WO2003026576A2 - Induction of brown adipocytes by transcription factor nfe2l2 - Google Patents

Abstract

New methods have been developed to treat obesity or other human disorders by increasing the degree of brown adipose thermogenesis, by both increasing the growth and differentiation of brown adipose tissue and increasing its activity. A new use for the transcription factor NFE2l2 was developed to increase BAT thermogenesis by increasing the expression of Ucp1. For the first time, NFE2l2 was found to interact with the NF-E2 motif and to stimulate the expression of Ucp1 (Figure 9). By modulating the expression of the Nfe2l2 gene or the concentration of NFE2l2 protein, changes in brown adipose tissue thermogenesis can be achieved to treat various weight disorders. Additionally, the direct involvement of CREB binding to CRE2 to regulate Ucp1 expression was demonstrated. Furthermore, transient transfection assays of luciferase reporter constructs and site-directed mutagenesis indicated that CRE2 was involved in transcriptional regulation of the Ucp1 through interaction with a phosphorylated CREB. NFE2l2 can be combined with other known transcription factors, e.g., CREB, PGC1, RXR, RAR, and PPAR gamma, to increase BAT thermogenesis by increasing the expression of Ucp1.

Description

INDUCTION OF BROWN ADIPOCYTES BY TRANSCRIPTION FACTOR NFE212

[0001] The benefit of the 24 September 2001 filing date of United States provisional patent application serial number 60/324,400 is claimed under 35 U.S.C. § 119(e) in the United States, and is claimed under applicable treaties and conventions in all countries.

[0002] The development of this invention was partially funded by the Government under grant nos. R01-DK58152-01 from the National Institute of Diabetes and Digestive and Kidney Diseases and RO1-HD08431 from the National Institute of Child Health and Human Development, both divisions of the National Institutes of Health. The Government has certain rights in this invention.

TECHNICAL FIELD

[0003] This invention pertains to a new method to prevent or alleviate mammalian obesity by increasing the effective concentration of the transcription factor, NFE212, which will increase the number and activity of brown adipocytes, whose role is to burn fat to produce primarily heat.

BACKGROUND ART

Brown Adipose Tissue [0004] Mammals possess two forms of adipose tissue — white adipose tissue (WAT) and brown adipose tissue (BAT). White adipose tissue stores fat to be released upon demand for the nutritional and metabolic needs of the mammal, hi contrast, the cells of BAT (brown adipocytes) function to burn fat to release heat, and thus to help the mammal maintain or attain its body temperature. BAT is characterized by the presence of numerous mitochondria, hi muscles and other tissues with numerous mitochondria, the oxidation of fuel (e.g., sugars or fats) generates a proton gradient across the mitochondrial membrane; the energy of this gradient is coupled to the synthesis of adenosine triphosphate (ATP), a universal energy source for the body. This production of ATP occurs with relatively little loss of energy as heat, i BAT mitochondria, the oxidation of fat results in a proton gradient as in muscle and other tissues. However, in contrast to other tissues, the BAT mitochondria have a unique ability to generate a large amount of heat by uncoupling the normal mitochondrial process of oxidation of fat to produce ATP. This uncoupling is due to the presence of a unique protein found only in the membranes of BAT mitochondria, called "mitocondrial uncoupling protein 1 " or "UCP 1." UCP 1 uncouples the usual process of catabolism of fuel to form ATP by serving as a proton channel to decrease the proton concentration gradient across the mitochondrial membrane which normally (in mitochondria of non-BAT tissue) powers the production of ATP. See U.S. Patent No. 5,453,270. [0005] At various points in the life of a mammal, the growth and differentiation of BAT are important to the mammal's ability to maintain energy balance, body temperature, and prevent obesity; and the expression of UCP1 is essential for this function of BAT. Moreover, an increased level of transcription of the Ucpl gene is a critical event leading to elevated BAT activity, namely thermogenesis. Several rodent models of obesity (including leptin and lepin- receptor mutants) have diminished or defective BAT function. See B. Lowell et al, "Development of Obesity in Transgenic Mice after Genetic Ablation of Brown Adipose Tissue," Nature, vol. 366, pp. 740-742 (1993). Additionally, in rodents Ucpl expression is increased in response to cold stress and to administration of norepinephrine and other β-adrenergic receptor agonists. Four hours of cold stress increased Ucpl mRNA by seven fold in mice. See S. Rehnmark et al, "Alpha- and Beta-adrenergic Induction of the Expression of the Uncoupling Protein Thermogenin in Brown Adipocytes Differentiated in Culture," J. Biol. Chem., vol. 265, p. 16464-16471 (1990); and A. Jacobssonetα/., "Mitochondrial Uncoupling Protein from Mouse Brown Fat. Molecular Cloning, Genetic Mapping, and mRNA Expression," J. Biol. Chem., vol. 260, pp. 16250-16254 (1985). Mitochondrial Uncoupling Protein (UCP1) [0006] Increased thermogenesis of BAT can be induced by cold exposure and/or a high fat diet in brown adipose tissue (BAT) through the induction of the mitochondrial uncoupling protein (UCPl). See J.A. Levine et al, "Role of Nonexercise Activity Thermogenesis in Resistance to Fat Gain in Humans," Science vol. 283, pp. 212-214 (1999). Although four homologues of UCP have been identified, definitive proof establishing that an uncoupling protein is essential for thermogenesis has been shown only for UCPl. See S. Enerback et al, "Mice Lacking Mitochondrial Uncoupling Protein Are Cold-sensitive butNot Obese," Nature, vol.387, pp. 90-94 (1997). UCPl is located in the inner membrane of mitochondria, where it dissipates the mitochondrial membrane potential resulting in the generation of heat instead of ATP. Overexpression of Ucpl has been achieved pharmacologically by administration of thermo genie β₃-adrenergic receptor agonists, or genetically by using tissue-specific gene promoters to drive expression of Ucplm. transgenic mice, or by the increase in Ucpl transcription due to increase protein kinase A (PKA) activity in PKA Rllb knockout mice. See J. Himms-Hagen et al, "Effect of Cl-316,243, a Thermogenic Beta 3 -agonist, on Energy Balance and Brown and White Adipose Tissues in Rats," Am. J. Physiol., vol.266, pp. R1371-1382 (1994); andD.E. Cummings et al. , "Genetically Lean Mice Result from Targeted Disruption of the RU Beta Subunit of Protein Kinase A," Nature, vol. 382, pp. 622-626 (1996). Each of these experimental mammals with increased UCP 1 showed an increase in brown fat activity and energy expenditure, and a reduction in adiposity. Accordingly, determining mechanisms to increase UCPl has practical applications to the problem of obesity.

[0007] There are several important aspects of Ucpl expression. One is the molecular basis of its unique expression in BAT; and a second is the tightly controlled regulation by the hypothalamus via the sympathetic nervous system in response to cold and possibly diet. See A. Jacobsson et al, 1986. A third aspect is the cellular mechanisms that can increase the number of mitochondria in the adiopocyte; and a fourth is the cellular mechanisms that can increase the formation of new brown adipocytes. These mechanisms may not be mutually exclusive, i the rodent, obesity has been reduced by a high fat diet or by a mutant gene that increases the transcription of Ucpl in pre-existing brown adipocytes. This strategy is unlikely to succeed in humans because adult humans have lost the large number of brown adipocytes that were present at birth. It is unknown whether the brown adipocytes of newborn humans undergo apoptotic death or are converted to white adipocytes. Any effective increase in brown fat-based thermogenesis in adult humans must first increase or reactivate the brown adiopocytes. [0008] Many investigators of human obesity consider the number of brown adipocytes in the adult human to be too small to be effective as a mechanism for reducing obesity. See M.E. Lean et al, "Brown adipose tissue uncoupling protein content in human infants, children and adults," Clin. Sci., vol. 71, pp. 291-297 (1986). However, little is known about the capacity in humans for brown adipocyte expression, because in the absence of an appropriate stimulus a brown adipocyte will assume the morphology of a white adipocyte. Nonetheless, histological examination of post-mortem human fat depots always reveals the presence of small numbers of brown adipocytes. P. Huttunen et al, "The occurrence of brown adipose tissue in outdoor workers," Eur. J. Appl. Physiol, vol. 46, pp. 339-345 (1981). In addition, chronic exposure to high levels of catecholamines, e.g., secreted by a phaeochromocytoma tumor, in humans leads to a large increase in brown fat depots. M.E. Lean et al. , "Brown adipose tissue in patients with phaeochromocytoma," it. J. Obes., vol. 10, pp. 219-227 (1986). Human adipocytes in culture have also been stimulated to produce UCP 1 by treatment with thiazolidinediones, indicating that brown adipocytes are present in adult humans and that the number of brown adipocytes can be increased by β-adrenergic stimulation. See J.E. Digby et al, "Thiazolidinedione exposure increases the expression of uncoupling protein 1 in cultured human preadipocytes," Diabetes, vol. 47, pp. 138-141 (1998). Although brown adipoctyes were once though to be restricted to defined brown fat depots, recently the emergence of brown adipocytes in white fat depots was found in mice, rats, and dogs. The capacity to induce brown fat in white fat depots was found to be under genetic control and displayed a wide genetic variability, which also affected the ability of the mice to respond to drugs that reduce adiposity. See C. Guerra et al, "Emergence of brown adipocytes in white fat in mice is under genetic control. Effects on body weight and adiposity," J. Clin. Invest, vol. 102, pp.412-420 (1998). In mice, at least four genes were found that control this genetic variability, three of which conferred high levels of UCPl . The three gene locations were on Chromosomes 2, 3 and 8. See R.A. Koza etal, "Synergistic Gene Interactions Control the Induction of the Mitochondrial Uncoupling Protein (Ucpl) Gene in White Fat Tissue," J. Biol. Chem., vol. 275, pp. 34486-34492 (2000). [0009] The considerable body of information that has accumulated on the molecular basis of Ucpl expression in BAT indicates that in mice there is a 200 bp enhancer, located approximately 2.5 kb upstream of the transcription start site for mouse Ucpl. This enhancer is known to contain cw-acting elements that play a critical role in the regulation of mouse Ucpl expression. These cz^'s-acting elements include peroxisomal proliferator activator receptor binding motif (PPRE), thyroid hormone regulatory element /retinoic acid regulatory element (TRE/RARE), and cAMP responsive elements (CRE). See U.C. Kozak et al, "An Upstream Enhancer Regulating Brown-fat-specific Expression of the Mitochondrial Uncoupling Protein Gene," Mol. Cell Biol., vol. 14, pp. 59-67 (1994); LB. Sears et al, "Differentiation-dependent Expression of the Brown Adipocyte Uncoupling Protein Gene: Regulation by Peroxisome Proliferator-activated Receptor Gamma," Mol. Cell Biol., vol. 16, pp. 3410-3419 (1996); A.M. Cassard-Doulcier et al. , "In Nitro Interactions Between Nuclear Proteins and Uncoupling Protein Gene Promoter Reveal Several Putative Transactivating Factors Including Etsl, Retinoid X Receptor, Thyroid Hormone Receptor, and a CACCC Box-binding Protein," J. Biol. Chem., vol. 269, pp. 24335-24342 (1994); R. Rabelo et al, "Delineation of Thyroid Hormone-responsive Sequences Within a Critical Enhancer in the Rat Uncoupling Protein Gene," Endocrinology, vol. 136, pp. 1003-1013 (1995); and U.S. Patent Nos. 6,166,192 and 6,033,656. (See also, Table 1 for a listing of abbreviations used in the Specification and the Claims). Recently, it has been shown that synergism between retinoids, isoproterenol and thiazolidinedione regulates human Ucpl transcription in an enhancer region located 3.5 kb upstream of the gene. See M. del Mar Gonzalez-Barroso et al. , "Transcriptional Activation of the Human Ucp 1 Gene in a Rodent Cell Line. Synergism of Retinoids, Isoproterenol, and Thiazolidinedione Is Mediated by a Multipartite Response Element," J. Biol. Chem., vol. 275, pp. 31722-31732 (2000).

[0010] The control of expression of Ucpl has been studied with transient expression assays of reporter constructs in brown adiopocytes in tissue culture, suggesting the interaction of the transcription factors PPARγ, RXR, and PGC1 via the PPRE binding site. See U.S. Patent No. 6,166,192; see also Table 1 for definitions of abbreviations. Additional regulatory elements and transcription factors are likely to be involved. There is evidence that induction is initiated by norepinephrine action on G protein-coupled β_t and β₃ adrenergic receptors. See U.C. Kozak et al, "Adrenergic Regulation of the Mitochondrial Uncoupling Protein Gene in Brown Fat Tumor Cells," Mol. Endocrinol., vol. 6, pp. 763-772 (1992). It is also known that Pgcl mRNA levels are increased in BAT in response to cold exposure. See U.S. Patent No. 6,166,192; and P. Puigserver et al, "A Cold-inducible Coactivator of Nuclear Receptors Linked to Adaptive Thermogenesis," Cell, vol. 92, pp. 829-839 (1998). Thyroid hormones, retinoids, and thiazolidinediones (TZD) have been reported to increase transcription of the Ucpl in rodents, both in vivo and in vitro. See C. Guerra et al, "Triiodothyronine Induces the Transcription of the Uncoupling Protein Gene and Stabilizes its mRNA in Fetal Rat Brown Adipocyte Primary Cultures," J. Biol. Chem., vol. 271, pp. 2076-2081 (1996); P. Puigserver et al, "In Vitro and in Vivo Induction of Brown Adipocyte Uncoupling Protein (Thermogenin) by Retinoic Acid," Biochem. J., vol. 317, pp. 827-833 (1996) ; and J.E. Digby et al, (1998). [0011] hi mice, transient transfection analyses utilizing primary cell cultures from a SV40

T-antigen- induced brown adipocyte tumor have shown that mutations in two of four half-site CREs in a CAT-reporter construct carrying 3 kb of the 5'-flanking region almost completely abolished expression of Ucpl. See U.C. Kozak et al, 1994. These two sites, CRE2 and CRE4, were located in the enhancer region and just 5' of the TATA box region, respectively. Mutations to the other two sites, CREl and CRE3, only slightly reduced reporter activity. [0012] Recently, the human Ucpl gene was cloned and evidence describing key elements controlling its transcriptional regulation obtained. See M. del Mar Gonzalez-Barroso et al. , 2000. A 350 bp, hormone-sensitive region of the human gene showed significant (60.1%) similarity with the mouse BAT-specific enhancer element. This region in the human gene was able to bind the nuclear factors RARs, RXRs, CREB/ATF, and PPARγ, indicating that transcriptional regulation of the Ucpl gene in rodents and humans share mechanisms in common. It would be desirable to identify additional factors which could activate Ucpl expression and which could also promote an increase in both the number of brown adipocytes and the amount of BAT thermogenesis. NF-E2 Binding Proteins

[0013] NF-E2 was first discovered as a binding site in the beta-globin gene locus control region where the hematopoietic specific NF-E2 p45 subunit and the ubiquitously expressed small Maf protein, an important regulator of cell differentiation in various systems, form heterodimers. See P. Moi et al. , "Isolation of NF-E2-related Factor 2 (Nr£2), aNF-E2-like Basic Leucme Zipper Transcriptional Activator That Binds to the Tandem NF-E2/AP 1 Repeat of the Beta-globin Locus Control Region," Proc. Natl. Acad. Sci. USA, vol. 91, pp. 9926-9930 (1994); V. Blank et al, "The Maf Transcription Factors: Regulators of Differentiation," Trends Biochem. Sci., vol. 22, pp. 437-441 (1997); H. Motohashi et al, "Mesodermal- vs. Neuronal-specific Expression of MafK Is Elicited by Different Promoters," Genes Cells, vol. 1, pp. 223-238 (1996); and K. Igarashi et al. , "Regulation of Transcription by Dimerization of Erythroid Factor NF-E2 p45 with Small Maf Proteins," Nature, vol. 367, pp. 568-572 (1994). Transcription factors of the NF-E2 family, which were originally identified as having eryfhrocyte-specific DNA binding activity, belong to the cap'n'collar (CNC)-type basic region leucine zipper (bZIP) superfamily, which represents a class of transcription factors that bind DNA using a simple, dimeric, alpha-helical recognition motif. Cap'n'collar (CNC) is a homeotic gene involved in the development of the head and neck structure in Drosophila. See J. Mohler et al. , "Segmentally Restricted, Cephalic Expression of a Leucine Zipper Gene During Drosophila Embryogenesis," Mech. Dev., vol. 34, pp. 3-9 (1991); and J. Mohler et al, "Control ofDrosophila Head Segment Identity by the bZIP Homeotic Gene cnc, "Development, vol. 121, pp. 237-247 (1995); and P. Moi et α/.,1994. The nuclear DNA binding protein NF-E2 regulates expression of globin genes in developing erythroid cells. Two additional members of CNC-bZTP family, NFE211 and NFE212 (also known as NRF1 and NRF2), have been cloned; they are expressed ubiquitously in tissues, but with variable expression among these different tissues. See K. Chan et al, "NRF2, a Member of the NFE2 Family of Transcription Factors, Is Not Essential for Murine Erythropoiesis, Growth, and Development," Proc. Natl. Acad. Sci., vol. 93,ρp. 13943-13948 (1996);R. Yuetα/., "Activation of mito gen- activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism," J. Biol. Chem., vol. 275, pp. 39907-39913 (2000); and H.-C. Huang et al, "Regulation of the antioxidant response element by protein kinase C-mediated phosphorylation of NF-E2-related factor 2," Proc. Natl. Acad. Sci., vol. 97, pp. 12475-12480 (2000). NFE212 is a member of the CNC (cap'n'collar)-basic region leucine zipper (bZIP) superfamily.

[0014] The transcription factors, NFE212 and NFE211 (or NRF2 and NRFl) are not to be confused with the nuclear respiratory factors, NRF- 1 and NRF-2 which bind to DNA binding motifs (NRF-1 and NRF-2) in the regulatory regions of nuclear genes that encode proteins destined for the mitochondia. NRF-1 and NRF-2 are known to be different proteins. See J.V. Virbasius et al. , "Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: A potenital regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis," Proc. Natl. Acad. Sci., vol. 91, pp. 1309-1313 (1994); and Z. Wu et al, "Mechanisms controlling mitochondrial biogenesis and respiration through the termogenic activator PGC-1," Cell, vol. 98, pp. 115-124 (1999).

[0015] Protein-protein interactions between NFE212 and other bZIP proteins including

CREB, Fos, and Jun have been reported. See R. Venugopal et al. , "Nrf2 and Nrfl in Association with June Proteins Regulate Antioxidant Response Element-mediated Expression and Coordinated Induction of Genes Encoding Detoxifying Enzymes," Oncogene, vol. 17, pp. 3145- 3156 (1998); T. Nguyen et al, "Transcriptional Regulation of the Antioxidant Response Element," J. Biol. Chem., vol.275, pp. 15466-15473 (2000); andR. Venugopal etal, "Nrfl and Nrf2 Positively and c-Fos and Fral Negatively Regulate the Human Antioxidant Response Element-mediated Expression of NAD(P)H:quinone Oxidoreductasel Gene," Proc. Natl. Acad. Sci. U S A, vol. 93, pp. 14960-14965 (1996). PPARγ can also directly interact with NFE212 via the NF-E2/AP1 binding site (TGCTGATTCAT) of the thromboxane synthesis gene in macrophages. See Y. Hceda et al, "Suppression of Rat Thromboxane Synthase Gene Transcription by Peroxisome Proliferator-activated Receptor Gamma in Macrophages via an Interaction with NRF2," J. Biol. Chem., vol. 275, pp. 33142-33150 (2000). Also, in situ hybridization with sections from 15.5 -day-old embryos has shown that the Nfe2l2 gene is expressed in brown fat. See K. Chan et al, 1996.

[0016] NRf2 (NF-E2 -related factor 2) was discovered to be involved in the cellular response to oxidative stress. NRf2 is normally retained in the cytoplasm, and then is liberated in response to oxidative stress to translocate into the nucleus. See H.-C. Huang et al, 2000. NRF2 (or NFE212) has been shown to be phosphorylated by protein kinase C which triggers nuclear translocation of Nrf2 and increases the binding to the NF-E2 binding motif. Compounds known to activate protein kinase C are known to increase the concentration and binding of NRF2 in the nucleus, e.g., phorbol esters (e.g., phorbol 12-myristate 13-acetate), tert- butylhydroquinone, and β-naphthoflavone.

[0017] NRF2 has not been previously implicated as a transcription factor to affect Ucpl expression and increase BAT thermogenesis.

DISCLOSURE OF THE INVENTION

[0018] We have discovered a new method to treat obesity or other human disorders by increasing the degree of brown adipose thermogenesis, by both increasing the growth and differentiation of brown adipose tissue and increasing its activity. We have found a new use for the transcription factor NFE212 to increase BAT thermogenesis by increasing the expression of Ucpl. For the first time, NFE212 binding to the NF-E2 motif was found to stimulate the expression of Ucpl. By modulating the expression of the Nfe2l2 gene or the concentration of NFE212 protein, changes in brown adipose tissue thermogenesis can be achieved to treat various weight disorders. Additionally, the direct involvement of CREB binding to CRE2 to regulate Ucpl expression was demonstrated. Furthermore, transient transfection assays of luciferase reporter constructs and site-directed mutagenesis indicated that CRE2 was involved in transcriptional regulation of the Ucpl through interaction with a phosphorylated CREB. NFE212 can be combined with other known transcription factors, e.g., CREB, PGC1, RXR, RAR, and PPARγ, to increase BAT thermogenesis by increasing the expression of Ucpl.

Brief Description of the Drawings [0019] Fig. 1 illustrates the nucleotide sequence of the 221 bp (4828/5048) of BAT tissue specific region of the mouse Ucpl gene (SEQ ID No: 1), with the enhancer elements shown inside boxes and the NF-E2 binding site shown as underlined bold letters. [0020] Fig.2A illustrates the binding of half site CRE sequences to nuclear extracts from various tissues of A/J mice exposed to cold (4°C overnight) in an autoradiogram using ³²P end- labeled CRE2 from mouse Ucpl gene, with arrows indicating the bands representing CREB (dark arrow) and free probes (light arrow).

[0021] Fig. 2B illustrates the competitive binding activity of CRE2 with half-site CREs from mouse Ucpl gene from nuclear extracts from BAT of A/J mice which were exposed to cold (4°C overnight), and a palindromic CRE from a somatostatin gene (CRE) in an autoradiogram showing only the CREB bands, with the percent competition of ³²P end-labeled CRE2 to CREB by a CRE sequences from mouse Ucpl and somatostatin genes calculated from the radioactivity of the slow migrating bands in the lane without (first lane) and with individual competitors

(calculations shown at the bottom).

[0022] Fig. 3 A illustrates a Western blot analysis showing the increase in CREB/ATFl phosphorylation in HIB-1B cells treated with norepinephrine (NE) for 0, 5, 10, 20, 30 and 60 minutes.

[0023] Fig. 3B illustrates the increase in CRE2 binding to nuclear proteins in HIB-1B cells treated with norepinephrine for 0, 10, and 60 min; and compared with intensity of BAT of

A/J mouse (cold, overnight) and for competition with cold CRE2. The arrows indicate CREB bands.

[0024] Fig.4 illustrates transient expression analyses of luciferase reporter constructs to determine the function of individual CREs, (CREl, CRE2, CRE3 and CRE4, represented by ovals) and of mutations of CRE2 and CRE3 (open ovals with cross marks).

[0025] Fig. 5 A illustrates the binding of NF-E2 sequences to nuclear extracts from HH3-

1B cells, in an autoradiogram using ³²P end-labeled NF-E2 (0.1 pmoles) from mouse Ucpl gene, and using nuclear extracts prepared from HIB-IB cells with (+) or without (-) norepinephine, with the bands representing NFE212 indicated with an arrow on the right.

[0026] Fig. 5B illustrates the binding of NF-E2 sequences to nuclear extracts from BAT of A/J mouse (cold, overnight) in an autoradiogram showing only the NFE212 bands using ³²P end-labeled NF-E2 (0.1 pmoles) with an antibody or a competitor.

[0027] Fig. 5C illustrates the increase in binding activity of NF-E2 sequence in BAT of

A/J mouse under cold stress in an autoradiogram showing only the NFE212 bands (indicated by arrow) using ³²P end-labeled NF-E2 probe.

[0028] Fig. 6 A illustrates the nucleotide sequences for the combined probe of NFCRE containing both NF-E2 and CRE2 binding sites (SEQ ID NO: 2), with the enhancer elements shown with underlined bold letters (NF-E2) or a box (CRE2), and with the nucleotide sequences for cold probes for NF-E2 (SEQ ID NO: 3) and CRE2 (SEQ ID NO: 4).

[0029] Fig. 6B illustrates the binding activity of NFCRE, NF-E2 and CRE2 with nuclear extracts from HIB-IB cells (exposed to norephinephrine for 60 min), using a ³²P end-labeled

NFCRE (SEQ ID NO: 2) , NF-E2 (SEQ ID NO: 3), and CRE2 (SEQ m NO: 4) probes. [0030] Fig. 7A illustrates thelOO bp (-3762Λ3662) of nucleotides sequences from the human 350 bp enhancer (SEQ ID NO. 5) and corresponding mouse (SEQ ID NO. 16) and rat (SEQ ID NO. 17) enhancer sequences, with the half-sites for ATF/CREB, a putative NF-E2 binding site, and PPRE labeled and shown within boxes, and with the similar bases among the three species shown in bold letters.

[0031] Fig.7B illustrates the binding of human NF-E2 sequences to nuclear extracts from

BAT of A/J mouse kept in RT or cold (4°C) for 7 days, using a ³²P end-labeled NF-E2 probe with competitors as indicated. Only the NFE212 bands are shown (arrow).

[0032] Fig. 8A illustrates the effect of NFE212 overexpression on the transcriptional activity of the luciferase reporter constructs containing mouse Ucpl NF-E2 using the luciferase reporter construct pGL3/3.1kb, that was cofransfected with expression vector which is empty (pCMV/tagl , open box), contains cDNA for Nfe2l2 correct (closed box), or contains cDNA for Nfe2l2 with reversed orientation (hatched box) into HIB-IB cells, incubated in medium with either cAMP, norepinephrine (NE), or troglitazone (Trog) for an additional 16 hrs. [0033] Fig. 8B illustrates the effect of NFE212 overexpression on the transcriptional activity of the luciferase reporter constructs containing mouse Ucpl NF-E2 using the luciferase reporter construct pGL3/CRE4pro/220, that was cofransfected with expression vector which is empty (pCMV/tagl, open box), contains cDΝA for Nfe2l2 correct (closed box), or contains cDΝA for Nfe2l2 with reversed orientation (hatched box) into HIB-IB cells, incubated in medium with either cAMP, norepinephrine (ΝE), or troglitazone (Trog) for an additional 16 hrs. [0034] Fig. 9 illustrates the effect of ΝFE212 overexpression on the transcriptional activity of the luciferase reporter constructs containing rat Ucpl NF-E2 using the luciferase reporter constructs (pGL/cre4pro/Rat221 and pGL/cre4pro/mRat221), that were cofransfected with expression vector which is empty (pCMV/tagl, Stratagen) or contains cDΝA for Nfe2l2 into HIB-IB cells, incubated in medium with cAMP for an additional 16 hrs. [0035] Fig. 10A illustrates the binding of ΝF-E2 sequences to nuclear extracts from BAT of B6 mice of different ages (from 19 days of gestation to 4 months after birth) in an autoradiogram showing only the NFE212 bands using ³²P end-labeled NF-E2 probe (0.1 pmoles) with an antibody or a competitor, after separation on 6% non-denaturing acrylamide gel. [0036] Fig. 10B illustrates a Western blot analysis forNFE212 from nuclear extracts from

BAT of B6 mice (ages from 19 days of gestation to 3 months after birth), using a specific antibody for NFE212 and after separation on a 10% SDS-polyacrylamine gel. [0037] Fig. 10C illustrates the binding of NF-E2 sequences to nuclear extracts from BAT of B6 mice at room temperature or a 4 C for 1 day or 7 days in an autoradiogram showing only the NFE212 bands using ³²P end-labeled NF-E2 (0.1 pmoles) with an antibody or a competitor, after separation on 6% non-denaturing acrylamide gel.

MODES FOR CARRYING OUT THE INVENTION Example 1 Materials and Methods

Cell culture and transfection: [0038] HIB-IB cells, obtained from the Dana-Farber Cancer Insitute (Boston,

Massachusetts), were maintained in Dulbecco' s modified Eagle' s medium (DMEM, 4,500 mg/L D-glucose, 584 mg/L L-glutamine, and 15 mg/L phenol red, GIBCO, InvitrogenCorp., Carlbad, California) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 0.1 mM non-essential amino acids. HIB-IB is an immortalized brown adipose cell line from hybridoma tissue that expresses Ucpl in response to retinoic acid and β-adrenergic agonists such as norepinephrine and isoproterenol. Medium was changed every two days. Reporter constructs were transiently transfected into HIB-IB cells using Lipofectamine Plus reagent (GIBCO) according to the manufacturer's protocol. The day before transfection, 2xl0⁵ cells were seeded into a 24- well cluster dish (Corning Inc., Acton, Massachusetts). Briefly, 0.5 μg of reporter construct was transfected with 50 ng of pRL/SV40 (Promega Corp., Madison, Wisconsin), a plasmid containing Renilla luciferase gene under control of SV40 promoter, in a mixture of PLUS and Lipofectamine reagent. For the co-expression experiment, each 0.3 μg of reporter construct and expression vector was transfected with 50 ng of pRL/SV40. Transfected cells were cultured in medium in the presence or absence of 1 mM of norepinephrine (Sigma) or 0.5 mM 8-Br-cAMP (Calbiochem-Novabiochem Corp., San Diego, California) for 16 hrs. Cell extracts were prepared, and the activity of both Photinus and Renilla luciferase was determined using the dual- luciferase reporter assay system (Promega). For each construct, the activity of the Photinus luciferase was divided by the activity of the Renilla luciferase to correct for transfection efficiency. Under each treatment, the corrected activity was again divided by activity from the empty vector, pGL3 /basic (Promega), to estimate the degree of increase for each construct. The degree of increase for the over-expression experiment was obtained by dividing the corrected activity by the empty vector (pCMV/tag). Each experiment was performed in duplicate dishes.

Subcloning of ^'5 '-flanking region of the mouse Ucpl gene and reporter constructs: [0039] The 3.1 kb of 5 '-flanking region containing the four cAMP responsive elements

(CRE) and the 220 bp BAT specific enhancer of the mouse Ucpl gene were obtained by PCR amplification. The 3.85 kb BgK fragment in pGEM, which was previously used in our characterization of Ucpl (note that nucleotide positions correspond to those in the Ucpl gene as described in GenBank U63418), was used as a template with forward and reverse primers, 5'- ggggtaCCGTGCACACTGCCAAATCATCTC (SEQ ID NO: 6) (4379/4355, anewXpnl siteis underlined) and 5'-gggaeCTCCTGCAGAGCCACCTGGGCTAGG (SEQ ID NO: 7) (7514/7538, a new Sαcl site is underlined), respectively, and subcloned into pGL3/basic using the Kpnl and Sacl restriction enzyme sites. See Kozak et al , 1994. To obtain the Ucpl promoter with or without CRE4, forward primers 5'-ggggatccGAGTGACGCGCGGCTGGG (SEQ ID NO: 8) (nucleotide sequences for CRE4 are shown as bold and a new BamHΪ site is underlined, 7261/7278) or 5'-ggggatcCGGCTGGGAGGCTTGCGCA (SEQ ID NO: 9) (anew BamHL site is underlined, 7271//7289) and reverse primer 5'-gggaagcttGGGCTAGGTAGTGCCAG (SEQ ID NO: 10) (a new HindlH site is underlined, 7504/7520) were used for PCR amplification and subcloned into pGL3/basic using BgKL and HindUL restriction enzyme sites. For the 220 bp of BAT specific enhancer region, the 3.85 kb BgK fragment was PCR amplified using primers 5'- ggggagCTCCTCTACAGCGTCACAGAGG (SEQ ID NO: 11) (Sαcl site is underlined, 4841/4862) and 5'-gggctogagAGTCTGAGGAAAGGGTTGA (SEQ ID NO: 12) (anewXhoϊ site is underlined, 5025/5045) and subcloned into luciferase reporter construct containing the Ucpl promoter. The structure of each fragment was verified by DNA sequencing. Nfe2l2 cDNA was kindly provided by Dr. Paul Ney (St. Jude Children' s Research Hospital, Memphis, Tennessee). A Nfe2l2 expression vector was made by cloning a Notl fragment into pCMN/tagl (Stratagene, La Jolla, California).

Site-directed mutagenesis for CRE2 and CRE3 [0040] CRE2 and CRE3 sequences in the 220 bp of B AT-specific-enhancer region were mutated using PCR and subcloned into the luciferase reporter plasmid, pGL3/basic. For CRE3 the forward primer was 5'-ggggagCTCCTCTACAGC α CAGAGG (SEQ ID NO: 13) (CRE3 shown in bold with lowercase italic letters which represent mutations; a new Sαcl site is underlined, 4841/4862) and the reverse primer was 5'-gggctcgagAGTCTGAGGAAAGGGTTGA

(SEQ ID NO: 12) (a nowXJioI site is underlined, 5025/5045). To mutate CRE2, two pairs of primers were required in separate amplifications. The first pair was 5'- ggggagCTCCTCTAC AGCGTCACAGAGG (SEQ ID NO: 11) (forward primer, a new Sad site is underlined, 4841/4862) and 5'-AGTGGAAAGGTtc GACTAGTTCAG (SEQ ID NO: 14)

(reverse primer, CRE2 is shown in bold with lowercase italic letters representing mutations,

4883/4907). The second pair was 5'-CTGAACTAGTC «ACCTTTCCACT (SEQ ID NO: 15)

(forward primer, CRE2 is shown in bold with lowercase italic letters representing mutations,

4883/4907) and 5'-gggctcgagAGTCTGAGGAAAGGGTTGA (SEQ ID NO: 12) (reverse primer, a new Xl ol site is underlined, 5025/5045). To generate the 220 bp of enhancer region with mutations in CRE2, aliquots (1 ul of each 50 ul PCR reactions) of the two PCR products were mixed and subjected to PCR amplification using primer pairs for intact 220 bp BAT specific enhancer region. The resulting mutations were confirmed by sequencing. To mutate both CRE2 and CRE3, the 220 bp fragment, which contains the mutation in CRE2, was subjected to PCR amplification using primer pairs described above to generate the CRE3 mutation: 5'- ggggagCJCCTCTACAGOs«ACAGAGG (SEQ ID NO: 13) (CRE3 shown in bold with lowercase italic letters which represent mutated sites; a new Sαcl site is underlined, 4841/4862) and 5'-gggctcgagAGTCTGAGGAAAGGGTTGA (SEQ ID NO: 12) (a new Xliol site is underlined, 5025/5045). After the mutations were verified by sequencing, the DNA fragments containing the mutated sites in CRE2 and/or CRE3 were subcloned into luciferase reporter plasmid containing Ucpl promoter with CRE4.

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA):

[0041] Nuclear extracts from various tissues of A/J mice (The Jackson Laboratory, Bar

Harbor, Maine) and HIB-IB cells were prepared as described in J.D. Dignam et al, Nucleic Acids Res., vol. 11, pp. 1475-1489 (1983), except that phosphatase inhibitors cocktail 1 and 2 (Sigma, St. Louis, Missouri) were added. The protein concentration was determined by the Lowry method using BSA as a standard as described in O.H. Lowry et al, J. Biol. Chem., vol. 239, pp. 18-30 (1964). To prepare probes for EMSA, single-stranded oligonucleotides were synthesized and purified (Operon Technologies, Inc., Alameda, California). 200 pmol each of the complementary oligonucleotides were annealed in 100 ul containing 100 mM NaCl to obtain a double-stranded probe. Five μg of nuclear extract were incubated initially for 10 min at room temperature in 29 μl containing 20 mM HEPES (pH 7.9), 100 mM KC1, 0.1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 1.5 μg of poly(dA-dT), and 5 mM MgCl₂. The mixture was then incubated for an additional 20 min after adding ³²P-labeled probe (4X10⁵ cpm/ul) with or without an unlabeled competitor or antibody for the supershift analysis. The antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California). The reaction was electrophoresed on a 6% polyacrylamide gel (Bio-Rad Laboratories, hie, Hercules, California) in 0.5X TBE buffer. The gel was then dried and exposed to a Phosphorfrnage screen. The radioactivity was visualized and quantified using Phosphorhnager and hnageQuant software from Molecular Dynamics (Sunnyvale, California).

Western blot analysis: [0042] Western blot analyses were performed with minor modifications as described in

U.K. Laem li, Nature, vol.227, pp. 680-685 (1970); andH. Towbinetα/., Proc. Natl. Acad. Sci. USA, vol. 76, pp. 4350-4354 (1979). Cell lysates from HIB-IB cells were prepared by adding SDS sample buffer containing 62.5 mM Tris-Cl (pH 6.8), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.1% w/v bromphenol blue with 1% v/v phosphatase inhibitor cocktail 1 and 2. All chemicals were from Sigma unless otherwise indicated. After cell lysates were separated on 8% SDS-polyacrylamide gel, protein was transferred onto a nitrocellulose membrane (Millipore Corp., Bedford, Massachusetts). The blots were then incubated with rabbit antibody against CREB (1:1,000 dilution, Santa Cruz Biotechnology), or phospho-CREB (Serl33, 1:1,000 dilution, New England Biolabs, Beverly, Massachusetts), overnight at 4°C with gentle agitation, followed by incubation with donkey anti-rabbit IgG as a secondary antibody (horseradish peroxide conjugated, Amersham Pharmacia Biotech, Piscataway, New Jersey). Bands were visualized by using the enhanced chemiluminescence reagent (Amersham Pharmacia Biotech) and exposed to X-Omat film (Eastman Kodak Co., Rochester, New York).

Example 2 Identification of CRE sequences for the binding of CREB

[0043] Four potential CRE sites are located in the 5' flanking region of Ucpl (Table 2).

All four CREs have half-site consensus sequences (CGTCA). Evidence that these half-sites are involved in the regulation of Ucpl is limited to loss of CAT reporter activity in transient expression assays in a BAT cell line. From this prior analysis CRE2 and CRE4 appeared to be essential; mutations to CREl showed no loss of expression and mutations to CRE3 only slightly reduced expression. See Kozak et al, 1994. This experiment was designed to establish the function of CRE2 located in the upstream enhancer. A CRE2 probe for EMSA was made with 5 bp of half-site CRE2 (CGTCA) flanked by 14 bp of 5' and 3'-flanking sequences as shown in Fig. 1. Fig. 1 shows the nucleotide sequence of the 221 bp (4828/5048) of BAT specific region of the mouse Ucpl gene, with the enhancer elements shown within boxes and the NF-E2 binding site underlined and in bold letters.

Table 2. Synthetic double-stranded CRE sequences used for electomobility shift assay. Each DNA containing half-site CRE motif (CGTCA) from mouse Ucpl or palindromic sequences from somatostatin gene was annealed as described under "Example 1." Consensus sequences for CRE are underlined (lowercase letters represent mutations).

Name Location Sequences SEQ ID NO:

CREl 4419/4437 TTATAGTGCCGTCACTAAC SEQ ID NO: 18 AATATCACGGCAGTGATTG

CRE2 4884/4902 TGAACTAGTCGTCACCTTT SEQ ID NO: 19 ACTTGATCAGCAGTGGAAA

CRE3 4843/4861 CCTCTACAGCGTCACAGAG SEQ ID NO: 20

GGAGATGTCGCAGTGTCTC

CRE4 7258/7276 TGGGAGTGACGCGCGGCTG SEQ ID NO: 21 ACCCTCACTGCGCGCCGAC

CRE (Somatostatin) TTGGCTGACGTCAGAGAGA SEQ ID NO: 22 AACCGACTGCAGTCTCTCT mlCRE2 AACTAGTCtgaACCTTT SEQ ID NO: 23 TTGATCAGactTGGAAA m2CRE2 AACTAtgaGTCACCTTT SEQ ID NO: 24 TTGATactCAGTGGAAA

[0044] Fig. 2A is an autoradiogram of an EMSA using ³²P end-labeled CRE2 (0.1 pmoles) from mouse Ucpl gene. Each lane (except for liver which was 1/10^th of the reaction) was loaded with a binding reaction containing 5 μg of nuclear extracts from A/J mice exposed to cold (4°C, overnight) with 2 pmoles of cold probe or antibody (1 μl) as indicated on 6% non- denaturing acrylamide gel. Slowly migrating bands representing CREB (dark arrow) and free probes (light arrow) are indicated on the right. However, probes prepared from the region just downstream of the CRE2 motif failed to form a similar retarded band (data not shown). The complex from liver was ~ 10-times stronger than that of other fat tissues (loading for liver was 1/10^th of the reaction; the second retarded band in liver is non-specific and can be seen with other probes, data not shown). Nuclear extracts, prepared from brown adipose tissue (BAT), retroperitoneal fat tissue (RP), inguinal fat tissue (IG), and liver of A/J mice kept in the cold (4°C) overnight, showed a maj or retarded band that was eliminated by competition with a 20-fold excess of cold CRE2 (specific shifted bands are shown with the dark arrow in Fig. 2A). [0045] To further characterize the binding sites of CRE2, competitive binding assays were performed with the same mutation, GTC to TGA, in two contiguous locations in the sequence. For the mlCRE2 probe, the mutation occurs in the middle of half-site CRE motif, while the m2CRE2 mutation only overlaps the first C in CRE motif (Table 2). In competitive EMSA, the mlCRE2, but not the m2CRE2 mutant oligonucleotide, lost the ability to compete with the labeled CRE2 probe (Fig. 2A). This indicates that the half-site CRE motif, but not the flanking 5' region, is active in binding the specific factor(s).

[0046] To identify the nuclear factor(s) which binds to CRE2, specific antibodies against

Fos, Jun, CBP, or CREB/ATF1 were applied in an EMSA reaction. Because of the sequence similarity of CRE and AP-1 binding sites for the Jun/Fos heterodimer (palidromic CRE, TGACGTCA; palindromic AP-1, TGA^e/_GTCA; half-site CRE sequences are shown underlined) and the known interaction between CREB and CBEB binding protein (CBP), the antibodies were tested in a super shift assay. The data (Fig. 2A) demonstrated both that the factors that bind to CRE2 arepart of the CREB/ATFl family, and that CREB/ATF1 does not interact with eifher/^'wn a άfos or CBP.

[0047] To quantify binding of the four half-site CREs to CREB/ATF 1 , the ability of each

CRE to compete with the CRE2 probe that binds to CREB/ATFl as described in Table 2 was measured. Fig. 2B shows an autoradiogram of an EMSA showing only the CREB bands. Each lane was loaded with a binding reaction containing 0.1 pmoles of ³²P end-labeled CRE2, 5 μg of nuclear extracts from BAT of A/J mice which were exposed to cold (4°C, overnight), and 0.4 pmoles of cold competitors as indicated on the top of Fig.2B. Percent competition of ³²P end- labeled CRE2 to CREB by a CRE sequences from mouse Ucpl and somatostatin (named CRE) gene was calculated (shown at the bottom) from the radioactivity of the slow migrating bands in the lane without (first lane) and in the lane with the individual competitors. Most of the labeled CRE2 probe complexed with proteins in nuclear extracts (as illustrated in Fig.2A) disappeared with a 40-fold excess (4 pmoles) of cold probe (data not shown). Under these conditions, palindromic CRE from somatostatin gene competes better than CRE2 itself (percent competition of 58.1% versus 29.1% in Fig. 2B) as expected. As shown in Fig. 2B, all the half-site CREs showed competition to CRE2 binding. This competition data together with the interference on probe binding upon addition of anti-CREB antibody indicates that CRE2 is a high affinity binding site that is enhanced by binding with CREB/ATF. The EMSA evidence for CRE2- CREB interactions corroborated the expression data to lead to the conclusion that CRE2 interacts with CREB to directly regulate Ucpl expression.

[0048] To test for a nuclear complex involving CBP or other CREB partners, a supershift assay was performed using nuclear extract from BAT of cold exposed A/J mouse (Fig.2A). Other than the strong interference in band formation found with CREB antibodies, there was no evidence that antibodies against CBP, c-Jun or c-Fos interfered with the interaction of the CRE2 probe with nuclear proteins in EMSA. Consistent with the lack of effects of these antibodies, a yeast two-hybrid screening for cDNA (4X10⁷ transformants were screened) from BAT of A J mouse with CREB as a bait, failed to detect positive clones (Data not shown) . This is conclusive evidence that CRE2 is a major site for the transcriptional activation of Ucpl expression by direct interaction with homodimers of CREB.

Example 3

Changes in CREB/ATFl phosphorylation and binding to CRE2 in response to norepinephrine

[0049] To assay for changes in CREB/ATFl binding to CRE2 in response to norepinephrine, HIB-IB cells were treated with fresh medium (control) or medium containing 1 mM norepinephrine (NE) for 0, 5, 10, 20, 30 and 60 minutes. Cell lysates were prepared and analyzed by a Western blot with phospho-CREB (Serl 33) specific antibody. The arrows on the right indicate the location of phosphorylated CREB (pCREB) and ATF 1 (p ATFl) with molecular weights of 43 KDa and 35 KDa, respectively. The treatment of HIB-IB cells with 1 mM norepinephrine significantly increased phosphorylation of both CREB and ATFl over a 60 min time course, whereas only a modest increase occurred with a change of culture medium (Fig.3 A). [0050] CRE2 binding to CREB/ATFl factors from HIB-IB cells was confirmed and quantified with EMSA using nuclear extracts from HIB-IB cells treated with 1 mM norepinephrine (NE). In Fig. 3B, nuclear extracts were isolated from HIB-IB cells treated with 1 mM of NE for 0, 10, and 60 min. Each lane was loaded with a binding reaction containing 5 μg of nuclear extracts and 0.1 pmoles of ³²P end-labeled CRE2 as indicated on the top. A lane for nuclear extracts from BAT of A/J mouse (cold, overnight) was added to compare intensity (lane BAT). Cold probe (2 pmoles) was added in the reaction for the competition (lane CRE2). Only the CREB bands are shown with arrows in Fig. 3B. The major thick band migrated to the same position on the gel as the single band from nuclear extract of cold exposed BAT of A/J mice (right lane). Treatment of HIB-IB cells with norepinephrine (1 mM) increased the intensity of the four retarded bands 20.6% and 24.2% after 10 and 60 min, respectively (from mean of 2 experiments). As shown in Fig. 3B, nuclear extracts from HIB-IB cells showed at least four retarded bands that were specifically removed with an excess of cold CRE2 (shown with arrows at right). These results indicate that norepinephrine (NE) induces phosphorylation and binding of CREB/ATFl proteins to CRE2.

[0051] As shown in Fig. 3 A, norepinephrine treatment in HIB-IB cell dramatically increased the phosphorylation of both CREB and ATFl within 5 min, followed by an increased binding of dimerized CREB or CREB/ATFl heterodimers to ³²P end-labeled CRE2 probe in EMSA (Fig. 3B). Similarly, the EMSA data showing an increase in complex formation with nuclear extracts from cells incubated with 1 uM NE indicated that phosphorylation of CREB and ATFl increase their binding affinity to CRE in Ucpl gene.

Example 4 Functional Characterization of CRE 1-4 [0052] To further characterize the functionality of CREs, a transient transfection assay was performed using luciferase reporter constructs and site-directed mutagenesis. The same site- directed mutations were introduced into CRE2 and CRE3 in the 220 bp of BAT-specific enhancer region as present in the probes used in the competitive EMSA (Table 2), since changes from GTC to TGA (mlCRE2 probe in Fig. 2A) eliminated the capacity of the oligonucleotide to compete with CRE2 probe. [0053] Luciferase reporter constructs (named in the left) were generated by subcloning the various fragments from 5' flanking region of mouse Ucpl gene into pGL3/basic vector (Promega). DNA fragments from mouse Ucpl gene are shown in Fig. 4 as thick lines with the position of individual CREs indicated as ovals. Mutations of CRE2 and/or CRE3 by mutating key nucleotide residues as described under Example 1 are indicated with open ovals with X marks. Each construct was transfected into HIB-IB cells with pRL/SV40 vector (Promega), and the cells were cultured under the medium containing 1 uM of norepinephrine (NE) or 0.5 mM of 8-Bro-cAMP (cAMP) another 16 hrs. Luciferase activity was measured from cell lysates using Dual-Luciferase assay system (Promega), and a fold increase of luciferase activity by NE or cAMP was calculated. Data is presented as the means and standard deviations of fold increase from three experiments. The restriction map shown at the top indicates the position of the restriction enymes — HindUL (H) ; Xbal (X); and BgK (B).

[0054] The promoter without CRE4 (pGL3/pro) had low basal promoter activity (Fig.4).

Addition of CRE4 (pGL3/CRE4pro) to the promoter construct showed about a 3-fold increase in luciferase activity in response to NE and cAMP. This level of transient expression was similar to that of the promoter construct containing 220 bp of BAT-specific enliancer region, but without CRE4 (pGL3 /pro/220). Importantly, the 220 bp of BAT-specific enliancer region together with CRE4 (pGL3/CRE4pro/220) showed a level of expression activity similar to the 3.1 kb of 5'- flanking region of Ucpl (pGL3/3.1 kb). This data suggests that CRE4 cooperates with the 220 bp BAT-specific enliancer region in determining the response to NE and cAMP. When each of CRE2 or CRE3 independently or together in pGL3/CRE4pro/220 were mutated to evaluate the contribution of CRE2 and CRE3 to the enhancer activity, the expression was diminished in assays with the mutant constructs, but it was less dramatic than had been previously observed with amore differentiated BAT cell line. See U.C. Kozak, et al. , Mol. Cell Biol., vol. 14, pp.59- 67 (1994).

[0055] An important conclusion that emerged from this analysis is the requirement for interactions between elements in the distal enhancer with CRE4 in the proximal promoter to confer high levels of expression. Overall, the results confirmed a major role for CRE2 in the enhancer activity. Example 5

NFE212 binds NF-E2 binding sites in the Upstream Enhancer of Mouse Ucpl [0056] A consensus NF-E2 binding motif, ACTAGTCGT, has been identified that partially overlaps the CRE-2 half-site in mice and is located 6 bp downstream of the peroxisomal prohferator activator receptor binding motif (PPRE)(Fig 1). A probe containing 10 bp of the NF- E2 binding motif with 3 bp of nonspecific flanking sequence (CCC) (SEQ ID NO: 2) was synthesized and incubated with nuclear extracts from HIB-IB cells (Fig 5A). Fig. 5A shows an autoradiogram of an EMSA using ³²P end-labeled NF-E2 (0.1 pmoles) from mouse Ucpl gene. Nuclear extracts were prepared from HIB-IB cells with (+) or without (-) NE (1 uM, 60 min). Each lane was loaded with a binding reaction containing 5 μg of nuclear extracts with 2 pmoles of cold probe or antibody (1 μl) as indicated in the figure on 6%o non-denaturing acrylamide gel. Slowly migrating bands representing NFE212 are indicated with an arrow on the right. Nuclear extracts from HIB-IB cells (extracted as described in Example 1) interacted with probes to the NF-E2 binding site (SEQ ID NO: 3) from mouse Ucpl gene to generate shifted bands that were eliminated in a competition assay with a 20-fold excess of cold probe (Fig 5 A). Nuclear extracts from the HIB-IB cells treated with 1 mM norepinephrine for 30 minute increased the intensity of the complex as did nuclear extracts prepared from brown adipose tissue of cold exposure mice (Fig. 5C). Fig. 5C illustrates that the binding activity of NF-E2 sequence is increased by cold exposure in BAT of A/J mouse. Nuclear extracts were isolated from BAT of A/J mouse from kept in RT or cold (4°C) for 7 days. 5 μg of nuclear extracts were incubated with ³²P end-labeled NF-E2 probe (0.1 pmoles), and then separated on 6% non-denaturing acrylamide gel. Only the NFE212 bands are shown with an arrow.

[0057] CRE2 probes (SEQ ID NO : 4) that interact with CREB showed a band shift with . a different mobility than the NF-E2 probe (Fig. 6B, below). When the ability of antibody against members of NF-E2 binding factors, including NF-E2 p45, NFE211 andNFE212, to interfere with the band shift was assessed, only the antibody from NFE212 interfered with the shifted bands (Fig. 5 A). In order to confirm the binding activity of the NFE212 from brown adipose tissue with NF-E2 binding sites, the same amount (5 μg) of nuclear extracts from BAT of A/J mouse were incubated with the NF-E2 probe. The effects of cold NF-E2 and antibody to NFE212 on the bands in the EMSA were very similar to that observed with extracts from HIB1B cells (Fig. 5B) [0058] Transcription factors of the NF-E2 family, which were originally identified as an erythrocyte-specific DNA binding activity, belong to the cap'n'collar (CNC)-type basic leucine zipper (bZDP) as described in P. Moi et al, Proc. Natl. Acad. Sci. USA, vol. 91, pp. 9926-9930 (1994). Protein-protein interactions between NFE212 and other bZIP proteins including, CREB, Fos, and Jun have been reported. PPARg can also directly interact with NFE212 via the NF- E2/AP1 binding site (TGCTGATTCAT) of the thromboxane synthesis gene in macrophages. Given the putative role for these transcription factors in Ucpl regulation, possible interactions between these transcription factors and NF-E2 in the Ucpl enhancer were evaluated by determining whether antibodies to these transcription factors interfere with the EMSA of the NF- E2 probe. Fig. 5B shows an autoradiogram illustrating the binding of NF-E2 sequences to nuclear extracts from BAT of A/J mouse (cold, overnight). Each lane was loaded with a binding reaction of ³²P end-labeled NF-E2 (0.1 pmoles) incubated with 5 μg of nuclear extracts from BAT of A/J mouse (cold, overnight) with 2 pmoles of cold probe or antibody (1 ul) as indicated on 6% non-denaturing acrylamide gel. Only the NFE212 bands are shown. Only antibodies to NFE212 interfered with the complexes between NF-E2 probes and nuclear factors from BAT (Fig. 5B).

[0059] These experiments demonstrate that the transcription factor NFE212 induced the expression of Ucpl in mice. The gene for NFE212 in mice is located on Chromosome 2. See M.P. Marcias etal. . Leukoc. Biol., vol. 67, pp. 567-76 (2000); andF. Yehiely etal, Neurobiol. Dis., vol. 3, pp. 339-55 (1997); and the Mouse Genomics Informatics website of The Jackson Laboratory, Bar Harbor, Maine (http://www.informatics.jax.org). Chromosome 2 in mice is the known site of a locus that increases the capacity to induce brown adipocytes in white adipose tissue, and to induce the expression of Ucpl. See, C. Guerra et al, 2000.

Example 6 Competition between NFE212 and CREB [0060] An overlap of the binding motif of NF-E2 with the half site CRE2 in mice suggested that competition for binding may exist between NFE212 and CREB. To test this, a 19 bp oligonucleotide probe, NFCRE, (SEQ ID NO: 2) was designed which covered both NF-E2 and CRE2, for a gel shift and super shift assay. Fig. 6A shows the nucleotide sequences for NFCRE containing both NF-E2 and CRE2 binding sites, with the enhancer elements shown with either underlined bold letters (NF-E2) or box (CRE2). The nucleotide sequences for cold probes for NF-E2 (SEQ ID NO: 3) and for CRE (SEQ ID NO: 4) are shown with underlined bold letters and the 3 bp of flanking sequences.

[0061 ] Fig. 6B illustrates the binding activity of NFCRE, NF-E2 and CRE2 with nuclear extracts from HIB-IB cells. Each lane was loaded with a binding reaction containing 5 μg of nuclear extracts (HIB-IB cells, lμM NE for 60 min for the treatment) with different concentrations of cold probe (2 pmole, 0.2 pmole) as indicated on 6%> non-denaturing acrylamide gel. Slowly migrating bands representing CREB and NFE212 complex with ³²P end-labeled probe are shown. The band shifts with the NFCRE probe were very similar to the pattern observed for CRE2, whereas the band shift with the NF-E2 probe migated slightly faster. The NF-E2 band that should have been formed with the NFCRE probe was not detected. Both cold CRE2 and NFCRE was able to compete with the NFCRE probe. However, NF-E2 could not compete away the band shifts with either CRE2 or NFCRE probe, but could with the NF-E2 probe. These findings indicated that CREB binds to CRE2 with a high affinity, and this CREB- CRE2 binding interfered in a competitive manner with the binding of NFE212 to the NF-E2 motif. However, since this overlap does not exist in the human gene (see below in Example 7), the competition between CREB and NFE212 probably does not occur. This suggests that in humans NFE212 may be more efficacious.

Example 7 Human Ucpl gene contains NF-E2 binding sites [0062] Recently, the human Ucpl gene was cloned and evidence describing key elements controlling its transcriptional regulation obtained. See M. del Mar Gonzalez-Barroso et al. , 2000. A 350 bp hormone-sensitive region of the human gene showed significant similarity with the mouse (60.1 %) and rat (62.5%) BAT-specific enhancer element. This region in the human gene was able to bind the nuclear factors, RARs, RXRs, CREB/ATF, and PPARγ indicating that transcriptional regulation of the Ucpl gene between rodents and human have mechanisms in common. A comparison of 100 bp (-3762/-3662 of human) of the human, rat, and mouse Ucpl gene is shown in Fig. 7A. Half-sites for ATF/CREB (CRE2 and CRE3), a putative NF-E2 binding site, and PPRE are shown within boxes. Bold letters represent bases which matched between the three species. A sequence similarity search indicates that NF-E2 binding site (TGCTGYCNCT) in the mouse, human and rat is located in a comparable location. (Fig. 7 A) However, unlike the mouse gene, neither the rat nor the human gene contain the downstream NF- E2 binding site that overlaps with CRE2 in the mouse. Using electromobility shift and supershift assays, the binding of NFE212 was identical for the NF-E2 binding sites for human and rodents. (Data not shown).

[0063] Binding activity of the putative human NF-E2 binding site was assayed with nuclear extracts from BAT of A/J mouse. Fig. 7B illustrates the binding of human NF-E2 sequences to nuclear extracts from BAT of A/J mouse. Nuclear extracts were isolated from BAT of A/J mouse kept in RT or cold (4°C) for 7 days. 5 μg of nuclear extracts were incubated with ³²P end-labeled NF-E2 probe (0.1 pmoles) corresponding to the human Ucpl gene, and separated on 6% non-denaturing acrylamide gel.2 pmoles of cold probe or antibody for NFE212 (1 μl) were added for competition and for the super shift assay, respectively. Only the NFE212 bands are shown (arrow). Nuclear extracts from cold exposed mouse (7 days at 4°C) showed an increase in binding activity (Fig. 7B) and a similar binding activity as the mouse NF-E2 probe (Fig. 5c). Mouse NF-E2 binding sites (mNF-E2) showed comparable competition with the human NF-E2 binding site (hNF-E2). Antibody against NFE212 (mouse, rat and human reactive) interacted effectively with the complex in the super shift assay. These results indicate that NFE212 regulates Ucpl expression in humans.

Example 8 Effects of Overexpression ofNFE2l2 on Mouse Ucpl Expression [0064] Since the binding activity of NFE212 to the corresponding NF-E2 sequences increased in response to cold exposure (Fig. 5C) and norepinephrine treatment (Fig. 5a), the effect of Nfe2l2 overexpression on reporter constructs containing the 5 ' regulatory region of Ucp 1 was examined. Previously, it has been reported that 3.1 kb of 5' flanking region (4380/7538) of the mouse Ucp 1 gene which contains 4 DNase I hypersensitive sites revealed strong CAT activity by adding norepinephrine to the cultures. See Kozak et al, 1994. To test the function of NFE212 on Ucp 1 gene promoter and transcription, a luciferase reporter construct regulated by the 3.1 kb of mouse Ucpl gene was co-expressed with CMN-controlled Nfe2l2 expression vector in HL3- 1 B cells. Fig. 8 shows the results of luciferase reporter constructs (pGL3/3.1kb, left and pGL3/CRE4pro/220, right) that were cofransfected with expression vector which is empty (pCMN/tagl , open box), containing cDΝA for Nfe2l2 correct (closed box), or containing cDΝA with reversed orientation (hatched box) into HIB-IB cells. Cells were cultured in medium with or without 0.5 mM of 8-Br-cAMP (cAMP), 1 uM of norepinephrine (ΝE) or luM troglitazone (Trog) for an additional 16 hrs. Luciferase activity was measured in cell lysates using the Dual- Luciferase assay system (Promega), and activity (fold increase) was obtained by dividing corrected activity (induction after treatment) by the activity from empty vector (pCMN/tagl). Data is presented as the mean and standard deviation of fold increase from two independent experiments.

[0065] As shown in Figure 8, overexpression of Nfe2l2 increased the luciferase activity of the 3.1 kb of Ucpl reporter construct by 3.9-fold (0.5 mM 8-Br-cAMP, 16 hrs) and 2.8-fold (ImM norepinephrine, 16 hrs) relative to the empty vector (pCMN/tagl). However, cells with 1 mM of troglitazone, a PPARγ ligand, showed no effect indicating that activation by ΝFE212 is dependent on β-adrenergic receptor activation and does not involve the upstream PPARγ regulatory site. Additionally, in Fig. 4 mutations to the first and second T and G nucleotides in NF-E2 which overlap with CRE2 decreased the luciferase activity with norepinephrine or cAMP treatment in the same cells. To focus on the NF-E2 binding site of mouse Ucpl gene, a similar experiment was performed with the truncated luciferase reporter construct (pGL3/CRE4/220), which was used in characterization of CRE2, as described in Figure 4. As expected from previous experiments, slightly higher activities were observed with pGL3/CRE4/220 luciferase reporter (Fig. 8, right). These results indicate that NFE212 has a role in regulating mouse Ucpl gene expression by interactions with a NF-E2 binding site that overlaps the CRE2 site in the enhancer region.

[0066] An EMSA probe designed from the sequence in the mouse Ucpl enhancer was found to form specific bands; however, supershift assays with antibodies showed that the proteins binding to the probe were not against the NF -E2 p45 subunit, but rather against NFE212, another member of the NF-E2 family. The binding of NFE212 probes was increased in brown fat cells isolated from following treatment with cold or norepinephrine; and coexpression studies of Nfe2l2 vectors with the Ucpl enhancer constructs indicated that transcription of Ucpl is mediated by NFE212.

[0067] Thus, binding activity of NFE212 was increased by norepinephrine treatment (in vivo) and cold exposure (in vitro). Furthermore, Nfe2l2 overexpression induced Ucpl promoter activity only with norepinephrine and a cAMP analog in HIB-IB cell. That no induction occurred with the PPARγ ligand, troglitazone, suggested that NFE212 activation is mediated by protein kinase A signaling pathway, but is independent of PPARγ. This activation does not depend on increased production of Nfe2l2 mRNA. Example 9 Effects of Overexpression ofNFE2l2 on Rat Ucpl Expression [0068] To further test the function of NFE212 on the Ucpl enhancer, luciferase reporter constructs containing the rat Ucpl enliancer with or without mutations to the NF-E2 site (pGL3/cre4pro/mRat221 andpGL3/cre4pro/Rat221) were co-expressed with a CMV-controUed Nfe2l2 expression vector in HIB-IB cells, similar to that described above for the mouse Ucpl enhancer. (Example 8). Luciferase reporter constructs (pGL/cre4pro/Rat221 and pGL/cre4piO/mRat221) were cofransfected with an expression vector which is either empty (pCMV/tagl, Stratagen) or contains cDNA for Nfe2l2 into HIB-IB cells. Cells were cultured in the medium with or without 0.5 mM of 8-Br-cAMP (cAMP) for an additional 16 hrs. Luciferase activity was measured in cell lysates using the Dual-Luciferase assay system (Promega). Data is presented in Fig.9 as the mean and standard deviation from two experiments. The luciferase reporter assay demonstrated that the NF-E2 binding site in the rat Ucpl promoter is responsible for its transcriptional activation in response to cAMP or norepinephrine treatment with sequence specific manner (data for norepinephrine not shown). As shown in Fig. 9, overexpression of Nfe2l2 increased Ucpl promoter activity but only with the wild-type NF-E2 element (pGL3/cre4pro/Rat221). These results further indicate that in mammals NFE212 participates in the BAT development as well as transcriptional activation of Ucpl in response to cold exposure or β-adrenergic stimulation.

Example 10 Analysis of mice deficient in NFE212 due to a targeted mutation of the gene. [0069] Mice are currently being bred to be deficient in NFE212 due to a target mutation of the gene. These mice will be analyzed for brown adipocyte expression in both brown and white fat depots following exposure to the cold. Mice that are homozygous for the Nfe2l2 target mutation will be exposed to the cold for periods of time that vary from 6 hr to 3 weeks. Adipose tissue from several depots will be removed and analyzed by the expression of Ucpl mRNA and for the increase in brown adipocytes by immunohistology. It is predicted that the mutation to Nfe2l2 will reduce, but not totally eliminate, the induction of Ucpl and brown adipocyte formation. Example 11 Analysis of mice with overexpression ø/NFE2I2

[0070] Transgenic mice in which Nfe2l2 is over-expressed in adipocytes have been generated by driving expression oϊNfe2l2 with the aP2 promoter. This promoter has been used extensively for fat specific over-expression of many genes. Mice carrying the transgene were exposed to cold (4°C) and were analyzed for the expression of Ucpl in white fat depots. A prelimary experiment indicated that some transgenic mice showed higher expression of Ucpl than control non-transgenic, cold-exposed mice, while other transgenic mice did not. These mice will also be analyzed for the number of brown adipocytes. They will also be fed a high fat diet to stimulate increased obesity.

[0071] We predict that the increase in the expression of Nfe2l2 will increase Ucpl expression and brown adipocyte differentiation and prevent obesity even with a diet high in fat.

Example 12

Interactions between loci on different chromosomes to achieve optimal expression of Ucpl.

[0072] Since the difference in Ucpl gene expression depends on variation in Nfe2l2 on

Chromosome 2, as well as genes on Chromosomes 3, 8 and 19, it is possible that effects due to

NFE212 may require specific interactions with alleles on these other chromosomes. To provide the proper genetic environment to detect the effects of a specific gene, including Nfe2l2, congenic mice have been constructed in which the alternative allele for each genetic locus associated with brown fat induction has been placed on the C57BL/6J background by 10 backcross generations. These congenic strains will be analyzed separately as well as in various combinations to identify the interactions between loci on different chromosomes to achieve optimal expression of Ucpl. Accordingly, the knowledge gained from the study of these special congenic lines will .enable the identification of the genetic environment that will optimize the effect of NFE212 on brown fat differentiation.

Example 13

NFE212 As a Transcription Factor in BAT Development and Differentiation.

[0073] Brown adipose tissue (BAT) develops during the perinatal period. Breeding pairs of C57BL16 J mice were purchased from the Jackson Laboratory. The breeding colon was then expanded to produce sufficient numbers of progeny to analyze during development. Numerous transcription factors, including peroxisome proliferator-activated receptor (PPAR), CCAAT enhancer-binding proteins (C/EBPs) and cAMP responsive element binding protein (CREB), are involved in the BAT development. Nuclear extracts from BAT isolated from C57BL16J mice ranging in age from 19 days of gestation to 4 months after birth were isolated. The binding of NF-E2 sequences to nuclear extracts from BAT was assayed by loading each lane with a binding reaction containing 5 ug of nuclear extract from C57BL16J mice. Only the NFE212 bands are shown in Fig. 10A. When we compared binding activity of adipogenic transcription factors during BAT development, NFE212 revealed the same pattern of binding activity. (Data for other factors not shown). As shown in Figure 2 A, binding activity of NFE212 onto its binding sites which derived from mouse Ucpl promoter region reached a maximum at 19-day fetus, but maintained a high level of binding until 1 -month-old age when BAT is actively developing. [0074] An Western blot analysis was conducted to measure protein level of NFE212 in the nucleus of C57BL16Jmice of various ages from 19 days of gestation to 3 months afterbirth. Nuclear extracts (10 μg) from BAT of C57BL16Jmice (ages as stated) were separated on a 10%> SDS-polyacrylamide gel, and a blot was applied with a specific antibody for NFE212. Only the NFE212 bands are shown. The amount of protein in the nucleus matched the binding activity in terms of highest level at 19 days of gestation and then decrease overtime. (Fig. 10B) [0075] It has been known that chronic stimulation of β-adrenergic receptors by cold exposure results in increased BAT development. To identify changes in NFE212 during cold adaptation, binding activity of NFE212 from BAT of cold exposed mice (lday and 7 day at 4°C) was measured by same technique. Nuclear extracts were isolated from BAT (as described in Example 1) of B6 mice kept at room temperature (approximately 20°C) or in the cold (4°C) for 1 and 7 days. 5 ug of nuclear extracts were incubated with ³²P end-labeled NF-E2 probe (0.1 pmoles), and separated on 6% non-denaturing acrylamide gel. The results are shown in Fig. IOC. These results indicate that binding activity of NFE212 increased gradually after cold exposure, a response consistent with other known transcription factors. These results clearly indicate that NFE212 is a transcription factor that plays an important role in both BAT development and differentiation. [0076] The term "therapeutically effective amount" as used herein refers to an amount of either NFE212 or a compound that will increase the expression of the Nfe2l2 gene sufficient to increase the expression of Ucpl and increase brown adipose tissue thermogenesis. The term "therapeutically effective amount" therefore includes, for example, an amount of either NFE212 or a compound that will increase the expression of the Nfe2l2 gene sufficient to increase brown adipose tissue thermogenesis to decrease obesity, preferably to reduce by at least 10%, and more preferably to reduce by at least 30%, the degree of obesity. The dosage ranges for the administration of either NFE212 or a compound that will increase the expression of the Nfe2l2 gene are those that produce the desired effect. Generally, the dosage will vary with the age, weight, condition, and sex of the patient. A person of ordinary skill in the art, given the teachings of the present specification, may readily determine suitable dosage ranges. The dosage can be adjusted by the individual physician in the event of any contraindications. In any event, the effectiveness of treatment can be determined by monitoring the weight of the patient by methods well known to those in the field. Moreover, either NFE212 or a compound that will increase the expression of the Nfe2l2 gene can be applied in pharmaceutically acceptable carriers known in the art.

[0077] Either NFE212 or a compound that will increase the expression of the Nfe2l2 gene may be administered to a patient by any suitable means, including parenteral, subcutaneous, intrapulmonary, topically, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, or mtraperitoneal administration. Either NFE212 or a compound that will increase the expression of the Nfe2l2 gene may also be administered transdermally, for example in the form of a slow-release subcutaneous implant, or orally in the form of capsules, powders, or granules.

[0078] Pharmaceutically acceptable carrier preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. The active therapeutic ingredient may be mixed with excipients that are pharmaceutically acceptable and are compatible with the active ingredient. Suitable excipients include water, saline, dextrose, glycerol and ethanol, or combinations thereof. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like.

[0079] The form may vary depending upon the route of administration. For example, compositions for injection maybe provided in the form of an ampule, each containing a unit dose amount, or in the form of a container containing multiple doses.

[0080] Either NFE212 or a compound that will increase the expression of the Nfe2l2 gene may be formulated into therapeutic compositions as pharmaceutically acceptable salts. These salts include the acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, or tartaric acid, and the like. Salts also include those formed from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

[0081] Controlled delivery may be achieved by admixing the active ingredient with appropriate macromolecules, for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, prolamine sulfate, or lactide/glycolide copolymers. The rate of release of either NFE212 or a compound that will increase the expression of the Nfe2l2 gene maybe controlled by altering the concentration of the macromolecule.

[0082] Another method for controlling the duration of action comprises incorporating either NFE212 or a compound that will increase the expression of the Nfe2l2 gene into particles of a polymeric substance such as a polyester, peptide, hydrogel, polylactide/glycolide copolymer, or ethylenevinylacetate copolymers. Alternatively, either NFE212 or a compound that will increase the expression of the Nfe2l2 gene maybe encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly(methyhnethacrylate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. [0083] The present invention provides a method of treating or ameliorating a disease that can be helped by increasing Ucpl expression, such as obesity, comprising administering to the patient, a therapeutically effective amount of either NFE212 or a compound that will increase the expression of the Nfe2l2 gene. The term "ameliorate" refers to a decrease or lessening of the symptoms of the disorder being treated. The symptoms that may be ameliorated include significant weight loss, and improved insulin senstivity. In addition, the use of either NFE212 or a compound that will increase the expression of the Nfe2l2 gene may be combined with the administration of another compound that are known to increase Ucpl expression, for example, a compound that is known to increase the activity or expression of other transcription factors, e.g., PKA, CREB, PGC1, RAR, RXR, PPARγ, ATF-1, and CBP. Examples of compounds known to increase the expression of Ucpl by one or more of these transcription factors, include norepinephrine, β-adrenergic receptor agonists, thiazolidinediones, isoproterenol, thyroid hormone, and retinoids.

[0084] Compounds that are known to increase the level or the activity of protein kinase

C can be used to increase the concentration and the binding of NFE212 to its DNA binding motif in the nucleus. These compounds include, but not limited to,phorbol esters (e.g., phorbol 12- myristate 13-acetate), tert-butylhydroquinone, and β-naphthoflavone.

[0085] The complete disclosures of all references cited in this specification are hereby incorporated by reference. Also, incorporated by reference is the following reference, which is not prior art to this application: J.S. Rim and L.P. Kozak, "Overlapping regulatory motifs for CREB and NFE212 transcription factors in the upstream enhancer of the mitochondrial uncoupling protein 1 gene," Journal of Biological Chemistry, vol.277, pp. 34589-34600 (2002). In the event of an otherwise irreconcilable conflict, however, the present specification shall control. SEQUENCE LISTING

<110> Board of Supervisors of Louisiana State University and Agricultural and Mechanical College

Kozak, Leslie P.

Rim, Jong S.

<120> Induction of Brown Adipocytes by Transcription Factor NFE2L2

<130> OOPOIW Kozak

<140> PCT/US02/_ <141> 2002-09-24

<150> US 60/324,400 <151> 2001-09-24

<160> 24

<170> Patenthi version 3.1

<210> 1 <211> 221 <212> DNA <213> Mus sp.

<220>

<221> misc_feature

<223> Mouse UCPl gene.

<400> 1 aagcttgctg tcactcctct acagcgtcac agagggtcag tcacccttga ccacactgaa 60

ctagtcgtca cctttccact cttcctgcca gaagagcaga aatcagactc tctggggata 120

tcagcctcac ccctactgct ctctccatta tgaggcaaac tttctttcac ttcccagagg 180

ctctgggggc agcaaggtca accctttcct cagactctag a 221

<210> 2 <211> 19 <212> DNA <213> Artificial

<220>

<223> Nucleotide sequences for the combined probe containing both NFE2 and CRE2 binding sites

<400> 2 tgaactagtc gtcaccttt 19

<210> 3 <211> 16 <212> DNA <213> Artificial

<220>

<223> Nucleotide sequences for cold probes for NF-E2.

<400> 3 gggactagtc gtcggg 16 <210> 4 <211> 16 <212> DNA <213> Artificial

<220>

<223> Nucleotide sequences for cold probes for CRE2.

<400> 4 gggctagtcg tcaggg 16

<210> 5

<211> 50

<212> DNA

<213> Homo sapiens

<400> 5 agaacttgct gccactcctt tgctacgtca taaagggtca gttgcccttg 50

<210> 6 <211> 30 <212> DNA <213> Artificial

<220>

<223> Forward primer used for PCR amplification of enhancer region of mouse UCpl gene.

<400> 6 ggggtaccgt gcacactgcc aaatcatctc 30 <210> 7 <211> 30 <212> DNA <213> Artificial

<220>

<223> Reverse primer for PCR amplification of enliancer region of mouse Ucpl gene.

<400> 7 gggagctcct gcagagccac ctgggctagg 30

<210> 8 <211> 26 <212> DNA <213> Artificial

<220>

<223> Forward primer for PCR amplification of promotor region of mouse Ucpl gene.

<400> 8 ggggatccga gtgacgcgcg gctggg 26

<210> 9 <211> 26 <212> DNA <213> Artificial

<220>

<223> Forward primer for PCR amplification of promoter region of mouse Ucpl gene.

<400> 9 ggggatccgg ctgggaggct tgcgca 26

<210> 10 <211> 26 <212> DNA <213> Artificial

<220>

<223> Reverse primer for PCR amplification of promoter region of mouse Ucpl gene.

<400> 10 gggaagcttg ggctaggtag tgccag 26

<210> 11 <211> 28 <212> DNA <213> Artificial

<220>

<223> Primer for PCR amplification of BAT specific enhancer region of m ouse Ucpl gene. <400> 11 ggggagctcc tctacagcgt cacagagg 28

<210> 12 <211> 28 <212> DNA <213> Artificial

<220>

<223> Primer for PCR amplification of BAT specific enhancer region of m ouse Ucpl gene.

<400> 12 gggctcgaga gtctgaggaa agggttga 28

<210> 13 <211> 28 <212> DNA <213> Artificial

<220>

<223> Forward primer to mutate CRE3 region in Bat specific enliancer of mouse Ucpl gene.

<400> 13 ggggagctcc tctacagctg aacagagg 28

<210> 14 <211> 25 <212> DNA <213> Artificial

<220>

<223> Reverse primer used to mutate CRE2 sequence in BAT specific enhan cer region of mouse Ucpl gene.

<400> 14 agtggaaagg ttcagactag ttcag 25

<210> 15 <211> 25 <212> DNA <213> Artificial

<220>

<223> Forward primer used to mutate CRE2 sequence in BAT specific enhan cer region of mouse Ucpl gene.

<400> 15 ctgaactagt ctgaaccttt ccact 25

<210> 16 <211> 50 <212> DNA <213> mus sp.

<400> 16 gaagcttgct gtcactcctc tacagcgtca cagagggtca gtcacccttg 50 <210> 17 <211> 50 <212> DNA <213> Rattus sp.

<400> 17 gaaccttgct gcctctcctt tgcgacgtca cagtgggtca gtcacccttg 50

<210> 18 <211> 19 <212> DNA <213> Mus sp.

<400> 18 ttatagtgcc gtcactaac 19

<210> 19 <211> 19 <212> DNA <213> Mus sp.

<400> 19 tgaactagtc gtcaccttt 19

<210> 20 <211> 19 <212> DNA <213> Mus sp. <400> 20 cctctacagc gtcacagag 19

<210> 21 <211> 19 <212> DNA <213> Mus sp.

<400> 21 tgggagtgac gcgcggctg 19

<210> 22

<211> 19

<212> DNA

<213> Homo sapiens

<400> 22 ttggctgacg tcagagaga 19

<210> 23 <211> 17 <212> DNA <213> Artificial

<220>

<223> First mutated sequence of CRE2 sequence of mouse Ucpl gene.

<400> 23 aactagtctg aaccttt 17 <210> 24 <211> 17 <212> DNA <213> Artificial

<220>

<223> Second mutated sequence of CRE2 sequence of mouse Ucpl gene.

<400> 24 aactatgagt caccttt 17

Claims

What is claimed:

1. A method of ameliorating or preventing, in a mammals, the symptoms of a disease treatable by increasing Ucpl expression, said method comprising administering to the mammal a therapeutically effective amount of a compound that causes an increase in the concentration of NFE212 protein.

2. A method of Claim 1 , wherein the disease is a weight disorder.

3. A method of Claim 2, wherein the weight disorder can be ameliorated or prevented with an increase in brown adipose tissue thermogenesis.

4. A method of Claim 1 , wherein the adminstered compound causes a change in Nfe2l2 gene expression.

5. A method of Claim 2, wherein the weight disorder is obesity.

A method of Claim 1, wherein the administered compound is NFE212.

7. A method of Claim 1, further comprising an initial treatment of the mammal with an amount of norepinephrine sufficient to increase the number of brown adiopocytes.

8. A method of Claim 1 , further comprising administering to the mammal other compounds that increase Ucpl expression, selected from the group consisting of norepinephrine, β- adrenergic receptor agonists, thiazolidinediones, isoproterenol, thyroid hormone, and retinoids.

9. A method of ameliorating or preventing, in a mammals, the symptoms of a disease treatable by increasing Ucpl expression, said method comprising administering to the mammal a therapeutically effective amount of a compound that increases the concentration of NFE212 protein in the nucleus.

10. A method of Claim 9, wherein the disease is a weight disorder.

11. A method of Claim 10, wherein the weight disorder can be ameliorated or prevented with an increase in brown adipose tissue thermogenesis.

12. A method of Claim 10, wherein the weight disorder is obesity.

13. A method of Claim 9, wherein the administered compound causes an increase in the concentration or activity of protein kinase C.

14. A method of Claim 13, wherein the administered compound is selected from the group consisting of ,phorbol esters, tert-butylhydroquinone, and β-naphthoflavone.

15. A method of Claim 14, wherein the phorbel ester is phorbol 12-myristate 13-acetate.

16. A method of Claim 9, further comprising an initial treatment of the mammal with an amount of norepinephrine sufficient to increase the number of brown adiopocytes.

17. Amethod of Claim 9, further comprising administering to the mammal other compounds that increase Ucpl expression, selected from the group consisting of norepinephrine, β- adrenergic receptor agonists, thiazolidinediones, isoproterenol, thyroid hormone, and retinoids