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ABSTRACT: Uncoupling protein (UCP) 2 is a member of the uncoupling-protein family, and it appears to function as an uncoupler of oxidative phosphorylation. To identify cis-acting regulatory elements controlling this gene's expression, we cloned an approx. 6.2-kb region upstream from the translation-initiation site of the mouse UCP2 gene and analysed its transcription activity using chimaeric mouse UCP2 promoter-placental-alkaline-phosphatase (PLAP) reporter-gene constructs. Sequence analysis showed that the 5'-flanking region of the mouse UCP2 gene was not similar to those of mouse UCP1 or UCP3. For the mouse UCP2, the region near the transcription-initiation site lacked the typical TATA box, but was GC-rich, resulting in presence of several potential specificity protein 1 (Sp-1), activator protein (AP)-1 and AP-2 binding sites. The putative regulatory motifs for muscle-regulatory protein (MyoD), brown-fat regulatory element, CCAAT box, cAMP-response element and Y box were also found in the mouse UCP2 promoter region by computer-assisted analysis. From the results of Northern-blot analysis and transient expression assay, we found that the mouse UCP2 gene responded to the cAMP-dependent protein kinase alpha-catalytic subunit signal activation at the transcription level. Additionally, deletion analysis of the UCP2 promoter-PLAP constructs indicated that the minimal region exhibiting the promoter activity was located between nt -33 and +100, and that a strong enhancer was present within 601 bp of the 5'-promoter region. In particular, the region from nt -233 to -34 significantly induced PLAP activity in the cell lines derived from various tissues and in the primary culture cells of rat brown adipose tissue, suggesting that this region is most important for the ubiquitous expression of mouse UCP2 mRNA. Furthermore, it was shown that two silencer elements were involved in the mouse UCP2 gene; one was located between nt -2746 and -602, and the other was identified in intron 1. These regions deprived the enhancer of the ability to induce PLAP activity. This study shows a fundamental role for positive and negative cis-acting DNA elements in regulating the basal and cAMP-induced transcription activity of the mouse UCP2 gene.
Biochemical Journal 07/1999; 340 ( Pt 2):397-404. · 4.90 Impact Factor
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ABSTRACT: Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters that dissipate the proton gradient, releasing stored energy as heat, without coupling to other energy-consuming processes. Therefore, the UCPs are thought to be important determinants of the metabolic efficiency. To elucidate relationships between the UCPs expressions and insulin sensitivity improvement, we treated KK-Ay mice with beta 3 adrenergic receptor agonist for 21 days and examined the changes of the UCPs mRNA expressions in various tissues. Chronic treatment of a specific beta 3 adrenergic receptor agonist, CL316,243 (0.2 mg/kg body weight/day s.c.) markedly increased the expressions of uncoupling protein 1 (UCP1), uncoupling protein 2 (UCP2), and uncoupling protein 3 (UCP3) by 14-fold, 6-fold, and 16-fold, respectively, in the brown adipose tissue (BAT). The UCP1 and UCP3 mRNA expressions in the white adipose tissue (WAT) were also increased by 12-fold and 9-fold, respectively, but the UCP2 mRNA expression was not changed in this tissue. Interestingly, the UCP2 and UCP3 mRNA expressions were strikingly decreased in the skeletal muscle and heart. Particularly, the UCP3 mRNA expression level in the skeletal muscle was dropped to 10% of that of the saline-treated control mice, indicating that the UCPs mRNA expressions are regulated in tissue-specific ways. The concentrations of plasma insulin and circulating free fatty acid (FFA) were significantly decreased, suggesting that they correlate with the reductions of the UCP2 and UCP3 mRNA expressions in the skeletal muscle and heart. It has been thought that the UCP1 and UCP3 mRNA expressions in the BAT and WAT are mainly controlled by the hypothalamus via the sympathetic nervous system, while the levels of insulin, FFA or both may play important roles in the control of the UCP2 and UCP3 mRNA expressions in the skeletal muscle an heart.
Biochemical and Biophysical Research Communications 01/1999; 253(1):85-91. · 2.48 Impact Factor
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ABSTRACT: Brown adipose tissue and skeletal muscle are important sites of non-shivering thermogenesis. It has been known that UCP1 and UCP2 function as the main effector of the thermogenesis: the former is expressed exclusively in brown adipose tissue, whereas the latter is distributed widely. Recently, the third UCP homologue was discovered in humans, which was designated as UCP3. We now report molecular cloning of full-length mouse UCP3 cDNA and its 5'-flanking genomic region. The mouse UCP3 cDNA sequence predicted a 308-amino acid protein, and the overall identity between the mouse and human UCP3 proteins was 85.6%. The mouse UCP3 amino acid sequence was 54.7% and 73.1% identical to the mouse UCP1 and UCP2, respectively. Expression of the mouse UCP3 was found to be abundant in skeletal muscle and somewhat less abundant in heart, but was minimally expressed in other critical organs. The sequences of 5'-flanking regions of the mouse UCP1 and UCP3 were very different, resulting in different distributions of putative transcriptional factor binding sites. The differences could reflect tissue-specific expression of the UCPs. The mouse Ucp3 gene was mapped near Ucp2 on chromosome 7, suggesting that the Ucp2 and Ucp3 are clustered genes. This region is boundary of synteny between human chromosome 11q13 and 11p15. As Solanes et al. reported that both human UCP2 and UCP3 genes are assigned to chromosome 11q13, the region where the mouse Ucp2 and Ucp3 are localized is syntenic to human chromosome 11q13.
Gene 08/1998; 215(1):77-84. · 2.34 Impact Factor
