Torrey Pines Institute for Molecular Studies, San Diego, California 92121, USA.
J Biol Chem. 2011 Feb 11;286(6):4589-97. doi: 10.1074/jbc.M110.182162. Epub 2010 Dec 8.
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 (∼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.
游离脂肪酸（FFA）跨心肌细胞质膜的转运对于心脏功能至关重要，但膜蛋白和 FFA 代谢在 FFA 转运中的作用仍不清楚。代谢被认为可维持细胞内 FFA 处于低水平，为 FFA 转运提供驱动力，但细胞内 FFA 水平尚未被直接测量。我们首次报告了心肌细胞内未结合游离脂肪酸浓度（FFA(i)）的测量结果。FFA 的荧光指示剂 ADIFAB（丙烯酰化标记的大鼠肠脂肪酸结合蛋白）被微注射到来自野生型（WT）和 FAT/CD36 缺失 C57B1/6 小鼠的分离心肌细胞中。ADIFAB 荧光的定量成像揭示了 FFA 内流和外流的时程。对于 WT 小鼠，流出的速率常数（约 0.02 s(-1)）是流入的两倍，并且稳态 FFA(i)比细胞外未结合 FFA（FFA(o)）大 3 倍以上。FFA(o)增加时，FFA 内流的浓度梯度和初始速率均饱和。油酸、棕榈酸和花生四烯酸也表现出类似的特征。FAT/CD36 缺失细胞也表现出类似的特征，只是流出比 WT 细胞慢 2-3 倍。用细胞内 ADIFAB 确定的速率常数通过测量细胞内 pH 得到了证实。用细胞外 ADIFAB 监测 FFA(o)，通过悬浮心肌细胞测定 FFA 的摄取，证实了从 FFA(i)测量中确定的内流速率常数，并表明 FFA 转运和 etomoxir 敏感代谢的速率是独立调节的。我们得出结论，心肌细胞中的 FFA 内流是由膜泵介导的，其转运速率常数可能受到 FAT/CD36 的调节。
