U.S. patent application number 11/799397 was filed with the patent office on 2007-09-06 for inhibition of jun kinase.
This patent application is currently assigned to Harvard University, President and Fellows of Harvard College. Invention is credited to Lufen Chang, Gokhan S. Hotamisligil, Michael Karin.
Application Number | 20070207137 11/799397 |
Document ID | / |
Family ID | 35375972 |
Filed Date | 2007-09-06 |
United States Patent
Application |
20070207137 |
Kind Code |
A1 |
Hotamisligil; Gokhan S. ; et
al. |
September 6, 2007 |
Inhibition of Jun Kinase
Abstract
A method of treating a metabolic disorder associated with
insulin resistance by administering to a mammal an inhibitor of a
NH2-terminal Jun Kinase (JNK), e.g., a compound or peptide which
inhibits JNK1 expression or enzymatic activity.
Inventors: |
Hotamisligil; Gokhan S.;
(Wellesley, MA) ; Karin; Michael; (La Jolla,
CA) ; Chang; Lufen; (La Jolla, CA) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Harvard University, President and
Fellows of Harvard College
Cambridge
MA
|
Family ID: |
35375972 |
Appl. No.: |
11/799397 |
Filed: |
May 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10475505 |
May 3, 2004 |
7232897 |
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PCT/US02/12687 |
Apr 24, 2002 |
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11799397 |
May 1, 2007 |
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60285966 |
Apr 24, 2001 |
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Current U.S.
Class: |
424/94.1 |
Current CPC
Class: |
A61K 31/675
20130101 |
Class at
Publication: |
424/094.1 |
International
Class: |
A61K 38/53 20060101
A61K038/53 |
Claims
1. A method of treating a metabolic disorder associated with
insulin resistance, comprising administering to a mammal an
inhibitor of a NH2-terminal Jun Kinase (JNK).
2. The method of claim 1, wherein said inhibitor binds to an ATP
binding site in JNK.
3. The method of claim 1, wherein said inhibitor binds to a
catalytic domain of JNK.
4. The method of claim 1, wherein said JNK is JNK1.
5. The method of claim 1, wherein said JNK is JNK2.
6. The method of claim 1, wherein said JNK is JNK1 and JNK2.
7. The method of claim 1, wherein said inhibitor is SP600125.
8. A method of improving insulin sensitivity, comprising
administering to a mammal an inhibitor of a NH2-terminal Jun Kinase
(JNK).
9. A method of treating or preventing the development of obesity in
an individual, comprising administering to said individual an
inhibitor of a NH2-terminal Jun Kinase (JNK).
10. A method of diagnosing insulin resistance or a risk of
developing insulin resistance, comprising measuring the level of
NH2-terminal Jun Kinase (JNK) activity in a tissue of a mammal,
wherein an increase in activity compared to a normal control
indicates that said mammal is suffering from or at risk of
developing insulin resistance.
11. A method of diagnosing insulin resistance or a risk of
developing insulin resistance, comprising measuring the level of
NH2-terminal Jun Kinase (JNK) expression in a tissue of a mammal,
wherein an increase in the level of expression compared to a normal
control indicates that said mammal is suffering from or at risk of
developing insulin resistance.
12. The method of claim 10 or 11, wherein said JNK is JNK1.
13. A method of inhibiting fat accumulation in liver tissue,
comprising contacting said tissue with an inhibitor of a JNK.
14. The method of claim 13, wherein said inhibitor binds to an ATP
binding site in JNK.
15. The method of claim 13, wherein said inhibitor binds to a
catalytic domain of JNK.
16. The method of claim 13, wherein said JNK is JNK1.
17. The method of claim 13, wherein said JNK is JNK2.
18. The method of claim 13, wherein said JNK is JNK1 and JNK2.
19. The method of claim 13, wherein said inhibitor is SP600125.
20. The method of claim 13, wherein said inhibitor preferentially
reduces enzymatic activity of JNK1 compared to JNK2.
Description
TECHNICAL FIELD
[0001] This invention relates to insulin resistance.
BACKGROUND OF THE INVENTION
[0002] An estimated one-half of adults in the country are either
overweight or obese. Obesity can least to a greater risk for
developing a host of diseases, including diabetes, heart disease,
stroke and certain cancers. Patients with non-insulin dependent
diabetes mellitus (NIDDM) may develop insulin resistance and
impaired glucose tolerance.
SUMMARY
[0003] The invention is based on the discovery that reduced
expression of a NH2-terminal Jun Kinase (JNK), e.g., JNK1, leads to
reduced weight and improved insulin sensitivity. Accordingly, the
invention features a method of treating a metabolic disorder
associated with insulin resistance by administering to a mammal an
inhibitor of JNK.
[0004] The mammal, e.g., a human patient, is identified as being
obese or at risk of becoming obese. By "obese" is meant having an
excess amount of adipose tissue. Standard clinical tests are used
to determine whether an individual is obese, e.g., by calculating
relative weight or body mass index (BMI) for an individual and
comparing the values to a predetermined standard of ideal or
desirable relative weight or BMI. For example, assessment of skin
fold thickness over various areas of the body is taken into
consideration together with height, weight, and age to determine
the amount of adipose tissue content in an individual. Excess of
adipose tissue content is determined by comparing the value against
average (or standard) values for an individual of comparable age.
For example, a 20% increase in mean relative weight or a BMI above
the 85.sup.th percentile for young adults constitutes a health risk
and may indicate therapeutic intervention, e.g., treatment with a
JNK inhibitor. The inhibitors are also administered to individuals
who are not obese, but wish to reduce their weight.
[0005] The mammal is identified as suffering from diabetes, is at
risk of developing diabetes, suffering from insulin resistance, or
at risk of developing insulin resistance. The term "diabetes,"
includes both insulin-dependent diabetes mellitus (i.e., IDDM, also
known as type I diabetes) and non-insulin-dependent diabetes
mellitus (i.e., NIDDM, also known as Type II diabetes). Preferably,
the mammal is suffering from or at risk of developing Type II
diabetes.
[0006] JNK inhibitors are compounds, which reduce the enzymatic
activity of a JNK, e.g., JNK1 or JNK2, or expression of a JNK
isotype. For example, compounds, which inhibit JNK enzymatic
activity, bind to an ATP binding site in JNK or bind to a catalytic
domain of JNK. The compound preferentially inhibits JNK1 compared
to JNK2 or other JNK isotypes. Alternatively, the compound inhibits
JNK2 or both JNK1 and JNK2. For example, the compound is SP600125.
Compounds, e.g., polypeptides, organic compounds, or inorganic
compounds, are isolated or purified. An "isolated" or "purified"
composition is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which it
is derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. A polypeptide that is
substantially free of cellular material includes preparations of
the polypeptide in which the polypeptide is separated from cellular
components of the cells from which it is isolated, e.g., the
polypeptide is recombinantly produced. Preferably, a preparation of
a therapeutic compound, e.g., a JNK inhibitor, is at least 75%,
more preferably 80%, more preferably 85%, more preferably 90%, more
preferably 95%, more preferably 98%, and most preferably 99 or 100%
of the dry weight of the preparation.
[0007] The invention also includes a method of improving insulin
sensitivity or alleviating a symptom of insulin resistance,
reducing the severity of insulin resistance, diabetics, or an
associated metabolic disorder, by administering to a mammal an
inhibitor of JNK expression or activity. Methods of treating or
preventing the development of obesity are also within the
invention. Metabolic conditions associated with insulin resistance
include high blood glucose levels, markedly elevated serum insulin
concentrations, and insensitivity to intravenously administered
insulin. Insulin resistance is defined as the requirement of 200 or
more units of insulin per day to control hyperglycemia and prevent
ketosis.
[0008] Compounds are administered at a dose that is therapeutically
effective. The term "therapeutically effective amount" as used
herein means that the amount of a compound(s) or pharmaceutical
composition elicits a beneficial biological or medicinal response
in a tissue, system, animal or human. For example, a
therapeutically effective amount of a JNK inhibitory compound is a
dose which leads to a clinically detectable improvement in insulin
sensitivity, weight loss, or a reduction in hepatic fat
content.
[0009] A method of identifying an individual that is at risk of
developing insulin resistance is carried out by measuring the level
of JNK activity in a tissue of a mammal. Measuring the level of JNK
activity in a tissue of a mammal is also useful to diagnose insulin
resistance, diabetes, or a predisposition to develop the disorders.
An increase in activity compared to a normal control indicates that
the mammal is suffering from or is predisposed to developing
insulin resistance. Insulin resistance or a predisposition thereto
is also diagnosed by measuring the level of JNK expression in a
tissue of a mammal. JNK expression is measured by detecting a gene
product, e.g., using an antibody or other specific ligand, or by
detecting gene transcription, e.g., using a standard Northern blot
assay or reverse transcriptase polymerase chain reaction (RT-PCR).
An increase in the level of expression compared to a normal control
indicates that the mammal is suffering from or is predisposed to
developing insulin resistance, or at risk of developing the
disorder.
[0010] The invention also includes a method of inhibiting fat
accumulation in liver tissue by contacting the tissue with an
inhibitor of a JNK. For example, the JNK inhibitor reduces JNK
enzymatic activity as described above, e.g., SP600125. The
inhibitor preferentially reduces enzymatic activity of JNK1
compared to JNK2. For example, the inhibitor reduces JNK1 activity
by at least 10%, more preferably 20%, 50%, 100%, and 200% compared
to the level of reduction of JNK2 activity. The method is useful to
prevent the development or slow the progression of fatty liver
disease or hepatosteosis. The method is carried out by identifying
an individual who is at risk of developing fatty liver disease,
e.g., by identifying one who consumes excessive amounts or alcohol,
one who is at least 10% above ideal body weight, one who is obese,
or one who has a family history of liver disease, and administering
to the individual an inhibitor of JNK1 activity. Liver tissue is
contacted directly in situ, e.g., by direct injection into the
liver, or systemically, e.g., by oral or intravenous
administration. Contacting liver tissue with a compound which
preferentially inhibits JNK1 activity leads to reduced accumulation
of fat in hepatic cells.
[0011] Other features, objects, and advantages of the invention
will be apparent from the description and drawings.
DESCRIPTION OF DRAWINGS
[0012] FIG. 1 is a line graph showing body weight in JNK-deficient
(JNK -/-) mice compared to wild type (JNK +/+) control mice.
JNK-deficient and control mice were put on a high fat diet for 12
weeks. The body weight was of JNK-deficient mice was consistently
and significantly reduced compared to control mice fed the same
diet.
[0013] FIG. 2A is a bar graph showing blood glucose levels in
JNK-deficient mice compared to control mice. Both groups of mice
were fed a high fat diet for 12 weeks. Blood glucose levels of
JNK-deficient mice were significantly reduced compared to wild type
mice.
[0014] FIG. 2B is a bar graph showing blood insulin levels in
JNK-deficient mice compared to control mice. Both groups of mice
were fed a high fat diet for 12 weeks. Blood insulin levels of
JNK-deficient mice were significantly reduced compared to wild type
mice. The reduction in blood glucose and blood insulin levels shown
in FIGS. 2A-B indicate improved insulin sensivity in JNK-deficient
mice compared to control mice.
[0015] FIG. 3A is a line graph showing the results of an insulin
tolerance test in JNK-deficient and control mice. Both groups of
mice were fed a high fat diet for 12 weeks. At t=0, animals were
injected with insulin. Blood levels of glucose and insulin were
monitored every 30 minutes.
[0016] FIG. 3B is a line graph showing the results of a glucose
tolerance test in JNK-deficient and control mice. Both groups of
mice were fed a high fat diet for 12 weeks. At t=0, animals were
injected with insulin. Blood levels of glucose and insulin were
monitored every 30 minutes. The data shown in FIGS. 3A-3B indicate
that a reduction in JNK is correlated with improved insulin
action.
[0017] FIG. 4 is a bar graph showing increased JNK activity in
obesity.
[0018] FIG. 5A is a photograph of an electrophoretic gel showing
the results of a solid-phase JNK assay measuring total JNK
activity. FIG. 5B is an photograph of an immunoblot showing
different JNK1/2 isoforms. FIG. 5C is a bar graph showing
means.+-.SEM of the quantitated and normalized activity. All mice
assayed were male, 16-week-old and on C57B1/6 background. Total JNK
activity and protein levels was measured in liver, muscle and
adipose tissues of lean and obese [dietary (obese-HF) and genetic
(ob/ob)] mice.
[0019] FIGS. 6A and 6C are line graphs showing weight gain over
time. FIGS. 6B and 6D are bar graphs representing means.+-.SEM of
body weights of male mice at 16 weeks of age. Development of
diet-induced obesity was measured in the Jnk2-/- (FIGS. 6A and 6B)
and Jnk1-/- (FIGS. 6C and 6D) mice. All data are collected from
male mice (n=10 in each group). Statistical significance
(p<0.05) in two-tailed Student t test comparing Jnk1-/- or
Jnk2-/- mice with controls is indicated by *.
[0020] FIGS. 7A-H are a series of bar graphs showing the results of
analyses of adipose tissue morphology and adiposity in Jnk1-/- mice
and wild type controls. Histological sections of epididymal fat
pads (FIG. 7A) and epididymal and subcutaneous fat pad weights
(FIG. 7B) of 16-week old male Jnk1-/- and Jnk1+/+mice (n=3 in FIGS.
7A and 9 in FIG. 7B). Total body composition (FIG. 7C), fecal lipid
content (FIG. 7D), daily food intake (FIG. 7E), core body
temperature (FIG. 7F) and serum adiponectin (FIG. 7G) and resistin
(FIG. 7H) levels of Jnk1-/- and Jnk1+/+mice. Total carcass lipid
analysis was performed to determine fat mass of individual mice
(n=6 in each group). Food intake was studied in 12 week-old male
mice on high fat diet (n=9 in Jnk1+/+ and n=6 in Jnk1-/-). Fecal
lipid content and core body temperature were measured in the same
group of mice in FIG. 7C. Statistical significance (p<0.05) in
two-tailed Student t test comparing Jnk1+/+ or Jnk1-/- mice is
indicated by *.
[0021] FIGS. 8A and 8C are bar graphs showing blood glucose levels
(a measure of glucose homeostasis) by fasting plasma glucose. FIGS.
8B and 8D are bar graphs showing blood insulin levels. FIGS. 8G/K
are line graphs and FIGS. 8H/L are bar graphs showing the results
of glucose tolerance tests in lean and obese Jnk1-/-, Jnk2-/- and
control male mice at 16-weeks of age. FIGS. 8E/I are line graphs
and FIGS. 8F/J are bar graphs showing the results of insulin
tolerance tests in lean and obese Jnk1-/-, Jnk2-/- and control male
mice at 16-weeks of age. In FIGS. 8F, 8H, 8J, and 8L, "AUC"
designates the area under curve for the glucose disposal curves in
FIGS. 8F, 8H, 8J and 8L. Investigation of the dynamics of the
responses to the tolerance tests were done by ANOVA repeated
measures analysis (Statview 4.01, Abacus Concepts, Berkeley,
Calif.) and demonstrated statistically significant differences
between Jnk1-/- and Jnk1+/+ mice indicated by * (p<0.001).
[0022] FIGS. 9A and 9B are bar graphs showing a reduction in
hepatomegaly in Jnk-/- mice.
[0023] FIGS. 10A-F are photomicrographs of liver tissue sections.
Tissue sections shown in FIGS. 10A-C were stained with a standard
eosin hematoxylin stain to visual tissue architecture. Tissue
sections shown in FIGS. 10D-F were stained with Oil-Red-O to
visualize fat deposits. Dark areas in the images shown in FIGS.
10D-F represent fat deposits. The amount of fat accumulation in
liver tissue of JNK1-deficient mice was greated reduced compared to
the amount observed in WT or JNK2-deficient mice.
[0024] FIGS. 11A-C are bar graphs showing body weight and glucose
homeostasis in Jnk1+/+ and Jnk1-/-ob/ob mice. FIG. 11A shows weight
gain over time. FIG. 11B shows plasma glucose levels, and FIG. 11C
shows plasma insulin levels. Body weight measurements and blood
sampling in the ob/ob group were performed at 4 and 8 weeks of age
and following a 6-h daytime food withdrawal. Statistical
significance (p<0.05) is indicated by *.
[0025] FIGS. 12A-G show JNK activity and insulin signaling in JNK1
deficient mice. FIG. 12A is a photograph of an electrophoretic gel
showing JNK kinase activity. FIG. 12B is a photograph of an
immunoblot showing JNK protein levels in liver, muscle and adipose
tissues of lean and obese, Jnk1+/+ (wt) and Jnk1-/- mice. FIG. 12C
is a bar graph showing JNK kinase activity as means.+-.SEM of the
quantitated and normalized JNK activity based on the data shown in
FIGS. 12A-B. FIG. 12D is a photograph of an electrophoretic gel
showing insulin receptor substrate-1 (IRS-1) phosphorylation at
serine 307; total and serine 307-phosphorylated IRS-1 levels were
determined in liver tissues from lean (L) and obese (O) mice. FIG.
12E is a bar graph showing the serine phosphorylation mean values.
FIG. 12F is a photograph of an immunoblots of insulin-stimulated
tyrosine phosphorylation (pTyr) of IR and IRS-1 in liver tissues of
Jnk1-/- and Jnk1+/+ mice in specific immunoprecipitates. IR and
IRS-1 tyrosine phosphorylation (pTyr) and total protein levels
after vehicle (-) or insulin (+) stimulation was determined by
immunoblot analyses. Each lane represents an individual mouse. FIG.
12G is a bar graph showing IR tyrosine phosphorylation mean
values.
DETAILED DESCRIPTION
[0026] TNF-alpha leads to serine phosphorylation of insulin
receptor substrate-1 (IRS-1) to induce insulin resistance. JNK
phosphorylates IRS-1 at a serine residue. Genetic ablation of JNK
was found to result in decreased body weight, increased systemic
insulin sensitivity, and reduced glucose and insulin levels.
Inhibitors of JNK are useful to treat obesity, insulin resistance,
and diabetes. Modulation of expression or activity of JNK
influences body weight, insulin resistance, and levels of insulin,
glucose, and lipids in vivo.
[0027] Insulin resistant mammals, e.g., humans, include mammals
suffering from non-insulin dependent diabetes mellitus (NIDDM) or
pre-NIDDM and other insulin resistant states such as glucose
intolerance. These conditions may be related to aging and
obesity.
[0028] Inhibiting JNK kinase activity or expression of JNK is used
to treat obesity and other metabolic disorders associated with
disregulation of JNK and/or insulin resistance. Treatment includes
the management and care of an individual for the purpose of
alleviating a symptom of a disease or pathological condition.
Treatment includes the administration of a compound to prevent the
onset of symptoms or complications of a clinical disorder,
alleviating the symptoms or complications, or eliminating the
disease, condition, or disorder. Treating an insulin resistant
mammal includes increasing insulin sensitivity and/or insulin
secretion to prevent islet cell failure.
[0029] JNK inhibition is also useful to alleviate the symptoms of
other conditions associated with insulin resistance such as cancer
cachexia, HIV-1 infection, polycystic ovarian syndrome,
atherosclerosis, and severe burns. In the latter case, acute phase
burn victims are given a JNK inhibitor shortly after the burn
incident to prevent or decrease the development of insulin
resistance.
[0030] Improvement of Conditions Related to Obesity and Insulin
Resistance
[0031] JNK-deficient mice were generated using methods known in the
art. The mice contain a null mutation in a gene encoding a JNK, and
therefore, fail to express the corresponding gene product or
express a non-functional gene product. FIG. 1 shows that
JNK-deficient mice fed a high fat diet weigh less than wild type
control mice fed the same diet. FIGS. 2A-B and FIGS. 3A-B indicate
that JNK-deficient mice have improved insulin sensitivity compared
to wild type control mice. In most tissues, inhibition of one
isotype of JNK leads to an increase in expression of another
isotype. However, in certain tissues, e.g., liver, white adipose
tissue, and muscle, JNK2 does not compensate for a decrease or loss
of JNK-1 expression or activity. Taken together, these data
indicate that contacting cells or a tissue of a mammal with an
inhibitor of JNK expression or an inhibitor of JNK enzyme activity
improves insulin sensitivity. The data also indicate that such
compounds are useful to treat or prevent the development of
obesity.
[0032] Therapeutic Administration
[0033] Mammals such a humans, which are overweight, obese, or at
risk of becoming so, are treated with compounds which decrease JNK
expression or activity. In addition, humans, who are at risk of
developing hepatosteosis, also benefit by intervention to reduce
JNK expression or activity. By "activity" is meant kinase enzyme
activity.
[0034] Methods of determining whether or not an individual is
overweight or obese are known in the art. For example, Body mass
index (BMI) is measured (kg/m.sup.2 (or lb/in.sup.2.times.704.5)).
Alternatively, waist circumference (estimates fat distribution),
waist-to-hip ratio (estimates fat distribution), skinfold thickness
(if measured at several sites, estimates fat distribution), or
bioimpedance (based on principle that lean mass conducts current
better than fat mass (i.e. fat mass impedes current), estimates %
fat) is measured. The parameters for normal, overweight, or obese
individuals is as follows: Underweight: BMI <18.5; Normal: BMI
18.5 to 24.9; Overweight: BMI=25 to 29.9. Overweight individuals
are characterized as having a waist circumference of >94 cm for
men or >80 cm for women and waist to hip ratios of .gtoreq.0.95
in men and .gtoreq.0.80 in women. Obese individuals are
characterized as having a BMI of 30 to 34.9, being greater than 20%
above "normal" weight for height, having a body fat percentage
>30% for women and 25% for men, and having a waist circumference
>102 cm (40 inches) for men or 88 cm (35 inches) for women.
Individuals with severe or morbid obesity are characterized as
having a BMI of .gtoreq.35.
[0035] Individuals who are at risk of developing hepatosteosis
include overweight persons as well as those who consume excessive
amounts of alcohol, e.g., greater than two drinks per day for
females, and greater than 3 drinks per day for males. Candidates
for JNK inhibitory treatment are also identified by examining the
liver by ultrasonography to detect hepatomegaly or excessive fat
accumulation or by biopsy to detect fat deposits. Administration of
a JNK inhibitory compound protects agains the development of
hepatosteosis and/or slows its progression. The inhibitor is
administered locally or systemically as described below.
[0036] Inhibitors of JNK kinase activity are known in the art,
e.g., SP-600125 (Signal Pharmaceuticals Inc., San Diego, Calif.).
SP-600125 is a selective JNK inhibitor, which inhibits the
phosphorylation of c-Jun in a dose-dependent manner. This inhibitor
is selective for JNK compared to other kinases and other enzymes.
Other compounds, which inhibit JNK activity, include genistein,
herbimycin A,
4-amino-5-(4-chlorophenol)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (or
PP2). EGFR specific inhibitor, tyrphostin AG1478 also inhibits
c-JNK activation.
[0037] Expression of JNK is inhibited by inducing expression of an
endogenous JNK inhibitor, e.g., Hsp72 (Park et al., 2001, EMBO J.
20:446-56). Cell-permeable peptide inhibitors of JNK (Bonny et al.,
2001, Diabetes 50:77-82). Inhibition of JNK may be accomplished by
inducing expression of JNK interacting protein (JIP-1), e.g., by
inducing overexpression of the JNK binding domain of JIP-1.
[0038] A peptide inhibitor includes amino acids 33-79 of c-Jun
(U.S. Pat. No. 6,193,965). This peptide is a competitive inhibitor,
which decreases the amount of c-Jun activation by JNK. Antibodies
or other ligands, which bind to an ATP binding site or catalytic
domain of JNK are used to inhibit JNK kinase activity. The amino
acid sequence, nucleotide sequence, and domains of JNK1 are
described in U.S. Pat. No. 6,193,965.
[0039] Inhibitory compounds are formulated with conventional
excipients, i.e., pharmaceutically acceptable organic or inorganic
carrier substances suitable for parenteral, enteral (e.g., oral or
inhalation) or topical application. Suitable pharmaceutically
acceptable carriers include but are not limited to water, salt
solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,
polyethylene glycols, gelatin, carbohydrates such as lactose,
amylose or starch, magnesium stearate, talc, viscous paraffin,
perfume oil, fatty acid esters, hydroxy methylcellulose, polyvinyl
pyrrolidone, etc. Compounds are administered using conventional
methods. For parenteral application, inhibitors are in the form of
injectable, sterile solutions, e.g., oily or aqueous solutions, as
well as suspensions, emulsions, or implants. Other formulations
suitable for parenteral administration include tablets, liquids,
drops, suppositories, or capsules. Sustained or directed release
compositions can be formulated, e.g., liposomes or those wherein
the active component is protected with differentially degradable
coatings, e.g., by microencapsulation, multiple coatings, etc.
Administration by injection, e.g. subcutaneous, intramuscular or
constant infusion by intravenous drip is also useful. The compounds
are also administered by transdermally, e.g., by transdermal patch,
to allow administration over a long period of time, e.g., over days
or weeks. The compounds are at doses of 50 to about 150 .mu.g/kg in
a pharmaceutically acceptable carrier per unit dosage. Doses are
adjusted depending upon the response of the mammal to the drug.
[0040] Diagnosis of Pathological Conditions
[0041] Patients suffering from or at risk of developing a
pathological condition such as aberrant glucose metabolism are
identified by measuring JNK activity and/or proteins level. FIG. 4
shows increased JNK activity in tissues derived from obese
individuals compared to non-obese individuals. An increase in JNK
enzyme activity, e.g., JNK1, in bodily tissues or fluids indicates
a diagnosis of insulin resistance, diabetes, or a predisposition
thereto. An increase also indicates a predisposition to
obesity.
[0042] Diagnostic assays are carried out by obtaining a tissue
sample or sample of bodily fluid from an individual and measuring
JNK activity using a standard kinase assay. For example, the data
in FIG. 4 was generated using a standard solid phase kinase assay.
The assay was carried out by contacting tissue or a cell suspension
(e.g., 600 .mu.g tissue) with 20 .mu.l of glutathione S-transferase
(GST)-agarose resin suspension to which 5 .mu.g of GST-c-Jun (amino
acids 1-79) is bound. The mixture is agitated at 4.degree. C.
overnight, pelleted by centrifugation, and washed in a buffer
containing 10 mM HEPES pH 7.7, 50 mM NaCl, 2.5 mM MgCl.sub.2. The
pelleted beads were subjected to an in vitro kinase assay described
by Hibi et al. Upon staining with Coomassie Blue R250 and
autoradiography, the bands corresponding to GST-c-Jun were
quantified by PhosphorImager. This assay measures total JNK enzyme
activity.
[0043] An increase in JNK activity of at least 10% compared to a
normal control indicates a diagnosis of diabetes, insulin
resistance, or a risk of developing diabetes, insulin resistance,
or obesity. A greater increase over a normal level (e.g., 20%, 25%,
30%, 40%, or 50%) indicates a greater risk of developing a disorder
or greater severity of disease. Preferably, the increase in
activity is at least 2-fold that of a normal control value.
[0044] To determine the kinase activity of JNK1 (independent of
other JNK isotypes such as JNK1 or JNK2), a tissue sample is
obtained from a test subject. Cells are lysed, and proteins are
extracted from the tissue sample. The membrane fraction is removed
by centrifugation. The supernatant is subjected to
immunoprecipitation using a JNK1 specific antibody. JNK1 specific
antibodies are known in the art and commercially available (e.g.,
MAb 333.8 from Pharmingen, Inc., La Jolla, Calif.). Following
immunoprecipitation of JNK1, a standard kinase assay is performed
as described above. A preferential increase in JNK1 (relative to
other JNK isotypes) compared to a normal control JNK1 value
indicates a diagnosis of diabetes, insulin resistance, or
predisposition to develop diabetes, insulin resistance, or
obesity.
[0045] Fatty liver disease or a risk of developing the disease is
carried out by measuring the level of JNK1 expression or activity
in a liver tissue sample. The tissue sample is obtained by biopsy.
An increase in the amount of JNK1 expression (e.g., measured by
detecting JNK protein or by detecting JNK gene transcripts)
indicates that the individual from which the tissue was obtained is
suffering from or at risk of developing a condition of excessive
fat accumulation in the liver.
[0046] Identification of JNK Inhibitors
[0047] Inhibitors of JNK enzymatic activity are identified by
contacting a JNK with a candidate compound. A control assay is run
in parallel; the control assay include a JNK in the absence of the
candidate compound. Kinase activity is measured using methods known
in the art (e.g., as described by Hibi et al., 1993, Genes Dev.
7:2135-2148). A decrease in enzyme activity in the presence of the
compound compared to the level in the absence of the compound
indicates that the compound is a JNK inhibitor.
[0048] In addition to a standard solid phase kinase assay, an
in-gel kinase assay may be used. The in-gel assay is carried out
using known methods, e.g., as described by Kameshita and Fujisawa,
Anal. Biochem., 1989, Anal. Biochem. 183:139-143. c-Jun binding
proteins were isolated from whole cell extracts by using
GSH-agarose beads containing GST-c-Jun. Proteins are eluted in a
standard SDS-PAGE sample buffer and resolved on 10%
SDS-polyacrylamide gel, which was polymerized in the absence or
presence of GST-c-Jun. After electrophoresis, the gel washed and
incubated in 200 ml of 6M urea. After incubation in a buffer
containing 0.05% Tween 20 and either 3M, 1.5M or 0.75M urea, the
gel washed and incubated with kinase buffer containing
.sup.32P-ATP. After the reaction, the gel washed with 100 ml of 5%
tricholoroacetic acid and 1% sodium pyrophosphate at room
temperature several times, followed by drying and
autoradiography.
[0049] Inhibitors of JNK expression are identified by incubating a
JNK promoter region operably linked to a reporter sequence with a
candidate compound. An decrease in transcription of the reporter
gene (or an increase in the amount of the reporter gene product) in
the presence of the candidate compound compared to the level in the
absence of the compound indicates that the compound decreases JNK
expression.
[0050] JNK1 Plays a Central Role in Obesity and Insulin
Resistance
[0051] Obesity and type 2 diabetes are associated with a state of
chronically enhanced inflammatory response characterized by
abnormal cytokine production, increased circulating acute-phase
reactants and other stress-induced molecules. Many of these
alterations seem to be initiated and reside within adipose tissue,
an unusual site for inflammatory responses. Elevated production of
TNF.alpha. by adipose tissue was found in a variety of experimental
obesity models and in obese humans, and free fatty acids (FFAs),
are also implicated in the etiology of obesity-induced insulin
resistance. Since both TNFa and FFAs are potent JNK activators,
experiments were carried out to determine whether obesity is
associated with alterations in stress-activated and inflammatory
responses through in this signaling pathway and whether JNKs are
causally linked to aberrant metabolic control in this state.
[0052] Mice deficient in JNK1 and JNK2 were made using known
methods (e.g., Davis et al. 2000, Cell 103:239-252). In the studies
described herein, Jnk1-/- mice on C57BL/6/129 mixed genetic
background were backcrossed for 3 generations to C57BL/6 prior to
experiments. These mice were intercrossed with Jnk2-/-mice on
C57BL/6 background to produce mice heterozygous for mutations in
both JNK1 and JNK2. All mice were generated from intercrosses
between these double heterozygotes and groups were derived from
littermates. ob/ob-Jnk1+/+ and ob/ob-Jnk1-/-mice were generated by
intercrossing Jnk1-/- and OB/ob animals to generate double
heterozygotes and with subsequent crosses with OB/ob breeders to
create double homozygous mutant mice.
[0053] Diet study and metabolic measurements were carried out as
follows. Male mice of different genotypes were housed in a barrier
free facility and placed on a high fat/high carbohydrate diet ad
libidum (Diet F3282, Bioserve, N.J.) at 4 weeks of age and were
followed for a period of twelve weeks. Parallel groups were left on
standard rodent chow to serve as controls. Total body weight
measurements were initiated at 4-week of age. Blood samples were
collected after a 6-hour, daytime fast at indicated ages and
biochemical measures were conducted using 12-week-old animals.
Standard glucose and insulin tolerance tests were performed on
conscious mice following a 6 hour fast.
[0054] Total JNK Enzymatic Activity and Total JNK Protein
Levels
[0055] JNK activity was measured in liver, muscle and adipose
tissues of various models of obesity compared to the lean controls
to determine whether obesity activates this pathway. Measurement of
JNK activity and protein levels were carried out as follows. Tissue
extracts (600 .mu.g protein) were mixed with 20 .mu.l of
glutathione S-transferase (GST)-agarose resin suspension (Sigma) to
which 5 .mu.g of GST-c-Jun1-79 were bound. The mixture was agitated
at 40.degree. C. for overnight, pelleted by centrifugation, washed
twice and JNK activity was measured using known methods, e.g., as
described by Yuan et al., 2001, Immunity 14:217-230. Upon staining
with Commassie Brilliant Blue R250 and autoradiography, the bands
corresponding to GST-c-Jun were quantified by Molecular Dynamics
PhosphorImager.
[0056] In both dietary and genetic (ob/ob) models of obesity, there
was a significant increase in total JNK activity in all of the
tissues tested (FIGS. 5A-C.). In these tissues, there was no
difference in the level of either JNK1 or JNK2 proteins, suggesting
that the activity of one or both of these kinases is increased in
response to obesity.
[0057] Diet-Induced Obesity is Inhibited by Reduction in JNK
Level
[0058] JNK activity was further evaluated to test the functional
significance of the observed increase in the pathogenesis of
obesity, insulin resistance and type 2 diabetes. To address this
question, obesity was induced in mice lacking either JNK1 (Jnk1-/-)
or JNK2 (Jnk2-/-). Jnk1-/- or Jnk2-/- mice and their control
littermates (Jnk1++ or Jnk1+/- and Jnk2+/+ or Jnk2+/-) were placed
on a high fat (50% of total calories derived from fat) and high
caloric diet (5286 kcal/kg, Bioserve, N.J.) along with a control
group in each genotype on standard diet. On high fat diet, both
controls and Jnk2-/- mice developed marked obesity as compared to
mice kept on standard diet (FIGS. 6A and 6B). The weight gain
curves of these animals were indistinguishable on either standard
or high fat diet. However, weight gain on both standard and high
fat diets was significantly reduced for the Jnk1-/- group (FIGS. 6C
and 6D). Animals with one targeted allele of Jnk1 (Jnk1+/-)
displayed intermediary body weight between wild type and Jnk1-/-
mice maintained on either diet (FIG. 6D).
[0059] Inhibition of JNK1 Activity and Reduced Adipocity
[0060] Studies were carried out to determine whether the
differences in weight gain are related to alterations in adiposity.
Adipose tissue sections obtained from Jnk1-/- mice exhibited
reduced adipocyte size relative to wild type controls (FIG. 7A).
This reduction was not observed in Jnk2-/- adipose tissue. The fat
pad weights of Jnk1+/+, Jnk1+/- and Jnk1-/- mice were similar in
the lean group at both subcutaneous and epididymal fat depots.
However, in the obese group, the average weight of the subcutaneous
fat depot was reduced by 33% in Jnk1-/- mice compared to the wild
type controls (FIG. 7B). Surprisingly, the weight of the epididymal
fat pad was even higher in the obese Jnk1-/- group compared to the
wild type (FIG. 7B). No difference in fat pad weight was evident
between Jnk2-/- and wild type mice in either condition. To
investigate systemic alterations in adiposity, total body
composition was examined. These studies demonstrated significantly
reduced total body adiposity in Jnk1-/- mice compared to controls
(FIG. 7C). In contrast, body composition of Jnk2-/- group was
indistinguishable from wild type controls.
[0061] To address alternative causes for reduced body weight in
Jnk1-/- mice, lipid metabolism, food intake, intestinal lipid
absorption and core body temperature of Jnk1-/- and Jnk1+/+ mice
were compared. No significant difference was observed in plasma
triglyceride, cholesterol and FFA levels between genotypes.
Examination of fecal lipid content also did not reveal any
differences between genotypes, thus, excluding changes in
intestinal lipid absorption (FIG. 7D). There was a very small
decrease in daily food intake (0.46 g/day) in obese Jnk1-/- mice
compared to wild type, but this difference did not approach
statistical significance (FIG. 7E). There was also a small increase
(0.32.degree. C.) in core body temperature in obese Jnk1-/- mice,
which was also statistically insignificant (FIG. 7F). The results
indicate that the JNK1-deficiency leads to decreased adipocyte size
and reduced adiposity and adipose redistribution in the context of
dietary obesity without other metabolic abnormalities. These
results also indicate that Jnk1-/- mice metabolize lipids more
efficiently than wild type animals.
[0062] Adipose tissue can have a substantial impact on systemic
glucose homeostasis through the production of various bioactive
molecules. Serum levels of adipocyte-derived secreted proteins were
examined to evaluate their roles in obesity and insulin action.
ACRP30/Adiponectin levels in the obese Jnk1-/- mice were found to
be significantly higher compared to obese Jnk1+/+ controls (FIG.
7G). In contrast, the levels of resistin protein were lower in
Jnk1-/- mice compared to Jnk1+/+ animals (FIG. 7H). Adiponectin has
been shown to act as a mediator of fatty acid oxidation and hepatic
insulin sensitivity, and resistin may have a role in insulin
resistance. The alterations in adiponectin and resistin could also
impact systemic insulin sensitivity.
[0063] Inhibition of JNK1 Protects Against Development of
Obesity-Induced Insulin Resistance
[0064] To test the role of JNK in insulin sensitivity, glucose
homeostasis in Jnk1-/- and Jnk2-/- mice was evaluated and compared
to wild type controls. Measurement of fasting blood glucose levels
demonstrated that obese Jnk1+/+ mice developed mild hyperglycemia
compared to lean wild type controls (224.+-.20 vs. 126.+-.11 mg/dl,
P<0.001). In contrast, obese Jnk1-/- mice had significantly
lower blood glucose compared to obese Jnk1+/+ mice (FIG. 8A). At 12
weeks of age, the blood glucose in obese Jnk1-/- mice was
indistinguishable from that of lean Jnk1+/+ or Jnk1-/- animals
(148.+-.15 vs. 126.+-.11 and 127.+-.8 mg/dl, mean.+-.SEM, p=0.8).
Obese wild type mice also developed significant fasting
hyperinsulinemia compared to those on standard diet (5.5.+-.1.5 vs.
0.69 ng/ml P<0.001). Blood insulin levels in obese Jnk1-/- mice
were significantly lower compared to obese Jnk1+/+ animals (FIG.
8B) and indistinguishable from either Jnk1+/+ or Jnk1-/- lean mice
(0.63.+-.0.18 vs. 0.69.+-.0.16 and 0.57.+-.0.13 ng/ml, p=0.8).
Blood glucose and insulin levels in Jnk1+/- mice were intermediate
between those of Jnk1+/+ and Jnk1-/- animals but these differences
were statistically insignificant (FIGS. 8A and 8B). Obese Jnk2-/-
mice developed a similar degree of hyperglycemia and
hyperinsulinemia as obese wild type animals. Blood glucose and
insulin levels were indistinguishable between the Jnk2-/-, Jnk2+/-
and Jnk2+/+ groups (FIGS. 8C and 8D). The rise in blood glucose and
insulin in animals on high fat diet indicates obesity-induced
insulin resistance and progression to type 2 diabetes. The data
indicate that the JNK1- but not JNK2-deficient animals are
protected from development of obesity-induced insulin
resistance.
[0065] To further investigate this point, intraperitoneal insulin
(IITT) and glucose (IGTT) tolerance tests were performed. The
hypoglycemic response to insulin was lower in obese Jnk1+/+ mice
throughout the experiment than in obese Jnk1-/- animals (FIG. 8E).
Again, the glucose disposal curves of obese Jnk1-/- mice were
indistinguishable from those of lean animals. Integration of the
area under the glucose disposal curves (AUC) illustrated an overall
difference of 40% between Jnk1+/+ and Jnk1-/- mice (FIG. 8F). IGTT
also revealed a higher degree of hyperglycemia in obese Jnk1+/+
animals throughout the experiment than in obese Jnk1-/- mice (FIG.
8G). In this test, however, the responses recorded in obese Jnk1-/-
mice did not reach those of lean controls, especially in the early
phases, indicating residual insulin resistance. In IGTT,
quantitation of the AUC illustrated an overall difference of 27%
between Jnk1+/+ and Jnk1-/- mice (FIG. 8H). Interestingly,
increased responsiveness in IGTT was even evident in lean Jnk1-/-
mice at the early phase of the experiment. In contrast, obese
Jnk2-/- animals exhibited marked insulin resistance in both IITT
(FIGS. 8I and 8J) and IGTT (FIGS. 8K and 8L). The response curves
of obese Jnk2-/- mice were essentially identical to those of obese
wild type animals. Both tests confirmed that the inhibition of Jnk1
gene or gene product dramatically reduces the development of
insulin resistance associated with dietary obesity.
[0066] Genetically obese mice (ob/ob) with targeted mutations in
Jnk1 gene were generated to test the action of JNK1 in a different
and more severe model of obesity. This experiment included two
additional generations of backcrossing into the C57B1/6 genetic
background. Ob/ob mice developed early onset and severe obesity
(FIG. 11A). In contrast, the extent of weight gain was
significantly lower in ob/ob-Jnk1-/-mice than in
ob/ob-Jnk1+/+animals. Furthermore, at both 4- and 8-weeks of age
the blood glucose levels were significantly lower in the
ob/ob-Jnk1-/- mice compared to ob/ob-Jnk1+/+ animals (FIG. 11B).
The ob/ob-Jnk1+/+ animals also displayed a severe and progressive
hyperinsulinemia during the course of the study (18.4.+-.6.2 and
26.4.+-.7.1 ng/ml at 4 and 8 weeks of age, respectively; FIG. 11C).
However, the ob/ob-Jnk1-/- displayed significantly lower plasma
insulin levels throughout the study (5.7.+-.2.1 and 7.7.+-.2.3
ng/ml at 4 and 8 weeks of age, respectively; FIG. 11C) compared to
the ob/ob animals with functional JNK1. IITT analysis also
demonstrated significantly increased insulin sensitivity in
ob/ob-Jnk1-/- compared to ob/ob-Jnk1+/+ animals. These experiments
demonstrated that the effects of JNK1-deficiency on obesity and
insulin resistance were not dependent on leptin and that inhibition
of JNK1 prevents weight gain. The data indicate that inhibition of
JNK1 prevents the development of insulin resistance even under
conditions of severe obesity.
[0067] The sharp contrast in the behaviors of Jnk1-/- and Jnk2-/-
animals in the context of obesity and type 2 diabetes is
intriguing. In many but not all functions mediated by JNK,
redundancy and molecular compensation have been observed. To seek a
mechanistic explanation for the unique involvement of JNK1 isoforms
in obesity-related insulin resistance, total JNK activity was
measured in liver, muscle and adipose tissues of obese Jnk1-/- and
Jnk2-/- mice and compared to obese wild type controls. These
experiments demonstrated that JNK1-deficiency significantly reduces
the obesity-induced increase in total JNK activity at all sites
examined (FIG. 12A-C). No such reduction was observed in Jnk2-/-
mice. Similar observations were also made following treatment of
wild type, Jnk1-/- and Jnk2-/- mice with lipopolysaccharide and
using wild type, Jnk1-/- and Jnk2-/- mouse embryo fibroblasts.
Thus, the JNK1 isoforms account for most, if not all, of the
increased total JNK activity in the target tissues relevant for
obesity-induced insulin resistance.
[0068] Mechanisms of Protection Against Development of Insulin
Resistance
[0069] Experiments were carried out to elucidate molecular
mechanisms by which JNK1 inhibition reduces or slows the
development of insulin resistance. Many aspects of insulin
signaling are defective in obesity-diabetes syndromes, including
changes in insulin-sensitive glucose transporters, alterations in
the secreted proteins interfering with insulin action and reduced
signaling output of the insulin receptor (IR). Significant
alterations in expression of the Glut1 glucose transporter were not
detected in either muscle or adipose tissue, but Glut4 expression
in muscle was mildly elevated in obese Jnk1-/- mice compared to
obese Jnk1+/+ controls. This change was not apparent in adipose
tissue. Hence regulation of glucose transporters is not likely to
be a major contributor to the observed phenotype.
[0070] A more direct involvement of JNK in insulin signaling was
suggested to be at the level of IRS-1 serine phosphorylation, which
uncouples this important adaptor protein from insulin receptor
thereby reducing IRS-1 tyrosine phosphorylation and insulin
receptor signaling. Inhibitory serine phosphorylation of IRS-1 has
been shown to be a mechanism for both TNF.alpha. and FFA-induced
insulin resistance.
[0071] Experiments were carried out to determine whether this
mechanism is involved in obesity-induced insulin resistance in
vivo, and elucidate a mechanistic explanation for the protective
effect of the JNK1 inhibition.
[0072] In vivo measurement of insulin receptor and IRS-1
phosphorylation was carried out as follows. After an overnight
fast, mice were anaesthetized and 25 mIU/kg insulin (Eli Lilly) or
an equal volume of vehicle were administered through the portal
vein. Tissues were collected 120 seconds after injection in liquid
nitrogen. IRS-1 serine phosphorylation was studied in livers
collected from mice without any treatment. Protein extracts from
the tissue samples were prepared using standard methods. Protein
extracts (1 mg) were immunoprecipitated for 3 hours at 4.degree. C.
with 1 .mu.g/ml rabbit anti-IR (Santa Cruz, Calif.) or 4 .mu.g/mg
anti-IRS-1 (Upstate Biotechnology, Lake Placid, N.Y.) antibodies.
Immune complexes were collected, washed, electrophoresed and
transferred to nitrocellulose membranes. Immunoblot analysis was
performed using a 1:2000 dilution of a monoclonal
anti-phosphotyrosine (Santa Cruz, Calif.), 1:2000 dilution of
polyclonal anti-IR (Santa Cruz, Calif.) or 1 .mu.g/mg polyclonal
anti-IRS-1 or anti-IRS-1-pSer307 antibodies (Upstate Biotechnology,
Lake Placid, N.Y.), followed by 1:2000 dilution of horse radish
peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary
antibodies (Amersham Pharmacia Biotech Inc., Piscataway, N.J.) for
detection.
[0073] JNK-mediated IRS was phosphorylated in the liver tissue of
lean and obese, Jnk1+/+ and Jnk1-/- mice was evaulated, and the
level of phosphorylation analyzed using a phospho-specific
antibody. Examination of IRS-1 phosphorylation at serine 307,
revealed that the extent of serine 307 phosphorylation was markedly
increased in wild type obese mice relative to the lean controls
(FIGS. 12D-E). Most importantly, no such increase could be detected
in obese Jnk1-/- mice demonstrating that serine 307 of IRS-1 is a
relevant target for JNK action in vivo (FIGS. 12D-E). The extent of
insulin-induced IRS-1 tyrosine phosphorylation was strongly
enhanced in the livers of obese Jnk1-/- mice in comparison to obese
Jnk1+/+ controls. An improvement in insulin-induced phosphorylation
of the 95-kD b subunit of IR in Jnk1-/- mice was also observed
(FIGS. 12F-G). Nevertheless, the increase in IRS-1 tyrosine
phosphorylation was far more dramatic and consistent with reduced
serine phosphorylation. These results indicate that the reduction
or absence of JNK1 enhances IR signaling capacity of the IR, at
least in part, through its effects on IRS-1 phosphorylation.
[0074] The data described herein provide evidence that obesity is
associated with abnormally elevated JNK activity, predominantly
JNK1 activity and that inhibition of JNK1 activity protects against
the development of insulin resistance. Importantly, JNK1 provides a
critical link between obesity and insulin resistance in the mouse
and its ablation prevents obesity-induced insulin resistance in two
different models. One mechanism for JNK action involves the
phosphorylation of IRS-1 at serine 307, a site where
phosphorylation causes the uncoupling of IRS-1 from IR.
[0075] The data provide strong evidence that JNK1 is a critical
component of the biochemical pathway responsible for
obesity-induced insulin resistance in two in vivo models. There is
also genetic evidence suggesting that the JNK scaffold protein JIP1
is involved in type 2 diabetes in humans. Selective inhibition of
JNK1 activity is a novel approach for the treatment of obesity,
insulin resistance and type 2 diabetes.
[0076] JNK1 and Accumulation of Fat in Liver Tissue
[0077] Excessive fat accumulation in liver tissue is termed fatty
liver or steatosis. Fatty liver with liver inflammation is called
or steatohepatitis. Steatosis and steatohepatitis can be caused by
alcohol and other drugs and can also occur in patients with
diabetes mellitus. Steatohepatitis not caused by alcohol is
sometimes referred to as non-alcoholic steatohepatitis or "NASH".
Most people who do not abuse alcohol and have fatty liver are
obese. The patient is usually 10% or more above ideal body weight.
Steatohepatitis can lead to scarring of the liver and cirrhosis,
which may be life-threatening.
[0078] The role of JNK in development of fatty liver was evaluated.
JNK1- and JNK2-deficient mice as well as wild type control mice
were fed a high fat diet (55% fat) for 20 weeks. Liver tissue was
excised and assayed. Gross liver weight was determined, and tissue
sections were stained to visualize fat deposition. FIGS. 9A-9B show
that liver weight is reduced in JNK1-deficient mice compared to JNK
wild type mice. Liver tissue sections were stained with
eosin/hematoxylin to visualize the tissue architecture and with
Oil-Red O to visualize fat deposits. FIGS. 10A-F demonstrate a
striking reduction in the amount of fat accumulation in liver
tissue from JNK1-deficient mice compared to JNK2-deficient and JNK
wild type mice. The data indicate that inhibition of JNK1 results
in decreased fat accumulation in liver tissue and that contacting
liver tissue with a compound that preferentially inhibits JNK1
protects against development of fatty liver disease.
[0079] Other embodiments are within the following claims.
* * * * *