Name: measurement of basal and forskolin-stimulated lipolysis in inguinal adipose fat pads
Uploaded: 2017-07-21T12:30:00
Description: Watch this Scientific Journal Video about Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads at JoVE.com

这项工作得到AHA Award 15BGIA23250030，NIH国家综合医学科学研究所的支持，获得了第8P20 GM103432-12的奖学金和怀俄明州大学授予BT援助。

所有协议均遵循怀俄明大学的动物保健指南。 动物饲养注意：根据机构动物护理和使用委员会（IACUC）批准的方案，在研究动物设施中培育成年雄性野生型小鼠（C57BL / 6）（12至24周）。 <ol><li>从第6周开始，将小鼠分为四组，分为笼子，随机将其分配到非传染性疾病或高血压（±0.01％CAP）的喂养组，直到第38周岁。 注：CAP是在脂肪组织<...

<ol>
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I., Langin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+lipolysis.">Adipose tissue lipolysis.</a> Curr Opin Clin Nutr Metab Care. 13 (4), 377-381 (2010).</li><li>Baskaran, P., Krishnan, V., Ren, J., Thyagarajan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsaicin+induces+browning+of+white+adipose+tissue+and+counters+obesity+by+activating+TRPV1+channel-dependent+mechanisms.">Capsaicin induces browning of white adipose tissue and counters obesity by activating TRPV1 channel-dependent mechanisms.</a> Br J Pharmacol. 173 (15), 2369-2389 (2016).</li><li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRPV1+by+dietary+capsaicin+improves+endothelium-dependent+vasorelaxation+and+prevents+hypertension.">Activation of TRPV1 by dietary capsaicin improves endothelium-dependent vasorelaxation and prevents hypertension.</a> Cell Metab. 12 (2), 130-141 (2010).</li><li>Ding, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+lipolysis+response+to+adipose+afferent+reflex+involved+in+impaired+activation+of+adrenoceptor-cAMP-PKA-hormone+sensitive+lipase+pathway+in+obesity.">Reduced lipolysis response to adipose afferent reflex involved in impaired activation of adrenoceptor-cAMP-PKA-hormone sensitive lipase pathway in obesity.</a> Sci Rep. 6, 34374 (2016).</li><li>Ohyama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combination+of+exercise+and+capsinoid+supplementation+additively+suppresses+diet-induced+obesity+by+increasing+energy+expenditure+in+mice.">A combination of exercise and capsinoid supplementation additively suppresses diet-induced obesity by increasing energy expenditure in mice.</a> Am J Physiol Endocrinol Metab. 308 (4), E315-E323 (2015).</li><li>Beylot, M., Martin, C., Beaufrere, B., Riou, J. 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TGL的击穿过程为甘油和脂肪酸被by ATGL 9 basal脂解期间催化和由蛋白质的阵列编排包括腺苷酸环化酶/ PKA-依赖性途径的激脂解21，22，23在活化。脂解作用增强增加脂肪酸的血浆水平为运输和能源使用24。脂肪酸被线粒体吸收为乙酰CoA，用于产生能量。 脂肪分解的程度和程?...

脂肪组织为所需的产热3,4脂肪2和脂肪酸氧化存储能量。通过饮食摄取的脂肪酸与脱蛋白一起包装到乳糜微粒中，并通过血液循环递送到身体的不同组织。虽然大多数细胞在体内储存能量的储备，脂肪组织储存多余的能量为脂肪5,6。脂肪组织中的脂肪分解是由复杂过程调节的，脂肪分解的分子细节仍然模糊7 。 脂肪分解是通过脂肪甘油三酯脂肪酶（ATGL） 8将存储在脂肪组织中的甘油三酯（TGL）水解以产生甘油和脂肪酸（FA）的过程。基础和刺激脂肪分解的改变是肥胖的特征。 basal脂肪分解由ATGL活化9调节，其将TGL转化为二酰基甘油（DAG），随后水解成单酰甘油（MAG）。 通过腺苷酸环化酶激活激素敏感性脂肪酶（HSL）激活环磷酸腺苷（cAMP）依赖性蛋白激酶A（PKA）刺激并引起脂肪分解。因此，脂肪分解，基础和刺激的测量对于分析涉及该过程的蛋白质的活性是重要的。另外，解开脂肪分解的分子调控可能有利于开发针对肥胖的新型治疗策略10 。因为刺激脂肪分解和脂肪酸氧化的分子是降低储存在贮库中的脂肪的潜在候选者，所以重要的是采用鲁棒的重现性测定法。 以前发表的数据表明，CAP在白色脂肪组织中表达的TRPV1蛋白的激活增强了基础和FSK（腺苷酸环化酶激活剂） - 腹股沟脂肪垫中的刺激性脂肪分解11 。以前的研究还表明，通过CAP长期激活TRPV1的激活PKA 12。因为PKA的活化刺激脂解13，14，测量从NCD或HFD（±CAP）32周供给各自的饮食后喂食的小鼠分离的腹股沟脂肪垫基础和PKA依赖性刺激脂解作用将验证TRPV1的作用激活脂解。 本文介绍了确定基础和刺激的脂肪分解的有效方法。虽然这采用glycerol和繁琐的高效液相色谱法或气相色谱法/质谱法测量15，16是可用的放射性同位素等方法，这种方法提供了一种更直接的，简单且成本有效的确定脂肪组织脂肪分解的技术。

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

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

脂解是脂肪组织中作为甘油三酯存储的脂质水解成甘油和脂肪酸的过程。本文介绍了从正常食物饮食（NCD），高脂肪饮食（HFD）或含有0.01的高脂肪饮食（HFD）的野生型小鼠分离的腹股沟脂肪垫中测量基础和毛喉素（FSK）刺激的脂肪分解的方法百分比的辣椒素（CAP;瞬时受体潜在的香草素亚科1（TRPV1）激动剂）32周。本文所述的用于进行离体脂解的方法采用Schweiger 等人 1我们提供了一个详细的方案，通过紫外 - 可见（UV / VIS）分光光度法测量甘油水平。这里描述的方法可用于成功分离腹股沟脂肪垫用于脂肪分解测量以获得一致的结果。腹股沟脂肪垫描述的方案可以容易地扩展到其他组织中的脂肪分解。

为了评估CAP对基础和受刺激的脂肪分解的影响，本研究测量了从NCD或HFD（±CAP）野生型小鼠分离的腹股沟脂肪脂肪垫中的脂肪分解。腹股沟脂肪垫的基础和FSK刺激的脂肪分解的代表性结果在表I中给出。在Triacsin C存在下，基础和FSK刺激的甘油释放，其抑制酰基辅酶A合成酶并阻止TGL的再生。 如图1所示，HFD抑制了FSK刺激的脂肪分解，CAP增加?...

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

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

该方案描述了从正常食物饮食（NCD）或高脂肪饮食（HFD）±辣椒素喂养的野生型小鼠获得的腹股沟脂肪垫中测定基础和毛喉素刺激的脂肪分解的方法。作为脂肪分解的指标，从腹股沟脂肪脂肪垫测量甘油释放。

测量基底和福斯柯林刺激的脂肪分解在腹股沟脂肪脂肪垫

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

Research

JoVE Journal

Biochemistry

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

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

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

科研

生物化学

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

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

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

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

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

Mouse Adipose Tissue Collection and Processing for RNA Analysis

小鼠脂肪组织的采集和 RNA 分析处理

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

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

用最小组织量测定果蝇透纤维中线粒体耗氧量

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

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

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

就地生物发光细菌细胞密度与光发射的测量与相关性研究

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

Cortisol Measurement in Koala (Phascolarctos cinereus) Fur

Cortisol Measurement in Koala Phascolarctos cinereus Fur

科蒂醇测量在考拉 （菲斯科拉托斯西尼鲁斯） 毛皮

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

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

使用基于化学探针的免疫测定直接测量KDM1A目标参与

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

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

Measurement of Fatty Acid ÃƒÅ½Ã‚Â²-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

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 ...

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

骨骼肌线粒体蛋白质导入能力的测定

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...