An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene

Abstract

Previous studies on the regulation of a Ucp minigene in transgenic mice demonstrated that the sequences necessary for brown-fat-specific expression and inducibility by norepinephrine were located in the 5' flanking region between 1 and 2.8 kb from the transcriptional start site. We have investigated this region in more detail in cultured mouse brown adipocyte tumor cells. Deletion analysis of two types of chloramphenicol acetyltransferase reporter gene constructs under control of either the Ucp promoter or a heterologous herpes simplex virus-tk promoter defined an enhancer in a 220-bp HindIII-XbaI fragment which was essential for both brown fat specificity and norepinephrine inducibility. Site-directed mutagenesis of the reporter gene constructs established that independent mutations to a cyclic AMP-responsive element (CRE-2) or one of two TTCC motifs (BRE [brown fat regulatory element]), all within 17 bp, eliminated transient expression. Competitive DNA mobility shift assays with probes of the CRE and BRE motifs indicate that nuclear proteins interact with these motifs in a cooperative, synergistic manner. While these CRE-BRE probes do not show changes in binding which is dependent on norepinephrine treatment, a probe containing a third TTCC motif located 130 bp downstream of BRE-1 does show this dependency. The results indicate that a complex interaction of the CRE and BRE motifs, which cannot be functionally separated, control Ucp expression.

Browning of the white adipose tissue regulation: new insights into nutritional and metabolic relevance in health and diseases

“…These characteristics are consequences of the transcriptional control mediated by trans-acting factors on regulatory regions found in the 5’ non-coding region of the UCP1 gene. The proximal regulatory region, which is found immediately upstream of the transcription start site, contains cAMP response element-binding protein (CREB) [ 50 , 51 ] and CCAAT-enhancer-binding protein (C/EBP) [ 52 ] binding sites. Also in the proximity of the site of transcription start, activating transcription factor-2 (ATF2)-binding site interacts with transcriptional coregulators, such as PGC-1α, impacting UCP1 gene transcription [ 9 ].…”

Tecido adiposo marrom em adultos como alvo de estudo no desenvolvimento de novas terapias para o manejo e tratamento da obesidade: uma revisão integrativa

“…In this sense, this uncoupling protein is essential for the heat production process, that is, the facultative thermogenesis independent of tremor5. Thus, from the exclusive expression of the gene that encodes the UCP protein, an induction of temperature response is noted 19 .…”

“…Analysis of UCP expression in transgenic mice provided the information that the gene contained regulatory information for specific brown fat expression and cold induction. In this context, the induction of this protein gene is centrally controlled from the hypothalamus through the sympathetic nervous system, with evidence suggesting that noradrenaline, an adrenergic neurotransmitter, binds to beta receptors to initiate an adenosine signal 3 ',5'-cyclic monophosphate (cyclic AMP) 19 .…”

Synthesis and Human β-Adrenoceptor Activity of 1-(3,5-Diiodo-4- methoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-6-ol Derivatives in Vitro

“…Active PKA stimulates a hormonesensitive lipase thereby releasing necessary free fatty acids (FFAs) from the triglyceride stores in BAT and WAT and phosphorylates cAMP response element binding protein (CREB) to increase uncoupling protein (UCP) 7 gene transcription. 8,9 The FFAs act not only as substrates for β-oxidation but also to overcome the restraint on respiration exerted by purine nucleotides, such as GDP, binding to UCP. Activation of UCP leads to dissipation of the hydrogen gradient across the inner mitochondial membrane and conversion of the gradient energy to heat as a byproduct rather than ATP, thereby uncoupling ATP synthesis from respiration.…”

“…The β 3 -adrenergic receptor (β 3 -AR) plays a major role in mediating adipocyte lipolysis (breakdown of fat) in white adipocyte tissue (WAT) and thermogenesis in brown adipocyte tissue (BAT). − Thermogenesis in BAT is initiated by the sympathetic release of noradrenaline from sympathetic nerve endings or treatment with β 3 -AR agonists which act predominantly via β 3 -ARs, to cause an activation of adenylyl cyclase which, by increasing the concentration of cAMP, regulates protein kinase A (PKA) and protein phosphorylation. Active PKA stimulates a hormone-sensitive lipase thereby releasing necessary free fatty acids (FFAs) from the triglyceride stores in BAT and WAT and phosphorylates cAMP response element binding protein (CREB) to increase uncoupling protein (UCP) gene transcription. , The FFAs act not only as substrates for β-oxidation but also to overcome the restraint on respiration exerted by purine nucleotides, such as GDP, binding to UCP. Activation of UCP leads to dissipation of the hydrogen gradient across the inner mitochondial membrane and conversion of the gradient energy to heat as a byproduct rather than ATP, thereby uncoupling ATP synthesis from respiration. , …”