Regulation Of UCP1 Research Articles

Alternative Polyadenylation and Differential Regulation of Ucp1: Implications for Brown Adipose Tissue Thermogenesis Across Species.

Brown adipose tissue (BAT) is a thermogenic organ owing to its unique expression of uncoupling protein 1 (UCP1), which is a proton channel in the inner mitochondrial membrane used to dissipate the proton gradient and uncouple the electron transport chain to generate heat instead of adenosine triphosphate. The discovery of metabolically active BAT in human adults, especially in lean people after cold exposure, has provoked the “thermogenic anti-obesity” idea to battle weight gain. Because BAT can expend energy through UCP1-mediated thermogenesis, the molecular mechanisms regulating UCP1 expression have been extensively investigated at both transcriptional and posttranscriptional levels. Of note, the 3′-untranslated region (3′-UTR) of Ucp1 mRNA is differentially processed between mice and humans that quantitatively affects UCP1 synthesis and thermogenesis. Here, we summarize the regulatory mechanisms underlying UCP1 expression, report the number of poly(A) signals identified or predicted in Ucp1 genes across species, and discuss the potential and caution in targeting UCP1 for enhancing thermogenesis and metabolic fitness.

2130-P: Transcription Factor NF-kB Regulates Thermogenic Function of Brown Adipocytes in UCP1-Independent Manner

NF-kB is a transcription factor that is activated upon cellular response to endotoxin, TNF-a or oxidative stress, etc. The transcription factor is formed by two subunits, p65 and p50, in most cells. The transcription activity is mainly determined by the p65 subunit for the transactivation domain. The p50 subunit does not contain a transactivation domain in the protein structure. NF-kB is expressed in brown adipocytes and the expression of p65 is increased by differentiation of the adipocytes. However, the activity of NF-kB remains unknown in brown adipocytes, especially in the thermogenic function of the adipocytes. To address this issue, we generated brown adipocyte-specific NF-kB transgenic mice with gain-of-function and loss-of-function using the Cre-LoxP strategy. The gain-of-function was achieved by over expression of the p65 subunit in a knockin mice, and the loss-of-function was made by inactivation of p65 subunit in a floxed mice. The gene modification was achieved by crossing the p65 mice with UCP1-CreER mice. The gain-of-function led to an elevation of mitochondrial function in brown adipocytes with an increase in uncoupling activity. The gene alteration preserved the brown fat function in DIO mice. In contrast, NF-kB loss-of-function made the mice intolerant to cold exposure in the lean condition and obese condition. The thermogenic function was significantly decreased in the mice. UCP-1 is a dominant uncoupling protein in the mitochondria of brown adipocytes to mediate the cold-induced heat production. UCP-1 expression and post-translational modification were not altered in brown adipocytes of the two lines of transgenic mice. These results suggest that NF-kB is required for the thermogenic function of brown adipocytes, and its activity is not associated with regulation of UCP-1. Disclosure J. Ye: None.

Research and thinking of acupuncture on promoting white adipose tissue browning

Acupuncture has become an effective approach in clinic for treating obesity, but its mechanism has not been clarified yet. A large number of researches have been conducted on the obesity mechanism in the aspects of neurophysiological regulation, feeding center regulation and peripheral digestion and absorption regulation at home and abroad. But, regarding the main storage site of excess energy, i．e. the remodeling and functional regulation of white adipose tissue (WAT), is still a new field in research. In the paper, focusing on the new filed of weight loss, in view of the promotion of WAT browning through the re-gulation of UCP1 and PPARγ signal pathway with acupuncture, the potential peripheral mechanism of acupuncture was explored on weight loss.

Adipose TBX1 regulates β-adrenergic sensitivity in subcutaneous adipose tissue and thermogenic capacity in vivo

ObjectiveT-box 1 (TBX1) has been identified as a genetic marker of beige adipose tissue. TBX1 is a mesodermal development transcription factor essential for tissue patterning and cell fate determination. However, whether it plays a role in the process of adipose beiging or how it functions in adipose tissue has not been reported. Here, we examined the function of TBX1 in adipose tissue as well as adipose-derived stem cells from mice and humans. MethodsAdipose-specific TBX1 transgenic (TBX1 AdipoTG) and adipose-specific TBX1 knockout (TBX1 AdipoKO) mice were generated to explore the function of TBX1 in the process of adipose beiging, metabolism and energy homeostasis in vivo. In vitro, we utilized a siRNA mediated approach to determine the function of TBX1 during adipogenesis in mouse and human stem cells. ResultsAdipose-specific overexpression of TBX1 was not sufficient to fully induce beiging and prevent diet-induced obesity. However, adipose TBX1 expression was necessary to defend body temperature during cold through regulation of UCP1 and for maintaining β3-adrenergic sensitivity and glucose homeostasis in vivo. Loss of adipose TBX1 expression enhanced basal lipolysis and reduced the size of subcutaneous iWAT adipocytes. Reduction of TBX1 expression via siRNA significantly impaired adipogenesis of mouse stromal vascular cells but significantly enhanced adipogenesis in human adipose derived stem cells. ConclusionsAdipose expression of TBX1 is necessary, but not sufficient, to defend body temperature during cold via proper UCP1 expression. Adipose TBX1 expression was also required for proper insulin signaling in subcutaneous adipose as well as for maintaining β-adrenergic sensitivity, but overexpression of TBX1 was not sufficient to induce adipocyte beiging or to prevent diet-induced obesity. TBX1 expression is enriched in adipose stem cells in which it has contrasting effects on adipogenesis in mouse versus human cells. Collectively, these data demonstrate the importance of adipose TBX1 in the regulation of beige adipocyte function, energy homeostasis, and adipocyte development.

Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation.

Brown adipose tissue (BAT) is rich in mitochondria and plays important roles in energy expenditure, thermogenesis, and glucose homeostasis. We find that levels of mitochondrial protein succinylation and malonylation are high in BAT and subject to physiological and genetic regulation. BAT-specific deletion of Sirt5, a mitochondrial desuccinylase and demalonylase, results in dramatic increases in globalprotein succinylation and malonylation. Mass spectrometry-based quantification of succinylation reveals that Sirt5 regulates the key thermogenic protein in BAT, UCP1. Mutation of the two succinylated lysines in UCP1 to acyl-mimetic glutamine and glutamic acid significantly decreases its stability and activity. The reduced function of UCP1 and other proteins in Sirt5KO BAT results in impaired mitochondria respiration, defective mitophagy, and metabolic inflexibility. Thus, succinylation of UCP1 and other mitochondrial proteins plays an important role in BAT and in regulation of energy homeostasis.

A dual Ucp1 reporter mouse model for imaging and quantitation of brown and brite fat recruitment

ObjectivesBrown adipose tissue (BAT) dissipates nutritional energy as heat through uncoupling protein 1 (UCP1). The discovery of functional BAT in healthy adult humans has promoted the search for pharmacological interventions to recruit and activate brown fat as a treatment of obesity and diabetes type II. These efforts require in vivo models to compare the efficacy of novel compounds in a relevant physiological context. MethodsWe generated a knock-in mouse line expressing firefly luciferase and near-infrared red florescent protein (iRFP713) driven by the regulatory elements of the endogenous Ucp1 gene. ResultsOur detailed characterization revealed that firefly luciferase activity faithfully reports endogenous Ucp1 gene expression in response to physiological and pharmacological stimuli. The iRFP713 fluorescence signal was detected in the interscapular BAT region of cold-exposed reporter mice in an allele-dosage dependent manner. Using this reporter mouse model, we detected a higher browning capacity in female peri-ovarian white adipose tissue compared to male epididymal WAT, which we further corroborated by molecular and morphological features. In situ imaging detected a strong luciferase activity signal in a previously unappreciated adipose tissue depot adjunct to the femoral muscle, now adopted as femoral brown adipose tissue. In addition, screening cultured adipocytes by bioluminescence imaging identified the selective Salt-Inducible Kinase inhibitor, HG-9-91-01, to increase Ucp1 gene expression and mitochondrial respiration in brown and brite adipocytes. ConclusionsIn our mouse model, firefly luciferase activity serves as a bona fide reporter for dynamic regulation of Ucp1. In addition, by means of iRFP713 we are able to monitor Ucp1 expression in a non-invasive fashion.

Intrinsic Properties of Brown and White Adipocytes Have Differential Effects on Macrophage Inflammatory Responses.

Obesity is marked by chronic, low-grade inflammation. Here, we examined whether intrinsic differences between white and brown adipocytes influence the inflammatory status of macrophages. White and brown adipocytes were characterized by transcriptional regulation of UCP-1, PGC1α, PGC1β, and CIDEA and their level of IL-6 secretion. The inflammatory profile of PMA-differentiated U937 and THP-1 macrophages, in resting state and after stimulation with LPS/IFN-gamma and IL-4, was assessed by measuring IL-6 secretion and transcriptional regulation of a panel of inflammatory genes after mono- or indirect coculture with white and brown adipocytes. White adipocyte monocultures show increased IL-6 secretion compared to brown adipocytes. White adipocytes cocultured with U937 and THP-1 macrophages induced a greater increase in IL-6 secretion compared to brown adipocytes cocultured with both macrophages. White adipocytes cocultured with macrophages increased inflammatory gene expression in both types. In contrast, macrophages cocultured with brown adipocytes induced downregulation or no alterations in inflammatory gene expression. The effects of adipocytes on macrophages appear to be independent of stimulation state. Brown adipocytes exhibit an intrinsic ability to dampen inflammatory profile of macrophages, while white adipocytes enhance it. These data suggest that brown adipocytes may be less prone to adipose tissue inflammation that is associated with obesity.

The Effect of Resveratrol and Quercetin Treatment on PPAR Mediated Uncoupling Protein (UCP-) 1, 2, and 3 Expression in Visceral White Adipose Tissue from Metabolic Syndrome Rats.

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier superfamily involved in the control of body temperature and energy balance regulation. They are currently proposed as therapeutic targets for treating obesity and metabolic syndrome (MetS). We studied the gene expression regulation of UCP1, -2, and -3 in abdominal white adipose tissue (WAT) from control and MetS rats treated with two doses of a commercial mixture of resveratrol (RSV) and quercetin (QRC). We found that UCP2 was the predominantly expressed isoform, UCP3 was present at very low levels, and UCP1 was undetectable. The treatment with RSV + QRC did not modify UCP3 levels; however, it significantly increased UCP2 mRNA in control and MetS rats in association with an increase in oleic and linoleic fatty acids. WAT from MetS rats showed a significantly increased expression of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ when compared to the control group. Furthermore, PPAR-α protein levels were increased by the highest dose of RSV + QRC in the control and MetS groups. PPAR-γ expression was only increased in the control group. We conclude that the RSV + QRC treatment leads to overexpression of UCP2, which is associated with an increase in MUFA and PUFA, which might increase PPAR-α expression.

IRE1α-XBP1 is a novel branch in the transcriptional regulation of Ucp1 in brown adipocytes.

The unfolded protein response (UPR) not only resolves endoplasmic reticulum (ER) stress, but also regulates cellular physiological functions. In this study, we first linked the UPR to the physiological roles of brown adipose tissue (BAT). BAT is one of the tissues that control energy homeostasis in the body. Brown adipocytes are able to dissipate energy in the form of heat owing to their mitochondrial protein, uncoupling protein 1 (UCP1). We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1. Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression. However, the activation of the IRE1α-XBP1 pathway by ER stress never upregulated Ucp1. On the other hand, the activation of protein kinase A (PKA) induced Ucp1 transcription through the activation of IRE1α-XBP1. The inhibition of PKA abrogated the activation of IRE1α-XBP1 pathway, while the inhibition of a p38 mitogen activated protein kinase (p38 MAPK), which is one of the downstream molecules of PKA, never suppressed the activation of IRE1α-XBP1 pathway. These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

Thyroid hormone status affects expression of daily torpor and gene transcription in Djungarian hamsters (Phodopus sungorus)

Thyroid hormones (TH) play a key role in regulation of seasonal as well as acute changes in metabolism. Djungarian hamsters (Phodopus sungorus) adapt to winter by multiple changes in behaviour and physiology including spontaneous daily torpor, a state of hypometabolism and hypothermia. We investigated effects of systemic TH administration and ablation on the torpor behaviour in Djungarian hamsters adapted to short photoperiod. Hyperthyroidism was induced by giving T4 or T3 and hypothyroidism by giving methimazole (MMI) and sodium perchlorate via drinking water. T3 treatment increased water, food intake and body mass, whereas MMI had the opposite effect. Continuous recording of body temperature revealed that low T3 serum concentrations increased torpor incidence, lowered Tb and duration, whereas high T3 serum concentrations inhibited torpor expression. Gene expression of deiodinases (dio) and uncoupling proteins (ucp) were analysed by qPCR in hypothalamus, brown adipose tissue (BAT) and skeletal muscle. Expression of dio2, the enzyme generating T3 by deiodination of T4, and ucps, involved in thermoregulation, indicated a tissue specific response to treatment. Torpor per se decreased dio2 expression irrespective of treatment or tissue, suggesting low intracellular T3 concentrations during torpor. Down regulation of ucp1 and ucp3 during torpor might be a factor for the inhibition of BAT thermogenesis. Hypothalamic gene expression of neuropeptide Y, propopiomelanocortin and somatostatin, involved in feeding behaviour and energy balance, were not affected by treatment. Taken together our data indicate a strong effect of thyroid hormones on torpor, suggesting that lowered intracellular T3 concentrations in peripheral tissues promote torpor.

Regulation of UCP1 in the Browning of Epididymal Adipose Tissue by β3-Adrenergic Agonist: A Role for MicroRNAs.

Background. White adipose tissue browning may be a promising strategy to combat obesity. UCP1 is strongly induced in White adipose tissue with β3-adrenergic agonist treatment, but the causes of this increase have not been fully elucidated. This study aims to explore more miRNAs involved in the process of browning of visceral adipose tissue. Methods. Total of fourteen mice were randomly divided into control and study group. Study group mice were injected intraperitoneally with CL316243 once daily for seven days; meanwhile the control group were treated with 0.9% NaCl. After a 7-day period, the expression of genes involved in WAT browning and potential UCP1-targeting miRNAs in adipose tissues was analyzed by qPCR. Results. qPCR analysis revealed that UCP1, DIO2, CIDEA, and CPT1B in epididymal adipose tissue were overexpressed in CL316243 group. Furthermore, potential UCP1-targeting miR-9 and miR-338-3p in epididymal adipose tissue were significantly decreased in CL316243 group. Conclusion. This suggests that potential UCP1-targeting miR-9 and miR-338-3p may be involved in the browning of epididymal adipose tissue by regulating UCP1 gene expression. In this study, we demonstrated that this increase of UCP1 is due, at least in part, to the decreased expression of certain UCP1-targeting miRNAs in epididymal adipose tissue compared to control.

PGC-1α Is Required for Exercise- and Exercise Training-Induced UCP1 Up-Regulation in Mouse White Adipose Tissue

BackgroundThe aim of the present study was to test the hypotheses that 1) a single exercise bout increases UCP1 mRNA in both inguinal (i)WAT and epididymal (e)WAT, 2) UCP1 expression and responsiveness to exercise are different in iWAT and eWAT, 3) PGC-1α determines the basal levels of UCP1 and PRDM16 in WAT and 4) exercise and exercise training regulate UCP1 and PRDM16 expression in WAT in a PGC-1α-dependent manner.MethodsWhole body PGC-1α knockout (KO) and wildtype (WT) littermate mice performed a single treadmill exercise bout at 14 m/min and 10% slope for 1 hour. Mice were sacrificed and iWAT, eWAT and quadriceps muscle were removed immediately after, 2, 6 and 10 hours after running, and from sedentary mice that served as controls. In addition, PGC-1α KO mice and WT littermates were exercise trained for 5 weeks with sedentary mice as untrained controls. Thirty-six-37 hours after the last exercise bout iWAT was removed.ResultsUCP1 mRNA content increased 19-fold in iWAT and 7.5-fold in eWAT peaking at 6 h and 0′ of recovery, respectively, in WT but with no changes in PGC-1α KO mice. UCP1 protein was undetectable in eWAT and very low in iWAT of untrained mice but increased with exercise training to 4.4 (AU) in iWAT from WT mice without significant effects in PGC-1α KO mice.ConclusionThe present observations provide evidence that exercise training increases UCP1 protein in iWAT through PGC-1α, likely as a cumulative effect of transient increases in UCP1 expression after each exercise bout. Moreover, the results suggest that iWAT is more responsive than eWAT in exercise-induced regulation of UCP1. In addition, as PRDM16 mRNA content decreased in recovery from acute exercise, the present findings suggest that acute exercise elicits regulation of several brown adipose tissue genes in mouse WAT.

Editorial: Molecular Endocrinology Articles in the Spotlight for October 2012

The October issue of Molecular Endocrinology is packed with excellent science. I would like to highlight a few articles especially recommended for your reading. In the article, “Cell-Autonomous Regulation of Brown Fat Identity Gene UCP1 by Unliganded Vitamin D Receptor,” Malloy et al demonstrate that regulation of the critical brown fat gene UCP1 by vitamin D receptor (VDR), which regulates brown fat identity genes in human cells is direct, resulting in cell autonomous control of this mediator of energy metabolism. The regulation of UCP1 by VDR is ligand independent, suggesting that hormonal intake or stores of vitamin D are unlikely to impact the beiging phenotype of adipocyte tissue. The ligand independence of the regulation will stimulate development of selective VDR antagonists (SVDRMs) that promote cellular beiging but do not impair other VDR activities like the regulation of calcium homeostasis. This study also suggests that mutations or polymorphisms in the VDR gene could impact the amount of beige or brown fat in humans and may therefore be worthy of investigation. “Targeting Stromal Androgen Receptor Suppresses Prolactin-Driven Benign Prostatic Hyperplasia (BPH)” by Lai et al describes the first stromal fibromuscular androgen receptor (AR) knockout, which used a probasin promoter driven prolactin transgenic (Pb-PRL tg) mouse, to study stromal AR function in BPH development. Specifically, elimination of stromal fibromuscular AR led to reduced prostate size, diminished epithelial/stromal cell proliferation, and alleviated microenvironmental changes including extracellular matrix remodeling and immune cells infiltration. Furthermore, stromal fibromuscular AR was able to modulate an epithelial-stromal interaction through the epithelial prolactin/prolactin receptor-granulocyte macrophage-colony stimulating factor-stromal signal transducer and activator of transcription 3 axes to facilitate the stromal cell growth. The report by Laryea et al, “Disrupting Hypothalamic Glucocorticoid Receptors Causes HPA Axis Hyperactivity and Excess Adiposity” (freely available at http://mend. endojournals.org/) demonstrates the essential role of glucocorticoid receptor-mediated negative feedback regulation in the paraventricular nucleus of the hypothalamus. Although chronic exposure to elevated glucocorticoids is often associated with anxiety and despair-related behavioral changes, the elevation resulting from disrupted paraventricular nucleus (PVN) glucocorticoid receptor (GR) does not. This may reflect adaptive mechanisms in other brain regions that attenuate glucocorticoid receptor activation. Alternatively, the normal circadian rhythm that is maintained in these animals may allow compensation to occur. Importantly, the loss of GR in the PVN is somehow protective against high circulating glucocorticoid levels. This study highlights the substantial differences in phenotypes that can emerge with conditional deletion of different versions of the same gene. Congratulations to all the authors in this issue for their fine work and research. Donald B. DeFranco, PhD Editor-in-Chief, Molecular Endocrinology E D I T O R I A L

Uncoupling protein 1 expression and high-fat diets

Uncoupling protein 1 (Ucp1) is the key component of β-adrenergically controlled nonshivering thermogenesis in brown adipocytes. This process combusts stored and nutrient energy as heat. Cold exposure not only activates Ucp1-mediated thermogenesis to maintain normothermia but also results in adaptive thermogenesis, i.e., the recruitment of thermogenic capacity in brown adipose tissue. As a hallmark of adaptive thermogenesis, Ucp1 synthesis is increased proportionally to temperature and duration of exposure. Beyond this classical thermoregulatory function, it has been suggested that Ucp1-mediated thermogenesis can also be employed for metabolic thermogenesis to prevent the development of obesity. Accordingly, in times of excess caloric intake, one may expect a positive regulation of Ucp1. The general impression from an overview of the present literature is, indeed, an increased brown adipose tissue Ucp1 mRNA and protein content after feeding a high-fat diet (HFD) to mice and rats. The reported increases are very variable in magnitude, and the effect size seems to be independent of dietary fat content and duration of the feeding trial. In white adipose tissue depots Ucp1 mRNA is generally downregulated by HFD, indicating a decline in the number of interspersed brown adipocytes.

Effect of epinephrine deficiency on cold tolerance and on brown adipose tissue

Catecholamines are involved in thermogenesis. We investigated the specific role of epinephrine in regulation of temperature homeostasis in mice. We subjected adult wildtype (WT) and phenylethanolamine N-methyl transferase knock out mice ( Pnmt −/−) lacking epinephrine to cold for 24 h. Body temperature and thyroid hormone levels were not different between WT and Pnmt −/− mice. Although temperature was normal in Pnmt −/− mice, the brown fat response to cold was abnormal with no increase in Ucp-1 or Pgc-1α mRNA levels (but with an exaggerated cold-induced lipid loss from the tissue). Our results show that epinephrine may have a role in brown fat mitochondrial uncoupling through regulation of Ucp-1 and Pgc-1α, although this is not required to maintain a normal temperature during acute cold exposure. We conclude that epinephrine may have an important role in induction of Ucp-1 and Pgc-1α gene expression during cold stress.

Gene expression survey of mitochondrial uncoupling proteins (UCP1/UCP3) in gilthead sea bream (Sparus aurata L.)

The aim of this work is to underline the biological significance of mitochondrial uncoupling proteins (UCPs) in ectothermic fish using the gilthead sea bream (Sparus aurata L.) as an experimental model. A contig of 1,990 bp in length was recognized as a UCP1 ortholog after initial searches in the gilthead sea bream AQUAFIRST database ( http://www.sigenae.org/aquafirst ). Additional searches were performed in skeletal muscle by RT-PCR, and the amplified PCR product was recognized as UCP3 after sequence completion by 5'- and 3'RACE. UCP1 expression was mostly detected in liver, whereas UCP3 transcripts were only found in skeletal and cardiac muscle fibres (white skeletal muscle > red skeletal muscle > heart). Specific gene regulation of UCP1 (liver) and UCP3 (white skeletal muscle) was addressed in physiological models of age, seasonal growth and energy-metabolic unbalances. Both the increase in energy demand (stress confinement) and the reduction in energy supply during adaptive cold response in winter down-regulated UCP1 expression. Conversely, transcript levels of UCP3 were higher with age, seasonal fattening and dietary deficiencies in essential fatty acids leading to the increase in fatty acid flux towards the muscle. This close association between UCP1 and UCP3 with the oxidative and metabolic tissue status is perhaps directly related to the ancestral protein UCP function, and allows the use of UCPs as lipotoxicity markers in ectothermic fish.

Tough love: left out in the cold, but not abandoned, by UCP3

In rodents, the challenge of maintaining core body temperature in a cold environment is partially met by increased activity of mitochondrial uncoupling protein (UCP) 1 in brown adipose tissue (BAT) ([1][1]). The energy that would normally be preserved by coupling ATP synthesis with the translocation

Transcriptional synergy and the regulation of Ucp1 during brown adipocyte induction in white fat depots.

Induction of brown adipocytes in white fat depots by adrenergic stimulation is a complex genetic trait in mice that affects the ability of the animal to regulate body weight. An 80-fold difference in expression of the mitochondrial uncoupling gene (Ucp1) at the mRNA and protein levels between A/J and C57BL/6J (B6) mice is controlled by allelic interactions among nine quantitative trait loci (QTLs) on eight chromosomes. Overlapping patterns of these QTLs also regulate expression levels of Pgc-1alpha, Pparalpha, and type 2 deiodinase. Independent validation that PPARalpha is associated with Ucp1 induction was obtained by treating mice with the PPARalpha agonist clofibrate, but not from the analysis of PPARalpha knockout mice. The most upstream sites of regulation for Ucp1 that differed between A/J and B6 were the phosphorylation of p38 mitogen-activated protein kinase and CREB and then followed by downstream changes in levels of mRNA for PPARgamma, PPARalpha, PGC-1alpha, and type 2 deiodinase. However, compared to Ucp1 expression, the two- to fourfold differences in the expression of these regulatory components are very modest. It is proposed that small variations in the levels of several transcriptional components of the Ucp1 enhanceosome interact synergistically to achieve large differences in Ucp1 expression.

Dietary fat interacts with QTLs controlling induction of Pgc-1 alpha and Ucp1 during conversion of white to brown fat.

To identify novel regulatory factors controlling induction of the brown adipocyte-specific mitochondrial uncoupling protein (Ucp1) mRNA in the retroperitoneal white fat depot, we previously mapped quantitative trait loci (QTLs) that control this trait to chromosomes 2, 3, 8, and 19. Since the peroxisome proliferator activator receptor-gamma coactivator-1alpha (PGC-1alpha) regulates Ucp1 and other genes of energy metabolism, we have evaluated whether the QTLs controlling Ucp1 mRNA levels also modulate Pgc-1alpha mRNA levels by analysis of backcross progeny from the A/J and C57BL/6J strains of mice. The results indicate that a locus on chromosome 3 orchestrates expression of Pgc-1alpha and Ucp1 in retroperitoneal fat of mice fed a low-fat diet; however, the effect of this locus on Pgc-1alpha is lost, and a significant correlation between Ucp1 and Pgc-1alpha is severely reduced in mice fed a high-fat diet. An additional QTL located on chromosome 5 has also been identified for the selective regulation of Ucp1 mRNA levels. Similar to the effects of a high-fat diet on the chromosome 3 QTL, linkage of the chromosome 5 QTL is also lost in mice on a high-fat diet. Thus dietary fat has a profound influence on PGC-1alpha-regulated pathways controlling energy metabolism in white fat. The allelic variation observed in the regulation of Ucp1 and Pgc-1alpha expression in brown adipocytes of white fat but not interscapular brown fat suggests that fundamentally different regulatory mechanisms exist to control the thermogenic capacities of these tissues.

Regulatory Motifs for CREB-binding Protein and Nfe2l2 Transcription Factors in the Upstream Enhancer of the Mitochondrial Uncoupling Protein 1 Gene

Thermogenesis against cold exposure in mammals occurs in brown adipose tissue (BAT) through mitochondrial uncoupling protein (UCP1). Expression of the Ucp1 gene is unique in brown adipocytes and is regulated tightly. The 5'-flanking region of the mouse Ucp1 gene contains cis-acting elements including PPRE, TRE, and four half-site cAMP-responsive elements (CRE) with BAT-specific enhancer elements. In the course of analyzing how these half-site CREs are involved in Ucp1 expression, we found that a DNA regulatory element for NF-E2 overlaps CRE2. Electrophoretic mobility shift assay and competition assays with the CRE2 element indicates that nuclear proteins from BAT, inguinal fat, and retroperitoneal fat tissue interact with the CRE2 motif (CGTCA) in a specific manner. A supershift assay using an antibody against the CRE-binding protein (CREB) shows specific affinity to the complex from CRE2 and nuclear extract of BAT. Additionally, Western blot analysis for phospho-CREB/ATF1 shows an increase in phosphorylation of CREB/ATF1 in HIB-1B cells after norepinephrine treatment. Transient transfection assay using luciferase reporter constructs also indicates that the two half-site CREs are involved in transcriptional regulation of Ucp1 in response to norepinephrine and cAMP. We also show that a second DNA regulatory element for NF-E2 is located upstream of the CRE2 region. This element, which is found in a similar location in the 5'-flanking region of the human and rodent Ucp1 genes, shows specific binding to rat and human NF-E2 by electrophoretic mobility shift assay with nuclear extracts from brown fat. Co-transfections with an Nfe2l2 expression vector and a luciferase reporter construct of the Ucp1 enhancer region provide additional evidence that Nfe2l2 is involved in the regulation of Ucp1 by cAMP-mediated signaling.