U.S. patent application number 11/924272 was filed with the patent office on 2009-12-24 for method of modifying glucose activity using polypeptides selectively expressed in fat tissue.
This patent application is currently assigned to UNIVERSITY OF MARYLAND, BALTIMORE. Invention is credited to DA-WEI GONG, JOHN MCLENITHAN, ALAN SHULDINER, RONGZE YANG.
Application Number | 20090317393 11/924272 |
Document ID | / |
Family ID | 33313293 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090317393 |
Kind Code |
A1 |
GONG; DA-WEI ; et
al. |
December 24, 2009 |
METHOD OF MODIFYING GLUCOSE ACTIVITY USING POLYPEPTIDES SELECTIVELY
EXPRESSED IN FAT TISSUE
Abstract
Isolated omentin polypeptides that selectively express in
omental fat tissue and methods for the use of the polypeptides. The
polypeptides can be used in a method for modifying insulin action
and/or glucose metabolism in an animal. The polypeptides can be
used to promote glucose uptake by animal adipocytes and other
cells, tissues, and/or organs. The polypeptides can also used to
provide a therapeutic treatment for diseases of or related to
glucose metabolism and adipose tissues. The polypeptides are also
incorporated into diagnostic tests and testing kits for diagnosing
or detecting a disease or condition involving animal tissue that
contains, uses, or expresses the polypeptide in an animal suspected
of having the disease or condition.
Inventors: |
GONG; DA-WEI; (OLNEY,
MD) ; MCLENITHAN; JOHN; (BALTIMORE, MD) ;
SHULDINER; ALAN; (COLUMBIA, MD) ; YANG; RONGZE;
(BALTIMORE, MD) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
UNIVERSITY OF MARYLAND,
BALTIMORE
BALTIMORE
MD
|
Family ID: |
33313293 |
Appl. No.: |
11/924272 |
Filed: |
October 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10785720 |
Feb 24, 2004 |
7312197 |
|
|
11924272 |
|
|
|
|
60449489 |
Feb 24, 2003 |
|
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Current U.S.
Class: |
424/139.1 |
Current CPC
Class: |
C07K 14/575 20130101;
A61K 38/1709 20130101; A61P 3/00 20180101; A61P 3/08 20180101 |
Class at
Publication: |
424/139.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Goverment Interests
GOVERNMENT INTEREST
[0002] This invention was made with government support under NIH
Grant Number DK57835, awarded by the National Institutes of Health.
The U.S. government has certain rights in this invention.
Claims
1. A method of decreasing glucose metabolism in an animal,
comprising interfering with an activity of the omentin polypeptide
of SEQ ID NO:1 or the omentin polypeptide of SEQ ID NO:3 in an
animal in need of treatment, wherein upon interfering with an
activity of said polypeptide, glucose metabolism in the animal is
decreased.
2. A method of decreasing insulin activity in an animal, comprising
interfering with an activity of the omentin polypeptide of SEQ ID
NO:1 or the omentin polypeptide of SEQ ID NO:3 in an animal in need
of treatment, wherein upon interfering with an activity of said
polypeptide, insulin activity in the animal is decreased.
3. A method of treating obesity or type 2 diabetes in an animal,
comprising interfering with an activity of the omentin polypeptide
of SEQ ID NO:1 or the omentin polypeptide of SEQ ID NO:3 in an
animal in need of treatment, wherein upon interfering with an
activity of said polypeptide, obesity or type 2 diabetes in the
animal is treated.
4. The method of claim 1, wherein the interfering comprises
administering a molecule that inhibits a signaling activity of the
omentin polypeptide.
5. The method of claim 2, wherein the interfering comprises
administering a molecule that inhibits a signaling activity of the
omentin polypeptide.
6. The method of claim 3, wherein the interfering comprises
administering a molecule that inhibits a signaling activity of the
omentin polypeptide.
7. The method of claim 1, wherein the interfering comprises
administering a molecule that inhibits a metabolic activity of the
omentin polypeptide.
8. The method of claim 2, wherein the interfering comprises
administering a molecule that inhibits a metabolic activity of the
omentin polypeptide.
9. The method of claim 3, wherein the interfering comprises
administering a molecule that inhibits a metabolic activity of the
omentin polypeptide.
10. The method of claim 4, wherein the molecule inhibits binding of
omentin to a receptor.
11. The method of claim 5, wherein the molecule inhibits binding of
omentin to a receptor.
12. The method of claim 6, wherein the molecule inhibits binding of
omentin to a receptor.
13. The method of claim 4, wherein the molecule is an anti-omentin
antibody.
14. The method of claim 5, wherein the molecule is an anti-omentin
antibody.
15. The method of claim 6, wherein the molecule is an anti-omentin
antibody.
16. The method of claim 7, wherein the molecule is an anti-omentin
antibody.
17. The method of claim 8, wherein the molecule is an anti-omentin
antibody.
18. The method of claim 9, wherein the molecule is an anti-omentin
antibody.
19. The method of claim 1, wherein the activity of the omentin
polypeptide of SEQ ID NO:1 or the omentin polypeptide of SEQ ID
NO:3 is a signaling activity or a metabolic activity.
20. The method of claim 2, wherein the activity of the omentin
polypeptide of SEQ ID NO:1 or the omentin polypeptide of SEQ ID
NO:3 is a signaling activity or a metabolic activity.
21. The method of claim 3, wherein the activity of the omentin
polypeptide of SEQ ID NO:1 or the omentin polypeptide of SEQ ID
NO:3 is a signaling activity or a metabolic activity.
22. A method of decreasing glucose metabolism in an animal,
comprising administering to an animal in need of treatment a
molecule that inhibits a signaling activity of the omentin
polypeptide of SEQ ID NO:1 or the omentin polypeptide of SEQ ID
NO:3, wherein upon inhibition of omentin polypeptide signaling
activity, glucose metabolism in the animal is decreased.
23. A method of decreasing glucose metabolism in an animal,
comprising administering to an animal in need of treatment a
molecule that inhibits a metabolic activity of the omentin
polypeptide of SEQ ID NO:1 or the omentin polypeptide of SEQ ID
NO:3, wherein upon inhibition of omentin polypeptide metabolic
activity, glucose metabolism in the animal is decreased.
24. The method of claim 10, wherein the molecule is an anti-omentin
antibody.
25. The method of claim 11, wherein the molecule is an anti-omentin
antibody.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit as a divisional of U.S.
patent application Ser. No. 10/785,720, filed Feb. 24, 2004, which
in turn claims benefit of U.S. provisional patent application No.
60/449,489, filed Feb. 24, 2003, both of which are hereby
incorporated by reference in their entireties.
FIELD OF INVENTION
[0003] The present invention relates to isolated polypeptides that
selectively express in fat tissue, as well as methods for use of
the polypeptides.
BACKGROUND OF THE INVENTION
[0004] Obesity affects a growing number of the U.S. population and
often is closely associated with insulin resistance, type 2
diabetes, cardiovascular disease, and dyslipidemia. Obesity itself
is typically a heterogeneous condition, due to regional
distribution of fat tissue. Central obesity generally refers to fat
accumulation in omental or visceral cavity, whereas peripheral
obesity generally refers the subcutaneous fat accumulation.
Epidemiological studies have established that central obesity is
associated with a higher degree of risk than peripheral obesity to
the above-mentioned diseases, however, the underlying mechanism(s)
are generally not well understood. Presumably, distinctive
biological properties of omental fat, in addition to its unique
anatomical location, contribute to the increased pathogenecity of
central obesity. It has been discovered that an excess of cortisol
can cause central obesity and that treatment of HIV patients with
protease inhibitor can lead to accumulation of omental fat
accumulation but depletion of subcutaneous fat. In vitro studies
have also demonstrated that abdominal visceral fat pads can be
relatively resistant to the anti-lipolytic effect of insulin and
susceptible to the lipolytic effect of catecholamine. At a
molecular level, omental fat has been shown to have increased gene
expression or secretion of interleukin 6, plasminogen activator
inhibitor (PAI-1), and angiotensinogen, compared to subcutaneous
fat. These observations indicate the existence of biological
difference between omental and subcutaneous fat depots.
[0005] Adipose, i.e., fat, tissue plays a critical role in the
pathogenesis of obesity and its associated diseases, but the
molecular mechanisms for these associations generally remain
unclear. Adipose tissue is generally recognized as an important
endocrine organ that communicates actively with the central nervous
system and other peripheral tissues through the release of a
variety of bioactive factors that regulate glucose and lipid
homeostasis. These factors, collectively known as adipocytokines,
include leptin, tumor-necrosis factor .alpha. (TNF.alpha.),
plasminogen activator inhibitor-1 (PAI-1),
adiponectin/ACRP30/adipoQ, and resistin. Adipocytokines have been
demonstrated to play a key role in the pathogenesis of obesity and
its associated diseases. Nevertheless, current knowledge of known
genes generally cannot fully explain the pathophysiology of
obesity, and effective treatment for these diseases is still
lacking.
[0006] Adipose tissue also plays a central role in energy
homeostasis. Its primary function is to store and mobilize energy
in the form of triglycerides in response to caloric excess and
deprivation, respectively. Adipose tissue can be divided into white
adipose tissue (WAT) and brown adipose tissue (BAT). WAT and BAT
are different in morphology and biological function; WAT is
monolocular and mainly functions in storing triglyceride, while BAT
is multilocular, rich in mitochondria, and designed to burn energy.
BAT is mainly present in hibernating animals, rodents and new-born
humans, indicating evolutional adaptation of adipose tissue to
environment. Because the significance of the BAT to adult
physiology is relatively not clear, references herein to adipose
tissue or fat cells generally refer to WAT. Central obesity
generally refers to intra-abdominal fat accumulation in visceral or
omental adipose tissues, although, strictly speaking, omental fat
is a subset of visceral fat.
[0007] Obesity is due to the excess accumulation of triglyceride in
intra-abdominal (omental) and subcutaneous adipose tissue. Studies
of animal models have provided some mechanistic understandings of
obesity, likely a communication disorder between the central
nervous system and the peripheral tissues, particularly adipose
tissue. In mice, certain single gene mutations, such as db/db
(leptin receptor mutation) and A.sup.y (the agouti protein is
homologous to melanocyte stimulating hormone), can cause obesity,
suggesting that a defect involving the central nervous system is
the cause for the obesity in these animals. Leptin mutations also
can cause obesity in mice (ob/ob), and in humans. However, obesity
caused by single gene mutations in humans is rare, and obesity in
the general population is thought to be of polygenic origin.
[0008] The association of type 2 diabetes with obesity has been
observed for a long time. Evidence from epidemiological, clinical
and experimental studies has demonstrated that obesity is generally
the greatest risk factor for insulin resistance and type 2 diabetes
and, moreover, visceral obesity is associated with a higher degree
of risk than peripheral obesity. The mechanism for the close
association is generally not well understood, but it is generally
accepted that an excess of fat leads to increasing insulin
resistance and/or impaired glucose disposal, which can predispose
someone to type 2 diabetes. The pancreas, liver, muscle, fat
tissue, and central nervous system are the principal organs
involved in regulating glucose and fat metabolism and are likely to
participate in the pathogenesis of obesity and type 2 diabetes.
However, recent experimental studies indicate that fat tissue can
play a relatively major role in the etiology of type 2 diabetes.
For example, surgical excision of visceral fat tissue in the rat
has been shown to increase insulin sensitivity suggesting that
excess fat is a causative factor for type 2 diabetes. In addition,
lipodystrophic patients and fat-depleted mice have developed
hyperinsulinemia and type 2 diabetes, and surgical implantation of
adipose tissue reverses the diabetic phenotype. Also, adipocyte
size may be a determinant of body insulin sensitivity, as it has
been proposed that small adipocytes confer insulin sensitivity
while large ones result in insulin resistance. These and other
studies strongly support the premise that adipose tissue can play a
central role in the regulation of insulin sensitivity, and in the
pathogenesis of type 2 diabetes.
[0009] As discussed above, fat cells generally play an active role
in energy storage, fatty acid metabolism and glucose homeostasis.
To perform this specialized function, the adipocyte expresses a
special subset of genes to communicate with the central nervous
system and peripheral tissues, and to respond to various neuronal,
metabolic and hormonal signals. The adipocyte secretes a number of
bioactive substances, collectively known as adipocytokines, such
as, for example, leptin, TNF.alpha., PAI-1, adiponectin, and
resistin. These adipocytokines function as endocrine, paracrine,
and autocrine factors, and have been implicated in obesity and its
associated diseases. Some of these adipocytokines are discussed
briefly below.
[0010] Leptin is a hormone secreted from fat tissue into the
circulation that acts to reduce food intake and increase energy
expenditure mainly through binding to leptin receptors in the
hypothalamus. Leptin secretion is regulated by the energy supply;
starvation decreases its expression and secretion, while
overfeeding or increased adiposity induces leptin expression.
Leptin is therefore a key molecule linking this adipose tissue to
the central nervous system and regulating energy homeostasis.
[0011] Tumor necrosis factor-alpha (TNF.alpha.) is a cytokine
produced not only by inflammatory cells but also by adipocytes.
TNF.alpha. expression has been shown to be elevated in the fat
tissue of obese animals and humans. TNF.alpha. appears to induce
insulin resistance by interfering directly and/or indirectly with
insulin signaling pathways in an autocrine or paracrine fashion.
The absence of TNF.alpha. results in significantly improved insulin
sensitivity in obese mice, the mice lacking TNF.alpha. receptors
appears protected against diabetes to a certain degree, implying
that there might be a yet uncharacterized pathway involved in
TNF.alpha.-induced insulin resistance.
[0012] Plasminogen activator inhibitor-1 (PAI-1) is a key
pathogenic factor for thrombotic vascular disease. Plasma PAI-1
levels are closely correlated with visceral fat, and gene
expression is highly elevated in visceral fat during the
development of obesity. TNF.alpha. has been shown to induce adipose
PAI-1 expression, providing a possible explanation for the
association of obesity with cardiovascular disease.
[0013] Adiponectin is a hormone secreted exclusively from adipose
tissue and is also referred to as ACRP30, AdipoQ, apM1, or GBP28.
Adiponectin has been demonstrated to have promising activities
potentially for the treatment of obesity and diabetes. Its
expression is reduced in the states of obesity and type 2 diabetes,
and its replenishment improves insulin sensitivity and prevents
diet-induced obesity in rodents, probably by increasing fat
oxidation and decreasing triglyceride content in muscle and liver.
This effect can result from increased expression of molecules
involved in both fatty-acid combustion and energy dissipation in
muscle. The mechanisms for these actions are generally not clear.
Adiponectin consists of collagenous repeats and a globular domain
homologous to complement C1q, and shares structural similarity to
TNF.alpha.. Interestingly, PPAR.gamma. induces, whereas TNF.alpha.
suppresses, the expression and secretion of adiponectin, suggesting
that adiponectin may be a target molecule relaying insulin
sensitivity.
[0014] Resistin is a hormone typically isolated from differentiated
3T3-L1 adipocytes by screening for genes regulated by the
PPAR.gamma. agonist rosiglitazone. During adipocyte
differentiation, resistin is increasingly expressed but is
suppressed by treatment with rosiglitazone. Moreover, ob/ob mice
secrete increased amounts of resistin, and recombinant resistin
induces insulin resistance. Resistin has therefore been proposed to
be a link between obesity and insulin resistance. However,
conflicting results have been reported, in which resistin
expression was reduced in several obese animal models and was
induced by PPAR.gamma. agonists. In addition, unlike the high
expression of mouse resistin in adipose tissue, the expression of
the human counterpart is very low.
[0015] There are additional adipocyte-specific/abundant genes, such
as is adipsin and angiotensin, acylation stimulating protein, PGAR,
and interleukin-6, whose functions in obesity and type 2 diabetes
are generally less understood. Nevertheless, the discovery of a
myriad of adipose secreted factors has generally established
adipose tissue as an endocrine organ. The dysregulation of adipose
tissues autocrine, paracrine and endocrine function is likely to
disturb energy homeostasis and lead to obesity, type 2 diabetes,
dyslipidemia and hypertension.
[0016] Abdominal fat is generally more pathogenic than subcutaneous
fat. An obvious explanation for this may simply relate to its
anatomical location. Visceral adipose tissue drains via the portal
venous system, such that liver is fully exposed to and functionally
affected by bioactive substances released from this depot. In
addition, differences in physiology, biochemistry and gene
expression have been observed between omental and subcutaneous fat
tissues. Abdominal obesity is predominant in males whereas
subcutaneous fat mass is mostly involved in female obesity,
indicating that sex hormones may play a role in these differences.
Moreover, an excess of cortisol is known to cause central obesity.
Finally, a selective increase in visceral fat is a common feature
of aging. It has been suggested that these two adipose tissue
depots differ in important ways. Omental adipose fat is more
metabolically active with respect to lipolysis and lipogenesis.
Compared to subcutaneous fat, abdominal fat pads have greater
secretion of interleukin 6, plasminogen activator inhibitor
(PAI-1), angiotensinogen, and the rate of apoptosis is greater. In
contrast, leptin expression is higher in subcutaneous fat tissue
than omental fat tissue. Yet, whether these changes discussed above
can explain features of insulin resistance syndrome generally
remains unclear. Because the pathophysiological basis of this
syndrome is likely to be complex, several genes/gene products and
pathways may participate in the disease process.
[0017] Insulin signaling is a complex and coordinated process
involving protein modification, translocation, and
compartmentalization. Insulin action is initiated through binding
of insulin to the .alpha. subunit of insulin receptor (IR), which
activates the beta subunit intrinsic receptor tyrosine kinase,
resulting in autophosphorylation of insulin receptor .beta. subunit
and tyrosine phosphorylation of intracellular target proteins such
as IR substrates (IRS-1-4) and Shc, Cb1, Gab-1. Three major
signaling pathways are initiated by these intracellular targets: 1)
IRS/PI 3-kinase/Akt; 2) CAP/Cb1; and 3) Shc(or Gab)/Ras/MAP
kinase.
[0018] In the first major pathway, tyrosine-phosphorylated IRS-1 or
IRS-2 binds to src-homology 2 domains of intracellular proteins,
including p85, a regulatory subunit of phosphatidylinositol
3-kinase (PI 3-kinase). The interaction of IRS and p85 subunits
results in the activation of the p110 catalytic subunit of PI
3-kinase, which raises phosphatidylinositol 3,4-bisphosphate and
phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels. These
second messengers activate phosphoinositide-dependent kinase-1
(PDK-1) to phosphorylate and hence activate Akt (also called
protein kinase B) and atypical PKC isozymes.
[0019] In the second major pathway, c-Cbl-associated protein (CAP)
recruits c-Cbl to the insulin receptor where it is phosphorylated.
This protein complex subsequently localizes to lipid raft domains
of the plasma membrane called caveola. The SH2-containing adapter
protein CRKII and C3G, a guanine nucleotide exchange factor, are
then targeted to phosphorylated c-Cbl at the lipid raft. C3G may
activate TC 10, a G-protein of the rho family, which is expressed
in adipose and muscle tissue. The IRS/PI 3-kinase/Akt and CAP/Cbl
pathways are generally believed to function in concert to
upregulate glucose transport in response to insulin.
[0020] The third major pathway involves the activation of the
p42/44 MAP kinase (mitogen activated protein kinase) cascade.
Insulin receptor phosphorylation of both Shc and Gab-1 adaptor
proteins leads to Ras activation of multiple kinases resulting in
activation of MAP kinase (Erk1 and 2). This pathway is more
involved in the mitogenic function of insulin.
[0021] Many other factors interact with and modify the efficiency
of insulin signaling in a positive or negative manner, which
include protein kinases, e.g., AMP-activated kinase, protein kinase
C, and IKK.beta., phosphatases, e.g., PTP1B, SHIP2, PTEN, and
modulators of IR activity, e.g., PC-1.
[0022] There is a need for methods of detecting and treating
diseases of or relating to adipose tissue and glucose metabolism,
such as obesity and type 2 diabetes.
SUMMARY OF THE INVENTION
[0023] An object of this invention is to provide a method for
modifying insulin action and/or glucose metabolism in an animal,
such as, for example, a human.
[0024] Another object of this invention is to provide a method for
inducing glucose uptake by animal adipocytes.
[0025] An additional object of this invention is to provide a
therapeutic treatment for diseases of or related to metabolism
and/or adipose tissues.
[0026] Yet another object of this invention is to provide a method
of diagnosing or detecting a disease or condition involving animal
tissue that contains, uses, or expresses omentin polypeptide in an
animal suspected of having the disease or condition.
[0027] Yet another object of this invention is to provide a method
and/or a diagnostic kit for detecting a polypeptide specific to
particular fat tissues, such as omental fat tissue, in bodily
fluids of an animal.
[0028] One object of the invention can be attained, at least in
part, through a method of modifying at least one of insulin action
and glucose metabolism in an animal. The method includes modifying
the amount of omentin polypeptide in the animal. The amount of
active omentin polypeptide can be increased in the animal, such as
by administering omentin polypeptide to the animal, or decreased in
the animal by interfering with the metabolic function of at least a
portion of the omentin polypeptide in the animal. In one embodiment
of this invention, the amount of omentin polypeptide in the animal
is first determined before any modification.
[0029] The invention further comprehends a method of detecting
omentin polypeptide in bodily fluids of an animal. The method
includes contacting a sample of the bodily fluids with at least one
antibody that specifically binds to the omentin polypeptide. The
antibody bound to the omentin polypeptide in the sample is then
detected.
[0030] The invention still further comprehends a method of
diagnosing or detecting a disease or condition involving animal
tissue that contains, uses, or expresses omentin polypeptide in an
animal suspected of having the disease or condition. The method
includes first contacting a sample of bodily fluid from the animal
with a plurality of antibodies adapted to specifically bind omentin
polypeptide. The antibody bound to omentin polypeptide in the
sample is detected and an amount of omentin polypeptide in the
bodily fluid is measured. The amount of omentin polypeptide is
compared to a control to diagnose or detect the disease or
condition.
[0031] The invention still further comprehends a diagnostic kit for
use in diagnosing damage, a condition, or disease in tissue
containing or expressing omentin polypeptide. The diagnostic kit
includes a measurer of an amount of omentin polypeptide in a sample
of bodily fluids and an indicator for determining if a measurement
taken by the measurer is in a predetermined range associated with
damage, a condition, or disease in the tissue.
[0032] The invention still further comprehends a method of inducing
glucose uptake by animal cells, tissues, and/or organs, such as,
for example, adipocytes or adipose tissue. The method includes
administering an omentin polypeptide to at least one of the animal
and the adipocytes. The administered omentin polypeptide can
enhance insulin-mediated glucose transport in the adipocytes and/or
activate, either directly or indirectly, the polypeptide kinase
Akt/PKB, also referred to herein as "Akt kinase."
[0033] As discussed above, obesity is a heterogeneous condition and
can be divided into central (omental or visceral) obesity and
peripheral (subcutaneous) obesity, based on the location of fat
accumulation. Central obesity is more closely associated with
insulin resistance, type 2 diabetes and cardiovascular disease than
peripheral obesity, but the underlying mechanisms are generally not
known, presumably due to the biological and anatomical difference
between the two fat depots. In this invention, fat depot-specific
secretory factors have been identified, such as, for example,
proteins having the amino acid sequences of SEQ ID NO:1 and SEQ ID
NO:3, as well as variants thereof, generally referred to herein as
"omentin" or "omentin polypeptides." Omentin polypeptides are
expressed in the stromal vascular cells derived from omental, but
generally not subcutaneous, adipose tissue. Omentin polypeptides
enhance insulin-mediated glucose uptake by adipocytes in vitro and
improve glucose disposal in vivo. Furthermore, omentin polypeptides
activate Akt kinase, both alone and synergistically with insulin.
Omentin polypeptides are also detectable in human blood.
[0034] Insulin is a pleiotropic hormone with a broad spectrum of
biological functions. In particular, it plays a vital role in
regulating glucose, lipid and protein metabolism. The maintenance
of glucose homeostasis depends on a precise balance between the
release of insulin from the pancreas, glucose production from the
liver, and insulin-stimulated glucose transport by muscle and
adipose tissue. A number of biological factors can alter insulin
sensitivity via different mechanisms. Adipocytokines have been
demonstrated to either positively, e.g., leptin and adiponectin, or
negatively, e.g., TNF.alpha., modulate insulin sensitivity. Omentin
polypeptides can enhance insulin-stimulated glucose in 3T3-L1
adipocytes.
[0035] As discussed above, adipocytokines generally play a role in
regulating energy metabolism and insulin action. Omentin
polypeptides are physiological regulators in this regard as well.
Omentin polypeptides are secretory factors from stromal vascular
cells in the omental depot and are detectable in human blood.
Omentin polypeptides are biologically active in enhancing insulin
action in vitro and in vivo. Therefore, the omentin polypeptide is
an adipokine that can regulate the adipose biology in a
depot-dependent manner.
[0036] In identifying and purifying omentin polypeptides, 10,437
expressed sequence tags (EST) from a human omental fat library were
sequenced. Bioinformatics analysis revealed that one frequently
sequenced EST was a potential secretory factor and Northern
analyses revealed that this EST was expressed in omental, but not
in subcutaneous, adipose tissue both in humans and Rhesus monkeys.
In one embodiment of this invention, the omentin polypeptide is 313
amino acids in length. Omentin polypeptides are secreted proteins
when expressed in mammalian cells. In addition, omentin polypeptide
can be detected in human blood by Western blotting.
Immunofluorescence microscopy demonstrates that omentin
polypeptides are generally expressed by stromal vascular cells in
omental fat.
[0037] In one embodiment of this invention, omentin polypeptides
enhance insulin-mediated glucose transport in 3T3-L1 adipocytes. In
another embodiment of this invention, omentin polypeptides activate
the protein kinase Akt/PKB, both in the presence and absence of
insulin. Omentin polypeptides also stimulate insulin-mediated
glucose transport and Akt phosphorylation in vitro. This activity
is believed to explain, at least in part, why the omental adipose
tissue continues to accumulate fat despite systemic insulin
resistance in obesity. Without intending to be bound by theory, it
is believed that omentin polypeptides sensitize adipocytes to
insulin by activating components of the insulin signaling pathway
and/or inhibiting negative regulators of the pathway. Furthermore,
omentin polypeptide levels can be correlated with visceral fat,
increased glucose disposal, and increased prevalence of type 2
diabetes.
[0038] In one embodiment of this invention, the gene encoding the
omentin polypeptide provides a positional candidate gene for type 2
diabetes because this gene localizes to a region on chromosome 1q22
that has been linked to type 2 diabetes susceptibility in the Old
Order Amish and in at least four other populations
independently.
[0039] As used herein, the terms "omentin," "omentin protein," or
"omentin polypeptide" generally refer to a polypeptide having an
amino acid sequence with at least 70% identity, preferably at least
80% identity, more preferably at least 90% identity, and desirably
at least 95% identity, to the amino acid sequence of either SEQ ID
NO:1 or SEQ ID NO:3.
[0040] As used herein, the term "animal" is intended to include
humans.
[0041] Other objects and advantages will be apparent to those
skilled in the art from the following detailed description taken in
conjunction with the appended claims.
DESCRIPTION OF THE DRAWINGS
[0042] FIGS. 1A and 1B are blots from Northern analyses. FIG. 1A is
a multiple tissue Northern analysis blot from a human. FIG. 1B is a
Northern blot of adipose tissues from omental and subcutaneous fat
depots of five individual humans.
[0043] FIGS. 2A and 2B are blots from Northern analyses. FIG. 2A is
a Northern blot of paired omental (O) adipose tissue and inguinal
subcutaneous (S) adipose tissue from five individual Rhesus
monkeys. FIG. 2B is a Northern blot that shows regional differences
in omentin expression in a single Rhesus monkey.
[0044] FIG. 3 is a schematic structure of the omentin polypeptide
of SEQ ID NO:1.
[0045] FIG. 4A is a structural representation of a His (6)-tagged
omentin polypeptide cDNA in a PET-28 plasmid.
[0046] FIG. 4B is a Coomassie Blue stain of bacterial cell lysates
expressing recombinant human omentin polypeptide.
[0047] FIG. 4C is a Western blot of recombinant human omentin
polypeptide.
[0048] FIG. 5 is a chromosome map showing the location of the
omentin gene by its peak linkage to diabetes phenotypes in
chromosome 1q21-q23.
[0049] FIG. 6 is an immunoblot of proteins from conditioned medium
and cell lysate of example HEK-293 cells stably transfected with an
omentin-F vector (+).
[0050] FIG. 7 is a Western blot of three plasma samples
immunoprecipitated with omentin antibodies ("Om") and pre-immune
antibodies ("Cont").
[0051] FIGS. 8A-D are photographs of immunofluorescent stained fat
tissue. FIG. 8A is omental tissue treated with pre-immune antibody.
FIG. 8B is omental tissue treated with omentin antibody. FIG. 8C is
subcutaneous tissue treated with omentin antibody. FIG. 8D is
omental tissue treated with omentin antibody shown at a greater
magnification (200.times.) than the sample of FIG. 8B
(100.times.).
[0052] FIG. 9 is a graphical representation of the results of a
glucose transport assay.
[0053] FIG. 10 is a graphical representation of the results of a
glucose to transport assay.
[0054] FIG. 11 is a Western blot showing Akt kinase phosphorylation
by acute and chronic exposure to omentin-F.
[0055] FIG. 12 is a graphical representation of the results of an
in vivo glucose transport test.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0056] The present invention relates to polypeptides, referred to
herein generally as omentin or omentin polypeptide, selectively
expressed in human and other animal omental fat depots, and
generally not in subcutaneous fat depots, as well as methods of use
of the polypeptides.
[0057] In one embodiment of the invention, the omentin polypeptide
includes an amino acid sequence of SEQ ID NO:1. The nucleotide
sequence for the complimentary DNA (cDNA) of SEQ ID NO:1 is SEQ ID
NO:2. The present invention also includes variants of the
polypeptide of SEQ ID NO:1, i.e., polypeptides that vary by
conservative amino acid substitutions, whereby a residue is
substituted by another with like characteristics.
[0058] In one embodiment of this invention, the variant
polypeptides for use in the methods of this invention have at least
70% identity, preferably at least 80% identity, more preferably at
least 85% identity, more preferably at least 90% identity, and
desirably at least 95% identity, to the amino acid sequence of SEQ
ID NO:1, over the entire length of SEQ ID NO:1. One preferred
homolog of this invention and usable in the methods of this
invention is a polypeptide including the amino acid sequence of SEQ
ID NO:3. The polypeptide of SEQ ID NO:3 has about 86% similarity
and about 84% identity with the polypeptide of SEQ ID NO:1. The
nucleotide sequence for the complimentary DNA (cDNA) of SEQ ID NO:3
is SEQ ID NO:4.
[0059] Polypeptides for use in the methods of this invention can be
prepared in any suitable manner. Such polypeptides include isolated
naturally occurring polypeptides, recombinantly produced
polypeptides, synthetically produced polypeptides, and/or
polypeptides produced by a combination of these methods. As will be
appreciated by one skilled in the art following the teachings
herein provided, various means for preparing such polypeptides are
available in the art.
[0060] The omentin polypeptide of this invention was isolated and
purified from adipose, i.e., fat, tissue. Messenger RNA encoding
omentin polypeptide is relatively highly and selectively expressed
in omental fat tissue. FIG. 1A shows the results of a multiple
tissue Northern analysis blot from a human. As shown in the
Northern blot of FIG. 1A, messenger RNA encoding omentin
polypeptide is selectively expressed in omental fat tissue, much
less in lung and heart, and not at all in muscle, liver, kidney,
breast, and brain. FIG. 1B is a Northern blot of adipose tissues
from omental and subcutaneous depots of five individual humans. As
shown in the Northern blot of FIG. 1B, messenger RNA encoding
omentin polypeptide is relatively highly expressed in omental fat,
but not in subcutaneous fat. In the Northern analysis represented
in FIG. 1B, the blot was reprobed with leptin, showing that leptin
is preferentially expressed in subcutaneous fat, and generally not
in omental fat, as is generally known in the art. For the Northern
analyses resulting in FIGS. 1A and 1B, adipose RNA was prepared
with TRIZOL, available from Invitrogen, Carlsbad, Calif., from
human tissue. The other RNA samples were obtained from Clontech,
Palo Alto, Calif. The Northern analysis was performed by loading 15
.mu.g of total RNA per lane. The human omentin probe, corresponding
to bases 263 to 1270 of AY549722 in GenBank, was random-labeled,
available from Stratagene, La Jolla, Calif., with .sup.32P-dCTP.
Hybridization was carried out at 65.degree. C. in Rapid-hyb buffer,
available from Amersham Biosciences, Piscataway, N.J., and blots
were washed twice with 0.5.times.SSC/1% SDS at 65.degree. C. The
RNA loadings were revealed by ethidium bromide staining.
[0061] To eliminate the possibility of individual variations in
gene expression among different subjects, Northern analysis was
also conducted using samples from Rhesus monkeys in the manner
described above. FIG. 2A is a Northern blot of paired omental (O)
adipose tissue and inguinal subcutaneous (S) adipose tissue from
five individual Rhesus monkeys. As shown in the Northern blot of
FIG. 2A, messenger RNA encoding omentin polypeptide was
predominantly expressed in omental fat compared to subcutaneous fat
from the same animals. FIG. 2B is a Northern blot that shows
regional differences in omentin expression in a single Rhesus
monkey. To help rule out concern of potential contamination from
omental structures other than omental fat, adipose tissues from
different regions were obtained from a single monkey for Northern
analysis. As shown in FIG. 2B, omentin is expressed in
intrathoracic as well as in omental fat tissue, but not in the
subcutaneous fat tissues from neck and inguina, confirming the
visceral pattern of omentin expression.
[0062] The omentin polypeptide of SEQ ID NO:1 contains 313 amino
acids. The estimated molecular weight is 35 kDa. FIG. 3 is a
schematic structure of the omentin polypeptide of SEQ ID NO:1. The
N-terminal portion (the end at amino acid 1) of omentin
polypeptides contain a hydrophobic region that is typical of a
signal sequence for protein secretion, followed by a region
homologous to a fibrinogen-related domain (FBG). Such domains are
globular structures found in proteins, such as the .beta. and
.gamma. chains of fibrinogen, PGAR(PPAR.gamma. angiopoietin
related), and tenasin. Full-length clones of omentin were obtained
from EST sequencing of an omental fat cDNA library, Cat# HL-5028t,
available from Clontech. The EST clone was used as a template for
PCR amplification of protein-coding regions with appropriate
primers. The resultant PCR products were subcloned into proper
expression vectors and verified without mutation by sequencing.
[0063] In one embodiment of this invention, omentin polypeptide is
expressed using a His (6)-tagged omentin cDNA in a bacteria host
cell as shown in FIG. 4A. Omentin polypeptide without the signal
peptide was cloned into a PET28 vector, available from Novagen, San
Diego, Calif., under the control of the T7 promoter. The target
gene was introduced into Tuner (DE3) pLacI cells, expressed by IPTG
induction, and purified with a nickel column. The
omentin-containing bacteria were grown with (+) or without (-) IPTG
induction. Cell lysates, shown in the first two lanes of FIG. 4B,
and purified protein, shown in lane 3 of FIG. 4C, were analyzed on
4-20% SDS-PAGE and stained with Coomassie Blue.
[0064] In one embodiment of this invention, antibodies are formed
from the omentin polypeptide. As will be appreciated by one skilled
in the art following the teachings herein provided, various methods
are available in the art for forming antibodies. For antibody
production, cDNA encoding amino acids 73-313 of SEQ ID NO:1 was
amplified with primer 1120 and 1121 using high-fidelity PCR system,
available from Boehringer Mannheim, Mannheim, Germany, and
subcloned into a PET28 vector, available from Novagen, to create
the plasmid G6329, which was verified by sequencing and transformed
into Escherichia coli, available from Novagen. The His(6)-tagged
protein was over-expressed by IPTG induction (Yang R. Z., Blaileanu
G., Hansen B. C., Shuldiner A. R., and Gong D. W., cDNA cloning,
genomic structure, chromosomal mapping, and functional expression
of a novel human alanine aminotransferase, Genomics, 2002 March;
79(3):445-50, herein incorporated by reference in its entirety),
and purified with Ni2+-NTA resin, available from QIAGEN Inc.,
Valencia, Calif., under denaturing condition using 8M urea for
polyclonal antibody production in rabbits and monoclonal antibody
production by AnaSpec, San Jose, Calif.
[0065] A representative immunoblot utilizing antibodies obtained in
this manner is shown in FIG. 4C. The indicated amount of
recombinant omentin polypeptide was blotted to a nitrocellulose
membrane and probed with omentin antiserum or pre-immune serum,
followed by a second antibody, such as alkaline phosphatase-labeled
goat against rabbit IgG, stained with BCIP/NBT. A clear induction
of a recombinant protein was observed at the expected size (about
35 kd) and purified to homogeneity. As shown in FIG. 4C, at about a
1 to 5,000 dilution factor, the antiserum reacts with recombinant
omentin polypeptide in a dose-dependent manner, whereas the
pre-immune serum shows no staining.
[0066] The gene encoding omentin polypeptides is localized on a
region of human chromosomes generally known to be linked to type 2
diabetes. The omentin gene consists of eight exons and localizes to
chromosome 1q22 at 160.1 cM, close to STS marker SHGC-31641. This
region of human chromosomes has been linked with type 2 diabetes.
FIG. 5 shows, by the arrow, the location of the omentin gene
relative to the peak linkage signal at 1q21-q23, known to be linked
to diabetes in the Old Order Amish population. As will appreciated
by one skilled in the art reviewing FIG. 5, evidence for linkage in
this region is stronger for the combined trait of diabetes and
impaired glucose homeostasis (lod=2.4) than for diabetes alone
(lod=0.92). This region on chromosome 1 is known to represent the
most strongly replicated linkage peak for type 2 diabetes. Lod
scores reported from other studies corresponding to this region
range from 4.3 in Utah Mormons, to 3.0 in collection of French
families, to 2.5 in Pima Indians.
[0067] As discussed above, omentin polypeptide is a secretory
protein. Bacteria-expressed omentin polypeptide is generally
insoluble and renaturing efforts generally failed to recover the
protein in a soluble fraction. In one embodiment of the invention,
to obtain the soluble protein, a flag peptide sequence is tagged to
the coding region of omentin polypeptide at the carboxy terminus by
polymerase chain reaction (PCR). The PCR product is subcloned into
a mammalian expression vector, such as, for example, pcDNA3,
available from Invitrogene Corporation, Carlsbad, Calif., driven by
a CMV promoter. The resultant plasmid, referred to as omentin-F, is
confirmed by sequencing and used to transiently transfect HEK-293T
cells using Transfectamine Plus, available from Invitrogene
Corporation. The cells are grown in DMEM with 10% FBS. The culture
medium was collected and cells are lysed 48 hours after
transfection for immunoprecipitation. The fractions are
immunoprecipitated with anti-flag M2 affinity beads, available from
Sigma-Aldrich, St. Louis, Mo., and blotted with omentin antibody.
As a result, omentin-F is detected in both culture media and cell
lystate from the cells transfected with omentin-F plasmid, but not
from empty vector-transfected control cell. FIG. 6 is an immunoblot
of proteins from conditioned medium and cell lysate of example
HEK-293 stable transfected with an omentin-F vector (+) according
to the above described method, or an empty vector (-). Culture
medium, in the amount of about 5 milliliters, and one third of the
cell lysates from a 10 centimeter dish were immunoprecipitated with
M2-Flag antibody beads. The precipitates were separated on 10%
SDS-polyacrylamide gel electrophoresis, immunoblotted with omentin
antibodies, and detected with ECL, available from Amersham
Biosciences.
[0068] Omentin polypeptide was detected in human plasma by
immunoprecipitating 3 milliliters of each of three individual
plasmas. The three plasma samples were immunoprecipitated with
omentin antibodies ("Om") or pre-immune antibodies ("Cont"). The
precipitates were analyzed by Western blotting using omentin
antiserum or pre-immune serum. FIG. 7 is a Western blot showing the
results of the analysis. The arrow indicates the specific band at
the expected size (about 38 Kd) of omentin. Cell lysates or
immunoprecipitates were separated by SDS-PAGE using 4-20%
polyacrylamide gels, available from Gradipore Inc., Hawthorne, N.Y.
Following electrophoresis, proteins were transferred onto PVDF
membranes and bound proteins were detected by blotting with primary
antibody. Immunodetection was achieved by chemiluminescence using
ECL, available from Pierce Biotechnology, Rockford, Ill. For human
fat explant testing, human biopsy adipose tissues were minced and
cultured in 199M medium for 4 hours. The cultured media were
concentrated 30 times before being subjected for Western blot
analysis with monoclonal anti-omentin 3GXX. For detection of
circulating omentin in humans, human plasmas (3 ml, from the Blood
Bank of the University of Maryland) were incubated with 20 .mu.l of
omentin antiserum or pre-immune (control) and protein A Sepharose
beads at 4.degree. C. for 4 hours. The beads were precipitated,
washed and subjected to immunoblotting with anti-omentin polyclonal
antibody.
[0069] Immunofluorescent staining, as shown in FIGS. 8A-D,
demonstrates that omentin polypeptide is actually expressed in
vivo, and defines the cellular localization. Human adipose tissues
was cryosectioned (tissue samples were about 20 .mu.m thick), fixed
with 3% paraformaldehyde and permeabilized with 0.5% cold Triton
X-100. The tissue slides were then incubated with polyclonal
anti-omentin or pre-immune IgG (2.5 mg/ml in PBS) at 1:1000
dilution, washed and re-incubated with a second antibody, goat
anti-rabbit IgG-Alexa 568, available from Molecular Probes, Eugene,
Oreg. The slides were counterstained with nuclei (DAPI). FIGS. 8A-D
show the immunofluorescent staining of the samples. As shown in
FIGS. 8A-D, the omentin staining illustrates the unique structure
of the adipocyte, with a large central triglyceride storage droplet
(round empty circle) and a thin rim of cytoplasm and cell membrane.
The fluorescence (lighter portions) is clearly visible in the
omentin antibody-stained tissue and much less evident in tissue
stained with the pre-immune antibodies. In addition, smaller cells
appear to be more intensively stained than the large adipocytes. It
is believed that these smaller cells represent stromal vascular
cells. FIGS. 8A-D also illustrate that omental, but not
subcutaneous, adipose tissue contains immunoreactive omentin
polypeptides by immunofluorescence staining. Again it is shown that
omentin polypeptides are selectively expressed in the omental
adipose tissue.
[0070] The selective expression of omentin polypeptide in omental
fat tissue and the metabolic effect of omentin polypeptide allow
for the use of omentin polypeptides in diagnosing and treating
injuries or diseases of or related to adipose tissue. In one
embodiment of this invention, a method of modifying at least one of
insulin action and glucose metabolism in an animal includes
determining an amount of omentin polypeptide in the animal. The
amount of omentin polypeptide in the animal can be modified as
needed to obtain a desired result. The methods of this invention
can include one or more of any of the omentin polypeptides
disclosed herein, including the polypeptides having an amino acid
sequence of one of SEQ ID NO:1 and SEQ ID NO:3, and homologous
variants thereof.
[0071] In one embodiment of the invention, modifying the amount of
the omentin polypeptide in the animal includes increasing the
amount of omentin polypeptide in the animal. The amount of omentin
polypeptide can be increased by administering omentin polypeptide
to the animal. In another embodiment of the invention, the amount
of omentin polypeptide in the animal can be modified by decreasing
or interfering with the omentin polypeptide in the animal. The
metabolic function of at least a portion of the omentin polypeptide
in the animal can be interfered with by introducing an amount of a
molecule that regulates signaling or functioning of the omentin
polypeptide. As will be appreciated by one skilled in the art
following the teachings herein provided, in one embodiment of this
invention, the molecule is any molecule that interferes with
receptor binding of the omentin polypeptide. Examples of such
molecules, include, for example, antibodies or other proteins or
polypeptides. By increasing or decreasing the effective amount of
omentin, the method of this invention can be used to
therapeutically treat an undesirable condition of tissue or a
disease, such as obesity and type 2 diabetes, such as by modifying
or manipulating glucose metabolism.
[0072] In another embodiment of this invention, the omentin
polypeptide is used in a method of inducing glucose uptake by at
least one of animal cells, tissues, and organs, such as, for
example adipose tissue, adipocytes, the liver and cells and tissues
thereof, the brain and cells and tissues thereof, muscles and cells
and tissues thereof, the kidney and cells and tissues thereof. The
method includes administering an omentin polypeptide to either the
animal in vivo, or adipocytes in vitro. The omentin polypeptide can
include one or more of any of the variants of omentin polypeptide
described above. In one embodiment of the invention, the
administered omentin polypeptide enhances insulin-mediated glucose
transport in the adipocytes, thereby inducing glucose uptake by
animal adipocytes. In another embodiment the administered omentin
polypeptide activates, either directly or indirectly, the
polypeptide kinase Akt/PKB, thereby inducing glucose uptake by
animal adipocytes.
[0073] In one embodiment of this invention, omentin polypeptide can
be used to increase insulin-stimulated 2-deoxyglucose transport. To
demonstrate omentin polypeptide effect on insulin-stimulated
2-deoxyglucose transport, a cassette of pIRES2-hrGFP, available
from Stratagene, was subcloned in pcDNA3 backbone via appropriate
shuttle vectors, thereby creating the plasmid G6422. The plasmid
was transfected into mammalian HEK-293T cells and the top 10% of
cells with highest fluorescence, sorted by fluorescence-activated
cell sorting (FACS), were collected and cultured. The cells were
grown in 10% FBS DMEM medium, available from Invitrogen, Carlsbad,
Calif., to 80% confluency and then in serum-free DMEM for 5 days.
The conditioned medium was subjected to SuperQ anion ion exchange
chromatography, available from Tosoh Biosciences, Montgomeryville,
Pa., and the omentin-containing fractions were pooled for
galactose-affinity chromatography, available from Pierce
Biotechnology, Inc. The protein was eluted with a buffer containing
10 mM Tris-1 mM EDTA (at about pH 8) and used for analysis and the
elution buffer was used as control. Typically, 3 liters of cell
culture media yield about 100 .mu.g omentin-F. The secretion of
omentin into the medium was verified by Western blotting with
omentin antibody. Cells with the highest omentin expression were
selected and then cultured first in growth medium (DMEM/10% FBS)
and then replaced with serum-free medium (DMEM only) for 48
hours.
[0074] The conditioned media were collected and concentrated 100
times with CENTRICON-80 filters (Millipore, 10 kDa filter) for a
glucose transport assay using differentiated 3T3-L1 adipocytes. The
glucose transport assay was conducted according to Kashiwagi A.,
Verso M. A., Andrews J., Vasquez B., Reaven G., Foley J. E., In
vitro insulin resistance of human adipocytes isolated from subjects
with noninsulin-dependent diabetes mellitus, J Clin Invest. 1983
October; 72(4): 1246-54, herein incorporated by reference in its
entirety. Briefly stated, human adipocytes were isolated by
collegenase digestion and centrifugation. The cells were incubated
with omentin (500 ng/ml) for 30 minutes and stimulated by insulin
(60 nM) for 5 min. The glucose transport was determined by
measuring the uptake of a trace concentration of glucose. The
results of the glucose transport assay are represented in FIG. 9.
As shown in FIG. 9, omentin polypeptide stimulates the glucose
uptake at all insulin concentrations tested. The omentin
polypeptide exhibited its largest effect (1.5 to 2-fold increase)
on insulin-stimulation between 2 and 20 nM.
[0075] To further demonstrate the affect of omentin polypeptide on
insulin-signaling, adipocytes from human fat tissue were isolated
and exposed to insulin with or without pretreatment of omentin
polypeptide. FIG. 10 is a graphical representation of the results
of the analysis. As shown in FIG. 10, insulin stimulated the uptake
of [14C]2-deoxyglucose about 50%. An additional 20-30% more glucose
uptake was observed with the pretreatment of omentin (30 minutes
before insulin).
[0076] Omentin polypeptides stimulate Akt kinase acutely and
synergistically with insulin. Akt kinase is activated by insulin
and other growth factors, and is a central molecule mediating cell
growth, proliferation, and apoptosis. Activation of Akt kinase
requires phosphorylation at threonine (T308) and serine (S473)
residues. To demonstrate omentin polypeptide affect on Akt
phosphorylation, isolated adipocytes were incubated with omentin
polypeptide for various times and then treated with or without
insulin (60 nM). The reaction was stopped by adding lysis buffer
containing 2% SDS, 62.5 mM Tris-HCl, pH 6.8 at 90.degree. C.,
followed by sonication for 15 seconds. The resultant lysates were
subjected to immunoblotting analysis with a mixture of two
phosphor-specific antibodies against the phosphorylated Akt or with
general Akt antibody for total Akt in the lysates. Omentin
polypeptide activated Akt kinase acutely, within about 15 minutes,
by a treatment with serum-free conditioned medium (about 200 ng/ml
omentin). Additional activation of Akt kinase by omentin
polypeptide was observed in the presence of insulin, indicating a
synergistic effect between the two factors. FIG. 11 shows Akt
kinase phosphorylation by acute and chronic exposure to omentin-F.
Adipocytes (3T3-L1) were exposed to omentin-F for 15 minutes and 2
hours, followed by stimulation with 0, 0.5, or 2 nM insulin for
five minutes. Cell lysates were immunoblotted with Akt antibodies
for Western blotting. As shown in the Western blot of FIG. 11,
after a two-hour exposure to omentin polypeptide, when maximal
effects on glucose transport were observed, activation of Akt
kinase was lower but still elevated over the control ("None").
[0077] To demonstrate the impact of omentin polypeptide on glucose
uptake in adipocytes in vivo, mice were administered
intraperitoneally with omentin polypeptide (1 .mu.g/g body weight)
or control vehicle (PBS) 30 minutes prior to administering glucose.
To obtain large quantity of omentin for animal study, the signal
peptide of omentin (16 amino acids at the amino-terminal) was
replaced with a His (6) tag and the fusion protein was expressed in
bacteria using a similar approach as described in Yang et al.,
previously incorporated by reference in its entirety. The
bacterially-expressed omentin was an inclusion body and insoluble.
The inclusion body was purified and was dissolved in CAPS,
available from Novagen, according to the manufacture's protocol.
The solublized protein was dialyzed and further purified by gel
filtration with Hload 16/60 Superdex 200, available from Amersham
Biosciences, into homogeneity. C57/BL mice were obtained from
Jackson Laboratory, Bar Harbor, Me. The mice were housed on a
12-hour light/dark cycle and allowed free access to standard mouse
food and water. The mice were used for testing were at 7-10 weeks
of age. For testing, the mice fasted overnight and received an
intraperitoneal injection of omentin (1 .mu.g/g body weight) or
only vehicle, as a control, 30 min before an intraperitoneal
injection of glucose load at 2 mg/g body weight. Blood samples were
obtained via tail veins at the indicated times before and after the
glucose injection. Blood glucose concentrations were measured with
an ACCU-CHEK blood glucometer, available from Roche Diagnostics,
Indianapolis, Ind.
[0078] FIG. 12 is a graphical representation of the results of the
analysis. Upon intraperitoneal glucose injection, blood glucose
levels in the mice rose rapidly from roughly 100 to 400 mg/dl
within 15 minutes in both groups. However, the glucose levels in
the control group (dotted line) peaked at about 30 minutes and
decreased thereafter. In contrast, the glucose levels in
omentin-administered group (solid line) began to drop from 30
minutes, and were lower than the control group over the two hour
period. This result demonstrates that omentin polypeptide improves
glucose disposal in vivo.
[0079] Omentin polypeptide thus enhances insulin action by
stimulating insulin-mediated glucose uptake or disposal and
improves glucose tolerance.
[0080] The invention additionally relates to a method of diagnosing
or detecting a disease or condition involving animal tissue that
contains, metabolically uses, or expresses omentin polypeptide in
an animal suspected of having the disease or condition. In one
embodiment of this invention, a first step of the method includes
contacting a sample of bodily fluid from the animal with a
plurality of antibodies adapted to specifically bind omentin
polypeptide. The antibodies bound to omentin polypeptides in the
sample can then be detected and measured. As will be appreciated
from the discussions above, levels of omentin polypeptide vary
within different humans or other animals. By comparing the measured
amount to a control, the omentin polypeptide can be used to
diagnose or detect a disease or condition, particularly those of or
related to particular tissues, such as, for example, liver tissue,
brain tissue, muscle tissue, adipose tissue, and kidney tissue. The
bodily fluid from the animal used for testing can include, without
limitation, blood, serum, lymph, urine, sweat, mucus, sputum,
saliva, semen, spinal fluid, interstitial fluid, synovial fluid,
cerebrospinal fluid, gingival fluid, vaginal fluid, and pleural
fluid.
[0081] As omentin polypeptides are secreted and measurable in a
human or other animal, another aspect of this invention includes
diagnostic tests and kits for detecting and/or measuring the amount
or level of omentin polypeptide in a human or other animal. The
omentin polypeptide level in a human or other animal can be used
for testing for particular diseases, such as, for example, obesity
and type 2 diabetes, or a susceptibility to such diseases.
[0082] One embodiment of this invention includes a method of
detecting omentin polypeptide, or the amount thereof, in bodily
fluids of an animal. In one particularly preferred embodiment of
this invention, the method includes first contacting a sample of
the bodily fluids with at least one antibody that specifically
binds to the omentin polypeptide. Then, the antibody bound to the
omentin polypeptide in the sample is detected and, optionally,
measured, such as by means available and know in the art. The
bodily fluid to be tested can be any bodily fluid including blood,
serum, lymph, urine, sweat, mucus, sputum, saliva, semen, spinal
fluid, interstitial fluid, synovial fluid, cerebrospinal fluid,
gingival fluid, vaginal fluid, and pleural fluid. The omentin
polypeptide can be any omentin polypeptide disclosed herein,
including variants thereof not particularly disclosed.
[0083] The invention additionally includes a diagnostic kit for use
in diagnosing damage or disease in tissue containing or expressing
omentin polypeptide. In one particularly preferred embodiment of
this invention, the diagnostic kit includes a measurer of an amount
of omentin polypeptide in a sample of bodily fluids. The measurer,
for example, can include a biologic assay, an antibody-based assay,
an enzyme linked immunosorbent assay, a Western blot, a rapid
immunoassay, and a radioimmunoassay. The diagnostic kit also
includes an indicator, such as, for example, a control or a list of
predetermined values for comparison, for determining if a
measurement taken by the measurer is in a predetermined range
associated with damage or disease in the tissue.
[0084] Thus, the invention provides isolated polypeptides that
selectively express in fat tissue as well as methods for use of the
polypeptide. The polypeptides can be used therapeutically to
regulate or modify glucose metabolism, thereby providing a
therapeutic agent for diseases, or in a diagnostic test or kit for
detecting diseases.
[0085] The invention illustratively disclosed herein suitably may
be practiced in the absence of any element, part, step, component,
or ingredient which is not specifically disclosed herein.
[0086] While in the foregoing detailed description this invention
has been described in relation to certain preferred embodiments
thereof, and many details have been set forth for purposes of
illustration, it will be apparent to those skilled in the art that
the invention is susceptible to additional embodiments and that
certain of the details described herein can be varied considerably
without departing from the basic principles of the invention.
Sequence CWU 1
1
41313PRTHomo sapiens 1Met Asn Gln Leu Ser Phe Leu Leu Phe Leu Ile
Ala Thr Thr Arg Gly1 5 10 15Trp Ser Thr Asp Glu Ala Asn Thr Tyr Phe
Lys Glu Trp Thr Cys Ser 20 25 30Ser Ser Pro Ser Leu Pro Arg Ser Cys
Lys Glu Ile Lys Asp Glu Cys 35 40 45Pro Ser Ala Phe Asp Gly Leu Tyr
Phe Leu Arg Thr Glu Asn Gly Val 50 55 60Ile Tyr Gln Thr Phe Cys Asp
Met Thr Ser Gly Gly Gly Gly Trp Thr65 70 75 80Leu Val Ala Ser Val
His Glu Asn Asp Met Arg Gly Lys Cys Thr Val 85 90 95Gly Asp Arg Trp
Ser Ser Gln Gln Gly Ser Lys Ala Val Tyr Pro Glu 100 105 110Gly Asp
Gly Asn Trp Ala Asn Tyr Asn Thr Phe Gly Ser Ala Glu Ala 115 120
125Ala Thr Ser Asp Asp Tyr Lys Asn Pro Gly Tyr Tyr Asp Ile Gln Ala
130 135 140Lys Asp Leu Gly Ile Trp His Val Pro Asn Lys Ser Pro Met
Gln His145 150 155 160Trp Arg Asn Ser Ser Leu Leu Arg Tyr Arg Thr
Asp Thr Gly Phe Leu 165 170 175Gln Thr Leu Gly His Asn Leu Phe Gly
Ile Tyr Gln Lys Tyr Pro Val 180 185 190Lys Tyr Gly Glu Gly Lys Cys
Trp Thr Asp Asn Gly Pro Val Ile Pro 195 200 205Val Val Tyr Asp Phe
Gly Asp Ala Gln Lys Thr Ala Ser Tyr Tyr Ser 210 215 220Pro Tyr Gly
Gln Arg Glu Phe Thr Ala Gly Phe Val Gln Phe Arg Val225 230 235
240Phe Asn Asn Glu Arg Ala Ala Asn Ala Leu Cys Ala Gly Met Arg Val
245 250 255Thr Gly Cys Asn Thr Glu His His Cys Ile Gly Gly Gly Gly
Tyr Phe 260 265 270Pro Glu Ala Ser Pro Gln Gln Cys Gly Asp Phe Ser
Gly Phe Asp Trp 275 280 285Ser Gly Tyr Gly Thr His Val Gly Tyr Ser
Ser Ser Arg Glu Ile Thr 290 295 300Glu Ala Ala Val Leu Leu Phe Tyr
Arg305 31021284DNAHomo sapiens 2ggcattgtgc caggggaggg tgaggctgga
aaccttggtt ggccccactg ggcttcctcc 60ataaagcttt ctgcacctca ttccacatca
ggagcgtttt tggagaaagc tgcactctgt 120tgagctccag ggcgcagtgg
agggagggag tgaaggagct ctctgtaccc aaggaaagtg 180cagctgagac
tcagacaaga ttacaatgaa ccaactcagc ttcctgctgt ttctcatagc
240gaccaccaga ggatggagta cagatgaggc taatacttac ttcaaggaat
ggacctgttc 300ttcgtctcca tctctgccca gaagctgcaa ggaaatcaaa
gacgaatgtc ctagtgcatt 360tgatggcctg tattttctcc gcactgagaa
tggtgttatc taccagacct tctgtgacat 420gacctctggg ggtggcggct
ggaccctggt ggccagcgtg catgagaatg acatgcgtgg 480gaagtgcacg
gtgggcgatc gctggtccag tcagcagggc agcaaagcag actacccaga
540gggggacggc aactgggcca actacaacac ctttggatct gcagaggcgg
ccacgagcga 600tgactacaag aaccctggct actacgacat ccaggccaag
gacctgggca tctggcacgt 660gcccaataag tcccccatgc agcactggag
aaacagctcc ctgctgaggt accgcacgga 720cactggcttc ctccagacac
tgggacataa tctgtttggc atctaccaga aatatccagt 780gaaatatgga
gaaggaaagt gttggactga caacggcccg gtgatccctg tggtctatga
840ttttggcgac gcccagaaaa cagcatctta ttactcaccc tatggccagc
gggaattcac 900tgcgggattt gttcagttca gggtatttaa taacgagaga
gcagccaacg ccttgtgtgc 960tggaatgagg gtcaccggat gtaacactga
gcaccactgc attggtggag gaggatactt 1020tccagaggcc agtccccagc
agtgtggaga tttttctggt tttgattgga gtggatatgg 1080aactcatgtt
ggttacagca gcagccgtga gataactgag gcagctgtgc ttctattcta
1140tcgttgagag ttttgtggga gggaacccag acctctcctc ccaaccatga
gatcccaagg 1200atggagaaca acttacccag tagctagaat gttaatggca
gaagagaaaa caataaatca 1260tattgactca aaaaaaaaaa aaaa
12843325PRTHomo sapiens 3Met Leu Ser Met Leu Arg Thr Met Thr Arg
Leu Cys Phe Leu Leu Phe1 5 10 15Phe Ser Val Ala Thr Ser Gly Cys Ser
Ala Ala Ala Ala Ser Ser Leu 20 25 30Glu Met Leu Ser Arg Glu Phe Glu
Thr Cys Ala Phe Ser Phe Ser Ser 35 40 45Leu Pro Arg Ser Cys Lys Glu
Ile Lys Glu Arg Cys His Ser Ala Gly 50 55 60Asp Gly Leu Tyr Phe Leu
Arg Thr Lys Asn Gly Val Val Tyr Gln Thr65 70 75 80Phe Cys Asp Met
Thr Ser Gly Gly Gly Gly Trp Thr Leu Val Ala Ser 85 90 95Val His Glu
Asn Asp Met Arg Gly Lys Cys Thr Val Gly Asp Arg Trp 100 105 110Ser
Ser Gln Gln Gly Asn Lys Ala Asp Tyr Pro Glu Gly Asp Gly Asn 115 120
125Trp Ala Asn Tyr Asn Thr Phe Gly Ser Ala Glu Ala Ala Thr Ser Asp
130 135 140Asp Tyr Lys Asn Pro Gly Tyr Tyr Asp Ile Gln Ala Lys Asp
Leu Gly145 150 155 160Ile Trp His Val Pro Asn Lys Ser Pro Met Gln
His Trp Arg Asn Ser 165 170 175Ala Leu Leu Arg Tyr Arg Thr Asn Thr
Gly Phe Leu Gln Arg Leu Gly 180 185 190His Asn Leu Phe Gly Ile Tyr
Gln Lys Tyr Pro Val Lys Tyr Arg Ser 195 200 205Gly Lys Cys Trp Asn
Asp Asn Gly Pro Ala Ile Pro Val Val Tyr Asp 210 215 220Phe Gly Asp
Ala Lys Lys Thr Ala Ser Tyr Tyr Ser Pro Tyr Gly Gln225 230 235
240Arg Glu Phe Val Ala Gly Phe Val Gln Phe Arg Val Phe Asn Asn Glu
245 250 255Arg Ala Ala Asn Ala Leu Cys Ala Gly Ile Lys Val Thr Gly
Cys Asn 260 265 270Thr Glu His His Cys Ile Gly Gly Gly Gly Phe Phe
Pro Gln Gly Lys 275 280 285Pro Arg Gln Cys Gly Asp Phe Ser Ala Phe
Asp Trp Asp Gly Tyr Gly 290 295 300Thr His Val Lys Ser Ser Cys Ser
Arg Glu Ile Thr Glu Ala Ala Val305 310 315 320Leu Leu Phe Tyr Arg
32541150DNAHomo sapiens 4gcaggggagc tccgagtgtc cacaggaagg
gaactatcag ctcctggcat ctgtaaggat 60gctgtccatg ctgaggacaa tgaccagact
ctgcttcctg ttattcttct ctgtggccac 120cagtgggtgc agtgcagcag
cagcctcttc tcttgagatg ctctcgaggg aattcgaaac 180ctgtgccttc
tccttttctt ccctgcctag aagctgcaaa gaaatcaagg aacgctgcca
240tagtgcaggt gatggcctgt attttctccg caccaagaat ggtgttgtct
accagacctt 300ctgtgacatg acttctgggg gtggcggctg gaccctggtg
gccagcgtgc acgagaatga 360catgcgtggg aagtgcacgg tgggtgatcg
ctggtccagt cagcagggca acaaagcaga 420ctacccagag ggggatggca
actgggccaa ctacaacacc tttggatctg cagaggcggc 480cacgagcgat
gactacaaga accctggcta ctacgacatc caggccaagg acctgggcat
540ctggcatgtg cccaacaagt cccccatgca gcattggaga aacagcgccc
tgctgaggta 600ccgcaccaac actggcttcc tccagagact gggacataat
ctgtttggca tctaccagaa 660atacccagtg aaatacagat cagggaaatg
ttggaatgac aatggcccag ccatacctgt 720ggtctatgac tttggtgatg
ctaagaagac tgcatcttat tactcaccgt atggtcaacg 780ggaatttgtt
gcaggattcg ttcagttccg ggtgtttaat aacgagagag cagccaacgc
840cctttgtgct gggataaaag ttactggctg taacactgag catcactgca
tcggtggagg 900agggttcttc ccacagggca aaccccgtca gtgtggggac
ttctccgcct ttgactggga 960tggatatgga actcacgtta agagcagctg
cagtcgggag ataacggagg cggctgtact 1020cttgttctat agatgagaca
gagctctgcg gtgtcagggc gagaacccat cttccaaccc 1080cggctatttg
gagacggaaa aactggaatt ctaacaagga ggagaggaga ctaaatcaca
1140tcaatttgca 1150
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