荷尔斯坦奶牛肝线粒体耗氧量和质子泄漏动力学评价线粒体呼吸
Summary
在这里, 我们分享测量线粒体耗氧量的方法, 营养能量学的一个决定性概念, 和质子泄漏, 这是线粒体产生 atp 效率低下的主要原因。这些结果可以占营养利用损失能量的 30%, 以帮助评估线粒体功能。
Abstract
耗氧量、质子动力 (pmf) 和质子泄漏是线粒体呼吸的测量, 或线粒体能够将 nadh 和 fadh 转化为 atp 的程度。由于线粒体也是氧气使用和营养氧化的主要场所, 二氧化碳和水, 他们如何有效地使用氧气和产生 atp 直接关系到营养代谢的效率, 动物的营养需求,动物的健康。该方法的目的是检查线粒体呼吸, 可用于检查不同药物、饮食和环境对线粒体代谢的影响。结果包括作为质子依赖性呼吸 (状态 3) 测量的耗氧量和质子泄漏依赖性呼吸 (状态 4)。国家 3/状态4呼吸的比例被定义为呼吸控制比 (rcr), 可以表示线粒体的能量效率。线粒体质子泄漏是通过从 adp 中分离氧化磷酸化来消除线粒体膜电位 (mmp) 的过程, 从而降低了 atp 合成的效率。氧和 trmp + 敏感电极与线粒体底物和电子传输链抑制剂被用来测量状态3和状态4呼吸, 线粒体膜 pmf (或产生 atp 的潜力) 和质子泄漏。这种方法的局限性是肝脏组织必须尽可能新鲜, 所有活检和检测必须在不到10小时内进行。这将一个人一天可以收集和处理的样本数量限制在大约5个。然而, 只需要1克的肝脏组织, 所以在大型动物, 如奶牛, 所需的样本量是小的相对于肝脏大小, 几乎不需要恢复时间。
Introduction
线粒体对压力非常敏感, 它们的细胞环境会导致多种代谢疾病。线粒体中的耗氧量和质子泄漏是线粒体健康的指标。本文所描述的基于有质子泄漏和无质子泄漏的耗氧量的 rcr 估计线粒体能量效率的方法。这些结果可以占营养利用1所损失能量的 30%.氧气消耗和质子泄漏的变化可以识别线粒体功能障碍, 从而导致代谢疾病, 并导致能源效率下降。这些方法也可用于检查不同治疗方法对线粒体呼吸的影响。测量线粒体耗氧量和质子泄漏动力学的总体目标是评估线粒体功能和能量效率。
肝线粒体功能障碍与奶牛的几种疾病有关。早期泌乳时, 当面对能量不足时, 细胞代谢在碳水化合物和脂类燃料之间切换的能力受到细胞2中线粒体数量和功能的影响.线粒体适应能量需求增加和β氧化增加的能力存在缺陷, 可导致与胰岛素抵抗相关的细胞内脂质积累, 并可能导致早期哺乳奶牛脂肪肝的形成。线粒体作为酮体生产和使用的场所, 在奶牛3中的酮症中发挥着关键作用。线粒体或线粒体功能障碍的缺乏将影响外围燃料的供应, 并反映在氧气消耗或 rcr 的变化上。
线粒体耗氧量随炎症而变化。7天龄的肉鸡随机分为感染 eimeria 最大值的组和对照组 4。没有经历过球虫病挑战的肉鸡由于质子泄漏而耗氧量较低, rcr 较高表明肝脏线粒体通过增加质子泄漏来应对免疫挑战。虽然质子泄漏和活性氧的产生曾经被认为是线粒体膜功能障碍的表现, 对能量效率不利, 但现在大家都知道, 将蛋白质和钙导入线粒体很重要。, 并为热的产生1。
从呼吸链的电子泄漏使线粒体容易产生活性氧, 并对线粒体膜蛋白、脂质和线粒体 dna 造成氧化损伤。随着线粒体年龄的增加, 损伤尤其会积累到 mtdna 中, 导致线粒体代谢的进一步功能障碍, 并使奶牛对疾病的易感性更强。实际上, 许多牲畜动物被喂养高水平的补充剂, 如铜, 锌和锰, 以提高抗氧化功能。然而, 由于质子泄漏 (状态4呼吸), 摄入高水平的铜、锌和锰会减少牛奶产量, 增加耗氧量.
此前关于线粒体功能在牛能量效率中的作用的研究主要集中在线粒体耗氧量和质子泄漏的变化上。在奶牛中发表的研究很少, 大多数论文将剩余饲料摄入量 (rfi) 的生产效率与肉牛的线粒体功能进行了比较。通过测定哺乳期荷斯坦奶牛和哺乳牛牛 (安格斯、布拉古斯和赫里福德) 肝脏中的3、4和 rcr 状态, 检测线粒体呼吸速率的变异性.研究人员没有发现肉牛线粒体呼吸与生长或挤奶性状有任何相关性, 但确实报告了线粒体呼吸与霍尔斯坦挤奶性状之间的相关性。在两项研究中, 将牛牛的 rfi 与肌肉线粒体 9、10的线粒体呼吸速率 (状态3、状态4和 rcr) 进行了比较。线粒体呼吸率随着 dmi 的变化而变化, 低呼吸率与效率较低的牛肉转向有关。在另一项研究中, 将高或低 rfi 公牛的蒸手的 rfi 与两组后代11之间的线粒体呼吸速率和质子泄漏动力学进行了比较.差异的原因是证实了增益不影响肉牛线粒体呼吸的结论。
本文通过一项实验, 对奶牛饲养3种抗氧化矿物质的肝脏 rcr 进行了研究, 阐述了用方法测量4号和3号州呼吸和 pmf 期间的耗氧量。
Protocol
Representative Results
Discussion
该协议中最关键的一点是获得具有代表性的肝脏组织样本, 并在活检后尽快开始分离线粒体。呼吸测量的变化很低 (表 1), 原因是从奶牛到实验室的运输时间很短。为了减少运输时间, 在奶牛场办公室设立了一个小型实验室, 并在收集每个样本时将肝脏样本送到办公室实验室, 以便在活检10分钟内分离线粒体。用 ph 计设置和测试呼吸室和电极 (氧气, tpmp +), 在采集和处理样品的前一天记录?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究得到了 alltech 和 usda 舱口基金通过加州大学戴维斯分校兽医学院食品动物健康中心的支持。
Materials
| Liver Biopsy | |||
| Equipment | |||
| Schackelford-Courtney bovine liver biopsy instrument | Sontec Instruments Englewood CO | 1103-904 | |
| Suture | Fisher Scientific | 19-037-516 | |
| Suture needles | NA | NA | Included with Suture |
| Scalpels | Sigma – Aldrich | S2896 / S2646 | # for handle and blades |
| Surgery towels | Fisher Scientific | 50-129-6667 | |
| Falcon tubes 50 mL | Fisher Scientific | 14-432-22 | |
| Tweezers | Sigma – Aldrich | Z168750 | |
| 50 mL syringes | Fisher Scientific | 22-314387 | |
| Injection needles (22, 2 1/2) | VWR | MJ8881-200342 | |
| Cow halter | Tractor Supply Co. | 101966599 | |
| Cotton swabbing | Fisher Scientific | 14-959-102 | |
| cotton gauze squares (4×4) | Fisher Scientific | 22-246069 | |
| Medical scissors | Sigma – Aldrich | Z265969 | |
| Chemicals | |||
| Coccidiosis Vaccine 0.75 bottle/cow | Provided by Veterinarian | ||
| Clostridia Vaccine | Provided by Veterinarian | ||
| Liver biopsy antibiotics excenel 2 cc/100 lbs for 3 days | Provided by Veterinarian | ||
| Providone Scrub | Aspen Veteterinary Resources | 21260221 | |
| Ethanol 70% | Sigma – Aldrich | 793213 | |
| Xylazine hydrochloride 100 mg/mL IV at 0.010-0.015 mg/kg bodyweight | Provided by Veterinarian | ||
| 2% lidocaine HCl (10-15 mL) | Provided by Veterinarian | ||
| 1 mg/kg IV injection of flunixin meglumine | Provided by Veterinarian | ||
| Isolation of Mitochondria (liver) | |||
| Equipment | |||
| Wheaton vial 30 mL with a Teflon pestle of 0.16 mm clearance | Fisher Scientific | 02-911-527 | |
| Homogenizer Motor | Cole Parmer | EW-04369-10 | |
| Homogenizer Probe | Cole Parmer | EW-04468-22 | |
| Auto Pipette (10 mL) | Cole Parmer | SK-21600-74 | |
| Beaker (500 mL) with ice | Fisher Scientific | FB100600 | |
| Refrigerated microfuge | Fisher Scientific | 75-002-441EW3 | |
| Microfuge tubes (1.5 mL) | Fisher Scientific | AM12400 | |
| Chemicals | |||
| Bicinchoninic acid (BCA) protein assay kit (microplates for plate reader) | abcam | ab102536 | |
| Sucrose | Sigma – Aldrich | S7903-1KG | |
| Tris-HCl | Sigma – Aldrich | T1503-1KG | |
| EDTA | Sigma – Aldrich | EDS-1KG | |
| BSA (fatty acid free) | Sigma – Aldrich | A7030-50G | |
| Mannitol | Sigma – Aldrich | M4125-1KG | |
| Deionized water | Sigma – Aldrich | 38796 | |
| Hepes | Sigma – Aldrich | H3375-500G | |
| Use to create mitochondria isolation media: 220 mM mannitol, 70 mM sucrose, 20 mM HEPES, 20 mM Tris-HCl, 1 mM EDTA, and 0.1% (w/v) fatty acid free BSA, pH 7.4 at 4 °C, will last 2 days in refrigerator | |||
| Mitochondrial Oxygen Comsuption | |||
| Equipment | |||
| Oxygraph Setup + Clark type oxygen electrode | Hansatech (PP Systems) | OXY1 | |
| Thermoregulated Water Pump | ADInstruments | MLE2001 | |
| Clark type Oxygen electrode | NA | NA | |
| Autopipette (1 mL) | Cole Parmer | SK-21600-70 | Included with Oxy1 |
| Small magnetic stir bar | Fisher Scientific | 14-513-95 | |
| Micropipette (10 μL) | Cole Parmer | SK-21600-60 | |
| pH meter | VWR | ||
| Chemicals | |||
| KCl | Sigma – Aldrich | P9333-1KG | |
| Hepes | Sigma – Aldrich | H3375-500G | |
| KH2PO4 | Sigma – Aldrich | P5655-1KG | |
| MgCl2 | Sigma – Aldrich | M1028-100ML | |
| EGTA | Sigma – Aldrich | E3889-100G | |
| Use to make mitochondrial oxygen consumption media: 120 mM KCL, 5 mM KH2PO4, 5 mM MgCl2, 5 mM Hepes and 1 mM EGTA, pH 7.4 at 30 °C with 0.3% defatted BSA | |||
| Rotenone (4 mM solution) | Sigma – Aldrich | R8875-5G | |
| Succinate (1 M solution) | Sigma – Aldrich | S3674-250G | |
| ADP (100 mM solution) | Sigma – Aldrich | A5285-1G | |
| Oligomycin (solution of 8 μg/mL in ethanol) | Sigma – Aldrich | 75351 | |
| FCCP | Sigma – Aldrich | C2920 | |
| Mitochondrial Membrane Potential and Proton Motive Force | |||
| Equipment | |||
| TPMP electrode | World Precision Instruments. | DRIREF-2 | |
| Chemicals-solutions do not need to be fresh but they do need to be kept in a freezer between runs | |||
| Malonate (0.1 mM solution) | Sigma – Aldrich | M1296 | |
| Oligomycin (8 μg/mL in ethanol), keep in freezer | Sigma – Aldrich | 75351 | |
| Nigericin (80 ng/mL in ethanol), keep in freezer | Sigma – Aldrich | N7143 | |
| FCCP | Sigma – Aldrich | C3920 | |
| TPMP | Sigma – Aldrich | T200 | |
| TPMP solution: 10 mM TPMP, 120 mM KCL, 5 mM Hepes and 1 mM EGTA, pH 7.4 at 30 °C with 0.3% defatted BSA | |||
Referencias
- Brand, M. D., Divakaruni, A. S. The regulation and physiology of mitochondrial proton leak. Physiology. 26, 192-205 (2011).
- Stephenson, E. J., Hawley, J. A. Mitochondrial function in metabolic health: A genetic and environmental tug of war. Biochimica et Biophysica Acta. 1840, 1285-1294 (2014).
- Bartlett, K., Eaton, S. Mitochondrial B oxidation. European Journal of Biochemistry. 271, 462-469 (2004).
- Acetoze, G., Kurzbard, R., Klasing, K. C., Ramsey, J. J., Rossow, H. A. Oxygen Consumption, Respiratory Control Ratio (RCR) and Mitochondrial Proton Leak of broilers with and without growth enhancing levels of minerals supplementation challenged with Eimeria maxima (Ei). Journal of Animal Physiology and Animal Nutrition. 101, e210-e215 (2016).
- Wallace, D. C., Fan, W. Energetics, epigenetics, mitochondrial genetics. Mitochondrion. 10, 12-31 (2010).
- Paradies, G., Petrosillo, G., Paradies, V., Ruggiero, F. M. Oxidative stress, mitochondrial bioenergetics and cardiolipin in aging. Free Radicals in Biology and Medicine. 48, 1286-1295 (2010).
- Acetoze, G., Champagne, J., Ramsey, J. J., Rossow, H. A. Liver mitochondrial oxygen consumption and efficiency of milk production in lactating Holstein cows supplemented with Copper, Manganese and Zinc. Journal of Animal Physiology Animal Nutrition. 102, e787-e797 (2017).
- Brown, D. R., DeNise, S. K., McDaniel, R. G. Mitochondrial respiratory metabolism and performance of cattle. Journal of Animal Science. 66, 1347-1354 (1988).
- Golden, M. S., Keisler, J. W., H, D. The relationship between mitochondrial function and residual feed intake in Angus steers. Journal of Animal Science. 84, 861-865 (2006).
- Lancaster, P. A., Carstens, G. E., Michal, J. J., Brennan, K. M., Johnson, K. A., Davis, M. E. Relationships between residual feed intake and hepatic mitochondrial function in growing beef cattle. Journal of Animal Science. 92, 3134-3141 (2014).
- Acetoze, G., Weber, K. L., Ramsey, J. J., Rossow, H. A. Relationship between liver mitochondrial respiration and proton leak kinetics in low and high RFI steers from two lineages of RFI Angus bulls. ISRN Vet Sci. 2015 (194014), (2015).
- Halliwell, B., Gutteridge, J. M. C. Protection against oxidants in biological systems: The superoxide theory of oxygen toxicity. Free Radicals in Biology and Medicine. , 186-187 (1989).
- National Research Council. . Nutrient Requirements of Dairy Cattle. , (2001).
- Ramsey, J. J., Harper, M. E., Weindruch, R. Restriction of energy intake, energy expenditure, and aging. Free Radical Biology and Medicine. 29, 946-968 (2000).
- Mehta, M. M., Weinberg, S. E., Chandel, N. S. Mitochondrial control of immunity: beyond ATP. Nature. 17, 608-620 (2017).
- Kirby, D. M., Thorburn, D. R., Turnbull, D. M., Taylor, R. W. Biochemical assays of respiratory chain complex activity. Methods in Cell Biology. 80, 93-119 (2007).
- Alex, A. P., Collier, J. L., Hadsell, D. L., Collier, R. J. Milk yield differences between 1x and 4x milking are associated with changes in mammary mitochondrial number and milk protein gene expression, but not mammary cell apoptosis or SOCS gene expression. Journal of Dairy Science. 98, 4439-4448 (2015).
- Lossa, S., Lionetti, L., Mollica, M. P., Crescenzo, R., Botta, M., Barletta, A., Liverini, G. Effect of high-fat feeding on metabolic efficiency and mitochondrial oxidative capacity in adult rats. British Journal of Nutrition. 90, 953-960 (2003).
- Boily, G., Seifert, E. L., Bevilacqua, L., He, X. H., Sabourin, G., Estey, C., Moffat, C., Crawford, S., Saliba, S., Jardine, K., Xuan, J., Evans, M., Harper, M. E., McBurney, M. W. SirT1 regulates energy metabolism and response to caloric restriction in mice. PloS One. 3 (3), e1759 (2008).
- Chen, Y., Hagopian, K., Bibus, D., Villaba, J. M., Lopez-Lluch, G., Navas, P., Kim, K., McDonald, R. B., Ramsey, J. J. The influence of dietary lipid composition on liver mitochondria from mice following 1 month of calorie restriction. Bioscience Reports. 33, 83-95 (2013).
- Chacko, B. K., Kramer, P. A., Ravi, S., Benavides, G. A., Mitchell, T., Dranka, B. P., Ferrick, D., Singal, A. K., Ballinger, S. W., Bailey, S. M., Hardy, R. W., Zhang, J., Zhi, D., Darley-Usmar, V. M. The bioenergetic health index: a new concept in mitochondrial translational research. Clinical Science. 127, 367-373 (2014).
