stimulated by bTSH was dramatically reduced by SQ22536 in L-02 cells (p< 0.001).(2) As a positive control, forskolin induced a 2.2-fold increase in the luciferase activity. Meanwhile,1.5-and 2.0-fold increases in activity were detected upon 0.4 and 4μM TSH treatment, respectively. Furthermore, we mutated the CRE binding site of this HMGCR promoter (TGACGTAG to TAAAAGGG). As a results, forskolin and TSH could not up-regulate its trans-activation, which strongly indicated that the CRE site is essential for TSH’s regulation of HMGCR.(3) In comparison to the control, forskolin (a positive control) markedly increased phosphorylated CREB protein (Serl33 pCREB) binding capacity (p＜0.001). Importantly, pCREB binding increased approximately 2.3-fold in response to TSH (4μM) (p＜0.001). The PKA inhibitor H-89 dramatically down-regulated this activation by TSH (p=0.019 vs. control). Likewise, knockdown of TSHR by TSHR-RNAi inhibited TSH-induced CREB activation (p=0.002 vs. TSH).(4) EMSA results showed that treatment with TSH resulted in strong CREB-specific gel retarded bands in nuclear samples extracted from L-02 cells relative to untreated cells. To investigate whether PKA is also involved in increased CERB-DNA-binding activity stimulated by TSH, PKA inhibitor H89 (20μM) were added, and faint gel retarded bands were found in the nuclear extracts, indicating involvement of PKA in the effect of TSH on CREB.6. TSH induced hepatic HMGCR expression in vivo and an increase in serum TC(1) Circulating T4 was reduced to an undetectable level while serum TSH was dramatically elevated, and this was accompanied by a significant increase in plasma TC (n=12, p= 0.041) in Tx rats compared with the sham-operated rats. When hepatic tissue proteins from Tx and Sh rats were analyzed for HMGCR and LDLR proteins, the expected bands of approximately 90 kDa and 160 kDa, respectively, were observed, with a significant increase (n=12,p=0.004) in the former and a significant decrease (n=12,p=0.038) in the latter in Tx rats relative to Sh animals.(2) Administration of T4 to Tx rats reduced the elevated serum TC to levels similar to those observed in sham-operated control rats. We then administered exogenous bTSH to these Tx rats at 0.05 and 0.3 IU/rat daily for seven days while they received daily T4,. There was no significant difference in the serum T4 levels in the group of Tx rats receiving only exogenous T4 compared with the group of Tx rats receiving both exogenous T4 and TSH (p>0.05). We observed an increase in serum TC after administration of exogenous TSH although this increase marginally failed to reach statistical significance (n=12, p=0.084 and 0.067) when comparing the group of Tx rats receiving only exogenous T4 with the group of Tx rats receiving both exogenous T4 and TSH. However, in the same group of Tx rats constantly receiving exogenous T4, a significant increase in serum TC was observed after TSH injection compared with pre-injection, with 1.2-and 1.5-fold increases with injections of 0.05 IU TSH (p=0.017) and 0.3 IU TSH (p=0.002), respectively.(3) In Tx rats consistently receiving T4, administration of exogenous TSH, particularly at the higher dose, significantly increased the protein level of hepatic HMGCR. In contrast, although the level of hepatic LDLR protein in Tx rats was increased by administration of T4, no further increase was observed after additional administration of exogenous TSH at either dose.Conclusion:1. In this article, we have examined that TSH involve in expression of HMGCR, which can be validly investigated by means of the available in vitro and vivo experimental models. In vitro experiments, TSH, in large part or totally through adenylyl cAMP/PKA/CREB signaling pathway induce cell HMGCR expression and this effect was dependent of TSHR. Hypothyroidism animal model have been extensively used for some functional studies. In agreement with our vitro findings, the present study also showed that increased serum cholesterol levels seen in hypothyroidism animals are associated with increased expression of hepatic HMGCR. Moreover, the relationship between the increase in hepatic HMGCR and increase in serum cholesterol were in response to an increase of TSH level.2. Our findings provide a novel mechanism in the pathogenesis of hypercholesterolemia in certain human diseases, particularly in hypothyroidism, a common endocrine disorder. Therefore, this study has important pathological and clinical implications for the understanding and management of hypercholesterolemia in hypothyroidism. Background:In humans, ethanol consumption is associated with the increased circulating glucose concentration, glucose intolerance, and glucose homeostasi. It has been reported that short-term ethanol treatment in rats led to glucose intolerance similar to that reported in humans. As to organs, long-term ethanol feeding to rats decreased glucose utilization in isolated hepatocytes and skeletal muscles. Moreover, it has been shown that chronic ethanol administration impaired hepatic insulin receptor signaling. Previous studies have stated that during hyperinsulinemic-euglycemic clamping, ethanol decreased insulin-stimulated glucose utilization in the heart and the skeletal muscles of overnight-fasted rats. Taken together, these data suggested that ethanol could not only result in glucose intolerance in hepatocytes and skeletal muscles, but could also lead to alcoholic cardiomyopathy. However, the relationship between the dysfunctional cardiac muscle insulin signals and the development of alcoholic cardiomyopathy has not been given much attention. Nevertheless, Xu et al. have reported that ethanol probably impaired the pathways of insulin signaling and glucose transport, due to the decrease in glucose oxidation and utilization. However, the underlying molecular mechanisms still remain unknown.Insulin plays a key role in the regulation of various aspects of cardiac muscle metabolism and function. The binding of insulin to its receptor initiates the regulation of diverse physiologic effects, such as increased uptake of glucose, induction of gene expression, and cellular proliferation. The insulin receptor (IR) is a tetrameric enzyme comprised of two extracellular a-subunits and two transmembraneβ-subunits. On the other hand, the binding of insulin to the extracellular part of IR activates the intrinsic tyrosine kinase activity of the P-subunits of the receptor. Once activated and phosphorylated, IR binds via its phosphotyrosine residues, along with a series of downstream elements, including the IR substrate (IRS) family and Shc. However, most studies on insulin signaling in ethanol consumption dealt with skeletal muscles or adipose tissues, and fewer studies have been conducted on cardiac muscles.The uptake of glucose depends on the presence of glucose transporters in the plasma membrane. Between Glutl and Glut4, the two glucose transporters expressed in the heart, Glut4 is considered to be the main contributor for the regulation of glucose uptake by insulin. Manipulation of Glut4 levels in transgenic mice has revealed that glucose homeostasis is highly sensitive to the level of Glut4 expression.Here, we prepared rats which received ethanol, to observe the effects of chronic ethanol intake on the expression of IRβ,IRS-1, and Glut4 in rat cardiac muscles.Objective:To investigate the effect of chronic ethanol intake on the expression of insulin receptorp-subunit (IRP), insulin receptor substrate-1 (IRS-1), and glucose transporter 4 (Glut4) in rat cardiac muscle.Methods:1. Animal feeding:Seventy-two male Wistar rats, divided into six groups, received either distilled water (C, control group) or edible ethanol, which was administered by gastric tube with a single daily dose:5 g-kg-1 (H, high dose group), 2.5 g·kg-1 (M, middle dose group) and 0.5 g-kg-1 (L, little dose group), The total feeding time was 22 weeks.2. Evaluation of insulin sensitivity:Fasting plasma glucose levels were monitored during fed period. At the end of 22 weeks, fasting serum insulin levels were measured and oral glucose tolerance test (OGTT) were performed, and calculate the HOMA-IR, repectively.3. Effects of ethanol on classical insulin signal pathway:the mRNA levels of IR, IRS1, IRS2 and GLUT4 of rat heart muscle were measured using RT-PCR method, the protein level of IR, IRS1, IRS2 as well as their phosphorylation and GLUT4 was detected by western blotting method.Results:1. Evaluation of insulin sensitivity at body level:with being treated for 22 weeks, for fasting plasma glucose, fasting serum insulin and 2 h plasma glucose levels after oral glucose load, we failed to detect the significant differences between control and each treated group.2. Compared with the control group, the IRβ, IRS-1, and Glut4 mRNA levels in groups H and M decreased remarkably. In addition, the protein levels of IRβ, IRS, and Glut4 showed a corresponding decline in ethanol-treated groups, especially in group H.3. The PY-IR p and PY-IRS-1 protein levels decreased in the hearts of ethanol-treated rats with corresponding changes in the IR P and IRS-1 protein levels.Conclusions:The present study showed that chronic ethanol intake could down-regulate the expression levels of IRP, IRS-1, and Gut4 in rat cardiac muscles.更多还原