A prominent hypothesis considers uncoupling protein 3 (Ucp3) to be a crucial component of lipid metabolism with implications for the regulation of body weight and composition . This role is further substantiated by the identification of polymorphisms/alleles in the human Ucp3 gene that are associated with an elevated body mass index . A more detailed analysis of the machinery regulating Ucp3 transcription is therefore of importance for identifying regulatory networks controlling energy partitioning.
Our comparison of previously characterized Ucp3 promoter elements in rat, mouse and human with the hamster sequence, shows full conservation of the binding sites for MyoD, PPARα/RXRα and TR/RXRα heterodimers. The sequence alignment furthermore demonstrates that two TATA-like boxes present in the human promoter  are absent in rodent sequences including the hamster. This might prove to be crucial considering, that in the study of Riquet and coworkers (2003) the activities of human constructs were investigated in murine tissue.
The TSS of the human Ucp3 gene is quite variable and displays a distinct tissue specificity, whereas in mouse the TSS is located at a single site . Our result of variable TSS in P. sungorus demonstrates that the constancy in mouse does not reflect a common trait of rodent species. However, in the hamster the TSS did not show a distinct tissue specificity as found in humans.
By in silico analysis we identified Coup-TFII as a candidate transcription factor for the regulation of Ucp3 expression. Coup-TFII is a 45 kD nuclear orphan receptor and member of the COUP-TF family. The amino acid sequence of the ligand binding and the DNA binding domain is conserved across species to a very high extent (human vs. Drosophila ~90%) indicating an important role for these domains. Coup-TFII has mainly been described as a crucial factor in developmental processes [21, 22].
By qPCR we measured Ucp3 and Coup-TFII transcript levels in a panel of nine different tissues. We confirmed that BAT and SKM are the major sites of Ucp3 mRNA expression. Coup-TFII was detected in both tissues, but in line with previous data on human adult tissue distribution  is rather ubiquitously expressed with highest levels in kidney, liver and heart. Contrary to reports that Coup-TFII is expressed in preadipocytes and myoblasts and downregulated during differentiation [23, 31], we were able to detect considerable amounts of Coup-TFII transcript in BAT and SKM tissue. Apparantely cell culture systems are devoid of the appropriate physiological stimuli promoting Coup-TFII mRNA expression in tissues. This is supported by the absence of Coup-TFII in human embryonal kidney cells as shown in our Western blot experiment (Fig. 5B) despite its presence in human kidney tissue in vivo . It has been demonstrated that Coup-TFII plays an important role in the regulation of several genes encoding key metabolic enzymes [18, 23–25], which are certainly regulated in terminally differentiated cells.
Ucp3 gene expression was upregulated in SKM of food deprived hamsters. In line with the function of Coup-TFII in metabolic regulation we also observed a significant increase of mRNA expression in response to this challenge. Probably owing to the short duration of cold exposure in our study we did not observe a significant cold induced increase in Ucp3 mRNA expression in BAT as published previously [5, 6]. However, Coup-TFII and Ucp3 mRNA expression in BAT displayed a similar cold-induced increase in variation. The resemblance of expression levels in SKM and BAT and the response to physiological stimuli culminates in a highly significant correlation of Coup-TFII and Ucp3 mRNA abundancy under challenged conditions (food deprivation and cold). The absence of such a correlation in the control group suggests that Coup-TFII requires additional factors in order to enhance Ucp3 mRNA expression which must be recruited and/or activated beforehand.
We could support this model in reporter gene assays, in which Coup-TFII strongly coactivated Ucp3 promotor activity in synergy with PPARα, MyoD, RXRα and/or p300, while the effect of Coup-TFII was much lower alone. These constituents of the well described basal transcription factor complex in our experiments affected Ucp3 expression as described previously , i.e. strong activation by PPARα/RXRα and p300, dependent on agonist stimulation and presence of MyoD. Coup-TFII specifically enhanced expression in synergy with these factors.
In contrast to our data, Coup-TFII has been shown to negatively interact with MyoD and p300 in SKM, reducing their potential to activate E-box driven reporter gene constructs . This discrepancy may be explained by a differential recruitment of the multiple COUP-TF family cofactors in different physiological environments [e.g. N-Cor, SMRT, RIP140, SRC-1; for a review see ]. The specific complex of transcription factors, to which Coup-TFII is recruited, may determine the final function as described for interaction with the glucocorticoid receptor . In general, COUP-TF proteins display a conflicting pattern of positive or negative interaction with nuclear receptors like PPARs or the estrogen receptor depending on the target gene [discussed in ]. There are genes where transcription is increased by PPARs and decreased by COUP-TFs [e.g. malic enzyme ] as well as genes for which the situation is opposite [e.g. transferrin ] or at which both PPARs and COUP-TFs act synergistically [e.g. lipoprotein lipase ]. Even a complete reversal of the effect of Coup-TFII on a single target has been reported ; transcription of the phosphoenolpyruvate carboxykinase gene is induced or repressed by Coup-TFII in a tissue specific manner.
Interestingly, specific PPARγ agonists upregulate Coup-TFII in the heart  and also Ucp3 gene expression in BAT and SKM [34, 35], although to our knowledge no direct interaction of PPARγ with the Ucp3 promoter has been verified so far. This effect might therefore be due to PPARγ mediated transactivation of Coup-TFII, which in turn enhances Ucp3 expression.
We were able to confine the sequence element mediating the Coup-TFII effect to -1307 to -664 by utilizing reporter gene deletion constructs. -2244UCP3 luc was the only construct being induced by Coup-TFII on a background of MyoD, RXRα and p300 and was exclusively activated high above an unspecific effect by Coup-TFII alone.
Of five candidate elements A-E within this region tested by electrophoretic mobility shift assays we confirmed specific binding of Coup-TFII to element C. Notably, element C did not only show complex formation with overexpressed Coup-TFII but also with nuclear extracts of hamster SKM (Fig. 5D). Extracts of all six animals tested were able to form a complex of comparable size and food deprivation of hamsters led to an increase in band intensity. The element is located at -816 to -840 and constitutes a functional repeat structure. By mutational analyses we were able to confine Coup-TFII binding to the 5' half-site of this element. In reporter gene assays disruption of this site surprisingly led to a complete loss of activity and responsiveness to any treatment tested. While this fact certainly underlines the importance of this element, its indifference to MyoD, RXRα and p300 treatment is in conflict with the retained effect of this treatment on -664UCP3 luc and -243UCP3 luc, that are also devoid of this element. It seems that the element exhibits a function beyond direct transactivation by Coup-TFII, that depends on the presence of the surrounding region. These characteristics point towards a model of Coup-TFII deactivating or displacing a so far unknown repressor of Ucp3 transcription. This repressor could be expected to bind in proximity to the 5' half-site of elementC, possibly even to the 3' half-site of this same element. In the light of this hypothetical model several formerly negligible seeming facts gain relevancy: Mutated construct -2244Cmut3' luc displayed an overall increase of luciferase activity as compared to -2244UCP3 luc (Fig. 7). Deletion construct -664UCP3 luc had a higher basal activity and could be activated by MyoD, RXRα and p300 to a higher extent than -2244UCP3 luc (Fig. 4B). The complex size of bandshift probe Cmut5' was slightly changed (Fig. 6A).
This model alone, however, does not account for the activation of -2244UCP3 luc by MyoD, RXRα, p300 and PPARα in the absence of cotransfected Coup-TFII. Therefore there would have to be an endogenous Coup-TFII expression in HEK293 cells that we can either not detect with our Western blots or is induced by transfection of factors like MyoD and p300. At present we cannot decide wether repressor displacement/deactivation itself or subsequent direct coactivation is the dominant means of Coup-TFII mediated transcriptional induction.
The conservation of the inverse repeat structure and its Coup-TFII binding site across rodent species suggests an important role for this element; its preservation in the murine promoter despite an obvious event of rearrangement during evolution supports this role. The upstream location of the binding element and its synergy with PPARα resembles a comparable situation, where Coup-TFII directly coactivates the lipoprotein lipase gene in cooperation with PPAR/RXR heterodimers as described previously . Notably lipoprotein lipase is a crucial determinant of fatty acid uptake from circulating triglycerides.
An explanation of the link between increased lipid utilization and the recruitment of Coup-TFII in BAT and SKM is hampered by the lack of knowledge about a specific ligand and the posttranslational mechanism of activation. It is beyond the scope of this study to clarify whether Coup-TFII is directly regulated in response to FFA levels and thus possibly conveys FFA-dependent Ucp3 transcription. Our qPCR data indicate that de novo synthesis of Coup-TFII is the dominant means of target gene upregulation rather than ligand binding or posttranslational modification. So far, transcription of Coup-TFII has been linked to MAP kinase pathway activity  and the presence of ETS family transcription factors . Further research is required to clarify whether these mechanisms represent a link between lipid utilization and Coup-TFII upregulation.
On the functional level beyond its role in organismic development Coup-TFII has been assumed to be an integral part of the glucose response complex, where it functions as an inhibitor of glucose dependent activators . This hypothesis is based on the ability of Coup-TFII to inhibit upstream stimulatory factor (USF)-dependent transactivation of a glucose response element in the L-type pyruvate kinase gene (PKLR) and a similar finding regarding the ATPA gene encoding the alpha subunit of the F1F0 ATP synthase complex . Recently, Bardoux and coworkers (2005), after discovering a role for Coup-TFII in insulin secretion and sensitivity, assigned Coup-TFII as an important regulator of glucose homeostasis . Our finding of Coup-TFII upregulation in states of augmented lipid oxidation confirms these models and extends them to a new aspect. The inhibition of glucose induced gene transcription may occur in conjunction with a positive regulation of lipid metabolism genes like Ucp3 and lipoprotein lipase.