Istituto di Fisica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy.
Istituto di Fisica, Università Cattolica del Sacro Cuore, Largo Francesco Vito 1, 00168 Rome, Italy.
Biochim Biophys Acta Mol Cell Biol Lipids. 2018 Jul;1863(7):783-793. doi: 10.1016/j.bbalip.2018.04.004. Epub 2018 Apr 11.
Organisms store fatty acids in triacylglycerols in the form of lipid droplets, or hydrolyze triacylglycerols in response to energetic demands via activation of lipolytic or storage pathways. These pathways are complex sets of sequential reactions that are finely regulated in different cell types. Here we present a high spatial and temporal resolution-based method for the quantification of the turnover of fatty acids into triglycerides in live cells without introducing sample preparation artifacts. We performed confocal spectral imaging of intracellular micropolarity in cultured insulin secreting beta cells to detect micropolarity variations as they occur in time and at different pixels of microscope images. Acquired data are then analyzed in the framework of the spectral phasors technique. The method furnishes a metabolic parameter, which quantitatively assesses fatty acids - triacylglycerols turnover and the activation of lipolysis and storage pathways. Moreover, it provides a polarity profile, which represents the contribution of hyperpolar, polar and non-polar classes of lipids. These three different classes can be visualized on the image at a submicrometer resolution, revealing the spatial localization of lipids in cells under physiological and pathological settings. This new method allows for a fine-tuned, real-time visualization of the turnover of fatty acids into triglycerides in live cells with submicrometric resolution. It also detects imbalances between lipid storage and usage, which may lead to metabolic disorders within living cells and organisms.
生物体以脂滴的形式将脂肪酸储存为三酰基甘油，或者通过激活脂肪分解或储存途径来响应能量需求水解三酰基甘油。这些途径是复杂的一系列连续反应，在不同的细胞类型中受到精细调节。在这里，我们提出了一种基于高时空分辨率的方法，用于定量检测活细胞中脂肪酸转化为甘油三酯的周转率，而不会引入样品制备artifact。我们对培养的胰岛素分泌β细胞中的细胞内微极性进行共聚焦光谱成像，以检测微极性随时间和显微镜图像不同像素的变化。然后，在光谱向位器技术的框架内分析获得的数据。该方法提供了一个代谢参数，定量评估脂肪酸-三酰基甘油周转率以及脂肪分解和储存途径的激活。此外，它还提供了一个极性分布，代表了超极化、极性和非极性脂质类别的贡献。这三个不同的类别可以在亚微米分辨率的图像上可视化，揭示生理和病理条件下细胞中脂质的空间定位。这种新方法允许以亚微米分辨率实时精细观察活细胞中脂肪酸转化为甘油三酯的周转率。它还可以检测脂质储存和使用之间的失衡，这可能导致活细胞和生物体中的代谢紊乱。
