Name: determination of fatty acid oxidation and lipogenesis in mouse primary hepatocytes
Uploaded: 2015-08-27T15:30:00.000+00:00
Duration: 12 min 11 s
Description: Watch this Scientific Journal Video about Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes at JoVE.com

We would like to acknowledge Susan Gray and Umadevi Chalasani for their help with technical aspects of the hepatocyte isolation protocol. This work was supported by NIDDK grant 5R01DK089185 (to M.P. Cooper) and the DERC Pilot and Feasibility Program at UMMS (to M.P. Cooper).

すべての動物実験は、ローカルおよび連邦規制に従って及び制度IACUCと放射線安全管理の承認を得て実施されるべきです。 1.準備<ol><li>数日前、アッセイに、肝臓ダイジェストミディアム（LDM）の500ミリリットル瓶を解凍し、50mlのコニカルチューブに〜35ミリリットルのアリコートを再凍結。必要になるまで-20℃で保存してください。 </li><li>アッセイの1日前に、オートクレーブによりクリーンな解剖ツールを事前に滅菌します。 </li><li>アッセイの日に、コラーゲンとの24ウェル培養プレートの必要な量を扱います。 <ol><li> PBSの50部で3 mg / mlのラット尾コラーゲン1部を混合します。 24ウェルプレートの各ウェルに約500μLを加え、室温（RT）で3~5分間インキュベートします。コラーゲンを外し、プレートをフード内空気乾燥することができます。少なくとも一つの追加の時間を繰り返します。 注意：コラーゲンコーティングが広告に1週間まで行うことができますヴァンス、プレートをプラスチックラップで密封し、4℃で保存しました。 </li></ol></li><li> 37℃に解凍LDMと暖かいと1N KOHを使用してpHを7.4に調整します。アッセイのLDMを準備します。再循環を42℃の水浴中で、0.2μmのシリンジフィルターと場所を使用してフィルタ。 </li><li>水浴を再循環で42°Cまでの肝灌流中のウォーム25ミリリットル。 </li><li>ポリビニルピロリドンの溶液を塗布し、90％のコロイダルシリカを準備します。ポリビニルピロリドンで被覆されたコロイド状シリカの9ミリリットルに10倍のDPBS 1ミリリットルを追加します。 0.1 N HClを用いてpHを7.4に調整します。滅菌0.2μmのシリンジフィルターでろ過します。室温で保管してください。 </li><li> 37°Cまでのめっき中暖かいです。 </li></ol>初代マウス肝細胞の単離2。 <ol><li>蠕動ポンプ、チューブ、および解剖テーブルを設定します。滅菌水10ml、続いて蒸留水で5ミリリットルの70％エタノールで洗浄することによりチューブを滅菌します。埋めるために肝灌流培地でチューブと実行ポンプを配置しますチューブの全長。 </li><li>制度承認の方法により、マウスを生け贄に捧げます。 </li><li>腹腔を開き、解剖70％エタノールで自由にマウスの腹部スプレー。平滑末端はさみを使用して、真皮を通って腹部の長さを正中切開を行い、横方向に反映しています。内臓を露出するために腹膜に同様の切開を行います。 <ol><li>鈍器を使用して、静かに腎静脈の遠位に、腹部下大静脈（IVC）を見つけて、血管の下に縫合糸を配置腹部血管系を露出するように腸を変位</li></ol></li><li>遠位縫合糸に、縫合糸のレベルを超えて、それを進める、IVCに針やカテーテルを配置します。まだ場所に針で、所定の位置に保持するために、カテーテルの周りに縫合糸を結びます。慎重に針を取り除きます。カテーテルを通して流れに血液、正しく行われた場合。 </li><li>ピペットを使用することで、確実に、灌流媒体とカテーテル内の残りの領域を埋めます空気が存在しません。細心の注意を払って、カテーテルにチューブを取り付けます。 </li><li>肺の空洞を開く解剖：優しくダイヤフラムを露出するために、優れた肝葉を反映しています。慎重に胆嚢と胸膜血管系を避けるように注意しながら、胸膜腔を露出するために、横方向の切開を行い、その後、鋭い先端のハサミで隔膜を穿刺。肝静脈のすぐ近位胸部下大静脈の周りブルドッグクランプを配置します。 </li><li>門脈をカットし、3でポンプの電源をオンに - 4ミリリットル/分。灌流培地の約20ミリリットルを用いて5分間肝灌流培地で肝臓を灌流。 注：肝臓はすぐにグレー/黄褐色に赤から変更する必要があります。肝臓の部分が赤のままである場合、これはおそらく乏しい灌流を示し、成功した分離の可能性が大幅に減少します。灌流の間に、媒体が出て実行されず、気泡がチューブに入らないことを保証するように注意してください。 </li><li> 5分間のインキュベーションに続いて、停止しますポンプおよび肝臓ダイジェスト中にチューブを移します。 （媒体がなくなるまで）15分 - ポンプを再起動して、別の10のために肝臓を灌流。 </li><li>灌流の終了時に、ポンプを停止します。肝臓はピンクがかった色合いを持っており、やや拡大表示されます。慎重な切開により、肝臓を切除。胆嚢を削除し、10cmの組織培養皿に肝臓を転送します。 </li><li>バイオセーフティキャビネットでは、肝臓にメッキミディアム（ 表1）の10ミリリットルを追加します。静かに肝細胞を除去するために、いずれかのピンセットやメスを用いて肝臓をこすり。 50mlコニカルチューブに100μmのセルストレーナー、転送を使用してサスペンションをフィルタリングします。 50mlコニカルチューブにめっき中、プールの追加10mlでプレートを洗浄します。 </li><li> 350×gで、4℃で5分間の遠心分離により細胞をペレット化。 </li><li>めっき中の10ml中の培地を吸引し、再懸濁ペレットとPOでコーティングされた90％のコロイダルシリカの10ミリリットルを追加しますlyvinylpyrrolidone。穏やかに混合し、ステップ2.11のように遠心します。生細胞が底にペレット化する一方遠心分離した後、死細胞の層は、混合物の上に浮遊されます。 </li><li>吸引し死んだ細胞および培地。メッキ中、2.11のように遠心20mlで2回洗浄します。 </li><li>めっき中の10ml中に再懸濁細胞。血球計数器で細胞をカウントし、コラーゲン処理した24ウェル培養皿に9×10 4細胞/ウェルで置き。 2時間、37℃の組織培養インキュベーター中でインキュベートします。各プレートは、脂肪酸酸化アッセイまたは脂質生成アッセイのいずれかのために使用することができます。 </li><li>必要に応じて、37℃で維持培地（ 表1）と文化に井戸を変更します。アッセイの結果に影響を与えずに3日 - 細胞を2ために培養することができます。 </li></ol> 3.脂肪酸酸化アッセイ警告：放射能の使用は危険です。すべての購買、保管、取り扱い、およびジ放射性物質のsposalは、制度的状態、および連邦の規制やガイドラインに従って行われるべきです。 <ol><li> 16から20時間アッセイの前に、温かいPBSで細胞を2回洗浄します。 20 nMのグルカゴンで無血清血清飢餓培地（ 表1）に細胞を変更し、37℃で一晩インキュベートします。 注意：ウシ胎児血清は、代謝ホルモンの未知の濃度（ 例えば、インスリン 、グルカゴン）を含んでいるので、血清これはアッセイに持つかもしれない交絡の影響を除去するために、細胞を飢えさせます。細胞は&gt; 24時間血清飢餓培地中で生存可能です。グルカゴン治療は、肝細胞内の脂肪酸酸化を刺激するために使用されます。 </li><li>アッセイの朝に、前培養培地を準備し、10分間70℃で100mMの溶液と熱を加えるために超純水にナトリウムパルミチン酸の適切な量を再懸濁します。一方で、必要量（24ウェルプレートのウェルあたり0.5 ml）を調製25mMのHEPES、1％のBSAフラクションV、および20 nMのグルカゴンを含むDMEMの。 37℃に加熱します。 <ol><li>可溶化した後、培地に250μmの最終濃度にパルミチン酸を追加します。プレインキュベーション中に肝細胞を変更し、37℃で2時間インキュベートします。後で使用するために、37℃で、いくつかのプレインキュベーション中を予約。 </li></ol></li><li>インキュベーションの間、窒素ガス下で蒸発させて14 C-パルミチン酸（0.5μCiの/ウェル）の適切な量を乾燥させます。 <ol><li>例えば、24ウェルプレートで24のサンプルを測定して1.5 mlチューブに0.1μCiの/ mlの14 C-パルミチン酸120μlのを転送します。ゆっくりとヒュームフード3-5センチの距離からの溶液上に窒素ガスを吹き付けてエタノール溶媒を蒸発させます。チューブの底に乾燥14 C-パルミチン酸を残し、40分-溶媒が約30を蒸発させる必要があります。 </li></ol></li><li>約15分間のインキュベーションの終了前に、14を再懸濁0.1 N NaOH中のC-パルミチン酸（12.5μL/マイクロCi）。 70℃で10分間インキュベートします。暖かいプレインキュベーション中の三巻を追加し、ピペッティングにより混和します。 </li><li>希釈された14 C-パルミチン酸の25μlの各ウェルをスパイク。静かにプレートをロッキングすることによって混合し、37℃で90分間インキュベートします。これは、アッセイプレートです。 </li><li>インキュベーションの間、フィルターペーパープレート（ 図1）を作製 。 <ol><li>滅菌24ウェルプレートからカバーを取り外します。各ウェルの底にろ紙の2センチメートル×2センチピースを置きます。 4 &quot;×7&quot;パラフィルムの切れ端でプレートをオーバーレイ。 </li><li>このようなピペットチップボックスとして、大きな長方形のオブジェクトを使用して、ウェル開口部にパラフィルムを穿孔し、プレートの残りの部分の上にシールを作成するために、ウェル上にパラフィルムをこします。今井戸をカバーするパラフィルムの穴あき円を削除します。プレートは現在、しっかりウェルOを除くすべての地域でパラフィルムでカバーされるべきですpenings。 </li></ol></li><li>インキュベーションの終了の10分前に、ろ紙は、液体の全てを吸収し確認して、フィルターペーパープレートの各ウェルに3 N NaOHを200μlを添加します。 <ol><li>必要に応じて前に新鮮な24ウェルプレートにメディアを転送し、凍結します。 PBSで1回洗浄した後、0.1 N HCl中の残りの細胞を溶解し、BCAアッセイによってタンパク質含量を算出します。 </li></ol></li><li>インキュベーションの終了時に、スナップ凍結アッセイプレートを、液体窒素中で。各ウェルは、先に進む前に、完全に凍結されていることを確認するように注意してください。 </li><li>アッセイプレートの各ウェルに70％過塩素酸を100μlのを追加します。すぐにフィルターペーパープレートでカバーしています。室温で2時間80 rpmでの軌道速度でオービタルシェーカーと岩の上にプレートを置きます。 </li><li>インキュベーション後、サンプルを処理します。 <ol><li>シンチレーション4 mlの液体シンチレーション液をろ紙の正方形を転送する、CO 2の画分を測定します14 C信号をバイアルおよび測定します。 </li><li>酸可溶性物質を測定するために、1.5mlマイクロチューブに培地を400μlのを転送します。 10分間最大速度で遠心します。簡単に1クロロホルム - メタノール（v / v）であり、渦：2の500μlに得られた上清を100μlを追加します。 <ol><li>再び混合物に水を250μl加え、ボルテックス。 3000×gで10分間遠心サンプル。シンチレーションバイアルに液体シンチレーション流体4ミリリットルに上相の200μLを移し、14 C信号を測定します。 </li></ol></li></ol></li></ol> 4.脂質生成アッセイ<ol><li>アッセイの前に開始する夜は、温かいPBSで細胞を2回洗浄します。 100nMのインスリンで血清飢餓中に肝細胞を変更します。 37℃で一晩インキュベートします。 ウシ胎児血清は代謝ホルモンの未知の濃度が含まれているので（ 例えば、インスリン 、グルカゴン注：）、血清がこのアッセイに持つかもしれない交絡の影響を除去するために、細胞を飢えさせます。細胞は&gt; 24時間血清飢餓培地中で生存可能です。インスリンは、脂質生成を刺激するために、このアッセイで使用されます。 </li><li> 100nMのインスリン、10μMの冷たい酢酸および0.5μCiの3 H-アセテートウェル当たりで血清飢餓培地：脂質生成中にします。脂質生成中に細胞を変更し、37℃で2時間インキュベートします。試験しようとする目的の任意の化合物が含まれます。 </li><li>インキュベーション期間の後、PBSで細胞を2回洗浄します。 0.1 N HClを120μlの掻き落とし行ない、細胞を溶解します。リザーブ10タンパク質アッセイ用μL（BCAアッセイにより評価）、および1.5mlマイクロチューブに100μlのを転送します。 </li><li> 1クロロホルム - メタノール（v / v）の：2の500μlに添加することにより、脂質を抽出します。渦は簡単にし、5分間室温でインキュベートします。水、渦の250μLを加え、さらに5分間室温でインキュベートします。遠心サンプル3000×gで、室温で10分。慎重にシンチレーションバイアルに4 mlの液体シンチレーション流体に下相を転送し、3 H活性を測定します。 </li></ol>

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The authors indicate they have no conflicts of interest.

犠牲から灌流までの時間は、肝臓の理想的な灌流およびコラゲナーゼ消化のために3分未満でなければなりません。灌流培地で灌流が開始されると、肝臓はすぐに淡赤からに外観を変更する必要があります。 LDMとのインキュベーションの約10分後、肝臓が腫れてピンク色に表示されます。潅流が不十分である場合には、肝臓はこれらの変化を示さないことがあり、これは典型的にはより低い肝...

Non-alcoholic fatty liver disease is one of the leading causes of liver disease in Westernized cultures1,2. Lipid accumulation within the liver is associated with cell death, fibrosis, and liver failure via yet unknown mechanisms3-6. In fatty liver disease, hepatocyte-mediated &#946;-fatty acid oxidation and de novo lipogenesis are important determinants of net lipid accumulation7,8. This article will, therefore, focus on hepatocyte isolation, followed by quantification of &#946;-fatty acid oxidation and de novo lipogenesis.
Numerous methodologies have been developed to interrogate hepatocyte lipid metabolism. Though it is possible to measure metabolism of fat in vivo using stable isotopes9,10, these methods are costly, and require large numbers of animals. Additionally, the ability to investigate the effect of exogenous chemicals is limited due to the nature of in vivo experimentation. In contrast, the isolation of primary hepatocytes from mouse liver provides an affordable avenue to pursue11. Furthermore, studying hepatocytes in culture allows investigators to study the effects of varying chemicals on lipid processing while circumventing the difficulties of in vivo experimentation. Finally, isolated hepatocytes avoid any confounding from varying genetics since they are derived from the liver of a single animal.
Here we isolate and culture of hepatocytes, and we measure &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled palmitate. The protocol detailed below is straight forward, effective, and reproducible.

<table><tbody><tr><td>Liver Perfusion Medium</td><td>Life Technologies</td><td>17701038</td><td></td></tr><tr><td>Liver Digest Medium</td><td>Life Technologies</td><td>17703034</td><td>Aliquot and store at -20 &amp;deg;C</td></tr><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>10X DPBS</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>FBS</td><td>Life Technologies</td><td>26140079&amp;nbsp;</td><td></td></tr><tr><td>Collagen</td><td>Life Technologies</td><td>A1048301&amp;nbsp;</td><td></td></tr><tr><td>Colloidal silica coated with polyvinylpyrrolidone</td><td>GE Life Sciences</td><td>17-0891-01</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Cellgro</td><td>25-000-CI</td><td></td></tr><tr><td>Penicillin / Streptomycin</td><td>Cellgro</td><td>30-001-CI</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I0516-5ML</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D2915-100MG</td><td></td></tr><tr><td>Albumin (BSA), Fraction V</td><td>MP Biomedicals</td><td>103703</td><td></td></tr><tr><td>24-Well Culture Dish</td><td>Corning Falcon</td><td>353047&amp;nbsp;</td><td></td></tr><tr><td>Tygon S3 Tubing&amp;nbsp;</td><td>Cole Parmer</td><td>06460-34</td><td></td></tr><tr><td>Male Leur Lock to 200 Barb Connectors</td><td>Cole Parmer</td><td>45518-00</td><td></td></tr><tr><td>24 G x 3/4&quot; Catheter</td><td>SurFlo</td><td>SROX2419CA</td><td></td></tr><tr><td>Perma-Hand Silk Suture</td><td>Ethicon</td><td>683G</td><td></td></tr><tr><td>Cell Strainer</td><td>Corning Falcon</td><td>08-771-2</td><td></td></tr><tr><td>IsoTemp 3013HD Recirculating Water Bath</td><td>Fisher</td><td>13-874-3</td><td></td></tr><tr><td>MasterFlex C/L Peristaltic Pump</td><td>MasterFlex</td><td>HV-77122-24</td><td></td></tr><tr><td>Microclamp</td><td>Roboz</td><td>RS-7438</td><td>Pre-sterilize in autoclave</td></tr><tr><td>5&amp;rdquo; Straight, Blunt-Blunt Operating Scissors</td><td>Roboz</td><td>RS-6810</td><td>Pre-sterilize in autoclave</td></tr><tr><td>24 mm Blade Straight, Sharp-point Microdissecting Scissors</td><td>Roboz</td><td>RS-5912</td><td>Pre-sterilize in autoclave</td></tr><tr><td>4&amp;rdquo; 0.8 mm Tip Microdissecting Forceps</td><td>Roboz</td><td>RS-5130</td><td>Pre-sterilize in autoclave</td></tr><tr><td>4&amp;rdquo; 0.8 mm Tip Full Curve Microdissecting Forceps</td><td>Roboz</td><td>RS-5137</td><td>Pre-sterilize in autoclave</td></tr><tr><td>60 ml Syringe</td><td>Becton Dickinson</td><td>309653</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Corning Falcon</td><td>352070</td><td></td></tr><tr><td>BCA Protein Assay</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Biosafety Cabinet</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2 &lt;/sub&gt;Incubator</td><td></td><td></td><td></td></tr><tr><td>Serological pipets</td><td></td><td></td><td></td></tr><tr><td>1,000, 200, 20 &amp;mu;l pipet and tips</td><td></td><td></td><td></td></tr></tbody></table>

determination of fatty acid oxidation and lipogenesis in mouse primary hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

3×10 7の全細胞-肝細胞の単離は、一般的に1になります。一晩のインキュベーションの後、細胞は、六角形の表示され、その多くは（ 図2）二核れます。健康な細胞は、細胞死の指標である顆粒またはブレブ、を欠いでなければなりません。 一般的には、脂肪酸酸化アッセイは、試験化合物ごとに3〜4回繰り返して実行されます。 CO 2のサンプ?...

Watch this Scientific Journal Video about Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes at JoVE.com

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

デノボ脂質生成およびβ脂肪酸酸化は、肝細胞に脂肪肝疾患を含むいくつかの代謝障害に摂動された経路を主要な代謝経路を構成しています。ここでは、マウス初代肝細胞の単離を実証し、β - 脂肪酸の酸化および脂質生成の定量化について説明します。

マウス初代肝細胞における脂肪酸酸化および脂質生成の決意

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

determination-fatty-acid-oxidation-lipogenesis-mouse-primary

Research

JoVE Journal

Biology

20.2K ビュー.  University of Massachusetts Medical School.  デノボ脂質生成およびβ脂肪酸酸化は、肝細胞に脂肪肝疾患を含むいくつかの代謝障害に摂動された経路を主要な代謝経路を構成しています。ここでは、マウス初代肝細胞の単離を実証し、β - 脂肪酸の酸化および脂質生成の定量化について説明します。 

ビデオ: Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

単離したてのマウス肝細胞の懸濁液中の脂肪酸β酸化の測定

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

生化学

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

の最適化分析<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

医学

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

非放射性 L azidohomoalanine ラベル法による新生タンパク質合成解析用プライマリ マウス肝細胞の分離

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

分化HepaRG細胞における大胞性脂肪症の誘導と特徴付け

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

マウスにおける全身脂質処理能力の評価

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease worldwide due to its relationship with obesity, diabetes type 2, and dyslipidemia. Hepatic steatosis, the accumulation of lipid droplets in the liver parenchyma, is a key feature of the disease preceding the inflammation observed in steatohepatitis, fibrosis, and end-stage liver disease. Lipid accumulation in hepatocytes might interfere with proper metabolism of xenobiotics and endogenous molecules, as well as to induce cellular processes leading to the advance of the disease. Although the experimental study of steatosis can be performed in vivo, in vitro approaches to the study of steatosis are complementary tools with different advantages. Hepatocyte culture in lipid overload-conditioned medium is an excellent reproducible option for the study of hepatic steatosis allowing the identification of cellular processes related to lipid accumulation, such as oxidative and reticular stresses, autophagia, proliferation, cell death, etcetera, as well as other testing including drug effectiveness, and toxicological testing, among many other possible applications. Here, it was aimed to describe the methodology of hepatocyte cell culture in lipid overload-conditioned medium. HepG2 cells were cultured in RMPI 1640 medium conditioned with sodium palmitate and sodium oleate. Importantly, the ratio of these two lipids is crucial to favor lipid droplet accumulation, while maintaining cell proliferation and a moderate mortality rate, as occurs in the liver during the disease. The methodology, from the preparation of the lipid solution stocks, mixture, addition to the medium, and hepatocyte culture is shown. With this approach, it is possible to identify lipid droplets in the hepatocytes that are readily observable by Oil-red O staining, as well as curves of proliferation/mortality rates.

A Model of Experimental Steatosis In Vitro: Hepatocyte Cell Culture in Lipid Overload-Conditioned Medium

This protocol is intended to be a tool to study steatosis and the molecular, biochemical, cellular changes produced by the over exposure of hepatocytes to lipids in vitro.

実験性脂肪化のモデル :脂質過負荷状態培地における肝細胞細胞培養

Metabolic dysfunction-associated fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

マウス初代肝細胞単離のための時間と労力の効率的なプロトコルの改善

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

生物学

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large extracellular matrix proteins that compose bone tissue and then concentrate the ions necessary for its mineralization. Data on the metabolic requirements of bone formation are emerging rapidly. While much remains to be learned, it is expected that derangements in the intermediary metabolism contribute to skeletal disease. Here, a protocol is outlined to assess the capacity of osteoblastic cells to oxidize 14C-labeled fatty acids to 14CO2 and acid-soluble metabolites. Fatty acids represent a rich-energy reserve that can be taken up from the circulation after feeding or after their liberation from adipose tissue stores. The assay, performed in T-25 tissue culture flasks, is helpful for the study of gene gain or loss-of-function on fatty acid utilization and the effect of anabolic signals in the form of growth factors or morphogens necessary for the maintenance of bone mass. Details on the ability to adapt the protocol to assess the oxidation of glucose or amino acids like glutamine are also provided.

The present protocol describes an assay to assess the capacity for fatty acid oxidation in cultures of primary bone cells or relevant cell lines.

Bone formation by differentiating osteoblasts is expected to require significant energetic input as these specialized cells must synthesize large ...

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are sequentially cleaved from the glycerol backbone, resulting in the release of free fatty acids and glycerol. Due to the low expression of glycerol kinase in white adipocytes, glycerol re-uptake rates are negligible, while fatty acid re-uptake is dictated by the fatty acid binding capacity of media components such as albumin. Both glycerol and fatty acid release into media can be quantified by colorimetric assays to determine the lipolytic rate. By measuring these factors at multiple time points, one can determine the linear rate of lipolysis with high confidence. Here, we provide a detailed protocol for the measurement of lipolysis in in vitro differentiated adipocytes and ex vivo adipose tissue from mice. This protocol may also be optimized for other preadipocyte cell lines or adipose tissue from other organisms; considerations and optimization parameters are discussed. This protocol is designed to be useful in determining and comparing the rate of adipocyte lipolysis between mouse models and treatments.

Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro

Triglyceride lipolysis in adipocytes is an important metabolic process resulting in the liberation of free fatty acids and glycerol. Here, we provide a detailed protocol to measure basal and stimulated lipolysis in adipocytes and ex vivo adipose tissue from mice.

in vitroで分化したマウス脂肪組織および初代前駆脂肪細胞における脂肪分解の速度の測定

Adipocytes store energy in the form of triglycerides in lipid droplets. This energy can be mobilized via lipolysis, where the fatty acid side chains are ...

マウス白脂肪組織の半自動単離

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

著者スポットライト:組織解離器を使用したマウス白脂肪組織からの間質血管画分の半自動分離  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

マウス初代肝細胞における脂肪酸酸化および脂質生成の決意

要約

さらに動画を探す

この動画の章

単離したてのマウス肝細胞の懸濁液中の脂肪酸β酸化の測定

の最適化分析

非放射性 L azidohomoalanine ラベル法による新生タンパク質合成解析用プライマリマウス肝細胞の分離

分化HepaRG細胞における大胞性脂肪症の誘導と特徴付け

マウスにおける全身脂質処理能力の評価

実験性脂肪化のモデル :脂質過負荷状態培地における肝細胞細胞培養

マウス初代肝細胞単離のための時間と労力の効率的なプロトコルの改善

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

in vitroで分化したマウス脂肪組織および初代前駆脂肪細胞における脂肪分解の速度の測定

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

マウス初代肝細胞における脂肪酸酸化および脂質生成の決意

要約

さらに動画を探す

この動画の章

単離したてのマウス肝細胞の懸濁液中の脂肪酸β酸化の測定

の最適化分析

非放射性 L azidohomoalanine ラベル法による新生タンパク質合成解析用プライマリ マウス肝細胞の分離

分化HepaRG細胞における大胞性脂肪症の誘導と特徴付け

マウスにおける全身脂質処理能力の評価

実験性脂肪化のモデル :脂質過負荷状態培地における肝細胞細胞培養

マウス初代肝細胞単離のための時間と労力の効率的なプロトコルの改善

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells

in vitroで分化したマウス脂肪組織および初代前駆脂肪細胞における脂肪分解の速度の測定

組織解離剤を用いたマウス白色脂肪組織からの間質血管画分の半自動単離(英語)

非放射性 L azidohomoalanine ラベル法による新生タンパク質合成解析用プライマリマウス肝細胞の分離