この研究は、AH賞第15BGIA23250030号、国立医学総合研究所NIHの受賞番号8P20 GM103432-12およびワイオミング大学助成金助成金BTの支援を受けて行われました。

すべてのプロトコルは、ワイオミング大学の動物保護指針に従っています。 1.動物の住宅と給餌注：成体雄性野生型マウス（C57BL / 6）（12〜24週齢）は、施設動物実験および使用委員会（IACUC）承認プロトコルに従って研究動物施設で飼育した。 <ol><li> 6週目から開始して、4匹のグループのマウスを別々のケージに収容し、38才までNCDまたはHFDの摂食群（±0.01％CAP）にランダムに割り当てる。 注：CAPは、脂肪組織11、17で表さTRPV1チャネルタンパク質のアゴニストです。フード内部ブレンダーにHFDを有するキャップを混合し、2つのパーティションに小さい1を含有するトレイにブレンドした混合物を移します。トレイを-20℃の冷凍庫の中に保管してください。 24時間後にHFD + CAP飼料を入れたトレイを冷凍庫から取り出し、-20＆＃176;冷凍庫を使用するまで。 </li><li> （22.8±2.0℃、湿度45〜50％）、12/12ライト/ダークサイクルで、指定食および水を自由に使用できるハウスマウス。 </li><li> 38週の終わりに、鼠径脂肪組織を切開し、脂肪分解実験に使用する（セクション2-7）。 </li></ol> 2.実験のためのマウスの準備<ol><li>ケタミンとキシラジンの混合物（それぞれ10mg / kgおよび80mg / kg体重）を注射することによってマウスを麻酔する。混合物0.01mL /マウス体重10gを注射する。 </li><li>徹底的なつま先で深い麻酔を確認する。ペダル・リフレックスがある場合は、少なくとも30秒後に再度マウスをテストしてください。 </li><li>麻酔中の眼の乾燥を防ぐために、獣眼軟膏を使用する。いずれの処置中もマウスを放置しないでください。 </li><li>高用量のケタミンおよびキシラジン混合物注射（それぞれ10mg / kgおよび80mg / kg体重）の混合物を注射することによってマウスを安楽死させるe（0.01ml / 10g体重）、続いて頚部脱臼。 注：安楽死のこの方法は、ワイオミング大学のIACUCによって承認されています。 </li></ol> 3.腹腔脂肪脂肪パッドの分離<ol><li>手順のために左に横たわるマウス（NCDまたはHFD（±CAP）を32週間与えた）を置く。 注：マウスを左に置くことによって、左前肢および左後肢は解剖用プラットフォーム上にあり、右前肢および右後肢はプラットフォームから離れている。 </li><li> 70％エタノール約2.5mLのに浸し2インチ2ガーゼパッドで皮膚表面を殺菌。下にある脂肪層を明らかにするために、メスを使用して皮膚を通して2〜3mmの側方切開を行う。 </li><li> 2つの外側の切開を結合する背側表面を横切って胸郭の真下にある外科用メスを使用して、皮膚を通して1cmの切断（マウスのサイズに依存する）を行う。 </li><li>慎重にドラッグして皮弁を剥がす滅菌鉗子を使用し、脂肪パッドを切断しないことによって皮下パッドをそのまま残す。これは、皮膚の下に横たわっている太ったパッドです。 </li><li>脂肪のパッドを下の筋肉と筋膜から慎重に解剖し、はさみを使用してください。下の筋肉からカットされて脂肪パッドを引き出します。 注：脂肪パッドの重量とサイズは、マウスの種類（NCDまたはHFD（±CAP）が授与された）に依存します。 </li><li>室温（〜15分）で脂肪分解実験までリン酸緩衝生理食塩水（PBS）を含むペトリ皿にピンセットを移す。 </li><li>ステップ3.2-3.6で説明したように、脂肪パッドをマウスの右側から分離します。 </li></ol> 4.鼠径脂肪パッドからの基礎グリセロール放出<ol><li>シャープなはさみを使用して約20 mgの脂肪組織を5〜8個に切断します。 </li><li> 鼠径脂肪パッドのカットピースを200μLのインキュベーション培地（2％脂肪酸を含むダルベッコ変法イーゲル培地（DMEM））にインキュベートする。37℃、5％CO 2および95％湿度で60分間、無血清ウシ血清アルブミン（BSA）中で培養した。 <ol><li>インキュベーション培地を回収し、脂肪分解アッセイのために-80℃で凍結する。 注：切片は、脂質抽出後のタンパク質濃度の測定に使用されます。 </li><li>切断された脂肪片をピンセットを用いて1 mLの抽出溶液（クロロホルム：メタノール（2：1、v / v）および1％氷酢酸）に移し、100℃で激しく振盪しながら37℃で60分間インキュベートするrpm。脂肪抽出溶液を捨てる。 注：この手順は、カットピースから脂肪を抽出します。脂肪抽出溶液はタンパク質の測定を妨げるため、廃棄してください。 </li><li> 500μLの溶解液（0.1％ドデシル硫酸ナトリウムを含有する0.3N NaOH; SDS）を含有する滅菌した微量遠心管にピンセットを用いて組織を移し（4.2.2）、激しく振盪しながら55℃で一晩インキュベートする100rpmで測定した。 </li></ol></li><li>標準18としてビシンコニン酸（BCA）試薬及びBSAを用いて組織のタンパク質濃度（ステップ4.2.3）を決定します。 </li><li> 氷上で凍結媒体（ステップ4.2.1）を解凍し、プロトコルセクション6及び7 6に記載したように遊離グリセロール試薬を用いて培地のグリセロール含量を決定します。 <ol><li>標準曲線から試料中のグリセロールの濃度を外挿するグリセロール標準19およびh 20当たりナノモルグリセロール当たりmgタンパク質として明示脂肪分解を使用してプロットしました。 </li></ol></li></ol> FSK刺激脂肪分解<ol><li> 2％の脂肪酸を含まないBSA、10μMのFSKおよび5μMのトライアクシンCを含有する200μLのDMEM中、NCDまたはHFD（±CAP）を与えた野生型マウスから得られた約20mgの鼠径脂肪パッド（5〜8切片）を、 5％CO 2および湿度95％で37℃で60分間インキュベートした。 </strong&gt; <ol><li>同じ培地にピンセットを使用して組織片を移し、37℃、5％CO 2および95％湿度でさらに60分間インキュベートする。インキュベーション培地を回収し、脂肪分解アッセイまで-80℃で保存する。 注：脂肪分解の刺激は、脂肪酸およびグリセロールの急速な放出を15分以内に引き起こし、これは最初の1時間の間は直線的であり、その後は平坦化する。誘導脂肪分解の速度はFSK刺激の第一及び第二の時間の間に高く、安定しているので、このプロトコルは、以前に1記載したように第2のインキュベーション期間後、刺激状態における脂肪分解を測定しました。したがって、実験ステップ（5.1および5.1.1）の両方が必要である。 </li></ol></li><li>鼠径脂肪パッドから脂肪を抽出するには、抽出溶液（クロロホルム：メタノール（2））1mLにピンセットを用いてステップ5.1の鼠径脂肪パッド（NCDまたはHFD（±CAP） ：1。v /v）および1％氷酢酸）で洗浄し、100rpmで激しく振盪しながら37℃で60分間インキュベートする。抽出脂肪を捨てる。 </li><li> 500μLの溶解液（0.1％SDSを含む0.3N NaOH）を含む滅菌微量遠心チューブにピンセットを用いて組織を移し（55℃、100rpmで激しく振盪しながら）、一晩（12時間）インキュベートする。 </li><li>ステップ4.3および4.4にそれぞれ記載されているように、組織のタンパク質濃度およびグリセロール含量を決定する。 </li></ol> 6.遊離グリセロール試薬の調製<ol><li>琥珀色のガラスバイアル中で40mLの脱イオン水中にグリセロール試薬19を再構成し、バイアルにストッパーを置き、10回反転させてよく混合する。振り混ぜはしないでください。 </li><li>バイアル瓶をアルミホイルで完全に覆うことによって、光から保護された冷蔵庫の中に4℃で保存します。 </li><li>グリセロール標準液の調製（セクション7）に進んでください。 </&gt; </ol> 7.グリセロール標準の調製およびグリセロール含有量の測定<ol><li> 35μLの供給ストックを65μLの脱イオン水で希釈して1mMのストックを調製する。 注：製造業者が供給するグリセロール標準品は2.8mMグリセロールです。 </li><li> 1cmの経路長の使い捨てメタクリレートキュベットを5本取る。マーカーを0,1.25,2.5,5,10 nmolの標準としてキュベットに標識する。 </li><li> 0mM、1.25mM、2.5mM、5μLおよび10μLの1mMグリセロール標準液をそれぞれの標識化キュベットに加える。脱イオン水で各キュベットの容量を10μLにします。 </li><li> 10μLのサンプルを90μLの脱イオン水に新鮮な500μLの前標識遠心チューブに加えることにより、すべてのサンプル（ステップ4.4または5.1.1）の1〜10倍希釈を行います。各サンプル識別番号でラベルされたキュベットに10μLの希釈サンプルを加えます。 </li><li> UV-VIS分光光度計のスイッチを入れ、波を設定します長さ〜540nm。 </li><li>バイアル（ステップ6.2）を室温で15分間保持することにより遊離グリセロール試薬を室温に温める。 </li><li>ブランク「0」nmol標準（ステップ7.3）、標準（ステップ7.3）およびサンプル（ステップ7.4）として一連の標識キュベットを設定する。 </li><li>スタンダードとサンプルのそれぞれ10μLをそれぞれの標識キュベットに加えます。ブランクとして &quot;0&quot; nmolスタンダードを使用してください。 </li><li> 1mLのピペットを用いてグリセロール標準を含有する各キュベットに0.8mLの遊離グリセロール試薬を加える。 </li><li>キュベットを1.5cm角のプラスチックパラフィンフィルムで覆い、キュベットを3回反転させて内容物を混合し、室温で10分間放置する。 </li><li>キュベットをUV-VIS分光光度計に置き、吸光度（波長540 nm）を記録する。 </li><li> X軸の標準濃度（nmol）およびY軸の記録吸光度を用いて標準曲線をプロットします。 </li><li> glの濃度を計算する（7.12）でプロットされた検量線を用いて外挿により試料中のγ-イセノールを定量する（nmol）。 </li><li>希釈倍率10（ステップ7.4参照）および20（全容積）でサンプルの濃度（nmol /キュベット）を掛け、 </li><li> BCA法で計算したタンパク質mgで、各サンプルのグリセロール濃度を除算する（ステップ7.13）。 注：鼠径脂肪パッドのサンプルは60分間インキュベートされているので、nmolのグリセロール/ mg蛋白質/ hとして結果を表します。 </li></ol>

<ol>
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Hormonal regulation of lipolysis in adipose tissue.</a> Am J Physiol Gastrointest Liver Physiol. 293 (1), G1-G4 (2007).</li><li>Jeppesen, J., Kiens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+limitations+to+fatty+acid+oxidation+during+exercise.">Regulation and limitations to fatty acid oxidation during exercise.</a> J Physiol. 590 (5), 1059-1068 (2012).</li><li>Nakamura, M. T., Yudell, B. E., Loor, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+energy+metabolism+by+long-chain+fatty+acids.">Regulation of energy metabolism by long-chain fatty acids.</a> Prog Lipid Res. 53, 124-144 (2014).</li><li>Murray, A. J., Panagia, M., Hauton, D., Gibbons, G. F., Clarke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+free+fatty+acids+and+peroxisome+proliferator-activated+receptor+alpha+in+the+control+of+myocardial+uncoupling+protein+levels.">Plasma free fatty acids and peroxisome proliferator-activated receptor alpha in the control of myocardial uncoupling protein levels.</a> Diabetes. 54 (12), 3496-3502 (2005).</li><li>Barbera, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+proliferator-activated+receptor+alpha+activates+transcription+of+the+brown+fat+uncoupling+protein-1+gene.+A+link+between+regulation+of+the+thermogenic+and+lipid+oxidation+pathways+in+the+brown+fat+cell.">Peroxisome proliferator-activated receptor alpha activates transcription of the brown fat uncoupling protein-1 gene. A link between regulation of the thermogenic and lipid oxidation pathways in the brown fat cell.</a> J Biol Chem. 276 (2), 1486-1493 (2001).</li><li>Leung, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsaicin+as+an+anti-obesity+drug.">Capsaicin as an anti-obesity drug.</a> Prog Drug Res. 68, 171-179 (2014).</li><li>Hursel, R., Westerterp-Plantenga, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermogenic+ingredients+and+body+weight+regulation.">Thermogenic ingredients and body weight regulation.</a> Int J Obes (Lond). 34 (4), 659-669 (2010).</li><li>Kang, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+capsaicin+reduces+obesity-induced+insulin+resistance+and+hepatic+steatosis+in+obese+mice+fed+a+high-fat+diet.">Dietary capsaicin reduces obesity-induced insulin resistance and hepatic steatosis in obese mice fed a high-fat diet.</a> Obesity (Silver Spring). 18 (4), 780-787 (2010).</li><li>Winkler, B., Steele, R., Altszuler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+glycerol+uptake+to+plasma+glycerol+concentration+in+the+normal+dog.">Relationship of glycerol uptake to plasma glycerol concentration in the normal dog.</a> Am J Physiol. 216 (1), 191-196 (1969).</li><li>Dugan, C. E., Cawthorn, W. P., MacDougald, O. A., Kennedy, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+microfluidic+enzyme+assays+for+simultaneous+detection+of+lipolysis+products+from+adipocytes.">Multiplexed microfluidic enzyme assays for simultaneous detection of lipolysis products from adipocytes.</a> Anal Bioanal Chem. 406 (20), 4851-4859 (2014).</li></ol>

著者は何も開示することはない。

グリセロールと脂肪酸にTGLの破壊プロセスは、基礎脂肪分解中ATGL 9によって触媒および誘導脂肪分解21、22、23間にアデニリルシクラーゼ/ PKA依存性経路の活性化を含むタンパク質のアレイによって調整されます。脂肪分解の強化は、輸送およびエネルギー使用のための血漿中の血漿レベルを増加させる<sup...

fat 2及び脂肪酸酸化が熱発生3,4のために必要とされるような脂肪組織は、エネルギーを蓄えます。ダイエットによって摂取された脂肪酸は、アポ蛋白質と共にキロミクロンにパッケージングされ、血液循環によって身体の異なる組織に送達される。本体ストアにおけるほとんどの細胞fat 5、6、エネルギーの予備、脂肪組織を格納する余分なエネルギーありません。脂肪組織における脂質分解は複雑なプロセスによって調節され、脂肪分解の分子的詳細はまだ曖昧なままである7 。 脂肪分解は、脂肪組織に貯蔵されたトリグリセリド（TGL）を加水分解して、酵素脂肪トリグリセリドリパーゼ（ATGL） 8によってグリセロールおよび脂肪酸（FA）を生成するプロセスである。基礎および刺激脂肪分解における変化は、肥満の特徴である。 The basal脂肪分解は、TGLをジアシルグリセロール（DAG）に変換し、続いてモノアシルグリセロール（MAG）に加水分解するATGL活性化9によって調節される。アデニリルシクラーゼを介して、ホルモン感受性リパーゼ（HSL）の活性化は、環状アデノシン一リン酸（cAMP）依存性プロテインキナーゼA（PKA）の刺激を活性化し、脂肪分解を引き起こします。従って、このプロセスに関与するタンパク質の活性を分析するためには、脂肪分解、基礎および刺激の測定が重要である。また、脂肪分解の分子調節を解明することは、肥満に対する新規な治療戦略を開発するために有益であり得る10 。脂肪分解および脂肪酸酸化を刺激する分子は、貯蔵所に貯蔵される脂肪を減少させる潜在的な候補であるので、再現性のために頑強なアッセイを用いることが重要である。 以前に公開されたデータは、CAPによる白色脂肪組織で発現されるTRPV1タンパク質の活性化が、および鼠径脂肪パッドにおけるFSK（アデニリルシクラーゼ活性化剤）刺激脂肪分解11 。前の研究はまた、CAPによるTRPV1の長期的な活性化はPKA 12を活性化させることを示唆しています。 PKAの活性化は、TRPV1の役割を検証するNCDまたは（CAP±）HFDそれぞれの食餌を給餌の32週間後-fedマウスから単離した鼠径脂肪パッドにおける基礎とPKA依存性刺激脂肪分解の両方を測定する脂肪分解13,14を刺激するので脂肪分解における活性化。 この記事では、基礎脂質分解および刺激脂肪分解を効率的に測定する方法について説明します。測定15のためのグリセロールおよび退屈な高速液体クロマトグラフィーまたはガスクロマトグラフィー/質量分析法の放射性同位体を使用する他の方法が、16が利用可能であり、この方法は、より直接的簡単かつコスト効率を提供します脂肪組織における脂肪分解を決定する技術。

<table border="0" cellpadding="0" cellspacing="0" width="694">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Capsaicin</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">M2028</td>
			<td style="width:189px;">TRPV1 agonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forskolin</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">F6886</td>
			<td>Adenylyl cyclase activator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM</td>
			<td style="width:171px;">GE healthcare and life sciences, UT, USA</td>
			<td style="width:119px;">SH30081.01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High fat diet</td>
			<td style="width:171px;">Research diets, New Brunswick, USA</td>
			<td style="width:119px;">D12492</td>
			<td>Abbreviated as HFD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:171px;">Amresco, USA</td>
			<td style="width:119px;">O497</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:171px;">Thermofisher Scientific</td>
			<td style="width:119px;">BP358-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium deoxycholate</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">D6750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">D9163</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium orthovanadate</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">S6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Free Glycerol reagent</td>
			<td style="width:171px;">Sigma USA</td>
			<td style="width:119px;">F6428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">D8779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triacsin C</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">T4540</td>
			<td>Acyl CoA transferase inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine serum albumin</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:119px;">31998-8</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methanol</td>
			<td style="width:171px;">Thermofisher Scientific, USA</td>
			<td style="width:119px;">A412-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hydroxide</td>
			<td style="width:171px;">Amresco, USA</td>
			<td style="width:119px;">O583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium dodecyl sulfate</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">L3371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bicinchoninic acid reagent</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">BCA1-1KT</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UV-VIS Spectrophotometer</td>
			<td style="width:171px;">Pharmacia Biotech, NJ, USA</td>
			<td style="width:119px;">Ultrospec 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal chow diet</td>
			<td style="width:171px;">Labdiet.com</td>
			<td style="width:119px;">500I</td>
			<td>abreviated as NCD</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">C57BL/6 mice</td>
			<td style="width:171px;">Jackson Laboratory, CT, USA</td>
			<td style="width:119px;">Stock number000664</td>
			<td>wild type mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:171px;">Heathrow Scientific, USA</td>
			<td style="width:119px;">HS 234526A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol standard</td>
			<td style="width:171px;">Sigma, USA</td>
			<td style="width:119px;">G7793</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of basal and forskolin-stimulated lipolysis in inguinal adipose fat pads

脂肪分解は、脂肪組織中にトリグリセリドとして貯蔵された脂質を加水分解してグリセロールおよび脂肪酸にするプロセスである。この記事では、正常食餌（NCD）、高脂肪食（HFD）または0.01を含む高脂肪食餌を与えた野生型マウスから単離した鼠蹊脂肪パッドにおける基礎およびフォルスコリン（FSK）刺激脂肪分解の測定方法を記載するカプサイシン（CAP;一過性受容体ポテンシャルバニロイドサブファミリー1（TRPV1）アゴニスト）を32週間投与した。 生体外脂肪分解を実施するために本明細書に記載される方法は、Schweiger et al。 1 UV-Visible（UV / Vis）分光光度法によりグリセロールレベルを測定するための詳細なプロトコールを提示します。本明細書に記載の方法は、脂肪分解測定のための鼠径脂肪パッドを首尾よく分離して、一貫した結果を得るために使用することができる。鼠径脂肪パッドについて記載されたプロトコルは、他の組織における脂肪分解を測定するために容易に拡張することができる。

基礎および刺激脂肪分解に対するCAPの効果を評価するために、本研究では、NCDまたはHFD（±CAP）飼育野生型マウスから単離された鼠蹊脂肪細胞の脂質分解を測定した。鼠径脂肪パッドの基礎およびFSK刺激脂肪分解の代表的な結果を表Iに示す 。アシルcoAシンテターゼを阻害し、TGLの再生を妨げるトリアクシンCの存在下での基礎およびFSK刺激グ?...

Watch this Scientific Journal Video about Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads at JoVE.com

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

このプロトコールは、正常飼料（NCD）または高脂肪食（HFD）+カプサイシン摂食野生型マウスから得られた鼠径脂肪パッドにおける基礎およびフォルスコリン刺激脂肪分解を決定する方法を記載する。脂肪分解の指標として、鼠径脂肪パッドからグリセロール放出を測定した。

腹腔脂肪脂肪パッドにおける基礎およびフォルスコリン刺激脂質測定の測定

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes ...

measurement-basal-forskolin-stimulated-lipolysis-inguinal-adipose-fat

Research

JoVE Journal

Biochemistry

9.5K ビュー.  University of Wyoming. このプロトコールは、正常飼料（NCD）または高脂肪食（HFD）+カプサイシン摂食野生型マウスから得られた鼠径脂肪パッドにおける基礎およびフォルスコリン刺激脂肪分解を決定する方法を記載する。脂肪分解の指標として、鼠径脂肪パッドからグリセロール放出を測定した。 

ビデオ: Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

鶏の羽根や化粧品への応用からケラチンを加水分解物の準備

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

生化学

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

分離とシロイヌナズナ由来のミトコンドリアの呼吸測定

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

活性酸素・過酸化水素とフラビン含有ミトコンドリア脱水素酵素による NADH 生産の同時測定

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

マウス脂肪組織の採取と RNA 解析のための処理

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

その場で測定と細胞密度の相関、発光細菌の発光

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial species, the koala (Phascolarctos cinereus), faces prolonged exposure to anthropogenic-induced stressors and assessment of chronic stress in wild populations is urgently warranted. One of the most effective ways to measure chronic stress is through analyzing the glucocorticoid hormone cortisol in hair or fur, as it supports physiological and behavioral responses. This laboratory validation study aims to test current techniques to validate an optimal hormone extraction method to be used as a non-invasive measure of cortisol in koala fur. It is recognized that using non-invasive techniques to measure stress hormones is preferred over traditional, invasive techniques due to their ideal practical and ethical standpoints. Additionally, it is comparatively easier to acquire fur from koalas than it is to acquire samples of their blood. This study used samples of koala fur acquired from the Adelaide Koala and Wildlife Hospital to run a number of hormone extraction techniques in an attempt to validate an optimal cortisol extraction method. Results showed that 100% methanol provided the most optimal solvent extraction compared to 100% ethanol or 100% isopropanol based on parallelism results. In conclusion, this method of cortisol extraction from koala fur provided a reliable non-invasive assay that could be used to study chronic stress in koalas.

Cortisol Measurement in Koala Phascolarctos cinereus Fur

We present a protocol to determine the optimal extraction solvent to measure cortisol from koala fur. The solvents used in this protocol are methanol, ethanol and isopropanol. Determining an optimal extraction solvent will aid in reliably measuring fur to determine the impact of chronic stress on koalas.

コアラでのコルチゾール測定(ファスコラルコトス・シネレウス)毛皮

Optimal methods of hormone extraction used to measure stress in animals across sample types are not always the same. Australia&#39;s iconic marsupial ...

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the interpretation of the biological activity of any compound in drug development or in basic research projects. In epigenetics, target engagement is most often assessed by the analysis of proxy markers instead of measuring the union of the compound to the target. Downstream biological readouts that have been analyzed include the histone mark modulation or gene expression changes. KDM1A is a lysine demethylase that removes methyl groups from mono- and dimethylated H3K4, a modification associated with the silencing of gene expression. Modulation of the proxy markers is dependent on the cell type and function of the genetic make-up of the cells investigated, which can make interpretation and cross-case comparison quite difficult. To circumvent these problems, a versatile protocol is presented to assess the dose effects and dynamics of the direct KDM1A target engagement. The assay described makes use of a KDM1A chemoprobe to capture and quantify uninhibited enzyme, can be broadly applied to cells or tissue samples without the need for genetic modification, has an excellent window of detection, and can be used both for basic research and analysis of clinical samples.

Here, we present a protocol to measure KDM1A target engagement in a human or animal cell, tissue or blood samples treated with KDM1A inhibitors. The protocol employs chemoprobe tagging of the free KDM1A enzyme and direct quantification of the target occupation using chemoprobe-based immunoassays and can be used in preclinical and clinical studies.

ケモプローブベースの免疫アッセイを用いてKDM1A標的エンゲージメントを直接測定

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

アミロイドフィブリルスによる脳ミトコンドリアの相互作用と膜透過性

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 &#176;C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

The purity and integrity of the isolated RNA is a vital step in RNA dependent assays. Here, we present a practical, rapid, and inexpensive method to extract RNA from a small quantity of undamaged pancreatic tissue.

ラット膵臓組織の迅速かつ費用対効果の高いRNA抽出

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective ...