Sandoval Angel, Fraisl Peter, Arias-Barrau Elsa, Dirusso Concetta C, Singer Diane, Sealls Whitney, Black Paul N
Center for Metabolic Disease, Ordway Research Institute, 150 New Scotland Avenue, Albany, NY 12208, USA.
Arch Biochem Biophys. 2008 Sep 15;477(2):363-71. doi: 10.1016/j.abb.2008.06.010. Epub 2008 Jun 20.
These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 x 10(6)s(-1)M(-1) to 1.35 x 10(6)s(-1)M(-1)).
这些研究使用定量聚合酶链反应（qPCR）确定了参与脂肪酸转运、激活和运输的基因的表达模式，并确定了脂肪酸转运的动力学常数，以确定向量酰化是否代表不同细胞类型（3T3-L1成纤维细胞和脂肪细胞、Caco-2和HepG2细胞以及三种内皮细胞系（b-END3、HAEC和HMEC））中的一种常见机制。正如预期的那样，脂肪酸转运蛋白（FATP）1和长链酰基辅酶A合成酶（Acsl）1是脂肪细胞中表达的主要异构体，这与其在将外源脂肪酸转运和激活以甘油三酯形式储存中的作用一致。在参与脂肪酸加工的细胞中，包括Caco-2（肠样）和HepG2（肝样）细胞，FATP2是主要的异构体。这两种细胞类型中Acsl的表达模式不同，Caco-2细胞中Acsl3和Acsl5占主导，而HepG2细胞中Acsl4占主导。在内皮细胞系中，FATP1和FATP4是表达最高的异构体；不同Acsl异构体的表达模式在不同的内皮细胞系之间变化很大。荧光长链脂肪酸C(1)-BODIPY-C(12)在3T3-L1成纤维细胞和3T3-L1脂肪细胞中的转运遵循典型的米氏动力学；脂肪细胞分化后，该过程的表观效率（k(cat)/K(T)）增加了2倍多（2.1×10(6)-4.5×10(6)s(-1)M(-1)）。Caco-2和HepG2细胞中脂肪酸转运的V(max)值基本相同，但Caco-2细胞中的效率高55%（2.3×10(6)s(-1)M(-1)对1.5×10(6)s(-1)M(-1)）。三种内皮细胞类型中脂肪酸转运的动力学参数表明，它们是该过程中效率最低的细胞类型，其V(max)值比3T3-L1脂肪细胞、Caco-2细胞和HepG2细胞定义的值低近4倍。相同的细胞脂肪酸转运效率降低（范围从0.82×10(6)s(-1)M(-1)到1.35×10(6)s(-1)M(-1)）。
