JP2011080908A - METHOD OF EVALUATING OR SELECTING Adipo R2 EXPRESSION REGULATING AGENT - Google Patents
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Abstract
Description
本発明は、AdipoR2発現調節剤の評価又は選択方法に関する。 The present invention relates to a method for evaluating or selecting an AdipoR2 expression regulator.
アディポネクチン(adiponectin)は、主に脂肪細胞から分泌される因子（アディポカイン）であるが、近年インスリン抵抗性改善作用や抗動脈硬化作用があることが報告されている（非特許文献１）。アディポネクチン以外のアディポカインとして、TNF-α、レジスチン、遊離脂肪酸などが知られているが、これらはいずれもインスリン抵抗性を惹起することが明らかとなっている。アディポネクチンは多くのアディポカインとは異なりインスリン抵抗性を改善する善玉アディポカインであることから、最近、非常に注目されている。 Adiponectin is a factor (adipokine) secreted mainly from adipocytes, but has recently been reported to have an insulin resistance improving action and an anti-arteriosclerotic action (Non-patent Document 1). As adipokines other than adiponectin, TNF-α, resistin, free fatty acid and the like are known, and it has been clarified that all of them induce insulin resistance. Adiponectin, which is a good adipokine that improves insulin resistance, unlike many adipokines, has recently received much attention.
アディポネクチンはその作用だけではなく、血中アディポネクチン量の低下が糖尿病発症の予知マーカーになることも臨床データとして示されている（非特許文献２）。また、血中アディポネクチン量は肥満度と逆相関を示すことが報告されており（非特許文献３）、肥満によるインスリン抵抗性、糖尿病、循環器疾患の発症、増悪においてアディポネクチンの低下が重要な役割を果たしていると推察されている。従って、アディポネクチンの分泌を促進する物質は、これら各疾病の予防・改善に有用であると考えられている。 It has been shown as clinical data that not only the action of adiponectin but also a decrease in blood adiponectin level is a predictive marker for the onset of diabetes (Non-patent Document 2). In addition, it has been reported that the amount of adiponectin in the blood shows an inverse correlation with the degree of obesity (Non-patent Document 3), and a decrease in adiponectin is an important role in the onset and exacerbation of insulin resistance, diabetes and cardiovascular disease due to obesity. It is presumed that Therefore, substances that promote the secretion of adiponectin are considered to be useful for the prevention and improvement of these diseases.
アディポネクチンの生体での作用にはアディポネクチン受容体を介した細胞内シグナル伝達が関与していることが知られている。現在、アディポネクチンには２種類の受容体（AdipoR1、AdipoR2）が知られており、AdipoR1からのシグナルは５’−ＡＭＰ−活性化プロテインキナーゼ（ＡＭＰＫ）を介して脂肪酸酸化を促進し、インスリン感受性を向上させると考えられている。またAdipoR2からのシグナルはペルオキシゾーム増殖因子−活性化受容体（ＰＰＡＲ）αの活性化を介して抗炎症作用や抗酸化ストレス作用を起こし、インスリン感受性を向上させると考えられている（非特許文献４）。従って、アディポネクチン受容体の発現を活性化する物質を有効成分とする肥満、インスリン抵抗性、糖尿病、循環器疾患、メタボリックシンドローム等の疾患の予防又は治療剤が提唱されている（特許文献１、２）。 It is known that intracellular signal transduction through an adiponectin receptor is involved in the action of adiponectin in the living body. Currently, two types of receptors (AdipoR1 and AdipoR2) are known for adiponectin, and the signal from AdipoR1 promotes fatty acid oxidation via 5'-AMP-activated protein kinase (AMPK) to increase insulin sensitivity. It is thought to improve. The signal from AdipoR2 is considered to cause an anti-inflammatory action and an antioxidant stress action through activation of peroxisome growth factor-activated receptor (PPAR) α, thereby improving insulin sensitivity (non-patent literature). 4). Therefore, prophylactic or therapeutic agents for diseases such as obesity, insulin resistance, diabetes, cardiovascular disease, metabolic syndrome and the like, which use substances that activate the expression of adiponectin receptor as active ingredients have been proposed (Patent Documents 1 and 2). ).
アディポネクチンやアディポネクチン受容体の賦活剤をスクリーニングするための方法がいくつか知られている。in vitro の系としては、培養細胞を用いる方法（特許文献１、非特許文献５）、腹部の手術の際に摘出された脂肪細胞を用いる方法（非特許文献６）、アディポネクチンのエンハンサーエレメントとレポータージーンを保持した細胞を用いる方法(特許文献３）等が知られている。 Several methods for screening for adiponectin and adiponectin receptor activators are known. Examples of in vitro systems include a method using cultured cells (Patent Document 1, Non-Patent Document 5), a method using fat cells removed during abdominal surgery (Non-Patent Document 6), an adiponectin enhancer element and a reporter gene. There is known a method using a cell that retains (Patent Document 3) and the like.
一方、in vivoでの評価方法としては、KKAyマウス（非特許文献７）、db/dbマウス（非特許文献８）、ob/obマウス（非特許文献９）、C57BL/6マウス（非特許文献１０）、Zucker fattyラット（非特許文献１１）などの肥満糖尿病モデル動物を用いる方法が知られている。これらの動物は高脂肪食下で肥満に伴い血中アディポネクチン量が低下する。また、特許文献１には、肥満糖尿病モデル動物でAdipoR1及びAdipoR2の発現が摂食量やインスリンレベルに伴って増減することが報告されている。従って、これらの動物はアディポネクチンやアディポネクチン受容体の賦活剤のスクリーニングに用いることができる。 On the other hand, in vivo evaluation methods include KKAy mice (Non-patent document 7), db / db mice (Non-patent document 8), ob / ob mice (Non-patent document 9), C57BL / 6 mice (Non-patent document). 10), a method using a model animal for obesity diabetes such as Zucker fatty rat (Non-patent Document 11) is known. In these animals, the amount of adiponectin in blood decreases with obesity under a high fat diet. Patent Document 1 reports that the expression of AdipoR1 and AdipoR2 increases and decreases with food intake and insulin level in obese diabetes model animals. Therefore, these animals can be used for screening for adiponectin and adiponectin receptor activators.
しかしながら、上記のような肥満糖尿病モデル動物を用いる場合、アディポネクチン賦活剤の生体内での効果をより直接的に評価できるという利点がある一方で、動物を血中アディポネクチン量やアディポネクチン受容体発現量が低下するまで高脂肪食下で少なくとも数週間予め飼育する必要があるため、スクリーニング結果を得るまでに多大な時間を要する。 However, when using an obese diabetes model animal as described above, there is an advantage that the in vivo effect of the adiponectin activator can be evaluated more directly, while the animal has a blood adiponectin level and adiponectin receptor expression level. Since it is necessary to keep in advance under a high fat diet for at least several weeks until it decreases, it takes a long time to obtain a screening result.
本発明は、in vivoでアディポネクチン受容体２（AdipoR2）発現を調節する物質を簡易且つ迅速に評価又は選択する方法を提供することに関する。 The present invention relates to providing a method for simply and rapidly evaluating or selecting a substance that regulates expression of adiponectin receptor 2 (AdipoR2) in vivo.
本発明者らは、睡眠障害モデル動物において、AdipoR2の発現が睡眠障害負荷後、短期間で顕著に低下すること、当該モデルを用いることによりアディポネクチンAdipoR2発現調節剤の迅速な評価又は選択が可能となることを見出した。 In the sleep disorder model animal, the present inventors can significantly reduce the expression of AdipoR2 in a short period after sleep disorder load, and by using this model, it is possible to quickly evaluate or select an adiponectin AdipoR2 expression regulator. I found out that
すなわち、本発明は、下記の１）〜４）に係るものである。
１）以下の（Ａ）〜（Ｃ）の工程を含むことを特徴とする、アディポネクチン受容体２（AdipoR2）発現調節剤の評価又は選択方法。
（Ａ）被験物質を実験動物に投与する工程
（Ｂ）当該動物に睡眠障害を負荷する工程
（Ｃ）当該動物におけるAdipoR2発現量を測定し、その変化を評価する工程
２）睡眠障害の負荷がレム睡眠阻害である１）記載の方法。
３）レム睡眠阻害がプラットホーム法による睡眠阻害である２）記載の方法。
４）実験動物がラット又はマウスである１）〜３）のいずれか１に記載の方法。
That is, the present invention relates to the following 1) to 4).
1) A method for evaluating or selecting an adiponectin receptor 2 (AdipoR2) expression regulator, comprising the following steps (A) to (C):
(A) A step of administering a test substance to an experimental animal (B) A step of loading the animal with sleep disorder (C) A step of measuring the expression level of AdipoR2 in the animal and evaluating the change 2) A load of sleep disorder The method according to 1), which is REM sleep inhibition.
3) The method according to 2), wherein the REM sleep inhibition is sleep inhibition by a platform method.
4) The method according to any one of 1) to 3), wherein the experimental animal is a rat or a mouse.
本発明によれば、睡眠障害モデル動物におけるAdipoR2発現の変化を観察することにより、生体内のアディポネクチン活性に影響を調節し得る物質を短期間にスクリーニングすることが可能になる。本発明の方法は、肥満糖尿病モデル動物に高脂肪食を接触させることを必要とする従来のスクリーニング方法と比較してより迅速且つ簡便である。また本発明によれば、高価な肥満糖尿病モデル動物を使用する必要がないためより安価なスクリーニングが実現できる。従って、本発明は、糖質代謝異常、脂質代謝異常等の代謝異常症候群及びそれに起因する各種疾患の予防又は改善剤の簡便、迅速且つ安価なスクリーニング法として有用である。 According to the present invention, by observing changes in AdipoR2 expression in a sleep disorder model animal, it becomes possible to screen for a substance capable of regulating the influence of adiponectin activity in vivo in a short time. The method of the present invention is quicker and simpler than conventional screening methods that require a high fat diet to be contacted with an obese diabetes model animal. Moreover, according to the present invention, since it is not necessary to use an expensive obese diabetes model animal, a cheaper screening can be realized. Therefore, the present invention is useful as a simple, rapid and inexpensive screening method for preventive or ameliorating agents for metabolic disorders such as abnormal carbohydrate metabolism and abnormal lipid metabolism and various diseases resulting therefrom.
後記実施例に示すとおり、睡眠障害を誘発した動物モデルにおいて、肝臓におけるアディポネクチン受容体２（AdipoR2）の発現量は睡眠障害負荷後短期間で低下した。一方、アディポネクチン受容体１（AdipoR1）の発現量には変化が生じなかった。従来、アディポネクチン受容体の発現を睡眠障害モデルで調べた研究は報告されていない。また従来、前述のように、AdipoR1とAdipoR2からのアディポネクチンの作用は異なることがわかっていたが、AdipoR1とAdipoR2とを個別に調節することは提唱されていなかった。一方、この睡眠障害動物モデルを用いた本発明の方法によれば、アディポネクチンの多様な作用の中でAdipoR2からの作用を特異的に促進することができる物質を評価または選択することができる。 As shown in Examples described later, in an animal model in which sleep disorder was induced, the expression level of adiponectin receptor 2 (AdipoR2) in the liver decreased in a short period after sleep disorder loading. On the other hand, there was no change in the expression level of adiponectin receptor 1 (AdipoR1). Heretofore, there has been no report on studies in which the expression of adiponectin receptor was examined using a sleep disorder model. Conventionally, as described above, it has been known that the actions of adiponectin from AdipoR1 and AdipoR2 are different, but it has not been proposed to individually regulate AdipoR1 and AdipoR2. On the other hand, according to the method of the present invention using this animal model of sleep disorder, a substance capable of specifically promoting the action from AdipoR2 among various actions of adiponectin can be evaluated or selected.
本発明のAdipoR2発現調節剤の評価又は選択方法は、（Ａ）被験物質を実験動物に投与する工程；（Ｂ）当該動物に睡眠障害を負荷する工程；及び（Ｃ）当該動物におけるAdipoR2発現量を測定し、その変化を評価する工程、を含むことを特徴とする。本発明において用いられる実験動物としては、睡眠障害のモデルとして使用可能な実験小動物であればよいが、例えばマウス、ラット、モルモット等げっ歯類が好ましい。 The method for evaluating or selecting the AdipoR2 expression regulator of the present invention comprises: (A) a step of administering a test substance to an experimental animal; (B) a step of loading the animal with sleep disorders; and (C) an AdipoR2 expression level in the animal. And measuring the change. The experimental animal used in the present invention may be an experimental small animal that can be used as a sleep disorder model, but for example, rodents such as mice, rats, guinea pigs are preferred.
工程（Ａ）で動物に投与される被験物質としては、AdipoR2発現調節作用を有することが期待される物質であれば、特に限定されない。被験物質の投与時期は、目的に応じて適宜選択すれば良く、実験動物に睡眠障害を負荷する前でも後でもよく、或いは睡眠障害の負荷と並行して行うことでも良い。例えば、被験物質を実験動物に睡眠障害を負荷する前に投与した場合、AdipoR2発現低下予防剤を評価又は選択することができ、被験物質を実験動物に睡眠障害を負荷する後に投与した場合、AdipoR2発現促進剤又はAdipoR2発現低下改善剤を評価又は選択することができるが、本発明の方法によるAdipoR2発現低下予防・改善剤の評価または選択の手順は上記に限定されない。投与方法は、経口投与、及び経皮投与、皮下投与、皮内投与、筋肉内投与、尾静脈投与、腹腔内投与等の非経口投与の何れでもよいが、好ましくは経口投与である。 The test substance administered to the animal in the step (A) is not particularly limited as long as it is a substance expected to have an AdipoR2 expression regulating action. The administration time of the test substance may be appropriately selected according to the purpose, and may be before or after the sleep disorder is loaded on the experimental animal, or may be performed in parallel with the load of the sleep disorder. For example, if the test substance is administered before the sleep disorder is loaded on the experimental animal, an AdipoR2 expression lowering preventive agent can be evaluated or selected, and if the test substance is administered after the test animal is loaded with the sleep disorder, AdipoR2 Although an expression promoter or an AdipoR2 expression decrease improving agent can be evaluated or selected, the procedure for evaluating or selecting an AdipoR2 expression decrease preventing / ameliorating agent according to the method of the present invention is not limited to the above. The administration method may be any of oral administration and parenteral administration such as transdermal administration, subcutaneous administration, intradermal administration, intramuscular administration, tail vein administration, intraperitoneal administration, etc., but oral administration is preferred.
工程（Ｂ）において動物に負荷される「睡眠障害」としては、睡眠の生体における意義を検討する目的で考案されている公知の睡眠障害方法によって誘発される睡眠障害が挙げられ、このうちレム睡眠を阻害するものが好ましい。
公知の睡眠障害方法としては、以下に示すような方法が挙げられるが、中でもプラットホーム法を用いるのが好ましい。
１）プラットホーム法（Youngblood BD et al. Physiol Behav. 67(5) 643-649,(1999)）。プラットホーム法は、フラワーポット法とも呼ばれ、代表的な睡眠阻害方法である。この方法は睡眠のなかでもレム睡眠を比較的特異的に阻害できる方法である。この方法は、ケージの中に水を張り、実験動物がのることができる小さな円柱の台（プラットホーム）を設置するものである。動物はプラットホーム上で休むことができるが、レム睡眠に入ると筋肉が弛緩するため体勢が崩れ水面に体が触れることから、動物はノンレム睡眠をとることはできるが、レム睡眠をとることができない状態になる。
具体的な例としては、ラットを用いる場合、直径6-7cmのステンレス製の円柱をラット飼育用アクリル樹脂性ケージに入れ、円柱の上部より1-2cm下まで水を張る。これに、250〜400gのラットを1匹ずつ入れ、飼育する。このときラットが餌と飲用水を自由に摂取することができるようにする。
Examples of the “sleep disorder” loaded on the animal in the step (B) include a sleep disorder induced by a known sleep disorder method devised for the purpose of examining the significance of sleep in the living body. What inhibits is preferable.
Examples of known sleep disorder methods include the methods described below. Among them, the platform method is preferably used.
1) Platform method (Youngblood BD et al. Physiol Behav. 67 (5) 643-649, (1999)). The platform method is also called a flower pot method and is a typical sleep inhibition method. This method is a method capable of relatively specifically inhibiting REM sleep among sleep. In this method, water is placed in a cage and a small cylindrical platform (platform) on which experimental animals can be placed is installed. Animals can rest on the platform, but when they enter REM sleep, the muscles relax and the body collapses and the body touches the water surface, so the animal can take non-REM sleep, but cannot take REM sleep It becomes a state.
As a specific example, when a rat is used, a stainless steel cylinder having a diameter of 6-7 cm is placed in an acrylic resin cage for rearing a rat, and water is applied to 1-2 cm below the upper part of the cylinder. One 250-400 g rat is put in this and reared. At this time, rats should be able to freely consume food and drinking water.
２）トレッドミル又はディスク法（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).）。この方法は、ケージの中に、トレッドミル又は回転するディスクを入れておき、定期的にトレッドミルまたはディスクを稼動させることにより、睡眠を阻害する方法である。 2) Treadmill or disk method (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) .). In this method, a treadmill or a rotating disk is placed in a cage, and the treadmill or disk is periodically operated to inhibit sleep.
３）飼育中に騒音を出して睡眠を阻害する方法（ Rabat A et al. Brain Res. 1059:82-92 (2005)）。この方法は、振動数が20-300ヘルツで強さが70-80デシベルの音を不定期にスピーカーより流し、睡眠を阻害するものである。 3) A method of disturbing sleep by making noise during breeding (Rabat A et al. Brain Res. 1059: 82-92 (2005)). In this method, a sound with a frequency of 20-300 Hz and a strength of 70-80 decibels is played irregularly from the speaker, and sleep is disturbed.
４）動物に対するハンドリングにより睡眠を阻害する方法（Toru M et al. Pharmacol Biochem Behav. 20(5):757-761 (1984)）。この方法は、動物が睡眠に入ろうとするときに手で触ることにより睡眠を阻害するものである。 4) A method of inhibiting sleep by handling animals (Toru M et al. Pharmacol Biochem Behav. 20 (5): 757-761 (1984)). This method inhibits sleep by touching an animal when the animal is about to go to sleep.
睡眠障害負荷の期間は、睡眠障害の方法によっても異なり、通常１日〜５日程度であるが、プラットホーム法を用いた場合、AdipoR2発現は、睡眠障害負荷後２日で有意に低下することから、２日程度であればよい。 The sleep disorder load period varies depending on the sleep disorder method, and is usually about 1 to 5 days. However, when the platform method is used, AdipoR2 expression decreases significantly two days after the sleep disorder load. It may be about 2 days.
工程（Ｃ）におけるAdipoR2発現量の測定は、生体から採取した試料中のAdipoR2発現量を任意の方法で測定すればよい。試料としては、脂肪組織および筋肉、肝臓、心臓、血管等の組織が挙げられ、肝臓が好ましい。AdipoR2発現量測定法としては、ｍＲＮＡ、タンパク質発現を測定する方法が挙げられ、ｍＲＮＡ発現量の測定が好ましい。発現量の測定方法としては、各種イムノアッセイ、ノサンブロット、ＲＴ−ＰＣＲ、リアルタイム定量ＰＣＲ、ディファレンシャルデスプレイ、ＤＮＡマイクロアレイ、プロテインマイクロアレイ、ＳＡＧＥ等の任意の公知の方法が挙げられる。 The measurement of the expression level of AdipoR2 in the step (C) may be performed by measuring the expression level of AdipoR2 in a sample collected from a living body by an arbitrary method. Examples of the sample include adipose tissue and tissues such as muscle, liver, heart, blood vessel and the like, and the liver is preferable. Examples of the AdipoR2 expression level measurement method include a method for measuring mRNA and protein expression, and measurement of mRNA expression level is preferable. Examples of the method for measuring the expression level include any known methods such as various immunoassays, Nosan blot, RT-PCR, real-time quantitative PCR, differential display, DNA microarray, protein microarray, SAGE and the like.
測定したAdipoR2発現量を、被験物質投与前後、あるいは被験物質投与群と被験物質非投与又は対照物質投与群（対照群）の間で比較することで、AdipoR2発現に対する被験物質の効果を評価することができる。例えばAdipoR2発現量が対照群と比較して被験物質投与群で統計的に有意に高い又は低い場合、該被験物質をAdipoR2発現調節剤として評価又は選択することができる。また例えば、AdipoR2発現量が対照群と比較して被験物質投与群で統計的に有意に高い場合、該被験物質をAdipoR2発現促進剤として評価又は選択することができる。 To evaluate the effect of the test substance on AdipoR2 expression by comparing the measured AdipoR2 expression level before and after administration of the test substance, or between the test substance administration group and the test substance non-administration group or the control substance administration group (control group) Can do. For example, when the AdipoR2 expression level is statistically significantly higher or lower in the test substance administration group than in the control group, the test substance can be evaluated or selected as an AdipoR2 expression regulator. For example, when the AdipoR2 expression level is statistically significantly higher in the test substance administration group than in the control group, the test substance can be evaluated or selected as an AdipoR2 expression promoter.
斯くして選択されたAdipoR2発現調節剤は、生体内におけるAdipoR2発現を調節し、結果として生体内におけるアディポネクチンの活性を調節し得る物質である。斯かるAdipoR2発現調節剤は、アディポネクチン量の変化によって引き起こされる疾患または症状の予防、治療又は改善に有用である。特に、AdipoR2発現促進剤は、低アディポネクチン状態によって引き起こされる疾患又は症状、例えば、低アディポネクチン血症、耐糖能障害、糖尿病、２型糖尿病、インスリン抵抗性症候群、糖尿病合併症、高血糖症、動脈硬化症、アテローム性動脈硬化症、心臓血管疾患、脳血管障害、血管狭窄、末梢血管疾患、動脈瘤、高脂血症、高コレステロール血症、肥満等の予防、治療又は改善のための医薬品、医薬部外品又は食品に有効成分として配合して使用するための素材となり得るものである。 The AdipoR2 expression regulator thus selected is a substance that can regulate AdipoR2 expression in vivo and consequently regulate the activity of adiponectin in vivo. Such an AdipoR2 expression regulator is useful for preventing, treating or ameliorating a disease or symptom caused by a change in the amount of adiponectin. In particular, AdipoR2 expression promoter is a disease or symptom caused by a low adiponectin state, such as hypoadiponectinemia, impaired glucose tolerance, diabetes, type 2 diabetes, insulin resistance syndrome, diabetic complications, hyperglycemia, arteriosclerosis , Drugs for the prevention, treatment or improvement of atherosclerosis, cardiovascular disease, cerebrovascular disorder, vascular stenosis, peripheral vascular disease, aneurysm, hyperlipidemia, hypercholesterolemia, obesity, etc. It can be a material for blending and using an quasi-drug or food as an active ingredient.
以下、実施例に基づき本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail based on examples.
実施例１：睡眠障害誘発動物におけるAdipoR2発現量の測定
１.方法
ＳＤラット（１０〜１１週齢、日本ＳＬＣより購入）を体重が等しくなるように群分けした。レム睡眠阻害群（Ｎ＝６）は、ラットが休息できる直径６．０ｃｍ高さ２．５ｃｍの円柱プラットホームが設置され、プラットホームの１ｃｍ下まで水を張ったケージで個別（１匹/１ケージ）飼育した。コントロール群（Ｎ＝４）は通常のケージで個別飼育した。飼育期間は２日間であった。飼育後、ラットをフォーレン（大日本住友製薬）による深麻酔下で脱血した後、肝臓を採取し、RNAlater(キアゲン)中で使用まで-２０℃で保存した。
保存された肝臓よりRNeasy Mini kit（キアゲン）を用いてＴｏｔａｌ ＲＮＡの抽出を行った。抽出したＴｏｔａｌ ＲＮＡからオリゴｄＴプライマー（インビトロジェン）とMMLV RT（インビトロジェン）を用いて逆転写反応(３７℃、１時間)を行い、ｃＤＮＡを作製した。作製したｃＤＮＡからTaqMan（登録商標）プローブ及びPCRプライマーを用いたReal-Time fast PCR法によりAdipoR2とAdipoR1のｍＲＮＡ発現量を測定した。なお発現量は３６Ｂ４遺伝子（内部コントロール）量に対する相対値として検出した。またTaqMan（登録商標）プローブ及びPCRプライマーはTaqMan（登録商標）gene expression assay （アプライドバイオシステムズ）提供のものを用いた。使用したTaqMan（登録商標）プローブ及びPCRプライマーは以下のとおり：AdipoR2; Rn01463173_m1、AdipoR1; Rn01114954_g1、36B4; Rn00821065_g1。
Example 1: Measurement of AdipoR2 expression level in sleep disorder induced animals 1. Method SD rats (10-11 weeks old, purchased from Japan SLC) were divided into groups so that their body weights were equal. In the REM sleep inhibition group (N = 6), a cylindrical platform with a diameter of 6.0 cm and a height of 2.5 cm, in which the rat can rest, is installed, and individually (1 animal / cage) in a cage with water up to 1 cm below the platform. Raised. The control group (N = 4) was individually housed in a normal cage. The breeding period was 2 days. After breeding, the rats were exsanguinated under deep anesthesia with Foren (Dainippon Sumitomo Pharma Co., Ltd.), and then the livers were collected and stored in RNAlater (Qiagen) at −20 ° C. until use.
Total RNA was extracted from the stored liver using RNeasy Mini kit (Qiagen). From the extracted total RNA, reverse transcription reaction (37 ° C., 1 hour) was performed using oligo dT primer (Invitrogen) and MMLV RT (Invitrogen) to prepare cDNA. From the prepared cDNA, mRNA expression levels of AdipoR2 and AdipoR1 were measured by Real-Time fast PCR method using TaqMan (registered trademark) probe and PCR primers. The expression level was detected as a relative value with respect to the 36B4 gene (internal control) level. TaqMan (registered trademark) probes and PCR primers were provided by TaqMan (registered trademark) gene expression assay (Applied Biosystems). TaqMan® probes and PCR primers used were as follows: AdipoR2; Rn01463173_m1, AdipoR1; Rn01114954_g1, 36B4; Rn00821065_g1.
２．結果
コントロール群に比べてレム睡眠阻害群の肝臓ではAdipoR2の遺伝子発現量が有意に低下した。一方、AdipoR1遺伝子発現量には変化が認められなかった(図１)。
2. Results AdipoR2 gene expression level was significantly decreased in the liver of the REM sleep inhibition group compared to the control group. On the other hand, no change was observed in the expression level of AdipoR1 gene (FIG. 1).
Claims (4)
（Ａ）被験物質を実験動物に投与する工程
（Ｂ）当該動物に睡眠障害を負荷する工程
（Ｃ）当該動物におけるAdipoR2発現量を測定し、その変化を評価する工程 A method for evaluating or selecting an adiponectin receptor 2 (AdipoR2) expression regulator, comprising the following steps (A) to (C):
(A) A step of administering a test substance to an experimental animal (B) A step of loading the animal with sleep disorders (C) A step of measuring AdipoR2 expression level in the animal and evaluating the change
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