We appreciate Feifei Zhang for the helpful discussions. We are grateful to Jing Gao and Yixuan Sun for the technical assistance and to Zhengshuai Liu and Fengguang Ma for the animal studies.

所有的动物实验方案通过在研究院营养科学学院，上海生命科学研究院，中国院士（中国上海）的机构动物护理和使用委员会批准。 1. DIO小鼠模型<ol><li> HFHS喂养 <ol><li>饲料八周龄雄性C57BL / 6小鼠用含有40千卡％的脂肪和40千卡％蔗糖的HFHS。房子在他们12小时暗光循环条件。 </li><li>保持在HFHS饮食喂养条件的小鼠4周至16周。检查每周体重。 </li></ol></li><li> 身体成分分析和伪彩色图像分析 <ol><li>若使小鼠身体成分分析仪分析。 <ol><li>在塑料管安全意识，不麻醉的小鼠。测量身体脂肪量，肌肉量，并使用核磁共振系统的流体，如前面2中所述。 </li></ol></li><li>使用扫描MRI系统每只小鼠的全身。 <ol><li>麻醉通过腹腔注射（25μL/ g）的1％阿佛丁的小鼠和专门的玻璃管进行成像分析。 注:成像的方法，持续每只小鼠近0.5小时。当完成测量时，返回的小鼠其笼子。 </li></ol></li><li>扫描背侧，中间，和腹侧层小鼠的整个机构;扫描在垂直方向上的肝脏。使用0.55磁铁t和下面的仪器参数:K空间= 192×256微米，截面厚度= 3毫米，TE = 14.8毫秒，TR = 400毫秒，FOVRead = 100毫米，FOVPhase = 100毫米，扫描次数= 8.扫描使用相同的磁场条件小鼠的所有组。 </li><li>根据信号强度9灰度图像映射到的伪彩色图象。 </li></ol></li><li> 鼠标安乐死 <ol><li>消毒dissectiNG工具，如镊子和剪刀。 </li><li>倒在纸巾上足够的异氟烷，并让异氟烷在玻璃罐挥发。确保将小鼠通过异氟烷安乐死。 </li><li>固定在手术台上的小鼠。喷每个腹部用75％乙醇，使在用剪刀剑突的真皮的切口，并通过与手中线纵向撕裂真皮暴露腹膜。 </li><li>夹紧钳剑突切腹膜纵横通过肋的底部，以暴露内脏。 </li><li>通过心脏穿刺收集在EDTA涂层管血。 </li><li>沿切肝隔膜，小心地取出使用细剪刀enterocoelia肝脏。切三块肝组织为以下脂质测量:一）6×3×3毫米的H＆E染色，B）6×3×3毫米油红O染色，和c）对氯仿20-40毫克肝脏/甲醇提取。 </li></ol></li><li> H＆E染色 <ol><li>固定在4％磷酸盐缓冲的福尔马林乙酸肝脏在4℃下过夜，然后在石蜡中嵌入它。 </li><li>切石蜡切片（5微米），并安装它们在载玻片上为H＆E染色，如前所述3,10。 </li></ol></li><li> 油红O染色 <ol><li>丽晶准备<ol><li>制备具有10％的福尔马林，60％异丙醇，苏木和甘油胶冻油红O工作溶液。溶解0.5油状物红O粉末用100mL异丙醇和其加热在60℃的浴中，使0.5％的储备溶液。 </li><li> 2比（毫升DDH 2 O的的储备溶液3毫升和2）:在3用双蒸水（DDH 2 O）稀释。过滤工作溶液，静置在室温下至少10分钟。 </li></ol></li><li>嵌入在AP最佳切削温度（OCT）化合物中的肝拉斯蒂克嵌入框。在-20℃30分钟将其冻结。把它切成8微米的部分在冷冻切片机，装入组织上滑动，并将其放置在室温下30分钟。 </li><li>放置纸巾在染色槽的底部，并倒10％福尔马林的少量润湿纸巾。放置在染色槽内的滑动和固定在甲醛蒸气冰冻切片在4℃5分钟。 </li><li>浸在3秒染色桶60％的异丙醇幻灯片;重复一次。 </li><li>添加的油红O工作溶液几滴到滑动覆盖所有组织孵育15分钟。在室温下避光组织。 </li><li>冲洗在3秒染色桶新的60％的异丙醇幻灯片;重复两次。 </li><li>轻轻漂洗通过蒸馏水滑动两次并取出组织周围的水。一定不要干组织。 </li><li>放在苏木幻灯片1分钟，用自来水轻轻漂洗5-10分钟。轻轻冲洗蒸馏水中滑两次，并保持它的蒸馏水，直到准备把盖玻片上。 </li><li>烧热在微波甘油果冻，并把几滴在组织之上。轻轻地在上面放置盖玻片并注意不要形成气泡。 </li><li>观察染色在显微镜下，并使用20X和40X的目标捕捉特征的照片。 </li></ol></li><li> 肝脏脂类的测量 <ol><li>放置解剖肝组织中新的2毫升的塑料离心管中。均质在1毫升的PBS用均化和裂解物转移到一个新的15毫升玻璃管中。 </li><li>加入5毫升氯仿/甲醇（2:1，体积/体积）溶液，涡旋混合物剧烈1分钟，并静置在冰上2小时。 </li><li>离心在4℃1650 XG 10分钟;该混合物应分离成3层:上层甲醇层，中间蛋白光盘，和底氯仿和脂质。 </li><li>陈德良<html> sfer底部相入用玻璃巴斯德吸管新玻璃管没有必要获取底部馏分的每一滴。抛开管冰。 </li><li>添加氯仿4mM的氯化镁和1.5毫升的600μL到留在以前的管和涡大力上清液部分。在4℃下在冰上孵育30分钟，并离心于1650×g离心10分钟。 </li><li>结合的底部相用巴斯德吸移管的第一底部相管。丢弃上层和中层。 </li><li>蒸发在氮气下的氯仿中37℃浴中。以1％的Triton X-100 /氯仿200μL的彻底清洗的玻璃试管的侧面（体积/体积）。在氮气下蒸发。溶解与DDH 2 O的200μL，涡旋脂质很好地做最后的乳液。 </li><li>根据制造商的方案通过使用甘油三酯或胆固醇试剂盒测量总甘油三酯（TG）和胆固醇（TC）水平外部参照"&gt; 3。 注:脂质值归一化到那些在初始匀浆中的蛋白质浓度的，如通过蛋白质定量3确定。 </li></ol></li></ol> 2.原发性肝细胞鼠标型号<ol><li> 小鼠原代肝细胞的分离 <ol><li>麻醉每只小鼠用6％水合氯醛（10微升/ g的腹膜内注射）。 </li><li>隔离原代肝细胞，如前所述10。生长细胞对胶原涂覆的玻璃盖玻片在油红O染色的6孔板中。用于脂质提取，种子细胞到10厘米的细胞培养皿10。 </li></ol></li><li> 细胞治疗和油红O染色 <ol><li>替换用2mL饥饿培养基的培养基，并在37℃孵育24小时。 30毫米的葡萄糖和100nM胰岛素治疗。它孵育在24小时37℃培养箱内培养。 </li><li>吸出网上平台。加入PBS毫升2和漩涡轻轻冲洗介质;重复一次。 </li><li>修复盖玻片用橡皮筋的幻灯片。暴露附着于外表面的细胞。上面（步骤1.5）中所述进行油红O染色。 注:脂质蓄积的基础水平被刺激血清饥饿后的高葡萄糖加胰岛素治疗。脂质液滴通过油红O染色可视化。 </li></ol></li><li> 血脂测定 <ol><li>治疗与高糖加胰岛素在相同的条件下（步骤2.2.1）的肝细胞。添加1毫升的PBS每10厘米细胞培养皿。刮去细胞和收获它们变成新的15毫升的玻璃管，从该100微升的等分试样转移至新的1.5毫升管中为蛋白质定量。 </li><li>添加氯仿的4毫升/甲醇（2:1，体积/体积），以细胞的剩余900微升。超声处理约20哔声，然后涡大力释放脂在细胞中。在冰上孵育混合物2小时。执行脂质提取（步骤1.6）。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。 资金来源:这项工作是由中国国家自然科学基金（81270930号，31471129，和31671224）和百人计划中国的科学院计划（2013OHTP04），以YL资助项目

NAFLD是一系列与代谢综合征，肥胖，胰岛素抵抗或II型糖尿病（T2DM）11相关联的进行性肝脏疾病。 NAFLD的特点是脂肪变性，脂质在肝细胞中的积累。这里，提出来表征表型和使用的DIO小鼠和小鼠原代肝细胞脂肪肝的参数的方法的光谱。此过程可帮助阐明NAFLD和其他相关的代谢性疾病的分子机制。 在体内和体外实验相结合，这些方法提供了全面的...

肥胖是发达国家和发展中国家一个新兴的健康问题。据报道是经常与非酒精性脂肪肝病（NAFLD）相关联的共存条件之一，具有患病30％和100％的NAFLD患者1之间的范围内。由于脂肪肝和肥胖症之间的强相关性，膳食诱导的肥胖（DIO）小鼠模型被广泛用于研究与NAFLD 2，3，4，5，6的发展相关联的复杂的分子机制。肝脂肪变性是NAFLD的最早阶段，它可以前进到非酒精性脂肪性肝炎（NASH），肝硬化，最终，肝癌7。因此，这种方法的总体目标是产生的肝脂肪变性条件动物和细胞模型和PR奥维德的高效，准确的测量血脂详细的协议。这些模型和测量也可用于其他代谢疾病，如胰岛素抵抗和2型糖尿病的调查是有用的。 由于肥胖被确定成为NAFLD，高脂肪的主要危险因素之一，高糖饮食（HFHS）模仿西式高脂饮食是用于诱导的肥胖小鼠。接着，肝脂肪变性的程度可以用不同的方法来评估。首先，体重和核磁共振（NMR）身体成分的分析表明喂养时间内脂质积累。的脂肪量和瘦体重可以以非侵入性的和实时的方式进行量化。 此外，磁共振成像（MRI）是用来同时显示全身和脂肪肝的分布。在MRI分析的灰度级信号可以被转换成清晰的伪彩色图象，和强度在灰度和彩色是半定量的。该技术提供了独特的优势，为在活的动物脂肪积累的测量。第二，肝脏的组织学分析是最常用的方法来确定肝脂肪变性。苏木精和曙红（H＆E）染色的组织学提供的信息，如肝细胞形态和巨噬细胞浸润，而油红O染色显示肝细胞中脂质小滴的大小和位置。第三，用氯仿/甲醇萃取脂质含量分析是肝脂质的精确和定量的测量。总甘油三酯和胆固醇水平可与生化方法来测量。重要的是，脂质提取分析和油红O染色，也可在基因操作或药学治疗肝细胞使用。 本方法的优点是多种优化方法及其利用，以产生肝脂肪变性模型和全面表征表型在体内和体外 。该DIO小鼠模型可以概括人类脂肪肝疾病的病理和代谢表型。在人类其他代谢参数可以在这个模型以及8被复制。响应于高糖加胰岛素的脂肪肝的肝细胞模型的生成是有效的，有用的，并且克服了昂贵和耗时鼠标工作的限制。两者合计，这些方法是足够和必不可少的肝脏脂质功能障碍和胰岛素抵抗养分超载在研究。

<table><tbody><tr><td>O.C.T compound</td><td>SAKURA</td><td>4583</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma</td><td>O0625-25G</td><td></td></tr><tr><td>Infinity Triglycerides kit</td><td>Fisher&amp;nbsp;Scientific</td><td>TR22421</td><td></td></tr><tr><td>Infinity Cholesterol kit</td><td>Fisher&amp;nbsp;Scientific</td><td>TR13421</td><td></td></tr><tr><td>Collagen type I, Rat tail</td><td>Millipore</td><td>08-115</td><td></td></tr><tr><td>DMEM (low glucose)</td><td>Invitrogen</td><td>11885-092</td><td></td></tr><tr><td>Penicillin / Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10099141</td><td></td></tr><tr><td>PBS</td><td>cellgro</td><td>R21-040-CVR</td><td></td></tr><tr><td>HBSS</td><td>cellgro</td><td>20-021-CV</td><td></td></tr><tr><td>Insulin</td><td>TOCRIS Bioscience</td><td>3435</td><td>dissolve in PBS, 1 mM for stock</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270-100G</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX53</td><td></td></tr><tr><td>Peristaltic pump</td><td>Longerpump</td><td>BT100-2J</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>Corning</td><td>420167</td><td></td></tr><tr><td>6-well-plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>BCA assay</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>Nuclear Magnetic Resonance</td><td>Niumag technology</td><td>MiniQMR23-060H-I</td><td></td></tr><tr><td>High fat high surcose diet (HFHS)</td><td>Research Diets</td><td>D12327</td><td></td></tr></tbody></table>

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.

如在图1A中所示，小鼠体重为16周HFHS馈送，这大约是1.5倍食物饮食喂养组更高的后增加至45±1.2克核磁共振身体组成分析示出的脂肪质量和小鼠的瘦体质量指示（ 图1B）。全身和肝脏的脂肪分布是通过MRI测定，并在活，有意识的小鼠代表伪彩色图像在图1C-D中所示。体重和身体组成进行测定，在HFHS饮食喂养过程可视化。 <p class="jove_con...

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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 ...

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Universidad de Cartagena UDEC

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Medicine

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

15.8K 次观看.  Chinese Academy of Sciences. 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. 

视频: Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

可视化和肝脏微循环血流量和耗氧量分析：急性肝炎模型中的应用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

科研

医学

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

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 ...

生物化学

使用 3D 荧光脂滴重建评估肝脂肪变性

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

作者聚焦： 使用 3D 重建分析荧光染色脂滴用于肝脂肪变性评估  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

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 ...

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.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

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 ...

生物学

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 ...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

优化分析

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此视频中的章节

可视化和肝脏微循环血流量和耗氧量分析：急性肝炎模型中的应用

在体内 ^19女性 MRI对细胞跟踪

测量新鲜分离的小鼠肝细胞悬浮液中的脂肪酸β氧化

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

分化肝细胞中的血管性脂细胞病的诱导和特征

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

体外实验性硬化模型:脂质超载介质中的肝细胞培养

用于评估非酒精性脂肪肝进展的新型体内微计算机断层扫描成像技术

优化分析

摘要

探索更多视频

此视频中的章节

可视化和肝脏微循环血流量和耗氧量分析：急性肝炎模型中的应用

在体内 19女性 MRI对细胞跟踪

测量新鲜分离的小鼠肝细胞悬浮液中的脂肪酸β氧化

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

分化肝细胞中的血管性脂细胞病的诱导和特征

在小鼠原代肝细胞测定脂肪酸氧化和脂肪生成的

体外实验性硬化模型:脂质超载介质中的肝细胞培养

用于评估非酒精性脂肪肝进展的新型 体内 微计算机断层扫描成像技术

在体内 ^19女性 MRI对细胞跟踪

用于评估非酒精性脂肪肝进展的新型体内微计算机断层扫描成像技术