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).

所有的动物实验应按照当地和联邦法规和体制IACUC和辐射安全管理局批准进行。 1.准备<ol><li>该法的若干天前，解冻一瓶500毫升肝脏消化培养基（LDM），并重新冻结〜35毫升分装在50毫升锥形管。储存在-20℃直到需要。 </li><li>在试验前一天，通过高压灭菌前消毒清洁清扫工具。 </li><li>在测定当天，治疗的24孔培养板的必要的量与胶原蛋白。 <ol><li>混合1份3毫克/毫升鼠尾胶原与50份PBS中。加约500μl到一个24孔板的每个孔中，并孵育在室温下3-5分钟（RT）。取出胶原蛋白，并允许板晾干的引擎盖。重复的至少一个额外的时间。 注意:胶原涂层可以在广告中进行长达一周万斯和板保存在4℃的保鲜膜密封。 </li></ol></li><li> LDM准备用于分析:解冻LDM和温暖至37℃，并用1N KOH调节pH值至7.4。使用过滤0.2微米的注射器过滤器并将其放置在一个循环42℃水浴。 </li><li>温馨25毫升肝脏灌注中，以42℃的循环水浴。 </li><li>准备90％的胶体二氧化硅涂有聚乙烯吡咯烷酮的解决方案:加入1毫升的10倍DPBS到9毫升胶态氧化硅涂有聚乙烯吡咯烷酮。调整使用0.1N盐酸pH值至7.4。用无菌0.2微米的注射器过滤器进行过滤。存储在RT。 </li><li>温暖的电镀液至37℃。 </li></ol> 2.隔离主小鼠肝细胞的<ol><li>设置蠕动泵，管道和解剖表:通过用5毫升70％的乙醇在蒸馏水中，接着用10毫升无菌水中冲洗消毒管材。将油管肝脏灌注中，并运行泵填写油管的整个长度。 </li><li>通过体制认可的方法，牺牲鼠标。 </li><li>解剖打开腹腔:用70％乙醇宽松喷雾鼠标腹部。使用钝端剪刀，通过真皮进行中线切开腹部的长度和反映横向。做一个类似的切口，在腹膜，露出内脏。 <ol><li>用钝刀，轻轻地取代了肠子，露出腹部血管找到腹部下腔静脉（IVC），然后将血管下方的缝合线，远端肾静脉</li></ol></li><li>远端的缝合线，放置一个针头和导管插入下腔静脉，它前进超越了缝合线的水平。与针仍然就位，配合围绕导管缝合到保持在适当位置。小心取出针。如果做得正确，血液流经的导管。 </li><li>用移液管，填补灌注中导管的其余区域，确保没有空气存在。精雕细刻，连接管路的导管。 </li><li>解剖开肺腔:轻轻地反映上级肝叶，露出隔膜。仔细穿刺尖端剪刀隔膜，然后做一个横向切口，显露胸膜腔，注意避免胆囊及胸腔血管。周围放置胸下腔静脉刚好接近肝静脉牛头犬钳。 </li><li>切门静脉，然后打开泵在3 - 4毫升/分钟。使用约20 ml的灌注介质灌注肝灌注培养基肝5分钟。 注:肝应立即从红色变为灰/棕褐色。如果肝脏的部分保持为红色，这可能表示灌注不良，成功分离的可能性大大降低。在灌注，要小心，以确保没有用完的媒介，而没有气泡进入管。 </li><li>继5分钟孵化，停止泵和管道传送到肝消化物培养基。重新启动泵和灌注肝脏另一个10 - 15分钟（直到介质是耗尽）。 </li><li>在灌注结束时，停止泵。肝脏应该有一个粉红色的色调，并出现一定程度的放大。切除肝经仔细解剖。取出胆囊和肝脏转移到10cm组织培养皿。 </li><li>在生物安全柜，加入10 ml的电镀培养基（表1）到肝脏。轻轻刮去使用或者镊子或手术刀去除肝细胞肝癌。使用100微米的细胞过滤并转移到50毫升锥形管过滤悬浮液。一个额外的10毫升电​​镀液中和池50毫升的锥形管清洗板。 </li><li>通过在350×g离心，4℃离心5分钟沉淀细胞。 </li><li>吸中期和10毫升电​​镀液中悬浮颗粒和加入10毫升90％的胶体二氧化硅涂宝lyvinylpyrrolidone。轻轻离心混合的步骤2.11。离心后，一层的死细胞将浮在混合物的顶部，同时活细胞将沉淀在底部。 </li><li>吸死细胞和介质。用20毫升电镀液，离心机在2.11的洗净2次。 </li><li>悬浮细胞在10毫升平板培养基中。计数细胞，血球和放置9×10 4个细胞/孔在胶原处理的24-孔培养皿。孵育在37℃组织培养箱2小时。每个板可用于任一脂肪酸氧化测定法或在脂肪形成试验。 </li><li>或者，在37℃下改变井维护中（ 表1）和文化。细胞可培养2 - 3天没有影响检测结果。 </li></ol> 3脂肪酸氧化测试警告:使用放射性可能非常危险。所有的采购，仓储，装卸，以及二放射性物质的sposal应按照机构，州和联邦的条例和准则进行。 <ol><li> 16 - 20小时前的检测，洗涤细胞2次，温PBS。改变的细胞于无血清血清饥饿培养基（表1）与20nM的胰高血糖素孵育过夜，在37℃。 注意:因为胎牛血清包含未知浓度的代谢激素（例如胰岛素，胰高血糖素），血清饥饿细胞，以消除任何混杂影响，这可能对分析。细胞是可行的血清饥饿培养基为&gt; 24小时。胰高血糖素的治疗是用于刺激的肝细胞内的脂肪酸氧化。 </li><li>在测定的早晨，准备预孵化培养基:重悬棕榈酸钠适量在超纯水中，使得100mM的溶液，并加热到70℃10分钟。在此期间，准备所需量（每24孔板的0.5毫升）的DMEM用25mM HEPES，1％BSA级分V，且为20nM胰高血糖素。加热至37℃。 <ol><li>一旦溶解，棕榈酸酯添加到250微米到培养基中的最终浓度。改变肝细胞预孵育培养基，37℃下2小时。保留一些预孵育培养基，在37℃以备后用。 </li></ol></li><li>在培育期间，通过蒸发干燥在氮气14 C-棕榈酸适量（加入0.5uCi /孔）。 <ol><li>例如，为了测量24个样品在一个24孔板中，转移的0.1微居里/毫升14 C-棕榈酸酯120微升到1.5ml管中。慢慢蒸发乙醇通过在从3-5厘米的距离在通风橱中的溶液吹入氮气溶剂。溶剂应当蒸发在大约30 - 40分钟，留下干14 C-棕榈酸酯在管的底部。 </li></ol></li><li>在孵育结束前大约15分钟，重悬14C-棕榈酸酯在0.1N氢氧化钠（12.5微升/微居里）。孵育在70℃下10分钟。添加三卷温暖预培养介质，并通过上下抽吸混合。 </li><li> 25微升稀释14 C-棕榈酸酯以及穗每个。轻摇板混合并在37℃下90分钟。这就是试验板。 </li><li>在孵化，准备过滤纸碟（ 图1）。 <ol><li>从无菌24孔板取下盖子。放置一个2厘米×2厘米一片滤纸在每孔的底部。采用了一块4"×7"封口膜叠加的板块。 </li><li>采用大的矩形对象，诸如移液器尖端框，擦上井的封口膜穿孔的封口膜在井孔和形成密封在所述板的剩余部分。除去封口膜的穿孔圈现已覆盖的水井。板现在应盖紧封口膜在各个领域，除了良好Øpenings。 </li></ol></li><li> 10分钟孵化结束前，加入200微升3 N的NaOH滤纸板的每个孔，确保滤纸吸收所有的液体。 <ol><li>任选在冷冻前，将培养基转移到新鲜的24孔板中。用PBS洗涤一次后，裂解剩余的细胞在0.1N HCl和计算通过BCA测定蛋白质含量。 </li></ol></li><li>在孵育结束时，管理单元冷冻在液氮中的试验板。要小心，以确保每口井，然后再继续被完全冻结。 </li><li>加入100微升70％高氯酸的到测定板的每个孔中。立即盖上滤纸盘。将平板在轨道摇床中岩石在为80rpm轨道速度在RT 2小时。 </li><li>继温育后，处理该样品: <ol><li>为了测量CO 2的级分，在闪烁的滤纸方块转移到4ml液体闪烁流体小瓶和措施14 C信号。 </li><li>为了测量酸可溶性材料，400微升培养基的转移到1.5ml微量离心管中。以最大速度离心10分钟。加入100微升所得上清以500微升2:1氯仿 - 甲醇（体积/体积），和涡旋简要。 <ol><li>再次加入250微升水到混合物中，并涡旋。离心样品在3000×g的10分钟。转移200μl的上层相的到4ml液体闪烁液的闪烁小瓶，测量14 C信号。 </li></ol></li></ol></li></ol> 4.脂肪生成分析<ol><li>傍晚开始前测定，用温热的PBS洗涤细胞2次。改变肝细胞血清饥饿培养基与100纳米胰岛素。孵育过夜，在37℃。 注:由于胎牛血清含有未知浓度的代谢激素（ 如胰岛素，胰高血糖素），血清饥饿细胞，以消除任何混杂影响，这可能对分析。细胞是可行的血清饥饿培养基为&gt; 24小时。胰岛素是用在该测定刺激脂肪生成。 </li><li>使脂肪生成介质:血清饥饿培养基用100nM胰岛素，10μM冷醋酸和0.5微居里3 H-醋酸每口井。改变细胞脂肪生成培养基孵育在37℃下2小时。包括感兴趣的任何化合物进行测试。 </li><li>培养结束后，洗涤细胞2次，用PBS。裂解细胞刮中120微升0.1N盐酸。储备10微升的蛋白质检测（通过BCA法评估），并转移100微升至1.5 ml离心管中。 </li><li>提取脂质，通过加入500微升2:1氯仿 - 甲醇（体积/体积）。短暂涡旋并在室温下孵育5分钟。加入250微升水，涡旋并在室温下孵育另外5分钟。样品离心在3000 XG，室温10分钟。小心在闪烁瓶中下层相转移到4ml液体闪烁流体，并测量3 H活性。 </li></ol>

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The authors indicate they have no conflicts of interest.

从牺牲灌注时间应小于3分钟的理想灌注和肝脏的胶原酶消化。一旦灌注灌注中启动，肝应立即改变外观，从红色到浅。经过与LDM大约10分钟的潜伏期，会使肝脏出现浮肿和粉红色。倘灌注不足，肝脏可能不表现出这些变化，而这通常会导致较低的肝细胞的产率。 以下的洗涤步骤后，分离的肝细胞可以在电镀之前被存储数小时悬浮液在冰上。一旦镀金，培养肝细胞需要几个小时才能坚持和传播出去。继2小时培养的电镀液，肝细胞会保持小而圆。过夜温育后，肝细胞将在一个更特性六角形外形，其中许多将双核。如果准备是不健康的，则CELLS将展出大量的肉芽和偶尔的出泡，指示细胞死亡。为了最好地保持文化的活力，媒体应改为每24小时结果并注意尽量减少暴露在露天。 棕榈酸钠不溶于水在RT，然而，温育在70℃下它应完全溶解后。曝光至RT将引起脂质迅速固化，从而就必须迅速地工作。一旦棕榈酸已经溶解在预孵育培养基，关键是要保持在37℃的介质中以保持脂质溶解性。 如上所述，以确保从脂肪酸氧化测定准确的结果，介质必须在液氮中完全冻结。这防止CO 2的任何逃逸加入高氯酸到孔中。紧接在加入高氯酸到冷冻样品，测定板必须是紧紧Ç由滤纸板overed，确保对齐板的孔之间的气体转移。由于滤纸浸泡在过量的氢氧化钠，将反应的化学计量是能够捕获所释放的 CO 2作为碳酸氢钠的。按照此协议的实验已经产生可再现的结果，与具有典型的重复％CV≤10.如果需要的话，从脂肪酸氧化测定法或从脂肪形成试验中的细胞裂解物的酸可溶材料能够被存储在-80℃下和加工的以后不会对结果产生任何明显的影响。上述测定允许相对简单和时间高效的机制，以评估在原代肝细胞从小鼠肝脏中分离脂质代谢。通过使用离体培养的，这些方法允许检测对脂肪酸氧化和脂肪生成几个条件的影响。这个协议可以适于评估这些PROCE遗传改变的作用小规模企业，然而，肝细胞的分离是时间限制，因此在体内分析可以是更适合于调查某些转基因动物模型。如果多个肝细胞制剂分析是必要的，测定值的归一化蛋白质水平可作为任选步骤3.7.1描述和用于归一化来执行。我们建议多准备进行的试验进行比较的相对变化与一个适当的控制。 最后，我们还没有探讨了较长时期培养的选项，但是，肝细胞可在培养数天表现出相当的代谢特征。稍加修改，这个协议可以适于允许前评估对脂质代谢化合物的效果几天的治疗。

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.

肝细胞隔离通常会导致1 - 3×10 7个细胞。过夜温育后，将细胞会出现六角形，其中许多将双核（图2）。健康的细胞中不应有肉芽或泡，这是指示细胞死亡的。 在一般情况下，脂肪酸氧化法是运行在每个测试化合物，三时五十七重复。计数的CO 2的样品是大约五分之一的那些从酸可溶性物质衍生的。我们通常计算的 CO 2与酸可溶性材料作为完全氧化的量度的比率（即，通过柠檬酸循环氧化的量）。促进细胞呼吸的物质，如羰基氰化物-4-（三氟甲氧基）苯腙（FCCP）将这个比值移向的 CO 2，通过TCA循环指示多种氧化。呼吸链抑制剂将降低氧化在整体上（图3）。当驾驶该测定中，最好是包括一个无细胞对照，以验证该过程产生细胞特异性活性。 在脂肪生成分析是最经常被比作一个零时间点控制（即收获前与底物处理的细胞）。该试验应该通过至少四个小时的潜伏期是线性的随时间变化的。化合物增强脂肪酸的氧化，如FCCP，或减少ATP合成，如寡，将减少脂肪生成活性（ 图4）。 <img alt="图1" src="/files/ftp_upload/52982/52982fig1.jpg" /> 图1:过滤板要按照步骤3.6中所述准备过滤板的制作，从一个无菌的24孔板取下盖子。放置一个2厘米×2厘米一片滤纸在每个基本良好。用7"×4"片第叠加整个板块膜，擦板紧密地密封所述孔的顶部。这将生成的多家开口，应删除的封口膜穿孔圈。此外，高氯酸步骤3.9后，紧紧地将滤纸板放在检测板产生一个密封。 <img alt="图2" src="/files/ftp_upload/52982/52982fig2.jpg" /> 图2:原代肝细胞在培养阶段的镀覆在涂胶原蛋白的24孔板后16小时对比的健康和不健康的原代小鼠肝细胞的图像。比例尺，200微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52982/52982fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52982/52982fig3.jpg" /> 图3:脂肪酸氧化所小学<html>小鼠肝细胞在培养14 C-棕榈酸酯氧化为（A）中的 CO 2，（B）的酸可溶性物质，和（C） 的 CO 2，以酸可溶的材料从孵育车辆，FCCP，或寡霉素原代肝细胞的比率。数据为均值±SEM。 * P &lt;0.05，** P &lt;0.01与车辆由单尾学生的t检验。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52982/52982fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52982/52982fig4.jpg" /> 图4:脂肪生成培养的主小鼠肝细胞 （A）生脂活性随时间在3小时乙酸和车辆，寡，或FCCP存在（B）脂肪合成酶活性治疗原发性肝细胞。数据为均值±SEM。 *** P &lt;0.001对车辆通过单尾学生t试验。 内容"&gt;表1:培养基培养基组成为原代肝细胞的分离。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 表1 </td><td></td></tr><tr><td colspan="2">文化传媒</td></tr><tr><td></td><td></td></tr><tr><td colspan="2">电镀液</td></tr><tr><td></td><td></td></tr><tr><td> DMEM </td><td> 500毫升</td></tr><tr><td> FBS </td><td> 10％ </td></tr><tr><td>丙酮酸钠</td><td> 2毫米</td></tr><tr><td>笔/链球菌</td><td> 2％ </td></tr><tr><td>地塞米松</td><td> 1μM </td></tr><tr><td>胰岛素</td><td> 0.1μM </td></tr><tr><td></td><td></td></tr><tr><td></td><td></td></tr><tr><td colspan="2">维护中</td></tr><tr><td></td><td></td></tr><tr><td> DMEM </td><td> 500毫升</td></tr&gt; <tr><td> BSA V组分</td><td> 0.2％ </td></tr><tr><td>丙酮酸钠</td><td> 2毫米</td></tr><tr><td>笔/链球菌</td><td> 2％ </td></tr><tr><td>地塞米松</td><td> 0.1μM </td></tr><tr><td>胰岛素</td><td> 1纳米</td></tr><tr><td></td><td></td></tr><tr><td></td><td></td></tr><tr><td colspan="2">饥饿中</td></tr><tr><td></td><td></td></tr><tr><td> DMEM </td><td> 500毫升</td></tr><tr><td> BSA V组分</td><td> 0.2％ </td></tr><tr><td>丙酮酸钠</td><td> 2毫米</td></tr><tr><td>笔/链球菌</td><td> 2％ </td></tr></tbody></table>

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

从头脂肪合成和β-脂肪酸氧化构成肝关键代谢途径，被扰动在几种代谢疾病，包括脂肪肝疾病途径。在这里，我们证明了小鼠原代肝细胞的分离和描述了β-脂肪酸氧化和脂肪生成量化。

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

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

Nanyang Technological University

Research

JoVE Journal

Biology

20.2K 次观看.  University of Massachusetts Medical School.  从头脂肪合成和β-脂肪酸氧化构成肝关键代谢途径，被扰动在几种代谢疾病，包括脂肪肝疾病途径。在这里，我们证明了小鼠原代肝细胞的分离和描述了β-脂肪酸氧化和脂肪生成量化。 

视频: 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.

测量新鲜分离的小鼠肝细胞悬浮液中的脂肪酸β氧化

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

科研

生物化学

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.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</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 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

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;

分化肝细胞中的血管性脂细胞病的诱导和特征

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.

评估小鼠全身脂质处理能力

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.

体外实验性硬化模型:脂质超载介质中的肝细胞培养

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.

一种改进的小鼠原代肝细胞分离时间和劳动效率方案

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.

<meta charset="utf8"></head><body>培养骨细胞中脂肪酸氧化的评价

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

培养骨细胞中脂肪酸氧化的评价

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.

测量体外分化的离体小鼠脂肪组织和原代前脂肪细胞的脂肪分解率

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 ...

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

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此视频中的章节

测量新鲜分离的小鼠肝细胞悬浮液中的脂肪酸β氧化

优化分析

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

分化肝细胞中的血管性脂细胞病的诱导和特征

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