EP0535038A4 - Mammalian adipogenic factors - Google Patents

Abstract

Mammalian adipogenic factors capable of inducing the adipose differentiation of adipogenic cells are disclosed, as are antibodies to such factors. A method for determining the susceptibility of a subject to obesity by measuring the levels of one or more adipogenic factors in a biological fluid or tissue extract is also disclosed, as is a method for evaluating an anti-obesity drug which comprises contacting the drug with cells capable of producing one or more adipogenic factors and measuring the amount of the factors produced.

Description

MAMMALIAN ADIPOGENIC FACTORS

BACKGROUND OF THE INVENTION

Field of the Invention

The invention in the field of cell biology, physiology and medicine relates to purified mammalian adipogenic factors, genetic constructs thereof, antibodies thereto, and methods of using such factors to determine susceptibility to obesity and for evaluating efficacy of anti-obesity drugs.

Description of the Background Art Adipose differentiation of adipogenic cell lines is under the control of factors called adipogenic factors which either trigger or stimulate the process of differen¬ tiation. The isolation and complete identification of adipogenic factors is important as, 1) they are responsible for turning on the differentiation program; 2) reports in the literature have disclosed that abnormal levels of circulating adipogenic factors exist in the blood of obese patients (Lau, D.C. . et al., 1984 Proc. 7th International Congress Endocrinology Excerpta Medica, p. 866) . Adipo- genie factors have been found in fetal bovine serum and in human serum and plasma. Crude fetuin preparations have been characterized as having an adipogenic activity that is heat sensitive and acid (pH 1) sensitive; the activity was apparently attributed to the fetuin within the preparation. D. Gaillard et al (1985) Biochem. Biophys. Acta 846, 185- 191.

Additional reports of bovine or human factors in serum or plasma, in which there was little or not charac¬ terization of the physico-chemical properties of the fac- tors are Y.Y. Meada et al (1980) EXP. Cell. Res. 126, 99- 107; W. Kuri-Harcuch and H. Green (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 6107-6109; G. Serrero et al (1979); in "Hormone and Cell Culture", Cold Spring Harbor Confer¬ ence on Cell Proliferation, Vol. 6, (R. Ross and G. Sato, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); D. Gaillard et al (1984), In Vitro Cell. Dev. Biol. 2ϋ, 79-88; and G. Sypniewska et al (1986) Int. J. Obesity 10, 265-276.

Aproliferin, the factor purified from human plasma is a 45 kDa protein. [M. L. Weir and R. E. Scott, Am. J. Phvsiol. (1982), vol. 125, pp.546-554.] It induces loss of proliferative potential of 3T3-T proadipocytes. By its molecular weight and its mode of action (P. Grimaldi et al (1982) EMBO J. 1 , 687-692) , aproliferin is different from the adipogenic factors we have discovered. Addition¬ ally, an active fraction has been isolated from fetal calf serum [P. Grimaldi et al (1982) EMBO J. 1 , 687-692]. It is heat labile acid stable and protease stable. It is likely that the active component fraction is arachidonic acid, a fatty acid. D. Gaillard, et al (1989) Biochem. J., 257. 389-397. SUMMARY OF THE INVENTION

The present invention is directed to novel mamma¬ lian, including human, adipogenic factors. These adipo¬ genic factors which appear to play an important role in the generation of fat cells in mammals, have are useful in a method for determining the susceptibility to obesity in a subject. The adipogenic factors are also useful for evalu¬ ating the efficacy of anti-obesity drugs.

The invention is directed first to a mammalian adipogenic factor having an apparent molecular weight of about 150 to 230 kDa which is isolatable from liver cells and has adipogenic activity substantially greater than that of naturally occurring liver cells or hepatocytes in cul¬ ture on a per milligram protein basis. The adipogenic activity of this factor is susceptible to destruction by treatment with pronase, with high temperatures of about 100°C, with a pH of about 2.5, and with 0.2 M 2-mercapto- ethanol. In a preferred embodiment, the factor is of human origin and has specific adipogenic activity at least about 625 times that of the conditioned medium obtained from confluent cultures of HepG2 hepatocarcinoma cells. A preferred source of this adipogenic factor is a liver cell line or conditioned medium from such a line.

The invention is also directed to a mammalian adipogenic factor having an apparent molecular weight of about 660 kDa which is isolatable from serum and has adipo¬ genic activity substantially greater than that of serum on a per milligram protein basis. Its adipogenic activity is susceptible to destruction by treatment with pronase, high temperature of about 100°C, and resistant to treatment with 0.2 M 2-mercaptoethanol at 25°C for 6 hours at pH 2.5. In a preferred embodiment, this factor has a specific adipo- genie activity at least 10 times that of serum and of crude fetuin.

The invention is further directed to a mammalian adipogenic factor having an apparent molecular weight of about 230 kDa which is isolatable from serum and has adipo¬ genic activity substantially greater than that of serum on a per milligram protein basis. Its adipogenic activity is susceptible to destruction by treatment with pronase, with a pH of about 2.5 and about 11.0, and is susceptible to partial destruction by treatment with 0.2 M 2-mercapto- ethanol at 25°C for 6 hours. In a preferred embodiment, this factor has a specific activity at least 250 times that of serum and of crude fetuin.

The invention is also directed to a mammalian adipogenic factor isolatable from serum and having an apparent molecular weiςnt of about 50 to 69 kDa, the factor being different from the 69 kDa glycoprotein known as "pure fetuin." The adipogenic activity of this factor is sub¬ stantially greater than that of serum on a per milligram protein basis. In a preferred embodiment, this factor has a specific adipogenic activity at least 2 times that of serum or crude fetuin. The present invention also involves a method for determining the susceptibility of a subject to obesity which comprises removing a sample of a biological fluid or tissue from the subject and measuring the amount of one or more of the above-described adipogenic factors present in the fluid or tissue. The amount of the adipogenic factor is proportional to the susceptibility of the subject to obesity.

The invention also includes a method for evalu- ating the efficacy of an anti-obesity drug which comprises contacting the drug being evaluated with an adipogenic cell in vitro and measuring the amount of one or more of the above-described adipogenic factors produced by the cell.

Another embodiment of the invention is an anti¬ body, either polyclonal or monoclonal, specific for an adipogenic factor. Such antibodies are useful both in isolation and purification of the factors as well as in the methods of the invention directed to evaluating anti- obesity drugs or in determining susceptibility to obesity. The antibodies are also useful in methods for treating obesity wherein an antibody to an adipogenic factor is administered to a subject who is susceptible to obesity based on increased levels of the adipogenic factor.

The invention is further directed to polynucleo- tide molecules, including RNA and DNA which encode the adipogenic factors, as well as to vectors comprising the DNA encoding the adipogenic factors, and prokaryotic and eukaryotic host cells transformed or transfected, and capable of expressing, the DNA.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. SDS-PAGE Sephacryl S-300 fractions of a human adipogenic factor preparation. Starting from the left side of the figure, the first four gels represent material from the column fractions spanning 70-200 kDa, 200-220 kDa, 220-400 kDa, and >400 kDa. The fraction spanning 200-220 kDa contained the highest adipogenic activity. MS refers to ammonium sulfate-concentrated conditioned medium.

Figure 2. SDS-PAGE, in the absence (panel A) or presence of 2-mercaptoethanol (panel B) , of aliquots of crude fetuin (far left lane in each panel) , the flow- through fraction from a chromatofocusing column (second lane from the left) , the heparin-sepharose flow-through fraction (second lane from the right) , and the hepa in sepharose fraction eluted with 1.0 M NaCl.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The inventors have discovered and purified one human and two bovine adipogenic factors.

The use of the adipogenic cell line, 1246, which can be maintained in defined medium, and the use of fetuin as the starting material were the keys for purification of the bovine factors. Moreover, the use of 1246 cells has allowed the characterization of the human factor, since these cells, for proliferation, require only small amounts of the growth factors which are present in the fetuin but absent in human HepG2 conditioned medium (CM) , a preferred source of the human factor; other adipogenic cell lines (such as 3T3-L1 and Obl7) require greater amounts of the growth factors not found in conditioned medium (CM) of HepG2 cells, a human hepatocyte-like cell line (Knowles, B.B. et al. Science 209:497-499 (1980)), rendering the bioassay for the adipogenic factor in these latter cell lines more difficult to interpret.

For isolation and characterization of the adipo- genie factor, a bioassay measuring the induction of glycerol-3-phosphate dehydrogenase (G3PDH) during adipose differentiation is utilized (L. Wise and H. Green (1979) J.

Biol. Chem. , 254, 273-275) . The induction of this enzyme is extremely powerful (>100 fold) , easy to measure, and is correlated with the degree of cell differentiation. Other parameters that can be measured to assess adipogenic factor activity include the amount of triglyceride accumulated per cell and the "frequency" of differentiation (represented by the proportion of differentiated adipocytes of the total number of cells.)

Using the bioassay, the inventors discovered that an adipogenic factor is produced by normal rat hepatocytes in culture. That resulted in the identification of liver cells as the physiological source of the adipogenic factor in vivo, a discovery important for the subsequent discovery of the human adipogenic factor in the supernatant of the human hepatocyte-like cell line, HepG2. Additionally, a bovine adipogenic factor was isolated from fetuin, a bovine serum substitute known to stimulate proliferation and various functions in several different types of cells in vitro (D. Salomon et al. (1984) , in Cell Culture Methods for Molecular and Cell Biology. Vol 3 , D.W. Barnes et al. , Eds., Alan R. Liss Inc., New York, pp 125-153.)

The term "mammalian adipogenic factor" refers to a molecule which has the capability of inducing adipose differentiation of adipogenic cells. The adipogenic fac¬ tors contemplated within the scope of the present invention are not limited to those which are purified from liver cells or serum, but to proteins or glycoproteins having adipogenic activity which have been chemically synthesized (by chemical and biochemical techniques) or produced using recombinant DNA technology.

The term "adipogenic" refers to cells or factors which are "fat producing." Thus, an adipogenic cell is a cell which can become an adipocyte (fat cell) . An adipo¬ genic factor is a substance which can induce or stimulate the differentiation of cells which are precursors of adipo¬ cytes, such as preadipocytes, to adipocytes. Also intended by the term "adipogenic factor" is a substance which can stimulate proliferation of preadipocytes or adipocytes. Adipose differentiation can be measured in any of a number of ways which are known to those skilled in the art. A preferred way of measuring adipose differentiation is by the induction of the enzyme, G3PDH, as described herein. The assay can be done, without undue experimentation, by one of skill in the art.

The enzyme, glycerol-3-phosphate dehydrogenase (G3PDH) , represents a differentiation marker which is suitable for assaying the differentiation-inducing activity of the adipogenic factors of the present invention and is easy to quantitate.

This enzyme is inducible by adipogenic agents. In the presence of an adipogenic factor, the level of G3PDH in an adipogenic cell, such as, for example, in the 1246 cell line, is increased by about 3-10 fold. In the 3T3-L1 cell line the enzyme level is induced as high as 100 fold. Induction of this enzyme is also measurable in primary cultures of epididymal fat pads. The induction of high levels of G3PDH specific enzyme activity is therefore an extremely useful bioassay during purification of an adipo¬ genic factor. A 2-fold increase in the G3PDH activity is considered induction.

In assessing whether a preparation contains an adipogenic factor with adipogenic activity "substantially greater" than that of the naturally occurring cells or the serum, one compares the specific adipogenic activity in the preparation with the activity of a liver tissue homogenate or in the conditioned medium of a normal or transformed hepatocyte cell line. "Specific adipogenic activity" refers to the amount of activity per mg (or other weight unit) protein in the preparation.

As alternatives to purified or recombinant adipo¬ genic factor, functional derivatives of the adipogenic factor may be used.

By "functional derivative" is meant a "fragment," "variant," "analog," or "chemical derivative" of the adipo¬ genic factor, which terms are defined below. A functional derivative retains at least a portion of the function of the adipogenic factor which permits its utility in accor¬ dance with the present invention. A "fragment" of the adipogenic factor refers to any subset of the molecule, that is, a shorter peptide.

A "variant" of the adipogenic factor refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conve¬ niently prepared by direct chemical synthesis of the vari¬ ant peptide, using methods well-known in the art.

Alternatively, amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combina¬ tion of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and pref¬ erably will not create complementary regions that could produce secondary mRNA structure (see European Patent Publication No. EP 75,444).

At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis (as exemplified by Adelman et al.. DNA 2.:183 (1983)) of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. The variants typically exhibit the same qualitative biological activity as the nonvariant peptide.

An "analog" of the adipogenic factor refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.

A "chemical derivative" of the adipogenic factor contains additional chemical moieties not normally a part of the peptide. Covalent modifications of the peptide are included within the scope of this invention. Such modifi¬ cations may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamido ethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloro-mercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa- 1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Deriva- tization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include i idoesters such as methyl picolinimidate; pyridoxal phos¬ phate; pyridoxal; chloroborohydride; tri- nitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenyl- glyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK_a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studied extensively, with particular interest - lo ¬

in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitro- methane. Most commonly, N-acetylimidizol and tetranitro- methane are used to form O-acetyl Carboxy1 side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N-C-N-R¹) such as 1 cyclohexyl-3-(2- morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3 (4 azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequent¬ ly deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful for cross-linking the peptide to a water-insoluble support matrix or to other macromolecular carriers. Commonly used cross-linking agents include, e.g., l,l-bis(diazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobi- functional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succini idylpropionate) , and bifunctional maleimides such as bis-N-maleimido-l,8-octane. Derivatizing agents such as methyl-3-[ (p-azidophenyl) ] dithiopropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or theonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecule Properties, W.H. Freeman St Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups.

Such derivatized moieties may improve the solu- bility, absorption, biological half life, and the like. The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences. 16th ed. , Mack Publishing Co., Easton, PA (1980)

A "liver cell line" includes hepatocytes derived from a liver or a cell line having hepatocyte functions, such as hepatocarcinoma cell line, as exemplified by HepG2. "Conditioned medium" refers to any culture medium in which cells have been incubated. A specific example is described herein. Generally, media are chosen that do not have significant deleterious effects on cell viability and the ability of the cell to produce a product which is being purified or assayed in a bioassay.

For use as an antigen for induction of anti¬ bodies, a fraction of the HepG2 derived human adipogenic factor, or a serum-derived adipogenic factor, preferably a purified fraction, is obtained and used to immunize an animal. In a preferred embodiment, a mouse is immunized with this antigen. In a more preferred embodiment, the mouse is of the inbred strain, Balb/c. The term "antibody" refers both to monoclonal antibodies (mAbs) which are a substantially homogeneous population and to polyclonal antibodies which are heterogeneous populations. Polyclonal antibodies are derived from the sera of animals immunized with the above antigen stein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

Hybridoma εupernatants are screened for the presence of antibody specific for the adipogenic factor by any of a number of immunoassays, including dot blots and standard enzyme immunoassays (EIA or ELISA) , which are well-known in the art. Once a supernatant has been identi¬ fied as having antibodies, it may be further screened by Western blotting to identify the size of the antigen to which the antibody binds. One of skill in the art will know how to prepare and screen such hybridomas without undue experimen The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')₂, which are capable of binding the antigen. Fab and F(ab*)₂ fragments lack the Fc fragment of intact antibody, clear F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al.. J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab')₂ and other used for the detection and quantitation of adipogenic factors according in the same manner as an intact antibody. Such fragments are typically produced by proteolytic cleav¬ age, using enzymes such as papain (to produce Fab frag¬ ments) or pepsin (to produce F(ab')₂ fragments).

Polyclonal or monoclonal antibodies can be used in an immunoaffinity column to purify the adipogenic factor by a one step procedure, using methods known in the art. The antibodies of the invention are useful for detecting and quantitate the adipogenic factors in an immunoassay, such as, for example, radioimmunoassay (RIA) or enzyme immunoassay (EIA) . Such assays are well-known in the art, and one of skill will readily know how to carry out such assays using the antibodies and adipogenic factors of the present invention.

Such immunoassays are useful for detecting and quantitating an adipogenic factor in the serum or other biological fluid, or in a tissue sample or tissue extract, from a normal or obese subject. In a preferred embodiment, the concentration of one or more of the adipogenic factors of this invention is measured in a tissue extract or bio¬ logical fluid of a subject as a means for determining the susceptibility or the propensity of the subject for obesity. The susceptibility of a subject to obesity is said to be proportional to the level of the adipogenic factor. The term "proportional" as used herein is not intended to be limited to a linear or constant relationship between the level of the adipogenic factor and the suscep- tibility to obesity. The nature of the relationship between factor level and susceptibility or propensity to obesity may be highly complex. For example, the doubling of the concentration of an adipogenic factor is not neces¬ sarily indicative of a doubling in the susceptibility to obesity. The term "proportional" as used herein is intend¬ ed to indicate that an increased level of factor is related to an increased propensity to obesity at ranges of concen¬ tration of the factor that can be readily determined by one of skill in the art. Another embodiment of the invention is evaluating the efficacy of anti-obesity drug or agent by measuring the ability of the drug or agent being evaluated to inhibit the production of one or more of the adipogenic factors of this invention by a cell or cell line capable of producing such factors. The antibodies of the present invention are useful in the method for evaluating anti-obesity drugs in that they can be employed to determine the amount of the adipogenic factor in one of the above-mentioned immuno¬ assays. Alternatively, the amount of adipogenic factor produced is measured by bioassay, as described herein. The bioassay and immunoassay can be used in combination for a more precise assessment of the factor or factors present. One embodiment of the present invention is directed to polynucleotide molecules, particularly DNA, encoding the adi•pogenic factors. Another embodi•ment is directed to the preparation of the adipogenic factors using recombinant DNA techniques. Also intended are vectors comprising the DNA, and host cells transformed or trans- fected with the DNA encoding an adipogenic factor.

The DNA encoding the polypeptide portion of the adipogenic factors of the present invention is either

5 synthesized chemically, prepared as genomic DNA, or pre¬ pared as cDNA from cellular mRNA. DNA sequences encoding the adipogenic factor or a portion or a variant thereof are inserted into an appropriate vector, such as a plasmid or virus, and introduced into an appropriate host cell, either 0 prokaryotic or eukaryotic. Such techniques are set forth, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Second edition, Cold Spring Harbor Laboratory Press, 1989) , which is hereby incorporated by reference.

I⁵ Based on the amino acid sequence of the adipo¬ genic factor, oligonucleotide probes can be prepared and used to isolated DNA (genomic or cDNA) encoding the pro¬ tein. Techniques for synthesizing such oligonucleotides are disclosed by, for example, Wu, R. , et al.. Prog. Nucl.

20 Acid. Res. Molec. Biol. 21:101-141 (1978). Procedures for constructing recombinant molecules in accordance with the above-described method are disclosed by Sambrook, J. et al. (supra) . Molecules are fragmented as with cyanogen bro¬ mide, or with proteases such as papain, chymotrypsin,

25 trypsin, etc. (Oike, Y., et al.. J. Biol. Chem. 257:9751- 9758 (1982); Liu, C. , et al.. Int. J. Pept. Protein Res. .21:209-215 (1983)). Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the

30 Gene. 4th Ed., Benjamin/Cummings Publishing Co. Inc., Menlo Park, CA (1987)). Using the genetic code, one or more different oligonucleotides can be identified, each of which would be capable of encoding a portion of the adipogenic factor peptide. The probability that a particular oligo¬

35 nucleotide will, in fact, constitute the actual adipogenic factor peptide-encoding sequence can be estimated by con¬ sidering abnormal base pairing relationships and the fre¬ quency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells. Such "codon usage rules" are disclosed by Lathe, R. , et al. , J. Molec. Biol. 183:1-12 (1985). Using the "codon usage rules" of Lathe, a single oligonucleotide, or a set of oligonucleotides, that contains a theoretical "most proba¬ ble" nucleotide sequence capable of encoding the adipogenic factor peptide sequences is identified.

Although occasionally an amino acid sequences may be encoded by only a single oligonucleotide, frequently the amino acid sequence may be encoded by any of a set of similar oligonucleotides. Importantly, whereas all of the members of this set contain oligonucleotides which are capable of encoding the adipogenic factor peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment, only one member of the set contains the nucleotide sequence that is identical to the nucleotide sequence of the gene. Because this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.

The oligonucleotide, or set of oligonucleotides, containing the theoretical "most probable" sequence capable of encoding the adipogenic peptide is used to identify the sequence of a complementary oligonucleotide or set of oligonucleotides which is capable of hybridizing to the "most probable" sequence, or set of sequences. An oligo¬ nucleotide containing such a complementary sequence can be employed as a probe to identify and isolate the adipogenic factor gene (Sambrook, J. et al.. supra) .

A suitable oligonucleotide, or set of oligo¬ nucleotides, which is capable of encoding a fragment of the adipogenic factor gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identi¬ fied (using the above-described procedure) , synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing the adipogenic factor gene, such as, for example, HepG2. Single stranded oligo¬ nucleotide molecules complementary to the "most probable" adipogenic factor peptide encoding sequences can be syn- thesized using procedures which are well known to those of ordinary skill in the art (Belagaje, R. , et al. , J. Biol. Chem. 254:5765-5780 (1979); Maniatis, T. , et al.. In: Molecular Mechanisms in the Control of Gene Expression. Nierlich, D.P., et al. , Eds., Acad. Press, NY (1976); Wu, R. , et al. , Prog. Nucl. Acid Res. Molec. Biol. 21:101- 141 (1978); Khorana, R.G., Science 203:614-625 (1979)). Additionally, DNA synthesis may be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook, J. et al. (supra) and by Haymes, B.D., et al. (In: Nucleic Acid Hybridiza¬ tion. A Practical Approach. IRL Press, Washington, DC (1985) , which references are herein incorporated by refer¬ ence. Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for human aldehyde dehydrogenases (Hsu, L.C., et al. , Proc. Natl. Acad. Sci. USA 82:3771-3775 (1985), fibronectin (Suzuki, S., et al.. Eur. Mol. Biol. Organ. J. 4_.:2519- 2524 (1985), the human estrogen receptor gene (Walter, P., et al. , Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-type plasminogen activator (Pennica, D., et al. , Nature 301:214-221 (1983)) and human term placental alka¬ line phosphatase complementary DNA (Kam, W. , et al. , Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985).

In a alternative way of cloning the adipogenic factor gene, a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA (from a cell capable of expressing adipogenic factor, such as HepG2) into an expression vector. The library is then screened for mem¬ bers capable of expressing a protein which binds to anti- adipogenic factor antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as adipogenic factor, or fragments thereof. In this embodiment, DNA, or more pref¬ erably cDNA, is extracted and purified from a cell which is capable of expressing adipogenic factor antigen. The purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNA frag¬ ments. DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of expression vectors whose members each contain a unique cloned DNA or cDNA fragment. An "expression vector" is a vector which (due to the presence of appropriate transcriptional and/or transla- tional control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing a polypeptide or protein. Expres- sion of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expression vector is employed, then the appro¬ priate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Similarly, if a eukary- otic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequences. Importantly, since eukaryotic DNA may contain intervening sequences, and since such sequences cannot be correctly processed in prokaryotic cells, it is preferable to employ cDNA from a cell which is capable of expressing adipogenic factor in order to produce a prokaryotic genomic expression vector library. Proce¬ dures for preparing cDNA and for producing a genomic library are disclosed by Sambrook, J. et al. (supra) . Nucleic acid detection assays can be predicated on any characteristic of the nucleic acid molecule, such as its size, sequence, susceptibility to digestion by restric¬ tion endonucleases, etc. The sensitivity of such assays may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for exam- pie, assay sensitivity can be increased through the use of detectably labeled reagents. A wide variety of such labels have been used for this purpose. Kourilsky et al. (U.S. Patent 4,581,333) describe the use of enzyme labels to increase sensitivity in a detection assay. Radioisotopic labels are disclosed by Falkow et al. (U.S. Patent 4,358,535), and by Berninger (U.S. Patent 4,446,237). Fluorescent labels (Albarella et al.. EP 144914) , chemical labels (Sheldon III et al.. U.S. Patent 4,582,789; Albarella et al.. U.S. Patent 4,563,417), modified bases (Miyoshi et al.. EP 119448) , etc. have also been used in an effort to improve the efficiency with which detection can be observed.

One method for overcoming the sensitivity limita¬ tion of nucleic acid concentration is to selectively a pli- fy the nucleic acid whose detection is desired prior to performing the assay. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Patent 4,237,224), Maniatis, T. , et al. , etc.

Recently, an in vitro, enzymatic method has been described which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been referred to as the "polymerase chain reaction or "PCR" (Mullis, K. et al.. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al.. EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K. , EP 201,184; Mullis K. et al. , US 4,683,202; Erlich, H. , US 4,582,788; and Saiki, R. et al.. US 4,683,194).

The polymerase chain reaction provides a method for selectively increasing the concentration of a particu- lar nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.

The precise nature of the two oligonucleotide probes of the PCR method is critical to the success of the method. As is well known, a molecule of DNA or RNA possesses directionality, which is conferred through the 5'-3* linkage of the phosphate groups of the molecule. Sequences of DNA or RNA are linked together through the formation of a phosphodiester bond between the terminal 5' phosphate group of one sequence and the terminal 3 ' hydro¬ xyl group of a second sequence. Polymerase dependent ampli¬ fication of a nucleic acid molecule proceeds by the addition of a 5' nucleotide triphosphate to the 3 • hydroxyl end of a nucleic acid molecule. Thus, the action of a polymerase extends the 3' end of a nucleic acid molecule. These inherent properties are exploited in the selection of the oligonucleotide probes of the PCR. The oligonucleotide sequences of the probes of the PCR method are selected such that they contain sequences identical to, or complementary to, sequences which flank the particular nucleic acid sequence whose amplification is desired. More specifical¬ ly, the oligonucleotide sequences of the "first" probe is selected such that it is capable of hybridizing to an oligonucleotide sequence located 3 ' to the desired sequence, whereas the oligonucleotide sequence of the "second" probe is selected such that it contains an oligo¬ nucleotide sequence identical to one present 5^• to the desired region. Both probes possess 3' hydroxy groups, and therefore can serve as primers for nucleic acid synthesis.

In the polymerase chain reaction, the reaction conditions are cycled between those conducive to hybridiza¬ tion and nucleic acid polymerization, and those which result in the denaturation of duplex molecules. In the first step of the reaction, the nucleic acids of the sample are transiently heated, and then cooled, in order to dena- ture any double-stranded molecules which may be present. The "first" and "second" probes are then added to the sample at a concentration which greatly exceeds that of the desired nucleic acid molecule. When the sample is incu¬ bated under conditions conducive to hybridization and polymerization, the "first" probe will hybridize to the nucleic acid molecule of the sample at a position 3 * to the sequence to be amplified. If the nucleic acid molecule of the sample was initially double-stranded, the "second" probe will hybridize to the complementary strand of the nucleic acid molecule at a position 3 • to the sequence which is the complement of the sequence whose amplification is desired. Upon addition of a polymerase, the 3* ends of the "first" and (if the nucleic acid molecule was double- stranded) "second" probes will be extended. The extension of the "first" probe will result in the synthesis of an oligonucleotide having the exact sequence of the desired nucleic acid. Extension of the "second" probe will result in the synthesis of an oligonucleotide having the exact sequence of the complement of the desired nucleic acid.

The PCR reaction is capable of exponential ampli¬ fication of specific nucleic acid sequences because the extension product of the "first" probe, of necessity, contains a sequence which is complementary to a sequence of the "second" probe, and thus can serve as a template for the production of an extension product of the "second" probe. Similarly, the extension product of the "second" probe, of necessity, contains a sequence which is comple¬ mentary to a sequence of the "first" probe, and thus can serve as a template for the production of an extension product of the "first" probe. Thus, by permitting cycles 5 of polymerization, and denaturation, a geometric increase in the concentration of the desired nucleic acid molecule can be achieved. Reviews of the polymerase chain reaction are provided by Mullis, K.B. (Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Saiki, R.K. , et al. ° (Bio/Technology 3:1008-1012 (1985)); and Mullis, K.B., et al. (Met. Enzvmol. 155:335-350 (1987)).

The above-described recombinant molecules can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by ⁵ application of recombinant DNA techniques. Techniques for synthesizing such molecules are disclosed by, for example, Wu, R. , et al. (Prog. Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978)). Procedures for constructing recombi¬ nant molecules in accordance with the above-described 0 method are disclosed Sambrook et al. (supra)

The 3' terminus of the above-described recombi¬ nant molecule is preferably treated to render it unsuitable for polymerization. Such treatment may be accomplished by blocking the terminus by chemical means, or by modifying 5 the terminal bases such that they sterically interfere with polymerase action. In a preferred embodiment, such treat¬ ment is accomplished by immobilizing the 3* terminus, such as by coupling it to a solid support (such as, for example, glass, plastic, latex, etc.). The support may be of any 0 form (i.e. a sheet, rod, sphere, ovoid, etc. Procedures for such immobilization are well known to those of ordinary skill. In the most preferred embodiment, the 3' end of the recombinant molecule is covalently bound to the solid support. A spacer region may be used to extend the probe 5 outward from the solid support as long as (1) it will not sterically hinder any function or characteristic of the recombinant molecule, and (2) the sequence of the spacer region does not participate in the hybridization or poly¬ merization reactions of the assay. It is typically desir¬ able to immobilize several, and preferably, a large number of such recombinant molecule to the support. For expression of the DNA encoding the adipogenic factor of the present invention, a genetic construct is produced that possesses the necessary control elements to permit appropriate transcription and translation of the nucleic acid sequence. A promoter is a double-stranded DNA or RNA molecule which is capable of binding RNA polymerase and promoting the transcription of an "operably linked" nucleic acid sequence. As used herein, a "promoter sequence" is the sequence of the promoter which is found on that strand of the DNA or RNA which is transcribed by the RNA polymerase. A "promoter sequence complement" is a nucleic acid molecule whose sequence is the complement of a "promoter sequence." Hence, upon extension of a primer DNA or RNA adjacent to a single-stranded "promoter sequence complement" or, of a "promoter sequence," a double-stranded molecule is created which will contain a functional promot¬ er, if that extension proceeds a nucleic acid molecule which is operably linked to that strand of the double- stranded molecule which contains the "promoter sequence" (and not that strand of the molecule which contains the "promoter sequence complement") .

Certain RNA polymerases exhibit a high specifici¬ ty for such promoters. The RNA polymerases of the bacte- riophages T7, T3, and SP-6 are especially well characterized, and exhibit high promoter specificity. The promoter sequences which are specific for each of these RNA polymerases also direct the polymerase to utilize (i.e. transcribe) only one strand of the two strands of a duplex DNA template. The selection of which strand is transcribed is determined by the orientation of the promoter sequence. This selection determines the direction of transcription since RNA is only polymerized enzymatically by the addition of a nucleotide 5' phosphate to a 3' hydroxyl terminus. Two sequences of a nucleic acid molecule are said to be "operably linked" when they are linked to each other in a manner which either permits both sequences to be transcribed onto the same RNA transcript, or permits an RNA transcript, begun in one sequence to be extended into the second sequence. Thus, two sequences, such as a promoter sequence and any other "second" sequence of DNA or RNA are operably linked if transcription commencing in the promoter sequence will produce an RNA transcript of the operably linked second sequence. In order to be "operably linked" it is not necessary that two sequences be immediately adjacent to one another.

Thus, as indicated above, in order to function as a promoter, a promoter sequence must be present as a double-stranded molecule. For the purposes of the present invention, the two strands of a functional promoter sequence are referred to as a "transcript" strand and a "complement" strand. The "transcript" strand is that strand of the duplex which will be transcribed by the RNA polymerase (i.e. which serves as the template for tran¬ scription) . The "complement" strand is the strand which has a sequence complementary to the "transcript" strand, and which must be present, and hybridized to the "tran¬ script" strand, in order for transcription to occur. Thus, when the "transcript" strand of a promoter sequence is operably linked to a second sequence, hybridization of the "transcript" strand with the "complement" strand, will, in the presence of a polymerase, result in the transcription of the "transcript" strand, and will produce an RNA tran¬ script using the sequence of the "transcript" strand as a template.

The promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Suitable promoters are repressible, or, more preferably, constitu¬ tive. Examples of suitable prokaryotic promoters include promoters capable of recognizing the T4 (Malik, S. et al.. J. Biol. Chem. 263:1174-1181 (1984); Rosenberg, A.H. et al.. Gene 59:191-200 (1987); Shinedling, S. et al. , J. Molec. Biol. 195:471-480 (1987); Hu, M. et al.. Gene 42:21- 30 (1986), T3, Sp6, and T7 (Chamberlin, M. et al.. Nature 228:227-231 (1970); Bailey, J.N. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 80:2814-2818 (1983); Davanloo, P. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 81:2035-2039 (1984) poly¬ merases; the P_R and P_L promoters of bacteriophage 1 (The Bacteriophage Lambda. Hershey, A.D., Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1973) ; Lambda II. Hendrix, R.W. , Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (1980) ; the trp, recA, heat shock, and lacZ promoters of E. coli; the a-amylase (Ulmanen, I., et al. , J. Bacteriol. 162:176-182 (1985) and the s-28-specific promoters of B. subtilis (Gilman, M.Z., et al.. Gene 32:11- 20 (1984)); the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli. Academic Press, Inc., NY (1982)); Streptomyces promoters (Ward, J.M., et al.. Mol. Gen. Genet. 203:468-478 (1986)); the int promoter of bacteriophage 1; the bla promoter of the b-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc. Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987); Cenatiempo, Y. (Biochimie 68_.:505-516 (1986); Watson, J.D. et al. (supra) ; and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)).

Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Ha er, D., et al.. J. Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (Mc night, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C. , et al.. Nature (London) 290:304-310 (1981)); and the yeast ga!4 gene promoter (Johnston, S.A. , et al. , Proc. Natl. Acad. Sci. (USA) 79.:6971-6975 (1982); Silver, P. . , et al.. Proc. Natl. Acad. Sci. (USA) 11:5951-5955 (1984). All of the above listed references are incorporated by reference herein. Strong promoters are the most preferred promoters of the present invention. Examples of such preferred promoters are those which recognize the T3, SP6 and T7 polymerase promoters; the P_L promoter of bacteriophage 1; the recA promoter and the promoter of one which is capable of recognizing the T7 polymerase promoter. The sequences of such polymerase recognition sequences are disclosed by Watson, J.D. et al. (supra) .

For purification and characterization of the proteins of (SDS-PAGE) is performed in general according to the method of Laemmli (1974) using 7.5% acrylamide gels with a constant ratio of 2.6% bisacrylamide/total acryl¬ amide concentration. Protein samples are denatured at 100°C for 10 in in 20 M Tris containing 3.3% glycerol, and bromophenol blue tracking dye with or without proteins with 0.05% R250 Coomassie brilliant blue in 25% isopropanol for 2 h, and destained for 24 h in 20% methanol-7% acetic acid. As molecular weight markers, myosin (200 kDa) , beta- galactosidase (116 kDa) , phosphorylase B (97 kDa) , BSA (66 kDa) , and egg albumin (43 kDa) are used. Known modifica¬ tions and variations of the described method are also contemplated within the scope of this inventio' to the class Mammalia. The invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well. the treatment of human subjects, although it is intended for veterinary uses as well.

The following example is intended to be illustra¬ tive, but not to limit, the invention.

EXAMPLE

Conditions for the culture of 1246 cells useful for the bioassay of adipogenic factors are modifications of methods described previously (Serrero and Khoo (1982) , Anal. Biochem. 120, 351-359; G. Serrero, (1985), In Vitro Cell. Dev. Biol. 21, 537-540), and are hereby incorporated by reference.

1246 cells, derived from C3H mouse teratoma, were cultivated in tissue culture plasticware (Costar, Cam- bridge, MA) in Dulbecco's modified Eagle's medium/Ham's F12 nutrient mixture (1:1 mixture) (Gibco, Grand Island, NY) (referred to as DME/F12) supplemented with 1.2 g/1 sodium bicarbonate (Sigma, St. Louis, MO), 15 M HEPES pH 7.4 (Research Organics, Cleveland, OH) and 10% fetal calf serum (FCS) (Hylcone, Logan, UT) in humidified atmosphere of 95% air-50% C0₂ at 37°C.

Adipose differentiation assay. On day 0, subconfluent 1246 cells were plated at a density of 1.5 x 10⁴ cells per well (having a surface area of 4.5 cm²) in 12-well cluster plates (Costar) in DM/F12 medium supplemented with 2% FCS. At day 1, the medium was replaced by DME/F12 supplemented with insulin (10 μg/ml) (Sigma, St. Louis, MO) , transferrin (10 μg/ml) (Sigma) , and fibroblast growth factor (5 ng/ml) (Collaborative Research, Waltham MA) . Cells were exposed to dexamethasone (2 x 10"⁷ M) (Sigma), iso- butylmethylxanthine (2 x 10"⁴ M) (Aldrich Chemical Co., Milwaukee, WI) , and indomethacin (3 x 10"⁵ M) (Sigma) from day 4 to day 6. Cells were further incubated in DME/F12 containing insulin and transferrin, and were harvested at day 11. Adipose differentiation was examined by measure¬ ment of G3PDH specific activity as described above. Fetuin (Sigma) and/or partially purified fractions (from fetuin or HepG2-CM) were added at day 1, day 4 and day 6. Control plates correspond to cell cultivated in defined medium alone without fetuin. Sephacryl fractionation

Sephacryl S-300 (Pharmacia, Piscataway, NJ) column (2.5 cm x 95 cm) was equilibrated and run in 20 mM phosphate buffer- 0.1 M NaCl pH 7.0 at a flow rate of 20 ml/hr at 4°C. Thyroglobulin (669 kDa), ferritin (445 kDa), catalase (232 kDa) , and bovine serum albumin (BSA) (69 kDa) contained in the gel filtration calibration kit (Pharmacia) were used as molecular weight markers. Purification of human adipogenic factor

The starting material for large scale purifica- tion of the human adipogenic factor is the culture medium conditioned by the HepG2 cells. The HepG2 cell line is available from American Type Culture collection (ATCC HB 8065). For the isolation of this factor, see Aden, D.P. et al. (1979) Nature 282 615. HepG2 cells are cultivated in defined medium, RITC-807 medium + 10% FBS. RITC-807 medium is described in M. Kan, and I. Yamane (1982) J. Cell Phvsiol. 111. 155-162. At confluence, they are cultivated RITC-807 medium. In these conditions, the cells secrete several proteins in the culture medium including the adipo- genie factor.

Conditioned medium from HepG2 cells was concen¬ trated 25-fold by ultrafiltration with a 10,000 molecular weight cut-off Filtron membrane system. Ammonium sulfate precipitation was carried out as using standard procedures which are well-known in the art. The protein factor pre¬ cipitated by 30-50% (w/v) ammonium sulfate was resuspended in phosphate buffer (20 mM, pH 7.0) and diluted. The diluted fraction was chromatographed on a heparin-sepharose column equilibrated in 20 mM sodium phosphate buffer pH 7.0. The active fraction was eluted with a gradient of NaCl between 0.35 M - 1 M NaCl. Eluted fractions were loaded onto a concanavalin A Sepharose column equilibrated with 20 mM phosphate buffer pH 7.0 containing 0.5 M NaCl. The active fraction was eluted with 0.5 M (alpha) ethyl- mannoside in 20 mM phosphate buffer pH 7.0. The active fraction was then loaded on a Sephacryl S-300 column or on a Sepharose CL-6B column equilibrated in 20 mM phosphate buffer pH 7.0 containing 0.1 M NaCl. The active fraction was eluted with an apparent molecular weight of 150 kDa to 230 kDa. SDS-PAGE analysis of material from various column fractions is shown in Figure 1. TABLE 1

SPECIFIC ACTIVITY OF HUMAN ADIPOGENIC FACTOR DURING PURIFICATION FROM HEPG2 CONDITIONED MEDIUM.

Conditions Protein Recovery % Specific

Activity* Conditioned medium 100

1 Ammonium sulfate ppt. (30-50%) w/v 35 2.5

Heparin sepharose 2 25

Concanavalin sepharose 1 50

Sepharose CL6B 0.03 625

(or Sephacryl S-300)

* Measured by the induction of glycerol-3-phosphate dehydrogenase activity using the bioassay described herein.

For the HepG2 factor that underwent the purifica¬ tion procedure described here, only three major bands are detectable after PAGE (without SDS) after silver staining of the gel. The adipogenic factor represents at least 30% of the total protein in the fraction.

Characterization of human adipogenic factor

The human adipogenic factor, isolated as described above, was analyzed by SDS-PAGE. A major band has a molecular weight of 230 kDa and two minor bands are of lower molecular weight. (Figure 1 depicts a similar profile obtained from running an ammonium sulfate precipi¬ tate onf Sephacryl S-300. Additional experiments revealed that its adipogenic activity was destroyed by incubation with pronase (indicating it is a protein) , by heat treat¬ ment (100°C, 5 minutes) and by incubation at pH 2.5 for 24 hr at 4°C and by treatment with 0.2 M 2-mercaptoethanol at room temperature (about 25° C) for 6 hours, indicating the existence of disulfide bridges which are important for the maintenance of its biological activity. It is partially resistant [about 60% of the activity remained] after expo¬ sure to pH 11.0 for 24 hours at 4°C.

Purification of bovine adipogenic factors The starting point for purification of these factors is crude fetuin, prepared according to the method of Pedersen

(Nature 154:575-576 (1944)). Three different procedures were used to purify the factors:

(1) For routine purification, the crude fetuin was dialyzed against start buffer (25 mM imidazole-CH₃ COOH, pH 7.4) and then load it on a chromatofocusing polybuffer exchange column PBE 94 gel (sold by Pharmacia) that had been equilibrated with start buffer. Unbound proteins were washed out with the start buffer and collected in the "flow-through" fraction. Factor F_j was present in the flow-through fraction. The column was then washed with polybuffer 96-CH₃COOH, pH 6.0 (purchased from Pharmacia, chemical composition undisclosed) and subsequently washed with 1.0 M NaCl. Factor F_{j j} was eluted with 1.0 M NaCl.

(2) The procedure was as in (1) except that after collection of the flow-through fraction, a pH gradient (pH 9.0 to pH 7.0) made with polybuffer PB 94 was applied to the column. Proteins not eluted by the gradient were subsequently eluted with 1M NaCl. F_r adipogenic activity eluted with the flow-through fraction (pi >9.0).

F_j , eluted with 1 M NaCl (pH < 7.0).

F_{j J} was subsequently loaded on a heparin- sepharose column equilibrated with 20 mM phosphate buffer pH 7.0. The column was washed in a stepwise manner with 20 mM phosphate buffer pH 7.0, then with 0.3 M NaCl in 20 mM phosphate buffer pH 7.0 and finally with 1 M NaCl in phos- phate buffer pH 7.0. FII was eluted with 1 M NaCl. F_{r t} was subsequently loaded on lectin sepharose column equili¬ brated with 20 mM phosphate buffer, pH 7.0, containing 0.15 M NaCl. The active fraction was eluted with 0.5 M alpha- methyl mannoside, dialyzed against 20 mM phosphate buffer, pH 7.0, and then loaded on a Mono Q ion exchange column. Elution was performed with a NaCl gradient from 0.1 M to 0.5 M NaCl. The active fraction was chromatographed on a hydrophobic interaction phenyl sepharose column. Elution was performed with a descending gradient of NaCl.

The Fj fraction was loaded on a heparin sepharose column equilibrated with 20 M phosphate buffer at pH 7.0. The active fraction was eluted with the same buffer con¬ taining 0.1 M NaCl. By gel filtration on Sephacryl S-300 equilibrated with 20 mM phosphate buffer pH 7.0 containing 0.1 M NaCl, F_l eluted with a molecular weight of 660 kDa. F_{j j} had an apparent molecular weight of 230 kDa. These estimates of molecular weight were considered more reliable than the ones obtained by procedure (c) below. SDS-PAGE analysis of the various fractions is shown in Fig.2.

The procedure resulted in F_{r l} having a specific activity 250 to 500-fold that of crude fetuin and 5% of the adipogenic activity of crude fetuin. It resulted in an F_j preparation with a specific activity at least 10-fold that of crude fetuin.

(3) By gel filtration of crude fetuin on Sephacryl S-300 in 20 mM potassium phosphate, pH 7.4, the adipogenic activity eluted primarily in two distinct peaks. The factor (or group of factors) in the first peak, which contained molecules of apparent molecular weights greater than 669 kDA, was labeled F_r . The factor (or group of factors) in the second peak, which contained molecules with apparent molecular weights in the range 232 to 445 kDA, was labeled F_{j 1} . In addition, a minor activity eluted with an apparent molecular weight of 69 kDA. The majority of adipogenic activity in fetuin was contained in F_j , . Biochemical characterization of adipogenic factors

Partially purified fractions from fetuin and from HepG2 CM were used for biochemical characterization experi¬ ments. Acid sensitivity or alkali sensitivity was tested by incubating samples at pH 2.5 or pH 11.0 for 24 h at 4°C. Heat stability was examined by heating a factor in 20 mM phosphate buffer, pH 7.0 for 10 min. Sensitivity to disulfide-reducing agent was tested by incubating samples with 0.2 M 2-mercaptoethanol at room temperature for 6 h. All the treated samples were dialyzed against 20 mM phos¬ phate buffer (pH 7.0) before being assayed. Protease sensitivity was examined by incubating samples with immobi- lized pronase conjugated to agarose beads (Streptomyces griseus, Sigma) at 37°C for 6 h. Pronase was removed by centrifugation before use.

Characterization of bovine adipogenic factors

Biochemical characterization demonstrated that F, and F_{j t} are distinct factors. Bovine adipogenic factor,

Fj , was found to have a pi > 9.4, to be heat and alkaline labile, protease sensitive, and stable during treatment with 2-mercaptoethanol or acid. F_{x r} was found to have a pi

< 4.0, be heat and acid labile, protease sensitive and partially destroyed [about 50 % of the activity remained] by treatment with 2-mercaptoethanol.

It is understood that the isolelectric point determinations of F_j and F_{j j} are of preparations containing some impurities, which may contribute to a greater or lesser degree to the observed pi. Furthermore, the glyco- protein nature of these factors, and the possibility that other sugars or proteoglycans are present in the fractions, may also contribute to the pi. The key point is the fact that two distinct adipogenic factors are discernible and capable of separation by chromatofocusing.

Comparison of bovine factors with known bovine substances From crude fetuin two factors have been isolated by others an acidic glycoprotein having a molecular weight of 69 kDa (Spiro, R. G. (1960) J. Biol. Chem. 235, 2860-

2869) and a large molecular weight factor called embryonin similar to alpha-2 macroglobulin (D. S. Saloman et al (1982) J. Biol. Chem. 257. 14093-14101) . We found that the two factors do not have adipogenic activity in the G3PDH assay. Therefore, "pure fetuin", a 69 kDa acidic glyco- protein, is not responsible for this minor adipogenic activity we observed at 69 kDa.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in con¬ nection with specific embodiments thereof, it will be understood that it is capable of further modifications.

This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or custom- ary practice within the art to which the invention pertains and as may be applied to the essential features herein¬ before set forth as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A mammalian adipogenic factor, capable of inducing adipose differentiation of adipogenic cells, isolatable from liver cells, having an apparent molecular weight of about 150 to about 230 kDa, and having adipogenic activity that is susceptible to inactivation by treatment with pronase, with temperature as high as about 100°C, with pH as low as about 2.5, or with 0.2M 2-mercaptoethanol, said factor being of sufficient purity such that its adipo¬ genic activity per millligram protein is substantially greater than the adipogenic activity of liver cell condi¬ tioned medium or extract.

2. The factor of claim 1 that is of human origin.

3. The factor of claim 2 that having specific adipogenic activity at least 625 times that of the condi¬ tioned medium obtained from confluent cultures of HepG2 hepatocarcinoma cells.

4. The factor of claim 2 purified from a hepato- cyte cell line or from the conditioned medium of said cell line. 5. A mammalian adipogenic factor, capable of inducing adipose differentiation of adipogenic cells, isolatable from serum, having an apparent molecular weight of about 660 kDa, and having adipogenic activity that is susceptible to inactivation by treatment with pronase, with temperature as high as about 100°C or with pH as high as about 11.0, but is resistant to inactivation by treatment with 0.2 M 2-mercaptoethanol at 25°C for 6 hours or with pH as low as about 2.5, said factor being of sufficient purity such that its adipogenic activity per milligram protein is substantially greater than the adipogenic activity of whole serum.

6. The factor of claim 5 having specific adipo- genie activity at least 250 times that of whole serum.

7. A factor of Claim 5 having specific adipogenic activity at least 250 times that of crude fetuin.

8. A mammalian adipogenic factor, capable of inducing adipose differentiation of adipogenic cells, isolatable from serum, having an apparent molecular weight of about 230 kDa, and having adipogenic activity that is susceptible to inactivation by treatment with pronase, with pH as low as about 2.5, or with pH as high as about 11.0, and is partially susceptible to inactivation by treatment with 0.2 M 2-mercaptoethanol at 25°C for 6 hours, said factor being of sufficient purity such that it adipogenic activity per milligram protein is substantially greater than the adipogenic activity of whole serum.

9. The factor of Claim 8 having specific adipo¬ genic activity at least 10 times that of whole serum.

10. The factor of Claim 8 having specific adipo¬ genic activity at least 10 times that of crude fetuin.

11. A mammalian adipogenic factor, capable of inducing adipose differentiation of adipogenic cells, isolatable from serum, having an apparent molecular weight of about 69 kDa, and having adipogenic activity substan- tially greater than the adipogenic activity of whole serum.

12. The factor of claim 11 having specific adipo- geni.e acti.vi.ty at least 2 times that of whole serum.

13. The factor of Claim 11 having specific adipo¬ genic activity at least 2 times that of crude fetuin. 14. A method for determining the susceptibility of a subject to obesity which comprises removing a sample of a biological fluid or tissue from said subject and measuring the amount of the adipogenic factor of claim 1 in said fluid or tissue, the amount of said factor being proportional to said susceptibility.

15. A method for determining the susceptibility of a subject to obesity which comprises removing a sample of a biological fluid or tissue from said subject and measuring the amount of the adipogenic factor of claim 5 in said fluid or tissue, the amount of said factor being proportional to said susceptibility.

16. A method for determining the susceptibility of a subject to obesity which comprises removing a sample of a biological fluid or tissue from said subject and measuring the amount of the adipogenic factor of claim 8 in said fluid or tissue, the amount of said factor being proportional to said susceptibility.

17. A method for determining the susceptibility of a subject to obesity which comprises removing a sample of a biological fluid or tissue from said subject and measuring the amount of the adipogenic factor of claim 11 in said fluid or tissue, the amount of said factor being proportional to said susceptibility. 18. A monoclonal antibody specific for the adipogenic factor of claim 1.

19. A monoclonal antibody specific for the adipogenic factor of claim 5.

20. A monoclonal antibody specific for the adipogenic factor of claim 8.

21. A monoclonal antibody specific for the 5 adipogenic factor of claim 11.

22. A method for evaluating the efficacy of an o anti-obesity drug which comprises contacting said drug with an adipogenic cell in vitro and measuring the amount of the factor of claim 1 that is produced by said cell.

⁵ 23. A method for evaluating the efficacy of an anti-obesity drug which comprises contacting said drug with an adipogenic cell in vitro and measuring the amount of the factor of claim 5 that is produced by said cell. 0

24. A method for evaluating the efficacy of an anti-obesity drug which comprises contacting said drug with an adipogenic cell in vitro and measuring the amount of the factor of claim 8 that is produced by said cell. 25. A method for evaluating the efficacy of an anti-obesity drug which comprises contacting said drug with an adipogenic cell in vitro and measuring the amount of the factor of claim 11 that is produced by said cell.

26. The adipogenic factor of claim 1 isolatable by ammonium sulfate precipitation, heparin-Sepharose chromatography, Concanavalin-Sepharose chromatography, and Sepharose CL6B or Sephacryl S-300 chromatography.

27. The adipogenic factor of claim 5 isolatable by chromatofocusing on a chromatofocusing polybuffer exchange column and selecting the flow-through fraction, performing heparin-sepharose chromatography, and Sephacryl S-300 chromatography.

28 The adipogenic factor of claim 8 isolatable by chromatofocusing on a chromatofocusing polybuffer exchange column and eluting with 1M NaCl, performing heparin-Sepharose chromatography, Concanavalin A-Sepharose chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography.

29. The adipogenic factor of claim 11 isolatable by Sephacryl S-300 chromatography, selecting the peak containing molecules with apparent molecular of 50-69 kDa.