| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | August 5, 2003 |
| PATENT TITLE |
Uncoupling protein 4 (UCP-4) |
| PATENT ABSTRACT |
A novel uncoupling protein, which we have designated UCP-4, that is expressed in various tissues, including brain, heart, pancreas, and muscle tissue, and nucleic acid molecules which encode for said novel protein, are described. Methods of screening for compounds that regulate the expression and the activity of UCP-4 are described, as well as methods of treating diseases or conditions in which the regulation of thermogenesis or respiratory ATP synthesis is desired. Such conditions include obesity, diabetes, malignant hyperthermia, and fever. The construction of cell lines that express UCP-4 is also described. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 27, 2001 |
| PATENT CT FILE DATE | July 13, 1999 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | January 27, 2000 |
| PATENT REFERENCES CITED |
Sanchis et al. BMCP1, a novel mitochondrial carrier with high expression in the central nervous system of human and rodents and respiration uncoupling activity in recombinant yeast. J. Biol. Chem. 273:34611-34615, 1998.* Mao et al. UCP4, a novel brain-specific mitochondrial protein that reduces membrane potential in mammalian cells. FEBS Letters 443:326-330, 1999.* Apoorva Mandavilli, Protein folds shield different Roles, BioMednet News, Nov. 1, 2001.* Peer Bork and Eugene V. Koonin, Predicting functions from protein sequences--where are the bottlenecks? Nature Genetics 18:313-318, 1998.* Bairoch, A., et al, "The PROSITE database, its status in 1997," Nucleic Acids Research, 25(1):217-221 (1997). Bathgate, B., et al., "Functional expression of the rat brown adipose tissue uncoupling protein in Saccharomyces cerevisiae," Molecular Microbiology, 6(3):363-370 (1992). Bianco, A.C., et al, "Triiodothyronine Amplifies Norepinephrine Stimulation of Uncoupling Protein Gene Transcription by a Mechanism Not Requiring Protein Synthesis," Journal of Biological Chemistry, 263(34):18168-18175 (1988). Boss, O., et al, "Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression," FEBS Letters, 408:39-42 (1997). Bouillaud, F., et al, "A sequence related to a DNA recognition element is essential for the inhibition by nucleotides of proton transport through the mitochondrial uncoupling protein," EMBO Journal, 13(8):1990-1997 (1994). Ferguson, M.A.J., et al. "Cell-Surface Anchoring of Proteins Via Glycosylphosphatidylinositol Structures", Annu. Rev. Biochem. 57:285-320 (1988). Fleury, C., et al, "Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia," Nature Genetics 15:269-272 (1997). Garlid, K.D., et al, "On the Mechanism of Fatty Acid-induced Proton Transport by Mitochondrial Uncoupling Protein," Journal of Biological Chemistry, 271(5):2615-2620 (1996). Goeddel, D.V., et al, "Synthesis of human fibroblast interferon by E. coli," Nucleic Acids Research, 8(18):4057-4075 (1980). Gonzalez-Barroso, M.M., et al, "Activation of the uncoupling protein by fatty acids is modulated by mutations in the C-terminal region of the protein," Eur J Biochem 239:445-450 (1996). Kane, J.F., "Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli," Current Opinion in Biotechnology, 6:494-500 (1995). Klingenberg, M., "Mechanism and evolution of the uncoupling protein of brown adipose tissue," Trends Biochem. Sci., 15:108-112 (1990). Knopfel, T., et al, "Metabotropic Glutamate Receptors: Novel Targets for Drug Development," J Medicinal Chemistry, 38(9):1417-1426 (1995). Kopecky, J., et al, "Expression of the Mitochondrial Uncoupling Protein Gene from the aP2 Gene Promoter Prevents Genetic Obesity," J Clinical Investigation, 96:2914-2923 (1995). Larkin, S., et al, "Regulation of the Third Member of the Uncoupling Protein Family, UCP3, by Cold and Thyroid Hormone," Biochem Biophys Res Comm, 240:222-227 (1997). Leibel, R.L., et al, "Changes in Energy Expenditure Resulting from Altered Body Weight," New England Journal of Medicine 332(10):621-628 (1995). Lennon, G., et al, "The I.M.A.G.E. Consortium: An Integrated Molecular Analysis of Genomes and Their Expression," Genomics, 33:151-152 (1996). Lowell, B.B., "Development of obesity in transfenic mice after genetic ablation of brown adipose tissue," Nature 366:740-742 (1993). MacLennan, D.H., et al, "Discordance between phenotype and genotype inmalignant hyperthermia," Curr. Opin. Neurol., 8:397-401 (1995). Mickelson, J.R., et al., "Malignant Hyperthermia: Excitation-Contraction Coupling, CA.sup.2+ Release Channel, and Cell CA.sup.2+ Regulation Defects," Physiol. Rev., 76:537-92 (1996). Miller, D.A., "Human gene therapy comes of age," Nature 357(6378): 455-460 (1992). Mumberg, D., et al, "Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression," Nucleic Acids Research, 22(25):5767-5768 (1994). Murdza-Inglis, D.L., et al, "Functional Reconsitution of Rat Uncoupling Protein Following Its High Level Expression in Yeast," J Biol Chem, 260(18):11871-11875 (1991). Murdza-Inglis, D.L., et al, "A Single Mutation in Uncoupling Protein of Rat Brown Adipose Tissue Mitochondria Abolishes GDP Sensitivity of H Transport," J Biol Chem 269(10):7435-7438 (1994). Nicholls, D.G., et al, "Thermogenic Mechanisms in Brown Fat," Physiological Reviews, 64:2-40 (1984). Rehnmark, S., et al, ".alpha.- and .beta.-Adrenergic Induction of the Expression of the Uncoupling Protein Thermogenin in Brown Adipocytes Differentiated in Culture," J Biol Chem, 265(27):16464-16471 (1990). Ricquier, D., et al, "Expression of Uncoupling Protein mRNA in Thermogenic or Weakly Thermogenic Brown Adipose Tissue," J Biol Chem, 261(30):13905-13910 (1996). Rothwell, N.J., et al, "A role for brown adipose tissue in diet-induced thermogenesis," Nature 281:31-35 (1979). Steinfath, et al., Anasthesiol. Intensivmed Notfallmed Schmerzther, 31:334-43 (1996) [English abstract only] Article in German. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An isolated uncoupling protein 4 comprising the amino acid sequence of SEQ ID NO: 2. 2. The isolated uncoupling protein 4 of claim 1, wherein said isolated uncoupling protein 4 is isolated from a cell that comprises an endogenous nucleic acid molecule that encodes uncoupling protein 4. 3. The isolated uncoupling protein 4 of claim 1, wherein said uncoupling protein 4 is isolated from a cell that is transformed with a nucleic acid molecule that encodes uncoupling protein 4. 4. The isolated uncoupling protein 4 of claim 1, wherein said cell is selected from the group consisting of bacterial cells, insect cells, yeast cells, CHO cells, COS cells, NIH3T3 cells, HEK-293 cells, and 3T3L1 cells. 5. The isolated uncoupling protein 4 of claim 1, wherein said uncoupling protein 4 is chemically synthesized |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION A novel uncoupling protein, which we have designated UCP-4, that is expressed in various tissues, including brain, heart, pancreas, and muscle tissue, and nucleic acid molecules which encode said novel protein, are described. Methods of screening for compounds that regulate the expression and the activity of UCP-4 are described, as well as methods of treating diseases or conditions in which the regulation of thermogenesis, or respiratory ATP synthesis, is desired. Such conditions include obesity, diabetes, malignant hyperthermia, and fever. The construction of cell lines that express UCP-4 is also described. BACKGROUND Uncoupling protein (UCP-1; thermogenin) is a transmembrane proton-translocating protein present in the mitochondria of brown adipose tissue, a specialized tissue which functions in heat generation and energy balance (Nicolls, D. G., and Locke, R. M., Physiol. Rev. 64:2-40, (1984); Rothwell,. N. J. and Stock, M. J. Nature, 281:31-35 (1979)). Mitochondrial oxidation of substrates is accompanied by proton transport out of the mitochondrial matrix, creating a transmembrane proton gradient. Re-entry of protons into the matrix via ATP synthase is coupled to ATP synthesis. However, UCP-1 functions as a transmembrane proton transporter, permitting re-entry of protons into the mitochondrial matrix unaccompanied by ATP synthesis. Environmental exposure to cold evokes neural and hormonal stimulation of brown adipose tissue, which increases UCP-mediated proton transport, brown fat metabolic activity, and heat production. Recent studies with transgenic models indicate that brown fat and UCP-1 have an important role in energy expenditure in rodents. Transgenic mice in which brown adipocyte tissue was ablated by a toxin coupled to the UCP-promoter developed obesity and diabetes (Lowell, B. B., et al., Nature, 366:740 (1993)). Obesity in these transgenic animals developed in the absence of hyperphagia, suggesting that the uncoupled mitochondrial respiration of brown fat is an important component of energy expenditure. In a separate transgenic mouse model, ectopic expression of UCP-1 in white adipose tissue of genetically-obese mice led to a significant reduction in body weight and fat stores (Kopecky J., et al., J. Clin. Invest. 96:2914-23, (1995)). These studies indicate that activity of UCP-1 is accompanied by energy expenditure and weight loss in rodents. Two other UCP proteins have recently been cloned. The first uncoupling protein-like protein (UCPL) or UCP-2, is expressed in multiple tissues, and is enriched in tissues of the lymphoid lineage (Fleury, C., et al. Nature Genetics, 15:269-272, (1997)). The second, UCP-3, is predominantly localized to skeletal muscle (U.S. Ser. No. 60,043,407, filed Apr. 4, 1997, U.S. Ser. No. 60/046,154, filed May 8, 1997, and PCT/US98/005892 filed Mar. 25, 1998, all of which are hereby incorporated by reference herein; Boss, O., et al., (FEBS Lett. 408:3942, 1997). UCP-3 has been found to be regulated by cold and thyroid hormone (Larkin, S., et al., Biochem. Biophys. Res. Comm. 240:222-227, (1997)). Thermogenic protein activity, such as that found with UCP-1, may be useful in reducing, or preventing the development of excess adipose tissue, such as that found in obesity. Obesity is becoming increasingly prevalent in developed societies. For example, approximately 30% of adults in the U.S. were estimated to be 20 percent above desirable body weight--an accepted measure of obesity sufficient to impact a health risk. (Harrison's Principles of Internal Medicine 12th Edition, McGraw Hill, Inc. (1991) p. 411). The pathogenesis of obesity is believed to be multifactorial, but the basic problem is that in obese subjects food intake and energy expenditure do not come into balance until there is excess adipose tissue. Attempts to reduce food intake, or to decrease hypernutrition, are usually fruitless in the medium term because the weight loss induced by dieting results in both increased appetite and decreased energy expenditure. (Leibel et al., New England Journal of Medicine 322:621-28, (1995)). The intensity of physical exercise required to expend enough energy to materially lose adipose mass is too great for many obese people to undertake on a sufficiently frequent basis. Thus, obesity is currently a poorly treatable, chronic, essentially intractable metabolic disorder. In addition obesity carries a serious risk of co-morbities including, Type 2 diabetes, increased cardiac risk, hypertension, atherosclerosis, degenerative arthritis, and increased incidence of complications of surgery involving general anesthesia. An increased level in thermogenesis in obese individuals, or individuals with a predisposition toward obesity should help to reduce the level of adipose tissue, and therefore avoid the complications associated with obesity. Too high of a level of thermogenesis may also be detrimental for certain individuals, thus a method of decreasing the level of thermogenesis in such individuals is desirable. It would be desirable, for example, to treat or prevent conditions such as malignant hyperthermia, which occurs in approximately 1 in 50,000 anesthetic procedures, and can have about a 70% mortality rate. Studies in pigs, which are susceptible to malignant hyperthermia, have suggested that it may be caused by inappropriate activation of a sarcoplasmic reticulum Ca+ release channel (the ryanodine receptor) which then acts in a positive feedback manner to further release intracellular calcium and, thus affecting myotonic contraction and thermal overload (Mickelson, J. R., and Louis, C. F., Physiol. Rev. 76:537-92 (1996)). However, defects in the ryanodine receptor are found in only approximately 50% of patients with malignant hyperthermia (MacLennan, D. H., Curr. Opin. Neurol. 8:397-401 (1995)). There is discord between actual incidence and predicted genetic susceptibility based upon detection of mutant ryanodine receptors in some, but not all humans with malignant hyperthermia (MacLennan, D. H., Curr. Opin. Neurol., 8:397-401 (1995)). Four chromosomal loci linked to malignant hyperthermia have been identified in familiar studies, but for only one has the genetic defect been localized to mutations of a particular gene, the ryanodine calcium channel gene on chromosome 19 (Id.; Steinfath, M., et al., Anasthesiol. Intensivmed Notfallmed Schmerzther 31:334-43 (1996)). It would therefore be useful to identify the protein involved in human malignant hyperthermia, and to design methods of regulating its expression to prevent or treat malignant hyperthermia. Another condition where thermogenesis is increased is in fever, an increase in body temperature in response to infection or inflammation. Fever is observed not only in mammals and birds (warm-blooded animals; homeotherms), but also in some poikiothermic (cold-blooded) animals such as lizards, which increase their temperature behaviorally, by seeking warmer surroundings. Inoculated poikilothermic animals that are denied access to a warmer environment have a higher mortality, supporting a general advantage of being able to increase body temperature during infection. The mechanism underlying this advantage has been elusive, but may involve changes in properties of iron-binding proteins, resulting in a drop of free iron in body fluids, to which bacteria are particularly susceptible. However, there are clinical settings in which fever is dangerous or unpleasant. These include epileptic patients where fever may precipitate convulsions; elderly patients with cardiac or cerebrovascular disease; children, who are at risk for febrile convulsions (which may then predispose to later epilepsy); patients with hyponutrition and chronic fever, where the increased metabolic demand of maintaining a higher body temperature compromises body energy stores; patients with fluid balance disturbances where the sweating associated with rapid up and down resetting of body temperature can exacerbate salt loss and electrolyte disturbance. As opposed to the unregulated heat gain in malignant hyperthermia, exercise and heat-stroke, heat.gain in fever is a regulated event that recruits all of those mechanisms that are employed in normal autonomic and behavioral thermoregulation. From a control system perspective, there is a shift in body temperature set-point so that a higher body temperature is defended. Effector systems used to defend the higher body temperature in fever (shivering, non-shivering UCP-mediated thermogenesis, vasoconstriction, piloerection, warmth-seeking behaviors, heat-loss-lessening behaviors) are the same responses that are observed during cold exposure in non-febrile circumstances. Compounds that defeat one or more of these effector responses will be useful to reduce fever. The present invention concerns a novel isolated uncoupling protein and nucleic acids coding for this protein, having some properties that differ from the properties of previously-identified uncoupling proteins. SUMMARY OF THE INVENTION The present invention concerns a novel uncoupling protein, which we have designated UCP-4, that is expressed in brain, heart, pancreas, and muscle tissue, as well as kidney, placenta, liver, lung, ovary, and spinal cord tissue, isolated nucleic acid molecules that encode UCP-4, as well as methods of screening for compounds that regulate the expression and/or the activity of UCP-4. Compounds that regulate the activity of UCP-4 will regulate thermogenesis, respiratory ATP synthesis, and energy utilization in brain, heart, pancreas, and muscle tissue, as well as kidney, placenta, liver, lung, ovary, and spinal cord tissue, and will be useful in treating or preventing diseases or conditions in which regulation of thermogenesis will be beneficial. Such conditions include, but are not limited to, obesity, diabetes, malignant hyperthermia, and fever. Our discovery that UCP-4 is present in brain and pancreas is consistent with a role in fuel sensing. UCP-2 is also found in parts of the brain, such as hypothalamic nuclei and the area postrema, that have a fuel-sensing function. A fuel-sensing function is likely to require tissue to have a high metabolic rate so that its ionic milieu becomes susceptible to fuel availability. The output of fuel sensors in the brain and in other tissues, such as the pancreas, will ultimately drive responses that will tend to correct disturbances in body energy content. Compounds that act upon UCP-4 to mimic the sensing of hypernutrition (body energy excess) will trigger energy minimizing responses (e.g., thermogenesis, decreased energy intake) and will be useful in the treatment of metabolic diseases characterized by hypernutrition, such as diabetes and obesity. Compounds that act upon UCP-4 to mimic undernutrition will trigger energy-maximizing responses (such as food ingestion and conservation of body energy stores) and will be useful in treating conditions characterized by a depletion of body energy stores (e.g., anorexia nervosa, malnutrition and cachexia). Thus, one embodiment of the invention comprises isolated nucleic acid molecules which encode UCP-4. The encoded UCP-4 may be the UCP-4 sequence encoded in any eukaryotic cell, preferably a vertebrate cell, more preferably a mammalian cell. In one preferred aspect, the invention provides an isolated nucleic acid molecule which encodes rat UCP-4. In another preferred aspect is an isolated nucleic acid molecule which encodes mouse UCP-4. Especially preferred are nucleic acid molecules which encode human UCP-4. In more preferred aspects, said nucleic acid encodes an amino acid sequence comprising the amino acid sequence of FIG. 1. In most preferred aspects, said nucleic acid molecule comprises the nucleic acid sequence of FIG. 1. The term "UCP-4" includes, but is not limited to, all isoforms thereof, generated by alternative splicing of the primary transcript that gives rise to a nucleotide sequence that encodes the amino acid sequence shown in FIG. 1. In another embodiment of the present invention, a nucleic acid molecule encoding UCP-4 is operably linked to a promoter sequence, wherein said promoter sequence promotes the transcription of the coding region of said nucleic acid. Also provided in the present invention, are vectors comprising a nucleic acid molecule of the present invention. In preferred aspects, said vectors further comprise a promoter sequence which is operably linked to said nucleic said molecule. In more preferred embodiments, said vectors further comprise a 3' polyadenylation sequence which is operably linked to said nucleic acid molecule. Also within the scope of the present invention are host cells transformed with the nucleic acid molecules or vectors of the present invention. The UCP-4-expressing host cell is preferably a yeast cell, CHO cell, COS cell, NIH3T3 cell, HEK-293 cell, or 3T3L1 cell. In another embodiment of the invention, isolated UCP-4, is provided. The UCP-4 may be, for example, isolated from a cell that comprises an endogenous nucleic acid molecule that encodes UCP-4. The UCP-4 may be isolated from a cell that expresses UCP-4 from a heterologous nucleic acid molecule, such as in a transformed cell. The cell may be eukaryotic or prokaryotic. Preferably the cell is a bacterial, insect, yeast, CHO, COS, NIH3T3, HEK-293, or 3T3L1 cell. Alternatively, UCP-4 may be synthesized by methods known to those skilled in the art, including, but not limited to UCP-4 that is expressed in, and/or isolated from a cell-free translation system, or isolated through chemical synthesis. In another embodiment of the present invention anti-UCP-4 antibodies are provided. Preferably these antibodies bind to an amino acid sequence that is not completely homologous among all three UCPs. More preferably these antibodies bind to a region comprising amino acids 38-51 of said UCP-4 sequenceor to a region comprising amino acids 93-107 of said UCP-4 sequence. More preferably, the anti-UCP-4 antibody is a monoclonal antibody; In yet other embodiments of the present invention are provided methods of gene therapy comprising administering to a subject a nucleic acid molecule that encodes UCP-4. In one preferred aspect is provided a method of increasing thermogenesis in a subject, comprising administering to said subject a nucleic acid molecule which encodes UCP-4, wherein said administering of said nucleic acid molecule increases the level of UCP-4 expression in one or more tissues of said subject. Preferably, said tissue is brain, heart, pancreas, muscle, kidney, placenta, liver, lung, ovary, or spinal cord tissue. More preferably said tissue is brain, heart, pancreas, or muscle tissue. Said method is preferably for purposes of treating obesity, diabetes, and/or decreasing fat in a subject. Other methods of gene therapy provided in the present invention are methods for decreasing expression of UCP-4 in a subject by administering to said subject an antisense nucleic acid molecule wherein said administering of said nucleic acid molecule decreases the level of UCP-4 expression in one or more tissues of said subject. Preferably said method is used for decreasing malignant hyperthermia, or fever, in said subject. Preferably said tissue is brain, heart, pancreas, muscle, kidney, placenta, liver, lung, ovary, or spinal cord tissue. More preferably, said tissue is brain, heart, pancreas, or muscle tissue. Another embodiment of the invention comprises a method of screening for a compound that binds to or modulates the activity of UCP-4, comprising a) introducing said UCP-4 and one or more test compounds into an acceptable medium, and b) monitoring the binding or modulation by physically detectable means, c) thereby identifying those compounds that bind to or modulate the activity of said UCP-4. In one preferred aspect, UCP-4 is associated with a mitochondrial membrane. In another preferred aspect, said monitoring of the binding or modulation of said compound to UCP-4 further comprises monitoring the level of purine nucleotide binding of UCP-4 in the presence of said compound or compounds; and identifying the compounds that, when in the presence of UCP-4, alter the level of purine nucleotide binding to UCP-4. Preferably, said purine nucleotide is GDP. In other preferred aspects, the method further comprises monitoring the level of purine nucleotide binding of UCP-4 in the absence of said compound or compounds. In another preferred aspect of the invention, said monitoring of the binding to or modulation of UCP-4 by said compound further comprises monitoring the level of fatty acid binding of UCP-4 in the presence of said compound; and identifying the compounds that, when in the presence of UCP-4, alter the level of fatty acid binding to UCP-4. Preferably, said fatty acid is laurate. In other preferred aspects, said monitoring further comprises monitoring the level of fatty acid binding of UCP-4 in the absence of said compound. In another preferred embodiment of the invention, a method is provided for screening for a compound that binds to or modulates the activity of UCP-4, comprising monitoring the effect of said compound on a cell that expresses UCP-4. In one preferred aspect, the cell that expresses UCP-4 is present in brain, heart, pancreas, muscle, kidney, placenta, liver, lung, ovary, or spinal cord tissue. Preferably, the cell that expresses UCP-4 is present in brain, heart, pancreas, or muscle tissue. Preferably, the cell that expresses UCP-4 is transformed with a nucleic acid encoding UCP-4. The nucleic acid may preferably be operably limited to the UCP-4 native promoter, or to a heterologous promoter. By "heterologous promoter" is meant any promoter that allows the expression of UCP-4, that is not the endogenous UCP-4 promoter. More preferably, the nucleic acid encodes an amino acid sequence comprising the amino acid sequence of FIG. 1. In another preferred embodiment, the nucleic acid comprises the nucleic acid sequence of FIG. 1. In another preferred embodiment, the method further comprises monitoring the effect of said compound on a cell that does not express UCP-4. Preferably, said cell that does not express UCP-4 is otherwise substantially genetically identical to said cell that-expresses UCP-4. One preferred method of monitoring of the effect of said compound on said cell comprises monitoring the level of mitochondrial respiration of said cell. Other preferred methods comprise monitoring the level of mitochondrial respiration in isolated mitochondria. Most preferred methods comprise monitoring the level of mitochondrial respiration or whole animals, preferably mammals, more preferably rats or mice, and most preferably humans. In another preferred method, the monitoring of the effect of said compound on said cell comprises monitoring the level of mitochondrial membrane purine nucleotide binding, preferably GDP, of said cell. In another preferred method, said monitoring of the effect of said compound on said cell comprising monitoring the level of fatty acid, preferably laurate. In preferred embodiments, said UCP-4 of the present invention, and used in the methods of the present invention, as well as UCP-4 encoded by the nucleic acids used in the methods of present invention, is human UCP-4. Preferably, said UCP-4 comprises the amino acid sequence of FIG. 1. Most preferably, said UCP-4 is encoded by a nucleic acid molecule comprising the nucleic acid sequence of FIG. 1. The term "UCP-4" also includes the various isoforms of UCP-4 due to variations in the splicing of the RNA transcript coding for UCP-4. UCP-4s that contain post-translational modifications that are required for activity or modulate activity are also within the scope of the present invention. The compound that regulates UCP-4 activity may be found to either increase or decrease UCP-4 activity. In another preferred embodiment, a method is provided for screening for a compound that regulates the expression of UCP-4, comprising monitoring the effect of said compound on the level of expression of UCP-4 RNA in a cell that expresses UCP-4. Preferably, the cell that expresses UCP-4 is present in brain, heart, pancreas, muscle, kidney, placenta, liver, lung, ovary, or spinal cord tissue. More preferably the cell is present in brain, heart, pancreas, or muscle tissue. Preferably, the cell that expresses UCP-4 is transformed with a nucleic acid encoding UCP-4. More preferably, said nucleic acid encodes an amino acid sequence comprising the amino acid sequence of FIG. 1. Most preferably, the nucleic acid comprises the nucleic acid sequence of FIG. 1. In one preferred aspect, the nucleic acid molecule is operably linked to a UCP-4 promoter. In another preferred aspect, the nucleic acid molecule is operably linked to a heterologous promoter. In another preferred aspect, the method utilizes a yeast cell that is transformed with a nucleic acid that encodes UCP-4. In more preferred aspects, said cell is a transformed eukaryotic cell, preferably a vertebrate cell, more preferably a mammalian cell, such as those known to those of skill in the art, and those described herein. In other preferred aspects, said cell is a transformed yeast cell. Preferably, the methods involve determining whether the expression of said messenger RNA is increased or decreased compared to the expression of said messenger RNA in a cell that has not been exposed to said compound. Preferably, said UCP-4 is human UCP-4. More preferably, said UCP-4 is encoded by a nucleic acid sequence comprising the nucleic acid sequence of FIG. 1. In one preferred aspect, the level of UCP-4 RNA is determined by probing the messenger RNA expressed in said cell with a nucleotide probe that comprises a nucleotide sequence that is homologous to at least 15, preferably 30, more preferably 45, consecutive nucleotides of a UCP-4 nucleotide sequence. Preferably, said nucleotide probe does not substantially bind under high stringency conditions to any non-UCP-4 nucleotide sequence in the same tissue. By "high stringency hybridization conditions" is meant those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.0 15 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50 C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 C; or (3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate, pH 7.0, 5.times.Denhardt's solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42 C, with washes at 42 C in 0.2.times.SSC and 0.1% SDS. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides binding of said cell. In other methods of the present invention, the effect of a compound on the level of expression of UCP-4 is further monitored on a cell that does not express UCP-4 thus, providing a negative control. Preferably, said cell that does not express UCP-4 is otherwise substantially genetically identical to said cell that expresses UCP-4. The compound may either increase or decrease the expression of UCP-4. In other aspects, the compound may bind to a transcriptional regulatory sequence that increases or decreases the expression of UCP-4 RNA. In another embodiment of the invention, a method is provided for screening for a compound that regulates the expression of UCP-4 in a fuel-sensing tissue. Such fuel-sensing tissues include, but are not limited to brain tissue, such as, for example, hypothalamic nuclei and the area postrema. Compounds may increase or decrease UCP-4 activity in a fuel-sensing tissue. Thus, a method is provided for treating a condition or disorder that can be ameliorated by increasing energy-minimizing responses (such as heat-wasting or inhibition of food intake) in a subject comprising administering to the subject a therapeutically effective amount of a compound that increases UCP-4 activity in a fuel-sensing tissue. A method is also provided for treating a condition or disorder that can be ameliorated by increasing energy-maximizing responses (such as promoting food ingestion or conserving body energy stores) in a subject comprising administering to the subject a therapeutically effective amount of a compound that decreases UCP-4 activity in a fuel-sensing tissue. In another embodiment of the invention is provided a method for treating conditions or disorders that can be ameliorated by increasing the level of thermogenesis in a subject, for example obesity, comprising administering to said subject a therapeutically effective amount of a compound that increases the activity of UCP-4. In one aspect, said condition or disorder is obesity. In another aspect, said condition or disorder is diabetes. By condition or disorder is meant a disease, condition, or disorder, or a susceptibility to the same. In another embodiment of the invention is provided a method for treating conditions or disorders that can be ameliorated by decreasing the level of thermogenesis in a subject, for example, in a subject with a susceptibility to malignant hyperthermia, or fever, comprising administering to said subject a therapeutically effective amount of a compound that decreases the activity of UCP-4. Also provided within the scope of the present invention is a method of preventing or treating diseases or conditions related to a decrease in thermogenesis, such as obesity, in a subject comprising administering to said subject a therapeutically effective amount of a compound that increases UCP-4 activity in said subject. Preferably, increase in said UCP-4 activity occurs in a tissue, preferably the brain, heart, pancreas, muscle, kidney, placenta, liver, lung, ovary or spinal cord tissue, more preferably the brain, heart, pancreas, or muscle tissue of said subject. Said increase in UCP-4 activity may be associated with, for example, an increase in UCP-4 activation. Said compound may the alter post-translational modification of UCP-4. Alternatively, said increase in UCP-4 activity is associated with an increase in UCP-4 gene expression. Also provided within the scope of the present invention is a method of regulating insulin secretion by administering a compound that increases or decreases the activity of UCP-4. Preferably, said UCP-4 activity occurs in the pancreas or the area postrema. Such increase or decrease in UCP-4 activity may be associated with, for example, an increase or decrease in UCP-4 activation or in UCP-4 gene expression. A method is also provided of preventing or treating diseases or conditions related to thermogenesis such as malignant hyperthermia or fever in a subject comprising administering to said subject a therapeutically effective amount of a compound that decreases the activity of UCP-4. Said compound may alter the post-translational modification of UCP-4. Alternatively, said decrease in UCP-4 activity is associated with a decrease in UCP-4 gene expression. Diagnostic methods are also provided in the present invention. In one embodiment, a method is provided for determining whether a subject has a susceptibility to a condition or disorder related to thermogenesis, such as, for example, malignant hyperthermia or obesity, comprising: probing the messenger RNA expressed in a tissue of said subject with a nucleotide probe that comprises a nucleotide sequence that is homologous to at least 15, preferably at least 30, more preferably at least 45, consecutive nucleotides of a UCP-4 nucleotide sequence; and determining whether said messenger RNA expression is increased compared to the messenger RNA in a subject that does not have a susceptibility to malignant hyperthermia. Preferably the nucleotide probe does not bind to any non-UCP-4 nucleotide sequence in the same tissue. Preferably, the nucleotide sequence is homologous to at least 10 consecutive nucleotides of a human UCP-4 sequence. In other diagnostic methods of the present invention, techniques known to those skilled in the art, such as PCR and sequencing, may be used to locate one or more point mutations or deletions in the UCP-4 gene in subjects to determine whether the subject has a susceptibility to a condition or disorder related to thermogenesis. Said mutation or deletion may or may not affect the expression level of UCP-4 RNA, but may affect the activity of UCP-4. Thus, in one aspect of the invention, a method is provided for determining whether a subject has a condition or disorder related to UCP-4 structure comprising: a) probing the RNA in a tissue of said subject with a nucleotide probe that comprises a nucleotide sequence that is homologous to at least 15 consecutive nucleotides of a UCP-4 nucleotide sequence; b) isolating the RNA bound to said probe; c) obtaining the sequence of said RNA; and d) comparing said sequence to the UCP-4 RNA of a subject without said condition or disorder or a subject with said condition or disorder. A method is also provided for determining whether a subject has a condition or disorder related to a defect in the expression level of UCP-4 in a tissue of said subject comprising determining the level of UCP-4 present in said tissue and comparing said level of UCP-4 with the level of UCP-4 in a subject that does not have a condition or disorder related to a defect in the expression level of UCP-4. In a preferred aspect, said level of UCP-4 present in said tissue is determined by probing said tissue with an antibody, preferably a monoclonal antibody, that recognizes UCP-4. In preferred aspects, the subject is determined to have a condition or disorder related to obesity or diabetes if said defect in the expression level of UCP-4 in said tissue of said subject results in a decreased level of UCP-4 as compared to a subject that does not have a condition or disorder related to obesity or diabetes said subject is determined to have a susceptibility to hyperthermia if said defect in the expression level of UCP-4 in said tissue of said subject results in an increased level of UCP-4 as compared to a subject that does not have a susceptibility to hyperthermia. In other preferred aspects, said subject may be determined to have a condition or disorder related to thermogenesis if said subject is found to have one or more deletions or point mutations in the UCP-4 gene. Said deletion or mutation may or may not affect the expression level of UCP-4 RNA, but may affect the activity of UCP-4. |
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