使用14CO2捕获在体外评估能量底物氧化
Summary
该协议描述了一种易于使用的方法，通过跟踪14CO2的体外生产来检查底物氧化。
Abstract
线粒体是三羧酸（TCA）循环和电子传递链（ETC）的机器，其产生三磷酸腺苷（ATP）以维持能量稳态。葡萄糖，脂肪酸和氨基酸是大多数体细胞中促进线粒体呼吸的主要能量底物。有证据表明，不同的细胞类型可能对某些底物有明显的偏好。然而，骨架中各种细胞的底物利用尚未得到详细研究。此外，由于细胞代谢与生理和病理生理学变化相协调，对骨骼细胞底物依赖性的直接评估可能为骨骼疾病的发病机制提供重要的见解。
以下方案基于氧化磷酸化后底物分子释放二氧化碳的原理。通过使用含有放射性标记碳原子（14C）的底物，该方法为细胞培养物中的底物氧化速率提供了一种灵敏且易于使用的测定方法。一项以原代颅骨前成细胞与骨髓来源的巨噬细胞 （BMM） 为临床的案例研究显示，两种细胞类型之间主要底物的利用率不同。
Introduction
真核生物中的氧化磷酸化（OXPHOS）是营养物质在线粒体内部分解的过程，通过消耗氧气以ATP的形式释放化学能。 线粒体内部各种底物通过三羧酸（TCA）循环的分解代谢直接产生很少的ATP分子，而是通过还原电子载体烟酰胺腺嘌呤二核苷酸（NAD +）和黄素腺嘌呤二核苷酸（FAD +）来储存能量。然后，还原的载流子被位于线粒体内膜上的ETC氧化，以产生穿过膜的质子浓度梯度。质子最终通过ATP合酶沿着梯度流回线粒体基质中，产生ATP。OXPHOS是从能量基材生产ATP的最有效手段，通常在有氧环境中是优选的。以前，有氧糖酵解 – 在存在氧气的情况下从葡萄糖中产生乳酸盐 – 被认为是病理生理学的，通常是癌细胞的标志。越来越多的人发现，一些正常细胞类型使用有氧糖酵解的原因尚未完全破译。
代谢灵活性是细胞或生物体适应不断变化的能量需求和可用燃料来源的能力。例如，骨骼肌的能量需求主要由稳定状态下的OXPHOS满足，但在高强度运动中通过厌氧糖酵解满足1。随着运动持续时间的增加，葡萄糖和脂肪酸氧化对整体能量产生的贡献更大2.然而，基板的使用不仅取决于可用性，因为基底在氧化过程中具有拮抗性竞争。最值得注意的是，脂肪酸氧化已被证明可以抑制骨骼肌对葡萄糖的利用，这种现象称为Randle效应3。随后的研究4，5证明了互惠效应。此外，许多疾病与底物偏好的变化和细胞代谢不灵活性的发展有关。例如，与正常对照组相比，II型糖尿病患者的骨骼肌中的脂肪酸氧化减少6。疾病环境中的代谢变化是一个需要深入研究的主题，因为它们可能有助于发病机制。
骨骼细胞类型中的能量代谢相对较少，但近年来引起了人们的关注7.先前的研究表明，有氧糖酵解是颅变成骨细胞中的主要能量途径，而通过TCA循环的葡萄糖氧化在破骨细胞形成中起作用8，9。其他人已经提供了脂肪酸作为成骨细胞能量来源的证据10。谷氨酰胺分解代谢也被证明支持成骨细胞与祖细胞的分化11，12。然而，对各种骨骼细胞类型的底物利用的全面了解仍然缺乏。此外，细胞分化过程中细胞代谢的变化或对病理信号的反应预计将改变燃料底物的利用率。下面描述的是一种易于使用的方案，用于 在体外测定底物氧化。
Protocol
放射性物质（RAM）的使用需要事先得到每个机构的指定安全委员会的批准。该协议中使用的RAM已获得宾夕法尼亚大学环境健康与辐射安全（EHRS）的批准。使用动物需要事先获得家庭机构的机构动物护理和使用委员会（IACUC）的批准。以下研究由费城儿童医院的IACUC批准。 1. 14种C标记底物的储备溶液的制备 通过将11gBSA和33.1mL Dulbecco的磷酸盐缓冲盐水…
Representative Results
在本例中，CO2 捕获法用于比较原代颅骨前成细胞的底物氧化与 BBM，后者通常分别用于 体外 成骨细胞或破骨细胞分化。在cMEMα中传代和培养过夜后，它们通常达到汇合的80-90%，并表现出其特征形态。颅内前成骨细胞明显大于BMM（图2）。按照步骤4.1至4.7对每种细胞类型进行葡萄糖，谷氨酰胺和油酸盐的氧化测定。使用等式（1）分析结果。 <p clas…
Discussion
该协议提供了一种易于使用的方法来确定主要能量基板的氧化速率。它是其他方案的更简单的替代方案，这些方案使用包含中央孔并盖有橡胶塞14，15，16的烧瓶。尽管这里的示例研究是用细胞培养进行的，但该方法可以很容易地适用于含有完整线粒体的组织外植体或组织匀浆，如前所述17。
<p class="jove_conte…Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH拨款R01 AR060456（FL）的部分支持。我们感谢Michael Robinson博士和Elizabeth Krizman（费城儿童医院）对闪烁计数器的慷慨帮助。
Materials
| 0.22 µm filters | Sigma-Aldrich | SLGVM33RS | Used to filter BSA solution |
| 0.25% Trypsin-EDTA | Gibco | 25200056 | Dissociate cells from cell culture plates |
| 1.5 mL Eppendorf tubes | PR1MA | PR MCT17 RB | Used for reaction incubation |
| 10 cm plates | TPP | 93100 | Used for cell culture |
| 10 mL syringe | BD | 302995 | Used to flush marrow from long bones |
| 10% FBS | Atlanta biologicals | S11550 | For Cell culture medium preparation |
| 14C-Glucose | PerkinElmer | NEC042X050UC | Used to make hot media |
| 14C-glutamine | PerkinElmer | NEC451050UC | Used to make hot media |
| 14C-oleate | PerkinElmer | NEC317050UC | Used to make hot media |
| 23 G needle | BD | 305120 | Used to flush marrow from long bones |
| 24-well plates | TPP | 92024 | Used for cell culture |
| 70 μm cell strainers | MIDSCI | 70CELL | Used to filter supernatant during cavarial digestion |
| Acridine Orange/Propidium Iodide (AO/PI) dye | Nexcelom Biosciences | CS2-0106 | Stains live cells to determine seed density |
| Bovine Serum Ablumin | Proliant Biologicals | 68700 | Used for fatty acid conjugation |
| Cellometer Auto 2000 | Nexcelom Biosciences | Determine the number of viable cells | |
| Centrifuge | Thermo Fisher | Legend Micro 21R | Used to pellet cells |
| Collagenase type II | Worthington | LS004176 | Dissociate cells from tissue |
| Custom MEM alpha | GIBCO | SKU: ME 18459P1 | Used to create custom hot media |
| Dulbecco's Phosphate-Buffered Saline | Gibco | 10010023 | Used to dissolve and dilute reagents, and wash culture dishes |
| Filter Paper | Millipore-Sigma | WHA1001090 | Traps CO2 with sodium hydroxide |
| Glucose | Sigma-Aldrich | g7528 | Used to make custom media |
| HEPES | Gibco | 15630080 | Traps CO2 during cell culture |
| L-carnitine | Sigma-Aldrich | C0283 | Supplemented for fatty acid oxidation |
| L-Glutamine | Sigma-Aldrich | g3126 | Used to make custom media |
| MEM alpha | Thermo | A10490 | Cell culture medium |
| Parafilm | Pecheney Plastic Packaging | PM998 | Used to seal cell culture dishes |
| Penicillin-Streptomycin | Thermo Fisher | 15140122 | Prevents contamination in cell culture |
| Perchloric Acid | Sigma-Aldrich | 244252 | Releases CO2 during metabolic assay |
| Pyruvate | Sigma-Aldrich | p5280 | Used to make custom media |
| Scintillation Counter | Beckman Coulter | LS6500 | Determines radioactivity from the filter paper |
| Scintillation Fluid | MP Biomedicals | 882453 | Absorb the energy emitted by RAMs and re-emit it as flashes of light |
| Scintillation Vial | Fisher Scientific | 03-337-1 | Reaction containers for scintillation fluid |
| Sodium carbonate | Sigma-Aldrich | S5761 | Balance buffer for medium |
| Sodium Hydroxide | Sigma-Aldrich | 58045 | Traps CO2 during metaboilc assay |
| Sodium oleate | SANTA CRUZ | SC-215879 | BSA conjugated fatty acid preparation |
| Vaccum filtration 1000 | TPP | 99950 | Filter cMEMα |
References
- Hargreaves, M., Spriet, L. L. Skeletal muscle energy metabolism during exercise. Nature Metabolism. 2 (9), 817-828 (2020).
- Jeukendrup, A. E. Regulation of fat metabolism in skeletal muscle. Annals of the New York Academy of Sciences. 967, 217-235 (2002).
- Randle, P. J., Garland, P. B., Hales, C. N., Newsholme, E. A. The glucose fatty-acid cycle. Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet. 1 (7285), 785-789 (1963).
- Randle, P. J., Newsholme, E. A., Garland, P. B. Regulation of glucose uptake by muscle. 8. Effects of fatty acids, ketone bodies and pyruvate, and of alloxan-diabetes and starvation, on the uptake and metabolic fate of glucose in rat heart and diaphragm muscles. Biochemical Journal. 93 (3), 652-665 (1964).
- Taegtmeyer, H., Hems, R., Krebs, H. A. Utilization of energy-providing substrates in the isolated working rat heart. Biochemical Journal. 186 (3), 701-711 (1980).
- Cha, B. S., et al. Impaired fatty acid metabolism in type 2 diabetic skeletal muscle cells is reversed by PPARgamma agonists. American Journal of Physiology. Endocrinology and Metabolism. 289 (1), 151-159 (2005).
- Lee, W. C., Guntur, A. R., Long, F., Rosen, C. J. Energy metabolism of the osteoblast: implications for osteoporosis. Endocrine Reviews. 38 (3), 255-266 (2017).
- Li, B., et al. Both aerobic glycolysis and mitochondrial respiration are required for osteoclast differentiation. FASEB Journal. 34 (8), 11058-11067 (2020).
- Lee, W. C., Ji, X., Nissim, I., Long, F. Malic enzyme couples mitochondria with aerobic glycolysis in osteoblasts. Cell Reports. 32 (10), 108108 (2020).
- Kim, S. P., et al. Fatty acid oxidation by the osteoblast is required for normal bone acquisition in a sex- and diet-dependent manner. JCI Insight. 2 (16), 92704 (2017).
- Yu, Y., et al. Glutamine metabolism regulates proliferation and lineage allocation in skeletal stem cells. Cell Metabolism. 29 (4), 966-978 (2019).
- Karner, C. M., Esen, E., Okunade, A. L., Patterson, B. W., Long, F. Increased glutamine catabolism mediates bone anabolism in response to WNT signaling. The Journal of Clinical Investigation. 125 (2), 551-562 (2015).
- Takeshita, S., Kaji, K., Kudo, A. Identification and characterization of the new osteoclast progenitor with macrophage phenotypes being able to differentiate into mature osteoclasts. Journal of Bone and Mineral Research. 15 (8), 1477-1488 (2000).
- Itoh, Y., et al. Dichloroacetate effects on glucose and lactate oxidation by neurons and astroglia in vitro and on glucose utilization by brain in vivo. Proceedings of the National Academy of Sciences of the United States of America. 100 (8), 4879-4884 (2003).
- Oba, M., Baldwin, R. L. 4. t. h., Bequette, B. J. Oxidation of glucose, glutamate, and glutamine by isolated ovine enterocytes in vitro is decreased by the presence of other metabolic fuels. Journal of Animal Science. 82 (2), 479-486 (2004).
- Esen, E., Lee, S. Y., Wice, B. M., Long, F. PTH promotes bone anabolism by stimulating aerobic glycolysis via IGF signaling. Journal of Bone and Mineral Research. 30 (11), 1959-1968 (2015).
- Huynh, F. K., Green, M. F., Koves, T. R., Hirschey, M. D. Measurement of fatty acid oxidation rates in animal tissues and cell lines. Methods in Enzymology. 542, 391-405 (2014).
- Hodson, L., Skeaff, C. M., Fielding, B. A. Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake. Progress in Lipid Research. 47 (5), 348-380 (2008).
- Abdelmagid, S. A., et al. Comprehensive profiling of plasma fatty acid concentrations in young healthy Canadian adults. PLoS One. 10 (2), 0116195 (2015).
Cite This Article
Song, C., Valeri, A., Long, F. Assessing Energy Substrate Oxidation In Vitro with 14CO2 Trapping. J. Vis. Exp. (181), e63568, doi:10.3791/63568 (2022).