U.S. patent application number 12/927750 was filed with the patent office on 2011-11-24 for methods of identifying adipocyte specific genes, the genes identified, and their uses.
This patent application is currently assigned to HMGene Inc.. Invention is credited to Hena Ashar, Kiran K. Chada, Roland Chouinard, Abu Sayed.
Application Number | 20110288034 12/927750 |
Document ID | / |
Family ID | 31191224 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110288034 |
Kind Code |
A1 |
Chada; Kiran K. ; et
al. |
November 24, 2011 |
Methods of identifying adipocyte specific genes, the genes
identified, and their uses
Abstract
Disclosed is a method of identifying genes that are
over-expressed in adipose tissue as compared to pre-adipocyte
tissue or other tissues comprising performing differential gene
expression analysis between the white adipose tissue (WAT) or
stromal vascular tissue (SVT) from any two different mice selected
from the group consisting of wild-type, HMGI-C-/-, ob/ob, and
HMGI-C-/- ob/ob genotype mice. Based on this method a number of
nucleotide sequences are identified whose expression is adipocyte
specific. The identified nucleotide sequences and their
corresponding polypeptides are then used to prevent adipogenesis,
to treat diabetes, and to screen for small-molecules that can
modulate or prevent adipogenesis and to treat diabetes.
Inventors: |
Chada; Kiran K.; (New York,
NY) ; Chouinard; Roland; (Piscataway, NJ) ;
Ashar; Hena; (Edison, NY) ; Sayed; Abu;
(Cincinnati, OH) |
Assignee: |
HMGene Inc.
|
Family ID: |
31191224 |
Appl. No.: |
12/927750 |
Filed: |
November 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10630423 |
Jul 29, 2003 |
7879544 |
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12927750 |
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60478206 |
Jun 12, 2003 |
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60398785 |
Jul 29, 2002 |
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Current U.S.
Class: |
514/21.2 ;
435/29; 435/325; 435/6.12; 435/69.1; 436/501; 514/44R; 530/350;
530/387.9; 536/23.5 |
Current CPC
Class: |
A61P 3/06 20180101; G01N
33/5041 20130101; C07K 14/4702 20130101; C07K 14/47 20130101; C12Q
1/6883 20130101; C12Q 2600/158 20130101; C07K 14/72 20130101; A61P
3/04 20180101; C12N 9/00 20130101; C07K 16/18 20130101; G01N
33/6893 20130101 |
Class at
Publication: |
514/21.2 ;
536/23.5; 530/350; 530/387.9; 514/44.R; 435/325; 435/69.1;
435/6.12; 435/29; 436/501 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07H 21/02 20060101 C07H021/02; C07K 14/435 20060101
C07K014/435; C07K 16/18 20060101 C07K016/18; A61P 3/04 20060101
A61P003/04; C12N 5/10 20060101 C12N005/10; C12P 21/00 20060101
C12P021/00; C12Q 1/68 20060101 C12Q001/68; C12Q 1/02 20060101
C12Q001/02; G01N 33/566 20060101 G01N033/566; C07H 21/04 20060101
C07H021/04; A61K 31/711 20060101 A61K031/711 |
Claims
1-46. (canceled)
47. An isolated polynucleotide comprising a) a nucleotide sequence
of SEQ ID NO:103; b) a nucleotide sequence coding for the
polypeptide of SEQ.ID.NO. 603; c) a nucleotide sequence that has at
least. 91% identity over its entire length to a nucleotide sequence
encoding the sFRP-5 polypeptide of SEQ ID NO:603 said identity
being over the entire region encoding SEQ ID NO:603; or d) a
nucleotide sequence complementary to the isolated nucleic acid
molecule.
48. The isolated polynucleotide of claim 47 wherein expression of
said gene upregulates its own expression in an autocrine fashion in
adipocytes.
49. The isolated polynucleotide of claim 47 comprising a nucleotide
sequence that is at least 91% identical with SEQ ID NO:103, said
identity being over the entire length of SEQ ID NO:103.
50. The isolated polynucleotide of claim 47 comprising the
nucleotide sequence of SEQ ID NO:103.
51. An isolated polynucleotide comprising a nucleotide sequence
which, by virtue of redundancy of the genetic code, encodes for the
amino acid of SEQ ID NO:603.
52. The polynucleotide of any one of claim 47 which is DNA or
RNA.
53. The isolated polynucleotide of any one of claim 47, further
comprising an expression system that is capable of transcribing the
nucleic acid sequence and producing the corresponding amino acid
sequence.
54. A host cell comprising a polynucleotide sequence of as defined
in any one of claim 47.
55. The host cell of claim 54, wherein the polynucleotide sequence
is stably incorporated into the genome of the host cell.
56. A process for producing an sFRP-5 polypeptide comprising
culturing a host of claim 54 under conditions sufficient for the
production of said polypeptide and recovering the polypeptide from
the culture.
57. A process for producing a cell which produces an sFRP-5
polypeptide comprising transforming or transfecting a host cell
with the polynucleotide of claim 53 such that the host cell, under
appropriate culture conditions, produces an sFRP-5 polypeptide.
58. An sFRP-5 polypeptide comprising an amino acid sequence which
is at least 91% identical to the amino acid sequence of
SEQ.ID.NO:603 over its entire length.
59. The polypeptide of claim 58 which comprises the amino acid
sequence of SEQ ID NO:603.
60. An antibody immunospecific for the sFRP-5 polypeptide of claim
58.
61. A method for the treatment of a subject in need of enhanced
activity or expression of the sFRP-5 polypeptide of claim 58
comprising: (a) administering to the subject a therapeutically
effective amount of an agonist to the polypeptide; and/or (b)
administering to the subject an isolated polynucleotide comprising
a nucleotide sequence that has at least 91% identity to a
nucleotide sequence encoding the sFRP-5 polypeptide of
SEQ.ID.NO.603 over its entire length, or a nucleotide sequence
complementary to said nucleotide sequence in a form so as to effect
production of such polypeptide in vivo; and/or (c) administering to
a subject a therapeutically effective amount of the sFRP-5
polypeptide.
62. A method for the treatment of a subject in need of decreased
activity or expression of the sFRP-5 polypeptide of claim 58
comprising: (a) administering to the subject a therapeutically
effective amount of an antagonist to said polypeptide; and/or (b)
administering to the subject a nucleic acid molecule that inhibits
the expression of the nucleotide sequence encoding said
polypeptide; and/or (c) administering to the subject a
therapeutically effective amount of a polypeptide that competes
with said polypeptide for its ligand, substrate, or receptor.
63. A process for diagnosing a susceptibility to obesity in a
subject related to expression or activity of the sFRP-5 polypeptide
of claim 58 in a subject comprising: (a) determining the presence
or absence of a mutation in the nucleotide sequence encoding said
sFRP-5 polypeptide in the genome of said subject; and/or (b)
analyzing for the presence or amount of the sFRP-5 polypeptide
expression in a sample derived from said subject.
64. A method for identifying compounds which antagonize or agonize
the sFRP-5 polypeptide of claim 58 comprising: (a) contacting a
candidate compound(s) with cells which express the sFRP-5
polypeptide (or cell membrane expressing the sFRP-5 polypeptide or
the sFRP-5 polypeptide or a fragment thereof bound to a solid
matrix) or respond to the sFRP-5 polypeptide; and (b) observing the
binding, or stimulation or inhibition of a functional response; or
comparing the ability of the cells (or cell membrane) which were
contacted with the candidate compound(s) with the same cells which
were not contacted for sFRP-5 polypeptide activity.
65-66. (canceled)
67. A recombinant host cell produced by a method of claim 57 or a
membrane thereof expressing an sFRP-5 polypeptide.
68. A method for screening for modulators of a target protein,
wherein the target protein is sFRP-5 and comprises a sequence that
has greater than 91% amino acid identity to SEQ ID NO:603 as
measured using a sequence comparison algorithm, the method
comprising the steps of a) contacting the target protein with a
candidate agent at a first concentration and determining a level of
activity of the target protein; and b) contacting the target
protein with a candidate agent at a second concentration and
determining a level of activity of the target protein; wherein a
difference between the level of activity of the target protein
contacted with the first concentration of the candidate agent and
the level of activity of the target protein contacted with the
second concentration of the candidate agent indicates that the
candidate agent modulates the activity of the target protein.
69-116. (canceled)
Description
[0001] This application claims benefit of U.S. Provisional
Application No. 60/398,785, filed Jul. 29, 2002, and U.S.
Provisional Application No. 60/478,206, filed Jun. 12, 2003, the
contents of both of which are hereby incorporated by reference.
[0002] Throughout this document various publications or patents are
referenced to describe the state of the art to which the invention
pertains. Each of the referenced publications and patents is
incorporated by reference herein.
[0003] Nucleotide and amino acid sequences referenced in this
document as "SEQ ID NO: ______" are submitted in computer readable
form as a Sequence Listing under 37 C.F.R. .sctn..sctn.1.821(c) and
(e). The contents of the compact disk labeled with the details of
this application containing the file name "69014-PRO2.ST25",
created on Jul. 29, 2003, and having a file size of 1,142
kilobytes, which is the computer readable form of the Sequence
Listing, is incorporated by reference herein. (Nucleotide sequences
are numbered as SEQ.ID.NO. 1-279, and their corresponding amino
acid sequences hear a SEQ.ID.NO. that is 500+ the number of the
nucleotide sequence. For example, the amino acid sequence
SEQ.ID.NO. 501 is encoded by nucleotide sequence SEQ.ID.NO.1.)
FIELD OF THE INVENTION
[0004] The present invention relates to the field of molecular
biology, human metabolism and physiology. In particular, this
invention identifies which known genes are adipose tissue specific,
and provides novel genes that exert influence directly in the
adipose tissue and which play an important role in the regulation
of obesity and activity in adipocyte differentiation and
obesity.
BACKGROUND OF THE INVENTION
[0005] Obesity, or an excess of body fat relative to lean body
mass, is a serious health problem in the United States and abroad.
A person is clinically obese if he or she has excess adipose
tissue. More particularly, for purposes of the present invention, a
person is obese if the person's body mass index equals or exceeds
27 kg/m.sup.2 and the person has excess adipose tissue.
[0006] Statistics suggest that more than 25% of the United States
population and 27% of the Canadian population are overweight.
Complications of obesity include, among others, diabetes mellitus,
hypertension, hyperlipoproteinemia, cardiac diseases
(atherosclerotic disease, congestive heart failure, etc.),
pulmonary diseases (e.g., sleep apnea, restrictive lung disease),
cerebrovascular injury, cancers (including breast, uterine, colon,
and prostate), gall bladder disease (stones, infection), toxemia
during pregnancy, risks during surgery (e.g., pneumonia, wound
infection, thrombo-phlebitis), gout, decreased fertility,
degenerative arthritis, and early mortality. Psychological
complications of obesity include poor self-image and poor
body-image. Social complications of obesity include discrimination
in jobs, education and marriage. Despite the known associated
risks, a significant portion of the population is unable to lose
weight or maintain weight loss. Obesity is now considered the
second leading cause of preventable death in the United States,
second only to smoking, with an estimated 300,000 deaths annually.
Accordingly, reduction of the prevalence of obesity in the adult
population to less than 20% is included by the US Department of
Health and Human Services among the national health objectives.
[0007] The human tragedy notwithstanding, the monetary costs of
obesity are staggering. The total cost attributable to obesity in
1995 has been estimated to be in excess of $99 billion, with
approximately $51.64 billion paid in direct medical costs. Overall,
the direct costs associated with obesity represent 5.7% of the
annual United States national health expenditure. Thus, it is clear
that the magnitude of this problem produces a significant demand
for safe and effective treatments for obesity. Obesity has a number
of known and suspected etiologies. See A. Sclafani, "Animal Models
of Obesity: Classification and Characterization," Int. J. Obesity
8, 491-508 (1984); G. A. Bray, "Classification and Evaluation of
the Obesities," Med. Clin. N. Am. 73, 161-184 (1989). While it is
generally known that overeating and inactivity are factors that
lead to obesity, there is substantial evidence of genetic
contribution to obesity. Although the molecular characterization of
genetic pathways associated with obesity is incomplete, several
recent advances into the elucidation of these pathways have been
made. Research indicates that there are several genes that act
independently or in combination to modulate metabolic pathways
associated with excess adipose tissue accumulation. The presence of
these various pathways suggests a complex system of obesity
regulation, a system that has not yet been fully defined.
[0008] Some mouse models for obesity include obese (ob/ob), agouti
(Ay/a), tubby (tub), fat (fa/fa) and diabetes (db/db). These models
have proven to be effective in the molecular characterization of
these genetic loci because of their ability to simplify the
heritability of complex traits.
[0009] One gene responsible for the autosomal recessive mouse
obesity mutation tub has been identified by positional cloning and
shown to be associated with maturity-onset obesity (U.S. Pat. No.
5,776,762). Identification of the tub gene and the protein it
encodes may lead to the development of agents that will function to
modulate either the protein or gene expression. However, a
disadvantage of this system is the ubiquitous nature of the gene,
in that the gene is expressed in high levels in the brain, eye and
testis, and at lower levels in various adult and fetal tissues,
including small and large intestine, ovary and adipose tissue.
Although the gene may be used as a probe for identification of
other tubby polypeptides, development of agents to modulate the
expression of these polypeptides would not be specific to a
particular tissue.
[0010] Similarly, the ob gene has recently been cloned. The ob gene
encodes a protein known as leptin, which has been implicated in an
energy feedback loop responsible for controlling vertebrate energy
balance. Serum levels of leptin are increased proportionately to
excess adipose accumulation as a result of increased expression in
hypertrophic fat cells in obese patients. In vitro studies have
indicated that insulin and glucocorticoids upregulate leptin mRNA
expression in a synergistic manner. The subsequent expression of
the protein product thereby functions to stimulate metabolic
activity. The promoter of the ob gene has been cloned and is a
candidate for pharmacological control (U.S. Pat. No.
6,124,448).
[0011] In addition to cloning the promoter of the ob gene, attempts
at obesity regulation have also been made through modulation of the
ob gene. The ob/ob mouse is a model of obesity and diabetes that
carries an autosomal recessive trait linked to a mutation in the
sixth chromosome (Yiying Zhang et al. Nature 372: 425-32 (1994).
Pharmacological agents have therefore been developed to mimic the
action of the ob gene encoded protein and assist in regulation of
appetite and metabolism. However, the majority of obese humans
actually have normal or somewhat elevated levels of leptin as
compared to lean humans leading some to hypothesize that human
obesity may be more related to leptin resistance rather than leptin
deficiency. Recent clinical trials have shown that leptin may be
useful for a certain subset of patients, but not for the treatment
of obesity generally (Gura, T., Science 1999, 286 (5441):
881-2).
[0012] Using molecular and classical genetic markers, the ob and db
genes have been mapped to proximal chromosome 6 and midchromosome
4, respectively (Bahary et al., Proc. Nat. Acad. Sci. USA,
87:8642-8646 (1990); Friedman et al., Genomics, 11:1054-1062
(1991)). In both cases, the mutations map to regions of the mouse
genome that are syntenic with human, which suggested that if there
were human homologs of ob and db, they would likely map,
respectively, to human chromosomes 7q and 1p. In fact, the human
homologs have been positionally cloned--OB (the human homolog for
ob) has been cloned to human chromosome 7q31.3 (Isse, et al. J Biol
Chem 1995 Nov. 17; 270 (46): 27728-33). LEPR (the human homolog for
db) has been mapped to human chromosome 1p31 (Thompson, et al.
Genomics 1997; 39(2):227-30). Defects in the leptins receptor gene
results in obesity in other mammalian species: the fa gene in the
rat encodes the leptin receptor.
[0013] Further, a method for detecting differential expression of
specific gene loci has been suggested as a method for identifying a
compound that modulates gene expression, but specific proteins and
pathways have not yet been identified.
[0014] Traditionally, pharmacological approaches to weight loss or
prevention of weight gain have relied either on reduction of food
intake or on reduction of nutrient absorption. Drugs of the first
group, which include Redux (American Home Products) and Meridia
(Knoll Pharmaceuticals), affect neurotransmitter activity in the
brain, resulting in appetite suppression and decreased food intake.
While effective in producing a moderate weight loss in some
proportion of patients these medications are associated with a
number of adverse side effects.
[0015] Drugs of the second group, including Xenical (Hoffmann-La
Roche), reduce total absorption of fat from the gastrointestinal
tract. However, inhibition of fat absorption by this drug can lead
to avitaminosis since successful uptake of fat soluble vitamins
from the intestines is impaired in the absence of fat.
Additionally, these drugs produce unpleasant side-effects, such as
steatorrhea, which reduce patient compliance. Other health problems
have been shown to stem directly or indirectly from use of the drug
as well such as an increased incidence of breast cancer.
[0016] Consequently, focus has since shifted away from these group
one and two pharmaceuticals and instead towards targeting
suppression of gene expression or protein inhibition. Some
pharmaceutical examples are leptin (Amgen), leptin receptor
(Progenitor) and tubby (Millennium Pharmaceuticals). However,
expression of these genes is not limited to adipose tissue and many
specifically act on the brain to stimulate or decrease adipose
accumulation. Therefore, it is possible that development of drugs
to specifically target the central nervous system (CNS) to
interfere with the CNS-active pathways in obese patients may
produce similar side effects to those appetite suppressors that are
currently available. Thus, it is extremely beneficial to target
adipose specific genes or the proteins encoded by such genes.
[0017] Recently, high mobility group I-C protein (HMGI-C) has been
associated with obesity. Obesity induced by two independent methods
of obesity induction, a high fat diet and leptin deficiency, is
alleviated by a partial or complete absence of HMGI-C making HMGI-C
a candidate target molecule for obesity. For example, studies
indicate that HMGI-C is epistatic to Leptin (Lep) in fat tissue,
where Leptin deficiency has been associated with the induction of
weight gain (U.S. Pat. No. 6,124,448).
[0018] However, closer observation reveals that ob/ob mice are not
the classic epistatic relationship because other phenotypic
features of ob/ob mice are still observed on the HMGI-C.sup.-/-
background. This suggests that HMGI-C and leptin function in
independent genetic pathways. Proliferative expansion of
undifferentiated pre-adipocytes requires HMGI-C expression. The
adipose tissue of mice deficient in both HMGI-C and leptin is
composed almost completely of differentiated adipocytes, whereas
mice deficient only in HMGI-C have adipose tissue composed almost
exclusively of preadipocytes. As described herein, these mice have
been found to provide a model system to dissect the genetic
pathways in adipogenesis and to thereby identify genes associated
with obesity.
SUMMARY OF THE INVENTION
[0019] This invention provides a method of identifying genes that
are over-expressed in adipose tissue as compared to non-adipose
tissue comprising performing differential gene expression analysis
between the white adipose tissue (WAT) or stromal vascular tissue
(SVT) from any two different mice selected from the group
consisting of wild-type, HMGI-C-/-, ob/ob, and HMGI-C-/- ob/ob
genotype mice. Based on this method a number of nucleotide
sequences are identified whose expression is adipocyte specific.
The identified nucleotide sequences and their corresponding
polypeptides are then used to prevent adipogenesis, to treat
diabetes, and to screen for small-molecules that can modulate or
prevent adipogenesis and to treat diabetes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The following drawings relate to an embodiment of the
invention:
[0021] FIG. 1 represents the tissue distribution of sFRP-5
expression in wild-type mice
[0022] FIG. 2 represents the dependence of sFRP-5 expression on
HMGI-C in the white adipose tissue (WAT) of wild-type mice versus
HMGI-C-/- mice
[0023] FIG. 3 represents the differential expression of sFRP-5 in
the adipose of wild type versus obese mice
[0024] FIG. 4 represents the differential expression of sFRP-5 in
cells of the stromal vascular fraction (preadipocytes) versus
differentiated adipocytes.
[0025] FIG. 5 represents the increased sFRP-5 expression in CHO
cells harboring an sFRP-5 expression plasmid.
[0026] FIG. 6 represents changes sFRP-5 expression levels in
response to different doses of conditioned medium containing
sFRP-5.
DETAILED DESCRIPTION OF THE INVENTION
[0027] This invention provides a method of identifying adipocyte
specific genes and proteins encoded thereby. "Adipocyte specific"
refers to genes that are preferentially expressed at a higher level
in adipocytes when compared to their expression, if any, in other
tissue. An aspect of the invention relates to methods for using
adipocyte specific polypeptides and polynucleotides, including the
treatment of obesity, hypertension, cardiovascular disease and
diabetes. The present invention is also implicated in diseases and
conditions such as obesity, hypertension, cardiovascular disease
and diabetes, among others. In still another aspect, the invention
relates to methods to identify agonists and antagonists using the
materials provided by the invention, and treating diseases or
conditions associated with obesity, hypertension, cardiovascular
disease and diabetes with the identified compounds. Methods of
using the inventive polynucleotides and polypeptides as
pharmaceutical formulations in the treatment of obese patients are
also provided. Still further provided in the present invention are
methods of screening individuals for predisposition to obesity
using polynucleotides and polypeptides of the present
invention.
[0028] As described, obesity is a condition resulting from an
overaccumulation (hyperplasia) and hypertrophy of fat cells. Due to
an increase in energy intake, preadipocytes enter into a program of
proliferation and differentiation to mature adipocytes. This
differentiation of preadipocytes into mature adipocytes is
accompanied by adipocyte hypertrophy resulting from an increase in
glucose uptake, resulting in increased lipid synthesis, primarily
triglyceride, and accumulation of that lipid as a droplet in the
cytoplasm. The process of the development of obesity is complex,
and there are several levels at which the development and
maintenance of obesity may be affected. These could be at the level
of differentiation from preadipocytes to adipocytes/cell cycle,
accumulation of glucose/lipogenesis, lipolysis (the breakdown of
fats to be burned as energy), signal transduction, apoptosis etc.
The method described herein to identify obesity targets has
identified obesity targets in the categories described below.
Genes Known to Play a Role in Adipocyte Biology
Seq ID Nos. 113-114. Kruppel-Like Factor 15 (KLF15).
[0029] The entry of glucose into the adipocyte requires the
activity of the insulin sensitive glucose transporters. GLUT4 is
the main insulin sensitive glucose transporter and is expressed
predominantly in adipose and muscle tissue. KLF15 is highly
expressed in adipocytes in vivo and is induced when 3T3-L1 are
induced to differentiate into adipocytes in vivo. Overexpression of
KLF15 significantly increases the expression of GLUT4, therefore
KLF15 is an important regulator of GLUT4 and thus entry of glucose
into the adipocyte in response to insulin (Gray, S., M. W.
Feinberg, et al. (2002). J Biol Chem 277(37): 34322-8). Therefore,
agonists of KLF15 would be expected to increase glucose entry into
the adipocyte and therefore increase lipid storage as the glucose
is converted to triglyceride. Conversely, KLF15 antagonists would
restrict glucose entry into the adipocytes and reduce energy i.e.
fat storage.
Seq ID Nos. 72-73. Nitric Oxide Synthase 3 (NOS3).
[0030] Nitric Oxide Synthase has been shown to be increased in the
subcutaneous adipose tissue of obese humans as compared to
non-obese humans. There is evidence to show that increased
production of nitric oxide by nitric oxide synthase, coupled with a
decrease in hormone sensitive lipase, may cause decreased lipolysis
in obesity. Therefore decreasing NOS3 would be expected to increase
lipolysis in adipocytes resulting in reduced obesity.
Seq ID Nos. 89-90. Carbonic Anhydrase III.
[0031] Carbonic anhydrase plays a major role in energy metabolism
by facilitating transport of CO2 produced by oxidative
phosphorylation. Metabolic rate plays an important role in adipose
accumulation. If the metabolic rate is low and few calories are
burned as energy, any excess calories are stored as lipid in the
differentiated adipocyte. Likewise, an increase in metabolism
results in increased lipolysis and a reduction of the fat stored in
adipocytes. Therefore an increase in carbonic anhydrase would
stimulate metabolism and result in an increase in
lipolysis/decrease in adipocyte hypertrophy.
Seq ID Nos. 154-155. Lactotransferrin.
[0032] Lactotransferrin acts as an inhibitor of carbonic anhydrase
(see above). Therefore, inhibition of lactotransferrin would lead
to an increase in carbonic anhydrase activity thus stimulating
metabolism and result in an increase in lipolysis/decrease in
adipocyte hypertrophy.
Seq ID Nos. 1-3. S3-12.
[0033] Plasma membrane S3-12 is a plasma membrane protein that is
highly induced after adipogenesis (Scherer, P. E., P. E. Bickel, et
al. (1998). Nat Biotechnol 16(6): 581-6). Lipid is stored in
adipocytes in protein coated lipid droplets, and generally these
proteins which coat the droplet are members of the perilipin
family. S3-12 is a member of this family. Prior to incubation with
fatty acids, S3-12 is spread diffusely throughout the adipocyte.
Upon incubation with fatty acids, S3-12 begins to coalesce around
lipid droplets and by 240 minutes of incubation the S3-12 has
become incorporated into perilipin coated droplets. Thus, S3-12
plays a role in lipid storage in the adipocyte (Wolins, N. E., J.
R. Skinner, et al. (2003). J Biol Chem).
Seq ID Nos. 101-102 Carboxylesterase (p62/CE).
[0034] As stated above, GLUT4 plays a major role for entry of
glucose into the adipocyte in response to insulin. GLUT4 is
sequestered into intracellular sites and is recruited to the cell
surface by insulin. P62/CE is found in all insulin sensitive GLUT4
intracellular compartments, and inhibition of p62/CE by an
anti-p62/CE antibody abolished insulin recruitment of GLUT4 to the
plasma membrane without changing the intracellular GLUT4
distribution. Therefore, p62/CE plays an crucial role for insulin
recruitment of GLUT4 to the plasma membrane (Lee, W., J. Ryu, et
al. (2000). J Biol Chem 275(14): 10041-6).
Seq ID Nos. 12-13. Fsp-27.
[0035] FSP27 is an adipocyte specific gene whose expression is
regulated by C/EBP as is seen in many genes involved in adipocyte
biology. Fsp27 is a member of a family of cell-death-inducing
DFF40-like effectors (CIDEs) (Danesch, U., W. Hoeck, et al. (1992).
J Biol Chem 267(10): 7185-93). The CIDEs have been shown to induce
DNA fragmentation which is a hallmark of apoptosis. Thus, FSP27
likely plays a role in the programmed cell death of mature
adipocytes. Thus, agonizing FSP27 would be expected to result in an
increase in adipocyte apoptosis resulting in a reduced adipose mass
and reduced adiposity.
Seq ID Nos. 120-121. Galectin 12.
[0036] Galectin-12 has recently been shown to be a predominantly
adipocyte-expressed protein which is stimulated by
insulin-sensitizing thiazolidinediones and possesses
apoptosis-inducing activity (Fasshauer, M., J. Klein, et al.
(2002). Eur J Endocrinol 147(4): 553-9). Caloric restriction and
treatment of obese animals with troglitazone results in an increase
in Galectin 12 expression and a decrease in adipocyte size (Hotta,
K., T. Funahashi, et al. (2001). J Biol Chem 276(36): 34089-97).
Induction of Galectin 12 expression is by troglitazone is in
parallel with an increase in apoptotic cells, and transfection of
293 cell with Galectin 12 results in an induction of apoptosis.
Thus, agonizing Galectin 12 would be expected to result in an
increase in adipocyte apoptosis resulting in a reduced adipose mass
and reduced adiposity.
Seq ID Nos. 158-159. ABCD2 (ALDR).
[0037] ABCD2 is regulated by the cholesterol sensitive sterol
regulatory element binding proteins (SREPBs) which are potent
transcription factors that are expressed in the differentiating
adipocyte and that regulate many genes involved in cholesterol and
lipid metabolism. ABCD2 is a peroxisomal protein involved in the
beta oxidation of very long chain fatty acids. Peroxisome
proliferation is a major event in the differentiation of
adipocytes, and interference with peroxisomal function would likely
disrupt that process.
Seq ID Nos. 167. Ras Like GTPase TC10.
[0038] Expression of this protein is rapidly induced in 3T3-L1
preadipocytes upon addition of induction medium. Antisense molecule
to this protein which inhibit expression of TC10 also block the
differentiation of 3T3-L1 Preadipocytes into adipocytes in response
to differentiation medium. NIH-3T3 cells, which do not usually
differentiate into adipocytes, show clear signs of lipid droplet
formation and accumulation when TC10 is overexpressed in these
cells. The cells also show an increase in expression of PPAR gamma
regulated genes in the presence of PPAR gamma ligand. Taken
together, these results indicate an essential role for TC10 in the
early stages of adipocyte differentiation which may be linked to
the PPAR gamma pathway (Nishizuka, M., E. Arimoto, et al. (2003). J
Biol Chem 278(17): 15279-84).
Seq ID Nos. 173-174. Proteasome Subunit Beta-5.
[0039] The proteasome is an essential pathway for the degradation
of proteins. The proteasome consists of three "particles": the core
particle, which consists of 2 copies of each of 14 different
proteins assembled in four stacked rings, and two identical
regulatory particles each of which is made of 14 different proteins
(distinct from those of the core particle) of which 6 are ATPases.
Proteins destined for degradation are conjugated with a short (76
amino acid) protein called ubiquitin. Additional molecules of
ubiquitin are then bound to the first ubiquitin molecule forming a
chain. The ubiquitin-protein complex binds to ubiquitin recognizing
sites on the regulatory particle and the protein is unfolded by the
ATPases. The unfolded protein is then transported into the central
cavity of the core particle where the protein is then cleaved into
peptide chains averaging 8 amino acids long which are then degraded
by proteases in the cytosol. The proteasome plays an important role
in adipocyte biology. For example, insulin rapidly stimulates the
tyrosine kinase activity of the insulin receptor. This leads to
phosphorylation of the insulin receptor substrates which then
activates phosphoinositol 3 kinase (PI 3-kinase) resulting in
increased glucose uptake. It has been demonstrated that
interference with proteasome function prolongs sustains the
tyrosine phosphorylation of insulin receptor substrate 1 (IRS1)
potentiating glucose intake by the adipocyte, leading to a greater
accumulation of energy (Rondinone, C. M. and D. Kramer (2002).
Biochem Biophys Res Commun 296(5): 1257-63.). Also, it has been
demonstrated that tumor necrosis factor alpha (TNF alpha)
stimulates lipolysis, in part, through inhibitory G proteins
(G(i)). Inhibition of proteasome activity blocks the ability of TNF
alpha to stimulate lipolysis through the G(i) pathway (Botion, L.
M., A. R. Brasier, et al. (2001). Endocrinology 142(12): 5069-75).
Thus, alteration of proteasome function would affect the ability of
adipocytes to take up glucose in response to insulin and hydrolyze
lipids in response to TNF-alpha.
Seq ID Nos. 202-203. Resistin-Like Molecule Alpha (RELM Alpha).
[0040] RELM alpha belongs to a family of cysteine rich proteins
similar to the molecule resistin ("resitant to insulin"). RELM
alpha has no effect on the proliferation of undifferentiated 3T3-L1
preadipocytes, and pretreatment of 3T3-L1 preadipocytes has no
effect on insulin induced mitogenesis or insulin induced IRS-1
phosphorylation and glucose transport. However, treatment of 3T3-L1
cells with resistin-like alpha inhibits their differentiation into
adipocytes (Blagoev, B., I. Kratchmarova, et al. (2002). J Biol
Chem 277(44): 42011-6). Since resistin like alpha is a secreted
protein expressed highly by adipose tissue, it is an important
regulator of adipocyte differentiation.
Seq ID Nos. 204-205. Thyroid Hormone Responsive SPOT14.
[0041] SPOT14 is a transcription factor which plays a key role in
tissue specific regulation of lipid metabolism. SPOT14 was
initially found to be responsive to thyroid hormone, but was later
found also to be responsive to dietary stimuli, including glucose
and polyunsaturated fats, as well as other energy related hormones
including insulin and glucagons (Cunningham, B. A., J. T. Moncur,
et al. (1998). Thyroid 8(9): 815-25). SPOT14 regulates the
expression of key metabolic enzymes including those for the
synthesis of long chain fatty acids. Inhibition of SPOT14 would
lead to decreased lipogenesis in adipocytes and therefore decreased
fat accumulation.
Seq ID Nos. 38-39. Matrix Metalloproteinase 9 (MMP9).
[0042] The growth of fat requires adipocyte hyperplasia and
hypertrophy as well as angiogenesis, and changes of the
extracellular matrix components often accompany this cellular
remodeling. Also, 3T3-L1 cells change their morphology upon)
differentiation, from an extended fibroblast-like phenotype to a
more rounded one (Croissandeau, G., M. Chretien, et al. (2002).
Biochem J 364(Pt 3): 739-46), again as a result of extracellular
matrix remodeling. MMP9 expression is upregulated both in 3T3-L1
and 3T3F442A preadipocyte upon induction of differentiation.
However, inhibition of MMP9 results in a failure of these cells to
differentiate in response to the differentiation medium (Lijnen, H.
R., E. Maquoi, et al. (2002). Arterioscler Thromb Vasc Biol 22(3):
374-9). Therefore, inhibition of MMP9 would reduce adipocyte
differentiation, one of the key events in the development of
obesity.
Seq ID Nos. 40-41. PTPase.
[0043] Obesity is often accompanied by a state of insulin
resistance, and this insulin resistance is often reduced in
response to weight loss. Reductions in PTPase have been shown to
correlate with improved insulin sensitivity in obese subjects in
response to weight loss (Ahmad, F., R. V. Considine, et al. (1997).
Metabolism 46(10): 1140-5). Insulin dependent oxidative inhibition
of PTPase plays an important role in permitting the transmission of
the signal from insulin to glucose transport (Wu, X., V. E. Hardy,
et al. (2003). Metabolism 52(6): 705-12). Furthermore, inhibition
of PTPase potentiates insulin signal transduction in 3T3-L1 cells
(Wu, X., V. E. Hardy, et al. (2003). Metabolism 52(6): 705-12).
Seq ID Nos. 10-11. Vap-1.
[0044] Vap-1 belongs to a family of semicarbazide sensitive amine
oxidases (SSAO). Vap-1 is upregulated in 3T3-L1 and 3T3-F442A
preadipocytes upon differentiation, and its enzymatic activity has
been shown to upregulate the PI-3 kinase pathway in these cell
types as well (Mercier, N., M. Moldes, et al. (2001). Biochem J
358(Pt 2): 335-42). This upregulation of the PI-3 kinase pathway
mimics the effects of insulin (i.e. increased glucose uptake and
decreased lipolysis) (Zorzano, A., A. Abella, et al. (2003).
Biochim Biophys Acta 1647(1-2): 3-9). This same effect is also seen
in human adipocytes. Furthermore, Vap-1 activity stimulates the
acquisition of adipocyte morphology (upregulation of GLUT4, GLUT2,
aP2, and glycerol-3-phosphate dehydrogenase) in 3T3-F442A cells.
Thus, inhibition of Vap-1 would result in a decrease in fat cell
differentiation and therefore the accumulation of fat in
adipocytes.
Seq ID Nos. 34-35. Early B-Cell Factor (O/E-1).
[0045] O/E-1 is a transcription factor that is expressed in adipose
tissue and is upregulated during adipogenesis. Forced expression of
O/E-1 in either 3T3-L1 preadipocytes of mouse embryonic fibroblasts
increases adipocyte differentiation (Akerblad, P., U. Lind, et al.
(2002). Mol Cell Biol 22(22): 8015-25). Furthermore, constitutive
expression of O/E-1 in uncommitted 3T3NIH fibroblasts, which do not
normally differentiate into adipocytes leads to the initiation of
differentiation to adipocytes. Repression of O/E-1 suppresses
3T3-L1 differentiation. Thus, O/E-1 is an important regulator of
adipocyte differentiation and inhibition of O/E-1 would lead to a
decrease in differentiated adipocytes and therefore a decrease in
adipocyte hypertrophy.
Seq ID Nos. 64-65. Sonic Hedgehog Homolog.
[0046] The hedgehog family of proteins regulate varying aspects of
tissue development.
[0047] Sonic Hedgehog (Shh) abolishes the differentiation of
C3H10T1/2 cells (Spinella-Jaegle, S., G. Rawadi, et al. (2001). J
Cell Sci 114(Pt 11): 2085-94). Short treatment of these cells with
Shh dramatically reduces the adipogenic transcription factors C/EBP
alpha and PPAR gamma. Therefore, inhibition of Shh would result in
a decrease in adipocyte differentiation and thus a decrease in
adipocyte hypertrophy.
G-Proteins/G-Protein Coupled Receptors/Signal Transduction
[0048] It has been well established that many medically significant
biological processes are mediated by proteins participating in
signal transduction pathways that involve guanyl-nucleotide-binding
proteins (G-proteins) and/or second messengers, e.g., cAMP. Some
examples of proteins that participate in G-protein signaling
pathways include the G-protein coupled receptors (GPCRs), such as
those adregenergic and dopaminergic receptors, G-proteins
themselves, effector proteins, e.g., phospholipase C, adenyl
cyclase, guanylyl cyclase, and phosphodiesterase, and actuator
proteins such as protein kinase A and protein kinase C.
[0049] An example of this type of G-protein signal transduction
would be binding of a hormone to a cell surface receptor activating
the enzyme adenyl cyclase. The hormone binds to its cell surface
receptor which in turn binds to a heterotrimeric G-protein in the
plasma membrane consisting of an alpha, a beta, and a gamma
subunit. The binding of the hormone receptor to the G-protein
causes the G-protein to exchange a bound molecule of GDP with a
molecule of GTP. The GTP bound form then interacts with and
activates adenyl cyclase which converts ATP to cyclic AMP (cAMP)
which acts as a second messenger to deliver the signal. The
G-protein is also a slow GTPase, and slowly converts the bound GTP
to GDP, thus returning the G-protein to its inactive form. In the
absence of hormone, the vast majority of G-proteins are bound to
GDP and are thus inactive. A single hormone bound receptor is
capable of activating many O-protein complexes, thus amplifying the
signal. Thus the G-protein acts in two ways: it transmits and
amplifies the signal from the receptor to the effector, and
determines the duration of the signal by the rate at which its
GTPase activity hydrolyzes GTP to GDP and inactivates itself.
[0050] G-protein coupled receptors can be intracellularly coupled
by heterotrimeric G-proteins to various intracellular enzymes, ion
channels and transporters. Different G-protein .alpha.-subunits
preferentially stimulate or inhibit particular effectors to
modulate various biological functions in a cell. Phosphorylation of
cytoplasmic residues of G-protein coupled receptors have been
identified as an important mechanism for the regulation of
G-protein coupling of some G-protein coupled receptors. G-protein
coupled receptors are found in numerous sites within a mammalian
host. Genes found by our method that participate in G-protein
signal transduction include:
Seq ID Nos.
143-144 CDC42
234-235 GPR18
[0051] 49-50 G-protein inhibitory alpha subunit 1
55-56 Guanylate Nucleotide Binding Protein 2
[0052] 59, 83 G-protein alpha 2 subunit 163-164 Ras p21 protein
activator 2 (Rasa2)
257-258 GPR127
[0053] The process of signal transduction by which signals from
outside of the cell are transmitted to the inside of the cell are
central to many biological processes. For example, the signal for
insulin to upregulate expression of the insulin sensitive glucose
transporters is key in the etiology of obesity and diabetes. Other
signal transduction molecules that were found using our method
include:
Seq ID Nos.
4-9 Neuronatin
136-137 Leukotriene C4 Synthase
185-186 Copine II
[0054] 208-209 Syntaxin 1B like molecule
Inflammation
[0055] There is evidence to indicate that obesity may be a chronic,
low-grade inflammatory response (Das, U. N., Nutritition, 2001;
17(11-12):956-966). Other examples of chronic inflammatory diseases
are arthritis, atherosclerosis, etc. Obese children and adults have
elevated markers of inflammatory response including C-reactive
protein, interleukin-6, and tumor necrosis factor alpha (TNF
alpha). In particular, TNF alpha is well known to have a profound
stimulatory effect on lipolysis in adipocytes. Inflammation factors
which affect obesity found using our method are:
Seq ID Nos.
[0056] 78-79 chemokine (C-C) receptor 2
14 MRP14
17-18 CD1d1
147 Isg12
[0057] 152-153 Decay accelerating factor 1 (DAF1)
29 Scya6
36-37 Scya 5 (RANTES)
[0058] 21-22 CD53 antigen
57-58 Proteinase 3
[0059] 225-226 D-6 beta chemokine receptor 80-81 Interferon
regulatory factor 4
Apoptosis
[0060] The term apoptosis was coined in a now-classic paper by
Kerr, Wyllie, and Currie (Brit J. Cancer 26:239) in 1972 as a means
of distinguishing a morphologically distinctive form of cell death
which was associated with normal physiology. Apoptosis was
distinguished from necrosis, which was associated with acute injury
to cells. Apoptosis is characterized by nuclear chromatin
condensation, cytoplasmic shrinking, dilated endoplasmic reticulum,
and membrane blebbing. Mitochondria remain unchanged
morphologically.
[0061] Apoptotic death can be triggered by a wide variety of
stimuli, and not all cells necessarily will die in response to the
same stimulus. Among the more studied death stimuli is DNA damage
(by irradiation or drugs used for cancer chemotherapy), which in
many cells leads to apoptotic death via a pathway dependent on p53.
Some hormones such as corticosteroids lead to death in particular
cells (e.g., thymocytes), although other cell types may be
stimulated. Some cells types express Fas, a surface protein which
initiates an intracellular death signal in response to
crosslinking. In other cases cells appear to have a default death
pathway which must be actively blocked by a survival factor in
order to allow cell survival.
[0062] Apoptosis in adipocytes (lipoapoptosis) is an important
regulator of fat mass. If lipoapoptosis is defective, then an
overaccumulation of fat cells will occur and will contribute to the
development and maintenance of obesity. Factors affecting
lipoapoptosis identified using our method include:
Seq ID Nos.
[0063] 80-81 Interferon regulatory factor 4
12-13 Fsp27
120-121 Galectin 12
Angiogenesis
[0064] Angiogenesis is the process by which growing tissues
generate new blood vessels (vascularizartion) in order to ensure an
adequate blood supply. Prevention of angiogenesis can be used to
treat human diseases. For example, certain types of cancer may be
treated by inhibiting vascular endothelial growth factor (VEGF) so
that the tumor cannot grow new blood vessels and thus cannot
expand. Angiogenesis must also occur in growing adipose tissue.
Leptin normally inhibits angiogenesis (Yamagishi, et al., 2003,
Microvasc Res, 65(3):186-190) although this mechanism fails in
obesity. Therefore, genes that affect the ability of adipose tissue
to vascularize as it expands would be useful for the treatment of
obesity. The genes involved in angiogenesis that we have found
using our method includes:
Seq ID Nos.
15-16 Peg1/MEST
[0065] 19-20 Synuclein gamma
38-39 MMP9
Differentiation
[0066] Differentiation of preadipocytes into adipocytes is one of
the first events in obesity. In a response to excess calorie
intake, preadipocytes multiply (hyperplastic obesity) and
differentiate. The cells enter a program in which they uptake
glucose from the plasma and convert it into fat, primarily
triglyceride. This fat is store in the cytoplasm as a lipid
droplet. As the droplet accumulates lipid, the adipocytes swell
until they are no longer able to increase in size (hypertrophic
obesity). Thus, interference with the ability of preadipocytes to
differentiate would aid in the treatment of obesity. Genes involved
in adipocyte differention identified using our method include:
Seq ID Nos.
158-159 ABCD2
167 GTPase TC10
[0067] 202-203 Resistin like molecule alpha 206-207 cyclin M-3
40-41 CD45
34-35 Early B Cell Factor (O/E-1)
[0068] 60-61 Colony Stimulating Factor 2 Receptor (CSF2 receptor)
64-65 Sonic Hedgehog homolog
165-166 Arl4
[0069] 212-213 limb-bud and heart gene (LBH)
[0070] The nucleic acid sequences not specifically referred to
above but disclosed herein are also relevant in adipogenesis by
virtue of being identified by the method described.
Polynucleotides
[0071] Included in the present invention are polynucleotides
encoding polypeptides which have at least 91% identity, preferably
at least 92% identity, more preferably at least 993% identity, yet
more preferably at least 94% identity, even more preferably at
least 96-99% identity, to the amino acid sequence of any of the
adipocyte specific peptides over the entire length of the recited
amino acid sequence.
[0072] When the polynucleotides of the invention are used for the
recombinant production of the nucleic acid, the polynucleotide may
include the coding sequence for the mature polypeptide or a
fragment thereof, by itself; the coding sequence for the mature
polypeptide or fragment in reading frame with other coding
sequences, such as those encoding a leader or secretory sequence, a
pre-, or pro- or prepro-protein sequence, or other fusion peptide
portions. For example, a marker sequence which facilitates
purification of the fused polypeptide can be encoded. The
polynucleotide may also contain non-coding 5' and 3' sequences,
such as transcribed, non-translated sequences, splicing and
polyadenylation signals, ribosome binding sites and sequences that
stabilize mRNA.
[0073] Thus, this invention provides oligonucleotides (sense or
antisense strands of DNA or RNA) having sequences capable of
hybridizing with at least one sequence of a nucleic acid molecule
encoding the protein of the present invention. Such
oligonucleotides are useful as probes for detecting genes
identified by the adipocyte specific sequences or their
transcripts. In one preferred embodiment, oligonucleotides for use
as probes or primers are based on rationally-selected amino acid
sequences chosen from the sequence expressed by the adipocyte
specific nucleotide sequences. In preferred embodiments, the amino
acid sequence information is used to make degenerate
oligonucleotide sequences as is commonly done by those skilled in
the art. In other preferred embodiments the degenerate
oligonucleotides are used to screen cDNA libraries from human and
mouse.
[0074] The polynucleotides of the present invention may be prepared
by two general methods: (1) they may be synthesized from
appropriate nucleotide triphosphates, or (2) they may be isolated
from biological sources. Both methods utilize protocols well known
in the art. The availability of nucleotide sequence information,
such as the cDNA from this disclosure, enables preparation of an
isolated nucleic acid molecule of the invention by oligonucleotide
synthesis. Synthetic oligonucleotides may be prepared by the
phosphoramadite method employed in the Applied Biosystems 38A DNA
Synthesizer or similar devices. The resultant construct may be
purified according to methods known in the art, such as high
performance liquid chromatography (HPLC). Long, double-stranded
polynucleotides, must be synthesized in stages, due to the size
limitations inherent in current oligonucleotide synthetic methods.
Thus, for example, a long double-stranded molecule may be
synthesized as several smaller segments of appropriate
complementarity. Complementary segments thus produced may be
annealed such that each segment possesses appropriate cohesive
termini for attachment of an adjacent segment. Adjacent segments
may be ligated by annealing cohesive termini in the presence of DNA
ligase to construct an entire long double-stranded molecule. A
synthetic DNA molecule so constructed may then be cloned and
amplified in an appropriate vector. Relevant genes also may be
isolated from appropriate biological sources using methods known in
the art. In the exemplary embodiments of the invention, the genes
may be isolated from genomic libraries of human or mouse. In
alternative embodiments, cDNA clones of the genes may be isolated,
such as have been isolated from human, or murine cDNA libraries. A
preferred means for isolating genes is PCR amplification using
genomic or cDNA templates and gene specific primers. Genomic and
cDNA libraries are commerically available, and can also be made by
procedures well known in the art. In positions of degeneracy where
more than one nucleic acid residue could be used to encode the
appropriate amino acid residue, all the appropriate nucleic acid
residues may be incorporated to create a mixed oligonucleotide
population, or a neutral base such as inosine may be used. The
strategy of oligonucleotide design is well known in the art.
[0075] Alternatively, PCR primers may be designed by the above
method to match the coding sequences of a human or murine protein
and these primers used to amplify the native nucleic acids from
isolated cDNA or genomic DNA.
[0076] In accordance with the present invention, nucleic acids
having the appropriate level sequence homology with a part or with
all of the coding region, may be identified by using hybridization
and washing conditions of appropriate stringency. For example,
hybridizations may be performed, according to the method of
Sambrook et al., using a hybridization solution comprising: 1.0%
SDS, up to 50% formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium
citrate), 0.05% sodium pyrophosphate (pH 7.6), 5.times.Denhardt's
solution, and 100 microgram/ml denatured, sheared salmon sperm DNA.
Ilybridization is carried out at 37-42.degree. C. for at least six
hours. Following hybridization, filters are washed as follows: (1)
5 minutes at room temperature in 2.times.SSC and 1% SDS; (2) 15
minutes at room temperature in 2.times.SSC and 0.1% SDS; (3) 30
minutes to 1 hour at 37.degree. C. in 2.times.SSC and 0.1% SDS; (4)
2 hours at 45-55.degree. C. in 2.times.SSC and 0.1% SDS, changing
the solution every 30 minutes.
[0077] One common formula for calculating the stringency conditions
required to achieve hybridization between nucleic acid molecules of
a specified percent identity is set forth by (Sambrook et al.,
1989, supra):
T.sub.m=81.5.degree. C.+16.6 Log [Na.sup.+]+0.41(% G+C)-0.63(%
formamide)-600/#bp in duplex
As an illustration of the above formula, using [Na.sup.+]=[0.368]
and 50% formamide, with GC content of 42% and an average probe size
of 200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA
duplex decreases by 1-1.5.degree. C. with every 1% decrease in
homology. Thus, targets with greater than about 75% sequence
identity would be observed using a hybridization temperature of
42.degree. C.
[0078] The stringency of the hybridization and wash depend
primarily on the salt concentration and temperature of the
solutions. In general, to maximize the rate of annealing of the
probe with its target, the hybridization is usually carried out at
salt and temperature conditions that are 20-25.degree. C. below the
calculated T.sub.m of the of the hybrid. Wash conditions should be
as stringent as possible for the degree of identity of the probe
for the target. In general, wash conditions are selected to be
approximately 12-20.degree. C. below the T.sub.m of the hybrid. In
regards to the nucleic acids of the current invention, a moderate
stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and wash in
2.times.SSC and 0.5% SDS at 55.degree. C. for 15 minutes. A high
stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and wash in
1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes. A very
high stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/ml
denatured salmon sperm DNA at 42.degree. C., and wash in
0.1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes.
[0079] Nucleic acids of the present invention may be maintained as
DNA in any convenient cloning vector. In a preferred embodiment,
clones are maintained in plasmid cloning/expression vector, such as
pBluescript (Stratagene, La Jolla, Calif.), which is propagated in
a suitable E. coli host cell.
[0080] The adipocyte specific polynucleotides may be used for a
variety of purposes in accordance with the present invention. DNA,
RNA, or fragments thereof may be used as probes to detect the
presence of and/or expression of adipocyte specific genes. Methods
in which the polynucleotides may be utilized as probes for such
assays include, but are not limited to: (1) in situ hybridization;
(2) Southern hybridization (3) northern hybridization; and (4)
assorted amplification reactions such as polymerase chain reaction
(PCR).
[0081] The polynucleotides may also be utilized as probes to
identify related genes from other species. As is well known in the
art, hybridization stringencies may be adjusted to allow
hybridization of nucleic acid probes with complementary sequences
of varying degrees of homology.
[0082] As described above, adipocyte specific nucleic acids may be
used to produce large quantities of substantially pure proteins, or
selected portions thereof.
[0083] The nucleic acids of the present invention can be used to
identify and isolate other members of the growth regulatory
pathway(s) in which the protein is involved. A yeast two-hybrid
system can be used to identify proteins that physically interact
with the identified adipocyte specific protein, as well as isolate
their nucleic acids. In this system, the coding sequence of the
protein of interest is operably linked to the coding sequence of
half of an activator protein. This construct is used to transform a
yeast cell library which has been transformed with DNA constructs
that contain the coding sequence for the other half of the
activator protein operably linked to a random coding sequence from
the organism of interest. When the protein made by the random
coding sequence from the library interacts with the protein of
interest, the two halves of the activator protein are physically
associated and form a functional unit that activates the reporter
gene. In accordance with the present invention, all or part of the
human, mouse, bovine or rat coding sequence may be operably linked
to the coding sequence of the first half of the activator, and the
library of random coding sequences may be constructed with cDNA
from human, mouse, bovine or rat and operably linked to the coding
sequence of the second half of the activator protein. Several
activator protein/reporter genes are customarily used in the yeast
two hybrid system, the Gal4/LacZ system (see Clark et al., 1998
PNAS 95:5401-5406), among others.
[0084] The nucleotide sequences of the present invention are also
valuable for chromosomal localization. The sequence is specifically
targeted to, and can hybridize with, a particular location on an
individual eukaryotic chromosome. The mapping of relevant sequences
to chromosomes according to the present invention is an important
first step in correlating those sequences with gene associated
disease. Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. The relationship between
genes and diseases that have been mapped to the same chromosomal
region are then identified through linkage analysis (coinheritance
of physically adjacent genes).
[0085] In the present invention, mapping of the adipocyte specific
nucleic acid sequences to chromosomes would be an especially
beneficial contribution to the human obesity gene map. The human
obesity study has attempted to relate DNA sequence variation in
specific genes to obesity phenotypes. To date, 44 loci have linked
to obesity indicators in genomic scans and other linkage study
designs. The number of genes, markers, and chromosomal regions that
have been associated or linked with human obesity phenotypes
continues to increase and is now well above 200. (The Human Obesity
Gene Map: The 1999 Update, Obesity Research Vol 8 NO: 1 January
2000.)
[0086] The polynucleotides of the present invention can be used to
identify and further isolate other members of the adipose
accumulation pathways specific to adipose tissue. Identification of
proteins that interact with the protein encoded by identified genes
will introduce new agents to influence the pathways involved in
adipose accumulation.
Polypeptides
[0087] In another aspect, the present invention relates to human
adipocyte specific polypeptides (or proteins). The present
invention further provides for a polypeptide which comprises an
amino acid sequence which has at least 90% identity, more
preferably at least 91% identity, yet more preferably at least 92%
identity, yet more preferably at least 95% identity, most
preferably at least 96-99% identity, to that of the entire length
of any one of the sequences identified herein.
[0088] In one aspect of the invention, the polypeptides may be
produced in large amounts in order to aid in the identification of
ligands or substrates that bind to, or modulate the action of the
polypeptides. The action of purified protein may be characterized
by determination of its three dimensional crystal structure leading
to elucidation of the molecular binding sites further aiding
studies in computer modeling of intermolecular interactions within
substrate binding sites. Determination of specific sites of
activity within the protein structure aids in the design of
pharmaceuticals to directly interact with the protein to modulate
its activity.
[0089] In another aspect of the invention, characterization of the
protein aids in elucidation of the protein pathways involved in
adipose accumulation because the development of agents to minimize
protein activity may disrupt associated protein pathways and
promote discovery of associated proteins. Characterization assays
may be developed with the aid of labeled in vitro protein-protein
binding assays, electrophoretic mobility shift assays, immunoassays
for protein binding. In addition, chromophoric substitution within
the protein binding site may be used in fluorescence studies
measuring chromophoric quenching.
[0090] The present data indicates that the identified genes are
associated with adipose accumulation. The identified adipocyte
specific polypeptides may be in the form of the "mature" protein or
may be a part of a larger protein such as a fusion protein. It is
often advantageous to include an additional amino acid sequence
which contains secretory or leader sequences, pro-sequences,
sequences which aid in purification such as multiple histidine
residues, or an additional sequence for stability during
recombinant production.
[0091] Fragments of the polypeptides are also included in the
invention. A fragment is a polypeptide having an amino acid
sequence that entirely is the same as part, but not all, of the
amino acid sequence of the polypeptides. Preferred fragments
include, for example, truncation polypeptides having the amino acid
sequence of the polypeptides, except for deletion of a continuous
series of residues that includes the amino terminus, or a
continuous series of residues that includes the carboxyl terminus
or deletion of two continuous series of residues, one including the
amino terminus and one including the carboxyl terminus. Also
preferred are fragments characterized by structural or functional
attributes such as fragments that comprise alpha-helix and
alpha-helix forming regions, beta-sheet and beta-sheet-forming
regions, turn and turn-forming regions, coil and coil-forming
regions, hydrophilic regions, hydrophobic regions, alpha
amphipathic regions, beta amphipathic regions, flexible regions,
surface-forming regions, substrate binding regions, and high
antigenic index regions. Other preferred fragments are biologically
active fragments. Biologically active fragments are those that
mediate the peptides activity, including those with a similar
activity or an improved activity, or with a decreased undesirable
activity. Also included are those that are antigenic or immunogenic
in an animal, especially in a human. Preferably, all of these
polypeptide fragments retain the biological activity of the
identified peptide, including any antigenic activity. Variants of
the defined sequence and fragments also form part of die present
invention. Preferred variants are those that vary from the
referents by conservative amino acid substitutions.
[0092] The polypeptides of the invention can be prepared in any
suitable manner. If produced in situ, the polypeptides may be
purified from appropriate sources, e.g., cells from human or
mouse.
[0093] Alternatively, the availability of nucleic acid molecules
encoding the polypeptides enables production of the proteins using
in vitro expression methods known in the art. For example, a cDNA
or gene may be cloned into an appropriate in vitro transcription
vector, for in vitro transcription, followed by cell-free
translation in a suitable cell-free translation system. In vitro
transcription and translation systems are commercially available,
e.g., from Promega Biotech, Madison, Wis., or Invitrogen, Carlsbad,
Calif. While in vitro transcription and translation is not the
method of choice for preparing large quantities of the protein, it
is ideal for preparing small amounts of native or mutant proteins
for research purposes, particularly since it allows the
incorporation of radioactive nucleotides.
[0094] According to a preferred embodiment, larger quantities of
the encoded polypeptide may be produced by expression in a suitable
prokaryotic or eukaryotic system. For example, part or all of a DNA
molecule, such as the coding portion of an identified sequence may
be inserted into a plasmid vector adapted for expression in a
bacterial cell (such as E. coli) or a yeast cell (such as
Saccharomyces cerevisiae), or into a baculovirus vector for
expression in an insect cell. Such vectors comprise the regulatory
elements necessary for expression of the DNA in the host cell,
positioned in such a manner as to permit expression of the DNA into
the host cell. Such regulatory elements required for expression
include promoter sequences, transcription initiation sequences and,
optionally, enhancer sequences.
[0095] Secretion signals may be used to facilitate purification of
the resulting protein. The coding sequence for the secretion
peptide is operably linked to the 5' end of the coding sequence for
the protein, and this hybrid nucleic acid molecule is inserted into
a plasmid adapted to express the protein in the host cell of
choice. Plasmids specifically designed to express and secrete
foreign proteins are available from commercial sources. For
example, if expression and secretion is desired in E. coli,
commonly used plasmids include pTrcPPA (Pharmacia); pPROK-C and
pKK233-2 (Clontech); and pNH8a, pNH16a, pcDNAII and pAX
(Stratagene), among others.
[0096] The proteins produced by in vitro transcription and
translation or by gene expression in a recombinant prokaryotic or
eukaryotic system may be purified according to methods known in the
art. Recombinant proteins can be purified by affinity separation,
such as by immunological interaction with antibodies that bind
specifically to the recombinant protein or fusion proteins such as
His tags, as described below. Such methods are commonly used by
skilled practitioners.
[0097] As mentioned, the proteins can be produced and fused to a
"tag" protein in order to facilitate subsequent purification. These
fusion proteins are produced by operably-linking the nucleic acid
coding sequence of the "tag" protein to the coding sequence of the
protein of interest, and expressing the fused protein by standard
methods. Systems are commercially available that comprise a plasmid
containing an expression cassette with the "tag" protein coding
sequence and a polylinker into which a coding sequence of interest
can be operably ligated. These fusion protein systems further
provide chromatography matrices or beads which specifically bind
the "tag" protein thereby facilitating the fusion protein
purification. These fusion protein systems often have the
recognition sequence of a protease at or near the junction of the
"tag" protein and the protein of interest so that the "tag" protein
can be removed if desired. Fusion protein systems include, but are
not limited to, the His-6-tag system (Quiagen) and the
glutathione-S-transferase system (Pharmacia).
[0098] The proteins of the invention, prepared by one of the
aforementioned methods, may be analyzed according to standard
procedures. For example, the protein may be subjected to amino acid
composition, amino acid sequence, or protein concentration analysis
according to known methods.
[0099] Using appropriate amino acid sequence information, synthetic
proteins of the present invention may be prepared by various
synthetic methods of peptide synthesis via condensation of one or
more amino acid residues, in accordance with conventional peptide
synthesis methods. Preferably, peptides are synthesized according
to standard solid-phase methodologies, such as may be performed on
an Applied Biosystems Model 430A peptide synthesizer (Applied
Biosystems, Foster City, Calif.), according to manufacturer's
instructions. other methods of synthesizing peptides or
peptidomimetics, either by solid phase methodologies or in liquid
phase, are well known to those skilled in the art.
[0100] The protein can be used as a label in many in vitro
applications currently used. Purified proteins can be covalently
linked to other proteins by methods well known in the art, and used
as a marker protein. The purified protein can be covalently linked
to a protein of interest in order to determine localization. In
particularly preferred embodiments, a linker of 4 to 20 amino acids
is used to separate the protein from the desired protein. This
application may be used in living cells by micro-injecting the
linked proteins. The protein may also be linked to antibodies and
used thus for localization in fixed and sectioned cells. The
protein may be linked to purified cellular proteins and used to
identify binding proteins and nucleic acids in assays in vitro,
using methods well known in the art.
[0101] The proteins of the present invention can be used to
identify each of their binding partners in coimmunoprecipitation
(co-IP). In these assays, the first protein of interest is allowed
to form a physical interaction with the unknown binding protein(s),
often in a heterologous solution of proteins. The complex of
proteins is then isolated, and the nature of the protein complex is
determined. This procedure is greatly facilitated by a simple
method for isolating the protein. For example,
immunologically-specific antibodies can be used to precipitate the
proteins, or the proteins can be bound to beads that can be easily
purified. Such beads can be magnetized, or simply dense enough to
be separated form the non-associated protein by centrifugation.
Vectors, Host Cells, and Expression
[0102] The present invention also relates to vectors which comprise
a polynucleotide or polynucleotides of the present invention, and
host cells which are genetically engineered with vectors of the
invention and to the production of polypeptides of the invention by
recombinant techniques. Cell-free translation systems can also be
employed to produce such proteins using RNAs derived from the DNA
constructs of the present invention.
[0103] For recombinant production, host cells can be genetically
engineered to incorporate expression systems or portions thereof
for polynucleotides of the present invention. Introduction of
polynucleotides into host cells can be effected by methods
described in many standard laboratory manuals, such as Davis et
al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium
phosphate transfection, DEAE-dextran mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection.
[0104] Representative examples of appropriate hosts include
bacterial cells, such as streptococci, staphylococci, E. coli,
Streptomyces and Bacillus subtilis cells; fungal) cells, such as
yeast cells and Aspergillus cells; insect cells such as Drosophila
SL2 and Spodoptera S19 cells; mammalian cells such as CHO, COS,
HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant
cells. The selection of an appropriate host is deemed to be within
the scope of those skilled in the art from the teachings
herein.
[0105] More particularly, the present invention also includes
recombinant constructs comprising the cDNA sequence. The constructs
comprise a vector, such as a plasmid or viral vector, into which
the clone has been inserted, in a forward or reverse orientation.
In a preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,
phagescript, X174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A,
pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host. In addition, a complete mammalian
transcription unit and a selectable marker can be inserted into a
prokaryotic plasmid. The resulting vector is then amplified in
bacteria before being transfected into cultured mammalian cells.
Examples of vectors of this type include pTK2, pHyg and
pRSVneo.
[0106] A great variety of expression systems can be used. Such
systems include, among others, chromosomal, episomal and
virus-derived systems, e.g., vectors derived from bacterial
plasmids, from bacteriophage, from transposons, from yeast
episomes, from insertion elements, from yeast chromosomal elements,
from viruses such as baculoviruses, papova viruses, such as SV40,
vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and retroviruses, and vectors derived from combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. The expression
systems may contain control regions that regulate as well as
engender expression. Generally, any system or vector suitable to
maintain, propagate or express polynucleotides to produce a
polypeptide in a host may be used. The appropriate nucleotide
sequence may be inserted into an expression system by any of a
variety of well-known and routine techniques, such as, for example,
those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL (supra).
[0107] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol acetyl transferase) vectors or other vectors
with selectable markers. Two appropriate vectors are pKK232-8 and
pCM7. Particular named bacterial promoters include lacI, lacZ, T3,
T7, gpt, lambda PR, PL and tip. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs
from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0108] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the desired polypeptide. These signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0109] If a polypeptide is to be expressed for use in screening
assays, generally, it is preferred that the polypeptide be produced
at the surface of the cell. In this event, the cells may be
harvested prior to use in the screening assay. If the polypeptide
is secreted into the medium, the medium can be recovered in order
to recover and purify the polypeptide; if produced intracellularly,
the cells must first be lysed before the polypeptide is
recovered.
[0110] Polypeptides can be recovered and purified from recombinant
cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography (HPLC) or fast
protein liquid chromatography (FPLC) is employed for purification.
Well known techniques for refolding proteins may be employed to
regenerate active conformation when the polypeptide is denatured
during isolation and or purification.
Antibodies
[0111] The present invention also provides antibodies capable of
immunospecifically binding to polypeptides of the invention.
Polyclonal or monoclonal antibodies directed towards the
polypeptide encoded by any one of the identified adipocyte specific
sequences may be prepared according to standard methods. Monoclonal
antibodies may be prepared according to general hybridoma methods
of Kohler and Milstein, Nature (1975) 256:495-497), the trioma
technique, the human B-cell hybridoma technique (Kozbor et al.,
Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole
et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan
R. Liss, Inc., 1985). In a preferred embodiment, antibodies are
prepared, which react immunospecifically with various epitopes of
the encoded polypeptides. These above-described antibodies may be
employed to isolate or to identify clones expressing the
polypeptide or to purify the polypeptides by affinity
chromatography.
[0112] Antibodies that are immunologically specific to the
proteins, or specific epitopes thereof, may be utilized in affinity
chromatography to isolate the proteins, to quantify the protein
utilizing techniques such as western blotting and ELISA, or to
immuno-precipitate the proteins from a sample containing a mixture
of proteins and other biological materials. The
immuno-precipitation of a protein is particularly advantageous when
utilized to isolate binding partners of the protein, as described
above.
[0113] Monoclonal antibodies or other genetically engineered
antibodies with specificity for the peptide of the invention could
be used as therapeutic agents. The antibodies should then be
humanized to reduce the immunogenicity. Humanization is
accomplished by linking the complementarity determining region
(CDR) from the original murine antibody to the constant regions of
a human antibody. Various methods may be employed to ensure the
specificity and avidity of the linked antibody (Queen, C., et al.,
Proc. Natl. Acad. Sci. USA, 86, 10029, 1989 and Reichmann, et al.,
Nature, 332, 323, 1988).
[0114] Antibodies to the peptide of the invention may be used for
diagnostic assays, for determination of circulating levels the
peptide, and as antagonists to block peptide activity in vitro and
in vivo (see, for example, Immobilized Affinity Ligand Techniques,
Hermanson, et al., eds., Academic Press, San Diego, Calif., 1992,
pp 195-202).
[0115] The invention also provides a pharmaceutical composition,
comprising one or more vectors which comprise DNA constructs
comprising any of the nucleic acid sequences of SEQ ID NO: 1 to
277.
[0116] In another embodiment, the invention provides a
pharmaceutical composition comprising one or more peptides encoded
by any of the sequences SEQ ID NO: 1 to 277.
[0117] In yet another embodiment, the invention provides for a
pharmaceutical composition comprising an agonist to any of the
peptides encoded by any of the sequences SEQ ID NO: 1 to 277.
[0118] In still another embodiment, the invention provides for a
pharmaceutical composition comprising an antagonist to any of the
peptides encoded by any of the sequences SEQ ID NO: 1 to 277.
[0119] Pharmaceutical compositions comprising a peptide or DNA
construct of the invention may be formulated by any of the
established methods for formulating pharmaceutical compositions
(see, for example, Gennaro, A. R., Remington's Pharmaceutical
Sciences, 18.sup.th edition, 1985, Pharmaceutical Press). The
pharmaceutical composition may be in a form suitable for systemic
injection or infusion and may, as such, be formulated with a
suitable liquid carrier such as sterile water, isotonic saline or
isotonic glucose solution. The pharmaceutical composition may be
sterilized by conventional sterilization techniques well known in
the art, such as autoclaving, irradiation, or filter sterilization.
The resulting sterile aqueous solutions may be packaged for use or
filtered under asceptic conditions and lyophilized in which
circumstance the lyophilized material is combined with an
appropriate aqueous solution prior to administration. The
composition may contain pharmaceutically acceptable auxiliary
substances as required to approximate physiological conditions,
such as buffering agents, tonicity adjusting agents, etc., for
instance sodium acetate, sodium chloride, potassium chloride,
calcium chloride, etc. The pharmaceutical composition may also be
adapted for other routes of administration, such as nasal,
transdermal, pulmonal, or rectal. The pharmaceutically acceptable
carrier or diluent in the composition may be any conventional solid
carrier. Examples of solid carriers are lactose, terra alba,
sucrose, talc, gelatin, agar, pectin, acacia magnesium stearate,
and stearic acid. Similarly, the carrier or diluent may include any
sustained release material known in the art, such as glyceryl
monostearate or glyceryl distearate alone or mixed with a wax. In
another embodiment, the invention provides a pharmaceutical
composition in the form of a sustained release formulation. The
sustained release formulation may include microcapsules or
microparticles containing the polypeptide encapsulated or dispersed
in a pharmaceutically suitable biodegradable polymer such as
polylactic acid, polyglycolic acid, or a lactic acid/polyglycolic
acid copolymer.
[0120] For nasal administration, the preparation may contain the
peptide of the invention dissolved or suspended in a liquid
carrier, in particular an aqueous carrier, for aerosol
administration. The carrier may contain additives such as
solubilizing agents, for example propylene glycol, surfactants,
absorption enhancers such as phosphatidyl choline, or cyclodextrin,
or preservatives such as parabenes.
[0121] In general, the compounds of the present invention are
dispensed in unit dosage form comprising 0.5 mg-50 mg of the
peptide (assuming a 70 kg patient) together with a pharmaceutically
acceptable carrier per unit dosage. The peptide is considered to be
advantageous to use in weight reduction programs, such as for the
treatment of diseases or disorders of excess, morbid overweight.
Such diseases include obesity and non-insulin dependent diabetes
mellitus (NIDDM; Type II diabetes). The dosage of the peptide
administered to a patient will vary with the type and severity of
the condition being treated, but is generally in the range of from
approximately 0.01 mg/kg to about 5 mg/kg body weight.
[0122] The peptide or the DNA construct is considered to be
advantageous in the treatment of diseases or conditions resulting
from obesity including, for example, hypertension, atherosclerosis,
coronary artery disease, cerebrovascular disease, insulin
resistance, etc.
[0123] The peptide or the DNA construct may be administered in
combination with food.
[0124] In the pharmaceutical composition of the invention, the
peptide may be combined with an appetite suppressing or satiety
inducing agent. An example of such an agent is GLP-1 which has been
shown to be efficaceous in appetite suppression (Turton, M. D., et
al., Nature 379, 4 Jan. 1996, p. 69-72).
[0125] Animal model systems that elucidate the physiological and
behavioral role of the peptide or the peptide receptor of the
invention may be produced by creating transgenic animals in which
the activity of the peptide or the peptide receptor is either
increased or decreased, or the amino acid sequence of the expressed
peptide or peptide receptor is altered, by a variety of techniques
well known in the art. (see, for example, Molecular Biology and
Biotechnology (3.sup.rd ed.), Walker and Gingold eds., The Royal
Society of Chemistry, 1993). Examples of the techniques include,
but are not limited to: 1) insertion of normal or mutant versions
of DNA encoding a peptide or a peptide receptor by microinjection,
electroporation, retroviral transfection or other means well known
to those skilled in the art, into appropriate fertilized embryos in
order to produce a transgenic animal; or 2) Homologous
recombination of mutant or normal, human or animal versions of
these genes with the native locus in transgenic animals to alter
the regulation of expression or the structure of these peptide or
peptide receptor sequences. The technique of using homologous
recombination to create so-called "knockout" mice is well known and
practiced in the art.
[0126] In another embodiment, the polypeptide of the present
invention may be used in a screening process to identify compounds
that activate ("agonists") or deactivate ("antagonists";
"inhibitors") the polypeptide of the invention. The agonist and
antagonist may be isolated from, for example, cells, cell free
preparations, combinatorial libraries, and natural product
mixtures. They may be natural or modified substrate, ligands,
enzymes, receptors, kinases, phosphatases, etc. as the case may be
of the polypeptide of the invention. They may be structural or
functional mimetics of the polypeptide, including anti-idiotypic
antibodies.
[0127] One of the polypeptides identified by this invention is a
ligand for the class of seven passe transmembrane receptors, which
are known to be central to many biological processes and are
involved in many pathhologies, including heart disease,
hypertension, cardiovascular disease, diabetes, insulin resistance,
and obesity. Thus, the identification of chemical entities capable
of agonizing the activity of the polypeptide of the invention on
the one hand, and chemical entities capable of antagonizing the
activity of the polypeptide of the present invention on the other
hand, would be highly desirable in that they would be candidate
compounds for therapeutics for such diseases related to the peptide
of the present invention and its biological pathway.
[0128] Screening procedures may involve using appropriate cells
which express the protein or respond to the protein. Such cells
include cells from mammals, yeast, Drosophila, or E. coli. Cells
that express the protein are contacted with a test compound to
determine binding, or an increase or decrease in biological
activity. These cells are compared to cells that have not been
contacted by the compound for activity of the protein. In another
embodiment, the polypeptide or a fragment thereof may be bound to a
solid support, such as for example agarose beads, and a plurality
of compounds would be tested for binding to the polypeptide or a
fragment thereof. Those compounds shown to bind to the polypeptide
could then be tested for an effect on the biological activity of
the polypeptide as described using a cell based test as described
above.
[0129] The polypeptide of the present invention may be used to
screen for any receptors that bind to it. For example, the protein
can be produced in large quantities in bacterial and/or baculovirus
systems and the protein may be iodinated to search for interacting
receptors in classical binding assays. Additionally, the protein
may be used to identify potential inhibitors to its receptor
binding activity in classical competition experiments.
[0130] The nucleic acid sequence may also be used to generate
antisense molecules either synthetically in vitro or by
construction of suitable expression vectors such as retroviral
vectors which after introduction into desired tissues may direct
the synthesis of antisense RNA in vivo.
[0131] Synthetic antisense RNA molecules may be either DNA, PNA, or
RNA, or variants thereof. The term PNA (peptide nucleic acid) is
used to indicate a synthetic DNA-mimetic comprising a polyamide
backbone, bearing ligands at respective spaced locations along the
backbone, the ligands being naturally occurring nucleobases,
non-naturally occurring nucleobases, or nucleobase analogues (see,
for example WO92/20703 (Buchardt, et al.); WO96/02558; and
WO96/11205).
[0132] Another mechanism by which genes may be regulated at the
level of mRNA is the technique of RNA interference (RNAi). In this
technique, long double stranded RNAs (dsRNAs) can be used to
silence the expression of target genes. Upon introduction into the
cell, the long (generally >200 nt) dsRNAs enter into the RNAi
pathway. The dsRNAs get cleaved into 20-25 nt small interfering
RNAs (siRNAs) by Dicer, an RNAse III-like enzyme. This is the
initiation step. The siRNAsassemble into complexes containing
endoribonuclease known as RNA induced silencing complexes (RISCs).
The siRNA molecules are then unwound to activate the RISCs. The
siRNA strans guide the RISCs to complementary RNA molecules i.e.
from the target gene of interest where the endoribonuclease
activity digests the complementary RNA (effector step) Cleavage of
the target RNA takes place near the center of the region bound by
the siRNA.
[0133] The term "antisense molecule" is well known to practitioners
in the art. See, for example, Molecular Biology and Biotechnology
(Y' ed.) Walker and Gingold (eds.), The Royal Society of chemistry
(1993) or Crooke and Bennett (1996) Ann. Rev. Pharmacol. Toxicol.
36, p. 107. An antisense molecule is capable of specifically
hybridizing to a unique sequence within the sequence of a nucleic
acid sequence of a peptide of the present invention. As used
herein, the phrase "specifically hybridizing" means the ability of
a nucleic acid (or PNA) to recognize a nucleic acid sequence
complementary to its own and to form double helical (or triple
helical) segment through hydrogen bonding between complementary
base pairs.
Diagnostic Assays
[0134] The present invention also relates to the use of the
polynucleotides for use as diagnostic reagents. Detection of a
mutated form of a polypeptide that is associated with an alteration
of a biological function of the polypeptide can be used as a
diagnostic tool to diagnose a disease or a susceptibility to a
disease that results from the overexpression, underexpression, or
altered expression. Individuals carrying a mutation in the
corresponding gene may be identified by a number of methods well
known in the art.
[0135] Samples of nucleic acids for diagnosis may be obtained from
a subects cells, such as from blood, urine, saliva, tissue biopsy,
or autopsy material. Genomic DNA may be screened directly for
detection or may be amplified enzymatically using PCR or other
amplification prior to analysis. RNA or cDNA may be used in a
similar fashion. Deletion mutations or insertion mutations may be
detected by an alteration in size of the amplified product in
comparison to a product amplified from a sample containing a normal
genotype. Point mutations may be detected by hybridizing the
amplified product to labeled nucleotide sequences. Perfectly
matched duplexes can be distinguished from duplexes containing a
mismatch by RNAse digestion or by a change in the melting
temperature of the duplex. Alternatively, DNA sequence differences
can be detected by an alteration of electrophoretic mobility of DNA
fragments in gels, with or without denaturing agents. See for
example Myers, et al., Science (1985)230:1242. Sequence changes at
specific locations may be detected by nuclease protection assays
including RNAse and S1. Point mutations may also be detected by
single strand conformation polymorphism (SSCP) analysis.
[0136] The diagnostic assays offer a tool for diagnosing diseases
or susceptibility to diseases related to obesity including, but not
limited to, diabetes, hypertension, atherosclerosis, coronary
artery disease, gall bladder disease, and cerebrovascular disease
through the detection of mutation(s) in the adipocyte specific gene
by the methods described or other methods well known in the
art.
[0137] Additionally, a diagnosis of a disease or susceptibility to
a disease related to obesity, such as but not limited to those
mentioned hereinabove, may be performed by methods comprising
determining from a sample from a subject an aberrantly increased or
decreased expression of a polypeptide or its mRNA. Increases or
decreases in mRNA expression can be determined by several methods
well known in the art including, for example, Northern blot,
quantitative real time PCR, such as the TaqMan.TM. system (Applied
Biosystems), and RNAse protection assay. Assay methods that can
determine the aberrant increased or decreased expression of a
polypeptide at the protein level include, for example, Western
Blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassays,
and competitive binding assays.
[0138] Therefore, another aspect of the invention is a kit to
diagnose or diagnose the susceptibility to a disease related to
obesity in a subject which comprises:
[0139] (a) an adipocyte specific polynucleotide having the
nucleotide sequence of any one of SEQ ID NO: 1-277; or a fragment
thereof;
[0140] (b) a nucleotide sequence complementary to that of (a)
[0141] (c) an adipocyte specific polypeptide having the sequence
set forth herein, or a fragment thereof; or
[0142] (d) and antibody to the adipocyte specific polypeptide.
In any such diagnostic kit, (a), (b), (c), or (d) may be a
substantial component.
DEFINITIONS
[0143] Various terms relating to the biological molecules of the
present invention are used throughout the specification and
claims.
[0144] "Antibodies" as used herein includes polyclonal and
monoclonal antibodies, chimeric, single chain, and humanized
antibodies, as well as Fab fragments, including the products of an
Fab or other immunoglobulin expression library. With respect to
antibodies, the term, "immunologically specific" refers to
antibodies that bind to one or more epitopes of a protein of
interest, but which do not substantially recognize and bind other
molecules in a sample containing a mixed population of antigenic
biological molecules.
[0145] "Isolated" means altered "by the hand of man" from the
natural state. If an "isolated" composition or substance occurs in
nature, it has been changed or removed from its original
environment, or both. For example, a polynucleotide or a
polypeptide naturally present in a living animal is not "isolated,"
but the same polynucleotide or polypeptide separated from the
coexisting materials of its natural state is "isolated", as the
term is employed herein.
[0146] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications have been made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0147] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to both short chains, commonly referred to as peptides,
oligopeptides or oligomers, and to longer chains, generally
referred to as proteins. Polypeptides may contain amino acids other
than the 20 gene-encoded amino acids. "Polypeptides" include amino
acid sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification
techniques which are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. Modifications can
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It
will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched as a result of
ubiquitination, and they may be cyclic, with or without branching.
Cyclic, branched and branched cyclic polypeptides may result from
post-translation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cystine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino
acids to proteins such as arginylation, and ubiquitination. See,
for instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and
Wold, F., Posttranslational Protein Modifications: Perspectives and
Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983;
Seifter et al., "Analysis for protein modifications and nonprotein
cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al.,
"Protein Synthesis: Posttranslational Modifications and Aging", Ann
NY Acad Sci (1992) 663:48-62.
[0148] "Variant" as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains essential properties. A
typical variant of a polynucleotide differs in nucleotide sequence
from another, reference polynucleotide. Changes in the nucleotide
sequence of the variant may or may not alter the amino acid
sequence of a polypeptide encoded by the reference polynucleotide.
Nucleotide changes may result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptide
encoded by the reference sequence, as discussed below. A typical
variant of a polypeptide differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference polypeptide and the variant
are closely similar overall and, in many regions, identical. A
variant and reference polypeptide may differ in amino acid sequence
by one or more substitutions, additions, deletions in any
combination. A substituted or inserted amino acid residue may or
may not be one encoded by the genetic code. A variant of a
polynucleotide or polypeptide may be a naturally occurring such as
an allelic variant, or it may be a variant that is not known to
occur naturally. Non-naturally occurring variants of
polynucleotides and polypeptides may be made by mutagenesis
techniques or by direct synthesis.
[0149] The term "substantially the same" refers to nucleic acid or
amino acid sequences having sequence variation that do not
materially affect the nature of the protein (i.e. the structure,
stability characteristics, substrate specificity and/or biological
activity of the protein). With particular reference to nucleic acid
sequences, the term "substantially the same" is intended to refer
to the coding region and to conserved sequences governing
expression, and refers primarily to degenerate codons encoding the
same amino acid, or alternate codons encoding conservative
substitute amino acids in the encoded polypeptide. With reference
to amino acid sequences, the term "substantially the same" refers
generally to conservative substitutions and/or variations in
regions of the polypeptide not involved in determination of
structure or function.
[0150] The terms "percent identical" and "percent similar" are also
used herein in comparisons among amino acid and nucleic acid
sequences. When referring to amino acid sequences, "identity" or
"percent identical" refers to the percent of the amino acids of the
subject amino acid sequence that have been matched to identical
amino acids in the compared amino acid sequence by a sequence
analysis program. "Percent similar" refers to the percent of the
amino acids of the subject amino acid sequence that have been
matched to identical or conserved amino acids. Conserved amino
acids are those which differ in structure but are similar in
physical properties such that the exchange of one for another would
not appreciably change the tertiary structure of the resulting
protein. Conservative substitutions are defined in Taylor (1986, J.
Theor. Biol. 119:205). When referring to nucleic acid molecules,
"percent identical" refers to the percent of the nucleotides of the
subject nucleic acid sequence that have been matched to identical
nucleotides by a sequence analysis program.
[0151] "Identity" and "similarity" can be readily calculated by
known methods. Nucleic acid sequences and amino acid sequences can
be compared using computer programs that align the similar
sequences of the nucleic or amino acids thus define the
differences. In preferred methodologies, the BLAST programs (NCBI)
and parameters used therein are employed, and the DNAstar system
(Madison, Wis.) is used to align sequence fragments of genomic DNA
sequences. However, equivalent alignments and similarity/identity
assessments can be obtained through the use of any standard
alignment software. For instance, the GCG Wisconsin Package version
9.1, available from the Genetics Computer Group in Madison, Wis.,
and the default parameters used (gap creation penalty=12, gap
extension penalty=4) by that program may also be used to compare
sequence identity and similarity.
[0152] With respect to single-stranded nucleic acid molecules, the
term "specifically hybridizing" refers to the association between
two single-stranded nucleic acid molecules of sufficiently
complementary sequence to permit such hybridization under
pre-determined conditions generally used in the art (sometimes
termed "substantially complementary"). In particular, the term
refers to hybridization of an oligonucleotide with a substantially
complementary sequence contained within a single-stranded DNA or
RNA molecule of the invention, to the substantial exclusion of
hybridization of the oligonucleotide with single-stranded nucleic
acids of non-complementary sequence.
[0153] With respect to oligonucleotides, but not limited thereto,
the term "specifically hybridizing" refers to the association
between two single-stranded nucleotide molecules of sufficiently
complementary sequence to permit such hybridization under
pre-determined conditions generally used in the art (sometimes
termed "substantially complementary"). In particular, the term
refers to hybridization of an oligonucleotide with a substantially
complementary sequence contained within a single-stranded DNA or
RNA molecule of the invention, to the substantial exclusion of
hybridization of the oligonucleotide with single-stranded nucleic
acids of non-complementary sequence.
[0154] The term "substantially pure" refers to a preparation
comprising at least 50-60% by weight the compound of interest
(e.g., nucleic acid, oligonucleotide, protein, etc.). More
preferably; the preparation comprises at least 75% by weight, and
most preferably 90-99% by weight, the compound of interest. Purity
is measured by methods appropriate to the compound of interest
(e.g. chromatographic methods, agarose or polyacrylamide gel
electrophoresis, HPLC analysis, and the like).
[0155] A "coding sequence" or "coding region" refers to a nucleic
acid molecule having sequence information necessary to produce a
gene product, when the sequence is expressed.
[0156] The term "operably linked" or "operably inserted" means that
the regulatory sequences necessary for expression of the coding
sequence are placed in a nucleic acid molecule in the appropriate
positions relative to the coding sequence so as to enable
expression of the coding sequence. This same definition is
sometimes applied to the arrangement other transcription control
elements (e.g. enhancers) in an expression vector.
[0157] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell.
[0158] The terms "promoter", "promoter region" or "promoter
sequence" refer generally to transcriptional regulatory regions of
a gene, which may be found at the 5' or 3' side of the coding
region, or within the coding region, or within introns. Typically,
a promoter is a DNA regulatory region capable of binding RNA
polymerase in a cell and initiating transcription of a downstream
(3' direction) coding sequence. The typical 5' promoter sequence is
bounded at its 3' terminus by the transcription initiation site and
extends upstream (5' direction) to include the minimum number of
bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence is a
transcription initiation site (conveniently defined by mapping with
nuclease S1), as well as protein binding domains (consensus
sequences) responsible for the binding of RNA polymerase.
[0159] A "vector" is a replicon, such as plasmid, phage, cosmid, or
virus to which another nucleic acid segment may be operably
inserted so as to bring about the replication or expression of the
segment.
[0160] The term "nucleic acid construct" or "DNA construct" is
sometimes used to refer to a coding sequence or sequences operably
linked to appropriate regulatory sequences and inserted into a
vector for transforming a cell. This term may be used
interchangeably with the term "transforming DNA". Such a nucleic
acid construct may contain a coding sequence for a gene product of
interest, along with a selectable marker gene and/or a reporter
gene.
[0161] The term "selectable marker gene" refers to a gene encoding
a product that, when expressed, confers a selectable phenotype such
as antibiotic resistance on a transformed cell.
[0162] The term "reporter gene" refers to a gene that encodes a
product which is detectable by standard methods, either directly or
indirectly.
[0163] A "heterologous" region of a nucleic acid construct is an
identifiable segment (or segments) of the nucleic acid molecule
within a larger molecule that is not found in association with the
larger molecule in nature. Thus, when the heterologous region
encodes a mammalian gene, the gene will usually be flanked by DNA
that does not flank the mammalian genomic DNA in the genome of the
source organism. In another example, a heterologous region is a
construct where the coding sequence itself is not found in nature
(e.g., a cDNA where the genomic coding sequence contains introns,
or synthetic sequences having codons different than the native
gene). Allelic variations or naturally-occurring mutational events
do not give rise to a heterologous region of DNA as defined herein.
The term "DNA construct", as defined above, is also used to refer
to a heterologous region, particularly one constructed for use in
transformation of a cell.
[0164] A cell has been "transformed" or "transfected" by exogenous
or heterologous DNA when such DNA has been introduced inside the
cell. The transforming DNA may or may not be integrated (covalently
linked) into the genome of the cell. In prokaryotes, yeast, and
mammalian cells for example, the transforming DNA may be maintained
on an episomal element such as a plasmid. With respect to
eukaryotic cells, a stably transformed cell is one in which the
transforming DNA has become integrated into a chromosome so that it
is inherited by daughter cells through chromosome replication. This
stability is demonstrated by the ability of the eukaryotic cell to
establish cell lines or clones comprised of a population of
daughter cells containing the transforming DNA. A "clone" is a
population of cells derived from a single cell or common ancestor
by mitosis. A "cell line" is a clone of a primary cell that can
grow in vitro for many generations.
[0165] The following non-limiting examples are provided to describe
the invention in greater detail. Unless otherwise specified,
general cloning procedures, such as those set forth in Sambrook et
al., Molecular Cloning, Cold Spring Harbor Laboratory (1989)
(hereinafter "Sambrook et al.") or Ausubel et al. (eds) Current
Protocols in Molecular Biology, John Wiley & Sons (2000)
(hereinafter "Ausubel et al.") are used.
EXPERIMENTAL DETAILS
[0166] The process of adipogenesis i.e. the generation of fat
tissue occurs in order that the body is able to store energy in the
form of fat, which is much more efficient than energy storage as
sugar or protein. In response to an obesity inducing stimulus--such
as a high fat diet--the process of adipogenesis is set in motion.
Preadipocytes are cells which have not yet differentiated into
mature adipocytes and therefore store no fat, but have the capacity
to do so when stimulated under the proper conditions. There appear
to be two connected processes occurring: there is an expansion of
the number of preadipocytes via cellular proliferation, and the
formation of fully developed adipocytes through differentiation.
There is in fact a third process, adipocyte hypertrophy, in which
the maximal amount of triglyceride has been stored in a given
differentiated adipocyte which then presumably signals back to
promote the proliferation of more preadipocytes and to move them
through the differentiation pathway (though that mechanism remains
unclear). Identifying molecular markers expressed in adipocytes,
but not in preadipocytes, would be attractive targets for the
prevention and treatment of obesity i.e. to prevent the formation
of differentiated adipocytes and thus block the ability of these
cells to accumulate fat.
[0167] The HMGI-C knockout (KO) mice were originally studied
because they present with the pygmy phenotype. That is, the mice
weigh only about 40% of their wild-type littermates, and this
reduction in size is as a result of fewer cell number, not cell
size. There are, in fact, two tissues in these animals that are
exceptions: the brain, which is normal sized, and the white adipose
tissue (WAT) which is decreased 8-fold. This data led us to explore
a direct role for HMGI-C in adipogenesis and obesity.
[0168] We first looked at the expression of HMGI-C in the WAT of
wild-type (wt) mice fed a normal chow diet versus WAT from wt mice
fed a high fat diet. While HMGI-C expression was undetectable by
Northern blot in the chow-fed mice, HMGI-C expression was readily
detectable in the high-fat fed mice after only one week of feeding,
long before any weight gain or overt phenotype can be observed.
Similarly, we looked at HMGI-C expression in the genetically obese
ob/ob and db/db mice. As observed in the feeding experiment, HMGI-C
expression was undetectable by Northern blot in the WAT of the wt
mice, but was readily detectable in the WAT of the ob/ob and db/db
mice.
[0169] However, these experiments were only correlative in nature,
and we thus carried out experiments to identify a causative role
for HMGI-C and adipogenesis. We fed wt or HMGI-C KO mice a normal
chow diet or an obesity inducing high fat diet (as above). Within
32 weeks, wt mice led the high fat diet showed a significant
increase in weight gain as compared to wt mice fed the chow diet.
By contrast, HMGI-C KO mice fed a high fat diet and HMGI-C mice fed
a chow diet exhibited no difference in weight gain. Thus, absence
of HMGI-C was capable of preventing the weight gain induced by the
physiological obesity inducing stimulus of a high fat diet.
[0170] The obesity inducing stimulus of leptin deficiency (i.e. the
ob/ob mice) is much stronger than that of a high fat diet. Whereas
the high fat diet may induce a 20% increase in weight, leptin
deficiency induces up to a 300% gain in weight. We decided to see
what the effect of HMGI-C deficiency would be in the leptin
deficient mice. Mice deficient in both HMGI-C and leptin weighed
the same as wt mice. This is in fact a statistically greater weight
than the HMGI-C KO mice alone, but this increase was much less than
would have been anticipated. [If we need to include these studies
as background to the invention, they can be incorporated by
reference--Anand, et al., In vivo modulation of HMGIc reduces
obesity. Nat. Genet. 2000 April; 24(4):377-80].
[0171] RNA isolated from the adipose tissue from mice of the
various genotypes was assayed for the expression levels of a number
of preadipocyte markers (GATA3 and Pref-1, [Gregoire, 2001]) and
adipocyte markers (resistin, LPL, PEPCK, AdipoQ and adipsin,
[Gregoire, 2001]). Surprisingly, in comparison to wild-type adipose
tissue, we found that the Hmga2 null adipose tissue expressed
preadipocyte markers 10-fold higher relative to adipocyte markers.
The ratio was reversed in the double homozygous mice, i.e. the
ob/ob and HMGA1 mice, and this led to the following model. We
hypothesized that the adipose tissue in the A2 null was mainly
composed of preadipocytes and in the double homozygotes was
adipocytes. Therefore, our A2 mice of various genotypes provide an
enriched in vivo source of preadipocytes and adipocytes.
[0172] The next step was to perform differential gene expression
analysis on RNA isolated from the epididymal white adipose tissue
of the A2 null, double homozygous and wild-type mice using the
Affymetrix gene chip array system.
[0173] Thus, the white adipose tissue of the HMGI-C null mice is
composed predominantly of preadipocytes. This comes as a result of
a deficiency in the proliferation and differentiation of
preadipocytes. In the case of the HMGI-C knockout mice bred onto
the leptin deficient background the presence of an overwhelming
obesity stimulus forces the differentiation of those existing
preadipocytes into lipid filled adipocytes. Thus, the adipose depot
is almost exclusively adipocytes in these mice.
[0174] Many groups have attempted to identify obesity/diabetes
targets by using "DNA chip" microarray analysis to determine
differential gene expression between lean and obese mice. The
drawback with this type of analysis is that the cell populations
are mixed, which leads to high levels of both false positive and
false negative results which leave these experiments
uninterpretable.
[0175] In an initial screen we identified 46 candidate genes out of
10,000 genes contained on a single gene chip, and these results
have been verified in subsequent experiments. Validation of this
approach to target identification came during the analysis of these
results. Of the 46 genes identified, 8 (17%) were genes undergoing
active investigation as obesity/diabetes targets including AdipoQ,
adipsin, 13-3 adrenergic receptor, PEPCK, IGF-1, aP2, leptin, and
resistin. This positive strike rate is remarkable and, as far as we
know, unparalleled in other systems. The fact that these genes came
up as targets in our analysis validates this approach to identify
obesity targets.
[0176] We then isolated total RNA samples from the mice and
subjected them to differential gene expression analysis using the
Affymetrix system and then set up a numerical algorithm to arrive
at the results, as follows:
[0177] The sequences of the inventions were obtained by
differential expression analysis using the Affymetrix GeneChip
system.
[0178] Animals:
[0179] Total RNA was isolated from the white adipose tissue (WAT)
from mice of the following genotypes:
1) wild-type; 2) HMGI-C-/-; 3) ob/ob; and 4) HMGI-C-/-, ob/ob.
[0180] Total RNA was also isolated from the stromal vascular tissue
(SVT) of mice of the following genotypes:
1) wild-type 2) ob/ob 3) HMGI-C-/-, ob/ob
[0181] Isolation of the Total RNA
The total RNA was isolated using the RNeasy (Quiagen) kit as
follows: RNA cleanup (Qiagen Rneasy): add water to 100 ul
add 350 ul Buffer RLT, mix
[0182] add 250 ul 100% ETOH, mix, label column/cap and collection
tube apply to RNeasy column, wait 3 min, spin (all spins >10,000
rpm) 30 sec re-apply flow-through wait 3 min, spin 30 sec transfer
column into a new 2 ml collection tube, add 500 ul Buffer RPE, spin
30 sec discard flow-through, add 500 ul Buffer RPE, spin 2 min,
discard flow-through, spin additional 3 min uncapped, you may cut
off caps, make sure columns are labeled, transfer column to a 1.5
ml collection tube with both the tube and cap labeled let sit on
bench uncapped 3 min add 100 ul water, soak 3 min, spin 2 min take
2 ul for OD in 48 ul water freeze sample in -70.degree. C., speed
vac at low heat, do not dry completely resuspend pellet with water
to 1 ug/ul run 1 ug on gel
[0183] The Affymetrix GeneChip Analysis:
RNA cleanup (Qiagen Rneasy): add water to 100 ul
add 350 ul Buffer RLT, mix
[0184] add 250 ul 100% ETON, mix, label column/cap and collection
tube apply to RNeasy column, wait 3 min, spin (all spins >10,000
rpm) 30 sec re-apply flow-through wait 3 min, spin 30 sec transfer
column into a new 2 ml collection tube, add 500 ul Buffer RPE, spin
30 sec discard flow-through, add 500 ul Buffer RPE, spin 2 min,
discard flow-through, spin additional 3 min uncapped, you may cut
off caps, make sure columns are labeled, transfer column to a 1.5
ml collection tube with both the tube and cap labeled let sit on
bench uncapped 3 min add 100 ul water, soak 3 min, spin 2 min take
2 ul for OD in 48 ul water freeze sample in -70.degree. C., speed
vac at low heat, do not dry completely resuspend pellet with water
to 1 ug/ul run 1 ug on gel
[0185] Affymetrix Differential Expression Analysis
[0186] The Affymetrix differential expression analysis was
performed on the MG-U74 "A" chip using the detailed protocols in
the attached manual. Briefly, the Affymetrix analysis proceeds as
follows:
The following major steps outline GeneChip.RTM. Expression
Analysis:
1. Target Preparation
2. Target Hybridization
3. Experiment and Fluidics Station Setup
4. Probe Array Washing and Staining
5. Probe Array Scan
6. Data Analysis
[0187] Step 1: Target Preparation
Double-stranded cDNA is synthesized from total RNA or purified
poly(A)+ messenger RNA isolated from tissue or cells. An in vitro
transcription (IVT) reaction is then done to produce biotin-labeled
cRNA from the cDNA. The cRNA is fragmented before hybridization.
After fragmentation, the RNA is end-modified and conjugated with
biotin.
[0188] Step 2: Target Hybridization
A hybridization cocktail is prepared, including the fragmented
target, probe array controls, BSA, and herring sperm DNA. It is
then hybridized to the probe array during a 16-hour incubation. The
hybridization process is described in the respective sections for
the different probe array types.
[0189] Step 3: Experiment and Fluidics Station Setup
Specific experimental information is defined using Affymetrix.RTM.
Microarray Suite on a PC-compatible workstation with a Windows NT
operating system. The probe array type, sample description and
comments are entered in Microarray Suite and saved with a unique
experiment name. The fluidics station is then prepared for use by
priming with the appropriate buffers.
[0190] Step 4: Probe Array Washing and Staining
Immediately following hybridization, the probe array undergoes an
automated washing and staining protocol on the fluidics
station.
[0191] Step 5: Probe Array Scan
Once the probe array has been hybridized, washed and stained, it is
scanned. Each workstation running Affymetrix.RTM. Microarray Suite
can control one scanner. Each probe array is scanned twice, taking
up to ten minutes, depending on the array format. The software
calculates an average of the two images, defines the probe cells
and computes an intensity for each cell. The double scan improves
assay sensitivity and reduces background noise. Each complete probe
array image is stored in a separate data file identified by the
experiment name and is saved with a data image file (.dat)
extension.
[0192] Step 6: Data Analysis
Data is analyzed using Microarray Suite Expression Analysis window.
The .dat image is analyzed for probe intensities; results are
reported in tabular and graphical formats. The data collected were
transferred from Affymetrix Microarray Suite 4.0 to Genespring
4.1.1 (Silicon Genetics) for analysis. The following sets of genes
were then selected: 1. Genes showing an expression increase of
7.times. or greater comparing wild-type WAT vs. HMGI-C-/- WAT 2.
Genes showing an expression increase of 5.times. or greater
comparing wild-type SVT vs. wild-type WAT 3. Genes showing an
expression increase of 7.times. or greater comparing HMGI-C-/,
ob/ob WAT vs. HMGI-C-/- WAT.
[0193] A Venn diagram was then made to identify overlap in the
above three sets of selected genes. This resulted in 300 genes
being selected. The genes were then further filtered as follows.
Genes indicating an "absent" call according to the Affymetrix
analysis in both wild-type WAT and HMGI-C-/-, ob/ob WAT were
eliminated. All ESTs were eliminated and all Ig genes were
eliminated. Genes that showed an absolute expression level less
than 500 units by the Affymetrix analysis were also eliminated,
resulting in the list that you now have. The sequences from these
genes were available from GenBank (Affymetrix supplies the
accession numbers). Human homologs of the identified mouse
sequences were found via BLAST search of the NCBI database.
Variables in the Method
[0194] Parameters in this system can be varied. For example, we
know that the HMGI-C KO mice have WAT composed predominantly of
preadipocytes, and that leptin deficiency in these mice forces the
differentiation of these preadipocytes into adipocytes, but there
is still a blockade on the proliferation of new preadipocytes i.e.
the WAT is now composed predominantly of adipocytes. It is
possible, however, that there are other mouse models in which the
WAT is mostly preadipocytes and that adding leptin deficiency to
the background would result in mice with mostly adipocytes in the
WAT. We predict that crossing the HMGI-C KO mice with the db/db
mice would give exactly the same result, hence there may be other
KO or transgenic animals that when crossed with the HMGI-C KO mice
would result in mice with mostly adipocytes.
[0195] Isolation of total RNA is possible by many methods well
known to those in the art, from CsCl gradients to triazol reagents
to kits containing RNA affinity columns (such as the Rneasy kit
mentioned.
[0196] There are also alternatives to using the Affymetrix system
for the microarrays. There are other systems commercially available
e.g. the Rosetta Resolver system. Invitrogen will produce custom
arrays (albeit at great expense). It is even possible for one to
make "home made" arrays in the laboratory, though this is time
consuming, labor intensive, and is generally of much poorer quality
than those commercially available.
The cutoffs used in the experimental analysis are also variable: 1.
Genes showing an expression increase of 7.times. or greater
comparing wild-type WAT vs. HMGI-C-/- WAT Genes showing an
expression increase of 5.times. or greater comparing wild-type SVT
vs. wild-type WAT 3. Genes showing an expression increase of
7.times. or greater comparing HMGI-C-/, ob/ob WAT vs. HMGI-C-/-
WAT. Elimination of genes that showed an absolute expression level
less than 500 units
[0197] The cutoffs in the algorithm were chosen because they
include the 8 well characterized adipogenic genes. Reducing the
cutoffs or raising the bar on absolute expression would result in
more genes, but as the list grows it becomes harder to defend those
as legitimate obesity targets e.g if we raise the cutoff for
absolute expression from 500 to 80,000 we include essentially all
10,000 genes on the chip. Of course the converse is true; if we
raise the cutoff, the number of genes becomes less, though not all
8 adipogenic genes will be represented e.g. leptin was #26 on the
list, therefore increasing the cutoff to result in only 25 genes
eliminates all of the known adipogenic genes. While the inclusion
of all of the known adipogenic genes gives the method its validity,
once validity has been established, increasing the cutoff would
simply identity highly expressed adipogenic genes.
[0198] The sequences identified by this method of Affymetrix
differential gene expression analysis and real time quantitative
PCR are listed in this specification in the section immediately
preceding the claims.
Identified Adipocyte Specific Sequences 1-83
[0199] AF064748. Mus musculus S3-1 . . . [gi:3236367] (SEQ.ID.NO.
501)&(SEQ.ID.NO. 1) Human homolog XM.sub.--069431. Homo sapiens
simi . . . [gi:18564480](SEQ.ID.NO.502)&(SEQ.ID.NO. 2)
XM.sub.--069427. Homo sapiens simi . . . [gi:17464283](SEQ.ID.NO.
503)&(SEQ.ID.NO. 3)
Neuronatin-1
[0200] X83568. M. musculus mRNA f . . . [gi:619497](SEQ.ID.NO. 504)
&(SEQ.ID.NO. 4)
Human Homolog
[0201] XM.sub.--009686. Homo sapiens neur . . .
[gi:18591751](SEQ.ID.NO. 505)&(SEQ.ID.NO.5)
Neuronatin-2
[0202] X83569. M. musculus mRNA f . . . [gi:619499] (SEQ.ID.NO.
506) &(SEQ.ID.NO. 6)
Human Homolog
[0203] U25034. Human neuronatin . . . [gi:1244409d no](SEQ.ID NO
507)&(SEQ.ID.NO. 7)
Neuronatin-3
[0204] X83570. M. musculus mRNA f . . . [gi:619501](SEQ.ID.NO.
508)&(SEQ.ID.NO. 508)
Human Homolog
[0205] XM.sub.--009686. Homo sapiens neur . . .
[gi:18591751](SEQ.ID.NO. 509)&(SEQ.ID.NO.9)
VAP-1
[0206] AF078705. Mus musculus vasc . . . [gi:3603354] (SEQ.ID.NO.
510)&(SEQ.ID.NO. 10)
Human Homolog
[0207] AF067406. Homo sapiens vasc . . . [gi:3283369](SEQ.ID.NO.
511)&(SEQ.ID.NO. 11)
FSP27
[0208] M61737. M. musculus adipoc . . . [gi:201107] (SEQ.ID.NO.
12)
Human Homolog
[0209] NM.sub.--022094. Homo sapiens hypo . . .
[gi:11545806](SEQ.ID.NO.513)&(SEQ.ID.NO.13)
MRP14
[0210] AF240177. Mus musculus MRP1 . . . [gi:13877300] (SEQ.ID.NO.
14) NP.sub.--033140. S100 calcium-bind . . . [gi:6677837]
Peg1/Mest
[0211] AF017994. Mus musculus Peg1 . . .
[gi:2570405](SEQ.ID.NO.515)&(SEQ.ID.NO. 15) Human homolog
D87367. Homo sapiens PEG1 . . . [gi:2317667](SEQ.ID.NO. 516)&
(SEQ.ID.NO. 16) CD1d1 antigen M63695. Mouse CD1.1 mRNA, . . .
[gi:192471](SEQ.ID.NO.517)&(SEQ.ID.NO. 17)
Human Homolog
[0212] NM.sub.--001766. Homo sapiens CD1D . . .
[gi:4502648](SEQ.ID.NO.518)&(SEQ.ID.NO.18)
Gamma Synuclein
[0213] F017255. Mus musculus pers . . . [gi:4090844] (SEQ.ID.NO.
519)& (SEQ.ID.NO. 19) Human homolog AF411524. Homo sapiens synu
. . . [gi:15705904](SEQ.ID.NO.520)&(SEQ.ID.NO.20)
CD53 Antigen
[0214] 1: X97227. M. musculus mRNA f . . .
[gi:1279545](SEQ.ID.NO.21)&(SEQ.ID.NO.21) Human homolog M60871.
Human cell surfac . . . [gi:180140] (SEQ.ID.NO.
522)&(SEQ.ID.NO. 22) Beta 1,3-galactosyltransferase AF029791.
Mus musculus UDP- . . . [gi:2745736](SEQ.ID.NO.
523)&(SEQ.ID.NO. 23)
Human Homolog
[0215] Y15014. Homo sapiens mRNA . . . [gi:2791314](SEQ.ID.NO.
524)&(SEQ.ID.NO. 24)
Retinol Binding Protein 4
[0216] U63146. Mus musculus reti . . . [gi:1515449](SEQ.ID.NO.
525)&(SEQ.ID.NO. 25)
Human Homolog
[0217] NM.sub.--006744. Homo sapiens reti . . . [gi:8400727]
(SEQ.ID.NO. 526)&(SEQ.ID.NO. 26)
ATP-Binding Cassette, Sub-Family D, 2
[0218] Z48670. M. musculus mRNA f . . . [gi:1107731](SEQ.ID.NO.
527)&(SEQ.ID.NO. 27) Human homolog NM.sub.--005164. Homo
sapiens ATP- . . .
[gi:9945307](SEQ.ID.NO.528)&(SEQ.ID.NO.28)
Small Inducible Cytokine A6
[0219] NM.sub.--009139. Mus musculus smal . . .
[gi:6677882](SEQ.ID.NO.529)&(SEQ.ID.NO.29)
Myeloperoxidase
[0220] X15313. Mouse MPO mRNA fo . . . [gi:53203](SEQ.ID.NO.
530)&(SEQ.ID.NO. 30)
Human Homolog
[0221] NM.sub.--000250. Homo sapiens myel . . .
[gi:4557758](SEQ.ID.NO.531)&(SEQ.ID.NO.31)
Coronin, Actin-Binding Protein 1A
[0222] NM.sub.--009898. Mus musculus coro . . .
[gi:6753491](SEQ.ID.NO. 532)&(SEQ.ID.NO.32)
Human Homolog
[0223] X89109. H. sapiens mRNA fo . . . [gi:1 136139] (SEQ.ID.NO.
533)&(SEQ.ID.NO. 33)
Early B-Cell Factor (EBF)
[0224] L12147. Mus musculus (clo . . . [gi:309208](SEQ.ID.NO.
534)&(SEQ.ID.NO. 34) Human homolog XM.sub.--038441. Homo
sapiens earl . . .
[gi:18562089](SEQ.ID.NO.535)&(SEQ.ID.NO.35)
Small Inducible Cytokine A5
[0225] AF065947. Mus musculus stra . . . [gi:3158455](SEQ.ID.NO.
36)
Human Homolog
[0226] XM.sub.--035842. Homo sapiens smal . . . [gi:14773546]
(SEQ.ID.NO. 37)
Matrix Metalloproteinase 9
[0227] X72795. M. musculus mRNA f . . . [gi:433434](SEQ.ID.NO.
538)&(SEQ.ID.NO. 38)
Human Homolog
[0228] NM.sub.--004994. Homo sapiens matr . . .
[gi:4826835](SEQ.ID.NO. 539)&(SEQ.ID.NO.39)
PTPase Receptor Type C
[0229] M14343. Mouse Ly-5 (leuco . . . [gi:198918] (SEQ.ID.NO.
40)
Human Homolog
[0230] NM.sub.--080922. Homo sapiens prot . . .
[gi:18641363](SEQ.ID.NO.541) (SEQ.ID.NO.41)
Aspartic Protease-Like Protein
[0231] D88899. Mouse mRNA for ki . . . [gi:1906809](SEQ.ID.NO.
542)&(SEQ.ID.NO. 42)
Human Homolog
[0232] NM.sub.--004851. Homo sapiens pron . . .
[gi:4758753](SEQ.ID.NO. 543)&(SEQ.ID.NO.43)
Eosinophil--Associated Ribonuclease 1
[0233] 1: U72032. Mus musculus eosi . . . [gi:1695898](SEQ.ID.NO.
544)&(SEQ.ID.NO. 44)
Hematopoietic Cell Specific Lyn Substrate 1
[0234] X84797. M. musculus mRNA s . . . [gi:683480](SEQ.ID.NO.
545)&(SEQ.ID.NO. 45)
Human Homolog
[0235] NM.sub.--005335. Homo sapiens hema . . .
[gi:4885404](SEQ.ID.NO.546)&(SEQ.ID.NO.46)
Rhom-2
[0236] M64360. Mouse rhom-2 mRNA . . . [gi:200748](SEQ.ID.NO.
547)&(SEQ.ID.NO. 47) Human homolog NM.sub.--005574. Homo
sapiens LIM . . .
[gi:6633806](SEQ.ID.NO.548)&(SEQ.ID.NO.48)
G-Protein, Alpha Inhibiting 1
[0237] U38501. Mus musculus G pr . . . [gi:1353505](SEQ.ID.NO.
49)
Human Homolog
[0238] NM.sub.--002070. Homo sapiens guan . . .
[gi:4504040](SEQ.ID.NO.550)&(SEQ.ID.NO.50)
Nicotinamide N-methylransferase
[0239] U86108. Mus musculus nico . . . [gi:2738008] (SEQ.ID.NO.
551)&(SEQ.ID.NO. 51)
Human Homolog
[0240] NM.sub.--006169. Homo sapiens nico . . .
[gi:5453789](SEQ.ID.NO. 552)&(SEQ.ID.NO.52) LMP2iran allele
D44456. Mus musculus bact . . . [gi:2467350](SEQ.ID.NO.
553)&(SEQ.ID.NO. 53)
Human Homolog
: XM.sub.--165798[gi:20555527](SEQ.ID.NO. 554)&(SEQ.ID.NO.
54)
Guanylate Nucleotide Binding Protein 2
[0241] AJ007970. Mus musculus mRNA . . .
[gi:4158171](SEQ.ID.NO.555)&(SEQ.ID.NO.55)
Human Homolog
[0242] XM.sub.--001804. Homo sapiens guan . . .
[gi:18548646](SEQ.ID.NO.556)&(SEQ.ID.NO.56)
Proteinase 3
[0243] U43525. Mus musculus pre- . . . [gi:1165220] (SEQ.ID.NO.
557)&(SEQ.ID.NO. 57)
Human Homolog
[0244] XM.sub.--009264. Homo sapiens prot . . .
[gi:18590908](SEQ.ID.NO.558)&(SEQ.ID.NO.58)
G Protein Gamma 2 Subunit
[0245] AF098489. Mus musculus G pr . . . [gi:4323235](SEQ.ID.NO.
559)&(SEQ.ID.NO. 59) Human homolg XM.sub.--090816. Homo sapiens
simi . . . [gi:18597218](SEQ.ID.NO. 583) (SEQ.ID.NO. 83) CSF 2
Receptor, beta 1 M34397. Mouse interleukin . . . [gi:191821
(SEQ.ID.NO. 560)&(SEQ.ID.NO. 60) Human homolog NM.sub.--000395.
Homo sapiens colo . . . [gi:4559407](SEQ.ID.NO.
561)&(SEQ.ID.NO.61)
Leukocyte Common Antigen
[0246] : M23158. Mouse leucocyte c . . . [gi:198752] (SEQ.ID.NO.
562)&(SEQ.ID.NO. 62)
Human Homolog
[0247] Y00638. Human mRNA for le . . . [gi:34280] (SEQ.ID.NO.
563)&(SEQ.ID.NO. 63)
Sonic Hedgehog Homolog
[0248] X76290. M. musculus (C57BL . . . [gi:2597987] (SEQ.ID.NO.
564)&(SEQ.ID.NO. 64)
Human Homolog
[0249] L38518. Homo sapiens soni . . . [gi:663156] (SEQ.ID.NO.
565)&(SEQ.ID.NO. 65)
BIT
[0250] D85785. Mouse mRNA for BI . . . [gi:2190256](SEQ.ID.NO.
566)&(SEQ.ID.NO. 66)
Human Homolog
[0251] AB023430. Homo sapiens Bit . . . [gi:6518912](SEQ.ID.NO.
567)&(SEQ.ID.NO. 67)
Vanin 3
[0252] AJ132103. Mus musculus mRNA . . .
[gi:6102995](SEQ.ID.NO.568)&(SEQ.ID.NO.68)
Human Homolog
[0253] AJ238982. Homo sapiens mRNA . . .
[gi:7160972](SEQ.ID.NO.569)&(SEQ.ID.NO.69)
Kruppel-Like Factor 7
[0254] NM.sub.--033563. Mus musculus Krup . . .
[gi:15808991](SEQ.ID.NO.570)&(SEQ.ID.NO.70)
Human Homolog
[0255] NM.sub.--003709. Homo sapiens Krup . . .
[gi:4507828](SEQ.ID.NO.571)&(SEQ.ID.NO.71)
Nitric Oxide Sythase 3, Endothelial Cell
[0256] U53142. Mus musculus endo . . . [gi:1518955](SEQ.ID.NO.
572)&(SEQ.ID.NO. 72) Human homolog AF400594. Homo sapiens endo
. . . [gi:15077875](SEQ.ID.NO. 573)&(SEQ.ID.NO. 73)
Paired-Ig-Like Receptor A1
[0257] U96682. Mus musculus immu . . . [gi:2138352](SEQ.ID.NO.
574)&(SEQ.ID.NO. 74) Human homolog XM.sub.--017977. Homo
sapiens leuk . . . [gi:20546205](SEQ.ID.NO.575)&(SEQ.ID.NO.75)
Voltage-Dependent NA+ Channel beta-1 L48687. Mus musculus volt . .
. [gi:1162950](SEQ.ID.NO. 576)&(SEQ.ID.NO. 76)
Human Homolog
[0258] U12192. Homo sapiens volt . . . [gi:619588](SEQ.ID.NO.
577)&(SEQ.ID.NO. 77)
Chemokine (C-C) Receptor 2
[0259] 1: U56819. Mus musculus mcp- . . . [gi:1532146](SEQ.ID.NO.
578)&(SEQ.ID.NO. 78)
Human Homolog
[0260] NM.sub.--000648. Homo sapiens chem . . .
[gi:4757937](SEQ.ID.NO.579) (SEQ.ID.NO.79)
Interferon Regulatory Factor 4
[0261] U20949. Mus musculus lymp . . . [gi:972947] (SEQ.ID.NO.
580)&(SEQ.ID.NO. 80)
Human Homolog
XM.sub.--165802[gi:20556306](SEQ.ID.NO. 581)&(SEQ.ID.NO.
81)
TABLE-US-00001 [0262] Seq ID No. Accession No. Comments 89 AK017227
90 BC004897 Human homolog to 89 91 AK080414 92 AAG23122 Human
homolog to 91 94 NM_008728 95 M59305 Human homolog to 94 101
NM_144930 102 BAA84996 Human homolog to 101 103 NM_018780 104
NM_003015 Human homolog to 103 113 NM_023184 114 NM_014079 Human
homolog to 113 118 BC030733 119 NBHUA2 Human homolog to 118 120
NM_019516 121 BC028222 Human homolog to 120 126 NM_152801 127
BC039856 Human homolog to 126 134 XM_284386 136 NM_008521 137
NM_145867 Human homolog to 136 141 NM_133198 142 NM_002863 Human
homolog to 141 143 NM_026772 144 NM_006779 Human homolog to 143 146
AK039295 147 AY090098 152 NM_010016 153 AAA52168 Human homolog to
152 154 NM_008522 155 AAA58656 Human homolog to 154 156 NM_019549
157 NM_002664 Human homolog to 156 158 NM_011994 159 NM_005164
Human homolog to 159 161 NM_148938 162 NM_004172 Human homolog to
161 163 NM_053268 164 NM_006506 Human homolog to 163 165 NM_007487
166 BC039123 Human homolog to 165 167 NM_145491 168 XM_209429 Human
homolog to 167 279 NM_025829 169 BC031289 Human homolog to 279 170
NM_147220 171 NM_080283 Human homolog to 170 173 NM_011186 174
NM_002797 Human homolog to 173 185 NM_153507 186 NM_152727 Human
homolog to 185 193 NM_021886 194 NM_022909 Human homolog to 193 195
NM_007387 196 NM_001610 Human homolog to 195 197 NM_013739 198
NM_024872 Human homolog to 198 202 BC029248 203 AAG59827 Human
homolog to 202 204 NM_009381 205 BC031989 Human homolog to 204 206
NM_053186 207 NM_017623 Human homolog to 206 208 NM_019560 209
NM_052874 Human homolog to 208 212 NM_029999 213 NM_030915 Human
homolog to 212 225 AK028281 226 NP_001287 Human homolog to 225 230
AK089391 234 BC034872 235 NM_005292 Human homolog to 234 237
AK037228 238 XP_211590 Human homolog to 237 257 NM_139138 258
AY181245 Human homolog to 257 265 NM_130905 266 NM_021155 Human
homolog to 265 270 XM_286373 271 XM_211303 Human homolog to 270 274
NM_010260 275 AL832451 Human homolog to 274
Along with identifying known genes and known obesity targets, the
disclosed method also identified sequences that have not been
characterized and sequences whose function has until now not been
discovered.
[0263] The following table lists the Accession number for sequences
that have until now been "ESTs", i.e. unknown.
TABLE-US-00002 Seq ID/Accession-seq. unknown Seq ID. No. Accession
Number Comments 86 AI536451 87 AA880159 93 AI643872 96 AI876264 115
AK029114 116 AB046799 Human homolog to 115 117 AK084680 135
AI843070 140 AI429810 145 AI841502 148 AW123926 151 AI593942 160
AI131739 172 AA178128 179 XM_287203 180 NM_020182 Human homolog to
179 191 AV293830 192 BC040983 Human homolog to 191 201 AI661950 223
AW049306 224 AI839459 229 AI413635 236 AI465892 246 AI021175 255
NM_033524 256 NM_152594 Human homolog to 255 263 AA914137 264
AI842280 267 AV376576 268 AI593010 276 AK046027 277 AB020644
The following table lists the Accession numbers for sequences whose
function has until now been unknown:
TABLE-US-00003 Seq ID Accession Number Comments 84 NM_144936 85
NM_138788 Human homolog to 84 88 XM_110133 97 BC037624 98 AK057179
Human homolog to 97 99 BC026527 100 XP_085101 Human homolog to 99
105 AK090296 106 NM_000106 Human homolog to 105 107 AK046700 108
NM_032554 Human homolog to 107 109 AK087428 110 BAB85539 Human
homolog to 109 111 AK079549 112 NP_620145 Human homolog to 111 115
AK029114 116 AB046799 Human homolog to 115 117 AK084680 122
AK088119 123 BC035630 Human homolog to 122 124 XM_112637 125
XP_032996 Human homolog to 124 128 AK017670 129 BC027926 Human
homolog to 128 130 AK017670 131 BC022276 Human homolog to 130 132
AK032359 133 XP_087331 Human homolog to 132 138 AK045363 139
BC047588 Human homolog to 138 149 BC024883 150 BC039867 Human
homolog to 149 175 BC018156 176 XM_031536 Human homolog to 175 177
AK048450 178 BC039591 Human homolog to 177 181 BC024788 182
NM_024074 Human homolog to 181 183 XM_125708 184 BC035564 Human
homolog to 183 187 NM_027349 188 AL832314 Human homolog to 187 189
NM_025629 190 BC040620 Human homolog to 189 199 AK012494 200
XM_058800 Human homolog to 199 210 AK031989 211 XP_229579 Human
homolog to 210 214 NM_029509 215 BC008421 Human homolog to 214 216
AK029076 217 XM_113636 Human homolog to 217 218 AK048344 219
NM_145479 220 NM_032775 Human homolog to 219 221 NM_026170 222
AAG44724 Human homolog to 221 227 NM_139269 228 AF317086 Human
homolog to 227 232 BC031142 233 BC036468 Human homolog to 232 239
AK045646 240 NM_016644 Human homolog to 239 241 NM_027863 242
BC016653 Human homolog to 241 243 BC031419 244 BC039067 Human
homolog to 243 245 XM_147934 247 AK048759 248 NM_012414 Human
homolog to 247 249 NM_175035 250 NP_060854 Human homolog to 249 251
AK037116 252 BC049196 Human homolog to 252 253 BC027174 254
NM_015147 Human homolog to 254 255 NM_033524 256 NM_152594 Human
homolog to 256 259 AK038853 260 AY033237 Human homolog to 259 ' 261
AK084351 262 BC042872 Human homolog to 261 268 AI593010 272
NM_053247 273 NM_006691 Human homolog to 272
Identification of sFRP-5 as an Adipocyte Specific Gene
[0264] Of the identified genes, one identified gene modulated by
the presence or absence of HMGI-C, upregulated by itself in an
autocrine manner, which is involved in the regulation of adipose
accumulation, is SFRP-5. The gene was identified by the described
method.
[0265] "sFRP-5" refers generally to a polypeptide in accordance
with the present invention, which is described in detail throughout
the specification. "sFRP-5 gene" refers to a polynucleotide as
defined above in accordance with the present invention, which
encodes an sFRP-5 polypeptide.
[0266] "sFRP-5 activity or sFRP-5 polypeptide activity" or
"biological activity of the s-FRP-1 or sFRP-5 polypeptide" refers
to the metabolic or physiologic function of said sFRP-5 including
similar activities or improved activities or these activities with
decreased undesirable side-effects. Also included are antigenic and
immunogenic activities of said sFRP-5. In particular, sFRP-5 is a
protein that is in the pathway and that is upregulated in
adipogenesis and obesity. The sFRP-1 protein of the present
invention is dependent upon HMGI-C for normal levels of expression.
Moreover, the sFRP-5 of the present invention acts in an autocrine
fashion to upregulate its own expression in adipocytes.
[0267] As described above, the expression of this gene is dependent
on HMGI-C in normal adipose tissue and upregulates its own
expression in adipocytes. Computer analysis (BLAST search) of the
open reading frame of the gene revealed no significant similarity
to either the mammalian ob or tub genes.
[0268] The sFRP-5 polynucleotides of the present invention includes
isolated polynucleotides encoding the sFRP-5 polypeptides and
fragments, and polynucleotides closely related thereto. More
specifically, sFRP-5 polynucleotide of the invention include a
polynucleotide comprising the human nucleotide sequences contained
in SEQ ID NO:103 encoding a sFRP-1 polypeptide of SEQ ID NO: 603,
and polynucleotides having the particular sequence of SEQ ID
NO:103. sFRP-5 polynucleotides further include a polynucleotide
comprising a nucleotide sequence that has at least 91% identity
over its entire length to a nucleotide sequence encoding the sFRP-5
polypeptide of SEQ ID NO:603, and a polynucleotide comprising a
nucleotide sequence that is at least 91% identical to that of SEQ
ID NO:103, wherein identity is calculated over the entire length of
the reference polynucleotide or polypeptide, for example, SEQ ID
NO:103. In this regard, polynucleotides with at least 70% are
preferred, more preferably at least 80% identity, even more
preferably at least 90% identity, yet more preferably at least 95%
identity, 97% are highly preferred and those with at least 98-99%
are most highly preferred, with at least 99% being the most
preferred. Also included under sFRP-5 polynucleotides are a
nucleotide sequence which has sufficient identity to a nucleotide
sequence contained in SEQ ID NO:103 to hybridize under conditions
useable for amplification or for use as a probe or marker. The
invention also provides polynucleotides which are complementary to
such sFRP-5 polynucleotides. In preferred embodiments, the
polynucleotides are fully complementary to sFRP-5 over the entire
length of the reference polynucleotide.
TABLE-US-00004 A. SEQ ID NO: 103 1 ggcgggggtg ggaggtgagg gggaccagcg
gctgcagatt ggctagggaa cggcgaagcc 61 tgggctggag ccccgtggtg
ggaggcgcct ggatcactcc ggcagcggag cggagcagag 121 actgccacct
acttggacga ccctagtagc agccggcttc agggaacatc ccagccgggg 181
cgcacgccgg gcagcggggc gccgagcacc gagccgtcgg ggcaggccgc aacatccagc
241 catgtgggtg gcctggagcg cacggacggc cgcactggcg ttgctgctcg
gggcgctgca 301 tggggcgcca acacgcggcc aggagtacga ctactacggt
tggcaggccg agccgctgca 361 cggccgctcc tactccaagc caccgcagtg
cctcgacatc cccgccgatc tgccgctctg 421 tcacacggtg ggctacaagc
gcatgcggct gcccaacctg ctggagcacg agagcctggc 481 cgaggtgaag
cagcaggcaa gcagctggct gccactgctg gccaagcgct gccactcaga 541
cacccaggtc ttcctctgct cgctcttcgc tcccgtctgc ctggaccgac ccatctaccc
601 ctgccgctcg ctgtgcgaag ctgcgcgcgc cggctgcgct ccgctcatgg
aggcctacgg 661 tttcccttgg cccgagatgc tgcactgcca caagttcccc
ctggacaacg acctctgcat 721 cgcggtgcag ttcgggcacc tgcctgccac
cgcgcctcca gtgaccaaga tctgtgccca 781 gtgtgagatg gagcacagcg
ctgacggcct catggaacag atgtgctcca gtgactttgt 841 ggtcaagatg
cgcattaagg agatcaagat agacaacggg gaccgaaagt tgattggagc 901
ccagaagaag aagaagctgc tcaaggcagg ccccttaaag cgcaaggaca ccaagaagct
961 ggtcctgcat atgaagaacg gggcaagctg cccttgccca caattagaca
acctgacggg 1021 cagcttcctg gtcatgggcc gcaaagtaga gggacagctg
ctgctcacgg ccgtctaccg 1081 ctgggacaag aagaataagg agatgaagtt
tgcggtcaaa ttcatgttct cctatccctg 1141 ttccctctac tacccttttt
tctatggggc agctgaaccc cactga B. SEQ ID NO: 603
MWVAWSARTAALALLLGALHGAPTRGQEYDYYGWQAEPLEHGRSYSKPPQCLDIPADL
PLCHTVGYKRMRLPNLLEHESLAEVKQQASSWLPLLAKRCHSDTQVFLCSLFAPVCLD
RPIYPCRSLCEAARAGCAPLMEAYGFPWPEMLHCHKFPLDNDLCIAVQFGHLPATAPP
VTKICAQCEMEHSADGLMEQMCSSDFVVKMRIKEIKIDNGDRKLIGAQKKKKLLKAGP
LKRKDTKKLVLHMKNGASCPCPQLDNLTGSFLVMGRKVEGQLLLTAVYRWDKKNKEMK
FAVKFMFSYPCSLYYPFFYGAAEPH C. SEQ ID NO: 104 1 gaggcgccag gatcagtcgg
ggcacccgca gcgcaggctg ccacccacct gggcgacctc 61 cgcggcggcg
gcggcggcgg ctgggtagag tcagggccgg gggcgcacgc cggaacacct 121
gggccgccgg gcaccgagcg tcggggggct gcgcggcgcg accctggaga gggcgcagcc
181 gatgcgggcg gcggcggcgg cggggggcgt gcggacggcc gcgctggcgc
tgctgctggg 241 ggcgctgcac tgggcgccgg cgcgctgcga ggagtacgac
tactatggct ggcaggccga 301 gccgctgcac ggccgctcct actccaagcc
gccgcagtgc cttgacatcc ctgccgacct 361 gccgctctgc cacacggtgg
gctacaagcg catgcggctg cccaacctgc tggagcacga 421 gagcctggcc
gaagtgaagc agcaggcgag cagctggctg ccgctgctgg ccaagcgctg 481
ccactcggat acgcaggtct tcctgtgctc gctctttgcg cccgtctgtc tcgaccggcc
541 catctacccg tgccgctcgc tgtgcgaggc cgtgcgcgcc ggctgcgcgc
cgctcatgga 601 ggcctacggc ttcccctggc ctgagatgct gcactgccac
aagttccccc tggacaacga 661 cctctgcatc gccgtgcagt tcgggcacct
gcccgccacc gcgcctccag tgaccaagat 721 ctgcgcccag tgtgagatgg
agcacagtgc tgacggcctc atggagcaga tgtgctccag 781 tgactttgtg
gtcaaaatgc gcatcaagga gatcaagata gagaatgggg accggaagct 841
gattggagcc cagaaaaaga agaagctgct caagccgggc cccctgaagc gcaaggacac
901 caagcggctg gtgctgcaca tgaagaatgg cgcgggctgc ccctgcccac
agctggacag 961 cctggcgggc agcttcctgg tcatgggccg caaagtggat
ggacagctgc tgctcatggc 1021 cgtctaccgc tgggacaaga agaataagga
gatgaagttt gcagtcaaat tcatgttctc 1081 ctacccctgc tccctctact
accctttctt ctacggggcg gcagagcccc actgaagggc 1141 actcctcctt
gccctgccag ctgtgccttg cttgccctct ggccccgccc caacttccag 1201
gctgacccgg ccctactgga gggtgttttc acgaatgttg ttactggcac aaggcctaag
1261 ggatgggcac ggagcccagg ctgtcctttt tgacccaggg gtcctggggt
ccctgggatg 1321 ttgggcttcc tctctcagga gcagggcttc ttcatctggg
tgaagacctc agggtctcag 1381 aaagtaggca ggggaggaga gggtaaggga
aaggtggagg ggctcagggc accctgaggc 1441 ggaggtttca gagtagaagg
tgatgtcagc tccagctccc ctctgtcggt ggtggggcct 1501 caccttgaag
agggaagtct caatattagg ctaagctatt tgggaaagtt ctccccaccg 1561
cccctgtacg cgtcatccta gcccccctta ggaaaggagt tagggtctca gtgcctccag
1621 ccacaccccc tgccttcccc agcttgccca tttccctgcc ccaaggccca
gagctccccc 1681 cagactggag agcaagccca gcccagcctc ggcatagacc
cccttctggt ccgcccgtgg 1741 ctcgattccc gggattcatt cctcagcctc
tgcttctccc ttttatccca ataagttatt 1801 gctactgctg tgaggccata
ggtactagac aaccaataca tgcagggttg ggttttctaa 1861 tttttttaac
tttttaatta aatcaaaggt cgacgcgcgg ccgcg D. SEQ ID NO: 604
MRAAAAAGGVRTAALALLLGALHWAPARCEEYDYYGWQAEPLHGRSYSKPPQCLDIP
ADLPLCHTVGYKRMRLPNLLEHESLAEVKQQASSWLPLLAKRCHSDTQVFLCSLFAP
VCLDRPIYPCRSLCEAVRAGCAPLMEAYGFPWPEMLHCHKFPLDNDLCIAVQFGHLP
ATAPPVTKICAQCEMEHSADGLMEQMCSSDFVVKMRIKEIKIENGDRKLIGAQKKKK
LLKPGPLKRKDTKRLVLHMKNGAGCPCPQLDSLAGSFLVMGRKVDGQLLLMAVYRWD
KKNKEMKFAVKFMFSYPCSLYYPFFYGAAEPH
[0269] The sFRP-5 gene is a member of the sFRP family, which
inhibit the Wnt signal transduction pathway. Wnts are a family of
paracrine/autocrine factors which affect cell growth and
proliferation. Wnt ligand binds to its cell surface receptor
Frizzled. Frizzled then signals through Dishevelled to inhibit the
kinase activity of a complex which contains glycogen synthase
kinase 3 (GSK3), Axin, .beta.-catenin and other proteins. When the
complex is phosphorylated, .beta.-catenin is targeted for rapid
degradation. When the complex is hypophosphorylated due to Wnt
signaling, .beta.-catenin is stabilized and translocates to the
nucleus where it binds the TCF/LEF family of transcription factors
which in turn regulate the Wnt target genes. Ross, et al. (Science
289:950-953) has shown sFRP-1 that Writ signaling inhibits
adipogenesis. Thus, Wnt signaling inhibits adipogenesis. Inhibiting
sFRP-5 should modulate the Wnt-1 signaling pathway, and hence
modulate adipogenesis. This evidence, in combination with sFRP-5
being a secreted protein, makes sFRP-5 a prime candidate for
serving as a drug target in treating obesity.
[0270] Also included under sFRP-5 polynucleotides are a nucleotide
sequence which has sufficient identity to a nucleotide sequence
contained in SEQ ID NO:103 or SEQ ID NO:104 to hybridize under
conditions useable for amplification or for use as a probe or
marker. The invention also provides polynucleotides which are
complementary to such sFRP-5 polynucleotides. In preferred
embodiments, the polynucleotides are fully complementary to sFRP-5
over the entire length of the reference polynucleotide.
[0271] Mouse sFRP-5 of the present invention has a 91% identity
with human sFRP-5 at the DNA level and 89% at the amino acid level.
Accordingly, included in the present invention are polynucleotides
encoding polypeptides which have at least 91% identity, preferably
at least 92% identity, more preferably at least 93% identity, even
more preferably at least 94-99% identity, to the amino acid
sequence of murine sFRP-5 over the entire length of the recited
amino acid sequence.
[0272] The nucleotide sequences encoding the sFRP-5 polypeptide of
SEQ ID NO:603 may be identical to the polypeptide encoding sequence
contained in SEQ ID NO:103 or it may be a sequence, which as a
result of the redundancy (degeneracy) of the genetic code, also
encodes the polypeptide of SEQ ID NO:603.
[0273] The present invention provides a novel gene sFRP-5, whose
expression is upregulated in response to leptin deficiency. In
leptin deficient mice, sFRP-5 expression in the adipose tissue is
increased by 6.5-fold (see FIG. 2). The normal expression of sFRP-5
is HMGI-C dependent. Expression of sFRP-5 in white adipose tissue
(WAT) is decreased 10-fold in the lean HMGI-C knockout mice as
compared to wild-type mice (FIG. 1). sFRP-5 expression in WAT is
confined to adipocyte fraction with negligible expression in the
stromal vascular fraction (FIG. 3). sFRP-5 also acts in an
autocrine fashion to upregulate its own expression in adipocytes.
Conditioned medium from CHO cells containing sFRP-5 (FIG. 4)
increases endogenous expression of sFRP-5 in 3T3-L1 adipocytes by
greater than 1600-fold (FIG. 5). sFRP-5 expression in wild-type
mice is most prominent in the adult fat pads (The protein of the
present invention also plays a role in obesity. According to one
aspect of this invention, an isolated polynucleotide capable of
upregulating its own expression is provided.
[0274] Preferably, the polynucleotide comprises a sequence selected
from the following group: SEQ ID NO:103; an allelic variant of SEQ
ID NO:103; a sequence hybridizing with SEQ ID NO:103 or its
complement under moderate hybridization and washing conditions; a
sequence encoding a polypeptide having an amino acid sequence of
SEQ ID NO:603 with up to 5% conservative substitutions; SEQ ID
NO:104; an allelic variant of SEQ ID NO:104; a sequence hybridizing
with SEQ ID NO:104 or its complement under moderate hybridization
and washing conditions; a sequence encoding a polypeptide having an
amino acid sequence of SEQ ID NO:604 with up to 5% conservative
substitutions.
[0275] Another aspect of the invention features a recombinant DNA
molecule comprising a vector having an insert that includes part or
all of an sFRP-5 polynucleotide and cells transformed with the
recombinant DNA molecule. Preferably, the cells are murine or human
cells. Most preferably, the cells are adipose cells from one of the
aforementioned organisms.
[0276] The invention also features an isolated polypeptide produced
by expression of the sFRP-5 polynucleotides described above.
Antibodies immunologically specific for the protein, or one or more
epitopes thereof; are also provided.
[0277] In another aspect, the invention relates to methods for
using such polypeptides and polynucleotides, including the
treatment of obesity, hypertension, cardiovascular disease and
diabetes. The present invention is also implicated in diseases and
conditions such as obesity, hypertension, cardiovascular disease
and diabetes, among others. In still another aspect, the invention
relates to methods to identify agonists and antagonists using the
materials provided by the invention, and treating diseases or
conditions associated with obesity, hypertension, cardiovascular
disease and diabetes or sFRP-5 imbalance with the identified
compounds. Methods of using the inventive polynucleotides and
polypeptides as pharmaceutical formulations in the treatment of
obese patients are also provided. Still further provided in the
present invention are methods of screening individuals for
predisposition to obesity using polynucleotides and polypeptides of
the present invention.
[0278] The invention also provides a pharmaceutical composition,
comprising one or more vectors which comprise DNA constructs
comprising any of the nucleic acid sequences of SEQ ID NO: 103 and
SEQ ID NO: 104
[0279] In another embodiment, the invention provides a
pharmaceutical composition comprising one or more peptides encoded
by any of the sequences SEQ ID NO: 103 and SEQ ID NO: 104.
[0280] In yet another embodiment, the invention provides for a
pharmaceutical composition comprising an agonist to any of the
peptides encoded by any of the sequences SEQ ID NO: 103 and SEQ ID
NO: 104.
[0281] In still another embodiment, the invention provides for a
pharmaceutical composition comprising an antagonist to any of the
peptides encoded by any of the sequences SEQ ID NO: 103 and SEQ ID
NO: 104.
[0282] Therefore, another aspect of the invention is a kit to
diagnose or diagnose the susceptibility to a disease related to
obesity in a subject which comprises:
[0283] (a) an sFRP-5 polynucleotide, preferably the nucleotide
sequence of SEQ ID NO:103 or 104; or a fragment thereof;
[0284] (b) a nucleotide sequence complementary to that of (a)
[0285] (c) an sFRP-5 polypeptide, preferably a polypeptide of SEQ
ID NO: 603 or 604, or a fragment thereof; or
[0286] (d) and antibody to an sFRP-5 polypeptide, preferably to the
polypeptide of SEQ ID NO: 603 or 604.
[0287] In any such diagnostic kit, (a), (b), (c), or (d) may be a
substantial component.
sFRP-5 Subcloning
[0288] sFRP-5 cDNA was subcloned as follows. Total RNA from the
ob/ob mouse was reverse transcribed using superscript II RT reverse
transcriptase. A forward primer SEQ ID NO:278
(5'-CACCATGTGGGTGGCCTGGAGCGCACGG-3') and reverse primer SEQ ID
NO:279 (5'-CACAGCTGGCTGGTTGGGGCAA-3') were used to amplify the
sFRP-5 coding sequence. The product was purified on a 1% agarose
gel and subcloned into the pENTR/D-TOPO plasmid vector. The gene
was sequenced and then transferred into the pcDNA3.2/DEST plasmid
vector (Invitrogen) via in vitro recombination using the LR clonase
enzyme mix (Invitrogen) as recommended by the manufacturer. The
pcDNA3.2/sFRP-5 was amplified in E. coli and purified using the
Midi plasmid isolation kit (Quiagen).
Real Time PCR Analysis of sFRP-5 Expression
[0289] Snap-frozen tissues were used to isolate total RNA by the
RNeasy protocol (Qiagen Inc. CA). Briefly, tissues were homogenized
in the lysis buffer containing guanidine isothiocyanate and applied
to the RNeasy column. After several washes to remove contaminants,
RNA bound to the column matrix is eluted in distilled water. First
strand cDNA is made from 1 g of total RNA using reverse
transcriptase in a standard reaction.
[0290] Quantitative real-time PCR using the Applied Biosystems ABI
Prism 7900HT sequence detector provides an accurate method for the
determination of mRNA levels in a tissue sample. The quantitation
is based on the detection of a fluorescent signal produced
proportionally during the amplification of a PCR product (see FIG.
4, below). A probe (TaqMan) is designed to anneal to the sequence
of interest between the usual forward and reverse PCR primers. The
probe is labelled with a reporter fluorochrome at the 5' end and a
quencher either at any T position or at the 3' end. The probe is
designed to have a higher T.sub.m than the primers as the probe
must be 100% annealed at the extension phase for the assay to be
successful. As long as the reporter and the quencher are both
attached to the probe, the quencher blocks the fluorescence of the
reporter. However, as the Taq polymerase extends the primer, the
intrinsic 5'-3' exonuclease activity of the Taq polymerase degrades
the probe thereby releasing the reporter. The amount of
fluorescence released during the amplification cycle is
proportional to the amount of product generated in each cycle.
[0291] Quantitative Real-Time PCR (1) Forward and reverse primers
are extended with Taq polymerase as in a normal PCR reaction. A
probe containing a reporter (R) and a quencher (Q) anneals between
the two primers. (2) As the polymerase extends the primer, the
probe is displaced. (3) The intrinsic exonuclease activity of the
polymerase cleaves the reporter from the probe. (4) After release
of the reporter, a fluorescent signal is generated. Emissions are
measured every six seconds by a laser/charge-coupled device (CCD)
optics system.
[0292] The sensitivity of detection of this system allows for the
acquisition of data while the PCR reaction is in the exponential
phase. This is measured by identifying the cycle number at which
the reporter signal rises above the background noise. The cycle at
which this occurs is called the threshold cycle (C.sub.t.) The
C.sub.t is determined at the steepest part of the curve during the
exponential phase of the reaction and is more reliable than
end-point measurements of accumulated PCR products. The C.sub.t is
inversely proportional to the copy number of the target template,
thus the higher the template concentration the lower the threshold
cycle measured. An example of a typical output is shown in the
figure below.
This graph illustrates one of the possible outputs of the ABI Prism
7900HT. This window shows the amount of fluorescence in each
amplification cycle for each reaction. The threshold cycle
(C.sub.t) is shown by the darker horizontal line.
[0293] One significant advantage to this system is the ability to
multiplex quantitative PCR reactions. That is, up to three
fluorescent dyes can be used in the same reaction, so endogenous
controls can be run in the same well. For example, kits are
available from Applied Biosystems for 18S ribosomal RNA or for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to be used as
internal controls. In order to determine copy number, kits are
available to produce cRNA in order to generate a standard curve.
The cRNA must be validated in the RT-PCR reactions to ensure that
it is transcribed and amplified at the same efficiency as the
target mRNA present in a mixture of extracted RNAs. Other controls
to be used are no-amplification controls to test for the
contamination of the RNA with genomic DNA and no-template controls
to ensure that the reagents are not contaminated.
[0294] The design of TaqMan probes and primers is critical to the
success of the experiment. Using the Primer Design program, we have
designed primers and probes for human and mouse sFRP-5 mRNAs. The
parameters for effective primers and probes have been
well-documented and have been built into the program. The
parameters include a T.sub.m for the probe that is 10.degree. C.
higher than the primers, a primer T.sub.m between 58.degree. C. and
60.degree. C., an amplicon size of between 50 and 150 bases, the
absence of 5' Gs and primer length.
Cell Culture
[0295] Briefly, transient expression of sFRP-5 in Chinese hamster
ovary (CHO) cells was carried out as follows. CHO cells were seeded
in a dish of 6 cm diameter and cultured in Ham's F12K medium
adjusted to 2 mM L-glutamine and to 1.5 g/L of sodium bicarbonate
and containing 10% fetal bovine serum. The cells were maintained at
37.degree. C. in a humidified atmosphere of 5% CO.sub.2. culture
were grown to 90% confluence and were transfected with 8 .mu.g
pcDNA3.2/DEST-sfrp5 DNA or pcDNA3.2DEST as a negative control with
Lipofectamine 2000 (Invitrogen) as recommended by the manufacturer.
After 6 hours of incubation, the cells were washed with PBS and
growth medium was added for incubation for 48 hours, at which point
the conditioned medium containing secreted sFRP-5 was collected for
further study.
Assays for sFRP-5 in RNA Expression Levels:
[0296] 3T3-L1 cells were grown in 6 cm diameter plates in
Dulbecco's Modified Eagle Medium (DMEM) adjusted to 4 mM
L-glutamine, to 1.5 g/L of Sodium Bicarbonate, and to 4.5 g/L
glucose and containing 10% fetal bovine serum. The cells were
maintained at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2. At full confluence the cells were supplemented with
conditioned medium containing sFRP-5 in amounts described in FIG.
4. Maximal autocrine stimulation of sFRP-5 expression in 3T3-L1
cells was achieved at a dose of 500 .mu.l conditioned medium per 5
ml of growth medium. After 6 days of incubation, the cells were
collected and total RNA was isolated using the Rneasy kit
(Quiagen). Random primer cDNA was synthesized using Superscript II
(Invitrogen). Real time quantitative PCR (TaqMan.TM.) for sFRP-5
and the GAPDH control was performed in the Applied biosystems ABI
7900HT Thermocycler using the recommended reagents and protocols of
the manufacturer. The primers and TaqMan.TM. sequences are as
follows:
TABLE-US-00005 sFRP-5: (forward) SEQ ID NO: 280
5'-TGTGCCCAGTGTGAGATGGA-3' (reverse) SEQ ID NO: 281
5'-GCGCATCTTGACCACAAAGTC-3' (probe) SEQ ID NO: 282
6FAM-TGACGGCCTCATGGAACAGATGTGC-TAMRA GAPDH Control: (forward) SEQ
ID NO: 283 5'-CAACGGGAAGCCCATCAC-3' (reverse) SEQ ID NO: 284
5'-CGGCCTCACCCCATTTG-3' (probe) SEQ ID NO: 285
VIC-ATCTTCCAGGAGCGAGACCCCACTAACA-TAMRA
Similar experiments were conducted using conditioned medium from
pcDNA3.2/DEST-sfrp5 transfected 3T3-L1 cells with substantially
identical results. Identification of npr-3 as an Adipocyte Specific
Gene
[0297] The natriuretic peptide (NP) system is an important
component in the regulation of sodium and water balance, blood
volume and blood pressure. This system works through several
mechanisms, for example decreasing renin release and consequently
aldosterone release by the adrenal cortex, thereby decreasing
sodium and fluid retention in the kidneys. The NP system has also
been shown to inhibit vasopressin, again causing a decrease in
fluid retention in the kidney. These actions contribute to
reductions in blood volume, and therefore central venous pressure,
cardiac output, and arterial blood pressure. A third mechanism
appears to be arterial vasodilation in response to
hypervolemia.
[0298] The family of natriuretic peptides includes atrial
natriuretic peptide (ANP), brain natriuretic peptide (BNP) and
C-type natriuretic peptide (CNP). The primary signalling molecules
for these peptides are natriuretic peptide receptor A (nprA, npr1)
which binds ANP and BNP, and natriuretic peptide receptor B (nprB,
npr2) which binds CNP. The A and B receptors share approximately
62% identity at the amino acid level and have been classified as
guanylyl cyclase receptors. That is, their intracellular domains
possess a kinase-like domain and a guanylyl cyclase catalytic
domain. Upon ligand binding to the extracellular ligand binding
domain, the guanylyl cyclase catalytic domain is activated causing
and increase in intracellular cyclic GMP (cGMP) which potentiates
the physiological activity of the receptors. The A receptor has
been implicated in vasodilation, increased diuresis and
natriuresis, and decreased renin and aldosterone. The B receptor
has been implicated in vasodilation and increased long bone growth.
(for review see Levin, et al., New England Journal of Medicine
339(5): 321-328 (1998) and Potter, et al., J. Biol. Chem. 276(9):
6057-6060 (2001)).
[0299] There is a third receptor, natriuretic peptide receptor C,
(nprC, npr3) which shares only approximately 32% identity with nprA
and nprB over the length of the protein. The C receptor retains a
similar extracellular ligand binding domain and trans-membrane
domain, but has only a short intracellular domain (37 amino acid
residues) which lacks both the kinase-like domain and guanylyl
cyclase activation domain. ANP, BNP, and CNP all bind this receptor
with approximately equal affinity. The receptor is called the "C"
receptor in that it has been shown to participate in the local
clearance of the natriuretic peptides. The receptor binds the
ligand and is internalized. The ligand is degraded and the receptor
is retroendocytozed back to the cell surface (Nussenzveig, et al.,
J. Biol. Chem. 265(34) 20952-20958 (1990). The C receptor accounts
for approximately 50% of natriuretic peptide clearance, the other
half being carried out by cell surface neutral endopeptidases.
However, while nprA and nprB are often called "biologically active"
receptors to the exclusion of nprC, it has been suggested that nprC
has other biological activity other than simply natriuretic peptide
clearance. Several groups have shown that the c-terminal domain of
nprC can interact with inhibitory G-proteins (G.sub.i) that act to
downregulate adenylyl cyclase and thus reduce the level of
intracellular cyclic A MP (cAMP) (Palaparti, et al., Biochem. J.,
346:313-320 (2000) and Pagano, et al., J. Biol. Chem.
276(25):22064-22070 (2001)). Recently, it has been postulated that
the vasodilatory effects of endothelium-derived hyperpolarizing
factor (EDHF) may be attributed to such nprC mediated adenylyl
cyclase inhibition (Chauhan, et al., Proc. Natl. Acad. Sci. USA,
100(3):1426-1431 (2003).
[0300] ANP and BNP have been linked to lipolysis in a cGMP
dependent manner which does not depend on cAMP production or
phosphodiesterase inhibition (Sengenes, et al., FASEB J.,
14(10):1345-51 (2000)). Thus, ANP and BNP have been implicated in
the biology of the adipocyte, but not nprC. In this case, for
example, the authors have attributed the lipolytic effects as being
linked to cGMP production in which nprC does not participate.
[0301] Specific npr3 knockout mice were made to determine the
effect of an absence of nprC on water balance, salt balance, and
blood pressure (Matsukawa, et al., Proc. Natl. Acad. Sci. USA
96:7403-7408 (1999)). The animals have a moderately but
statistically significantly lowered blood pressure and with age
show an increase in daily water uptake with a significant increase
in urinary output. The knockout mice also have a defect in the
ability to concentrate their urine. The observed alterations in
renal function were interpreted as being the result of a failure of
local clearance of natriuretic peptides in the glomerular and
post-glomerular structures resulting in an increase in filtered
volume and a decrease in water reabsorption. The decrease in blood
pressure was attributed to simple hypovolemia. These animals
exhibit skeletal abnormalities including an overgrowth of the long
bones as well as other defects. The authors note that the animals
exhibit " . . . elongated femurs, tibias, metatarsal, and digital
bones, longer vertebral bodies, increase body length, and decreased
weight [emphasis added]." However, the authors did not account for
the decrease in weight nor did they make any examination of the
adipose tissue.
[0302] Several spontaneously occurring mutants in the npr3 have
been identified, the first of which was called longjohn (lgj) due
to the skeletal defects described above. A French group studied
them to examine and compare the skeletal defects among the three
strains (Jaubert, et al., Proc. Natl. Acad. Sci. USA 96:10278-10283
(1999). They note offhandedly that " . . . older mutant mice are
exceptionally thin and . . . at necropsy normal body fat deposits
are absent." Again, the authors do not make any more mention of the
animals' weight or adipose tissue.
[0303] In light of the genetic evidence above, inhibitors to nprC
should be useful to treat or prevent adipose accumulation. A group
of such inhibitors has been disclosed in PCT International
Publication No. WO 00/61631, the contents of which are hereby
incorporated by reference. The role of nprC in adipose biology is
unclear. One possibility is this: since ANP and BNP have been shown
to promote lipolysis, the lack of the clearance receptor i.e. nprC
causes an increased half-life of ANP and BNP and therefore results
in increased lipolysis. No alterations in weight gain have been
noted in patients receiving recombinant human BNP for acute
congestive heart failure, but these patients receive the medication
acutely as a bolus and thus there was not enough time to see an
effect on adipose mass.
The Genbank accession number for the mouse npr3 gene is
NM.sub.--008728 [online], Genbank. Retrieved from the Internet:
<URL:
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=nucleoti-
de&list_uids=6679115&dopt=GenBank> The peptide sequence
is as follows:
TABLE-US-00006 (SEQ. ID. NO. 594)
MRSFLLFTFSACVLLARVLLAGGASSGAGDTRPGSRRRAREALAAQKIE
VLVLLPRDITYLFSLARVRPAIEYALRSVEGNGTGRKLLPPGTRFQVAY
EDSDCGNRALFSLVDRVAAARGAKPDLILGPVCGYAAAPVARLASHWDL
PMLSAGALAAGFQHKDTEYSHLTRVAPAYAKMGEMMLALFRHHHWSRAA
LVYSDDKLERNCYFTLEGVHEVFQEEGLHTSAYNFDETKDLDLDDIVRY
IQGSERVVIMCASGDTIRRIMLAVHRHGMTSGDYAFFNIELFNSSSYGD
GSWRRGDKHDSEAKQAYSSLQTVTLLRTVKPEFEKFSMEVKSSVEKQGL
NEEDYVNMFVEGFHDAILLYVLALHEVLRAGYSKKDGGKIIQQTWNRTF
EGIAGQVSIDANGDRYGDFSVVAMTDTEAGTQEVIGDYFGKEGRFQMRS
NVKYPWGPLKLRLDETRIVEHTNSSPCKSSGGLEESAVTGIVVGALLGA
GLLMAFYFFRKKYRITIERRNQQEESNIGKHRELREDSIRSHFSVA
The DNA sequence is as follows
TABLE-US-00007 (SEQ. ID. NO. 94) 1 atgcggtcct ttctgctgtt cactttctcg
gcgtgcgtgc tgctggcccg ggtgctgctg 61 gctggcggcg cgagcagcgg
cgccggggac acccggccag gcagcaggcg ccgggcgaga 121 gaggcgctgg
cggctcaaaa gatcgaggtg cttgttctat tgccccggga cattacgtac 181
ttgttctcgc tggcccgggt gaggccggcc atcgagtacg cgctgcgtag cgtggagggc
241 aatggcaccg ggaggaagct gctgccgcca ggcactcgct tccaggtggc
ctacgaagac 301 tcggactgcg gcaaccgcgc gctcttcagt cttgtggacc
gcgtggcggc ggcgcgcggc 361 gccaagccgg atctcatcct ggggcccgtg
tgcgggtacg cggcggcgcc ggtggctcgg 421 ctggcgtctc actgggacct
gccgatgctg tccgcaggag cgctggccgc cggtttccag 481 cacaaggaca
cggaatactc gcacctcacg cgcgtggcgc ctgcctacgc caagatggga 541
gagatgatgc tcgctctgtt tcgccaccac cactggagcc gtgcagccct ggtctacagc
601 gacgacaaac tcgagaggaa ctgttatttc accctcgagg gggtccacga
ggtttttcag 661 gaggaggggt tgcacacgtc tgcctacaat ttcgacgaga
ccaaagactt ggacctggac 721 gacatagtgc gctacatcca aggcagcgag
cgagtggtga tcatgtgtgc cagtggtgac 781 accattcgga gaatcatgtt
ggcggtgcac agacacggca tgaccagtgg agactacgct 841 ttcttcaaca
ttgaactctt caacagttct tcctacggag atggctcgtg gaggagagga 901
gacaaacacg actctgaagc taaacaagca tactcgtccc tccaaacagt caccctactg
961 aggaccgtga aacctgagtt tgagaagttt tccatggagg tgaaaagttc
tgttgagaaa 1021 caagggctca atgaggagga ttacgtgaac atgtttgttg
aagggttcca tgacgccatc 1081 ctcctctacg ttctggcttt acatgaagta
ctcagagctg gctacagcaa gaaggatggg 1141 gggaaaatca tccagcagac
ttggaacagg acatttgaag gtatcgccgg gcaggtgtcc 1201 atagatgcca
acggggaccg gtatggggac ttctctgtgg ttgctatgac tgacactgaa 1261
gcaggcaccc aagaggtcat tggtgattac tttgggaaag aaggccggtt ccaaatgcga
1321 tcgaatgtca aatatccttg gggccctttg aaactgagac tagatgagac
cagaatcgtg 1381 gagcatacca acagctctcc ttgcaaatca tcaggtggcc
tagaagaatc tgcagtgaca 1441 ggaatcgttg tcggggccct actaggtgct
ggcttgctaa tggccttcta ctttttcagg 1501 aagaaataca gaataaccat
tgagaggcga aatcagcaag aggaaagcaa catcgggaag 1561 catcgagagc
tgcgagaaga ttccatcaga tcacattttt cggtggctta a
The Genbank accession number for the human receptor (NPR3) is
NM.sub.--000908 [online], Genbank. Retreived from the Internet:
<URL:
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=nucleoti-
de&list_uids=4505440&dopt=GenBank The protein sequence is
as follows:
TABLE-US-00008 (SEQ. ID. NO. 778)
MPSLLVLTFSPCVLLGWALLAGGTGGGGVGGGGGGAGIGGGRQEREALP
PQKIEVLVLLPQDDSYLFSLTRVRPAIEYALRSVEGNGTGRRLLPPGTR
FQVAYEDSDCGNRALFSLVDRVAAARGAKPDLILGPVCEYAAAPVARLA
SHWDLPMLSAGALAAGFQHKDSEYSHLTRVAPAYAKMGEMMLALFRHHH
WSRAALVYSDDKLERNCYFTLEGVHEVFQEEGLHTSIYSFDETKDLDLE
DIVRNIQASERVVIMCASSDTIRSIMLVAHRHGMTSGDYAFFNIELFNS
SSYGDGSWKRGDKHDFEAKQAYSSLQTVTLLRTVKPEFEKFSMEVKSSV
EKQGLNMEDYVNMFVEGFHDAILLYVLALHEVLRAGYSKKDGGKIIQQT
WNRTFEGIAGQVSIDANGDRYGDFSVIAMTDVEAGTQEVIGDYFGKEGR
FEMRPNVKYPWGPLKLRIDENRIVEHTNSSPCKSCGLEESAVTGIVVGA
LLGAGLLMAFYFFRKKYRITIERRTQQEESNLGKHRELREDSIRSHFSV A
The DNA sequence is as follows:
TABLE-US-00009 (SEQ. ID. NO. 278) 1 ggggggcaga gggcgagtcg
gcggcggcga gggcaagctc tttcttgcgg cacgatgccg 61 tctctgctgg
tgctcacttt ctccccgtgc gtactactcg gctgggcgtt gctggccggc 121
ggcaccggtg gcggtggcgt tggcggcggc ggcggtggcg cgggcatagg cggcggacgc
181 caggagagag aggcgctgcc gccacagaag atcgaggtgc tggtgttact
gccccaggat 241 gactcgtact tgttttcact cacccgggtg cggccggcca
tcgagtatgc tctgcgcagc 301 gtggagggca acgggactgg gaggcggctt
ctgccgccgg gcactcgctt ccaggtggct 361 tacgaggatt cagactgtgg
gaaccgtgcg ctcttcagct tggtggaccg cgtggcggcg 421 gcgcggggcg
ccaagccaga ccttatcctg gggccagtgt gcgagtatgc agcagcgcca 481
gtggcccggc ttgcatcgca ctgggacctg cccatgctgt cggctggggc gctggccgct
541 ggcttccagc acaaggactc tgagtactcg cacctcacgc gcgtggcgcc
cgcctacgcc 601 aagatgggcg agatgatgct cgccctgttc cgccaccacc
actggagccg cgctgcactg 661 gtctacagcg acgacaagct ggagcggaac
tgctacttca ccctcgaggg ggtccacgag 721 gtcttccagg aggagggttt
gcacacgtcc atctacagtt tcgacgagac caaagacttg 781 gatctggaag
acatcgtgcg caatatccag gccagtgaga gagtggtgat catgtgtgcg 841
agcagtgaca ccatccggag catcatgctg gtggcgcaca ggcatggcat gaccagtgga
901 gactacgcct tcttcaacat tgagctcttc aacagctctt cctatggaga
tggctcatgg 961 aagagaggag acaaacacga ctttgaagct aagcaagcat
actcgtccct ccagacagtc 1021 actctactga ggacagtgaa acctgagttt
gagaagtttt ccatggaggt gaaaagttca 1081 gttgagaaac aagggctcaa
tatggaggat tacgttaaca tgtttgttga aggattccac 1141 gatgccatcc
tcctctacgt cttggctcta catgaagtac tcagagctgg ttacagcaaa 1201
aaggatggag ggaaaattat acagcagact tggaacagaa catttgaagg tatcgccggg
1261 caggtgtcca tagatgccaa cggagaccga tatggggatt tctctgtgat
tgccatgact 1321 gatgtggagg cgggcaccca ggaggttatt ggtgattatt
ttggaaaaga aggtcgtttt 1381 gaaatgcggc cgaatgtcaa atatccttgg
ggccctttaa aactgagaat agatgaaaac 1441 cgaattgtag agcatacaaa
cagctctccc tgcaaatcat gtggcctaga agaatcggca 1501 gtgacaggaa
ttgtcgtggg ggctttacta ggagctggct tgctaatggc cttctacttt 1561
ttcaggaaga aatacagaat aaccattgag aggcgaaccc agcaagaaga aagtaacctt
1621 ggaaaacatc gggaattacg ggaagattcc atcagatccc atttttcagt
agcttaaagg 1681 aagcccccca cttttttttt tctgcctgag attctttaag
gagatagacg ggttgaaaga 1741 catcaatgaa ac
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110288034A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110288034A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References