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Pyruvate dehydrogenase kinase 4 (PDK4) is a key regulatory enzyme involved in switching the energy source from glucose to fatty acids in response to physiological conditions. Transcription of the PDK4 gene is activated by fasting or by the administration of a PPARα ligand in a tissue-specific manner. Here, we show that the two mechanisms are independent, and that ERRα is directly involved in PPARα-independent transcriptional activation of the PDK4 gene with PGC-1α as a specific partner. This conclusion is based on the following evidence. First, detailed mutation analyses of the cloned PDK4 gene promoter sequence identified a possible ERRα-binding motif as the PGC-1α responsive element. Second, overexpression of ERRα by cotransfection enhanced, and the knockout of it by shRNAs diminished, PGC-1α-dependent activation. Third, specific binding of ERRα to the identified PGC-1α responsive sequence was confirmed by the electrophoresis mobility shift assay. Finally, cell-type-specific responsiveness to PGC-1α was observed and this could be explained by differences in the expression levels of ERRα, however, ectopic expression of ERRα in poorly responsive cells did not restore PGC-1α responsiveness, indicating that ERRα is necessary, but not sufficient for the response.
Metabolic switching of oxidative fuel use from glucose and three-carbon compounds to fatty acids is a key adaptive mechanism to maintain blood glucose levels during starvation [1,2]. To limit the irreversible decarboxylation of pyruvate to acetyl-CoA, pyruvate dehydrogenase kinases (PDKs) are induced by starvation and phosphorylate the pyruvate dehydrogenase complex that catalyzes decarboxylation. Of four isoforms, PDK4 has been reported to be the most highly inducible . It is known that PDK4 is induced in the liver and muscles of mice by fasting [4,5] and also by the administration of a peroxisome proliferator-activated receptor α (PPARα) ligand .
We reported previously that hypolipidemic drug fibrates, PPARα ligands, rapidly induce PDK4 mRNA in various mouse tissues . This is in stark contrast to the relatively slow induction of the mRNAs for fatty acid utilization which are mostly limited to the liver. The observation that fibrates rapidly and efficiently induced mRNA at the whole-body level prompted us to propose a metabolic-switching hypothesis as the first step in the hypolipidemic effect of fibrates. Rapid and general inactivation of the pyruvate dehydrogenase complex by fibrate-induced PDK4 is followed by metabolic switching to limit oxidative fuel to fatty acids, thus enhancing fatty acid utilization. This is the driving force for fatty acid uptake from the blood, and uptake is further accelerated by induction of the enzymes involved in fatty acid utilization and repression of the inhibitor apoCIII for fatty acid-producing lipoprotein lipase. A metabolic-switching hypothesis predicts that these changes will contribute sequentially to the rapid and sustained hypolipidemic effect of fibrates .
The induction of PDK4 by fasting has been studied from the viewpoint of the action of insulin in inhibiting the ability of dexamethasone to stimulate PDK4 gene expression . The mechanism responsible for the insulin inhibition of glucocorticoid-stimulated human PDK4 gene expression was extensively analyzed by Harris and co-workers using human hepatoma HepG2 cells . They proposed a model in which glucocorticoid activates PDK4 gene expression via the glucocorticoid-responsive element located −820 from the transcriptional start site. Activation occurs in a glucocorticoid receptor-dependent manner in cooperation with forkhead box class O (FOXOs) bound at three sites in the promoter. Insulin activates protein kinase B to phosphorylate FOXOs [10,11], leading to the inhibition of glucocorticoid-stimulated PDK4 gene expression.
It is not known whether the regulatory mechanism for PDK4 gene expression in skeletal muscle is the same as in liver. Nor is the relationship between the PPARα-dependent mechanism and other physiological responses currently understood. The unique tissue distribution and kinetic characteristics of the PDK isoforms suggest the existence of tissue-specific regulation by PDKs [4,12], but the detailed mechanisms have not been elucidated. Sugden and co-workers reported that the PPARα pathway and other pathways that induce PDK4 were parallel or overlapping, and that there was, at least in part, a fatty acid-dependent, PPARα-independent pathway in liver and cardiac muscle [13,14]. Consistent with these studies, we report a PPARα-independent pathway that induces PDK4 in skeletal muscle, and we demonstrate that the orphan nuclear receptor ERRα is directly involved in the mechanism by recruiting PGC-1α as a coactivator.
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We identified ERRα as an essential partner of PGC-1α in the PPARα-independent activation of the mouse PDK4 gene promoter in C2C12 myotube cells. In addition to its previously known function in regulating the amounts of energy-creating enzymes , PGC-1α seems to play an essential role in metabolic switching by collaborating with the muscle-abundant transcription factor ERRα.
During preparation of this manuscript, Ma et al. reported activation of the rat PDK4 gene by PGC-1α. Using an adenovirus expressing PGC-1α, they showed that it activates PDK4 gene transcription in rat hepatocytes and ventricular myocytes. Further in vitro analyses were carried out in human hepatoma HepG2 cells and HNF4 was identified as a transcription factor that stimulates PDK4 promoter activity. However, HNF4 is not directly involved in the recruitment of PGC-1α, and Ma et al. suggested that FOXO1 might participate in the recruitment, based on the fact that a FOXO1-binding site is conserved among humans, mice and rats.
Our results on the PGC-1α-responsive site in the 2.3 kb mouse PDK4 gene promoter obtained using mutation analyses do not support speculation that FOXO1 is a partner of PGC-1α in this region (Fig. 3B), but they show that the partner is ERRα, as stated above. Our data do not contradict the model proposed by Kwon et al. that multiple FOXO1s are involved in the regulation with glucocorticoid receptor (GR) by binding to different regions . We concentrated on the identification of a direct PGC-1α partner and did not search for other independent factors. So, the possible independent involvement of HNF4 from PGC-1α on the gene activation remains, and at least two mechanisms are able to explain the independent involvement of ERRα and HNF4. One explanation is that the two factors act on the promoter in the same cell, each factor responding to a different signal. The other explanation is that a given cell type preferentially uses one factor rather than the other. We think that the latter is more likely because the expression profile of ERRα in various tissues differs from that of HNF4 . In particular, it is known that the expression level of ERRα is high, whereas that of HNF4 is low, in skeletal muscle, and vice versa in liver [23,24]. Ma et al. analyzed the mechanism using hepatoma cells and suggested that HNF4 will stimulate PDK4 gene expression, at least in liver . We analyzed it using kidney CV-1, myoblast and hepatoma cells, and concluded that ERRα enhances PDK4 expression with PGC-1α in myoblast but not hepatoma cells. Consequently, our model in skeletal muscle and the model proposed by Ma et al. for liver  seem to be compatible, as shown in Fig. 7. Both mechanisms may operate in either tissue to varying degrees, depending on the expression levels of ERRα and HNF4. FOXO1 is not included in the model because we have not directly studied its involvement in the mechanism. To understand the overall regulation of PDK4 expression, it is essential to study the relationship between the ERRα/PGC-1α and the FOXO1/GR pathways in detail.
Figure 7. Proposed model depicting the cell-type-specific response of the PDK4 promoter to PGC-1α in skeletal muscle and liver. (A) In skeletal muscle, PGC-1α is specifically recruited by ERRα and the complex activates transcription (this study). The pathway to activate ERRα expression is taken from Sato et al.. (B) In liver, PGC-1α and HNF4 independently activate transcription. The partner of PGC-1α is not yet identified. These liver-specific pathways are taken from Ma et al. .
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The attempt to identify the ERRα-regulated pathway using primary myocytes overexpressing ERRα conducted by Huss et al.  did not assign PDK4 as a gene that is upregulated by ERRα. Instead, pyruvate dehydrogenase phosphatase I, which reverses the action of PDK4 and activates pyruvate dehydrogenase complex, was identified as a being upregulated by ERRα. Although Huss et al.'s approach did not identify all the genes directly regulated by ERRα, they did identify a subset of PGC-1α target genes involved in multiple energy-production pathways. This may be because the difference in cell types (cardiac myocytes vs. skeletal myoblast) was critical and/or that transcriptional control of PDK4 gene expression is such a complex process that some complementary effects cancelled out the enhanced expression. Contrary to the overexpressing approach, Luo et al. generated ERRα-null mice and reported the differentially expressed genes only in the adipose tissue because of their lean phenotype . Interestingly, PDK4 expression was increased by 1.8-fold in the adipose tissue of ERRα-null mice. The expression of several genes directly linked to lipid and energy metabolism was also altered, resulting in a defect in triglyceride synthesis. Luo et al. suggested that the loss of ERRα might interfere with other nuclear signaling pathways. If so, the direct cause of their result showing increased PDK4 expression in the adipose tissue may not be the loss of ERRα. Thus, both the forced expression and loss-of-function studies using animal models to understand the in vivo role of ERRα are not straightforward, and the results reported by other groups do not necessarily conflict with our conclusion.
We also noticed that the PGC-1α and ERRα pathway leading to PDK4 activation was not simple. During the ERRα overexpressing experiment (Fig. 4A), dose-dependent enhancement of PGC-1α-dependent activation of the PDK promoter was observed only in C2C12 cells. Although the lack of responsiveness to PGC-1α in hepatoma Fao cells could be explained by the undetectable level of endogenous ERRα (Fig. 6), the positive effect of the cotransfected ERRα expression plasmid was not observed at various doses (not shown). It is conceivable that the expression level is important in reproducing the essential functions of a regulatory protein interacting with many other effector proteins. Furthermore, there is the possibility that another unidentified muscle-specific factor is needed to activate the PGC-1α and ERRα pathway. Thus, we conclude that ERRα is necessary but not sufficient for the PGC-1 responsiveness of PDK4 gene expression in the muscle.
Wide and essential roles of PGC-1α in regulating the metabolism, including the maintenance of glucose, lipid and energy homeostasis, have been revealed by recent studies . In skeletal muscle, the most characterized signals including PGC-1α would be the MEF2 pathway, which activates MEF2-dependent genes . In fact, we first examined the possibility that MEF2 was involved in PGC-1α-dependent activation of the PDK4 promoter. However, cotransfection of the MEF2 expression plasmid had a dose-dependent inhibitory effect on PGC-1α-dependent activation. We now think that the ERRα pathway leading to the activation of the PDK4 is independent of the MEF2 pathway and speculate that the inhibition was caused by competition for PGC-1α between the two factors. Further possible inhibitory interactions between ERRα and PGC-1α suggested by Ichida et al.  may also be involved in the complex regulation. Nevertheless, the two independent pathways use PGC-1α as a common and essential coactivator and each pathway has its own physiological significance. Therefore, there must be some mechanism to discriminate between the two pathways and we again emphasize the possibility that unidentified factors are essential to separately activate these PGC-1α pathways.
Wende et al.  reported that the PGC-1α-mediated transcriptional activation of the mouse PDK4 gene is independent of PPARα and FOXO1, but it is dependent on ERRα. Their conclusion is apparently the same as ours, but they suggested a different binding site for ERRα (AAGGACA/TGTCCTT; bp −357 to −352) from the one that we have identified (TGACATT; bp −389 to −383) (Fig. 3) and both sequences do not perfectly matched with the consensus sequence, TGACCTT . In contrast to our direct demonstration, Wende et al. used a long DNA fragment containing both possible sites in their binding studies. Their result showing the loss of the responsiveness to PGC-1α by the mutation in the suggested region can be explained by our result that the region is important for basal activity (Fig. 2C). Not ERRα, but an unidentified factor, should bind to the site suggested and may play an important role in basal transcription.
In this study, we characterized the PPARα-independent transcriptional activation of the mouse PDK4 gene and concluded that it is activated via the ERRα/PGC-1α pathway. We speculate that this pathway mediates the signals produced by fasting to induce PDK4 in the skeletal muscle. However, there is no direct evidence to indicate that the ERRα/PGC-1α pathway is responsible for transcriptional activation of the PDK4 gene in muscle in response to fasting. Further studies to directly link the result obtained using cultured cells and those that occur in animals are necessary to understand the in vivo mechanism and its physiological significance. In addition, characterization of PPARα-dependent activation of the PDK4 gene and its relation to other activation pathways remain important issues.