Department of Nephrology, Osaka University Graduate School of Medicine, Osaka, Japan.
Transdimensional Life Imaging Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka, Japan.
Am J Physiol Endocrinol Metab. 2023 Nov 1;325(5):E552-E561. doi: 10.1152/ajpendo.00147.2023. Epub 2023 Sep 20.
Mitochondrial fatty acid β-oxidation (FAO) plays a key role in energy homeostasis. Several FAO evaluation methods are currently available, but they are not necessarily suitable for capturing the dynamics of FAO in vivo at a cellular-level spatial resolution and seconds-level time resolution. FAOBlue is a coumarin-based probe that undergoes β-oxidation to produce a fluorescent substrate, 7-hydroxycoumarin-3-(-(2-hydroxyethyl))-carboxamide (7-HC). After confirming that 7-HC could be specifically detected using multiphoton microscopy at excitation/emission wavelength = 820/415-485 nm, wild-type C57BL/6 mice were randomly divided into control, pemafibrate, fasting (24 or 72 h), and etomoxir groups. These mice received a single intravenous injection of FAOBlue. FAO activities in the liver of these mice were visualized using multiphoton microscopy at 4.2 s/frame. These approaches could visualize the difference in FAO activities between periportal and pericentral hepatocytes in the control, pemafibrate, and fasting groups. FAO velocity, which was expressed by the maximum slope of the fluorescence intensity curve, was accelerated in the pemafibrate and 72-h fasting groups both in the periportal and the pericentral hepatocytes in comparison with the control group. Our approach revealed differences in the FAO activation mode by the two stimuli, i.e., pemafibrate and fasting, with pemafibrate accelerating the time of first detection of FAO-derived fluorescence. No increase in the fluorescence was observed in etomoxir-pretreated mice, confirming that FAOBlue specifically detected FAO in vivo. Thus, FAOBlue is useful for visualizing in vivo liver FAO dynamics at the single-cell-level spatial resolution and seconds-level time resolution. Fatty acid β-oxidation (FAO) plays a key role in energy homeostasis. Here, the authors established a strategy for visualizing FAO activity in vivo at the cellular-level spatial resolution and seconds-level time resolution in mice. Quantitative analysis revealed spatiotemporal heterogeneity in hepatic FAO dynamics. Our method is widely applicable because it is simple and uses a multiphoton microscope to observe the FAOBlue-injected mice.
线粒体脂肪酸 β-氧化 (FAO) 在能量平衡中起着关键作用。目前有几种 FAO 评估方法，但它们不一定适合在细胞水平的空间分辨率和秒级时间分辨率下捕捉体内 FAO 的动态。FAOBlue 是一种香豆素基探针，经过 β-氧化产生荧光底物 7-羟基香豆素-3-(-(2-羟乙基))-酰胺 (7-HC)。在用多光子显微镜在激发/发射波长 = 820/415-485nm 下确认可以特异性检测到 7-HC 后，将野生型 C57BL/6 小鼠随机分为对照组、培马弗林组、禁食（24 或 72 小时）组和 etomoxir 组。这些小鼠接受 FAOBlue 的单次静脉注射。使用多光子显微镜以 4.2s/帧的速度观察这些小鼠肝脏中的 FAO 活性。这些方法可以在对照组、培马弗林组和禁食组中可视化门静脉周围和中央肝细胞之间的 FAO 活性差异。FAO 速度，由荧光强度曲线的最大斜率表示，在培马弗林和 72 小时禁食组中均在门静脉周围和中央肝细胞中加速，与对照组相比。我们的方法揭示了两种刺激物（即培马弗林和禁食）激活 FAO 的方式的差异，即培马弗林加速了 FAO 衍生荧光的首次检测时间。在预先用 etomoxir 处理的小鼠中未观察到荧光增加，证实 FAOBlue 可特异性检测体内 FAO。因此，FAOBlue 可用于以单细胞水平的空间分辨率和秒级的时间分辨率可视化体内肝脏 FAO 动力学。脂肪酸 β-氧化 (FAO) 在能量平衡中起着关键作用。在这里，作者建立了一种在小鼠体内以细胞水平的空间分辨率和秒级的时间分辨率可视化 FAO 活性的策略。定量分析揭示了肝 FAO 动力学的时空异质性。我们的方法具有广泛的适用性，因为它简单且使用多光子显微镜观察注射了 FAOBlue 的小鼠。
