Fatty acids induce L-CPT I gene expression through a PPARα-independent mechanism in rat hepatoma cells
Fatty acids induce L-CPT I gene expression through a PPARα-independent mechanism in rat hepatoma cells
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DOI：
10.1093/jn/135.10.2313
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发表时间：
2005-10-01
影响因子：
4.2
通讯作者：
Pegorier, Jean-Paul
中科院分区：
文献类型：
--
作者：
Le May, Cedric;Cauzac, Michele;Pegorier, Jean-Paul
肝脏肉毒碱棕榈酰转移酶(L-CPT)I是长链脂肪酸氧化的关键调节酶，保证长链脂肪酸进入线粒体基质的第一步。在大鼠肝细胞中，我们先前已经证明，L-CPT I基因的表达既受低碳链脂肪酸的诱导，也受贝特类的诱导。本研究的目的是确定LCFA诱导的L-CPT I基因表达是否由PPARα介导。为此，我们构建了一个PPARα显性负性受体来抑制内源性PPARα信号转导。配体结合区螺旋12上高度保守的疏水和带电残基(Leu(459)和Glu(462))突变为丙氨酸。这些突变导致转录活性完全丧失，这是由于辅助激活因子招募受损所致。此外，竞争研究证实突变的PPARα受体取消了野生型PPARα受体的作用，从而发挥了强大的显性负性受体的作用。当用重组腺病毒在大鼠肝癌细胞(H4IIE)中过表达时，突变的PPARα受体可拮抗氯贝特诱导的L-CPT I基因的表达，而不影响LCFA诱导的L-CPT I基因的表达。这些结果首次直接证明了LCFAs通过PPARα不依赖的途径调控L-CPT I的转录，至少在肝癌细胞中是这样。
Liver carnitine palmitoyl transferase (L-CPT) I is a key regulatory enzyme of long-chain fatty acid (LCFA) oxidation that ensures the first step of LCFA import into the mitochondrial matrix. In rat hepatocytes, we showed previously that L-CPT I gene expression was induced by LCFAs as well as by fibrates. The aim of this study was to determine whether LCFA-induced L-CPT I gene expression was mediated by PPAR alpha. For this purpose, we constructed a PPAR alpha-dominant negative receptor to inhibit endogenous PPAR alpha signaling. Highly conserved hydrophobic and charged residues (Leu(459) and Glu(462)) in helix 12 of the ligand-binding domain were mutated to alanine. These mutations led to a total loss of transcriptional activity due to impaired coactivator recruitment. Furthermore, competition studies confirmed that the mutated PPAR alpha receptor abolished the wild-type PPAR alpha receptor action and thus acted as a powerful dominant negative receptor. When overexpressed in rat hepatoma cells (H4IIE) using a recombinant adenovirus, the mutated PPAR alpha receptor antagonized the clofibrate-induced L-CPT I gene expression, whereas it did not affect LCFA-induced L-CPT I. These results provide the first direct demonstration that LCFAs regulate L-CPT I transcription through a PPAR alpha-independent pathway, at least in hepatoma cells.