Fatty Acid (FFA) Transport in Cardiomyocytes Revealed by Imaging Unbound FFA Is Mediated by an FFA Pump Modulated by the CD36 Protein
Fatty Acid (FFA) Transport in Cardiomyocytes Revealed by Imaging Unbound FFA Is Mediated by an FFA Pump Modulated by the CD36 Protein
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DOI：
10.1074/jbc.m110.182162
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发表时间：
2011-02-01
影响因子：
4.8
通讯作者：
Kleinfeld, Alan M.
中科院分区：
文献类型：
--
作者：
Carley, Andrew N.;Kleinfeld, Alan M.
游离脂肪酸（FFA）通过心肌细胞质膜的转运对正常的心脏功能至关重要，但膜蛋白和FFA代谢在FFA转运中的作用仍不清楚。代谢被认为维持细胞内FFA在低水平，提供FFA运输的驱动力，但细胞内FFA水平尚未直接测量。我们报告了心肌细胞内游离FFA浓度（FFA（i））的第一次测量。将FFA的荧光指示剂ADIFAB（丙烯酰丹标记的大鼠肠脂肪酸结合蛋白）显微注射到来自野生型（WT）和FAT/CD 36缺失的C57 B1/6小鼠的分离的心肌细胞中。ADIFAB荧光定量成像显示FFA流入和流出的时间过程。对于WT小鼠，外排的速率常数（类似于0.02 s（-1））是内流的两倍，稳态FFA（i）比细胞外未结合FFA（FFA（o））大3倍以上。浓度梯度和FFA流入的初始速率随着FFA（o）的增加而饱和。观察到油酸酯、棕榈酸酯和花生四烯酸酯具有类似的特征。FAT/CD 36无效细胞显示出相似的特征，除了外排比WT细胞慢2-3倍。通过测量细胞内pH值确认了用细胞内ADIFAB确定的速率常数。通过使用细胞外ADIFAB监测FFA（o）确定的心肌细胞悬浮液的FFA摄取确认了由FFA（i）测量确定的流入速率常数，并证明FFA转运和依托莫西敏感性代谢的速率是独立调节的。我们的结论是，FFA流入心肌细胞介导的膜泵，其运输速率常数可能是由FAT/CD 36调制。
Free fatty acid (FFA) transport across the cardiomyocyte plasma membrane is essential to proper cardiac function, but the role of membrane proteins and FFA metabolism in FFA transport remains unclear. Metabolism is thought to maintain intracellular FFA at low levels, providing the driving force for FFA transport, but intracellular FFA levels have not been measured directly. We report the first measurements of the intracellular unbound FFA concentrations (FFA(i)) in cardiomyocytes. The fluorescent indicator of FFA, ADIFAB (acrylodan-labeled rat intestinal fatty acid-binding protein), was microinjected into isolated cardiomyocytes from wild type (WT) and FAT/CD36 null C57B1/6 mice. Quantitative imaging of ADIFAB fluorescence revealed the time courses of FFA influx and efflux. For WT mice, rate constants for efflux (similar to 0.02 s(-1)) were twice influx, and steady state FFA(i) were more than 3-fold larger than extracellular unbound FFA (FFA(o)). The concentration gradient and the initial rate of FFA influx saturated with increasing FFA(o). Similar characteristics were observed for oleate, palmitate, and arachidonate. FAT/CD36 null cells revealed similar characteristics, except that efflux was 2-3-fold slower than WT cells. Rate constants determined with intracellular ADIFAB were confirmed by measurements of intracellular pH. FFA uptake by suspensions of cardiomyocytes determined by monitoring FFA(o) using extracellular ADIFAB confirmed the influx rate constants determined from FFA(i) measurements and demonstrated that rates of FFA transport and etomoxir-sensitive metabolism are regulated independently. We conclude that FFA influx in cardiac myocytes is mediated by a membrane pump whose transport rate constants may be modulated by FAT/CD36.