JP2014217375A - Method for controlling expression of gip receptor - Google Patents
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Abstract
Description
本発明は、脂肪細胞におけるＧＩＰ受容体の発現制御及びその利用に関する。 The present invention relates to regulation of GIP receptor expression in adipocytes and use thereof.
消化管ホルモンの１つであるＧＩＰ（ｇｌｕｃｏｓｅ−ｄｅｐｅｎｄｅｎｔ ｉｎｓｕｌｉｎｏｔｒｏｐｉｃ ｐｏｌｙｐｅｐｔｉｄｅ）は、グルコース依存的に膵臓β細胞からインスリン分泌を促進し、血糖値の調節に寄与するインクレチンとして知られている。 GIP (glucose-dependent insulotropic polypeptide), one of the gastrointestinal hormones, is known as an incretin that promotes insulin secretion from pancreatic β cells in a glucose-dependent manner and contributes to the regulation of blood glucose level.
一方で、ＧＩＰはインクレチン作用以外に、脂肪組織、骨、神経細胞において様々な膵外作用を有することが報告されている（非特許文献１）。中でも、ＧＩＰと肥満の関連性が示唆されており、脂肪組織に対するＧＩＰの作用は特に着目されている。すなわち、脂肪細胞にはＧＩＰ受容体（ＧＩＰＲ）が発現し、ＧＩＰＲ欠損マウスにおいて高脂肪食負荷による肥満が認められないことが報告され（非特許文献２）、また、脂肪組織に対するＧＩＰの作用として、ＬＰＬ（ｌｉｐｏｐｒｏｔｅｉｎ ｌｉｐａｓｅ）活性化を介した脂肪酸取り込みの促進（非特許文献３）、Ａｋｔを介した糖取り込みの亢進（非特許文献４）、脂肪蓄積亢進（非特許文献５）、炎症性の亢進（非特許文献６）等が報告されている。 On the other hand, GIP has been reported to have various extrapancreatic actions in adipose tissue, bone, and nerve cells in addition to the incretin action (Non-patent Document 1). Among them, the relationship between GIP and obesity has been suggested, and the action of GIP on adipose tissue has attracted particular attention. That is, it has been reported that GIP receptor (GIPR) is expressed in adipocytes, and obesity due to high fat diet load is not observed in GIPR-deficient mice (Non-patent Document 2). , Promotion of fatty acid uptake through activation of LPL (Lipoprotein Lipase) (Non-patent Document 3), enhanced sugar uptake through Akt (Non-patent Document 4), increased fat accumulation (Non-patent Document 5), inflammatory Enhancement (Non-Patent Document 6) has been reported.
したがって、脂肪細胞におけるＧＩＰ受容体の発現や機能の解析を進めることは、肥満や炎症のメカニズム解明や受容体分子を標的とした抗肥満物質や抗炎症物質の開発につながり、重要である。
上記非特許文献６では、アデノウィスルベクターを用い、ＧＩＰ受容体を強制高発現させた脂肪細胞において、インターロイキン６（ＩＬ６）の発現・産生が促進し、炎症反応が亢進することが開示されているが、アデノウィスルベクターを用いＧＩＰ受容体を強制高発現させる系は、操作が煩雑であり、また非生理的であることから、受容体機能の解明やスクリーニング系として使用することには適さない。
Therefore, it is important to advance the analysis of the expression and function of GIP receptors in adipocytes, leading to the elucidation of obesity and inflammation mechanisms and the development of anti-obesity substances and anti-inflammatory substances targeting receptor molecules.
Non-Patent Document 6 discloses that the expression and production of interleukin 6 (IL6) is promoted and the inflammatory reaction is enhanced in adipocytes in which an adenovirus vector is used and the GIP receptor is forcibly highly expressed. However, a system for forcedly expressing the GIP receptor using an adenovirus vector is not suitable for use in elucidation of the receptor function or as a screening system because it is complicated and non-physiological. .
本発明は、脂肪細胞においてＧＩＰ受容体を効率よく発現できる系、及び当該発現系の利用法を提供するものである。 The present invention provides a system that can efficiently express a GIP receptor in adipocytes, and a method of using the expression system.
本発明らは、脂肪細胞におけるＧＩＰ受容体の発現について検討したところ、脂肪細胞をマクロファージと共培養することにより、ＧＩＰ受容体の発現が高まり且つ維持されることを見出した。 The present inventors examined the expression of the GIP receptor in adipocytes, and found that the expression of the GIP receptor was increased and maintained by co-culturing the adipocytes with macrophages.
すなわち、以下の１）−４）に係るものである。
１）脂肪細胞をマクロファージと共培養する、脂肪細胞におけるＧＩＰ受容体の発現増強方法。
２）脂肪細胞をマクロファージと共培養する、脂肪細胞におけるＧＩＰ受容体の機能解析方法。
３）脂肪細胞をマクロファージと共培養する工程を含む、ＧＩＰ受容体発現制御物質のスクリーニング方法。
４）脂肪細胞をマクロファージと共培養する工程を含む、ＧＩＰ受容体親和性物質のスクリーニング方法。
That is, it relates to the following 1) -4).
1) A method for enhancing GIP receptor expression in adipocytes, wherein adipocytes are co-cultured with macrophages.
2) A method for analyzing the function of GIP receptors in adipocytes, wherein adipocytes are co-cultured with macrophages.
3) A screening method for a GIP receptor expression regulator, comprising the step of co-culturing adipocytes with macrophages.
4) A method for screening a substance having affinity for GIP receptor, comprising a step of co-culturing adipocytes with macrophages.
本発明によれば、脂肪細胞におけるＧＩＰ受容体の発現を、アデノウィスルベクター等を用い、強発現させるような面倒な手間を掛けず、また、より生理的条件に近い系で効率よく行うことができる。したがって、脂肪細胞におけるＧＩＰ受容体の発現解析や機能解析を容易に行うことができ、これにより肥満や炎症のメカニズム解明や受容体分子を標的とした抗肥満物質や抗炎症物質等の探索が容易となる。 According to the present invention, the expression of the GIP receptor in adipocytes can be efficiently performed in a system that is closer to physiological conditions, without using the time-consuming labor of strong expression using an adenovirus vector or the like. it can. Therefore, expression analysis and functional analysis of GIP receptors in adipocytes can be easily performed, thereby making it possible to elucidate the mechanism of obesity and inflammation and to search for anti-obesity substances and anti-inflammatory substances targeting receptor molecules. It becomes.
本発明において、ＧＩＰ受容体（ＧＩＰＲ）の発現は、脂肪細胞とマクロファージを共培養することによって行われる。
ここで、「脂肪細胞」は、脂肪細胞の性質（例えば、脂肪細胞特有の細胞内代謝を行い、脂肪滴を多量に蓄積する）をもち、ＧＩＰ受容体を発現し得る細胞であればよく、ヒトやラットの初代培養細胞等の培養脂肪細胞の他、前駆脂肪細胞から分化誘導された細胞でもよい。前駆脂肪細胞としては、脂肪細胞に分化する能力を有する線維芽細胞として広く当該研究領域で使用されている３Ｔ３−Ｌ１細胞（Journal of Biological Chemistry (1996), 271(5), pp.2365-2368）、Ｏｂ１７７１（Biochemical and Biophysical Research Communications (1997), 238(2), pp.606-611）等が挙げられる。各種の動物に由来する脂肪前駆細胞は市販されており、適当な販社から購入することができる。例えばマウス由来の３Ｔ３−Ｌ１細胞、３Ｔ３−Ｆ４４２Ａ細胞はＤＳファーマバイオメディカル株式会社等から購入することができる。
前駆脂肪細胞から脂肪細胞への分化誘導は、公知の方法により前駆脂肪細胞に分化誘導刺激を加えることで行うことができる。例えば、３Ｔ３−Ｌ１細胞を脂肪細胞に誘導する場合、ウシ胎児血清（ＦＢＳ）、Ｉｎｓｕｌｉｎ、ＩＢＭＸ(３-Ｉｓｏｂｕｔｙｌ−１−ｍｅｔｈｙｌｘａｎｔｈｉｎｅ)、Ｄｅｘ（Ｄｅｘａｍｅｔｈａｓｏｎｅ）を含む脂肪細胞分化誘導カクテル等により当該前駆脂肪細胞の分化誘導を行うことができる（Wu Z. et
al., Genes & Dev. (1992), 9, pp.2350-2363.等）。
In the present invention, GIP receptor (GIPR) expression is performed by co-culturing adipocytes and macrophages.
Here, the “adipocyte” may be any cell that has the properties of an adipocyte (for example, performs intracellular metabolism peculiar to an adipocyte and accumulates a large amount of lipid droplets) and can express a GIP receptor, In addition to cultured adipocytes such as human and rat primary cultured cells, cells derived from preadipocytes may be used. As the preadipocytes, 3T3-L1 cells (Journal of Biological Chemistry (1996), 271 (5), pp.2365-2368) widely used in the research area as fibroblasts having the ability to differentiate into adipocytes. ), Ob1771 (Biochemical and Biophysical Research Communications (1997), 238 (2), pp. 606-611). Adipose precursor cells derived from various animals are commercially available and can be purchased from appropriate sales companies. For example, mouse-derived 3T3-L1 cells and 3T3-F442A cells can be purchased from DS Pharma Biomedical Corporation.
Differentiation induction from preadipocytes to adipocytes can be performed by applying differentiation induction stimulation to the preadipocytes by a known method. For example, in the case of inducing 3T3-L1 cells into adipocytes, the precursor fat is obtained by using a fat cell differentiation-inducing cocktail containing fetal bovine serum (FBS), Insulin, IBMX (3-Isobutyl-1-methylxanthine), Dex (Dexamethasone), or the like. Can induce cell differentiation (Wu Z. et
al., Genes & Dev. (1992), 9, pp.2350-2363.).
前駆脂肪細胞から脂肪細胞へ分化誘導する場合、参考例１に示すように、３Ｔ３−Ｌ１細胞の場合、ＧＩＰ受容体は分化初期及び成熟期において２相性の発現を示し、その発現量は成熟期（分化誘導後６日目頃）において顕著に増加する。したがって、前駆脂肪細胞を分化誘導して用いる場合は、成熟期以降の細胞を用いるのが好ましく、例えば３Ｔ３−Ｌ１細胞であれば分化誘導後６日目以降、例えば６〜１６日目の細胞を用いるのが好ましい。 In the case of inducing differentiation from preadipocytes to adipocytes, as shown in Reference Example 1, in the case of 3T3-L1 cells, the GIP receptor shows biphasic expression in the early differentiation stage and the mature stage, and the expression level thereof is in the mature stage. It increases markedly at around 6 days after differentiation induction. Therefore, when preadipocytes are differentiated and used, it is preferable to use cells after the maturation stage. For example, in the case of 3T3-L1 cells, cells after 6 days after differentiation induction, for example, cells 6 to 16 are used. It is preferable to use it.
本発明において用いられるマクロファージとしては、単球に由来するものであればよく、生物種は特に限定されず、例えばマウス、ラット、ヒト等の何れでもよい。好適には、例えば、マクロファージ様細胞として公知のＲＡＷ２６４細胞、ＲＡＷ２６４．７細胞、腹腔マクロファージ（ｐｒｉｍａｒｙ ｍａｃｒｏｐｈａｇｅｓ）等が挙げられる。
ＲＡＷ２６４細胞、ＲＡＷ２６４．７細胞は、ＤＳファーマバイオメディカル株式会社等から購入可能である。また、腹腔マクロファージは、公知の調製方法、例えば、チオグリコネート含有液をマウスの腹腔内に投与し、２−４日後に腹腔内を洗浄し、その洗浄液からマクロファージを回収することにより得ることができる。
Macrophages used in the present invention are not particularly limited as long as they are derived from monocytes, and for example, any of mouse, rat, human and the like may be used. Suitable examples include RAW264 cells, RAW264.7 cells, and peritoneal macrophages known as macrophage-like cells.
RAW264 cells and RAW264.7 cells can be purchased from DS Pharma Biomedical Co., Ltd. In addition, peritoneal macrophages can be obtained by a known preparation method, for example, by administering a thioglyconate-containing solution into the abdominal cavity of mice, washing the abdominal cavity after 2-4 days, and collecting the macrophages from the washing solution. it can.
マクロファージと脂肪細胞の共培養は、脂肪細胞とマクロファージが相互に接触する方法（接触法）、或いは脂肪細胞とマクロファージが同じ培養系に存在するが物質の行き来が可能な隔壁により隔てられ、細胞自体を物理的に接触させない方法（非接触法）の何れでもよい。非接触法としては、例えば、通常の細胞培養に用いられるカルチャーインサート等のフィルターを用いて両細胞を隔てて培養するトランスウェル法が挙げられ、本発明においては、マクロファージと脂肪細胞を個々に回収し、それぞれの遺伝子発現の解析を行うこと、また、接触による両細胞間での相互作用を回避する点から当該非接触法を用いるのが好ましい。
トランスウェル法において、フィルターの孔径は、0.1μｍ以上、好ましくは0.4μｍ以上で、3μｍ以下、好ましくは1μｍ以下である。このようなフィルターとしては、BD Falcon セルカルチャーインサート等を挙げることができる。脂肪細胞とマクロファージのどちらをフィルター上に培養しても構わないが、マクロファージをフィルター上で培養するのが好ましい。
The co-culture of macrophages and adipocytes is a method in which adipocytes and macrophages are in contact with each other (contact method), or the cells themselves are separated by a partition wall in which the fat cells and macrophages are present in the same culture system, but the substance can move back and forth. Any of the methods (non-contact method) that does not allow physical contact with the substrate may be used. Non-contact methods include, for example, a transwell method in which both cells are cultured separately using a filter such as a culture insert used for normal cell culture. In the present invention, macrophages and adipocytes are individually collected. However, it is preferable to use the non-contact method from the viewpoint of analyzing the expression of each gene and avoiding the interaction between both cells due to contact.
In the transwell method, the pore size of the filter is 0.1 μm or more, preferably 0.4 μm or more, 3 μm or less, preferably 1 μm or less. Examples of such filters include BD Falcon cell culture inserts. Either fat cells or macrophages may be cultured on the filter, but it is preferable to culture the macrophages on the filter.
培養培地としては、任意の液体培養培地、好ましくは哺乳動物細胞用培地を使用できる。細胞培養培地としては、例えば、イーグル培地のような最小必須培地(ＭＥＭ)、ダルベッコ改変イーグル培地(ＤＭＥＭ）、ＲＰＭＩ１６４０培地、１９９培地を用いることができる。(H. Sakaue et al., Mol. Endocrinol. (2004), 11, 1552.; Miki et al., Mol. Cell. Biol. (2001), 21, 2521.; H. Sakaue et al., Genes & Dev. (2002), 16, 908.を参照) As the culture medium, any liquid culture medium, preferably a medium for mammalian cells can be used. As the cell culture medium, for example, a minimal essential medium (MEM) such as Eagle medium, Dulbecco's modified Eagle medium (DMEM), RPMI 1640 medium, and 199 medium can be used. (H. Sakaue et al., Mol. Endocrinol. (2004), 11, 1552 .; Miki et al., Mol. Cell. Biol. (2001), 21, 2521 .; H. Sakaue et al., Genes & (See Dev. (2002), 16, 908.)
これらの培地には、ＧＩＰ受容体の発現に影響を与えない範囲で、適宜添加剤を添加することができる。 Additives can be appropriately added to these media as long as they do not affect the expression of the GIP receptor.
共培養の温度は３０℃〜４０℃、好ましくは３５℃〜３７℃である。培地のｐＨ値は、一般に６〜８、好ましくはｐＨ７〜７．５である。
共培養は、通常、約５％のＣＯ2雰囲気下で行われ、共培養期間は、例えば１時間以上、４８時間以下であり、好ましくは、１６時間以上、２４時間以下である。
The temperature of co-culture is 30 ° C to 40 ° C, preferably 35 ° C to 37 ° C. The pH value of the medium is generally 6 to 8, preferably 7 to 7.5.
The co-culture is usually performed in a CO 2 atmosphere of about 5%, and the co-culture period is, for example, 1 hour or more and 48 hours or less, preferably 16 hours or more and 24 hours or less.
また、共培養開始時の脂肪細胞とマクロファージの細胞数の割合は、特に限定されないが、例えば、５０：１〜１：１であり、好ましくは２０：１〜１：１であり、より好ましくは５：１〜２：１である。 The ratio of the number of adipocytes and macrophages at the start of co-culture is not particularly limited, but is, for example, 50: 1 to 1: 1, preferably 20: 1 to 1: 1, more preferably 5: 1 to 2: 1.
共培養後、共培養物から脂肪細胞を回収する。回収方法は特に限定されないが、例えば、トリプシンを用いることが出来る。また、ＲＮＡを回収する際は、ＲＮｅａｓｙ ｍｉｎｉ ５０（ＱＩＡＧＥＮ）付属のＬｙｓｉｓ Ｂｕｆｆｅｒを用いることが出来る。 After co-culture, adipocytes are collected from the co-culture. The recovery method is not particularly limited, but for example, trypsin can be used. In addition, when recovering RNA, a lysis buffer attached to RNeasy mini 50 (QIAGEN) can be used.
後記実施例に示すように、脂肪細胞をマクロファージと共培養すると、脂肪細胞におけるＧＩＰ受容体の発現量が増加し、且つその発現は維持される。
したがって、脂肪細胞とマクロファージの共培養系は、脂肪細胞におけるＧＩＰ受容体の発現増強のために使用できる。また、脂肪細胞とマクロファージの共培養系は、ＧＩＰ受容体の機能を解析するため、ＧＩＰ受容体発現制御物質又はＧＩＰ受容体親和性物質をスクリーニングするために使用できる。
As shown in Examples described later, when adipocytes are co-cultured with macrophages, the expression level of GIP receptors in adipocytes increases and the expression is maintained.
Therefore, the co-culture system of adipocytes and macrophages can be used for enhancing the expression of GIP receptors in adipocytes. In addition, the co-culture system of adipocytes and macrophages can be used for screening a GIP receptor expression controlling substance or a GIP receptor affinity substance in order to analyze the function of the GIP receptor.
ここで、ＧＩＰ受容体の発現増強とは、脂肪細胞においてＧＩＰ受容体の存在量及び／又は産生量が増加することを意味する。当該ＧＩＰ受容体の存在量又は産生量は、例えばＧＩＰ受容体遺伝子の発現量として測定でき、遺伝子発現量の測定は、公知の方法によりＲＮＡを抽出し、ｃＤＮＡ合成後、定量的リアルタイムＰＣＲ法等を用いることにより行うことができる。 Here, enhanced expression of the GIP receptor means that the abundance and / or production amount of the GIP receptor increases in adipocytes. The abundance or production amount of the GIP receptor can be measured, for example, as the expression level of the GIP receptor gene. The gene expression level can be measured by extracting RNA by a known method, synthesizing cDNA, quantitative real-time PCR, etc. It can be performed by using.
ＧＩＰ受容体の機能としては、ＧＩＰ受容体を介して引き起こされる各種細胞刺激活性や、糖取り込み促進作用、脂肪蓄積促進作用、脂肪分解抑制作用等が挙げられる。細胞刺激活性としては、例えば、細胞内ｃＡＭＰ生成、細胞内ｃＧＭＰ生成、細胞内タンパク質のリン酸化、炎症性サイトカイン（インターロイキン６（ＩＬ６）、ＴNＦα、ＭＣＰ−１、ＰＡＩ１）の産生等が挙げられる。 Examples of the function of the GIP receptor include various cell stimulating activities caused by the GIP receptor, sugar uptake promoting action, fat accumulation promoting action, lipolysis inhibiting action, and the like. Examples of the cell stimulating activity include intracellular cAMP generation, intracellular cGMP generation, phosphorylation of intracellular proteins, production of inflammatory cytokines (interleukin 6 (IL6), TNFα, MCP-1, PAI1), and the like. .
また、ＧＩＰ受容体発現制御物質とは、脂肪細胞においてＧＩＰ受容体の発現量を増加又は減少させる物質を意味する。ＧＩＰ受容体の発現量は、例えばＧＩＰ受容体遺伝子の発現量として測定できる。 The GIP receptor expression controlling substance means a substance that increases or decreases the expression level of the GIP receptor in adipocytes. The expression level of the GIP receptor can be measured, for example, as the expression level of the GIP receptor gene.
また、ＧＩＰ受容体親和性物質としては、ＧＩＰ受容体に結合して受容体を活性化する物質、ＧＩＰ受容体に結合するが受容体を活性化しない物質等が挙げられる。受容体活性としては、ＧＩＰ受容体を介した細胞刺激活性、例えば、細胞内ｃＡＭＰ生成、細胞内ｃＧＭＰ生成、細胞内タンパク質のリン酸化、ＩＬ６、ＴＮＦαの産生等が挙げられる。 Examples of GIP receptor affinity substances include substances that bind to the GIP receptor and activate the receptor, and substances that bind to the GIP receptor but do not activate the receptor. Examples of the receptor activity include cell stimulation activity via GIP receptor, for example, intracellular cAMP generation, intracellular cGMP generation, intracellular protein phosphorylation, IL6, TNFα production, and the like.
脂肪細胞とマクロファージの共培養を用いたＧＩＰ受容体発現制御物質のスクリーニング方法は、具体的には以下の工程（ａ）〜（ｃ）を含むものである。
（ａ）脂肪細胞をマクロファージと共培養する工程
（ｂ）被験物質を前記共培養系に接触させる工程
（ｃ）工程（ｂ）において被験物質を接触させた場合におけるＧＩＰ受容体の発現レベルと、被験物質を接触させない場合におけるＧＩＰ受容体の発現レベルとを比較する工程
A screening method for a GIP receptor expression regulator using co-culture of adipocytes and macrophages specifically includes the following steps (a) to (c).
(A) a step of co-culturing adipocytes with macrophages (b) a step of contacting a test substance with the co-culture system (c) an expression level of a GIP receptor when the test substance is contacted in step (b), A step of comparing the expression level of the GIP receptor when the test substance is not contacted
工程（ａ）の共培養は、前記したとおりである。工程（ｂ）被験物質の接触は、被験物質を共培養系へ、１時間〜２４時間程度接触させることが好ましい。工程（ｃ）では、ＧＩＰ受容体の発現レベルが評価されるが、当該ＧＩＰ受容体発現レベルは、例えばＧＩＰ受容体遺伝子の発現量を測定することにより行うことができる。発現レベルの比較は、例えば、有意差の有無に基づいて行われる。比較の結果、被験物質の非存在下に対して被験物質の存在下で、ＧＩＰ受容体発現レベルの抑制が確認できれば、その被験物質はＧＩＰ受容体発現抑制物質となり得る。 The co-culture in the step (a) is as described above. In the step (b), the test substance is preferably brought into contact with the co-culture system for about 1 to 24 hours. In step (c), the expression level of the GIP receptor is evaluated. The expression level of the GIP receptor can be determined, for example, by measuring the expression level of the GIP receptor gene. The comparison of the expression level is performed based on the presence or absence of a significant difference, for example. As a result of the comparison, if the suppression of the GIP receptor expression level can be confirmed in the presence of the test substance relative to the absence of the test substance, the test substance can be a GIP receptor expression inhibitor.
また脂肪細胞とマクロファージの共培養を用いたＧＩＰ受容体親和性物質のスクリーニング方法は、具体的には、例えば、以下の工程（ｄ）〜（ｆ）を含むものである。
（ｄ）脂肪細胞をマクロファージと共培養する工程
（ｅ）被験物質及びＧＩＰを前記共培養系に接触させる工程
（ｆ）工程（ｅ）において被験物質の存在下でＧＩＰを共培養系へ接触させた場合における受容体活性と、被験物質の非存在下でＧＩＰを共培養系へ接触させた場合における受容体活性とを比較する工程
Moreover, the screening method of a GIP receptor affinity substance using the co-culture of adipocytes and macrophages specifically includes, for example, the following steps (d) to (f).
(D) The step of co-culturing adipocytes with macrophages (e) The step of contacting the test substance and GIP with the co-culture system (f) The step of contacting the GIP with the co-culture system in the presence of the test substance in step (e) Comparing the receptor activity in the case of contact with GIP in the absence of the test substance to the co-culture system
工程（ｄ）の共培養は、前記したとおりである。工程（ｅ）被験物質及びＧＩＰの添加は、共培養系へ、１時間〜２４時間程度接触させることが好ましい。工程（ｆ）では、被験物質及びＧＩＰの存在下における受容体活性が測定される。同時に該受容体活性を、被験物質の非存在下の活性と比較する。受容体活性の比較は、例えば、有意差の有無に基づいて行われる。比較の結果、被験物質の非存在下に対して被験物質の存在下で、活性の促進が確認できれば、その被験物質はＧＩＰ受容体作動物質、活性の抑制が確認できれば、その被験物質はＧＩＰ受容体阻害物質となり得る。 The co-culture in the step (d) is as described above. Step (e) The test substance and GIP are preferably added to the co-culture system for about 1 to 24 hours. In step (f), receptor activity in the presence of the test substance and GIP is measured. At the same time, the receptor activity is compared with the activity in the absence of the test substance. Comparison of receptor activity is performed based on, for example, the presence or absence of a significant difference. As a result of comparison, if the promotion of activity can be confirmed in the presence of the test substance relative to the absence of the test substance, the test substance is a GIP receptor agonist, and if suppression of the activity is confirmed, the test substance is GIP receptor. Can be a body inhibitor.
上述した実施形態に関し、本発明においては以下の態様が開示される。
＜１＞脂肪細胞をマクロファージと共培養する、脂肪細胞におけるＧＩＰ受容体の発現増強方法。
＜２＞脂肪細胞をマクロファージと共培養する、脂肪細胞におけるＧＩＰ受容体の機能解析方法。
＜３＞脂肪細胞をマクロファージと共培養する工程を含む、ＧＩＰ受容体発現制御物質のスクリーニング方法。
＜４＞脂肪細胞をマクロファージと共培養する工程を含む、ＧＩＰ受容体親和性物質のスクリーニング方法。
With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> A method for enhancing GIP receptor expression in adipocytes, wherein the adipocytes are co-cultured with macrophages.
<2> A method for analyzing the function of GIP receptors in adipocytes, wherein adipocytes are co-cultured with macrophages.
<3> A screening method for a GIP receptor expression regulator, comprising a step of co-culturing adipocytes with macrophages.
<4> A method for screening a substance having affinity for a GIP receptor, comprising a step of co-culturing adipocytes with macrophages.
＜５＞以下の工程（ａ）〜（ｃ）を含む、＜３＞の方法。
（ａ）脂肪細胞をマクロファージと共培養する工程
（ｂ）被験物質を前記共培養系に接触させる工程
（ｃ）工程（ｂ）において被験物質を接触させた場合におけるＧＩＰ受容体の発現レベルと、被験物質を接触させない場合におけるＧＩＰ受容体の発現レベルとを比較する工程
＜６＞以下の工程（ｄ）〜（ｆ）を含む、＜４＞の方法。
（ｄ）脂肪細胞をマクロファージと共培養する工程
（ｅ）被験物質及びＧＩＰを前記共培養系に接触させる工程
（ｆ）工程（ｅ）において被験物質の存在下でＧＩＰを共培養系へ接触させた場合における受容体活性と、被験物質の非存在下でＧＩＰを共培養系へ接触させた場合における受容体活性とを比較する工程
＜７＞脂肪細胞が、３Ｔ３−Ｌ１細胞、３Ｔ３−Ｆ４４２Ａ細胞及びＯｂ１７７１細胞から選ばれる前駆脂肪細胞から分化誘導された細胞である、＜１＞〜＜６＞の方法。
＜８＞脂肪細胞として、３Ｔ３−Ｌ１細胞を分化誘導後、６〜１６日目の細胞を用いる＜１＞〜＜７＞の方法。
＜９＞マクロファージが、ＲＡＷ２６４細胞、ＲＡＷ２６４．７細胞又は腹腔マクロファージである＜１＞〜＜８＞の方法。
<5> The method of <3>, comprising the following steps (a) to (c).
(A) a step of co-culturing adipocytes with macrophages (b) a step of contacting a test substance with the co-culture system (c) an expression level of a GIP receptor when the test substance is contacted in step (b), <6> The method of <4>, comprising the following steps (d) to (f): <6> comparing the expression level of the GIP receptor when the test substance is not contacted.
(D) The step of co-culturing adipocytes with macrophages (e) The step of contacting the test substance and GIP with the co-culture system (f) The step of contacting the GIP with the co-culture system in the presence of the test substance in step (e) <7> adipocytes are 3T3-L1 cells, 3T3-F442A cells, and a step of comparing the receptor activity in the case of contact with GIP in the absence of the test substance in the presence of the test substance And <1> to <6>, which are cells induced to differentiate from preadipocytes selected from Ob1771 cells.
<8> The method according to <1> to <7>, wherein the cells on the 6th to 16th days are used as adipocytes after inducing differentiation of 3T3-L1 cells.
<9> The method according to <1> to <8>, wherein the macrophages are RAW264 cells, RAW264.7 cells, or peritoneal macrophages.
実施例１
（１）脂肪細胞とマクロファージの共培養によるＧＩＰＲの発現
３Ｔ３−Ｌ１（ＤＳ ファーマバイオメディカル株式会社）をＤＭＥＭ培地（１０％ＦＢＳ，Ｈｉｇｈ ｇｌｕｃｏｓｅ，Ｓｉｇｍａ）にて３７℃、５％ＣＯ2存在下で継代培養した後、２４ｗｅｌｌ ｐｌａｔｅに１×１０5ｃｅｌｌｓ／ｗｅｌｌ、若しくは６ｗｅｌｌ ｐｌａｔｅに１×１０6ｃｅｌｌｓ／ｗｅｌｌで播種した。２日後、Ｉｎｓｕｌｉｎ（Ｓｉｇｍａ）：１０μｇ／ｍｌ，ＩＢＭＸ（３−Ｉｓｏｂｕｔｙｌ−１−ｍｅｔｈｙｌｘａｎｔｈｉｎｅ，Ｓｉｇｍａ）：０．５ｍＭ，Ｄｅｘ（Ｄｅｘａｍｅｔｈａｓｏｎｅ，Ｓｉｇｍａ）：１μＭを含む上記ＤＭＥＭ培地に交換し、分化誘導を開始した。更に２日後Ｉｎｓｕｌｉｎ：５μｇ／ｍｌを含むＤＭＥＭ培地に交換し、２日間培養した。その後、ＤＭＥＭ培地にて２日毎に培地交換をし、分化誘導開始から合計６日間培養した。
マクロファージ様細胞（Ｒａｗ２６４，ＤＳ ファーマバイオメディカル株式会社）をＤＭＥＭ培地（１０％ＦＢＳ，Ｈｉｇｈ ｇｌｕｃｏｓｅ，Ｓｉｇｍａ）にて３７℃、５％ＣＯ2存在下で継代培養した後、０．４μｍ孔を有する多孔質膜からなる２４ｗｅｌｌ
ｐｌａｔｅ、若しくは６ｗｅｌｌ ｐｌａｔｅ用トランスウェルインサート（ＢＤファルコン）に１×１０4〜１×１０5ｃｅｌｌｓ／ｗｅｌｌで播種し、２４時間培養した。
分化誘導後６日目の３Ｔ３−Ｌ１と上記２４時間培養したＲａｗ２６４を図１（Ａ）に示すようにトランスウェルを用いた非接触法により共培養を行った。２４時間共培養後、下層ウェルの３Ｔ３−Ｌ１を採取し、下記（２）の方法で、ＧＩＰＲの遺伝子発現レベルの測定を行った。なお、対照培養法として、図１（Ｂ）に示すように、３Ｔ３−Ｌ１とＲａｗ２６４をそれぞれ単独で培養し、同様に遺伝子発現レベルの測定を行った。
Example 1
(1) Expression of GIPR by co-culture of adipocytes and macrophages 3T3-L1 (DS Pharma Biomedical Co., Ltd.) in DMEM medium (10% FBS, High glucose, Sigma) at 37 ° C. in the presence of 5% CO 2 After subculture, the cells were seeded at 1 × 10 5 cells / well on a 24 well plate or 1 × 10 6 cells / well on a 6 well plate. Two days later, the induction of differentiation was started by exchanging with the above DMEM medium containing Insulin (Sigma): 10 μg / ml, IBMX (3-Isobutyl-1-methylxanthine, Sigma): 0.5 mM, Dex (Dexamethasone, Sigma): 1 μM. did. Two more days later, the medium was replaced with DMEM medium containing Insulin: 5 μg / ml, followed by culturing for 2 days. Thereafter, the medium was changed every 2 days in the DMEM medium, and cultured for a total of 6 days from the start of differentiation induction.
Macrophage-like cells (Raw264, DS Pharma Biomedical Co., Ltd.) are subcultured in DMEM medium (10% FBS, High glucose, Sigma) at 37 ° C. in the presence of 5% CO 2 and then have a 0.4 μm pore. 24well made of porous membrane
plate or 6-well plate transwell insert (BD Falcon) was seeded at 1 × 10 4 to 1 × 10 5 cells / well and cultured for 24 hours.
As shown in FIG. 1 (A), 3T3-L1 on day 6 after differentiation induction and Raw 264 cultured for 24 hours were co-cultured by a non-contact method using a transwell. After co-culture for 24 hours, 3T3-L1 in the lower well was collected, and the gene expression level of GIPR was measured by the method (2) below. As a control culture method, as shown in FIG. 1 (B), 3T3-L1 and Raw264 were each cultivated alone, and the gene expression level was measured in the same manner.
（２）遺伝子発現量の測定
共培養した３Ｔ３−Ｌ１より、ＲＮｅａｓｙ ｍｉｎｉ ５０（ＱＩＡＧＥＮ）を用いてＲＮＡを抽出した。ｃＤＮＡ合成（Ｈｉｇｈ ｃａｐａｃｉｔｙ ＲＮＡ−ｔｏ−ｃＤＮＡ Ｋｉｔ，Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）後、定量的リアルタイムＰＣＲ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ ７５００ Ｆａｓｔ リアルタイムＰＣＲシステム）により遺伝子の発現定量を行い、内部標準として３６Ｂ４を用い、各遺伝子の発現量を補正した。なお、プライマーはＡｐｐｌｉｅｄ ＢｉｏｓｙｓｔｅｍｓのＴａｑｍａｎ Ｇｅｎｅ Ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓのＧＩＰ受容体(Ｍｍ０１３１６３４４＿ｍ１)、３６Ｂ４(Ｍｍ００７２５４４８＿ｓ１)を用いた。
(2) Measurement of gene expression level RNA was extracted from co-cultured 3T3-L1 using RNeasy mini 50 (QIAGEN). After cDNA synthesis (High capacity RNA-to-cDNA Kit, Applied Biosystems), gene expression is quantified by quantitative real-time PCR (Applied Biosystems 7500 Fast real-time PCR system), and 36B4 is used as an internal standard, and the expression level of each gene Was corrected. In addition, GIP receptors (Mm01316344_m1) and 36B4 (Mm00725448_s1) of Applied Biosystems Taqman Gene Expression Assays were used.
（３）結果
図２に示すように、共培養により３Ｔ３−Ｌ１におけるＧＩＰＲの発現が亢進した。また、遺伝子発現亢進の程度は、Ｒａｗ２６４の細胞数に依存した。
(3) Results As shown in FIG. 2, the expression of GIPR in 3T3-L1 was enhanced by co-culture. The degree of gene expression enhancement depended on the number of Raw264 cells.
実施例２ ＧＩＰＲの機能解析
実施例１の方法により発現されたＧＩＰＲの機能をＩＬ６の発現を指標として確認した。
（１）ＩＬ６遺伝子発現量
実施例１と同様の方法により、３Ｔ３−Ｌ１とＲａｗ２６４を２４時間共培養した後、ＧＩＰ，ＧＩＰ＋ｉｎｓｕｌｉｎを含む培地で刺激し、１，３，６時間後に下層ウェルの３Ｔ３−Ｌ１を採取し、ｃＤＮＡ合成（Ｈｉｇｈ ｃａｐａｃｉｔｙ ＲＮＡ−ｔｏ−ｃＤＮＡ Ｋｉｔ，Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）後、定量的リアルタイムＰＣＲ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ ７５００ Ｆａｓｔ リアルタイムＰＣＲシステム）により遺伝子の発現定量を行い、３６Ｂ４による補正を行った。なお、プライマーはＡｐｐｌｉｅｄ ＢｉｏｓｙｓｔｅｍｓのＴａｑｍａｎ Ｇｅｎｅ Ｅｘｐｒｅｓｓｉｏｎ ａｓｓａｙｓのＩＬ６（Ｍｍ００４４６１９０＿ｍ１）を用いた。その結果、図３に示すようにＧＩＰはインスリン存在下、非存在下いずれにおいても刺激後１時間においてＩＬ６の遺伝子発現を亢進した。
Example 2 Functional Analysis of GIPR The function of GIPR expressed by the method of Example 1 was confirmed using IL6 expression as an index.
(1) IL6 gene expression level 3T3-L1 and Raw264 were co-cultured for 24 hours in the same manner as in Example 1, then stimulated with a medium containing GIP and GIP + insulin, and after 1, 3 and 6 hours, 3T3 in the lower well -L1 was collected, and after cDNA synthesis (High capacity RNA-to-cDNA Kit, Applied Biosystems), gene expression quantification was performed by quantitative real-time PCR (Applied Biosystems 7500 Fast real-time PCR system), and correction by 36B4 was performed. . As a primer, IL6 (Mm00446190_m1) of Applied Biosystems Taqman Gene Expression Assays was used. As a result, as shown in FIG. 3, GIP enhanced IL6 gene expression 1 hour after stimulation in the presence or absence of insulin.
（２）ＩＬ６産生量
実施例１と同様の方法により、３Ｔ３−Ｌ１とＲａｗ２６４を２４時間共培養した後、ＧＩＰ，ＧＩＰ＋ｉｎｓｕｌｉｎを含む培地で刺激し、３時間後に培養上清を回収し、市販の固相酵素免疫検定法（Ｍｏｕｓｅ ＩＬ６ ＥＬＩＳＡ ＭＡＸ， Ｂｉｏｌｅｇｅｎｄ）を用いて、上清中ＩＬ６の定量を行った。
その結果、図４に示すようにＧＩＰによりＩＬ６の産生が亢進した。また、インスリン共存下においても、ＧＩＰ刺激によりＩＬ６の産生が亢進した。
(2) IL6 production amount 3T3-L1 and Raw264 were co-cultured for 24 hours by the same method as in Example 1, and then stimulated with a medium containing GIP and GIP + insulin. After 3 hours, the culture supernatant was collected, Quantification of IL6 in the supernatant was performed using a solid phase enzyme immunoassay (Mouse IL6 ELISA MAX, Biolegend).
As a result, IL6 production was enhanced by GIP as shown in FIG. In addition, IL6 production was enhanced by GIP stimulation even in the presence of insulin.
実施例３ ＧＩＰ受容体阻害物質のＩＬ６産生阻害作用
実施例１と同様の方法により、３Ｔ３−Ｌ１とＲａｗ２６４を２４時間共培養した後、ＧＩＰにより誘導される炎症性サイトカイン（ＩＬ６）の産生を抑制する物質として公知（Timper, K et al. Glucose-dependent insulinotropic polypeptide induces cytokine
expression, lipolysis, and insulin resistance in human adipocytes. Am J Physiol
Endocrinol Metab (2013), 304, E1-E13）のＨ−８９（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ
Ｔｅｃｈｎｏｌｏｇｙ Ｊａｐａｎ）２０ｎＭを１時間前処理した後、ＧＩＰ １００ｎＭを含む培地で刺激し、１時間後に下層ウェルの３Ｔ３−Ｌ１を採取し、ＩＬ６の遺伝子発現レベルの測定を行った。
その結果、図５に示すように、Ｈ−８９により、ＧＩＰによるＩＬ６の発現亢進が有意に阻害された。これより、本発明の共培養系は、Ｈ−８９のようなＧＩＰ受容体阻害物質をスクリーニングするための系として有用であることが確認された。
Example 3 IL6 Production Inhibitory Action of GIP Receptor Inhibitor After Incubating 3T3-L1 and Raw 264 for 24 Hours Using the Same Method as Example 1, Suppresses Production of Inflammatory Cytokine (IL6) Induced by GIP (Timper, K et al. Glucose-dependent insulinotropic polypeptide induces cytokine
expression, lipolysis, and insulin resistance in human adipocytes. Am J Physiol
Endocrinol Metab (2013), 304, E1-E13) H-89 (Cell Signaling)
(Technology Japan) 20 nM was pretreated for 1 hour, and then stimulated with a medium containing 100 nM GIP. After 1 hour, 3T3-L1 was collected from the lower well, and the gene expression level of IL6 was measured.
As a result, as shown in FIG. 5, H-89 significantly inhibited IL6 expression enhancement by GIP. From this, it was confirmed that the co-culture system of the present invention is useful as a system for screening a GIP receptor inhibitor such as H-89.
実施例 ４ ＧＩＰ受容体親和性物質の評価
実施例３と同様の方法により、分化誘導後６日目の３Ｔ３−Ｌ１とＲａｗ２６４を２４時間共培養した後、実施例３で用いたＨ−８９（Ｃｅｌｌ Ｓｉｇｎａｌｉｎｇ Ｔｅｃｈｎｏｌｏｇｙ Ｊａｐａｎ） ２０ｎＭ、オウゴンエキス０．００５％、ジンジャーエキス０．００５％を添加した培地にて１ｈ前処理した後、ＧＩＰ １００ｎＭを含む培地で刺激し、１時間後に下層ウェルの３Ｔ３−Ｌ１を採取し、ＩＬ６の遺伝子発現レベルの測定を行った。
その結果、図１に示すように、オウゴンエキス及びジンジャーエキスは、Ｈ−８９と同様に、ＧＩＰによるＩＬ６の発現亢進を有意に阻害した。また、結果は各剤を添加した刺激培地に対する相対値で示している。
したがって、オウゴンエキス及びジンジャーエキスは、ＧＩＰ受容体阻害物質であると評価できる。
Example 4 Evaluation of GIP Receptor Affinity Substance According to the same method as in Example 3, 3T3-L1 and Raw264 on day 6 after differentiation induction were co-cultured for 24 hours, and then H-89 ( Cell Signaling Technology Japan) Pre-treated for 1 h in a medium supplemented with 20 nM, 0.005% argon extract, and 0.005% ginger extract, then stimulated with a medium containing GIP 100 nM, and 1 hour later, 3T3-L1 of the lower well Were collected, and the gene expression level of IL6 was measured.
As a result, as shown in FIG. 1, Augon extract and ginger extract significantly inhibited the increase in IL6 expression by GIP, similar to H-89. Moreover, the result is shown by the relative value with respect to the stimulation culture medium which added each agent.
Therefore, it can be evaluated that an argon extract and a ginger extract are GIP receptor inhibitors.
参考例１
３Ｔ３−Ｌ１を実施例１の方法に従い、分化誘導させ、分化誘導後０ｍｉｎ、３０ｍｉｎ、２ｈ、４ｈ、８ｈ、２４ｈ、２ｄａｙ、３ｄａｙ、４ｄａｙ、６ｄａｙ、８ｄａｙ、１２ｄａｙ、１６ｄａｙにおいて上記方法によりＲＮＡ抽出、ＧＩＰ受容体の遺伝子発現解析を行った。
その結果、３Ｔ３−Ｌ１細胞の分化に伴い、ＧＩＰ受容体は分化後２ｈ、６日目に２相性の発現を示すことが確認された（図７）。
Reference example 1
3T3-L1 was induced to differentiate according to the method of Example 1, and RNA was extracted by the above method at 0 min, 30 min, 2 h, 4 h, 8 h, 24 h, 2 day, 3 day, 4 day, 6 day, 8 day, 12 day, 16 day after differentiation induction. GIP receptor gene expression analysis was performed.
As a result, it was confirmed that with differentiation of 3T3-L1 cells, the GIP receptor showed biphasic expression 2 days after differentiation and on the 6th day (FIG. 7).
Claims (9)
（ａ）脂肪細胞をマクロファージと共培養する工程
（ｂ）被験物質を前記共培養系に接触させる工程
（ｃ）工程（ｂ）において被験物質を接触させた場合におけるＧＩＰ受容体の発現レベルと、被験物質を接触させない場合におけるＧＩＰ受容体の発現レベルとを比較する工程 The method of Claim 3 including the following process (a)-(c).
(A) a step of co-culturing adipocytes with macrophages (b) a step of contacting a test substance with the co-culture system (c) an expression level of a GIP receptor when the test substance is contacted in step (b), A step of comparing the expression level of the GIP receptor when the test substance is not contacted
（ｄ）脂肪細胞をマクロファージと共培養する工程
（ｅ）被験物質及びＧＩＰを前記共培養系に接触させる工程
（ｆ）工程（ｅ）において被験物質の存在下でＧＩＰを共培養系へ接触させた場合における受容体活性と、被験物質の非存在下でＧＩＰを共培養系へ接触させた場合における受容体活性とを比較する工程 The method of Claim 4 including the following process (d)-(f).
(D) The step of co-culturing adipocytes with macrophages (e) The step of contacting the test substance and GIP with the co-culture system (f) The step of contacting the GIP with the co-culture system in the presence of the test substance in step (e) Comparing the receptor activity in the case of contact with GIP in the absence of the test substance to the co-culture system
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