CN102007410A - 脂联素分泌调节剂的评价或选择方法 - Google Patents
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Abstract
本发明提供一种简易且迅速地评价或选择脂联素分泌调节剂的方法。脂联素分泌调节剂的评价或选择方法，其特征在于：包含以下(A)～(C)的工序：(A)将受试物给予实验动物的工序；(B)对实验动物施加睡眠障碍的负担的工序；(C)测定血液中的脂联素浓度，评价其变化的工序。
Description
技术领域
本发明涉及脂联素分泌调节剂的评价或选择方法。
背景技术
脂联素(adiponectin)是主要从脂肪细胞中分泌的因子(脂肪细胞因子(adipocytokine))，近年来，据报道其具有胰岛素抵抗性改善作用和抗动脉硬化作用(非专利文献1)。作为脂联素以外的脂肪细胞因子，已知有TNF-α、抵抗素、游离脂肪酸等，已知它们都会产生胰岛素抵抗性。由于脂联素与多数脂肪细胞因子不同，是改善胰岛素抵抗性的好的脂肪细胞因子，因此近来受到极大注意。
脂联素不仅有该作用，而且作为临床数据表示血液中脂联素量的降低是糖尿病发病的预先标志(非专利文献2)。另外，报道了血液中脂联素的量显示出与肥胖度呈逆相关(非专利文献3)，推测在由肥胖导致的胰岛素抵抗性、糖尿病、心血管疾病的发病、恶化中，脂联素的降低起到了重要作用。因此，认为促进脂联素分泌的物质对上述各疾病的预防、改善是有用的。
确立了几个评价脂联素表达量的体外试验体系，例如，有报道使用使作为脂肪前体细胞株的3T3-L1细胞分化的体系，通过添加TNF-α(肿瘤坏死因子α)、胰岛素、地塞米松等，使脂联素表达量降低(非专利文献4)。
另外，在使用腹部手术时取出的脂肪细胞的试验体系中也可以评价脂联素表达量(非专利文献5)。另外，报道了通过使用保持有脂联素的增强子元件和报道基因的细胞，可以体外筛选脂联素表达诱导剂(专利文献1)。
另一方面，体内试验体系中，KKAy小鼠(非专利文献6)、db/db小鼠(非专利文献7)、ob/ob小鼠(非专利文献8)、C57BL/6小鼠(非专利文献9)、Zucker fatty(非专利文献10)等肥胖糖尿病模型动物由于食入高脂肪，引起肥胖、胰岛素抵抗性、高中性脂血症等，并且随着肥胖血液中脂联素量降低，因此，使用该肥胖糖尿病模型动物来评价脂联素分泌能力等。
但是，问题在于：食入高脂肪直至血液中脂联素的量降低需要至少数周的饲养，为了评价脂联素分泌量的变化而需要大量时间。
专利文献
专利文献1：国际公开第2005/094866号小册子
非专利文献
非专利文献1：Kadowaki，T.et al.，J.Clin.Invest.，116：1784.1792(2006).
非专利文献2：Spranger，J.et al.，Lancet.，361：226-228(2003).
非专利文献3：Arita，Y.et al.，Biochem.Biophys.Res.Commun.，257：79-83(1999).
非专利文献4：Fasshauer.et al.，Biochem.Biophys.Res.Commun.290：1084-1089(2002).
非专利文献5：Halleux，CM.et al.，Biochem.Biophys.Res.Commun.288：1102-1107(2001)
非专利文献6：Tsuchida A et al.，Diabetes.，54(12)：3358-3370(2005).
非专利文献7：Fujita H et al.，Endocrine J.，52：427-433(2005).
非专利文献8：Haluzik M et al.，Endocrinology.，145：3258-3264(2004).
非专利文献9：Araki K et al.，Hypertension.，48：51-57(2006).
非专利文献10：Galisteo M et al.，J.Nutr.135：2399-2404(2005).
发明内容
本发明涉及一种脂联素分泌调节剂的评价或选择方法，其特征在于，包含以下(A)～(C)的工序：
(A)将受试物给予实验动物的工序；
(B)对实验动物施加睡眠障碍的负担的工序；
(C)测定血液中的脂联素浓度，评价其变化的工序。
附图说明
图1是表示由于快速眼动睡眠(REM sleep)阻碍造成的血浆脂联素降低的图。*P＜0.05(Dunnet检验，vs休息组)
图2是表示绿原酸对于由于快速眼动睡眠阻碍造成的血浆脂联素的降低的效果的图。*P＜0.05(Fisher’s PLSD检验)
具体实施方式
本发明涉及提供一种简易且迅速地评价或选择体内脂联素分泌调节剂的方法。
本发明人研究讨论使用动物的筛选体系时，意外地发现在睡眠障碍模型中，负担睡眠障碍后血液中脂联素浓度短时间内降低，并且发现通过使用该模型，可以评价或选择脂联素分泌调节剂。
根据本发明，可以简易且迅速地评价或选择脂联素分泌调节剂。因此，本发明作为糖代谢异常、脂代谢异常等代谢异常综合症和由此引发的各种疾病的预防或改善剂的简易筛选法是有用的。
如后述实施例所示，在诱发睡眠障碍的动物模型中，负担睡眠障碍后，血液中脂联素浓度短时间内降低。而且，该血液中脂联素浓度的降低可以通过给予绿原酸而得到抑制，该绿原酸据报道具有脂联素分泌促进作用(日本特开2005-232059号公报)，而且具有体内胰岛素抵抗性改善效果、以及降低胆固醇和甘油三酯的作用(Journal of Nutritional Biochemistry 13(2002)，717-726)。因此，通过使用睡眠障碍模型体系，可以对促进或抑制脂联素分泌的脂联素分泌调节剂进行评价或选择。
以下，对于本发明脂联素分泌调节剂的评价或选择方法进行说明。
作为本发明中使用的实验动物，只要是作为睡眠障碍模型而可以使用的实验小动物即可，例如，优选小鼠、大鼠、豚鼠等啮齿类。
在本发明中，作为附加给动物的“睡眠障碍”，可以列举出以研讨在睡眠生物体中意义为目的而进行考察的由公知的睡眠障碍方法所诱导的睡眠障碍。其中，优选阻碍快速眼动睡眠的睡眠障碍。
作为公知的睡眠障碍方法，可以列举出如下所示的方法，其中，优选使用平台法(platform method)。
1)平台法(Youngblood BD et al.，Physiol Behav.67(5)：643-649，(1999))，平台法也称为花盆(flower pot)法，是代表性的睡眠阻碍方法。
该方法是在睡眠中比较特定地阻碍快速眼动睡眠的方法。
该方法是在笼中盛满水，并设置实验动物可以站的小圆柱台(平台)的方法。动物可以在平台上休息，但由于进入快速眼动睡眠时肌肉松弛而造成体势随便，身体接触水面，因此动物成为可以得到非快速眼动睡眠，但不可以得到快速眼动睡眠的状态。
具体而言，将直径6～7cm的不锈钢制圆柱放入大鼠饲养用丙烯酸树脂笼中，水加至比圆柱上部低1～2cm处。其中放入1只250～400g大鼠进行饲养。这时使大鼠可以自由摄取饲料和饮用水。
2)活动平板(treadmill)或圆盘(disk)法(Guzman-Marin R et al.Eur J Neurosci.22(8)：2111-2116(2005)、Everson CA et al.Am J Physiol Endocrinol Metab.286：1060-1070(2004))
该方法是在笼中放入活动平板或转动的圆盘，通过定时运转平板或圆盘来阻碍睡眠的方法。
3)饲养中放出噪音来阻碍睡眠的方法(Rabat A et al.Brain Res.1059：82-92(2005))
该方法是用扩音器不定期地放出频率为20～300赫兹且强度为70～80分贝的声音，从而阻碍睡眠的方法。
4)对于动物通过触摸来阻碍睡眠的方法(Toru M et al.Pharmacol Biochem Behav.20(5)：757-761(1984))
该方法是在动物进入睡眠时，通过用手触碰来阻碍睡眠的方法。
负担睡眠障碍的时间根据睡眠障碍的方法而不同，通常是1天～5天左右。在使用平台法的情况下，由于血液中脂联素浓度的降低在负担睡眠障碍后1天则显著降低，因此只要1～2天左右即可。
受试物的给予时间可以根据目的适当选择。也可以在实验动物负担睡眠障碍前或后给予，或者也可以与负担睡眠障碍同时给予。
给予方法可以是经口给予、经皮给予、皮下给予、皮内给予、肌内给予、尾静脉给予、腹腔内给予等，但优选为经口给予。
血清或血浆中脂联素的测定可以通过ELISA(酶抗体)法、放射免疫分析法、蛋白质印迹法等进行。
具体而言，可以使用大鼠脂联素ELISA试剂盒(AdipoGen公司)等进行。
通过以上所述，基于血液中脂联素浓度，可以评价或选择脂联素的分泌调节物质。在该情况下，例如如果其浓度与没有给予受试物的对照浓度相比有统计学的显著增高，则可以评价或选择该受试物作为脂联素分泌促进剂。
这里，作为脂联素分泌促进剂，可以列举出增强脂联素的表达等，并改善低脂联素状态的物质。这样的脂联素分泌促进剂可以成为用于预防、治疗或改善由于低脂联素状态而引起的疾病或症状，例如，低脂联素血症、糖耐量异常、糖尿病、2型糖尿病、胰岛素抵抗综合症、糖尿病并发症、高血糖、动脉硬化、动脉粥样硬化性心血管病、心血管疾病、脑血管疾病、血管狭窄、末梢血管疾病、动脉瘤、高血脂、高胆固醇血症、肥胖等的医药品或食品。
实施例
1.方法
将SD大鼠(雄性，10～11周龄)按照体重相等的方式进行分组。在笼中设置大鼠可以休息的直径为6.0cm、高为2.5cm的圆柱平台，水加至平台下1em处。在该笼中饲养1～4天(1只/1笼)。休息组是在放有通常的床的笼子中进行饲养(各组N＝4)。给予绿原酸的实验是在用于进行30小时快速眼动睡眠阻碍的笼中进行饲养，绿原酸(5-CQA，sigma公司)给予组以150mg/kg(300mg/kg/day)的用量每12小时进行3次利用探针的经口给予。对照组给予蒸馏水，休息组在放有通常的床的笼子中进行饲养并给予蒸馏水(各组N＝6)。在快速眼动睡眠阻碍结束后，直接在Forane(大日本制药)麻醉下，用VENOJECT真空采血管从腹大动脉采集血液，将得到的肝素血浆在-80℃下保存至使用。血浆脂联素通过大鼠脂联素ELISA试剂盒(AdipoGen公司)进行测定。将血浆用试剂盒中的稀释缓冲液稀释至1000倍后，按试剂盒规程测定脂联素浓度。
2.结果
(1)快速眼动睡眠阻碍对于血浆脂联素的影响
在快速眼动睡眠阻碍1天后，血浆脂联素与休息组相比已经显著下降，在快速眼动睡眠阻碍2、4天后，血浆脂联素与休息组相比显著下降。然而，血浆脂联素的降低在快速眼动睡眠阻碍期间无依存，在快速眼动睡眠阻碍的1～4天中下降的程度相同(图1)。
(2)绿原酸对由于快速眼动睡眠阻碍造成的血浆脂联素降低的效果
由于快速眼动睡眠阻碍造成血浆脂联素显著降低。绿原酸(CQA)显著地抑制了由于快速眼动睡眠阻碍造成的血浆脂联素的降低(图2)。
根据以上所述，由于快速眼动睡眠阻碍体系可以在非常短时间内使血中脂联素降低，因此，如果使用该体系，可以效率良好地筛选脂联素分泌调节剂。
Claims (4)
1.一种脂联素分泌调节剂的评价或选择方法，其特征在于，
包含以下(A)～(C)的工序：
(A)将受试物给予实验动物的工序；
(B)对实验动物施加睡眠障碍的负担的工序；
(C)测定血液中的脂联素浓度，评价其变化的工序。
2.如权利要求1所述的方法，其中，
所述睡眠障碍的负担是快速眼动睡眠阻碍。
3.如权利要求2所述的方法，其中，
所述快速眼动睡眠阻碍是由平台法造成的睡眠阻碍。
4.如权利要求1～3中任意一项所述的方法，其中，
所述实验动物为大鼠或小鼠。
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| EP1757302A1 (en) * | 2004-03-31 | 2007-02-28 | Toudaitlo, Ltd. | Adiponectin expression inducer and utilization of the same |
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