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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Assessment of mitochondrial energy coupling in vivo by ~13C/~31P NMR
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Assessment of mitochondrial energy coupling in vivo by ~13C/~31P NMR

机译：通过〜13C /〜31P NMR评估体内线粒体能量耦合

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The recently cloned uncoupling protein homolog UCP3 is expressed primarily in muscle and therefore may play a significant role in the regulation of energy expenditure and body weight. However, investigation into the regulation of uncoupling protein has been hampered by the inability to assess its activity in vivo. In this report, we demonstrate the use of a noninvasive NMR technique to assess mitochondrial energy uncoupling in skeletal muscle of awake rats by combining ~13C NMR to measure rates of mitochondrial substrate oxidation with ~31P NMR to assess unidirectional ATP synthesis flux. These combined ~31P/~13C NMR measurements were performed in control, 10-day triiodo-L-thyronine (T_3)-treated (model of increased UCP3 expression). and acute 2,4-dinitrophenol (DNP)-treated (pro- tonophore and mitochondrial uncoupler) rats. UCP3 mRNA and protein levels increased 8.1-fold (± 1.1) and 2.8-fold (± 0.8), respectively, in the T_3-treated vs. control rat gastrocnemius muscle. ~13C NMR measurements of tricarboxylic acid cycle flux as an index of mitochondrial substrate oxidation were 61 ± 21, 148 ± 25, and 310 ± 48 nmol/g per min in the control, T3, and DNP groups, respectively. ~31P NMR saturation transfer measurements of unidi- rectional ATP synthesis flux were 83 ± 14, 84 ± 14, and 73 ± 7 nmol/g per s in the control, T_3, and DNP groups, respectively. Together. these flux measurements, when normalized to the control group, suggest that acute administration of DNP (mito- chondrial uncoupler) and chronic administration of T_3 decrease energy coupling by ≈80/100 and ≈60/100, respectively, and that the latter treatment correlates with an increase in UCP3 mRNA and protein expression. This NMR approach could prove useful for exploring the regulation of uncoupling protein activity in vivo and elucidating its role in energy metabolism and obesity.
机译：最近克隆的解偶联蛋白同源物UCP3主要在肌肉中表达，因此可能在能量消耗和体重的调节中起重要作用。然而，由于无法评估其在体内的活性，阻碍了对解偶联蛋白调控的研究。在本报告中，我们证明了使用非侵入性NMR技术通过将〜13C NMR与〜31P NMR结合起来测量线粒体底物氧化速率以评估单向ATP合成通量来评估清醒大鼠骨骼肌中的线粒体能量解偶联。这些合并的〜31P /〜13C NMR测量是在对照，10天三碘代-L-甲状腺素（T_3）处理（UCP3表达增加的模型）下进行的。和急性2,4-二硝基苯酚（DNP）治疗的大鼠（原体细胞和线粒体解偶联剂）。与对照大鼠腓肠肌相比，经T_3处理的UCP3 mRNA和蛋白质水平分别提高了8.1倍（±1.1）和2.8倍（±0.8）。在对照组，T3和DNP组中，作为线粒体底物氧化指标的三羧酸循环通量的〜13C NMR测量分别为每分钟61±21、148±25和310±48 nmol / g。在对照组，T_3和DNP组中，单一ATP合成通量的〜31P NMR饱和转移测量值分别为83±14、84±14和73±7 nmol / g / s。一起。这些通量测量值在与对照组进行归一化后，表明急性给予DNP（线粒体解偶联剂）和长期给予T_3可使能量耦合分别降低≈80/ 100和≈60/ 100，并且后一种治疗与UCP3 mRNA和蛋白表达增加。这种NMR方法可用于探索体内解偶联蛋白活性的调控，并阐明其在能量代谢和肥胖中的作用。

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