Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes.
Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes.
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DOI：
10.3791/62904
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发表时间：
2021-09-09
影响因子：
1.2
通讯作者：
Leonardi, Roberta
中科院分区：
文献类型：
--
作者：
Vickers, Schuyler D.;Saporito, Dominique C.;Leonardi, Roberta
脂肪酸β-氧化是满足肝脏能量需求的关键代谢途径，并为生酮和糖异生等其他过程提供底物和辅助因子，这对于在禁食状态下维持全身葡萄糖稳态和支持肝外器官功能至关重要。脂肪酸 β-氧化发生在线粒体和过氧化物酶体内，并通过多种机制进行调节，包括脂肪酸的摄取和激活、酶表达水平以及辅酶 A 和 NAD+ 等辅助因子的可用性。在测量肝匀浆中脂肪酸β-氧化的测定中，细胞裂解和常见的超生理水平的辅助因子的添加掩盖了这些调节机制的影响。此外，匀浆中细胞器的完整性很难控制，并且在不同制剂之间可能存在显着差异。完整原代肝细胞中脂肪酸β-氧化的测量克服了上述缺陷。该协议描述了一种测量与 14C 标记的棕榈酸一起孵育的新鲜分离的原代小鼠肝细胞悬浮液中脂肪酸 β-氧化的方法。通过避免数小时至数天的培养，该方法的优点是可以更好地保留原始肝脏的蛋白质表达水平和代谢途径活性，包括与进食小鼠相比，在从禁食小鼠分离的肝细胞中观察到的脂肪酸β-氧化的激活。脂肪酸β-氧化是一种重要的代谢途径，负责在包括肝细胞在内的许多不同细胞类型中产生能量。在这里，我们描述了一种使用 14C 标记的棕榈酸测量新鲜分离的原代肝细胞中脂肪酸 β-氧化的方法。
Fatty acid β-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 β-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 β-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 β-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid β-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 β-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice. Fatty acid β-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 β-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.
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影响因子：
3.9
作者：
Baker A;Carrier DJ;Schaedler T;Waterham HR;van Roermund CW;Theodoulou FL
通讯作者：
Theodoulou FL
影响因子：
3.7
作者：
Leonardi R;Rehg JE;Rock CO;Jackowski S
通讯作者：
Jackowski S
影响因子：
8.2
作者：
Leonardi R;Rock CO;Jackowski S
通讯作者：
Jackowski S
影响因子：
1.2
作者：
Lee, Serene M. L.;Schelcher, Celine;Thasler, Wolfgang E.
通讯作者：
Thasler, Wolfgang E.
DOI：
10.1007/978-1-59745-019-5_13
发表时间：
2010-01-01
期刊：
MOUSE CELL CULTURE: METHODS AND PROTOCOLS
影响因子：
--
作者：
Li, Wan-Chun;Ralphs, Kate L.;Tosh, David
通讯作者：
Tosh, David