WO1995006411A1

WO1995006411A1 - Method and composition for weight reduction

Info

Publication number: WO1995006411A1
Application number: PCT/US1994/009799
Authority: WO
Inventors: David Kagan
Original assignee: LIPOCYTE Inc
Current assignee: LIPOCYTE Inc
Priority date: 1993-08-30
Filing date: 1994-08-29
Publication date: 1995-03-09
Anticipated expiration: 1996-02-29
Also published as: AU7717894A

Abstract

The invention provides a method of effecting weight reduction by utilizing the mitochondrial uncoupling protein, thermogenin, to convert calories to heat in brown adipose tissue resulting in a decrease in weight due to higher energy consumption.

Description

METHOD AND COMPOSITION FOR HEIGHT REDUCTION

Related Application

This is a continuation-in-part application of U.S. Serial No. 08/114,313 filed on August 30, 1993.

Field Of The Invention The present invention relates to a method of effecting weight reduction in warm-blooded animals, particularly in humans. In particular, the invention relates to a method of effecting weight reduction in which the gene coding for mitochondrial uncoupling protein (UCP) is isolated and inserted into mammalian cells to instigate thermogenesis.

Background Of The Invention It is known that during the periodic arousals from deep hibernation , animals rapidly warm themselves from hibernating body temperatures to a temperature close to that of a normal, warm-blooded animal. It has also been noted that all species which hibernate possess brown adipose tissue, sometimes also referred to as "brown fat" or "hibernation gland" although no secretion associated with hibernation has been attributed to this so-called gland. Moreover, the occurrence of brown adipose tissue, is not found only in animals which hibernate. A variety of non- hibernating animals, such as the monkey, rabbit, mouse, rat and human possess brown adipose tissue. However, other animal species, such as the cat, badger and raccoon, are believed to lack this tissue. Although the newborn human possesses tissue which histologically has the appearance of brown fat, a large percentage of such tissue becomes white adipose tissue in adulthood. However, a certain amount of brown adipose tissue does remain.

It has been reported in the literature that brown adipose tissue appears to contribute to heat production by means of oxidative processes which occur in the tissue itself, and also by release of stored lipids into circulation for use by other tissues. Brown adipose tissue in mice and rats, which are not hibernating species, contain an unusually high content of mitochondria and a paucity of other types of intracellular specializations.

The intracellular compositions of brown adipose tissue is just what one might expect to find in a tissue that has as its specialty, rapid heat production or thermogenesis. It has been noted from chemical studies that brown fat has an unusually high content of components of an electron transfer system. Present in high concentrations are mitochondria, cytochro e c and coenzyme Q (ubiguinone) which are believed to contribute to a high rate of oxygen consumption. Of interest, is that the heat production of brown adipose tissue of the mouse and rat, is much less than that of hibernating species such as the ground squirrel. Thermogenesis in brown adipose tissue involves the participation of many enzymes and proteins in the cell. The mitochondrial uncoupling protein (UCP) , also referred to as "thermogenin", is believed to be the brown adipose tissue specific protein necessary for uncoupled heat production in the mitochondria. The isolation of a cDNA clone for UCP as well as other cDNAs derived from brown adipose tissue RNAs has been reported by A. Jacobsson et al., in Journal of Biological Chemistry. Vol. 260, No. 30, pages 16250-16254 (1985).

It is disclosed in this reference that the mechanism for heat production in many non-hibernating species which are exposed to a cold environment is complex. Brown adipocytes, upon stimulation by nerve terminals from signals mediated by the hypothalamus, undergo coordinate changes in the expression of several genes which include enzymes of energy and lipid metabolism as well as the mitochondrial uncoupling protein. This protein is believed to be essential for heat production by the mitochondria. Thus, thermogenesis in brown adipose tissue requires the participation of many enzymes and proteins in the cell.

Jacobsson et al., cited above, indicated that as of 1985, only one brown adipocyte-specific gene product had been identified.

With the generation of heat in brown adipose tissue, there is a calorie consumption and therefore a corresponding loss in weight. Although humans do not possess as high a percentage of brown adipose tissue as some other species, by increasing the amount of tissue which behaves like brown adipose tissue in the generation of heat, and consequential calorie loss, there can be obtained an effective means of reducing weight.

Accordingly, one or more of the following objects will be achieved by the present invention. It is an object of the invention to provide a method of effecting weight reduction, particularly in humans. Another object of the invention is to provide a method of effecting weight reduction which utilizes the mitochondrial uncoupling protein. A further object is to provide a method of isolating the gene which encodes the mitochondrial uncoupling protein. Another object is to provide a method for the insertion of the mitochondrial uncoupling protein into cells such as fibroblasts so that they mimic brown adipose tissue. A still further object of the present invention is to provide a method for administering to humans a pharmaceutical composition which contains cells having the mitochondrial uncoupling protein. These and other objects will readily become apparent to those skilled in the art in light of the teachings disclosed herein.

Summary Of The Invention

In its broadest aspect, the present invention relates to a method for effecting weight reduction, particularly in humans, which utilizes the mitochondrial uncoupling protein. The invention also relates to pharmaceutical compositions containing mitochondrial uncoupling protein-containing cells which can be administered to humans to effect weight reduction. Processes for the preparation of such compositions are also encompassed by the invention.

The method of effecting weight reduction comprises administering to warm-blooded animals such as humans, a weight reducing effective amount of a composition comprised of a biologically acceptable carrier and mammalian cells in which the gene for the mitochondrial uncoupling protein has been expressed. Brief Description of the Drawing The following drawing is illustrative of an embodiment of the present invention and is not intended to limit the invention as encompassed by the claims forming part of the application. Figure 1 is a schematic diagram of the rat gene for mitochondrial uncoupling protein.

Detailed Description Of The Invention The mitochondrial uncoupling protein, which as indicated is also known as "thermogenin", is a 32 kd protein which is produced by brown fat cells. The protein causes the mitochondria of brown fat cells to uncouple, converting energy into heat. The consumption of calories results in a weight decrease due to the high energy consumption.

Brown fat is known as the flash heater, since it plays an important role in the central heating system that functions in an animal's arousal from hibernation and provides nonshivering thermogenesis in cold environments. Brown fat is responsive to adrenergic stimuli and is specialized for sympathetically regulated heat production associated with uncoupled oxidative phosphorylation in which energy provided by metabolic substrates, such as free fatty acids is used to produce heat.

The nucleotide and amino acid sequence for this protein is known and several variants of thermogenin are known for different species of animals. For example, Frederic Bouillard et al., "Complete cDNA-derived Amino Acid Sequence of Rat Brown Fat Uncoupling Protein", J. Bio. Che . , Vol. 261, No. 4, pages 1487- 1490 (February 5, 1986) discloses the nucleotide sequence and amino acid sequence of rat uncoupling protein and its similarities with hamster uncoupling protein. Anne-Marie Cassard et al., "Human Uncoupling Protein Gene: Structure, Comparison with Rat Gene, and Assignment in the Long Arm of Chromosome 4" discloses the complete nucleotide sequence of the 3.8 Kb 5' domain of human uncoupling protein gene. L.P. Kozak et al. reported in the Journal of Biological Chemistry, Vol. 263, No. 25, pages 12274-12277 (1988) , in an article entitled "The Mitochondrial Uncoupling Protein Gene", that the protein-sequence for mouse UCP has been obtained by reverse translation of the cDNA sequence.

The mouse, hamster and rat proteins all have 306 amino acids. The mouse protein is indicated to differ from the rat in 8 amino acids and from the hamster in 24 amino acids. Fig. 1 herein provides a schematic diagram of rat gene for mitochondrial uncoupling protein.

Sequence Listing No. 1 of the present application shows the amino acid sequence of the human uncoupling protein gene. The human and rat uncoupling protein are 79% homologous and a single probe can be used to probe for both species. Because of the functional similarity of uncoupling proteins of different species, any species may be used as a source of the uncoupling protein gene. Accordingly, the source of the uncoupling protein gene may come from human, mouse, rat, bovine, hamster and other sources. Mouse and rat uncoupling protein are preferred because brown adipose tissue is easier to obtain from these species. The nucleotide sequence of mouse uncoupling protein is disclosed in L.P. Kozak et al., "The Mitochondrial Uncoupling Protein Gene...", J. Biol. Che .. Vol. 263, pp. 12274-12277 (1988) .

The gene from any of the sources identified above may be isolated by probing a genomic library. A suitable DNA probe is shown in Sequence Listing No. 2. This probe represents a highly homologous sequence between human and rat mitochondrial uncoupling protein and can be used for either species.

The clones isolated may be counter-screened against brown adipose tissue oligo-dT RNA prepared from cold adapted rats to verify homology. On Northern blot the RNA runs at 16s and should translate a 32 Kd protein corresponding to mitochondrial uncoupling protein. The protein sequence for human mitochondrial uncoupling protein is shown in Sequence Listing No. 1.

The source of the brown adipose tissue is, for example, the interscapular brown fat pad in mice or peri-renal fat in humans. Brown adipocytes obtained from these sources are polygonal or fibroblastic with many fat droplets within the cytoplasm. They are readily differentiated from white adipocytes which contain only one or two fat droplets per cell.

Brown fat precursor cells may be isolated from the location of such cells by digesting the tissue with collagenase and subsequent growth in a culture medium supplemented with a solution of new-born calf serum and insulin. By way of illustration, the tissue may be obtained from rats or mice by aseptically dissecting the interscapular fat pad. The tissue is minced and incubated in a 0.2% collagenase solution and incubated at 37"C with gentle shaking. The digested slurry is filtered through two nylon screens (250u and 25u) to remove connective tissue and undigested material. The filtered material is then washed in two changes of medium by centrifuging at 200g for 5 minutes.

The cells are resuspended in Dulbecco's Modified Eagles Medium (DMEM) with non-essential amino acids supplemented with 10% fetal bovine serum, suitable antibiotics (e.g. penicillin- streptomycin or gentamicin) and lOU/ml insulin and plated onto tissue culture flasks. The cells have a polygonal or fibroblastic shape and contain numerous lipid vesicles.

An additional method of obtaining primary cells for culture is by tissue explant. For brown adipocytes, the interscapular fat would be dissected. The sample is placed on a dry tissue culture plate and allowed to rest undisturbed for about 10 minutes. The medium described above is carefully added to the culture plate so as not to dislodge the tissue. After several days, the cells migrate out of the tissue and the tissue is then removed. Various cell types including endothelial cells, fibroblasts and smooth muscle may be isolated with this method.

In the present invention, the gene sequence which encodes the mitochondrial uncoupling protein is cloned into a suitable expression vector or plasmid of bacterial origin by known techniques and the vector or plasmid is inserted into a suitable host cell. The vectors and host cells may be of mammalian, avian, reptilian, insect, yeast and bacterial origin.

The expression vectors which may be used as a recipient for the gene sequence are known and readily available from several sources and include those which are generally applicable to most eukaryotic cells. The preferred vectors have expression which are both constitutive and stable and include pDR2 which is available from Clontech Laboratories, Inc. Other vectors which may be used are pAD3, pCMV3, pSV? and pTK/?, are also available from, for example, Clontech Laboratories, Inc. Other appropriate expression vectors include, pSV2-dhfr, pSV3-dhfr, pSV5-dhfr, pSV2-neo, pSV3-neo, pSV5-neo and pSV2cat, all available from the American Type Culture Collection of Rockville, Maryland.

After construction of an expression vector containing the cDNA for the mitochondrial uncoupling protein, the vector is inserted in a host cell by one of several means such as transfection, electroporation, microinjection, viral infection and the like. Promoters to be employed include RSV, Ad2, HSV, chick beta-actin, SV40, CMV and the like.

The host cells which are employed in the present inventions include fibroblasts, white adipocytes and the like. The cells which may be of mammalian, avian or reptilian origin, containing the protein can be formulated into pharmaceutical compositions for administering to a patient desiring weight reduction, by incorporating the cells into a pharmaceutically acceptable carrier. The preferred carriers are those which provide physical protection for the cells. Physical protection is desirable to prevent rupture of the cells when they are transported and injected into the patient. An example of a preferred carrier is a solution of collagen of the type that has been used in recent years in collagen replacement therapy. Other carriers which may be used include isotonic saline which provides osmotic protection for the cells. The composition of the present invention may also include known additives such as preservatives, antioxidants and the like. The presence of the mitochondrial uncoupling protein may be determined by such methods as enzyme linked immunoabsorbent assay and western blot analysis as disclosed in Ellen L.P. Chan et al., "Measurement of Brown Adipose Tissue Uncoupling Protein by Enzyme Linked Immunosorbent Assay", Bioche . Int. , Vol. 23, No. 2, pp. 291-298 (January 1991) and Mary F. Henningfield et al., "Immunochemical Detection and Quantification of Brown Adipose Tissue Uncoupling Protein", Biochem. Cell. Bio., Vol. 65, pp. 245-251 (1987) .

Mitochondria are isolated from transfected cells by homogenizing in 5 times the cell pellet volume of 0.25M sucrose with 5mM K-TES (pH 7.2) in a centrifuge at 8,500g for 10 minutes. The pellet contains cell debris, nuclei and mitochondria and is resuspended to the original volume in the same medium. The resulting mixture is centrifuged at 700g for 10 minutes to pellet the nuclei and debris which is discarded. The supernatant is centrifuged at 8,500g for 10 minutes to pellet the mitochondria. Mitochondrial uncoupling protein is isolated from brown adipose tissue mitochondria or from transfected cells as prepared above. 500mg of mitochondrial protein is resuspended in 30ml (2% Brij , 20mM Na-T-SO,,, 10 mM MOPS, pH 6.7) by mixing well for 5 minutes at 0°C. The resulting suspension is centrifuged at 800g for 5 minutes and the supernatant discarded. The resulting pellet is resuspended in 20ml of 20mM Na₂S0₄ and lOmM MOPS at a pH of 6.1 and centrifuged at 8,000g for 5 minutes. The resulting pellet is shaken for 5 minutes in 30ml of 3% Triton-XlOO, 30mM Na₂S0₄, and 15mM of MOPS at a pH of 6.7. The resulting suspension is centrifuged at 10,000g for 20 minutes. The supernatant is added to a 70ml (3x10cm) hydroxylapatite column pre-equilibrated with 30mM Na₂S0₄ and 15mM MOPS at a pH of 6.7 and eluted with the same medium. The first 30ml are pooled and concentrated by pressure dialysis to a volume of from 5 to 8ml.

The resulting material can be used to coat microtiter plates for an enzyme linked immunosorbent assay as described hereinafter. Rabbits are immunized with rat-mitochondrial uncoupling protein and blood samples containing anti-rat- mitochondrial uncoupling protein antiserum are obtained. Alternatively, primary polyclonal rabbit anti-thermogenin antibody serum can be obtained from East Acres Biologicals of Southbridge, Massachusetts.

The mitochondrial uncoupling protein is subjected to sodium dodecyl sulfate-polyacryla ide gel electrophoresis to obtain a gel containing the protein. The protein is transferred from the gel onto nitrocellulose paper by electroblotting. The paper is then incubated with antiserum produced as described above. A paper containing the same protein is incubated with normal rabbit serum as a negative control.

An enzyme linked immunosorbent assay can be developed by adsorbing purified mitochondrial uncoupling protein to microtiter plates. After washing to remove unbound protein and blocking with bovine serum albumin, a dilute rabbit anti-mitochondrial uncoupling protein serum is added to the appropriate wells. The unbound antibody is then washed and an anti-rabbit serum conjugated to horseradish peroxidase or other suitable reporter molecule is added. The substrate is washed and developed. High titers of mitochondrial uncoupling protein will be reflected by high substrate conversion.

The function of the mitochondrial uncoupling protein employed in the present invention may be determined by first isolating mitochondria from transfected cells and then isolating the mitochondrial uncoupling protein as described above. The function of the mitochondrial uncoupling protein can be verified by photoaffinity labelling of the regulatory site of energy dissipation as described in G.M. Heaton et al. , "Brown Adipose- tissue Mitochondria: Photoaffinity Labeling of the Regulatory Site of Energy Dissipation", J. Biochem. , Vol. 82, pp. 515-521 (1976) . The activity of the uncoupling protein as a high conductance ion uniport through which H⁺ ion can cross the mitochondrial membrane without ATP synthesis. Since brown adipose mitochondria are uncoupled due to the mitochondria uncoupling protein, the addition of nucleotides in-vitro will bind to the uncoupling protein thereby inhibiting the uncoupling of the mitochondria.

A competitive assay may be established by adding [γ^"3P] 8- azido-adenosine 5'triphosphate which is cross-linked to the active site of the uncoupling protein and will compete with GDP (guanosine 5'-diphosphate) for binding to the uncoupling protein. The reduction in ATP counts will relate to the activity of the uncoupling protein in the sample. In order to effect weight reduction, the compositions prepared in accordance with the teachings of this invention, are administered to a patient preferably by subcutaneous injection. The dosage amount will vary over a wide range, given at appropriate intervals. It is preferred to provide the composition in a dose amount of at least 10⁶ cells.

The desired dosage may be estimated by calculating the uncoupling activity of a given preparation of cells and adjusting for the desired activity. The following Example is illustrative of the invention and is not intended to limit the invention as encompassed by the claims forming part of the application.

EXAMPLE

Construction Of A Plasmid/Vector Containing The UCP Gene And Expression Of The Gene In Cells

The pDR2 vector from Clontech Laboratories, Inc. with the gene for neomycin resistance is opened at a position in the polylinker and the cDNA for UCP is cloned in. The plasmid is then selected for on ampicillin and grown up as a maxi-prep in the following manner.

The bacterial stock containing the plasmid is inoculated into 250ml of Luria-Bertrani broth containing 50ug/ml Ampicillin. This preparation is grown overnight at 37°C with vigorous shaking. The broth is centrifuged at 5,000 rpm for 10 minutes to pellet the bacteria. The resulting pellet is resuspended in 10ml of TRIS 50mM/EDTA lOmM (pH8) . 10ml of NaOH 200mM/l% SDS are added to the suspension and gently mixed by inversion. 10ml of 3M KHOAc (pH 5.5) is added to the mixture. The resulting preparation is mixed well and centrifuged at 5,000 rpm for 10 minutes. The supernatant is precipitated in one volume of isopropanol and centrifuged at 10,000 rpm for 15 minutes. The resulting pellet is resuspended in 5ml of water. 5g of solid cesium chloride is mixed to dissolve the suspended material.

80ug of lOmg/ml of ethidium bromide is added to each one ml of DNA/cesium solution, mixed and placed in an ultracentrifuge tube. The tube is topped off with light paraffin oil, sealed and loaded into an ultracentrifuge.

The ultracentrifuge is operated at 70,000 rpm overnight. The lower closed circular band of DNA is obtained from the ultracentrifuge tube using a syringe. An equal volume of water saturated with 1-butanol is added to the thus removed DNA band and mixed to remove the ethidium bromide. The resulting mixture is centrifuged at 1500g to separate the phases. The lower aqueous phase is transferred to a new tube and the extraction of ethidium bromide is repeated as necessary until the aqueous portion no longer has a pink color. The DNA is then precipitated with ethanol, washed and resuspended in water to a concentration of one ug/ul.

To precipitate the DNA for transfection, 18ug of the DNA is added to a tube with 420ul of water and then is added 500ul of 2XHBS comprised of 280mM NaCl, lOmM KC1, 1.5mM Na₂HP0₄, 12mM dextrose and 50mM HEPES at a pH of 7.05. The tube is then vortexed at low speed while adding 63ul of 2M CaCl₂ by dropwise addition. The tube is then allowed to stand at room temperature for 30 minutes to allow precipitate to form.

The cells for transfection are trypsinized and 2.5 x 10⁶ cells are added to a tube pelleted at 200g for 5 minutes and the supernatant is then aspirated. The cell pellet is then resuspended in the DNA precipitate prepared above and incubated for 15 minutes at room temperature.

9 ml of DMEM + serum is added to the tube and the contents of the tube are then transferred (5ml each) to two 100mm tissue culture plates. The culture medium is changed every 24 hours. Cells are suitable as transients 48 hours after transfection or may be selected for stable expression. The presence of the gene product can be verified by ELISA or Western Blot. The selected cells now express the gene for mitochondrial uncoupling protein. These cells are trypsinized and washed and resuspended in saline for injection directly into a subcutaneous location in the patient.

Alternately, these cells may also be mixed with an isotonic solution of collagen and injected into a subcutaneous location in the patient.

Although the invention has been illustrated by the preceding example, it is not to be construed as being limited to the materials employed therein, but rather, the invention encompasses the generic area as herein before disclosed. Various modifications and embodiments thereof can be made without departing from the spirit and scope thereof. (1) GENERAL INFORMATION:

(i) APPLICANT: Kagan, David

(ii) TITLE OF INVENTION: Method and Composition for Weight

Reduction

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Watov & Kipnes, P.C.

(B) STREET: 186 Princeton-Hightstown Rd, PO Box 247

(C) CITY: Princeton Junction

(D) STATE: New Jersey

(E) COUNTRY: USA

(F) ZIP: 08550

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.50 inch, 800 Kb storage

(B) COMPUTER: IBM

(C) OPERATING SYSTEM: DOS 6.0

(D) SOFTWARE: WordPerfect 5.1 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: 08/249,179

(B) FILING DATE: 25 May 1994

(C) CLASSIFICATION: 514 (VU) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/114,313

(B) FILING DATE: 30 August 1993 (viϋ) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kipnes, Allen R.

(B) REGISTRATION NUMBER: 28,433

(C) REFERENCE/DOCKET NUMBER: 489.1.001A (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 609-243-0330

(B) TELEFAX: 609-275-1010

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: Linear (vi) ORIGINAL SOURCE:

(A) ORGANISM: Human

(G) CELL TYPE: Brown adipose (ix) FEATURE:

(D) OTHER INFORMATION: Cassard, A.M. et al. publication

Genbank - Locus 37607 (X) PUBLICATION INFORMATION:

(A) AUTHORS: Cassard, A.M. et al.

(B) TITLE: Human Uncoupling Protein Gene: Structure,

Comparison With (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Met Gly Gly Leu Thr Ala Ser Asp Val His Pro Thr Leu Gly Val 1 5 10 15

Gin Leu Phe Ser Ala Pro lie Ala Ala Cys Leu Ala Asp Val lie

20 25 30

Thr Phe Pro Leu Asp Thr Ala Lys Val Arg Leu Gin Val Gin Gly

35 40 45

Glu Cys Pro Thr Ser Ser Val lie Arg Tyr Lys Gly Val Leu Gly

50 55 60

Thr lie Thr Ala Val Val Lys Thr Glu Gly Arg Met Lys Leu Tyr

65 70 - 75

Ser Gly Leu Pro Ala Gly Leu Gin Arg Gin lie Ser Ser Ala Ser

80 85 90

Leu Arg lie Gly Leu Tyr Asp Thr Val Gin Glu Phe Leu Thr Ala

95 100 105

Gly Lys Glu Ser Lys Pro Leu Gly Ser Lys lie Leu Ala Gly Leu

110 115 120

Thr Thr Gly Gly Val Ala Val Phe lie Gly Gin Pro Ser Glu Val

125 130 135

Val Lys Val Arg Leu Gin Ala Gin Ser His Leu His Gly lie Lys

140 145 150

Pro Arg Tyr Thr Gly Thr Tyr Asn Ala Tyr Arg lie lie Ala Thr

155 160 165

Thr Glu Gly Leu Thr Gly Leu Thr Lys Gly Thr Thr Pro Asn Leu

170 175 180

Met Arg Ser Val lie lie Asn Cys Thr Glu Leu Val Thr Tyr Asp

185 190 195

Leu lie Lys Glu Ala Phe Val Lys Asn Asn lie Leu Ala Asp Asp

200 205 210

Val Pro Cys His Leu Val Ser Ala Leu lie Ala Gly Phe Cys Ala

215 220 225

Thr Ala Met Ser Ser Pro Val Asp Val Val Lys Thr Arg Phe lie

230 235 240

Asn Ser Pro Pro Gly Gin Tyr Lys Ser Val Pro Asn Cys Ala Met

245 250 255

Lys Val Phe Thr Asn Glu Gly Pro Thr Ala Phe Phe Lys Gly Leu

260 265 270 Val Pro Ser Phe Leu Arg Leu Gly Ser Trp Asn Val lie Met Phe

275 280 285

Val Cys Phe Glu Gin Leu Lys Arg Glu Leu Ser Lys Ser Arg Gin

290 295 300

Thr Met Asp Cys Ala Thr

305

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 186 base pairs

(B) TYPE: Nucleic acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: Linear (Vi) ORIGINAL SOURCE:

(A) ORGANISM: Rat (G) CELL TYPE: Brown adipose (X) PUBLICATION INFORMATION:

(A) AUTHORS: F. Bouillard et al.

(B) TITLE: Detection of Brown Adipose Tissue Uncoupling

Protein in mRNA in Adult Patients by a Human Genomic Probe

(C) JOURNAL: Clinical Science

(D) VOLUME: 75

(E) PAGES: 21-27

(F) DATE: (1988)

( i) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TTA GGA AGC AAG AAT TTA GCT GGT CTA ACG 30

ACT GGA GGA GTG GCC GTA TTC ATT GGG CAA 60

CCC TCA GAG GTC GTG AAA GTC AGA CTT CAA 90

CAG AGC CAT CTC CAC GGA ATC AAA CCT CGC 120

TAC ACG GGG ACT TAT AAT GCG TAC AGA ATA 150

ATA GCA ACA ACC GAA GGC TTG ACG GGT CTT 180

TGG AAA 186

Claims

WHAT IS CLAIMED IS:

1. A method of effecting weight reduction in a warm blooded animal which comprises administering to said warm blooded animal a weight reducing effective amount of a pharmaceutical composition comprised of a pharmaceutically acceptable carrier and mammalian cells in which the gene for mitochondrial uncoupling protein has been expressed.

2. The method of Claim 1 wherein the carrier is a saline solution or a collagen solution.

3. The method of Claim 1 wherein the composition is administered in a dose amount of at least about 10⁶ cells.

4. A pharmaceutical composition useful for effecting weight reduction comprised of a pharmaceutically acceptable carrier and mammalian cells in which the gene for the mitochondrial uncoupling protein has been expressed.

5. The composition of Claim 4 wherein the carrier is a saline solution or a collagen solution.

6. The composition of Claim 4 in a dosage form containing at least about 10⁶ cells.

7. The composition of Claim 6 wherein the dosage form is in from about 5 to 10 ml of saline solution.

8. A method for preparing a pharmaceutical composition useful for effecting weight reduction which comprises the steps of: (a) isolating the gene which encodes the mitochondrial uncoupling protein; (b) preparing a vector containing the gene for encoding the mitochondrial uncoupling protein; (c) transferring said vector into host cells for expression of the protein; and (d) contacting said cells with a pharmaceutically acceptable carrier.

9. The method of claim 8 wherein the vector is selected from pDR2, pAD3, pCMV/3, pSV3, pTKyS, pSV2-dhfr, pSV3-dhfr, pSV5- dhfr, pSV2-neo, pSV3-neo, pSV5-neo and pSV2cat.

10. The method of claim 8 wherein the host cells are selected from the group consisting of fibroblasts and white adipocytes.

11. The method of claim 8 wherein the pharmaceutical carrier is a saline solution or a collagen solution.

12. The method of claim 8 comprising obtaining fibroblasts or white fat cells from the warm blooded animal and transferring said vector into said cells for expression of the uncoupling protein.

13. The method of claim 12 wherein the warm blooded animal is a human.

14. A method of forming cultured brown adipocytes for administration to a warm blooded animal comprising:

(a) obtaining brown adipocyte precursor cells from a source;

(b) treating the precursor cells to form a slurry containing digested cells;

(c) filtering the slurry to remove undigested material to form a slurry of brown adipocyte cells;

(d) suspending the cells brown adipocyte in a nutrient medium containing an antibiotic; and

(e) placing the resulting cells on a suitable substrate.

15. The method of claim 14 wherein step (b) comprises treating the precursor cells with collagenase and a culture medium.

16. The method of claim 14 further comprising administering the resulting cells to a human.