Note: Descriptions are shown in the official language in which they were submitted.
WO 91/18924 PCT/US91/03868
20 84062
~sALIAN ADIPOGENIC FACTORS
BACRGRODND 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 Backctround 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.W. et al., 1984 Proc. 7th International
Congress Endocrinology Excerpta Medica, p. 866). Adipo-
genic 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);
WO 91/18924 ,ø 20 8 ~ ~ 6 2 PCT/US91/03868
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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.
20, 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. Physiol. (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.
S~Ry 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
WO 91/18924 PCT/US91/03868
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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-
genic 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 weight of about 50 to 69 kDa, the factor
being different from the 69 kDa glyc~~sprotein 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.
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20 a
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-
sting 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
WO 91/18924 PCT/US91/03868
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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 heparin
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
WO 91/18924 PCT/US91/03868
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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.
WO 91/18924 PCT/US91/03868
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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.
r
WO 91/18924 ~ ~ ~ 4 ~ ~ ~ PCT/US91/03868
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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
WO 91 / 18924 PCT/US91 /03868
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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 carboxyamidomethyl derivatives. Cysteinyl residues also
are derivatized by reaction with bromotrifluoroacetone,
alpha-bromo-beta-(5-imidozoyl) propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-vitro-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 6Ø
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
imidoesters 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 pKe 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 er
se has been studied extensively, with particular interest
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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 Carboxyl side groups
(aspartyl or glutamyl) are selectively modified by reaction
with carbodiimides (R'-N-C-N-R') such as 1 cyclohexyl-3-(2-
morpholinyl-(4-ethyl) carbodiimide or 1-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., 1,1-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 (succinimidylpropionate), and
bifunctional maleimides such as bis-N-maleimido-1,8-octane.
perivatizing 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
Beryl or theonyl residues, methylation of the alpha-amino
groups of lysine, arginine, and histidine side chains (T. E.
Creighton, Proteins: Structure and Molecule Properties,
WO 91 / 18924 PCT/ US91 /03868
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W.H. Freeman & 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 Reminctton'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 supernatants are screened for the
presence of antibody specific for the adipogenic factor by
WO 91 / 18924 PCT/ US91 /03868
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2~~~p62~
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')Z fragments lack the Fc
fragment of intact antibody, clear F(ab')z 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')Z 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')Z 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
WO 91 /18924 ~ ' " PCT/US91 /03868
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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 inear 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 b bioassa
Y y, 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 adipogenic factors. Another embodiment is
directed to the preparation of the adipogenic factors using
recombinant DNA techniques. Also intended are vectors
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WO 91/18924 PCT/US91/03868
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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
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
prokaryotic or eukaryotic. Such techniques are set forth,
for example, in Sambrook et al. (Molecular Cloningr: A
Laboratory Manual, Second edition, Cold Spring Harbor
Laboratory Press, 1989).
Based bn 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., Proq. Nucl.
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,
trypsin, etc. (Dike, Y., et al., J. Biol. Chem. 257:9751-
9758 (1982); Liu, C., et al., Int. J. Pelt. 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
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-
nucleotide will, in fact, constitute the actual adipogenic
WO 91/18924 _ _ _ _ ~ , or PCT/US91/03868
20 84062- 15 -
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
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WO 91/18924 PCT/US91/03868
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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., Proa. 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).
Techniques such as, or similar to, those described
above have successfully enabled the cloning of genes for
h~an aldehyde dehydrogenases (Hsu, L.C., et al., Proc.
Natl. Acad. Sci. USA 82:3771-3775 (1985), f ibronectin
(Suzuki, S., et al., hur. 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
WO 91/18924 z PCT/US91/03868
zos4os2 -1~-
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 assa s can be
Y predicated
on any characteristic of the nucleic acid molecule, such as
WO 91/18924 PCT/US91/03868
2p8~pp2.
- 18 -
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-
s ple, 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 ampli
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
Cohere -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 Sgrinct 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.
WO 91/18924 PCT/US91/03868
20 84062 - 19 - :_
et al., US 4,683,202; Erlich, H., US 4,582,788; arid 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
WO 91/18924 PCT/US91/03868
- 20 -
20 ~~062,
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"
WO 91/18924 PCT/US91/03868
20 84062. - 21 -
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
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.
(BioJ Technolocty 3:1008-1012 (1985)); and Mullis, K.B., et
al. (Met. Enzymol. 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
application of recombinant DNA techniques. Techniques for
synthesizing such molecules are disclosed by, for example,
Wu, R., et al. (Prod Nucl. Acid. Res Molec Biol
21:101-141 (1978)). Procedures for constructing- recombi-
nant molecules in accordance with the above-described
method are disclosed Sambrook et al. (su ra)
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
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
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
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
WO 91/18924 " J PCT/US91/03868
22
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
~A 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.
WO 91/18924 PCT/US91/03868
_ _ .. ms
N 4 v.
zos~os2 -23-
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-
five. Examples of suitable rokar otic
p y promoters include
promoters capable of recognizing the T4 (Malik, S. et al.,
J. Biol. Chem. 263:1174-1181 (1984); Rosenberg, A.H. et
CA 02084062 2000-12-20
WO 91/18924 - PCT/US91/03868
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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 PR and P~ promoters of bacteriophage 1 (The
Hacteriophage 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
~ottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)).
Preferred eukaryotic promoters include the promoter of the
mouse metallothionein I gene (Hamer, D., et al., J. Mol.
A~ppl. Gen..1:273-288 (1982)); the TK promoter of Herpes
virus (McKnight, S., Cell 31:355-365 (1982)); the SV40
early promoter (Benoist, C., et al., Nature SLondon)
290:304-310 (1981)); and the yeast gal4 gene promoter
(Johnston, S.A., et al., Proc. Natl. Acad. Sci. ~~USA)
79:6971-6975 (1982); Silver, P.A., et al., Proc. Natl.
Acad. Sci. (USA1 81:5951-5955 (1984).
3~ -
WO 91/18924 ~ a PCT/US91/03868
2o8~os2 -25-c
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
polymerise promoters; the P~ promoter of bacteriophage 1;
the recA promoter and the promoter of one which is capable
of recognizing the T7 polymerise promoter. The sequences
of such polymerise recognition sequences are disclosed by
Watson, J.D. et a (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 min in 20 mM Tris containing 3.3% glycerol,
and bromophenol blue tracking dye with or without proteins
with 0.05% 8250 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 a
gg albumin (43 kDa) are used. Known modifica-
tions and variations of the described method are also
contemplated within the scope of this invention
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
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Cell. Dev. Biol. 21, 537-540),
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 mM HEPES pH 7.4
(Research Organics, Cleveland, OH) and 10% fetal calf serum
(FCS)(Hylcone, Logan, UT) in humidified atmosphere of 95%
air-50% COZ at 37°C.
Adipose differentiation assay. On day O, subconfluent 1246
cells were plated at a density of 1.5 x 104 cells per well
(having a surface area of 4.5 cmz) 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 ~cg/ml) (Sigma, St. Louis, MO), transferrin
(10 ~,g/ml) -_(Sigma) , .and_.fibroblast .growth factor (5 ng/ml)
(Collaborative Research, Waltham MA). Cells were exposed
to dexamethasone (2 x 10'~-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 il. Adipose differentiation was examined by measure-
went 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)
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contained in the gel filtration calibration kit (Pharmacia)
were used as molecular weight markers.
Purification of human adipoqenic 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
Physiol. 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-
genic 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
7Ø 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) methyl-
mannoside in 20 mM phosphate buffer pH 7Ø 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.
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,_, 20 84062
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 adipoqenic 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
WO 91/18924 pCT/US91/03868
20 84062 - 29 -
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-CH3COOH,
pH 7.4) and then load it on a chromatofocusing polybuffer
exchange column PBE 94 gel (sold by Pharmacies) that had
been equilibrated with start buffer. Unbound proteins were
washed out with the start buffer and collected in the
"flow-through" fraction. Factor FI was present in the
flow-through fraction. The column was then washed with
polybuffer 96-CH3COOH, pH 6.0 (purchased from Pharmacies,
chemical composition undisclosed) and subsequently washed
with 1.0 M NaCl. Factor Fig 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. FI adipogenic
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WO 91/18924 PCT/US91/03868 _ .
- 30 -
activity eluted with the flow-through fraction (pI >9.0).
FII eluted with 1 M NaCl (pH < 7.0).
F~, was subsequently loaded on a heparin-
sepharose column equilibrated with 20 mM phosphate buffer
pH 7Ø 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-
l0
phate buffer pH 7Ø FII was eluted with 1 M NaCl. Fit
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 FI fraction was loaded on a heparin sepharose*
column equilibrated with 20 mM phosphate buffer at pH 7Ø
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, FI eluted with a molecular weight of 660 kDa.
FII 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.
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2084062 -31-
SDS-PAGE analysis of the various fractions is
shown in Fig.2.
The procedure resulted in FII 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 FI
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 Ft. 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 F11. In addition, a minor activity eluted with an
apparent molecular weight of 69 kDA. The majority of
adipogenic activity in fetuin was contained in F11.
Biochemical characterization of adipoctenic 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.
WO 91/18924 PCT/US91/03868
- 32 2
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
g~riseus, 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 FII are distinct factors. Bovine adipogenic factor,
F" 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. FII 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~ and FII 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
WO 91/18924 PCf/US91/03868
ZV ~ ~ ~ 6 ~ - 33 - ,
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.
