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黑腹果蝇中放射性标记底物二氧化碳生成量的测定

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster.

作者信息

Bland Michelle L

机构信息

Department of Pharmacology, University of Virginia;

出版信息

J Vis Exp. 2016 Jun 27(112):54045. doi: 10.3791/54045.

Abstract

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of (14)C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

摘要

果蝇遗传学的力量越来越多地应用于激素信号传导和代谢问题以及该生物体中人类疾病模型的开发。需要灵敏的方法来测量代谢率等参数,以推动对果蝇等小型动物生理学和疾病的理解。这里描述的方法评估了少量成年果蝇的燃料氧化情况,这些果蝇食用含有微量(14)C标记底物(如葡萄糖或脂肪酸)的食物。在喂食期和任何额外的实验操作之后,将果蝇转移到用网覆盖的短管中,然后将其放置在装有氢氧化钾饱和滤纸的玻璃瓶中,该滤纸捕获呼出的、由放射性标记底物氧化产生的放射性标记二氧化碳,并将其转化为碳酸氢钾(KHCO3)。通过闪烁计数法测量这种放射性标记的碳酸氢盐。这是一种用于研究燃料氧化的定量、可重复且简单的方法。使用放射性标记的葡萄糖、脂肪酸或氨基酸可以确定这些不同燃料来源在不同条件下(如进食和禁食)以及不同遗传背景下对能量代谢的贡献。这补充了用于测量体内能量代谢的其他方法,并应有助于进一步理解代谢调节。

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