U.S. patent application number 10/289172 was filed with the patent office on 2003-08-14 for methods and compositions for modulating carbohydrate metabolism.
Invention is credited to Chen, Hubert C., Farese, Robert V. JR..
Application Number | 20030154504 10/289172 |
Document ID | / |
Family ID | 38088303 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030154504 |
Kind Code |
A1 |
Farese, Robert V. JR. ; et
al. |
August 14, 2003 |
Methods and compositions for modulating carbohydrate metabolism
Abstract
Methods and compositions for modulating carbohydrate metabolism
in a host are provided. In the subject methods, diacylglycerol
acyltransferase (DGAT) activity (specifically DGAT1 activity) is
modulated, e.g., reduced or enhanced, to achieve a desired insulin
and/or leptin sensitivity, thereby modulating carbohydrate
metabolism, e.g., increasing or decreasing blood glucose levels,
glucose uptake into cells and assimilation into glycogen. Also
provided are pharmaceutical compositions for practicing the subject
methods. The subject methods and compositions find use in a variety
of applications, including the treatment of hosts suffering
conditions associated with abnormal carbohydrate metabolism, such
as obesity or diabetes.
Inventors: |
Farese, Robert V. JR.; (San
Francisco, CA) ; Chen, Hubert C.; (San Francisco,
CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
38088303 |
Appl. No.: |
10/289172 |
Filed: |
November 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10289172 |
Nov 5, 2002 |
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10040315 |
Oct 29, 2001 |
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10040315 |
Oct 29, 2001 |
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09339472 |
Jun 23, 1999 |
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10040315 |
Oct 29, 2001 |
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PCT/US98/17883 |
Aug 28, 1998 |
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PCT/US98/17883 |
Aug 28, 1998 |
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09103754 |
Jun 24, 1998 |
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6344548 |
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60107771 |
Nov 9, 1998 |
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Current U.S.
Class: |
800/18 ; 514/4.8;
514/5.8; 514/5.9; 514/6.8; 514/6.9; 514/7.4 |
Current CPC
Class: |
A61K 38/00 20130101;
G01N 33/5088 20130101; C12N 15/8247 20130101; C12P 7/64 20130101;
G01N 2800/042 20130101; C12N 9/1029 20130101 |
Class at
Publication: |
800/18 ; 514/2;
514/3 |
International
Class: |
A01K 067/027; A61K
038/28; A61K 038/17 |
Goverment Interests
[0002] This invention was made with Government support under Grant
Nos. DK-56084 and DK-26356 awarded by the National Institutes of
Health. The Government has certain rights in this invention.
Claims
What is claimed is:
1. A method of modulating sensitivity of a host to insulin and/or
leptin, said method comprising: administering to said host an
effective amount of one or more active agents that alter DGAT1
activity in said host to modulate sensitivity of said host to
insulin and/or leptin.
2. The method according to claim 1, wherein said agent decreases
DGAT1 activity and said method is a method of increasing
sensitivity of a host to insulin.
3. The method according to claim 1, wherein said agent decreases
DGAT1 activity and said method is a method of increasing
sensitivity of a host to leptin.
4. The method according to claim 1, wherein said agent increases
DGAT1 activity and said method is a method of decreasing
sensitivity of a host to insulin.
5. The method according to claim 1, wherein said agent increases
DGAT1 activity and said method is a method of decreasing
sensitivity of a host to leptin.
6. A method of modulating carbohydrate metabolism in a host, said
method comprising: administering to said host an effective amount
of one or more active agents that modulate DGAT1 activity in said
host and modulate carbohydrate metabolism in said host.
7. The method according to claim 6, wherein said carbohydrate is
glucose.
8. The method according to claim 7, wherein said active agent
modulates glucose uptake into cells of said host.
9. The method according to claim 8, wherein said cells are selected
from muscle and liver cells.
10. The method according to claim 7, wherein said active agent
increases glycogen synthesis in cells of said host.
11. The method according to claim 10, wherein said cells are
selected from muscle and liver cells.
12. The method according to claim 6, wherein said carbohydrate is
fat.
13. The method according to claim 12, wherein said active agent
modulates triacylglycerol synthesis.
14. The method according to claim 13, wherein said active agent
modulates levels of stored triacylglycerol in cells of said
host.
15. The method according to claim 14, wherein said cells are
adipocyte cells.
16. A method of treating a host suffering from a condition
associated with abnormal carbohydrate metabolism, said method
comprising: administering to said host an effective amount of one
or more active agents that modulate DGAT activity in said host and
treat said host for said condition.
17. The method according to claim 16, wherein said agent modulates
sensitivity to insulin and/or leptin.
18. The method according to claim 16, wherein said agent decreases
DGAT1 activity in said host and the condition is obesity.
19. The method according to claim 18, wherein said treatment
comprises a reduction of host weight and/or body fat.
20. The method according to claim 16, wherein said agent decreases
DGAT activity in said host and the condition is diabetes.
21. The method according to claim 20, wherein said agent treatment
comprises a stabilization of blood glucose levels.
22. The method according to claim 16, wherein said agent increases
DGAT1 activity in said host and said condition is anorexia.
23. The method according to claim 16, wherein said host is a
human.
24. The method according to claim 16, wherein said administering
step comprises co-administering insulin and/or leptin or an
activity mimetic thereof with an agent that modulates DGAT1
activity.
25. A pharmaceutical preparation comprising a DGAT1 activity
modulatory agent in a pharmaceutically acceptable vehicle.
26. The pharmaceutical preparation according to claim 25, wherein
said DGAT1 activity modulatory agent increases DGAT1 activity and
decreases insulin and/or leptin sensitivity.
27. The pharmaceutical preparation according to claim 25, wherein
said DGAT1 activity modulatory agent decreases DGAT1 activity and
increases insulin and/or leptin sensitivity.
28. The pharmaceutical preparation according to claim 25, further
comprising insulin, leptin or a mimetic thereof.
29. A kit comprising: (a) a pharmaceutical preparation comprising
at least one active DGAT1 modulatory agent; and (b) instructions
for treating a host suffering from a condition associated with
abnormal carbohydrate metabolism by administering to said host an
effective amount of one or more active DGAT1 modulatory agents to
treat said host for said condition.
30. The kit according to claim 29, wherein said one or more active
DGAT1 modulatory agents reduce DGAT1 activity and said instructions
are instructions for treating obesity and/or diabetes.
31. The kit according to claim 29, wherein said kit further
includes insulin and/or leptin.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 10/040,315 filed Oct. 29, 2001; which application is: (a)
a continuation-in-part of application Ser. No. 09/339,472 filed on
Jun. 23, 1999, which application claims priority to the filing date
of U.S. Provisional Patent Application Serial No. 60/107,771 filed
Nov. 9, 1998; and (b) a continuation-in-part of PCT application
Ser. No. PCT/US98/17883, filed Aug. 28, 1998, which application is
a continuation in part of application Ser. No. 09/103,754, now U.S.
Pat. No. 6,344,548, filed Jun. 24, 1998; the disclosures of which
applications are herein incorporated by reference.
INTRODUCTION
[0003] 1. Field of the Invention
[0004] The invention relates generally to methods of treating
conditions associated with abnormal carbohydrate metabolism, such
as obesity and diabetes. Specifically, the invention relates to
methods of modulating insulin and/or leptin sensitivity in a
host.
[0005] 2. Background of the Invention
[0006] A human that weighs greater than about 20% more than an
ideal weight is considered obese, and, as such, is highly
susceptible the health problems including coronary artery disease,
stroke, and certain cancers. Within the United States about 24% of
men and 27% of women are considered mildly to severely obese. While
partially effective treatments for obesity based on diet, lifestyle
and surgery have been developed, no effective drug-based treatment
for human obesity is currently available.
[0007] Obesity in humans is commonly associated with altered
sensitivity to insulin and/or leptin, which are circulatory
hormones that modulate energy homeostasis. In general, leptin, the
product of the obese gene, acts in the central nervous system to
regulate food intake, energy metabolism and body weight. Insulin,
however, mainly functions to regulate the concentration of blood
sugar and blood lipids through the promotion of glucose and lipid
intake into cells for utilization and storage.
[0008] Leptin is secreted by adipocytes and acts in the
hypothalamus through the leptin receptor (OB-R). Despite the fact
that rodents with mutations in either the leptin or OB-R genes are
profoundly obese and diabetic (Coleman et al., Diabetologia 14,
141-148, 1978), most obese humans do not have mutations in these
genes (Considine et al., Diabetes 19, 992-994, 1996). The
observation that the vast majority of obese humans have chronically
elevated serum leptin levels (Maffei, et al. (1995) Nature Med. 1,
1155-1161) has led to proposal that a primary cause of obesity is
"leptin resistance" in that obese individuals, while capable of
generating a large amount of circulatory leptin, are unable to
properly respond to it. The mechanisms by which a subject becomes
leptin resistant are not understood.
[0009] Insulin is secreted by the pancreas in response to an
increase in blood glucose, for example after glucose has entered
the bloodstream from the intestine after a carbohydrate-rich meal.
Insulin stimulates glucose uptake by muscle tissue, where the
glucose is converted to glucose 6-phosphate, which is then used to
make glycogen. As a consequence of the accelerated uptake of
glucose from the blood, blood glucose concentration falls to the
normal level, slowing insulin release from the pancreas. There is a
closely adjusted feedback relationship between the rate of insulin
secretion and blood glucose concentration, which holds blood
glucose concentration nearly constant despite large fluctuations in
dietary intake.
[0010] In addition to stimulating glucose uptake, insulin also
stimulates the storage of excess carbohydrates as fat. It activates
both the oxidation of glucose 6-phosphate to pyruvate via
glycolysis and the oxidation of pyruvate to acetyl-CoA. If not
oxidized further for energy production, this acetyl-CoA is used for
fatty acid synthesis in the liver, and these fatty acids are
exported as triglycerols of plasma lipoproteins (VLDLs) to adipose
tissue. Insulin stimulates triacylglycerol (TAG) synthesis in
adipocytes, using fatty acids released from the VLDL TAGs. The
fatty acids are ultimately derived from the glucose taken from the
blood by the liver.
[0011] As such, insulin regulates the conversion of excess blood
glucose into two storage forms: glycogen (in liver and muscle) and
TAG (in adipose tissue).
[0012] Many obese individuals have elevated levels of insulin as
well as elevated levels of blood sugar and/or blood lipid, and, as
such, are thought to be "insulin resistant" in that these
individuals, while capable of generating a large amount of
circulatory insulin, are unable to respond properly to the insulin.
The causative mechanisms of insulin resistance have not yet been
fully elucidated, and examples of diseases caused by insulin
resistance include diabetes, obesity, diabetic microangiopathies
(diabetic nephropathy, diabetic neuropathy, and diabetic
retinopathy), impaired glucose tolerance, hyperinsulinemia,
hyperlipemia, arteriosclerosis, hypertension, obesity, ischemic
heart diseases, ischemic brain disorders, and peripheral arterial
embolism (Tamio Teramoto, et al., (1995) Biomedicine &
Therapeutics 29, 8-96).
[0013] It is clear that insulin and leptin signaling play key roles
in maintaining energy homeostasis in normal individuals and that
most obese individuals have altered sensitivity to these hormones,
leading to excessive food consumption, low energy usage and weight
gain. A need therefore exists for therapies based on modulating the
sensitivity of a subject to leptin and/or insulin. This invention
meets these, and other, needs.
SUMMARY OF THE INVENTION
[0014] Methods and compositions for modulating carbohydrate
metabolism in a host are provided. In the subject methods,
diacylglycerol acyltransferase (DGAT) activity (specifically DGAT1
activity) is modulated, e.g., reduced or enhanced, to achieve a
desired insulin and/or leptin sensitivity, thereby modulating
carbohydrate metabolism, e.g., increasing or decreasing blood
glucose levels, glucose uptake into cells and assimilation into
glycogen. Also provided are pharmaceutical compositions for
practicing the subject methods, etc. The subject methods and
compositions find use in a variety of applications, including the
treatment of hosts suffering conditions associated with abnormal
carbohydrate metabolism, such as obesity or diabetes.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 is a graph showing decreased adipocyte size in
Dgat1.sup.-/- mice. Each circle represents the mean adipocyte
surface area of one female mouse. More than 100 adipocytes were
measured per mouse. For high-fat experiments, mice were fed a
high-fat diet for 10 weeks.
[0016] FIGS. 2a-2c are graphs showing increased insulin sensitivity
in Dgat1.sup.-/- mice. (FIG. 2a) Glucose tolerance test. (FIG. 2b)
Insulin tolerance test. (FIG. 2c) Hyperinsulinemic-euglycemic clamp
study. n=5-6 chow-fed male mice per genotype in each experiment.
*P<0.05 versus Dgat1.sup.+/+ mice.
[0017] FIGS. 3a-3d are graphs showing increased weight loss in
response to leptin infusion in Dgat1.sup.-/- mice. (FIG. 3a and
FIG. 3b) Body weight. (FIG. 3c and FIG. 3d) Food intake. Sex-,
age-, and weight-matched mice were used. n=6-8 chow-fed mice per
genotype. Error bars represent SEM. **P<0.01 versus
Dgat1.sup.+/+ mice receiving the same dose of leptin.
[0018] FIG. 4. is a panel of graphs showing expression of
leptin-regulated genes in Dgat1.sup.-/- mice. The expression of
UCP1 was examined in BAT. The expression of other genes was
examined in WAT. For PPAR. and leptin, results were obtained with
real-time PCR. For other genes, results were obtained with Northern
blotting. n=4-6 chow-fed male mice per genotype. *P<0.05 yersus
Dgat1.sup.+/+ mice.
[0019] FIGS. 5a-5j are a series of figures showing the effects of
DGAT1 deficiency on energy and glucose metabolism in Agouti yellow
(A.sup.Y/a) and leptin-deficient (ob/ob) mice. n=8-12 mice per
genotype for growth curves, n=5 chow-fed male mice per genotype for
fat pad content, and n=4-6 chow-fed male mice per genotype for
plasma glucose and insulin concentrations. *P<0.05, **P<0.01
versus Dgat1.sup.+/+ mice.
[0020] FIG. 6. is a graph showing increased DGAT2 mRNA expression
in WAT of leptin-deficient Dgat1.sup.-/- mice. Results were
obtained with real-time PCR. n=4-6 male mice per genotype.
*P<0.05 versus ob/ob Dgat1.sup.+/+ mice.
DEFINITIONS
[0021] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Still,
certain elements are defined below for the sake of clarity and ease
of reference.
[0022] As used herein, the terms "condition associated with
abnormal carbohydrate metabolism"and "disorder associated with
abnormal carbohydrate metabolism" are used interchangeably to refer
to any disorder that is caused by an alteration in carbohydrate
metabolism. Such disorders include, but are not limited to: blood
glucose-related disorders, including diabetic microangiopathies
(diabetic nephropathy, diabetic neuropathy, and diabetic
retinopathy), impaired glucose tolerance, hyperinsulinemia,
hyperlipemia, arteriosclerosis, hypertension, obesity, ischemic
heart diseases, ischemic brain disorders, peripheral arterial
embolism, diabetes mellitus, diabetes insipidus, gestational
diabetes, diabetes innocens, diabetes insipidus, nephrogenic
diabetes insipidus, diabetes intermittens, diabetes mellitus,
insulin-dependent diabetes mellitus, lipoatrophic diabetes
mellitus, non-insulin-dependent diabetes mellitus, type 1 diabetes
and type 2 diabetes; and disorders relating to obesity, including
diabetes, type 2 diabetes, hypertension, stroke, myocardial
infarction or congestive heart failure, prostate and colon cancer,
gallstones, cholecystitis, gout, gouty arthritis, osteoarthritis,
degenerative arthritis of the knees, hips, or the lower back,
apnea, and pickwickian syndrome. In general, a condition associated
with abnormal carbohydrate metabolism is caused, or is otherwise
associated with, an abnormal, e.g., decreased sensitivity to
insulin and/or leptin. In certain embodiments, a condition
associated with leptin and/or insulin over-activity, e.g.,
anorexia, is also encompassed by this term.
[0023] The term "phenomenon associated with carbohydrate
metabolism" as used herein refers to a structural, molecular, or
functional characteristic associated with carbohydrate metabolism,
particularly such a characteristic that is readily assessable in a
mammalian host. Such characteristics include, but are not limited
to: increased or decreased lipid levels, adipocyte size, blood
glucose levels, glucose disposal, glucose tolerance, glucose
uptake, glycogen synthesis rate, insulin tolerance, weight loss,
food intake, triacylglycerol levels, energy expenditure, weight
levels, weight gain, activity levels or an alteration in lipid
content and the like.
[0024] The term "phenomenon associated with sensitivity to insulin
and/or leptin" as used herein refers to a structural, molecular, or
functional characteristic associated with carbohydrate metabolism,
particularly such a characteristic that is readily assessable in a
mammalian host. Such characteristics include, but are not limited
to: increased or decreased lipid levels, adipocyte size, blood
glucose levels, glucose disposal, glucose tolerance, glucose
uptake, glycogen synthesis rate, insulin tolerance, weight loss,
food intake, triacylglycerol levels, energy expenditure, weight
levels, weight gain, activity levels or an alteration in lipid
content and the like.
[0025] As used herein, the terms "determining," "measuring," and
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations.
[0026] The terms "polypeptide" and "protein", used interchangeably
herein, refer to a polymeric form of amino acids of any length,
which can include coded and non-coded amino acids, chemically or
biochemically modified or derivatized amino acids, and polypeptides
having modified peptide backbones. The term includes fusion
proteins, including, but not limited to, fusion proteins with a
heterologous amino acid sequence, fusions with heterologous and
homologous leader sequences, with or without N-terminal methionine
residues; immunologically tagged proteins; fusion proteins with
detectable fusion partners, e.g., fusion proteins including as a
fusion partner a fluorescent protein, .beta.-galactosidase,
luciferase, etc.; and the like.
[0027] The terms "polynucleotide" and "nucleic acid molecule" are
used interchangeably herein to refer to polymeric forms of
nucleotides of any length. The polynucleotides may contain
deoxyribonucleotides, ribonucleotides, and/or their analogs.
Nucleotides may have any three-dimensional structure, and may
perform any function, known or unknown. The term "polynucleotide"
includes single-, double-stranded and triple helical molecules.
"Oligonucleotide" generally refers to polynucleotides of between
about 5 and about 100 nucleotides of single- or double-stranded
DNA. However, for the purposes of this disclosure, there is no
upper limit to the length of an oligonucleotide. Oligonucleotides
are also known as oligomers or oligos and may be isolated from
genes, or chemically synthesized by methods known in the art.
[0028] As used herein the term "isolated," when used in the context
of an isolated compound, refers to a compound of interest that is
in an environment different from that in which the compound
naturally occurs. "Isolated" is meant to include compounds that are
within samples that are substantially enriched for the compound of
interest and/or in which the compound of interest is partially or
substantially purified.
[0029] As used herein, the term "substantially pure" refers to a
compound that is removed from its natural environment and is at
least 60% free, preferably 75% free, and most preferably 90% free
from other components with which it is naturally associated.
[0030] By "transgenic animal" is meant a non-human animal, usually
a mammal, having a non-endogenous (i.e., heterologous) nucleic acid
sequence present as an extrachromosomal element in a portion of its
cells or stably integrated into its germ line DNA (i.e., in the
genomic sequence of most or all of its cells). Heterologous nucleic
acid is introduced into the germ line of such transgenic animals by
genetic manipulation of, for example, embryos or embryonic stem
cells of the host animal according to methods well known in the
art. A "transgene" is meant to refer to such heterologous nucleic
acid, e.g., heterologous nucleic acid in the form of an expression
construct (e.g., for the production of a "knock-in" transgenic
animal) or a heterologous nucleic acid that upon insertion within
or adjacent a target gene results in a decrease in target gene
expression (e.g., for production of a "knock-out" transgenic
animal).
[0031] A "knock-out" of a gene means an alteration in the sequence
of the gene that results in a decrease of function of the target
gene, preferably such that target gene expression is undetectable
or insignificant. Transgenic knock-out animals can comprise a
heterozygous knock-out of a target gene, or a homozygous knock-out
of a target gene. "Knock-outs" as used herein also include
conditional knock-outs, where alteration of the target gene can
occur upon, for example, exposure of the animal to a substance that
promotes target gene alteration, introduction of an enzyme that
promotes recombination at the target gene site (e.g., Cre in the
Cre-lox system), or other method for directing the target gene
alteration postnatally.
[0032] A "knock-in" of a target gene means an alteration in a host
cell genome that results in altered expression (e.g., increased
(including ectopic) or decreased expression) of a target gene,
e.g., by introduction of an additional copy of the target gene, or
by operatively inserting a regulatory sequence that provides for
enhanced expression of an endogenous copy of the target gene.
"Knock-in" transgenics can comprise a heterozygous knock-in of the
target gene or a homozygous knock-in of a target gene. "Knock-ins"
also encompass conditional knock-ins.
[0033] By "operably linked" is meant that a DNA sequence and a
regulatory sequence(s) are connected in such a way as to permit
gene expression when the appropriate molecules (e.g.,
transcriptional activator proteins) are bound to the regulatory
sequence(s).
[0034] By "operatively inserted" is meant that a nucleotide
sequence of interest is positioned adjacent a nucleotide sequence
that directs transcription and translation of the introduced
nucleotide sequence of interest.
[0035] The term "therapeutic agent" as used herein refers to any
molecule, e.g., protein or small molecule, pharmaceutical compound,
antibody, antisense molecule, ribozyme, and the like, useful in the
treatment of a disease or condition, e.g., a condition associated
with abnormal carbohydrate metabolism. For example, therapeutic
agents of the invention include molecules that inhibit, ameliorate,
or relieve symptoms of a condition associated with abnormal
carbohydrate metabolism.
[0036] The term "unit dosage form" as used herein refers to
physically discrete units suitable as unitary dosages for subjects
(e.g., animals, usually humans), each unit containing a
predetermined quantity of agent(s) in an amount sufficient to
produce the desired effect in association with a pharmaceutically
acceptable diluent, carrier or vehicle. The specifications for the
novel unit dosage forms of the present invention will depend on a
variety of factors including, but not necessarily limited to, the
particular agent employed and the effect to be achieved, and the
pharmacodynamics associated with each compound in the host.
[0037] The terms "treatment", "treating" and the like are used
herein to generally mean obtaining a desired pharmacologic and/or
physiologic effect. The effect may be prophylactic in terms of
completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete cure
for a disease and/or adverse effect attributable to the disease.
"Treatment" as used herein covers any treatment of a disease in a
mammal, particularly a human, and includes: (a) preventing the
disease from occurring in a subject which may be predisposed to the
disease but has not yet been diagnosed as having it; (b) inhibiting
the disease, i.e., arresting its development; or (c) relieving the
disease, i.e., causing regression of the disease.
[0038] The terms "subject," "host," "patient," and "individual" are
used interchangeably herein to refer to any mammalian subject for
whom diagnosis or therapy is desired, particularly humans. Other
subjects may include cattle, dogs, cats, guinea pigs, rabbits,
rats, mice, horses, and so on.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Methods and compositions for modulating carbohydrate
metabolism in a host are provided. In the subject methods,
diacylglycerol acyltransferase (DGAT) activity (specifically DGAT1
activity) is modulated, e.g., reduced or enhanced, to achieve a
desired insulin and/or leptin sensitivity, thereby modulating
carbohydrate metabolism, e.g., increasing or decreasing blood
glucose levels, glucose uptake into cells and assimilation into
glycogen, etc. Also provided are pharmaceutical compositions for
practicing the subject methods. The subject methods and
compositions find use in a variety of applications, including the
treatment of hosts suffering conditions associated with abnormal
carbohydrate metabolism, such as obesity or diabetes.
[0040] Before the subject invention is described further, it is to
be understood that the invention is not limited to the particular
embodiments of the invention described below, as variations of the
particular embodiments may be made and still fall within the scope
of the appended claims. It is also to be understood that the
terminology employed is for the purpose of describing particular
embodiments, and is not intended to be limiting. Instead, the scope
of the present invention will be established by the appended
claims.
[0041] In this specification and the appended claims, the singular
forms "a," "an" and "the" include plural reference unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs.
[0042] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the, context clearly dictates otherwise, between the upper
and lower limit of that range, and any other stated or intervening
value, in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0043] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0044] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the subject
components of the invention that are described in the publications,
which components might be used in connection with the presently
described invention.
[0045] In further describing the invention, representative methods
of modulating sensitivity of a host to insulin and/or leptin and
compositions for performing the methods are described first.
Following this, a detailed description of representative
applications in which the subject methods find use is provided.
Next, representative kits that find use in practicing the subject
methods are further described.
[0046] Methods of Modulating Sensitivity to Insulin and/or
Leptin
[0047] The invention provides methods of modulating sensitivity to
insulin and/or leptin in a host. In general, the methods involve
administering to a host an effective amount of one or more active
agents that modulate DGAT1 activity in the host to modulate
sensitivity to insulin and/or leptin in the host.
[0048] By DGAT1 activity is meant the activity of a DGAT1 protein,
where representative DGAT1 proteins are disclosed in Cases et al.,
Proc. Nat'l Acad. Sci. USA (1998) 95.13018-13023 and Genbank
Accession Nos.: AAC63997, AF059202; as well as U.S. Pat. Nos.
6,100,077 and 6,344,548 and the priority applications to the
present application (listed above); the disclosures-of which are
herein incorporated by reference.
[0049] As DGAT1 activity is modulated in certain embodiments of the
invention, DGAT1 activity is increased or decreased in these
embodiments. In many embodiments, DGAT1 activity is increased or
decreased by at least about 10%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
80%, at least about 90%, or more, as compared to a baseline DGAT1
activity level, e.g., that observed in the host prior to
administration of the active agent. In certain embodiments, DGAT1
activity may be decreased by at least about 100%, by at least about
300%, by at least about 5-fold, by at least about 10-fold, by at
least about 50-fold, or by at least about 100-fold, or more, as
compared to a baseline DGAT1 activity level, e.g., that observed in
the host prior to administration of the active agent.
[0050] Upon administration of a DGAT1 modulatory agent to a host,
insulin and/or leptin activity is usually increased or decreased by
at least about 10%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 80%, at
least about 90%, or more, when compared to a control in the absence
of an effective amount of the active agent. In certain embodiments,
upon administration of a DGAT1 modulatory agent, especially a DGAT1
inhibitory agent, to a host, insulin and/or leptin activity may be
increased by at least about 100%, by at least about 300%, by at
least about 5-fold, by at least about 10-fold, by at least 50-fold,
or by at least 100-fold as compared to a control in the absence of
an effective amount of the active agent.
[0051] Sensitivity to insulin and/or leptin is usually measured in
terms of an assessment of a phenomenon associated with carbohydrate
metabolism of a host, e.g., levels of blood glucose or fatty acids,
in response to a specific dose of insulin and/or leptin, especially
after consumption of carbohydrates.
[0052] In some embodiments where the desired modulation of
sensitivity to insulin and/or leptin is an increase in sensitivity
to insulin and/or leptin, one or more agents that decreases DGAT1
activity is administered to the host: For example, in certain
embodiments, one or more agents that decreases DGAT1 activity is
administered to the host. In these embodiments, the agent is
typically a DGAT1 inhibitor.
[0053] In some embodiments where the desired modulation of
sensitivity to insulin and/or leptin is a decrease in sensitivity
to insulin and/or leptin, one or more agents that increase DGAT1
activity is administered to the host. For example, in certain
embodiments, one or more agents that increases DGAT1 activity is
administered to the host.
[0054] For the modulation of DGAT 1 activity in a host, an
effective amount of an active agent(s) that modulates the activity,
e.g., reduces the activity of DGAT1 in vivo, is administered to the
host. The active agent may be one or a mixture of a variety of
different compounds, including: polynucleotide compositions (e.g.,
coding sequences, antisense compositions, siRNA compositions,
etc.), polypeptide, including antibody, compositions, naturally
occurring or synthetic small molecule compounds, etc.
[0055] In certain embodiments, the active agents administered to
the host are polynucleotide or nucleic acid compositions. The
nucleic acids may be coding sequences, e.g., genes, gene fragments
etc., which may be present in expression vectors, where such
vectors generally have convenient restriction sites located near
the promoter sequence to provide for the insertion of nucleic acid
sequences. Transcription cassettes may be prepared that include a
transcription initiation region, the target gene or fragment
thereof, and a transcriptional termination region.
[0056] The transcription cassettes may be introduced into a variety
of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus;
and the like, where the vectors are able to transiently or stably
be maintained in the cells, usually for a period of at least about
one day, more usually for a period of at least about several days
to several weeks.
[0057] In yet other embodiments of the invention, the active agent
is an agent that modulates, and generally decreases or down
regulates, the expression of DGAT1 in the host. Antisense molecules
can be used to down-regulate expression of a gene in cells. The
anti-sense reagent may be antisense oligonucleotides (ODN),
particularly synthetic ODN having chemical modifications from
native nucleic acids, or nucleic acid constructs that express such
anti-sense molecules as RNA. The antisense sequence is
complementary to the mRNA of the targeted gene, and inhibits
expression of the targeted gene products. Antisense molecules
inhibit gene expression through various mechanisms, e.g. by
reducing the amount of mRNA available for translation, through
activation of RNAse H, or steric hindrance. One or a combination of
antisense molecules may be administered, where a combination may
comprise multiple different sequences.
[0058] Antisense molecules may be produced by expression of all or
a part of the target gene sequence in an appropriate vector, where
the transcriptional initiation is oriented such that an antisense
strand is produced as an RNA molecule. Alternatively, the antisense
molecule is a synthetic oligonucleotide. Antisense oligonucleotides
will generally be at least about 7, usually at least about 12, more
usually at least about 20 nucleotides in length, and not more than
about 500, usually not more than about 50, more usually not more
than about 35 nucleotides in length, where the length is governed
by efficiency of inhibition, specificity, including absence of
cross-reactivity, and the like. It has been found that short
oligonucleotides, of from 7 to 8 bases in length, can be strong and
selective inhibitors of gene expression (see Wagner et al. (1996),
Nature Biotechnol. 14:840-844).
[0059] A specific region or regions of the endogenous sense strand
mRNA sequence is chosen to be complemented by the antisense
sequence. Selection of a specific sequence for the oligonucleotide
may use an empirical method, where several candidate sequences are
assayed for inhibition of expression of the target gene in an in
vitro or animal model. A combination of sequences may also be used,
where several regions of the mRNA sequence are selected for
antisense complementation.
[0060] Antisense oligonucleotides may be chemically synthesized by
methods known in the art (see Wagner et al. (1993), supra, and
Milligan et al., supra.) Preferred oligonucleotides are chemically
modified from the native phosphodiester structure, in order to
increase their intracellular stability and binding affinity. A
number of such modifications have been described in the literature,
which alter the chemistry of the backbone, sugars or heterocyclic
bases.
[0061] Among useful changes in the backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the
non-bridging oxygens are substituted with sulfur;
phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate,
3'-S-5'-O-phosphorothioate, 3'-CH.sub.2-5'-O-phosphonate and
3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the
entire ribose phosphodiester backbone with a peptide linkage. Sugar
modifications are also used to enhance stability and affinity. The
.alpha.-anomer of deoxyribose may be used, where the base is
inverted with respect to the natural .beta.-anomer. The 2'-OH of
the ribose sugar maybe altered to; form 2'-(-methyl or 2'-O-allyl
sugars, which provides resistance to degradation without comprising
affinity. Modification of the heterocyclic bases must maintain
proper base pairing. Some useful substitutions include deoxyuridine
for deoxythymidine, 5-methyl2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have
been shown to increase affinity and biological activity when
substituted for deoxythymidine and deoxycytidine, respectively.
[0062] As an alternative to anti-sense inhibitors, catalytic
nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc.
may be used to inhibit gene expression. Ribozymes may be
synthesized in vitro and administered to the patient, or may be
encoded on an expression vector, from which the ribozyme is
synthesized in the targeted cell (for example, see International
patent application WO 9523225, and Beigelman et al. (1995), Nucl.
Acids Res. 23:4434-42). Examples of oligonucleotides with catalytic
activity are described in WO 9506764. Conjugates of anti-sense ODN
with a metal complex, e.g. terpyridylCu(II), capable of mediating
mRNA hydrolysis are described in Bashkin et al. (1995), Appl.
Biochem. Biotechnol. 54:43-56.
[0063] Alternatively, gene expression can be modified by gene
silencing using double-strand RNA (Sharp (1999) Genes and
Development 13: 139-141). RNAi, otherwise known as double-stranded
RNA interference (dsRNAi) or small interfering RNA (siRNA), has
been extensively documented in the nematode C. elegans (Fire, A.,
et al, Nature, 391, 806-811, 1998) and an identical phenomenon
occurs in plants, in which it is usually referred to as
post-transcriptional gene silencing (PTGS) (Van Blokland, R., et
al., Plant J., 6: 861-877, 1994; deCarvalho-Niebel, F., et al.,
Plant Cell, 7: 347-358, 1995; Jacobs, J. J. M. R. et al., Plant J.,
12: 885-893, 1997; reviewed in Vaucheret, H., et al., Plant J., 16:
651-659, 1998). The phenomenon also occurs in fungi (Romano, N. and
Masino, G., Mol. Microbiol., 6: 3343-3353, 1992, Cogoni, C., et
al., EMBO J., 15: 3153-3163; Cogoni, C. and Masino, G., Nature,
399: 166-169, 1999), in which it is often referred to as
"quelling". RNAi silencing can be induced many ways in plants,
where a nucleic acid encoding an RNA that forms a "hairpin"
structure is employed in most embodiments. Alternative strategies
include expressing RNA from each end of the encoding nucleic acid,
making two RNA molecules that will hybridize. Current strategies
for RNAi induced silencing in plants are reviewed by Carthew et al
(Curr Opin Cell Biol. 2001 13:244-8). RNAi is also described in WO
02/44321 and WO 01/68836; the priority documents of which are
herein incorporated by reference.
[0064] Also of-interest are polypeptide, e.g., proteinaceous,
active agents. Specific polypeptide agents include proteins or
active fragments thereof, e.g., DGAT1 proteins, etc.
[0065] A specific type of polypeptide active agent of interest is
an antibody agent that modulates DGAT1 activity in the host. The
antibodies may be monoclonal or polyclonal, and produced according
to methods known in the art. Antibody fragments, such as Fv,
F(ab').sub.2 and Fab may be prepared by cleavage of the intact
protein, e.g. by protease or chemical cleavage.
[0066] Alternatively, a truncated gene is designed. For example, a
chimeric gene encoding a portion of the F(ab').sub.2 fragment would
include DNA sequences encoding the CH1 domain and hinge region of
the H chain, followed by a translational stop codon to yield the
truncated molecule.
[0067] Consensus sequences of H and L J regions may be used to
design oligonucleotides for use as primers to introduce useful
restriction sites into the J region for subsequent linkage of V
region segments to human C region segments. C region cDNA can be
modified by site directed mutagenesis to place a restriction site
at the analogous position in the human sequence.
[0068] Expression vectors include plasmids, retroviruses, YACs, EBV
derived episomes, and the like. A convenient vector is one that
encodes a functionally complete human CH or CL immunoglobulin
sequence, with appropriate restriction sites engineered so that any
VH or VL sequence can be easily inserted and expressed. In such
vectors, splicing usually occurs between the splice donor site in
the inserted J region and the splice acceptor site preceding the
human C region, and also at the splice regions that occur within
the human CH exons. Polyadenylation and transcription termination
occur at native chromosomal sites downstream of the coding regions.
The resulting chimeric antibody may be joined to any strong
promoter, including retroviral LTRs, e.g. SV-40 early promoter,
(Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcoma virus
LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murine
leukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native
Ig promoters, etc.
[0069] Naturally occurring or synthetic small molecule compounds of
interest as active agents include numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 daltons. Candidate agents include functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Of particular interest are those agents
identified by the screening assays of the subject invention, as
described above.
[0070] In certain embodiments, in addition to the DGAT1 modulatory
active agent, a leptin and/or insulin modulatory active agent,
e.g., an agent that enhances or inhibits leptin and/or insulin
activity, is administered. For example, in certain embodiments
where a DGAT1 inhibitory agent is administered, a leptin activity
enhancing agent (e.g. leptin or an activity mimetic thereof) may
also be administered, such that both a DGAT1 inhibitory agent and a
leptin activity enhancing agent are administered to the host. In
certain other embodiments where a DGAT1 inhibitory agent is
administered, an insulin activity enhancing agent (e.g. insulin or
an activity mimetic thereof) may also be administered, such that
both a DGAT1 inhibitory agent and an insulin activity enhancing
agent are administered to the host. Such embodiments include those
embodiments where one wishes to modulate sensitivity to insulin
and/or leptin in way that modulates a phenomenon associated with
carbohydrate metabolism, e.g., blood glucose levels.
[0071] In practicing the subject methods, an effective amount of
the active agent is administered to a host, where the term
"effective amount" means a dosage sufficient to produce a desired
result, where the desired result is the desired modulation, e.g.,
enhancement, reduction, of DGAT1 activity and a desired modulation
in sensitivity to insulin and or leptin.
[0072] In practicing the subject methods, the active agent or
agents are typically administered to the host in a physiologically
acceptable delivery vehicle, e.g., as a pharmaceutical preparation.
A variety of representative formulations, dosages, routes of
administration for candidate agents, nucleic acid delivery vehicles
and nucleic acid formulations for nucleic acid delivery are
described below.
[0073] Formulations, Dosages, and Routes of Administration
[0074] The invention provides formulations, including
pharmaceutical formulations, that include an agent which modulates
sensitivity to insulin and/or leptin in a host. In general, a
formulation comprises an effective amount of an agent that
modulates DGAT 1 (and/or leptin) activity in a host. An "effective
amount" refers to an amount that is sufficient to produce a desired
result, e.g., reduction of weight to normal levels, reduction of
blood glucose levels, stabilization of blood glucose levels etc. In
many embodiments, the desired result is at least a reduction or
increase in a phenotype as compared to a control such that the
phenotype is more similar to normal.
[0075] In certain embodiments, the agent that modulates sensitivity
to insulin and/or leptin is administered before, with, or after
administration of insulin and/or leptin, or mimetics thereof.
Mimetics of insulin and of leptin activity are well known the art
(see, for example, U.S. Pat. Nos. 6,444,641, 6,335,316, 5,952,297,
5,700,662, 5,698,669, 6,420,340, 7 6,355,635 and 3 6,420,339; the
disclosures of which are herein incorporated by reference).
[0076] Formulations
[0077] In the subject methods, the active agent(s) may be
administered to the host using any convenient means capable of
resulting in the desired reduction in of a carbohydrate
metabolism-related phenotype.
[0078] Thus, the agent can be incorporated into a variety of
formulations for therapeutic administration. More particularly, the
agents of the present invention can be formulated into
pharmaceutical compositions by combination with appropriate,
pharmaceutically acceptable carriers or diluents, and may be
formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants and
aerosols.
[0079] In pharmaceutical dosage forms, the agents may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination, with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0080] For oral preparations, the agents can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents; buffering agents, moistening agents, preservatives
and flavoring agents.
[0081] The agents can be formulated into preparations for injection
by dissolving, suspending or emulsifying them in an aqueous or
nonaqueous solvent, such as vegetable or other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0082] The agents can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0083] Furthermore, the agents can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0084] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier.
[0085] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0086] Other modes of administration will also find use with the
subject invention. For instance, an agent of the invention can be
formulated in suppositories and, in some cases, aerosol and
intranasal compositions. For suppositories, the vehicle composition
will include traditional binders and carriers such as, polyalkylene
glycols, or triglycerides. Such suppositories may be formed from
mixtures containing the active ingredient in the range of about
0.5% to about 10% (w/w), preferably about 1% to about 2%.
[0087] Intranasal formulations will usually include vehicles that
neither cause irritation to the, nasal mucosa nor significantly
disturb ciliary function. Diluents such as water, aqueous saline or
other known substances can be employed with the subject invention.
The nasal formulations may also contain preservatives such as, but
not limited to, chlorobutanol and benzalkonium chloride. A
surfactant may be present to enhance absorption of the subject
proteins by the nasal mucosa.
[0088] An agent of the invention can be administered as
injectables. Typically, injectable compositions are prepared as
liquid solutions or suspensions; solid forms suitable for solution
in, or suspension in, liquid vehicles prior to injection may also
be prepared. The preparation may also be emulsified or the active
ingredient encapsulated in liposome vehicles.
[0089] Suitable excipient vehicles are, for example, water, saline,
dextrose, glycerol, ethanol, or the like, and combinations thereof.
In addition, if desired, the vehicle may contain minor amounts of
auxiliary substances such as wetting or emulsifying agents or pH
buffering agents. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in the art. See, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 17th edition, 1985; Remington: The Science and
Practice of Pharmacy, A. R. Gennaro, (2000) Lippincott, Williams
& Wilkins. The composition or formulation to be administered
will, in any event, contain a quantity of the agent adequate to
achieve the desired state in the subject being treated.
[0090] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0091] Dosages
[0092] Although the dosage used will vary depending on the clinical
goals to be achieved, a suitable dosage range is one which provides
up to about 1 .mu.g to about 1,000 .mu.g or about 10,000 .mu.g of
an agent that modulates insulin and/or leptin resistance in a host,
and in embodiments, modulates carbohydrate metabolism or treats a
host suffering from a condition associated with abnormal
carbohydrate metabolism.
[0093] Those of skill will readily appreciate that dose levels can
vary as a function of the specific compound, the severity of the
symptoms and the susceptibility of the subject to side effects.
Preferred dosages for a given compound are readily determinable by
those of skill in the art by a variety of means.
[0094] Routes of Administration
[0095] Conventional and pharmaceutically acceptable routes of
administration include intranasal, intramuscular, intratracheal,
intratumoral, subcutaneous, intradermal, topical application,
intravenous, rectal, nasal, oral and other parenteral routes of
administration. Routes of administration may be combined, if
desired, or adjusted depending upon the agent and/or the desired
effect. The composition can be administered in a single dose or in
multiple doses.
[0096] The agent can be administered to a host using any available
conventional methods and routes suitable for delivery of
conventional drugs, including systemic or localized routes. In
general, routes of administration contemplated by the invention
include, but are not necessarily limited to, enteral, parenteral,
or inhalational routes.
[0097] Parenteral routes of administration other than inhalation
administration include, but are not necessarily limited to,
topical, transdermal, subcutaneous, intramuscular, intraorbital,
intracapsular, intraspinal, intrasternal, and intravenous routes,
i.e., any route of administration other than through the alimentary
canal. Parenteral administration can be carried to effect systemic
or local delivery of the agent. Where systemic delivery is desired,
administration typically involves invasive or systemically absorbed
topical or mucosal administration of pharmaceutical
preparations.
[0098] The agent can also be delivered to the subject by enteral
administration. Enteral routes of administration include, but are
not necessarily limited to, oral and rectal (e.g., using a
suppository) delivery.
[0099] Methods of administration of the agent through the skin or
mucosa include, but are not necessarily limited to, topical
application of a suitable pharmaceutical preparation, transdermal
transmission, injection and epidermal administration. For
transdermal transmission, absorption promoters or iontophoresis are
suitable methods. Iontophoretic transmission may be accomplished
using commercially available "patches" which deliver their product
continuously via electric pulses through unbroken skin for periods
of several days or more.
[0100] By treatment is meant at least: an amelioration of the
symptoms associated with the pathological condition afflicting the
host, where amelioration is used in a broad sense to refer to at
least a reduction in the magnitude of a parameter, e.g. symptom,
associated with the pathological condition being treated, such as a
obesity and psychological trauma associated therewith. As such,
treatment also includes situations where the pathological
condition, or at least symptoms associated therewith, are
completely inhibited, e.g. prevented from happening, or stopped,
e.g. terminated, such that the host no longer suffers from the
pathological condition, or at least the symptoms that characterize
the pathological condition.
[0101] A subject polynucleotide can be delivered as a naked
polynucleotide, or associated with (complexed with) a delivery
vehicle. "Associated with", or "complexed with", encompasses both
covalent and non-covalent interaction of a polynucleotide with a
given delivery vehicle.
[0102] Nucleic Acid Delivery Vehicles
[0103] In certain embodiment, an agent is a nucleic acid. Nucleic
acids may be delivered using several different vehicles, including
viral and non-viral delivery vehicles.
[0104] Viral Delivery Vehicles
[0105] A subject polynucleotide can be associated with viral
delivery vehicles. As used herein, a "viral delivery vehicle"
intends that the polynucleotide to be delivered is encapsidated in
a viral particle.
[0106] Numerous viral genomes useful in in vivo transformation and
gene therapy are known in the art, or can be readily constructed
given the skill and knowledge in the art. Included are replication
competent, replication deficient, and replication conditional
viruses. Viral vectors include adenovirus, mumps virus, a
retrovirus, adeno-associated virus, herpes simplex virus (HSV),
cytomegalovirus (CMV), vaccinia virus, and poliovirus, and
non-replicative mutants/variants of the foregoing. In some
embodiments, a replication-deficient virus is capable of infecting
slowly replicating and/or terminally differentiated cells, since
the respiratory tract is primarily composed of these cell types.
For example, adenovirus efficiently infects slowly replicating
and/or terminally differentiated cells. In some embodiments, the
viral genome itself, or a protein on the viral surface, is specific
or substantially specific for cells of the targeted cell. A viral
genome can be designed to be target cell-specific by inclusion of
cell type-specific promoters and/or enhancers operably linked to a
gene(s) essential for viral replication.
[0107] Where a replication-deficient virus is used, as the viral
genome, the production of virus particles containing either DNA or
RNA corresponding to the polynucleotide of interest can be produced
by introducing the viral construct into a recombinant cell line
which provides the missing components essential for viral
replication and/or production. Preferably, transformation of the
recombinant cell line with the recombinant viral genome will not
result in production of replication-competent viruses, e.g., by
homologous recombination of the viral sequences of the recombinant
cell line into the introduced viral genome. Methods for production
of replication-deficient viral particles containing a nucleic acid
of interest are well known in the art and are described in, for
example, Rosenfeld et al., Science 252:431-434, 1991 and Rosenfeld
et al., Cell 68:143-155, 1992 (adenovirus); U.S. Pat. No. 5,139,941
(adeno-associated virus); U.S. Pat. No. 4,861,719 (retrovirus); and
U.S. Pat. No. 5,356,806 (vaccinia virus). Methods and materials for
manipulation of the mumps virus genome, characterization of mumps
virus genes responsible for viral fusion and viral replication, and
the structure and sequence of the mumps viral genome are described
in Tanabayashi et al., J. Virol. 67:2928-2931, 1993; Takeuchi et
al., Archiv. Viro, 128:177-183, 1993; Tanabayashi et al., Virol.
187:801-804, 1992; Kawano et al., Virol., 179:857-861, 1990; Elango
et al., J. Gen. Virol. 69:2893-28900, 1988.
[0108] Non-Viral Delivery Vehicles
[0109] A subject polynucleotide can be administered using a
non-viral delivery vehicle. "Non-viral delivery vehicle" (also
referred to herein as "non-viral vector") as used herein is meant
to include chemical formulations containing naked or condensed
polynucleotides (e.g, a formulation of polynucleotides and cationic
compounds (e.g., dextran sulfate)), and naked or condensed
polynucleotides mixed with an adjuvant such as a viral particle
(i.e., the polynucleotide of interest is not contained within the
viral particle, but the transforming formulation is composed of
both naked polynucleotides and viral particles (e.g., adenovirus
particles) (see, e.g., Curiel et al. 1992 Am. J. Respir. Cell Mol.
Biol. 6:247-52)). Thus "non-viral delivery vehicle" can include
vectors composed of polynucleotides plus viral particles where the
viral particles do not contain the polynucleotide of interest.
"Non-viral delivery vehicles" include bacterial plasmids, viral
genomes or portions thereof, wherein the polynucleotide to be
delivered is not encapsidated or contained within a viral particle,
and constructs comprising portions of viral genomes and portions of
bacterial plasmids and/or bacteriophages. The term also encompasses
natural and synthetic polymers and co-polymers. The term further
encompasses lipid-based vehicles. Lipid-based vehicles include
cationic liposomes such as disclosed by Felgner et al (U.S. Pat.
Nos. 5,264,618 and 5,459,127; PNAS 84:7413-7417, 1987; Annals N.Y.
Acad. Sci. 772:126-139, 1995); they may also consist of neutral or
negatively charged phospholipids or mixtures thereof including
artificial viral envelopes as disclosed by Schreier et al. (U.S.
Pat. Nos. 5,252,348 and 5,766,625).
[0110] Non-viral delivery vehicles include polymer-based carriers.
Polymer-based carriers may include natural and synthetic polymers
and co-polymers. Preferably, the polymers are biodegradable, or can
be readily eliminated from the subject. Naturally occurring
polymers include polypeptides and polysaccharides. Synthetic
polymers include, but are not limited to, polylysines, and
polyethyleneimines (PEI; Boussif et al., PNAS 92:7297-7301, 1995)
which molecules can also serve as condensing agents. These carriers
may be dissolved, dispersed or suspended in a dispersion liquid
such as water, ethanol, saline solutions and mixtures thereof. A
wide variety of synthetic polymers are known in the art and can be
used.
[0111] "Non-viral delivery vehicles" further include bacteria. The
use of various bacteria as delivery vehicles for polynucleotides
has been described. Any known bacterium can be used as a delivery
vehicle, including, but not limited to non-pathogenic strains of
Staphylococcus, Salmonella, and the like.
[0112] Formulations for Nucleic Acid Delivery
[0113] The polynucleotide to be delivered can be formulated as a
DNA- or RNA-liposome complex formulation. Such complexes comprise a
mixture of lipids which bind to genetic material (DNA or RNA) by
means of cationic charge (electrostatic interaction). Cationic
liposomes which may be used in the present invention include
3.beta.-[N-(N', N'-dimethyl-aminoethane)- -carbamoyl]-cholesterol
(DC-Chol), 1,2-bis(oleoyloxy-3-trimethylammonio-pr- opane (DOTAP)
(see, for example, WO 98/07408), lysinylphosphatidylethanola- mine
(L-PE), lipopolyamines such as lipospermine,
N-(2-hydroxyethyl)-N,N-d-
imethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide, dimethyl
dioctadecyl ammonium bromide (DDAB), dioleoylphosphatidyl
ethanolamine (DOPE), dioleoylphosphatidyl choline (DOPC),
N(1,2,3-dioleyloxy) propyl-N,N,N-triethylammonium (DOTMA), DOSPA,
DMRIE, GL-67, GL-89, Lipofectin, and Lipofectamine (Thiery et al.
(1997) Gene Ther. 4:226-237; Felgner et al., Annals N.Y. Acad. Sci.
772:126-139, 1995; Eastman et al., Hum. Gene Ther. 8:765-7.73,
1997). Polynucleotide/lipid formulations described in U.S. Pat. No.
5,858,784 can also be used in the methods described herein. Many of
these lipids are commercially available from, for example,
Boehringer-Mannheim, and Avanti Polar Lipids (Birmingham, Ala.).
Also encompassed are the cationic phospholipids found in U.S. Pat.
Nos. 5,264,618, 5,223,263 and 5,459,127. Other suitable
phospholipids which may be used include phosphatidylcholine,
phosphatidylserine, phosphatidylethanolamine, sphingomyelin,
phosphatidylinositol, and the like. Cholesterol may also be
included.
[0114] Utility
[0115] The subject compositions and methods of modulating
sensitivity to insulin and/or leptin in a host find use in a
variety of protocols. In some embodiment, these protocols involve
administering to a host an effective amount of one or more active
agents that alter DGAT1 activity in the host and therby modulate
carbohydrate metabolism in the host. In other embodiments, these
protocols involve administering to a host suffering from a
condition associated with abnormal carbohydrate metabolism an
effective amount of one or more active agents that modulate DGAT
activity in the host and treat the host for the condition.
[0116] By treatment is meant at least an amelioration of a symptom
associated with the pathological condition afflicting the host,
where amelioration is used in a broad sense to refer to at least a
reduction in the magnitude of a parameter, e.g., symptom,
associated with the pathological condition being treated. Treatment
also includes outcomes where the pathological condition, or at
least symptoms associated therewith, are completely inhibited, e.g.
prevented from happening, or stopped, e.g., terminated, such that
the host no longer suffers from the pathological condition, or at
least the symptoms that characterize the pathological condition.
For example, where the disease condition is marked by the presence
of variable blood glucose levels, treatment includes at least a
reduction in the variability of blood glucose levels, including a
restoration of stable blood glucose levels of a normal host.
[0117] In certain other embodiments, insulin, leptin or a mimetic
thereof is administered before, at the same time as, or after the
DGAT activity modulating agent in order to modulate carbohydrate
metabolism or treat a condition associated with abnormal
carbohydrate metabolism. It is expected that such combinations,
e.g., a formulation including insulin and/or leptin and a DGAT
modulatory agent will be efficacious in treating a-disease relating
to abnormal carbohydrate metabolism.
[0118] A variety of hosts may be used in the-subject methods.
Generally such hosts are mammals or mammalian, where these terms
are used broadly to describe organisms which are within the class
mammalia, including the orders carnivore (e.g., dogs and cats),
rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g.,
humans, chimpanzees, and monkeys). In many embodiments, the hosts
will be humans.
[0119] Of particular interest is treatment and prevention of
diseases relating to abnormal carbohydrate metabolism associated
with increased sensitivity to insulin and/or leptin. Such diseases
include blood glucose-related disorders (diabetes) and obesity,
inculding, blood glucose-related disorders, including diabetic
microangiopathies (diabetic nephropathy, diabetic neuropathy, and
diabetic retinopathy), impaired glucose tolerance,
hyperinsulinemia, hyperlipemia, arteriosclerosis, hypertension,
obesity, ischemic heart diseases, ischemic brain disorders,
peripheral arterial embolism, diabetes mellitus, diabetes
insipidus, gestational diabetes, diabetes innocens, diabetes
insipidus, nephrogenic diabetes insipidus, diabetes intermittens,
diabetes mellitus; insulin-dependent diabetes mellitus,
lipoatrophic diabetes mellitus, non-insulin-dependent diabetes
mellitus, type 1 diabetes and type 2 diabetes; and disorders
relating to obesity, including diabetes, type 2 diabetes,
hypertension, stroke, myocardial infarction or congestive heart
failure, prostate and colon cancer, gallstones, cholecystitis,
gout, gouty arthritis osteoarthritis, degenerative arthritis of the
knees, hips, or the lower back, apnea, and pickwickian
syndrome.
[0120] The subject methods also find use in the modulation of
carbohydrate metabolism in hosts not suffering from a particular
condition but in which the modulation of carbohydrate metabolism is
nonetheless desired. Subject treatment methods are typically
performed on hosts with such disorders or on hosts with a desire to
avoid contracting such disorders. As such, the invention also
contemplates preventing or reducing the risk of a disease relating
to abnormal carbohydrate metabolism in a host by administering a
pharmaceutical composition, and for achieving a desired outcome
that is not abnormal, but otherwise desirable, for example weight
loss of a non-obese host, or a decrease in blood glucose levels in
a non-diabetic host.
[0121] In particular, modulation of glucose uptake and glycogen
synthesis in muscle or liver cells, or lipid (e.g.
triacylglycerol). transport, synthesis, and storage e.g. in
adipocyte cells is of interest.
[0122] Kits
[0123] Also provided by the subject invention are kits for
practicing the subject methods, as described above. The subject
kits at least include one or more of a pharmaceutical preparation
comprising at least one active agent that modulates DGAT activity,
such as those representative DGAT1 modulatory agents described
above. Other optional components of the kit include: a syringe or
another administration device, and glucose-monitoring devices such
as glucose test strips, etc. Also included in the kit may be
insulin, leptin, or an activity mimetic thereof. The various
components of the kit may be present in separate containers or
certain compatible components may be pre-combined into a single
container, as desired. In many embodiments, kits with unit doses of
the active agent, e.g., in oral or injectable doses, are provided.
In many embodiments the subject composition is contained within a
media, such as a media suitable for injection into a human
bloodstream.
[0124] In addition to above-mentioned components, the subject kits
typically further include instructions for using the components of
the kit to practice the subject methods treating a host suffering
from a disease relating to abnormal carbohydrate metabolism by
administering to said host an effective amount of one or more
active agents that modulate DGAT in the host to modulate
sensitivity to insulin and/or leptin sensitivity in the host and
treat the host for the condition. The instructions for practicing
the subject methods are generally recorded on a suitable recording
medium. For example, the instructions may be printed on a
substrate, such as paper or plastic, etc. As such, the instructions
may be present in the kits as a package insert, in the labeling of
the container of the kit or components thereof (i.e., associated
with the packaging or subpackaging) etc. In other embodiments, the
instructions are present as an electronic storage data file present
on a suitable computer readable storage medium, e.g. CD-ROM,
diskette, etc. In yet other embodiments, the actual instructions
are not present in the kit, but means for obtaining the
instructions from a remote source, e.g. via the internet, are
provided. An example of this embodiment is a kit that includes a
web address where the instructions can be viewed and/or from which
the instructions can be downloaded. As with the instructions, this
means for obtaining the instructions is recorded on a suitable
substrate.
[0125] Animal Models for Modulated Sensitivity to Insulin and/or
Leptin
[0126] The invention further provides a non-human animal model for
modulated sensitivity to insulin and/or leptin. In general, the
non-human animal model is characterized by having abnormal DGAT
activity.
[0127] A non-human animal may be any animal, e.g., a mammal or
avian species that can serve as an animal model for testing
therapies sensitivity to insulin and/or leptin. In many embodiments
the non-human animal is a laboratory animal, usually a rodent,
e.g., mouse, rat, hamster, guinea pig or the like. Other suitable
laboratory animals are rabbits, cats, dogs, small monkeys, and
apes. In addition, certain farm animals are also often employed as
laboratory animals, notably chickens, goats, sheep, and pigs. Mice
suitable for use in the present invention can be produced from any
of a variety of background strains including, but not necessarily
limited to, the strains C.B-17, C3H, BALB/c, C57131/6, AKR, BA,
B10, 129, etc. Non-human animals are readily available from
researchers or commercial suppliers, such as Jackson Laboratories
(Bar Harbor, Me.), Charles River Breeding Laboratories (Wilmington,
Mass.), Taconic Farms (Germantown, N.Y.), to mention a few such
suppliers.
[0128] DGAT activity, including DGAT1 and/or DGAT2 activity, may be
modified in animals by a variety of methods. In many embodiments,
these methods involve modifying the expression of DGAT in a
transgenic animal. In many embodiments, the expression of a DGAT
endogenous to the animal is reduced in an animal. This may be
accomplished through knockout strategies, where an nucleic acid
insertion into an endogenous gene inactivates the gene (described
in U.S. Pat. Nos. 5,487,992; 5,627,059; 5,631,153; and 6,204,061),
or by other methods e.g. antisense, inhibitory RNA (RNAi), ribozyme
or co-supression technologies, as is known in the art (e.g. Hannon
et al., Nature 418:244-51, 2002; Ueda, J Neurogenet. 15:193-204,
2001; Review. Lindenbach et al., Mol Cell. 9:925-7, 2002; Brantl,
Biochim Biophys Acta. 1575:15-25, 2002; Zhang et al., Ann N Y Acad
Sci. 923:210-33, 2000). In other embodiments, an endogenous or
exogenous DGAT is over-expressed in an animal. In these
embodiments, a DGAT, leptin or leptin receptor coding sequence (for
example, a coding sequence provided by one of the following NCBI
accession: NM.sub.--010046 (SEQ ID NO:1), XM.sub.--035370 (SEQ ID
NO:2), NM.sub.--053437 (SEQ ID NO:3), AJ318490 (SEQ ID NO:4),
AF221132 (SEQ ID NO:5), AF468649 (SEQ ID NO:6), AY093657 (SEQ ID
NO: 7), AF384161 (SEQ ID NO:8), NM.sub.--012079 (SEQ ID NO:9),
AF384163 (SEQ ID NO:10), AF384162 (SEQ ID NO:11), AF078752 (SEQ ID
NO:12), is cloned into an expression cassette in an appropriate
vector, and transferred into the genome of an animal to make a
transgenic animal. In certain embodiments, the animal is homozygous
for a defect in a DGAT gene (DGAT1 or DGAT2), and in many
embodiments, the subject animal is homozygous for a knockout in one
of these genes.
[0129] Cloning technology, cloning strategies, expression
cassettes, and suitable vectors for performing animal
transformation are well known in the art (Ausubel, et al, Short
Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995;
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, (1989) Cold Spring Harbor, N.Y.). Methods of generating
transgenic, non-human animals, particularly transgenic, non-human
mammals, are also known in the art. See, e.g., U.S. Pat. Nos.
6,268,545; 6,255;554; 6,222,094; 5,387,742, 4,736,866 and 5,565,186
and 6,204,43; "Transgenic Animal Technology" C. A. Pinkert, ed.
(1997) Acad. Press; "Gene Knockout Protocols" M. J. Tymms, et al.,
eds. (2001) Humana Press; and "Gene Targeting: A Practical
Approach" A. L. Joyner, ed. (2000) Oxford Univ. Press.
[0130] One method for producing a transgenic animal employs
embryonic stem (ES) cells obtained from an animal to be
transformed, e.g. mouse, rat, guinea pig, etc. In these methods, ES
cells are grown on an appropriate fibroblast-feeder layer or grown
in the presence of appropriate growth factors, such as leukemia
inhibiting factor (LIF). When ES cells have been transformed, they
may be used to produce transgenic animals. After transformation,
the cells are plated onto a feeder layer in an appropriate medium.
Cells containing the construct may be detected by employing a
selective medium. After sufficient time for colonies to grow, they
are picked and analyzed for the occurrence of homologous
recombination or integration of the construct. Those colonies that
are positive may then be used for embryo manipulation and
blastocyst injection. Blastocysts are obtained from 4 to 6 week old
superovulated females. The ES cells are trypsinized, and the
modified cells are injected into the blastocoel of the blastocyst.
After injection, the blastocysts are returned to each uterine horn
of pseudopregnant females. Females are then allowed to go to term
and the resulting litters screened for mutant cells having the
construct. By providing for a different phenotype of the blastocyst
and the ES cells, chimeric progeny can be readily detected. Progeny
of transgenic animals may be screened for the presence of the
modified gene and males and females having appropriate modified
genomes are mated to produce homozygous progeny.
[0131] In certain embodiments, transgenic animals may be
inter-crossed or may contain more than one genetic modification in
order to produce a subject animal model. For example, an animal
overexpressing DGAT1 may be bred with-an animal knockout of a
leptin-encoding gene to produce an a subject-animal model
containing an increase in DGAT activity and a decrease in leptin
activity and an animal overexpressing leptin may also be bred with
an animal knockout of a DGAT-encoding gene to produce an a subject
animal model containing a decrease in DGAT activity and an increase
in leptin activity, etc. Animals with abnormal DGAT expression may
be intercrossed with animals containing abnormal expression of
other genes (e.g. obese, agouti yellow) in order to produce a
subject animal model.
[0132] In embodiments where DGAT is over-expressed in a subject
animal, DGAT activity is increased more than about 1.5-fold, more
than about 2-fold, more than about 3-fold, more than about 5-fold,
more than about 10-fold or even more than about 100-fold in a
subject animal, as compared to an animal in which DGAT expression
is not increased.
[0133] In embodiments where DGAT expression is decreased in a
subject animal, DGAT expression is decreased by more than about
30%, more than about 50%, more than about 70%, more than about 90%,
more than about 95% or even more than about 98%, about 99% or 99.5%
in a subject animal, as compared to an animal in which DGAT is not
decreased.
[0134] The subject animals have abnormal sensitivity to insulin
and/or leptin relative to a normal animal of the same species. In
certain embodiments, the features exhibited by the subject animals
include, but are not limited to, increased or decreased blood
glucose, increased or decreased blood insulin, increased or
decreased body weight, increased or decreased physical activity,
increased or decreased gat pad content, and increased or decreased
sensitivity to insulin and/or leptin. In these embodiment, subject
animal features may be increased or decreased by about 10% or more,
about 20% or more, about 30% or more, about 50% or more, about 70%
or more, about 80% or more, about 90% or more, or even about 95% or
more as compared to a normal animal of the same species. Such
animals find use in a variety of applications, including the
screening methods described below.
[0135] Screening Assays
[0136] The invention further provides methods of screening a
candidate agent for an activity that modulates sensitivity to
insulin and/or leptin, e.g. stimulators or inhibitors of
sensitivity to insulin and/or leptin. These screening assays
typically provide for qualitative/quantitative measurements of a
phenomenon associated with sensitivity to insulin and/or leptin in
the presence of a particular candidate agent. The screening methods
be performed in vivo, in vitro or in a cell free system, which are
readily developed by those of skill in the art. Test agents that
have a desirable effect in any subject screening assay method find
use in a variety of applications, including modulating sensitivity
to insulin and/or leptin in a host, modulating carbohydrate
metabolism in a host, or treating a disorder associated with
carbohydrate metabolism.
[0137] Using the above screening methods, a variety of different
agents may be identified. Such agents may target the DGAT enzyme
itself, or an expression regulatory factor thereof. Such agents may
be inhibitors or promoters of DGAT activity, where inhibitors are
those agents that result in at least a reduction of DGAT activity
as compared to a control and enhancers result in at least an
increase in DGAT activity as compared to a control. Specific
screening assay methods are described below.
[0138] In vivo Assays
[0139] The invention provides in vivo methods of screening a
candidate agent for an activity that modulates sensitivity to
insulin and/or leptin. In general, the method involves
administering a candidate agent to an animal, particularly a
subject transgenic animal and determining the effect of the
candidate agent on sensitivity of the animal to insulin and/or
leptin.
[0140] The invention further provides in vivo methods of screening
a candidate agent for an activity that modulates carbohydrate
metabolism. In general, the method involves administering a
candidate agent to an animal, particularly a subject transgenic
animal and determining the effect of the candidate agent on a
phenomenon associated with carbohydrate metabolism.
[0141] The invention further provides in vivo methods of screening
a candidate agent for an activity that reduces a symptom of a
condition associated with abnormal carbohydrate metabolism. In
general, the method involves administering a candidate agent to an
animal, particularly a subject transgenic animal and determining
the effect of the candidate agent on a phenomenon associated with
carbohydrate metabolism.
[0142] In many embodiments the determining step of the in vivo
assay method involves measuring a certain phenomenon associated
with sensitivity to insulin and/or leptin or a, phenomenon
associated with carbohydrate metabolism including, but not limited
to: insulin sensitivity or tolerance, leptin sensitivity or
tolerance, lipid composition, adipocyte size, blood glucose levels,
glucose-disposal, glucose tolerance, insulin tolerance, weight
loss, food intake, triacylglycerol content or composition, energy
expenditure, weight, weight gain, activity or an alteration in
lipid content and the like.
[0143] In certain embodiments, the subject animals are placed on a
high fat diet, or a glucose-rich diet, before, during or after
administration of the candidate agent, and compared to similarly
fed controls.
[0144] In vivo assays of the invention include controls, where
suitable controls include a sample in the absence of the test
agent. Generally a plurality of assay mixtures is run in parallel
with different agent concentrations to obtain a differential
response to the various concentrations. Typically, one of these
concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection.
[0145] A candidate agent of interest is one that modulates, i.e.,
reduces or increases, DGAT activity, DGAT expression, lipid (e.g.
TAG) composition, adipocyte size, blood glucose levels, glucose
disposal, glucose tolerance, insulin tolerance, weight loss, food
intake, triacylglycerol content or composition, energy expenditure,
weight, weight gain, activity or alters lipid content etc., by at
least about 10%, at least about 20%, at least about 25%, at least
about 30%, at least about 35%, at least about 40%, at least about
45%, at least about 50%, at least about 55%, at least about 60%, at
least about 65%, at least about 70%, at least about 80%, at least
about 90%, or more, when compared to a control in the absence of
the test agent. In general, a candidate agent will cause a subject
animal to be more similar to an equivalent animal that is not
altered in DGAT activity.
[0146] In vitro Assays
[0147] The invention provides in vitro methods of screening a
candidate agent for an activity that modulates sensitivity to
insulin and/or leptin. In general, the methods involve contacting a
cell with abnormal DGAT activity with a candidate agent and
determining the effect of the agent on the cell in order to assess
the candidate agent's activity that modulates sensitivity to
insulin and/or leptin.
[0148] In many embodiments, the cell with abnormal DGAT activity is
an in which DGAT gene expression has been modified as compared to
an unaltered cell. Methods for altering gene expression in a cell
are well known to one of skill in the art (discussed in Ausubel, et
al, Short Protocols in Molecular Biology, 3rd ed., Wiley &
Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory
Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). These
methods may involve DGAT overexpression via introduction of a
genetic construct designed to express DGAT coding sequences, or may
involve downregulating DGAT expression via knockout strategies
(described in U.S. Pat. Nos. 5,487,992; 5,627,059; 5,631,153; and
6,204,061), or by other methods e.g. antisense, inhibitory RNA
(RNAi), ribozyme or co-supression technologies, as is known in the
art (e.g. Hannon et al., Nature 418:244-51, 2002; Ueda, J
Neurogenet. 15:193-204, 2001; Review. Lindenbach et al., Mol Cell.
9:925-7, 2002; Brantl, Biochim Biophys Acta. 1575:15-25, 2002;
Zhang et al., Ann N Y Acad Sci. 923:210-33, 2000).
[0149] In embodiments where DGAT is overexpressed in a cell, DGAT
expression is increased more than about 1.5-fold, more than about
2-fold, more than about 3-fold, more than about 5-fold, more than
about 10-fold or even more than about 100-fold in the cell, as
compared to an cell in which DGAT is not increased.
[0150] In embodiments where DGAT expression is decreased in a cell,
DGAT expression is decreased by more than about 30%, more than
about 50%, more than about 70%, more than about 90%, more than
about 95% or even more than about 98%, about 99% or 99.5% in the,
as compared to a cell in which DGAT is not decreased.
[0151] Methods for measuring insulin sensitivity in a cell are well
known in the art, and generally involve contacting a cell with
insulin or a mimetic, and assaying glucose uptake (or, for example,
2-deoxyglucose, 3-O-methylglucose, hexose, methylaminoisobutyric
acid etc.), glycogen synthesis, or the expression of certain genes
such as glycogen synthase. Many cell lines may be used for these
methods, including fibroblast cell lines, muscle cell lines, and
hepatoma cell lines. Exemplary publications describing insulin
sensitivity assays are: Klein et al. (Bioessays, 24:382-8, 2002),
Crook et al, (Diabetes 44:314-20, 1995), Saribia et al. (Biochem
Cell Biol 68:536-42, 1990), Meienhofer et al., (Eur J Biochem
169:237-43, 1987), Grunfeld et al. (Endocrinology 113:1763-70,
1983) and several other assays would be immediately apparent to
one-of skill in the art.
[0152] Methods for measuring leptin sensitivity are also well known
the art, and may be performed on a number of cell lines, including
human breast cells, pituitarys cells, hepatoma cells and pancreatic
cells. Exemplary publications describing insulin sensitivity assays
are: Smith et al., (Domest Anim Endocrinol. 22:145-54, 2002),
Tsumanuma et al., (Pituitary. 3:211-20, 2000), Kaser et al., (Int J
Obes Relat Metab Disord. 25:1633-9, 2001), Tanizawa et al.,
(Endocrinology. 138:4513-6, 1997) and Gainsford et al., (Proc Natl
Acad Sci 93:14564-8, 1996) and several other assays would be
immediately apparent to one of skill in the art.
[0153] In certain embodiments the subject cell is a cell from a
subject model animal. In these embodiments a cell from a subject
animal model is isolated and may be cultured to produce a cell that
has altered DGAT activity.
[0154] In certain embodiments the determining step of the in vitro
assay method involves measuring DGAT activity, DGAT expression,
lipid (e.g. TAG) biosynthesis, deposition or secretion and the
like.
[0155] In vitro assays of the invention include controls, where
suitable controls include a sample in the absence of the test
agent. Generally a plurality of assay mixtures is run in parallel
with different agent concentrations to obtain a differential
response to the various concentrations. Typically, one of these
concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection.
[0156] A test agent of interest is one that modulates, i.e.,
reduces or increases, DGAT activity, DGAT expression, lipid (e.g.
TAG) biosynthesis, deposition or secretion, or sensitivity to
insulin and/or leptin by at least about 10%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 45%, at least about 50%, at least about
55%, at least about 60%, at least about 65%, at least about 70%, at
least about 80%, at least about 90%, or more, when compared to a
control in the absence of the test agent. In general, a test agent
will cause a subject cell to be more similar to an equivalent cell
that is not altered in DGAT activity.
[0157] Cell Free Assays
[0158] The invention provides cell free methods of screening a
candidate agent for an activity that modulates sensitivity to
insulin and/or leptin. In general, the methods involve admixing an
extract of a cell (or a synthetic mimetic thereof) with DGAT
activity, or isolated DGAT polypeptide, with a candidate agent and
determining the effect of the agent on the extract in order to
assess the candidate agent's activity that modulates sensitivity to
insulin and/or leptin. In many embodiments the assay methods
involve measuring DGAT activity, lipid (e.g. TAG) biosynthesis, or
and the like,
[0159] Cell free assays of the invention include controls, where
suitable controls include a sample in the absence of the candidate
agent. Generally a plurality of assay mixtures is run in parallel
with different agent concentrations to obtain a differential
response to the various concentrations. Typically, one of these
concentrations serves as a negative control, i.e. at zero
concentration or below the level of detection.
[0160] A test agent of interest is one that modulates, i.e.,
reduces or increases, DGAT activity, lipid biosynthesis or the
like, by at least about 10%, at least about 20%, at least about
25%, at least about 30%, at least about 35%, at least about 40%, at
least about 45%, at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
80%, at least about 90%, or more, when compared to a control in the
absence of the test agent. In general, a candidate agent will cause
a subject extract to be more similar to an equivalent extract from
a cell that is not altered in DGAT activity.
[0161] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc that are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc. may be used.
[0162] Candidate Agents
[0163] The terms "candidate agent," "test agent," "agent",
"substance" and "compound" are used interchangeably herein and
describe a variety of agents that may be screened using the above
methods.
[0164] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. -The candidate agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic
or polyaromatic structures substituted with one or more of the
above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0165] Candidate agents include those found in large libraries of
synthetic or natural compounds. For example, synthetic compound
libraries are commercially available from Maybridge Chemical Co.
(Trevillet, Cornwall, UK), ComGenex (South San Francisco, Calif.),
and MicroSource (New Milford, Conn.). A rare chemical library is
available from Aldrich (Milwaukee, Wis.). Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available from Pan Labs (Bothell, Wash.) or are
readily producible. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs. New potential therapeutic agents may
also be created using methods such as rational drug design or
computer modeling. For example, numerous means are available for
random and directed synthesis of a wide variety of organic
compounds and biomolecules, including expression of randomized
oligonucleotides and oligopeptides.
[0166] Screening may be directed to known pharmacologically active
compounds and chemical analogs thereof, or to new agents with
unknown properties such as those created through rational drug
design.
[0167] A variety of other reagents may be included in screening
assays. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc that are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that improve the efficiency of the assay,
such as protease inhibitors, anti-microbial agents, etc. may be
used. The mixture of components is added in any order that provides
for the requisite binding. Incubations are performed at any
suitable temperature, typically between 4 and 40.degree. C.
Incubation periods are selected for optimum activity, but may also
be optimized to facilitate rapid high-throughput screening.
Typically between 0.1 and 1 hour will be sufficient.
[0168] Candidate agents may also include biopolymers, including
nucleic acids (e.g. DNA, RNA, cDNA, plasmids and this like), for
example those encoding DGAT1 or DGAT2, or antisense DGAT1 or DGAT2
nucleic acids and the like, carbohydrates, lipids (e.g. lipids that
inhibit the activity of DGAT) and proteins and polypeptides, (such
as DGAT1 or DGAT2 or an antibody specific for DGAT1 or DGAT2).
[0169] In particular embodiments, the candidate agent may be
niacin, or other agents known in the art, e.g. those described in
Lesnik et al. (Arch Dermatol Res 1992;284(2):100-5).
[0170] Agents that have an effect in an assay method of the
invention may be further tested for cytotoxicity, bioavailability,
and the like, using well known assays. Agents that have an effect
in an assay method of the invention may be subjected to directed or
random and/or directed chemical modifications, such as acylation,
alkylation, esterification, amidification, etc. to produce
structural analogs. Such structural analogs include those that
increase bioavailability, and/or reduced cytotoxicity. Those
skilled in the art can readily envision and generate a wide variety
of structural analogs, and test them for desired properties such as
increased bioavailability and/or reduced cytotoxicity and/or
ability to cross the blood-brain barrier.
[0171] The following examples are presented for purposes of
illustration only, and are not to be construed as limiting on the
scope of the invention in any way.
EXPERIMENTAL
[0172] Materials and Methods
[0173] Mice: Dgat1.sup.-/- mice (about 95% C57BL/6 and 5% 129/SvJae
background) were generated previously (Smith, et al.(2000) Nat.
Genet. 25:87-90). Wild-type (Dgat1.sup.+/+), ob/+, and A.sup.Y/a
mice (all in C57BL/6 background) were from the The Jackson
Laboratory (Bar Harbor, Me. USA). A.sup.Y/a mice are obese and
insulin resistant, reflecting the antagonism of
melanocyte-stimulating hormone in the hypothalamus. They are also
severely leptin resistant. Genotyping for Dgat1 and ob was
performed as described (Smith et al., supra). A.sup.Y/a mice were
identified by their yellow fur. Mice were housed in a pathogen-free
barrier facility (12-hour light/12-hour dark cycle) and fed rodent
chow (Ralston Purina Co., St. Louis, Miss., USA). For high-fat diet
experiments, mice were fed a Western-type diet containing 21% fat
by weight (Harlan Teklad Laboratory, Madison, Wis., USA) for 4
weeks unless stated otherwise. All experiments were approved by the
Committee on Animal Research of the University of California, San
Francisco.
[0174] Tissue lipid analysis. Tissue lipids were extracted and
separated by TLC as described (Smith et al., supra). Triglycerides
and diacylglycerol were scraped from the TLC plate, and
heptadecanoic acid (about 10% of total fatty acids) was added as an
internal standard. Methyl esters were synthesized with the addition
of methanolic HCl/toluene (4:1 vol/vol) to the TLC adsorbent for 12
hours at 37.degree. C., extracted with hexane, and analyzed with
gas-liquid chromatography (HP6890 Gas Chromatograph;
Hewlett-Packard, Palo Alto, Calif., USA). Methyl esters were
separated with a 10-ft glass column (10% SP-2330 on 100/120
Chromosorb WAW; Supelco, St. Louis, Mo., USA) at 175.degree. C. for
5 minutes and increasing to 210.degree. C. at a rate of 2.5.degree.
C./min. The weight of triglycerides and diacylglycerol in the
samples was calculated with reference to the internal standard.
[0175] Adipocyte size determination: Adipose tissue was obtained
from the reproductive fat pads of 14-week-old male mice. The
samples were fixed in paraformaldehyde, embedded in paraffin, cut
into 5-.mu.m sections, and stained with hematoxylin and eosin.
Images of the histology sections were analyzed with Adobe Photoshop
5.0.1 (Adobe Systems Inc., San Jose, Calif., USA) and Image Process
Tool Kit (Reindeer Games, Gainesville, Fla., USA) as described
(Chen and Farese, J. Lipid Res. 43:986-9, 2002).
[0176] Glucose metabolism studies: Glucose (1 g/kg body wt) or
bovine insulin (1 U/kg body wt; Sigma Chemical Co., St. Louis, Mo.,
USA) was injected intraperitoneally, and glucose concentrations
were measured with a glucometer (Accu-Chek; Roche Diagnostics
Corp., Indianapolis, Ind., USA). The hyperinsulinemic-euglycemic
clamp studies were performed as described (Ferriera Diabetes.
50:1064-1068, 2001) with slight modifications. Weight-matched 10-
to 14-week-old male mice were fasted for4 hours before the clamp
studies. Insulin was infused at 20 mU/kg/min, and plasma glucose
concentration was clamped at 120 mg/dl. For ob/ob and A.sup.Y/a
mice, nonfasting blood samples for glucose and insulin measurements
were obtained at noon. Insulin was measured with a rat insulin RIA
kit (Linco Research Inc., St. Charles, Mo., USA).
[0177] Leptin infusion studies: Mice were infused with recombinant
human leptin (gift from F. Chehab, ,University of California, San
Francisco) with a microosmotic pump (Alza model 1002; DURECT Corp.,
Cupertino, Calif., USA),inserted subcutaneously into the
interscapular region. Plasma leptin levels were measured by
AniLytics Inc. (Gaithersburg, Md., USA).
[0178] Northern blots. White adipose tissue (WAT) was obtained from
the reproductive fat pads and brown adipose tissue (BAT) from the
interscapular region of 10- to 14-week-old male mice. Total RNA was
isolated, and pooled RNA samples (10 .mu.g) were subjected to
electrophoresis and blot hybridization with .sup.32P-labeled cDNA
probes. Blots were rehybridized with a .beta.-actin probe (Ambion
Inc., Austin, Tex., USA) for loading normalization. Probes for
uncoupling protein 2 (UCP-2) and UCP-3 were generated by PCR with
WAT cDNA and the following primers: 5'-GTCGATTCCGCCCTCGGTG-3' (SEQ
ID NO:13), 5'-GAGGGAAAGTGATGAGATCT-3' (SEQ ID NO:14) (UCP-2);
5'-GTCGGACACAGCCTTCTGC-3', (SEQ ID NO:15)5'-ACCTTGGACCGCCAGCCGG-3'
(SEQ ID NO:16) (UCP-3). The remaining probes were gifts from M.
Reitman, NIH, Bethesda, Md., USA (UCP-1); B. Staels, Institut
National de la Sant et de la Recherche Mdicale (Lille, France)
(acyl CoA oxidase); B. Spiegelman, Dana-Farber Cancer Institutes,
Boston, Mass., USA (peroxisome proliferator-activated receptor);
and I. Shimomura, M. Brown, and J. Goldstein, University of Texas
Southwestern, Dallas, Tex., USA (fatty acid synthase). Signals were
quantified with a PhosphorImager (Bio-Rad Laboratories, Hercules,
Calif., USA).
[0179] Real-time PCR: Tissues were homogenized, and total RNA was
extracted. RNA (1 .mu.g) was reverse-transcribed in a 20-.mu.l
reaction containing oligo(dT).sub.12-18 primer and Superscript II
enzyme (Invitrogen Corp., Carlsbad, Calif., USA). Primer and probe
sequences (Table 1) were selected with Primer Express (Perkin-Elmer
Applied Biosystems, Foster City, Calif., USA). The PCR reaction (50
.mu.l) contained 1 .mu.l of cDNA, 1.times. Gold buffer, 4 mM
MgCl.sub.2, 500 .mu.M dNTP, primers (200 nM), 100 nM probe (labeled
with 6-carboxyfluorescein), and 1.25 U of AmpliTaq Gold DNA
polymerase (Perkin-Elmer Applied Biosystems). Real-time PCR was
performed with the ABI Prism 7700 System (Perkin-Elmer Applied
Biosystems). Expression levels were calculated by the comparative
cycle of threshold detection method, according to the manufacturer.
Expression of .beta.-actin was used for loading normalization.
[0180] Statistical methods: Data are shown as mean.+-.SD unless
stated otherwise. Measurements were compared with the t test or
Mann-Whitney rank-sum test. Differences in body weight or food
intake were compared with ANOVA, followed by the Tukey-Kramer
test.
1TABLE 1 Real-time PCR primer and probe sequences Table 1 Primer
pair or Gene: probe Sequence Actin 5' 5'-CATCTTGGCCTCACTGTCCA-3'
SEQ ID NO:17 3' 5'-GGGCCGGACTCATCGTACT-3' SEQ ID NO:18 Probe
5'-CTTCCAGCAGATGTGGATCAGCAAGC-3' SEQ ID NO:19 DGAT2 5'
5'-AGTGGCAATGCTATCATCATCGT-3' SEQ ID NO:20 3'
5'-AAGGAATAAGTGGGAACCCAGATCA-3' SEQ ID NO:21 Probe
5'-CCTGGCAAGAACGCAGTCACCCTG-3' SEQ ID NO:22 Leptin 5'
5'-TCTCCGAGACCTCCTCCATCT-3' SEQ ID NO:23 3'
5'-TTCCAGGACGCCATCCAG-3' SEQ ID NO:24 Probe
5'-TCCCTGCCTCAGACCAGTGGCCT-3' SEQ ID NO:25 PPAR.varies. 5'
5'-CAGGAGAGCAGGGATTTGCA-3' SEQ ID NO:26 3'
5'-CCTACGCTCAGCCCTCTTCAT-3' SEQ ID NO:27 Probe
5'-AGAGGGCCTCCCTCCTACGCTTGG-3' SEQ ID NO:28
EXAMPLE I
Preparation and Characterization of DGAT Knockout Mice
[0181] DGAT knockout mice were generated using standard techniques
of gene targeting. A mouse P1 clone containing the mouse DGAT gene
was isolated from a genomic 129/Sv library. Short and long arms of
homologous sequences were amplified by PCR from this clone and
subcloned intopNTKLoxP to generate a gene targeting vector. The
vector contained a neomycin resistance gene for positive selection
and a thymidine kinase gene for negative selection. Upon homologous
recombination, the vector was designed to interrupt the DGAT coding
sequences at amino acid 360 of the 498-amino acid murine protein.
The entire C-terminus, including a highly conserved region common
to all ACAT gene family members is deleted. The gene targeting
vector was electroporated into RF8 embryonic stem cells by
electroporation, and several targeted clones were identified by
Southern blotting (targeting frequency of .about.1 in 300).
[0182] One of these targeted clones was injected into C57BL/6
blastocysts and chimeras were generated; male chimeras subsequently
passed the DGAT knockout mutation through the germline to their
offspring. The resultant mice, which were heterozygous for the DGAT
gene disruption, were intercrossed to generate mice that were
homozygotes.
[0183] Inactivation of the DGAT-gene in the homozygote knockouts
was verified by examining DGAT mRNA which was found to be absent in
the knockout mice. In activation of the DGAT gene was also verified
by studying DGAT activity in tissues using an assay that measures
the incorporation of [14C]oleoyl.CoA into triglycerides. The
results from the activity assays show that DGAT activity is
virtually gone from every nearly every tissue tested.
EXAMPLE II
Altered Lipid Composition in Tissues of Dgat1.sup.-/- Mice
[0184] Dgat1.sup.-/- mice had a 30-40% reduction of triglyceride
levels in WAT and skeletal muscle (Table 2). Liver triglyceride
levels trended lower in chow-fed Dgat1.sup.-/- mice. On a high-fat
diet, however, Dgat1.sup.-/- mice had significantly lower liver
triglyceride levels than Dgat1.sup.+/+ mice (28.+-.16 versus
157.+-.28 mg/g tissue weight, P<0.05). Unexpectedly, levels of
diacylglycerol, a substrate for the DGAT reaction, were not
elevated, and in fact tended to be lower in Dgat1.sup.-/- tissues
(Table 2). DGAT1 deficiency also altered the fatty acid composition
of triglycerides in WAT and skeletal muscle, resulting in a
relative decrease in monounsaturated (16:1 and 18:1) fatty acids
and a relative increase in saturated (16:0 and 18:0) fatty acids
(Table 2).
2TABLE 2 Lipid composition of tissues in Dgat1 and Dgat1.sup.++
mice Fatty acid composition of triglycerides Triglycerides
Diacylglycerol (% of total fatty acids) (% of tissue weight) 16:0
16:1 18:0 18:1 18:2w6 18:3w6 18:3w3 White adipose tissue +/+ 32.4
.+-. 7.9 0.06 .+-. 0.02 20.0 .+-. 0.6 8.4 .+-. 1.2 1.8 .+-. 0.2
37.3 .+-. 1.2 25.3 .+-. 1.9 0.3 .+-. 0.3 0.7 .+-. 0.1 -/- 20.3 .+-.
3.0.sup.A 0.04 .+-. 0.01 26.4 .+-. 1.5.sup.B 4.5 .+-. 1.4.sup.B 3.8
.+-. 1.0.sup.B 34.2 .+-. 1,3.sup.A 25.3 .+-. 3.4 0.1 .+-. 0.3 0.9
.+-. 0.4 Skeletal muscle +/+ 4.3 .+-. 1.9 0.008 .+-. 0.002 21.0
.+-. 2.0 7,7 .+-. 1.1 <0.001 38.0 .+-. 1.9 24.3 .+-. 2.8 0.2
.+-. 0.2 1.1 .+-. 0.6 -/- 2.4 .+-. 0.5.sup.A 0.006 .+-. 0.003 27.3
.+-. 0.7.sup.B 3.1 .+-. 1.1.sup.B <0.001 33.5 .+-. 1.7.sup.A
23.3 .+-. 2.8 0.3 .+-. 0.2 1.3 .+-. 0.4 Liver +/+ 0.2 .+-. 0.1
0.013 .+-. 0.003 ND ND ND ND ND ND ND -/- 0.1 .+-. 0.0 0.003 .+-.
0.001.sup.A Twelve to sixteen-week-old male mice fed a chow diet
were used ND, not determined. .sup.AP < 0.05, .sup.BP < 0.01
versus Dgat1.sup.++ n = 3 per genotype.
EXAMPLE III
Decreased Adipocyte Size in Dgat1.sup.-/- Mice
[0185] Concomitant with the decreased triglyceride levels in WAT,
Dgat1.sup.-/- mice had smaller adipocytes than Dgat1.sup.+/+ mice
on both chow and high-fat diets (FIG. 1). Adipocytes from
Dgat1.sup.-/- mice developed minimal hypertrophy in response to a
high-fat diet, whereas those from Dgat1.sup.+/+ mice doubled in
size. This protection from diet-induced adipocyte hypertrophy in
Dgat1.sup.-/- mice mirrored their weight response to a high-fat
diet. In addition to having smaller adipocytes, male Dgat1.sup.-/-
mice had a lower mean DNA content in reproductive fat pads than
Dgat1.sup.+/+ mice (169.+-.36 versus 273.+-.50 .mu.g/fat pad,
P<0.05), showing that Dgat1.sup.-/- mice also had fewer
adipocytes.
EXAMPLE IV
Increased Insulin Sensitivity in Dgat1.sup.-/- mice
[0186] Glucose- and insulin-tolerance tests were performed.
Dgat1.sup.-/- mice had lower plasma glucose concentrations than
Dgat1.sup.+/+ mice after a glucose load (FIG. 2a) or an insulin
injection (FIG. 2b), showing that Dgat1.sup.-/- mice have increased
insulin sensitivity. This was confirmed in
hyperinsulinemic-euglycemic clamp studies, which showed that
Dgat1.sup.-/- mice required an approximately 20% higher glucose
infusion rate than Dgat1.sup.+/+ mice to maintain euglycemia (FIG.
2c).
EXAMPLE V
Increased Weight Loss in Response to Leptin Infusion in
Dgat1.sup.-/- Mice
[0187] Leptin was subcutaneously infused into Dgat1.sup.+/+ mice
and Dgat1.sup.-/- mice and measured their response in body weight
and food intake. Leptin infusion achieved comparable levels of
increase in plasma leptin levels in Dgat1.sup.+/+ and Dgat1.sup.-/-
mice (0.7.+-.0.1 versus 0.5.+-.0.2 ng/ml for 6 .mu.g/day,
P>0.05). In young (10- to 14-week-old) Dgat1.sup.+/+ mice,
leptin administration suppressed the normal weight gain seen in
control (PBS-infused) mice (FIG. 3a). The same doses of leptin
caused an additional 3% weight loss in age-matched Dgat1.sup.-/-
mice (FIG. 3a), indicating an enhanced response to leptin.
[0188] Dgat1.sup.-/- mice consistently ate more than Dgat1.sup.+/+
mice at baseline (25.3%.+-.1.6% versus 19.5%.+-.3.2% of body
weight, P<0.05). During leptin infusion, Dgat1.sup.-/- mice
continued to eat more than Dgat1.sup.+/+ mice (19.8%.+-.3% versus
15.8%.+-.2.4% of body weight after 5 days of leptin (6 .mu.g/day)
infusion, P<0.05). Expressed as percentage of change from
baseline, reduction in food intake in response to leptin infusion
was comparable in Dgat1.sup.-/- and Dgat1.sup.+/+ mice (FIG. 3, c
and d). Similarly, the absolute reduction in food intake per day
was similar in Dgat1.sup.+/+ and Dgat1.sup.-/- mice in response to
leptin infusion in several different experiments (not shown). These
results imply that the increased weight loss in leptin-treated
Dgat1.sup.-/- mice resulted from increased energy expenditure
rather than reduced food intake.
EXAMPLE VI
Expression of Leptin-Regulated Genes in Dgat1.sup.-/- Mice
[0189] The expression of several leptin-regulated genes in BAT and
WAT of Dgat1.sup.-/- mice was measured (FIG. 4). In Dgat1.sup.-/-
BAT, UCP 1 expression was increased by approximately 70% versus
controls. In WAT, Dgat1.sup.-/- mice had increased levels of UCP3
expression, and UCP2 expression levels trended higher. The
expression of genes involved in fatty acid oxidation (peroxisome
proliferator-activated receptor .alpha.[PPAR.alpha.] and acyl CoA
oxidase) was also higher in Dgat1.sup.-/- WAT. In contrast,
Dgat1.sup.-/- mice had decreased levels of expression for genes
involved in adipogenesis (PPAR.gamma.) and lipid synthesis (fatty
acid synthase) in WAT. Dgat1.sup.-/- mice also had an approximate
50% reduction in leptin (ob) mRNA expression. These results show
that Dgat1.sup.-/- mice have increased leptin sensitivity at
baseline.
EXAMPLE VII
DGAT1 Deficiency Protects Against Obesity and Insulin Resistance in
A.sup.Y/a Mice but not in ob/ob Mice
[0190] DGAT1 deficiency protects against obesity and insulin
resistance induced by high-fat feeding. We introduced DGAT1
deficiency into A.sup.y/a and ob/ob mice through breeding. DGAT1
deficiency protected against obesity in A.sup.Y/a mice (FIG. 5a),
decreasing body weight (FIG. 5b) and fat pad content (FIG. 5c) by
approximately 20% at 25 weeks. A.sup.Y/a Dgat1.sup.-/- and
A.sup.Y/a Dgat1.sup.+/+ mice had similar plasma glucose levels
(FIG. 5d), but DGAT1 deficiency reduced plasma insulin levels by
approximately 80% in A.sup.Y/a mice (FIG. 5e), most likely by
increasing their insulin sensitivity. In contrast, DGAT1 deficiency
had no apparent effects in ob/ob mice. In the setting of leptin
deficiency; both Dgat1.sup.-/- and Dgat1.sup.+/+ mice became obese
(FIG. 5f) and diabetic, with similar growth curves (FIG. 5g), fat
pad content (FIG. 5h), and plasma glucose (FIG. 5i) and insulin
(FIG. 5j) levels. DGAT1 deficiency also had no apparent effect on
the obesity and diabetes of mice lacking functional leptin
receptors (db/db mice, not shown).,
EXAMPLE VIII
Increased DGAT2 mRNA Expression in WAT of Leptin-Deficient
Dgat1.sup.-/- Mice
[0191] DGAT2 mRNA expression in WAT of Dgat1.sup.-/- mice in
different backgrounds and conditions was measured (FIG. 6). DGAT2
expression was not increased in Dgat1.sup.-/- mice at baseline
(chow) or after 15 weeks of a high-fat diet. DGAT2 expression was
also not increased in A.sup.Y/a Dgat1.sup.-/- mice. In contrast,
DGAT2 expression was elevated approximately threefold in
leptin-deficient Dgat1.sup.-/- mice. This suggests that leptin
normally downregulates DGAT2 expression in WAT and that the
upregulation of DGAT2 expression may compensate for the loss of
DGAT1 in leptin-deficient Dgat1.sup.-/- mice.
[0192] It is evident from the above results and discussion that the
subject invention provides an important method for modulating
sensitivity to insulin and/or leptin which method may be used for
the treatment of diseases and conditions associated with altered
sensitivity to leptin and/or insulin, e.g. conditions associated
with abnormal carbohydrate metabolism such as diabetes and obesity.
As such, the subject methods and systems find use in a variety of
different applications, including research, industry, and medicine.
Accordingly, the present invention represents a significant
contribution to the art.
[0193] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. The
citation of any publication is for its disclosure prior to the
filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by
virtue of prior invention.
[0194] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
Sequence CWU 1
1
4 1 495 PRT Arabidopsis thaliana 1 Met Glu Thr Lys Pro Asn Pro Arg
Arg Pro Ser Asn Thr Val Leu Pro 1 5 10 15 Tyr Gln Thr Pro Arg Leu
Arg Asp His Tyr Leu Leu Gly Lys Lys Leu 20 25 30 Gly Gln Gly Gln
Phe Gly Thr Thr Tyr Leu Cys Thr Glu Lys Ser Thr 35 40 45 Ser Ala
Asn Tyr Ala Cys Lys Ser Ile Pro Lys Arg Lys Leu Val Cys 50 55 60
Arg Glu Asp Tyr Glu Asp Val Trp Arg Glu Ile Gln Ile Met His His 65
70 75 80 Leu Ser Glu His Pro Asn Val Val Arg Ile Lys Gly Thr Tyr
Glu Asp 85 90 95 Ser Val Phe Val His Ile Val Met Glu Val Cys Glu
Gly Gly Glu Leu 100 105 110 Phe Asp Arg Ile Val Ser Lys Gly His Phe
Ser Glu Arg Glu Ala Val 115 120 125 Lys Leu Ile Lys Thr Ile Leu Gly
Val Val Glu Ala Cys His Ser Leu 130 135 140 Gly Val Met His Arg Asp
Leu Lys Pro Glu Asn Phe Leu Phe Asp Ser 145 150 155 160 Pro Lys Asp
Asp Ala Lys Leu Lys Ala Thr Asp Phe Gly Leu Ser Val 165 170 175 Phe
Tyr Lys Pro Gly Gln Tyr Leu Tyr Asp Val Val Gly Ser Pro Tyr 180 185
190 Tyr Val Ala Pro Glu Val Leu Lys Lys Cys Tyr Gly Pro Glu Ile Asp
195 200 205 Val Trp Ser Ala Gly Val Ile Leu Tyr Ile Leu Leu Ser Gly
Val Pro 210 215 220 Pro Phe Trp Ala Glu Thr Glu Ser Gly Ile Phe Arg
Gln Ile Leu Gln 225 230 235 240 Gly Lys Leu Asp Phe Lys Ser Asp Pro
Trp Pro Thr Ile Ser Glu Ala 245 250 255 Ala Lys Asp Leu Ile Tyr Lys
Met Leu Glu Arg Ser Pro Lys Lys Arg 260 265 270 Ile Ser Ala His Glu
Ala Leu Cys His Pro Trp Ile Val Asp Glu Gln 275 280 285 Ala Ala Pro
Asp Lys Pro Leu Asp Pro Ala Val Leu Ser Arg Leu Lys 290 295 300 Gln
Phe Ser Gln Met Asn Lys Ile Lys Lys Met Ala Leu Arg Val Ile 305 310
315 320 Ala Glu Arg Leu Ser Glu Glu Glu Ile Gly Gly Leu Lys Glu Leu
Phe 325 330 335 Lys Met Ile Asp Thr Asp Asn Ser Gly Thr Ile Thr Phe
Glu Glu Leu 340 345 350 Lys Ala Gly Leu Lys Arg Val Gly Ser Glu Leu
Met Glu Ser Glu Ile 355 360 365 Lys Ser Leu Met Asp Ala Ala Asp Ile
Asp Asn Ser Gly Thr Ile Asp 370 375 380 Tyr Gly Glu Phe Leu Ala Ala
Thr Leu His Met Asn Lys Met Glu Arg 385 390 395 400 Glu Glu Ile Leu
Val Ala Ala Phe Ser Asp Phe Asp Lys Asp Gly Ser 405 410 415 Gly Tyr
Ile Thr Ile Asp Glu Leu Gln Ser Ala Cys Thr Glu Phe Gly 420 425 430
Leu Cys Asp Thr Pro Leu Asp Asp Met Ile Lys Glu Ile Asp Leu Asp 435
440 445 Asn Asp Gly Lys Ile Asp Phe Ser Glu Phe Thr Ala Met Met Arg
Lys 450 455 460 Gly Asp Gly Val Gly Arg Ser Arg Thr Met Met Lys Asn
Leu Asn Phe 465 470 475 480 Asn Ile Ala Asp Ala Phe Gly Val Asp Gly
Glu Lys Ser Asp Asp 485 490 495 2 1747 DNA Arabidopsis thaliana 2
gatccgggta catattcttc ttcttcttca aatcgagatc gaagaagaac caacaaaaaa
60 ccaaaaatgg agacgaagcc aaaccctaga cgtccttcaa acacagttct
accatatcaa 120 acaccacgat taagagatca ttaccttctg ggaaaaaagc
taggccaagg ccaatttgga 180 acaacctatc tctgcacaga gaaatcaacc
tccgctaatt acgcctgcaa atcgatcccg 240 aagcgaaagc tcgtgtgtcg
cgaggattac gaagatgtat ggcgtgagat tcagatcatg 300 catcatctct
ctgagcatcc aaatgttgtt aggatcaaag ggacttatga agattcggtg 360
tttgttcata ttgttatgga ggtttgtgaa ggtggtgagc tttttgatcg gattgtttct
420 aaaggtcatt ttagtgagcg tgaagctgtc aagcttatta agacgattct
tggtgttgtt 480 gaggcttgtc attctcttgg tgttatgcat agagatctca
aacctgagaa tttcttgttt 540 gatagtccta aagatgatgc taagcttaag
gctaccgatt ttggtttgtc tgtcttctat 600 aagccaggac aatatttata
tgacgtagtt ggaagtccgt actatgttgc accagaggtg 660 ctaaagaaat
gttatggacc tgaaatagat gtgtggagtg ctggtgttat cctctacatt 720
ttactcagcg gtgttcctcc cttctgggca gagactgagt ctggaatctt tagacagata
780 ttgcaaggga agttagattt caaatctgac ccgtggccta ctatctcaga
agctgctaaa 840 gatttgatct ataaaatgct cgaaaggagc cccaagaaac
gcatttctgc tcatgaagcc 900 ttgtgtcacc catggattgt cgatgaacaa
gcagcaccag acaagcctct tgatccagca 960 gtcttatctc gtctaaagca
gttttctcaa atgaataaga ttaagaaaat ggcattacgg 1020 gtaattgctg
agagactttc agaggaagaa attggaggtc tgaaggaatt gttcaagatg 1080
atagacacag acaacagcgg aacgattact tttgaagagc tcaaagcggg tttgaagaga
1140 gtcggatctg aactgatgga atcagaaatc aagtctctca tggatgcggc
tgatatcgac 1200 aacagtggta caatagacta cggagaattc ctagcagcaa
ccttacacat gaacaagatg 1260 gagagagagg agattctggt ggctgcattt
tcggactttg acaaagacgg aagcggttat 1320 atcaccatcg atgagcttca
gtcagcttgc acagagtttg gtctatgtga tacacctctg 1380 gacgacatga
tcaaggagat tgatcttgac aatgacggga agatcgattt ctcggagttt 1440
acagcaatga tgaggaaagg agatggagtt gggagaagca gaaccatgat gaagaacttg
1500 aacttcaaca ttgctgatgc ttttggagtt gatggtgaaa aatctgatga
ctgactcatc 1560 attcttccac aatttctgtt ttttttctct ttaatttcgt
ttatattttg aattctaatt 1620 tctaaggata caaaaatata ttctggcttg
ttttttgctt tcctttttat ttttgtacat 1680 gagcaacttt ctaaattttt
atcctcatat ggataatttt tgcttcatat aaaagttttt 1740 gaattcc 1747 3 501
PRT Arabidopsis thaliana 3 Met Glu Lys Pro Asn Pro Arg Arg Pro Ser
Asn Ser Val Leu Pro Tyr 1 5 10 15 Glu Thr Pro Arg Leu Arg Asp His
Tyr Leu Leu Gly Lys Lys Leu Gly 20 25 30 Gln Gly Gln Phe Gly Thr
Thr Tyr Leu Cys Thr Glu Lys Ser Ser Ser 35 40 45 Ala Asn Tyr Ala
Cys Lys Ser Ile Pro Lys Arg Lys Leu Val Cys Arg 50 55 60 Glu Asp
Tyr Glu Asp Val Trp Arg Glu Ile Gln Ile Met His His Leu 65 70 75 80
Ser Glu His Pro Asn Val Val Arg Ile Lys Gly Thr Tyr Glu Asp Ser 85
90 95 Val Phe Val His Ile Val Met Glu Val Cys Glu Gly Gly Glu Leu
Phe 100 105 110 Asp Arg Ile Val Ser Lys Gly Cys Phe Ser Glu Arg Glu
Ala Ala Lys 115 120 125 Leu Ile Lys Thr Ile Leu Gly Val Val Glu Ala
Cys His Ser Leu Gly 130 135 140 Val Met His Arg Asp Leu Lys Pro Glu
Asn Phe Leu Phe Asp Ser Pro 145 150 155 160 Ser Asp Asp Ala Lys Leu
Lys Ala Thr Asp Phe Gly Leu Ser Val Phe 165 170 175 Tyr Lys Pro Gly
Gln Tyr Leu Tyr Asp Val Val Gly Ser Pro Tyr Tyr 180 185 190 Val Ala
Pro Glu Val Leu Lys Lys Cys Tyr Gly Pro Glu Ile Asp Val 195 200 205
Trp Ser Ala Gly Val Ile Leu Tyr Ile Leu Leu Ser Gly Val Pro Pro 210
215 220 Phe Trp Ala Glu Thr Glu Ser Gly Ile Phe Arg Gln Ile Leu Gln
Gly 225 230 235 240 Lys Ile Asp Phe Lys Ser Asp Pro Trp Pro Thr Ile
Ser Glu Gly Ala 245 250 255 Lys Asp Leu Ile Tyr Lys Met Leu Asp Arg
Ser Pro Lys Lys Arg Ile 260 265 270 Ser Ala His Glu Ala Leu Cys His
Pro Trp Ile Val Asp Glu His Ala 275 280 285 Ala Pro Asp Lys Pro Leu
Asp Pro Ala Val Leu Ser Arg Leu Lys Gln 290 295 300 Phe Ser Gln Met
Asn Lys Ile Lys Lys Met Ala Leu Arg Val Ile Ala 305 310 315 320 Glu
Arg Leu Ser Glu Glu Glu Ile Gly Gly Leu Lys Glu Leu Phe Lys 325 330
335 Met Ile Asp Thr Asp Asn Ser Gly Thr Ile Thr Phe Glu Glu Leu Lys
340 345 350 Ala Gly Leu Lys Arg Val Gly Ser Glu Leu Met Glu Ser Glu
Ile Lys 355 360 365 Ser Leu Met Asp Ala Ala Asp Ile Asp Asn Ser Gly
Thr Ile Asp Tyr 370 375 380 Gly Glu Phe Leu Ala Ala Thr Leu His Ile
Asn Lys Met Glu Arg Glu 385 390 395 400 Glu Asn Leu Val Val Ala Phe
Ser Tyr Phe Asp Lys Asp Gly Ser Gly 405 410 415 Tyr Ile Thr Ile Asp
Glu Leu Gln Gln Ala Cys Thr Glu Phe Gly Leu 420 425 430 Cys Asp Thr
Pro Leu Asp Asp Met Ile Lys Glu Ile Asp Leu Asp Asn 435 440 445 Asp
Gly Lys Ile Asp Phe Ser Glu Phe Thr Ala Met Met Lys Lys Gly 450 455
460 Asp Gly Val Gly Arg Ser Arg Thr Met Arg Asn Asn Leu Asn Phe Asn
465 470 475 480 Ile Ala Glu Ala Phe Gly Val Glu Asp Thr Ser Ser Thr
Ala Lys Ser 485 490 495 Asp Asp Ser Pro Lys 500 4 1657 DNA
Arabidopsis thaliana 4 atggagaaac caaaccctag aagaccctca aacagtgttc
ttccatacga aacaccaaga 60 ttaagagatc actatctcct cggcaaaaag
ctaggccaag gccaatttgg aacaacctat 120 ctctgtacag agaaatcatc
atcagctaat tacgcttgca aatcaatccc aaaacgtaag 180 cttgtatgtc
gtgaagacta cgaagatgta tggcgtgaga ttcagatcat gcatcatctc 240
tctgagcatc ctaatgttgt tagaatcaag ggtacttatg aagactctgt ttttgttcac
300 attgttatgg aagtttgtga aggtggtgag ctttttgatc ggattgtttc
taaagggtgt 360 tttagtgaac gtgaagctgc taagttgatt aagactattc
ttggtgttgt tgaggcttgt 420 cattctcttg gtgttatgca tagagatctt
aagcctgaga atttcttgtt tgatagtccc 480 agtgatgatg ctaagcttaa
agctacagac tttggtttgt ctgtcttcta caagccaggg 540 cagtatctgt
atgatgtagt tggaagtccg tattatgttg cacctgaggt tctgaagaaa 600
tgttatggac cagagataga cgtgtggagc gccggtgtta tcttgtacat cttactaagt
660 ggggttcctc ctttttgggc agaaaccgag tcaggaatct ttaggcagat
attgcaaggg 720 aagatagatt ttaaatctga tccgtggcct actatctcag
aaggtgctaa agatttgatt 780 tacaaaatgc tcgataggag ccccaagaaa
cgtatttctg cacatgaagc attgtgtcac 840 ccttggattg ttgatgaaca
tgctgcacca gacaagcctc tcgacccagc agtcttgtcg 900 cgacttaagc
agttctcgca aatgaataaa atcaagaaaa tggccttacg agtaatcgcg 960
gagagactct cggaggaaga gattggtggt ctgaaggaat tgttcaaaat gatagataca
1020 gacaacagtg gaacaatcac ctttgaagag cttaaagcag gtctaaagag
agttggatct 1080 gaattgatgg aatcagaaat caagtctctt atggatgcgg
cggatataga caacagtggg 1140 acaatagact acggtgaatt cctagcagcg
acattacata taaacaagat ggagagagaa 1200 gagaacttgg tggttgcgtt
ttcatacttt gataaagatg gtagcggtta tatcaccatt 1260 gacgagcttc
aacaagcctg cacagagttt ggtctctgtg acactcctct tgatgacatg 1320
atcaaagaga ttgatcttga taatgacggg aagattgatt tctcagagtt tactgctatg
1380 atgaagaaag gagatggtgt tgggaggagc agaactatga ggaacaactt
gaacttcaat 1440 atagctgaag cttttggagt tgaggacaca agcagcactg
ctaaatctga tgattcacca 1500 aagtaattat aatcatctat atacttggaa
ttgagaaatg agaactcaca aaagaaaaac 1560 tgaatctttc ctttttgttt
tcgtttccac ttttgtagat gagcaacttt ctcaattttg 1620 ttataagcat
ggataatttt gcttcatatt ttctgcg 1657
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