测量新鲜分离的小鼠肝细胞悬浮液中的脂肪酸β氧化
Summary
脂肪酸β氧化是负责在许多不同细胞类型（包括肝细胞）中产生能量的重要代谢途径。在这里，我们描述了一种使用 14个C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。
Abstract
脂肪酸β氧化是满足肝脏能量需求的关键代谢途径，并为其他过程（如生酮和糖异生）提供底物和辅助因子，这对于维持全身葡萄糖稳态和支持禁食状态下的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活化，酶表达水平以及辅酶A和NAD +等辅助因子的可用性。在测量肝脏匀浆中脂肪酸β氧化的测定中，细胞裂解和辅助因子的超生理水平的常见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难以控制，并且在制剂之间可能会有很大差异。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 14个C标记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，该方法具有更好地保存原始肝脏的蛋白质表达水平和代谢途径活性的优点，包括与喂养小鼠相比，在从禁食小鼠分离的肝细胞中观察到的脂肪酸β氧化的活化。
Introduction
脂肪酸β氧化是脂质代谢的必要过程，提供分解代谢途径来平衡脂肪酸合成和饮食中的摄入量。该过程为多个器官产生能量，包括心肌，肾皮层和禁食肝脏，并利用从饮食中获得的脂肪酸，脂肪组织脂肪分解和内部甘油三酯储存1，2。
脂肪酸通过β氧化途径的氧化导致脂肪酰基链一次由两个碳连续缩短，以乙酰辅酶A的形式释放，并且该过程同时发生在线粒体和过氧化物酶体中。虽然大多数脂肪酸只经历β氧化，但有些脂肪酸在进入该途径之前在不同的碳中被氧化。例如，3-甲基取代的脂肪酸，如植烷酸，在进入β氧化途径之前，通过过氧化物酶体中的α氧化除去一个碳。类似地，一些脂肪酸首先通过氧化内质网中的末端甲基（ω-氧化）转化为二羧基脂肪酸，然后通过β氧化3在过氧化物酶体中优先氧化。
无论特定的细胞器如何，脂肪酸必须首先转化为辅酶A（CoA）硫酯或酰基辅酶A，才能通过β氧化途径被氧化。线粒体基质中长链酰基辅酶A的β氧化需要肉碱穿梭进行其易位，其中肉碱棕榈酰转移酶1（CPT1）催化酰基辅酶A向酰基肉碱的转化，并且是该过程中的限速酶4。一旦转移到线粒体基质，酰基辅酶A被重新形成并作为线粒体β氧化机制的底物。在禁食状态下，肝线粒体中通过β氧化产生的乙酰辅酶A主要引导至生酮作用。过氧化物酶体是超长链，支链和二羧酸脂肪酸β氧化的主要位点。过氧化物酶体不需要肉碱穿梭来导入脂肪酸底物，而是通过ATP结合盒（ABC）转运蛋白ABCD1-3的活性导入相应的酰基辅酶A5。在过氧化物酶体内，酰基辅酶A随后被一组专用的酶氧化，这与线粒体脂肪酸β氧化机制不同。线粒体和过氧化物酶体也需要供应NAD + 和游离辅酶A来氧化脂肪酰基链。肝脏中的CoA水平已被证明随着空腹而增加，支持在这种状态下发生的脂肪酸氧化速率的增加6。此外，过氧化物酶体中CoA降解的增加导致过氧化物酶体脂肪酸氧化7的选择性降低。因此，细胞内脂肪酸氧化的过程受到参与脂肪酸活化，运输和氧化的酶的表达水平和活性的调节，以及整个多个亚细胞区室的辅助因子和其他代谢物的浓度。
使用组织匀浆来测量脂肪酸氧化的程序破坏了调节和支持该过程的细胞结构，导致数据收集不能准确反映体内代谢。虽然使用接种的原代肝细胞的技术保留了该系统，但长时间培养分离的细胞会导致细胞中仍然存在的体内基因表达谱的丧失，当它们仍然生活在动物体内时8，9。以下方案描述了一种分离原代肝细胞的方法，并使用[1-14C]棕榈酸在分离后立即和悬浮液测定其脂肪酸β氧化的能力。该测定基于测量与酸溶性代谢物（ASM）或产物（如乙酰辅酶A）相关的放射性，由[1-14 C]棕榈酸10，11的β氧化产生。
Protocol
小鼠（C57BL / 6J，雄性，9-11周龄）的所有实验程序均已获得西弗吉尼亚大学机构动物护理和使用委员会（IACUC）的批准。 1. 肝细胞分离 制备 在肝细胞分离前几天，制备 表1中列出的缓冲液和细胞培养基。设置一个水浴，温度设置为37°C，靠近将要进行手术的地方。 在肝细胞分离当天，在层流罩下，将35mL缓冲液1转移到无菌的50mL离心管中，将70…
Representative Results
这里描述的肝脏灌注通常产生3000-4000万个细胞/肝脏，平均活力为80%，通过台盼蓝排除估计（图2）。用于制备灌注缓冲液1和2的克雷布斯 – 亨赛莱特缓冲液（KHB）中葡萄糖的典型浓度为11 mM。当测量从禁食小鼠分离的肝细胞中的脂肪酸β氧化时，可以降低KHB中葡萄糖的浓度以更好地代表禁食状态。如图 2所示，将葡萄糖浓度降低到5.6mM对肝细胞的产量或活?…
Discussion
在肝脏灌注期间，避免引入气泡至关重要，因为它们会阻塞肝脏中的微毛细血管，防止或限制缓冲液循环并总体上降低肝细胞产量和活力20，21。预防措施，例如在IVC插管之前仔细检查填充缓冲液的入口管路，并避免将入口管从含有缓冲液1的管中抬起以切换到缓冲液2，如本文所述，可以成功减少失败灌注的数量（活力<70%）。在灌注系统中使用气泡捕集?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（National Institutes of Health）向罗伯塔·莱昂纳迪（Roberta Leonardi）授予R35GM119528的支持。
Materials
| (R)-(+)-Etomoxir sodium salt | Tocris Bioscience | 4539/10 | |
| [1-14C]-Palmitic acid, 50–60 mCi/mmol, 0.5 mCi/mL | American Radiolabeled Chemicals | ARC 0172A | |
| 1 M HEPES, sterile | Corning | 25060CI | |
| 10 µL disposable capillaries/pistons for positive displacement pipette | Mettler Toledo | 17008604 | |
| 1000 µL, 200 µL, and 10 µL pipettes and tips | |||
| 5 mL, 10 mL, and 25 mL serological pipettes | |||
| 50 mL sterile centrifuge tubes | CellTreat | 229421 | |
| 70% Perchloric acid | Fisher Scientific | A2296-1LB | |
| BSA, fatty acid-free | Fisher Scientific | BP9704100 | |
| CaCl2 dihydrate | MilliporeSigma | 223506 | |
| D-(+)-Glucose | MilliporeSigma | G7021 | |
| EGTA | Gold Biotechnology | E-217 | |
| Ethanol | Pharmco | 111000200CSPP | |
| Filter System, 0.22 μm PES Filter, 500 mL, Sterile | CellTreat | 229707 | |
| Gentamicin sulphate | Gold Biotechnology | G-400-25 | |
| HDPE, 6.5 mL scintillation vials | Fisher Scientific | 03-342-3 | |
| Hemocytometer | |||
| Hypodermic needles 22 G, 1.5 in | BD Biosciences | 305156 | |
| Isoflurane | VetOne | 502017 | |
| KCl | Fisher Scientific | BP366-1 | |
| KH2PO4 | MilliporeSigma | P5655 | |
| Liberase TM Research Grade | MilliporeSigma | 5401119001 | Defined blend of purified collagenase I and II with a medium concentration of thermolysin |
| M199 medium | MilliporeSigma | M5017 | |
| MgSO4 heptahydrate | MilliporeSigma | M1880 | |
| Microcentrifuge | Fisher Scientific | accuSpin Micro 17 | |
| Microdissecting Scissors | Roboz Surgical Instrument Co | RS-5980 | |
| NaCl | Chem-Impex International | 30070 | |
| NaHCO3 | Acros Organics | 424270010 | |
| Palmitic acid | MilliporeSigma | P0500 | |
| Penicillin/streptomycin (100x) | Gibco | 15140122 | |
| Phosphate buffered saline (PBS) | Cytiva Life Sciences | SH30256.01 | |
| Positive displacement pipette MR-10, 10 µL | Mettler Toledo | 17008575 | |
| Refrigerated centrifuge with inserts for 50 mL conical tubes | Eppendorf | 5810 R | |
| Round-bottom, 14 mL, polypropylene culture test tubes | Fisher Scientific | 14-956-9A | |
| Scintillation counter | Perkin Elmer | TriCarb 4810 TR | |
| ScintiVerse BD cocktail | Fisher Scientific | SX18-4 | |
| Shaking water bath, 30 L capacity | New Brunswick Scientific | Model G76 | |
| Sterile cell strainers, 100 µm | Fisher Scientific | 22363549 | |
| Thumb Dressing Forceps | Roboz Surgical Instrument Co | RS-8120 | |
| Trypan Blue | Corning | 25900CI | |
| Variable-flow peristaltic pump | Fisher Scientific | 138762 | |
| Water baths, 2–2.5 L capacity |
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Cite This Article
Vickers, S. D., Saporito, D. C., Leonardi, R. Measurement of Fatty Acid β-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes. J. Vis. Exp. (175), e62904, doi:10.3791/62904 (2021).