U.S. patent application number 13/962744 was filed with the patent office on 2013-11-28 for test agent for visceral obesity and use thereof.
This patent application is currently assigned to SHIONOGI & CO., LTD.. The applicant listed for this patent is RIKEN, SHIGA UNIVERSITY OF MEDICAL SCIENCE, SHIONOGI & CO., LTD.. Invention is credited to Minoru IKEDA, Atsunori KASHIWAGI, Yoshihiro KAWAMURA, Shiro MAEDA, Hiroshi MAEGAWA, Masatomo ROKUSHIMA, Minoru SUZUKI, Tatsuya TAKAHASHI, Tohru TANI.
Application Number | 20130317202 13/962744 |
Document ID | / |
Family ID | 43085077 |
Filed Date | 2013-11-28 |
United States Patent
Application |
20130317202 |
Kind Code |
A1 |
KAWAMURA; Yoshihiro ; et
al. |
November 28, 2013 |
TEST AGENT FOR VISCERAL OBESITY AND USE THEREOF
Abstract
Disclosed are: a method for detecting (diagnosing) visceral
obesity in a subject; a test agent useful for the method; a method
for searching for a substance that can be used as an active
ingredient for ameliorating visceral obesity; and an ameliorating
agent for visceral obesity or a medicinal agent for preventing a
metabolic disease developed as a result of the progression of
visceral obesity. As the test agent, a polynucleotide which
comprises at least 15 nucleotides and can hybridize with a
nucleotide sequence for coiled-coil domain containing protein 3
(CCDC3) gene or a nucleotide sequence complementary to the
nucleotide sequence under stringent conditions or an antibody
capable of recognizing CCDC3 protein is used.
Inventors: |
KAWAMURA; Yoshihiro; (Osaka,
JP) ; ROKUSHIMA; Masatomo; (Osaka, JP) ;
SUZUKI; Minoru; (Shiga, JP) ; TAKAHASHI; Tatsuya;
(Osaka, JP) ; IKEDA; Minoru; (Osaka, JP) ;
KASHIWAGI; Atsunori; (Shiga, JP) ; MAEGAWA;
Hiroshi; (Shiga, JP) ; TANI; Tohru; (Shiga,
JP) ; MAEDA; Shiro; (Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHIONOGI & CO., LTD.
RIKEN
SHIGA UNIVERSITY OF MEDICAL SCIENCE |
Osaka
Saitama
Shiga |
|
JP
JP
JP |
|
|
Assignee: |
SHIONOGI & CO., LTD.
Osaka
JP
RIKEN
Saitama
JP
SHIGA UNIVERSITY OF MEDICAL SCIENCE
Shiga
JP
|
Family ID: |
43085077 |
Appl. No.: |
13/962744 |
Filed: |
August 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13319717 |
Nov 10, 2011 |
8530392 |
|
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PCT/JP2010/058097 |
May 13, 2010 |
|
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13962744 |
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Current U.S.
Class: |
530/387.9 ;
536/24.5 |
Current CPC
Class: |
C12Q 2600/158 20130101;
A61K 31/7088 20130101; A61P 3/10 20180101; C07K 16/22 20130101;
C07K 2317/55 20130101; C07K 2317/76 20130101; C12Q 1/6883 20130101;
C07K 2317/92 20130101; G01N 33/6893 20130101; A61K 31/7105
20130101; C12N 15/113 20130101; C12N 2799/027 20130101; G01N
2800/044 20130101; C07K 2317/56 20130101; C07K 16/18 20130101; C12Q
2600/136 20130101; C07K 2317/565 20130101; A61K 2039/505 20130101;
A61P 3/04 20180101; C12N 2310/14 20130101 |
Class at
Publication: |
530/387.9 ;
536/24.5 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C12N 15/113 20060101 C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
May 13, 2009 |
JP |
2009-116323 |
Claims
1. An anti-visceral obesity agent that comprises as an active
ingredient a nucleic acid that suppresses CCDC3 gene expression or
CCDC3 protein function, or a neutralizing antibody against the
CCDC3 protein.
2. An antidiabetic agent that comprises as an active ingredient a
nucleic acid that suppresses CCDC3 gene expression or CCDC3 protein
function, or a neutralizing antibody against the CCDC3 protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/319,717, filed Nov. 10, 2011, which is a U.S. national stage
application and claims the benefit of PCT application number
PCT/JP2010/058097, filed May 13, 2010, which claims benefit of, and
priority to, Japanese patent application number 2009-116323, filed
on May 13, 2009, each of which is herein incorporated by
reference.
TECHNICAL FIELD
[0002] The present invention relates to visceral obesity test drugs
useful for the diagnosis of visceral obesity. More specifically,
the present invention relates to test drugs useful as a tool for
determining visceral obesity in a subject and detecting visceral
obesity individuals, and to methods for detecting (diagnosing)
visceral obesity with the test drugs. The present invention also
concerns pharmaceutical compositions for ameliorating visceral
obesity, or for preventing metabolic disease or cardiovascular
disease such as metabolic syndrome that develops from visceral
obesity, and methods (screening methods) for searching for an
active ingredient of such pharmaceutical compositions.
[0003] Further, the present invention relates to pharmaceutical
compositions effective for ameliorating diabetes by suppressing
hepatic glucose output, and for preventing diabetes complications
such as diabetic neuropathy that develop from diabetes, and to
methods (screening methods) for searching for an active ingredient
of such pharmaceutical compositions.
BACKGROUND ART
[0004] The increasing incidence of metabolic syndrome (hereinafter,
also referred to simply as "MS") arising from visceral obesity has
become a social issue. MS refers to conditions were visceral
obesity occurs in an individual with accumulations of other
conditions such as impaired glucose tolerance, hyperlipidemia, and
high-blood pressure, though the symptoms may be minor. The
importance of MS has thus been recognized as the background of
arteriosclerotic disease onset. According to the increasingly and
internationally accepted MS diagnosis criteria introduced by the US
National Cholesterol Education Program (NCEP), Adult Treatment
Panel III (ATP III), MS is determined when at least three of the
following criteria are met: (1) visceral fat diagnosed from a
circumference at navel, (2) hypertriglyceridemia, (3) low HDL
cholesteremia, (4) high-blood pressure, and (5) impaired glucose
tolerance (NPL 1). MS diagnosis for Japanese is based on the
criteria published by The Japanese Society of Internal Medicine in
2005. These criteria include visceral obesity as the essential
item, and MS is determined when two or more of the following are
met: high-blood pressure, abnormal lipid metabolism
(hypertriglyceridemia and/or low HDL-cholesteremia), and fasting
hyperglycemia (NPL 2).
[0005] The proposed criteria for determining visceral obesity as
the essential conditions for MS include (a) a visceral fat area of
100 cm.sup.2 or more in computed tomography (CT), and (b) an
abdominal circumference of 85 cm or more for men, and 90 cm or more
for women in abdominal circumference measurement (NPL 3). CT
measurement of visceral fat area can only be carried out in limited
facilities, and involves cost and exposure problems. For these
reasons, many facilities adopt abdominal circumference measurement
for the determination of visceral obesity.
[0006] However, while the abdominal circumference measurement is
simple, it is not necessarily sufficient in terms of measurement
error and accuracy (NPL 4, 5), and development of newer diagnosis
criteria is waited.
[0007] There are many reports concerning the involvement of genetic
factors in MS (for example, NPL 6, 7, and PTL 1 to 4). NPL 6
introduces chromosomal sites related to MS. Further, PTL 1
describes a method for detecting specific polymorphism in Klotho
gene as a method of determining the possibility of developing MS.
PTL 2 describes a method for detecting specific polymorphism in
angiopoietin-1 gene. PTL 3 describes a method for detecting
specific polymorphism in McKusick-Kaufman syndrome gene. PTL 4
describes a method for detecting specific polymorphism in myosin
IXA (MY09A) gene. However, a link to coiled-coil domain containing
protein 3 (hereinafter, "CCDC3") gene is not known.
[0008] PTL 5 reports FARS1 gene having the same sequence as the
CCDC3 gene. It is also reported that increased diet in mice
increases the body weight and FARS1 mRNA levels.
[0009] The anatomical location of the liver makes the liver the
first target organ of the insulin secreted by the pancreas, and an
organ that first uses the sugar that has entered the portal vein
after absorption in the digestive tract. The liver also has
contradictory functions synthesizing glycogen through the uptake of
sugar during ingestion, and, on the other hand, producing and
releasing sugar through glycogen decomposition and gluconeogenesis
during a period of fasting. These functions are regulated in an
effort to maintain the homeostasis in sugar metabolism. It is known
that the sugar production in liver is modulated through activation
of the insulin-induced glycogen synthesis in the hyperglycemia
state, and through activation of glycogen decomposition and
gluconeogenesis by the effects of substances such as glucagon,
catecholamine, and cortisol in the hypoglycemic state.
[0010] While the sugar production and release in liver are
regulated in this manner, there have been reports that liver
glucose release amounts increase in cases of diabetes, an abnormal
sugar metabolism disease. Specifically, an about 2-fold increase is
confirmed in gluconeogenesis and liver glucose release amount in
the diabetes model animal db/db mice (NPL 8), and an about 1.9-fold
increase in liver glucose release amount is confirmed in human
diabetes patients. It has been revealed that while 70% of liver
glucose release amount originates in glycogen decomposition and 30%
in gluconeogenesis in healthy individuals, 56% of the liver glucose
release amount originates in gluconeogenesis in diabetes patients.
These represent a strong indication that the increased liver
glucose release amounts due to the acceleration of the
gluconeogenesis system are involved in the hyperglycemia state of
diabetes (NPL 9). Taken together, regulation of liver glucose
release amount can be an important factor in considering diabetes
therapy.
CITATION LIST
Patent Literature
[0011] PTL 1: JP-T-2003-514513 [0012] PTL 2: JP-A-2007-54002 [0013]
PTL 3: JP-A-2009-27985 [0014] PTL 4: JP-A-2009-27986 [0015] PTL 5:
Published US Patent Application, No. 20030113889
Non-Patent Literature
[0015] [0016] NPL 1: Expert Panel on Detection, Evaluation, and
Treatment of High Blood Cholesterol in Adults: Executive Summary of
The Third Report of The National Cholesterol Education Program
(NCEP) Expert Panel on Detection, Evaluation, and Treatment of High
Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 285:
2486-2497, 2001. [0017] NPL 2: Metabolic Syndrome Diagnose Criteria
Exploratory Committee: Definition and Diagnose Criteria of
Metabolic Syndrome, The Japanese Society of Internal Medicine 94:
794-809, 2005 [0018] NPL 3: The Examination Committee of Criteria
for `Obesity Disease` in Japan, Japan Society for the Study of
Obesity: New criteria for `obesity disease` in Japan. Circ J 66:
987-992, 2002. [0019] NPL 4: S Yamada, Y Tsukamoto, J Irie: Waist
circumference in metabolic syndrome. Lancet 370: 1541-42, 2007.
[0020] NPL 5: S Yamamoto, T Nakagawa, S Kusano, M Takamura, K
Hattori, M Irokawa: Development of the Automated Diagnosis System
CT Screening for Visceral Obesity MEDIX 41: 15-20, 2004 [0021] NPL
6: Nat Clin Pract Cardiovasc Med 3:482-489 (2006) [0022] NPL 7:
Obesity Research 13:381-490 (2005) [0023] NPL 8: Aoki et al:
Dehydroepiandrosterone decreases elevated hepatic glucose
production in C57BL/KsJ-db/db mice. Life Sciences 74: 3075-3084,
2004 [0024] NPL 9: Consoli et al: Predominant Role of
Gluconeogenesis in Increased Hepatic Glucose Production in NIDDM.
Diabetes 38: 550-557, 1989
SUMMARY OF INVENTION
Technical Problem
[0025] As described above, there are implications that visceral
obesity represents a pathological component underlying the
metabolic syndrome (MS) that increases the chance of the onset of
cardiovascular diseases such as myocardial infarction. Accordingly,
there is a strong need for appropriately diagnosing visceral
obesity for early amelioration, and for preventing the onset of MS,
and, in turn, cardiovascular disease. Conventional diagnoses for
visceral obesity are either complicated or inaccurate, and a
detection marker that can accurately and conveniently determine
visceral obesity is needed. There is also a need for a
pharmaceutical composition effective at ameliorating visceral
obesity, and for a pharmaceutical composition that effectively
ameliorates diabetes caused by visceral obesity and prevents
diabetes complications.
[0026] In view of these problems, it is an object of the present
invention to provide a visceral obesity test drug that can
accurately determine visceral obesity by specifically reflecting
the disease. Another object is to provide a visceral obesity
detection method that uses the test drug.
[0027] Yet another object of the present invention is to provide a
pharmaceutical composition effectively at ameliorating visceral
obesity or diabetes, and preventing the onset of MS and
cardiovascular disease, or preventing diabetes complications. It is
still another object of the present invention to provide a method
(screening method) for searching for a substance that can be used
as an active ingredient of the pharmaceutical composition.
Solution to Problem
[0028] The present inventors conducted intensive studies to achieve
the foregoing objects, and found a gene (coiled-coil domain
containing protein 3 (CCDC3) gene) that resides in the visceral
adipose tissue of visceral obesity individuals and shows high
expression specific to visceral obesity individuals. It was also
found that the gene, while having increased expression specific to
the visceral fat of visceral obesity individuals, did not show high
expression in subcutaneous fat. It was confirmed that the gene was
a gene (visceral obesity-related gene) closely associated with
visceral obesity. The protein encoded by the gene had increased
expression specific to the visceral adipose cells in visceral
obesity, and was identified as a secretory protein secreted outside
of the cells. It was thus confirmed that the CCDC3 gene and the
product protein (hereinafter, referred to as "CCDC3 protein" to
distinguish from the gene) were useful as detection markers for
detecting visceral obesity. The CCDC3 protein was found to be
particularly useful as a detection marker for a blood test
sample.
[0029] The present inventors also found that CCDC3 protein
stimulations increase glucose output from liver cells, and that
CCDC3 protein stimulations significantly increase the mRNA
expression amounts of the gluconeogenesis rate-limiting enzymes
glucose-6-phosphatase (hereinafter, "G6 Pase") and
phosphoenolpyruvate carboxykinase (hereinafter, "PEPCK"). It was
thus confirmed that the CCDC3 protein has a glucose output
promoting effect in liver. Further, the present inventors found
that the body weight and glucose level of a transgenic mouse
designed to overexpress CCDC3 gene increase by overexpression of
CCDC3 mRNA. This suggest that the CCDC3 (hereinafter, collectively
refers to "CCDC3 gene" and "CCDC3 protein") is strongly involved in
visceral obesity and in metabolic syndrome (MS) caused by visceral
obesity, particularly in the onset of diabetes and diabetes
complications.
[0030] These findings assured that the CCDC3 gene was a visceral
obesity-related gene deeply involved in visceral obesity and the
resulting MS and cardiovascular disease, and that the CCDC3 gene
could be used as a target gene useful for the development of a
method for ameliorating visceral obesity or preventing MS and
cardiovascular disease. It was therefore considered that a
substance with the effect to suppress CCDC3 gene expression or
CCDC3 protein function (activity) could be used as an active
ingredient for ameliorating visceral obesity or preventing MS and
cardiovascular disease, or as an active ingredient for ameliorating
diabetes or preventing diabetes complications resulting from the
progression of the disease. Further, it was assured that a
screening system using these as an index would provide a new
mechanism for the effective development of a pharmaceutical
composition effective for ameliorating visceral obesity or
preventing metabolic disease and cardiovascular disease such as MS,
or a pharmaceutical composition effective for ameliorating diabetes
or preventing diabetes complications.
[0031] The present invention has been completed based on these
findings, and includes the following specific aspects.
(I) Visceral Obesity Test Drug and Use Thereof
[0032] (I-1)(1) A visceral obesity test drug, comprising:
[0033] (1) a polynucleotide with at least 15 contiguous bases of a
base sequence of CCDC3 gene, and/or a polynucleotide complementary
to the polynucleotide; or
[0034] (2) a polynucleotide of at least 15 bases that hybridizes
under stringent conditions with the CCDC3 gene base sequence or
with a base sequence complementary to the CCDC3 gene base
sequence.
(I-2) A visceral obesity test drug that includes an antibody that
recognizes CCDC3 protein. (I-3) A visceral obesity test drug of
(I-2) in which the antibody that recognizes CCDC3 protein is any
one of the following antibodies (i) to (vi):
[0035] (i) an antibody having amino acid sequences of SEQ ID
NOS:31, 32, and 33 for the CDR1, CDR2, and CDR3, respectively, of
the heavy-chain variable domain, and amino acid sequences of SEQ ID
NOS:35, 36, and 37 for the CDR1, CDR2, and CDR3, respectively, of
the light-chain variable domain;
[0036] (ii) an antibody having amino acid sequences of SEQ ID
NOS:39, 40, and 41 for the CDR1, CDR2, and CDR3, respectively, of
the heavy-chain variable domain, and amino acid sequences of SEQ ID
NOS:43, 44, and 45 for the CDR1, CDR2, and CDR3, respectively, of
the light-chain variable domain;
[0037] (iii) an antibody having amino acid sequences of SEQ ID
NOS:47, 48, and 49 for the CDR1, CDR2, and CDR3, respectively, of
the heavy-chain variable domain, and amino acid sequences of SEQ ID
NOS:51, 52, and 53 for the CDR1, CDR2, and CDR3, respectively, of
the light-chain variable domain;
[0038] (iv) an antibody of (i) having an amino acid sequence of SEQ
ID NO:30 for the heavy-chain variable domain, and an amino acid
sequence of SEQ ID NO:34 for the light-chain variable domain;
[0039] (v) an antibody of (ii) having an amino acid sequence of SEQ
ID NO:38 for the heavy-chain variable domain, and an amino acid
sequence of SEQ ID NO:42 for the light-chain variable domain;
and
[0040] (vi) an antibody of (iii) having an amino acid sequence of
SEQ ID NO:46 for the heavy-chain variable domain, and an amino acid
sequence of SEQ ID NO:50 for the light-chain variable domain.
(II) Visceral Obesity Detection Method
[0041] (II-1) A visceral obesity detection method that uses as an
index the CCDC3 mRNA expression amount or the amount of the product
CCDC3 protein in a test sample collected from a subject. (II-2)(1)
A visceral obesity detection method according to (II-1), comprising
the steps of:
[0042] (1) measuring the amount of CCDC3 mRNA expression or the
amount of the product CCDC3 protein in the test sample collected
from the subject;
[0043] (2) comparing the measured CCDC3 mRNA expression amount
(subject expression amount) or the measured amount of the product
CCDC3 protein (subject production amount) with the CCDC3 mRNA
expression amount (control expression amount) or the amount of the
product CCDC3 protein (control production amount) in a control
sample collected from a non-visceral obesity individual; and
[0044] (3) determining visceral obesity in the subject by using a
higher subject expression amount or a higher subject production
amount than the control expression amount or the control production
amount as an index.
(II-3) A visceral obesity detection method according to (II-1) or
(II-2), wherein the CCDC3 mRNA expression amount is measured by
using the test drug of (1-1). (II-4) A visceral obesity detection
method according to (II-1) or (II-2), wherein the CCDC3 production
amount (subject production amount) is measured by using the test
drug of (1-2) or (1-3). (III) Visceral Obesity Ameliorating Agent
or Preventing Agent for Diseases that Develop from Visceral Obesity
(III-1) A visceral obesity ameliorating agent or a preventing agent
for diseases that develop from visceral obesity (hereinafter,
collectively referred to as "anti-visceral obesity agent") that
includes as an active ingredient a nucleic acid that suppresses
CCDC3 gene expression or CCDC3 protein function, or a neutralizing
antibody against the CCDC3 protein. (III-2) An anti-visceral
obesity agent according to (III-1), wherein the nucleic acid that
suppresses CCDC3 gene expression or CCDC3 protein function is a
siRNA that specifically suppresses CCDC3 gene expression. (III-3) A
nucleic acid that suppresses CDC3 gene expression or CCDC3 protein
function, or a neutralizing antibody against the CCDC3 protein, for
use in ameliorating visceral obesity or preventing diseases that
develop from visceral obesity. (III-4) A nucleic acid that
suppresses CCDC3 gene expression or CCDC3 protein function
according to (III-3), wherein the nucleic acid is a siRNA that
specifically suppresses CCDC3 gene expression. (III-5) A method for
ameliorating visceral obesity or preventing diseases that develop
from visceral obesity, the method comprising the step of
administering to a visceral obesity individual a nucleic acid that
suppresses CDC3 gene expression or CCDC3 protein function, or a
neutralizing antibody against the CCDC3 protein. (III-6) A method
according to (III-5), wherein the nucleic acid that suppresses
CCDC3 gene expression or CCDC3 protein function is a siRNA that
specifically suppresses CCDC3 gene expression.
(IV) Diabetes Therapeutic Agent or Diabetes Complication Preventing
Agent
[0045] (IV-1) A diabetes therapeutic agent or a diabetes
complication preventing agent (hereinafter, collectively referred
to as "antidiabetic agent") that includes as an active ingredient a
nucleic acid that suppresses CCDC3 gene expression or CCDC3 protein
function, or a neutralizing antibody against the CCDC3 protein.
(IV-2) An antidiabetic agent according to (IV-1) wherein the
nucleic acid that suppresses CCDC3 gene expression or CCDC3 protein
function is a siRNA that specifically suppresses CCDC3 gene
expression. (IV-3) A nucleic acid that suppresses CDC3 gene
expression or CCDC3 protein function, or a neutralizing antibody
against the CCDC3 protein, for use in treating diabetes or
preventing diabetes complications. (IV-4) A nucleic acid that
suppresses CCDC3 gene expression or CCDC3 protein function
according to (IV-3), wherein the nucleic acid is a siRNA that
specifically suppresses CCDC3 gene expression. (IV-5) A method for
treating diabetes or preventing diabetes complications, comprising
the step of administering to a diabetes patient a nucleic acid that
suppresses CDC3 gene expression or CCDC3 protein function, or a
neutralizing antibody against the CCDC3 protein. (IV-6) A method
according to (IV-5), wherein the nucleic acid that suppresses CCDC3
gene expression or CCDC3 protein function is a siRNA that
specifically suppresses CCDC3 gene expression.
(V) Method for Screening for a Component Effective for Preventing
or Treating Visceral Obesity and Diabetes
[0046] (V-1) A screening method for a substance that suppresses
CCDC3 gene expression or CCDC3 protein production, the method
comprising the steps of:
[0047] (1) contacting a test substance to a CCDC3 gene expressible
cell or to a cell capable of producing CCDC3 protein;
[0048] (2) measuring the CCDC3 gene expression amount or the amount
of the product CCDC3 protein in the cell contacted to the test
substance; and
[0049] (3) selecting a test substance that makes the measured CCDC3
gene expression amount or the measured amount of the product CCDC3
protein smaller than in a control cell not brought into contact
with the test substance.
(V-2) A screening method according to (V-1), wherein the CCDC3 gene
expressible cell or the cell capable of producing CCDC3 protein is
a visceral adipose cell. (V-3) A method for screening a substance
that suppresses CCDC3 protein function or activity, the method
comprising the steps of:
[0050] (1') contacting a test substance, a cell that reacts with
CCDC3 protein, and CCDC3 protein;
[0051] (2') detecting the CCDC3 protein function or activity in the
cell contacted to the test substance; and
[0052] (3') selecting a test substance that makes the measured
CCDC3 protein function or activity lower than the CCDC3 protein
function or activity in a control cell contacted to the CCDC3
protein but not to the test substance.
(V-4) A screening method according to (V-3), wherein the cell that
reacts with CCDC3 protein is a liver-derived cell. (V-5) A
screening method according to (V-3) or (V-4), wherein the CCDC3
protein function or activity is a glucose output promoting effect.
(V-6) A screening method according to (V-3) or (V-4), wherein the
CCDC3 protein function or activity is an effect of increasing the
mRNA level of a gluconeogenesis rate-limiting enzyme gene. (V-7) A
screening method according to any one of (V-1) to (V-6), wherein
the method searches for an active ingredient for ameliorating
visceral obesity or preventing diseases that develop from visceral
obesity. (V-8) A screening method according to any one of (V-1) to
(V-6), wherein the method searches for an active ingredient for
ameliorating diabetes or preventing diabetes complications. (V-9) A
method for screening an active ingredient for ameliorating visceral
obesity or for preventing diseases that develop from visceral
obesity, or an active ingredient for ameliorating diabetes or
preventing diabetes complications, the method comprising the steps
of:
[0053] (a) administering a test substance to a CCDC3 transgenic
mouse overexpressing CCDC3 gene;
[0054] (b) measuring the body weight (subject weight) or glucose
level (subject glucose level) of the test substance-administered
CCDC3 transgenic mouse over a time course, and comparing the
measured body weight or glucose level with the body weight (control
body weight) or glucose level (control glucose level) measured over
a time course for a CCDC3 transgenic mouse not administered with
the test substance; and
[0055] (c) selecting a test substance that makes the subject weight
or subject glucose level lower than the control body weight or
control glucose level, the test substance being selected as an
active ingredient that ameliorates visceral obesity or
diabetes.
(VI) Antibody that Recognizes CCDC3 Protein (VI-1) An antibody that
recognizes CCDC3 protein, and is selected from the group of the
following antibodies (i) to (iii):
[0056] (i) an antibody having amino acid sequences of SEQ ID
NOS:31, 32, and 33 for the CDR1, CDR2, and CDR3, respectively, of
the heavy-chain variable domain, and amino acid sequences of SEQ ID
NOS:35, 36, and 37 for the CDR1, CDR2, and CDR3, respectively, of
the light-chain variable domain;
[0057] (ii) an antibody having amino acid sequences of SEQ ID
NOS:39, 40, and 41 for the CDR1, CDR2, and CDR3, respectively, of
the heavy-chain variable domain, and amino acid sequences of SEQ ID
NOS:43, 44, and 45 for the CDR1, CDR2, and CDR3, respectively, of
the light-chain variable domain; and
[0058] (iii) an antibody having amino acid sequences of SEQ ID
NOS:47, 48, and 49 for the CDR1, CDR2, and CDR3, respectively, of
the heavy-chain variable domain, and amino acid sequences of SEQ ID
NOS:51, 52, and 53 for the CDR1, CDR2, and CDR3, respectively, of
the light-chain variable domain.
(VI-2) An antibody according to (VI-1), wherein the antibody is an
antibody selected from the group of the following antibodies (iv)
to (vi):
[0059] (iv) an antibody having an amino acid sequence of SEQ ID
NO:30 for the heavy-chain variable domain, and an amino acid
sequence of SEQ ID NO:34 for the light-chain variable domain;
[0060] (v) an antibody having an amino acid sequence of SEQ ID
NO:38 for the heavy-chain variable domain, and an amino acid
sequence of SEQ ID NO:42 for the light-chain variable domain;
and
[0061] (vi) an antibody having an amino acid sequence of SEQ ID
NO:46 for the heavy-chain variable domain, and an amino acid
sequence of SEQ ID NO:50 for the light-chain variable domain.
(VI-3) An antibody that binds to an epitope bound to the antibody
of (VI-1) or (VI-2).
Advantageous Effects of Invention
[0062] The test drug and test method provided by the present
invention enable accurate detection of visceral obesity in a
subject by in vitro testing, without using conventional,
complicated and inaccurate measurement methods such as visceral fat
area measurement using computed tomography, and abdominal
circumference measurement. Further, by using the test drug and
detection method of the present invention, visceral obesity can
easily be detected in a subject in vitro, without having the need
to have the expert knowledge of physicians or other
specialists.
[0063] The subject having visceral obesity (visceral obesity
patient) found by the method of the present invention is given the
chance to take appropriate measures to ameliorate and eliminate the
visceral obesity by being informed of the disease. The present
invention is therefore very useful as a test method for
ameliorating visceral obesity at early stages, and for preventing
the progression of visceral obesity into metabolic disease and
cardiovascular disease such as metabolic syndrome (MS).
[0064] Further, the anti-visceral obesity agent provided by the
present invention can be effectively used as a pharmaceutical
composition effective for ameliorating visceral obesity, and thus
preventing the progression of visceral obesity into metabolic
disease and cardiovascular disease such as MS. Further, the
antidiabetic agent provided by the present invention can be
effectively used as a pharmaceutical composition effective for
suppressing glucose release in liver and ameliorating the
hyperglycemia state, and thus preventing progression into diabetes,
and preventing diabetes complications.
[0065] Further, the screening method of the present invention makes
it possible to obtain a component effective for ameliorating
visceral obesity, and preventing the progression of the disease
into metabolic disease and cardiovascular disease such as MS, or a
component effective for ameliorating the hyperglycemia state, and
preventing diabetes and diabetes complications caused by the
progression of diabetes. Specifically, the method can be
effectively used for the development of a novel medicinal agent
effective for ameliorating visceral obesity and preventing
metabolic disease and cardiovascular disease, and a novel medicinal
agent effective for ameliorating diabetes and preventing diabetes
complications.
BRIEF DESCRIPTION OF DRAWINGS
[0066] FIG. 1 represents the result of the comparison of CCDC3 gene
(mRNA) expression amount between the visceral adipose tissue and
subcutaneous adipose tissue of non-visceral obesity individuals (5
cases) and visceral obesity individuals (5 cases). The CCDC3 gene
expression amount in the visceral obesity individuals is presented
as the relative ratio with respect to the expression amount 1 of
the non-visceral obesity individuals. The CCDC3 gene expression
amount in the visceral fat of the visceral obesity individuals had
a 3.2-fold increase over the expression amount in the visceral
adipose tissue of the non-visceral obesity individuals (Example
1).
[0067] FIG. 2 represents the result of the comparison of CCDC3 gene
(mRNA) expression amount between the mesenteric adipose tissue,
subcutaneous adipose tissue, and periepididymal adipose tissue of
obesity model mice (db/db mice) and db/m mice (control group). The
CCDC3 gene expression amount in the obesity model mice (db/db mice)
is presented as the relative ratio with respect to the expression
amount 1 of the control group. The CCDC3 gene expression amount in
the mesenteric adipose tissue of the obesity model mice had a
2.1-fold increase over the expression amount in the mesenteric
adipose tissue of the control group (Example 2).
[0068] FIG. 3 represents the result of the western blotting
detection of CCDC3 protein secreted into medium from HEK293T cells
that had human CCDC3 overexpression vector pReceiver-CCDC3
introduced into the cells. The CCDC3 protein signals recognized in
the medium suggested that CCDC3 protein was a secretory protein
(Example 3).
[0069] FIG. 4 represents the result of the examination of the
influence of 100 nM human recombinant CCDC3 protein stimulations on
glucose output using isolated liver cells from rats. Increased
glucose output by 100 nM CCDC3 protein stimulation was confirmed
(CCDC3 0 nM vs 100 nM, 1.2-fold increase, n=6, p<0.01) (Example
5(2)).
[0070] FIG. 5 represents the result of the examination of the
influence of 100 nM CCDC3 protein stimulations on gene expression
for the gluconeogenesis rate-limiting enzymes glucose-6-phosphatase
(G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Significant
increases in the mRNA expression of the gluconeogenesis
rate-limiting enzymes G6Pase and PEPCK by 100 nM CCDC3 protein
stimulation were confirmed (CCDC3 0 nM vs 100 nM G6Pase, 2.6-fold
increase, PEPCK, 2.2-fold increase, n=5, p<0.01 for each enzyme)
(Experiment Example 5(3)).
[0071] FIG. 6 represents the result of examination concerning
suppression of CCDC3 gene (mRNA) expression by the introduction of
three CCDC3 siRNAs (QIAGEN, HP GenomeWide siRNA (#1, #2, #3)).
CCDC3 gene expression was suppressed by 80% or more by the siRNA
introduction (Example 6).
[0072] FIG. 7 represents time-dependent changes in body weight
under high-fat diet (HFD) in transgenic mice (CCDC3 Tg mice)
expressing CCDC3 gene in 4 times or greater amounts and in control
group mice (Example 7).
[0073] FIG. 8 represents glucose levels (mg/dl) of the blood
collected from CCDC3 Tg mice and control group mice on day 52 of
HFD ingestion (Example 7).
[0074] FIG. 9 represents human recombinant CCDC3 protein
displacement curves for the binding of human anti-CCDC3 antibodies
(6G12, 3H6, 11E10) to biotin-labeled human recombinant CCDC3
protein (Example 8).
[0075] FIG. 10 is the result of western blotting representing the
reactivity of human anti-CCDC3 antibodies (6G12, 3H6, 11E10) to
human recombinant CCDC3 protein (Example 9). The proteins at 30 kDa
and 60 kDa apparently correspond to the monomer and dimer,
respectively, of the CCDC3 protein from the deduced molecular
weight of the CCDC3 protein (also in FIG. 12).
[0076] FIG. 11 represents mouse recombinant CCDC3 protein
displacement curves for the binding of mouse anti-CCDC3 antibodies
(13A8, 13D4, 24F6) to immunogen peptide or biotin-labeled mouse
recombinant CCDC3 protein (Example 11).
[0077] FIG. 12 is the result of western blotting representing the
reactivity of mouse anti-CCDC3 antibodies (13A8, 13D4) to mouse
recombinant CCDC3 protein (Example 12).
[0078] FIG. 13 represents standard curves in the CCDC3 protein
immunoassay using two combinations of human anti-CCDC3 antibodies
(6G12-11E10, 3H6-11E10) (Example 13).
[0079] FIG. 14 represents the result of the comparison of blood
plasma CCDC3 protein concentrations between a non-visceral obesity
group with a visceral fat area below 100 cm.sup.2 and a visceral
obesity group with a visceral fat area of 100 cm.sup.2 or more
(Example 14).
[0080] FIG. 15 represents the result of the comparison of blood
plasma CCDC3 protein concentrations between a normal value group
with a HbA1c value of 5.8% or less and a high value group with a
HbA1c value above 5.8%, where HbA1c is an index reflecting a
long-term glucose level (Example 14).
[0081] FIG. 16 represents CCDC3 protein immunostained images of the
mesenteric fat and subcutaneous fat of control group mice (Lean)
and DIO mice (DIO) (Example 15).
DESCRIPTION OF EMBODIMENTS
1. Definitions of the Terms Used in the Present Invention
[0082] The abbreviations representing the base sequences
(nucleotide sequences), nucleic acids, and other elements used
herein follow the rules of IUPAC-IUB [IUPAc-IUB communication on
Biological Nomenclature, Eur. J. Biochem., 138; 9 (1984)], The
Guideline for Preparation of Minute Descriptions of Compounds
Including Base Sequence or Amino Acid Sequence (Japan Patent
Office), and the conventional notation used in the art.
[0083] As used herein, "CCDC3 gene" encompasses not only
human-derived CCDC3 gene represented by SEQ ID NO:1, but genes,
including ortholog genes conserved among different biological
species such as humans, mice, and rats, that encode proteins (for
example, homologs (including splice variants), mutants, and
derivatives) having biologically equivalent functions as the
human-derived protein (CCDC3 protein). For example, the genes that
encode the homologs of the human-derived CCDC3 protein may be the
genes of other biological species such as mice and rats,
corresponding to the human CCDC3 gene that encodes the CCDC3
protein. These genes (homologs) can be identified from HomoloGene
(http://www.ncbi.nlm.nih.gov/HomoloGene/). Specifically, human gene
data are found by searching specific human gene names (CCDC3 gene)
or base sequence accession numbers (GenBank Accession No.
NM.sub.--031455) in LocusLink
(http://www.ncbi.nlm.nih.gov/LocusLink/). The genes of other
biological species such as mice and rats, corresponding to the
human CCDC3 gene can then be selected as the genes (homologs) of
interest from a list of the gene homolog correlation between other
biological species and human genes displayed by accessing the
HomoloGene in the data link menu. A specific example of a mouse
homolog of human CCDC3 gene (GenBank Accession No. NM.sub.--031455;
SEQ ID NO:1) is the mouse CCDC3 gene (GenBank Accession No.
NM.sub.--028804) represented by SEQ ID NO:2.
[0084] Further, the "gene" as used herein does not distinguish
between regulatory region, coding region, exon, and intron, unless
otherwise stated.
[0085] As used herein, "DNA" encompasses double-stranded DNAs
including human genomic DNA, single-stranded DNAs (sense strands)
including cDNA and synthetic DNA, single-stranded DNAs (antisense
strands) having complementary sequences to the sense strands, and
fragments thereof, unless otherwise stated. As used herein, "RNA"
is intended to include not only single-stranded RNAs, but
single-stranded RNAs having complementary sequences, and
double-stranded RNAs formed from such RNAs, unless otherwise
stated. The RNA includes synthetic RNAs such as total RNA, mRNA,
rRNA, and siRNA.
[0086] Further, as used herein "nucleotide", "oligonucleotide", and
"polynucleotide" have the same meaning as the nucleic acid, and
include both DNA and RNA. These may be double-stranded or
single-stranded. By "nucleotide (or oligonucleotide,
polynucleotide) of a certain sequence", it inclusively means a
nucleotide (or oligonucleotide, polynucleotide) having a
complementary sequence, unless otherwise stated. Further, when the
"nucleotide" (or "oligonucleotide", "polynucleotide") is an RNA,
the letter "T" in the bases of the Sequence Listing should be read
as "U".
[0087] As used herein, "CCDC3 protein" encompasses not only
human-derived CCDC3 protein represented by SEQ ID NO:3, but
proteins translated from the ortholog genes conserved among
different biological species such as humans, mice, and rats, and
homologs (including splice variants), mutants, derivatives, and
amino acid-modified products, provided that such equivalents have
the same biological functions as such proteins. Examples of such
homologs include proteins of other biological species such as mice
and rats, corresponding to the human CCDC3 protein (SEQ ID NO:3),
and these can be deductively identified from the base sequences of
the genes identified in HomoloGene
(http://www.ncbi.nlm.nih.gov/HomoloGene/). The mutants may be
naturally occurring allele mutants, not naturally occurring
mutants, and mutants having amino acid sequences artificially
modified through deletion, substitution, addition, and insertion.
Note that the mutants may have at least 70%, preferably 80% or
more, more preferably 85% or more, further preferably 90% or more,
even more preferably 95% or more, particularly preferably 97% or
more amino acid sequence identify with unmutated proteins. An
example of a mouse homolog of the human CCDC3 protein is the mouse
CCDC3 protein represented by SEQ ID NO:4. Note that the term
"CCDC3" is also used to collectively refer to the CCDC3 gene and
the CCDC3 protein, without distinguishing the two.
[0088] As used herein, the nucleic acid that suppresses CCDC3 gene
expression means a nucleic acid that has the effect to suppress
mRNA expression from the CCDC3 gene or suppress translation of
CCDC3 mRNA into CCDC3 protein, and encompasses not only siRNAs for
the CCDC3 gene, but miRNAs, antisense poly(oligo)nucleotides,
ribozymes, and decoys.
[0089] Further, as used herein, the nucleic acid that suppresses
the function of the CCDC3 protein means a nucleic acid that has the
effect to suppress the function of CCDC3 protein, and encompasses
aptamers for CCDC3 protein.
[0090] As used herein, "antibody" encompasses polyclonal
antibodies, monoclonal antibodies, chimeric antibodies,
single-stranded antibodies, including antigen binding parts such as
Fab fragments, and fragments created from a Fab expression
library.
[0091] Obesity has two types: visceral obesity that involves fat
accumulation in the gut and the surrounding area, and subcutaneous
obesity that involves more subcutaneous fat and less visceral fat.
As used herein, "visceral obesity" refers to the former, a type of
obesity characterized by accumulation of large fat in the gut and
the surrounding area. The diagnosis criteria for visceral obesity
involve a visceral fat area of 100 cm.sup.2 or more at the navel
height in a CT measurement.
[0092] As used herein, "metabolic syndrome (MS)" refers to
conditions such as high-blood pressure, abnormal sugar metabolism,
and abnormal lipid metabolism, stemming from visceral obesity.
Conventionally, metabolic syndrome is diagnosed when a person with
an abdominal circumference (waist circumference) of 85 cm or more
for men and 90 cm or more for women (equivalent of a visceral fat
area of 100 cm.sup.2 or more) satisfies two or more of the
following: (1) abnormal serum lipid level (triglyceride value of
150 mg/dL or more, or an HDL cholesterol value of less than 40
mg/dL), (2) high blood pressure (maximum blood pressure of 130 mmHg
or more, or minimum blood pressure of 85 mmHg or more), and (3)
hyperglycemia (fasting glucose level of 110 mg/dL).
[0093] As used herein, "cardiovascular disease" means conditions
that involve a disturbed blood flow in the heart due to
cardiovascular abnormalities, lowered heart functions, and lesions.
Specific examples include diseases such as angina, myocardial
infarction, and heart failure. Further, as used herein, "metabolic
disease" means disorders caused by the excess or deficient
accumulation of metabolites in blood or in specific organs as a
result of failures, innate or acquired, in various stages of the
metabolic pathway. Aside from MS, other specific examples include
diseases such as hyperlipidemia, diabetes, and obesity.
[0094] As used herein, "diabetes complication" means diseases and
symptoms that develop from the diabetes itself. Specific examples
include diseases such as diabetic retinopathy, diabetic
nephropathy, and diabetic neuropathy.
[0095] Further, as used herein, "test drug" means agents directly
or indirectly used to determine the presence or absence of visceral
obesity, or to diagnose the extent of visceral obesity, and agents
directly or indirectly used for the screening of a candidate
substance effective for ameliorating visceral obesity or preventing
the progress of visceral obesity. In relation to visceral obesity,
the test drug encompasses (poly)(oligo)nucleotides and antibodies
that can specifically recognize and bind to the CCDC3 gene that has
increased expression in an organism, particularly in the visceral
adipose tissue, or to the expression product (CCDC3 protein). The
(poly)(oligo)nucleotides and antibodies of these properties can be
effectively used as probes for detecting the CCDC3 gene and CCDC3
protein, either in vivo or in vitro, expressed in the tissues and
cells of an organism. The (poly)(oligo)nucleotides also can be
effectively used as primers for amplifying the gene expressed in
the organism.
[0096] As used herein, the "organism tissue" and "organism sample"
under diagnosis encompass tissues that show increased expression of
the CCDC3 gene (mRNA) of the present invention in visceral obesity,
and tissues (including blood) in which the gene expression product
(CCDC3 protein) is produced or secreted. Preferred examples include
visceral adipose tissue and blood.
[0097] The following describes specific uses of the CCDC3 gene
(polynucleotide), the expression product (CCDC3 protein), and their
derivatives.
2. Visceral Obesity Test Drug
[0098] The present invention provides a test drug suitably used for
detecting visceral obesity in a subject. The test drug is also used
in a screening method (described later) for searching for an active
ingredient (candidate substance) of an ameliorating agent for
visceral obesity, or of a preventing agent for metabolic disease
and cardiovascular disease such as MS. The test drug also has use
in searching for an active ingredient (candidate substance) of a
diabetes ameliorating agent, or of a preparation for preventing
diabetes progression and diabetes complications.
(1) Poly(or oligo)Nucleotide
[0099] In the present invention, the detection (diagnosis) of
visceral obesity is performed by evaluating the presence or absence
of an increase in CCDC3 gene (mRNA) expression, or the expression
level (expression amount) of CCDC3 gene (mRNA) in the tissues,
particularly the visceral adipose tissue, of a subject organism.
Here, the test drug of the present invention can be used as a
primer that recognizes and amplifies the RNA produced by the
expression of the gene, or the polynucleotide derived from the RNA.
The test drug also can be used as a probe for specifically
detecting the RNA, or the polynucleotide derived from the RNA.
[0100] The test drug of the present invention may be a
polynucleotide with the base sequence (full-length sequence) of the
CCDC3 gene, or a polynucleotide having a complementary sequence
thereof, provided that it selectively (specifically) recognizes
CCDC3 gene or a polynucleotide derived from the gene. Further, the
test drug may be a polynucleotide with a partial sequence of the
full-length sequence, or a polynucleotide with a partial sequence
of the complementary sequence. In this case, the partial sequence
may be a polynucleotide with a length of at least 15 contiguous
bases arbitrarily selected from the base sequence of the
full-length sequence or of the complementary sequence.
[0101] Note that the phrase "selectively (specifically) recognize"
means, for example, specifically detecting CCDC3 gene or a
polynucleotide derived from CCDC3 gene in northern blotting, or
specifically producing CCDC3 gene or a polynucleotide derived from
CCDC3 gene in a RT-PCR method. However, the meaning of the phrase
is not limited to this, as long as the detected product or the
product obtained can be determined as being derived from CCDC3 gene
by a skilled artisan.
[0102] Specifically, the test drug of the present invention
includes a polynucleotide having at least 15 contiguous bases,
and/or a polynucleotide complementary thereto, in the base sequence
of CCDC3 gene. As used herein, "complementary polynucleotide
(complementary strand, opposite strand)" means a polynucleotide
with the base complementarity, based on base pairing such as A:T
and G:C, to the full-length sequence of the polynucleotide with the
CCDC3 gene base sequence, or to the partial sequence having a base
sequence with a length of at least 15 contiguous bases of the base
sequence (the full-length sequence and the partial sequences will
be referred to as "plus strands" for convenience). It should be
noted that the complementary strand is not limited to forming a
completely complementary sequence to the base sequence of the plus
strand of interest, and may have complementarity sufficient to
hybridize with the plus strand of interest under stringent
conditions. Note that stringent conditions may be decided based on
the melting temperature (Tm) of the conjugate or the nucleic acid
bound to a probe, as taught by Berger and Kimmel (1987, Guide to
Molecular Cloning Techniques Methods in Enzymology, Vol. 152,
Academic Press, San Diego Calif.). For example, typical
post-hybridization washing conditions may involve roughly
1.times.SSC, 0.1% SDS, and 37.degree. C. Preferably, the
complementary strand remains hybridized with the target plus strand
even after washing under these conditions. More severe
hybridization conditions may involve washing under roughly
0.5.times.SSC, 0.1% SDS, and 42.degree. C., even more severely
under roughly 0.1.times.SSC, 0.1% SDS, and 65.degree. C., though
the hybridization conditions are not limited to these. Specific
examples of such complementary strands include strands with the
base sequences completely complementary to the base sequences of
the target plus strands, and strands with the base sequences having
at least 90%, preferably 95% homology to such strands.
[0103] The test drug of the present invention may be designed, for
example, based on the base sequence of the human-derived CCDC3 gene
represented by SEQ ID NO:1, using, for example, a vector NTI
(Infomax). Specifically, a primer or probe candidate sequence, or a
sequence that includes at least such a candidate sequence, obtained
by processing the CCDC3 gene base sequence with vector NTI
software, may be used as a primer or a probe.
[0104] As described above, the test drug of the present invention
has a length of at least 15 contiguous bases. However, the length
may be appropriately selected and set according to the specific use
of the test drug.
[0105] The test drug of the present invention may be used as a
primer or a probe according to an ordinary method in the known
methods of specifically detecting specific genes, including
northern blotting, RT-PCR method, and in situ hybridization. The
use of the test drug enables evaluation of the presence or absence
of a CCDC3 gene expression, or the expression level (expression
amount) of CCDC3 gene in the visceral adipose tissue of a
subject.
[0106] The measurement sample (biological sample) may be selected
according to the type of the detection method used. For example,
the measurement sample may be a total RNA prepared according to an
ordinary method from the collected specimen, which may be blood, or
a part of the visceral adipose tissue collected from a subject by
biopsy. Various polynucleotides prepared from the RNA also may be
used as the measurement sample.
[0107] When the test drug of the present invention is used as a
primer for the detection (gene diagnosis) of visceral obesity, for
example, a polynucleotide of typically 15 bp to 100 bp, preferably
15 bp to 50 bp, more preferably 15 bp to 35 bp may be used as the
polynucleotide having at least 15 contiguous bases of the base
sequence of the CCDC3 gene, and/or a complementary polynucleotide
thereof. For use as a detection probe, the test drug may be, for
example, a polynucleotide of a length from typically 15 bp to the
whole base sequence, preferably 15 bp to 1 kb, more preferably 100
bp to 1 kb.
[0108] The test drug of the present invention (probe or primer) may
include suitable labels, for example, such as fluorescent dyes,
enzymes, proteins, radioisotopes, chemiluminescence substances, and
biotins, added to the test drug for the detection of CCDC3
gene.
[0109] The fluorescent dye used in the present invention may be
those generally used for the labeling and the detection or
quantification of nucleic acids. Non-limiting examples include HEX
(4,7,2',4',5',7'-hexachloro-6-carboxyl fluorescein, green
fluorescent dye), fluorescein, NED (commercially available from
Applied Biosystems; yellow fluorescent dye), 6-FAM (commercially
available from Applied Biosystems; yellow-green fluorescent dye),
and rhodamins or derivatives thereof [for example, tetramethyl
rhodamin (TMR)]. The nucleotide may be labeled with a fluorescent
dye using a method suitably selected from known methods [see Nature
Biotechnology, 14, 303-308 (1996)]. Commercially available
fluorescence labeling kits also may be used (for example, the
oligonucleotide ECL 3'-oligolabeling system from Amersham
Pharmacia).
[0110] Further, the probe (oligo or polynucleotide) may be used by
being immobilized on any solid phase. Accordingly, the test drug of
the present invention may be provided in the form of an immobilized
probe obtained by immobilizing the probe (oligo or polynucleotide)
(for example, a probe-immobilized DNA chip, cDNA microarray, oligo
DNA array, and membrane filter; hereinafter collectively referred
to as "DNA chips").
[0111] The solid phase used for immobilization is not particularly
limited, as long as the oligo or polynucleotide can be immobilized.
For example, glass plates, nylon membranes, microbeads, silicon
chips, capillaries, and other substrates can be used. The oligo or
polynucleotide may be immobilized on a solid phase by placing the
oligo or polynucleotide thereon after being synthesized, or by
synthesizing the oligo or polynucleotide of interest on a solid
phase. The immobilization method is known in the art, and can be
selected according to the type of immobilized probe [for example,
in situ synthesis of oligonucleotide using a photolithographic
technique (Affymetrix) or inkjet technique (Rosetta Inpharmatics)].
For example, in the case of a DNA microarray, the probe may be
immobilized using a commercially available spotter (Amersham).
[0112] The DNA chips immobilizing the test drug (probe) of the
present invention may be hybridized with the labeled DNA or RNA
prepared from the RNA collected from a biological tissue, and the
resulting conjugate formed by the hybridization of the probe with
the labeled DNA or RNA may be detected using the label of the
labeled DNA or RNA as an index. The presence or absence of CCDC3
gene expression, or the expression level (expression amount) of the
CCDC3 gene in a biological tissue can then be evaluated.
[0113] The DNA chips may include one or more test drugs (probes) of
the present invention that can bind to the CCDC3 gene. By using DNA
chips that include more than one test drug (probe), the presence or
absence of more than one gene, or the expression level of these
genes can be simultaneously evaluated for a single biological
sample.
[0114] The test drug of the present invention is useful for the
detection of visceral obesity in a subject (the presence or absence
of obesity, or the diagnosis of the extent of the disease).
Specifically, the test drug can be used to diagnose visceral
obesity by determining the difference in the CCDC3 gene expression
level between the biological tissue of a subject and the biological
tissue or sample of a normal individual (non-visceral obesity
individual). In this case, because the CCDC3 gene has increased
expression in the visceral fat of visceral obesity individuals, the
biological tissue or sample of the subject has increased expression
of the gene (mRNA). Thus, the subject has a high possibility of
having visceral obesity when the expression amount of the gene in
the subject is higher than that in the biological tissue or sample
of the normal individual (non-visceral obesity individual) by 50%
or more, preferably 100% or more, more preferably 200% or more.
(2) Antibody
[0115] The present invention provides an antibody that can be used
as a test drug to specifically recognize CCDC3 protein. A specific
example of the antibody is an antibody that can specifically
recognize the human-derived CCDC3 protein having the amino acid
sequence of SEQ ID NO:3.
[0116] As described above, the present invention is based on the
finding that CCDC3 gene expression specifically increases in the
visceral adipose tissue of visceral obesity individuals, and the
underlying idea of the invention is that visceral obesity or the
extent of visceral obesity can be detected in a subject by
detecting the presence or absence of a CCDC3 protein production
increase, or the extent of the increase in the subject.
[0117] Thus, the antibody can be used as a tool (test drug) with
which visceral obesity or the extent of visceral obesity can be
diagnosed in a subject through the detection of the presence or
absence of a CCDC3 protein production increase, or the extent of
the increase in the subject.
[0118] The form of the antibody of the present invention is not
particularly limited, and the antibody may be a polyclonal antibody
that uses the CCDC3 protein as the immunogen, or may be a
monoclonal antibody. The antibody of the present invention also
encompasses antibodies that show antigen binding to a polypeptide
of typically at least 8 contiguous amino acids, preferably 15
contiguous amino acids, more preferably 20 contiguous amino acids
of the CCDC3 amino acid sequence.
[0119] Producing methods of such antibodies are known, and the
antibody of the present invention can be produced using these
ordinary methods (Current protocols in Molecular Biology, Chapter
11.12 to 11.13 (2000)). Specifically, when the antibody of the
present invention is a polyclonal antibody, the antibody can be
obtained according to an ordinary method from the serum of an
immunized animal produced by immunizing a non-human animal such as
house rabbit with the CCDC3 protein purified after being expressed
in, for example, Escherichia coli using an ordinary method, or with
an oligopeptide synthesized using an ordinary method and including
a partial amino acid sequence of the CCDC3 protein. In the case of
monoclonal antibody, the antibody can be obtained from the
hybridoma cells prepared by fusing the spleen cells and myeloma
cells obtained by immunizing a non-human animal such as mouse with
the CCDC3 protein purified after being expressed in, for example,
Escherichia coli using an ordinary method, or with an oligopeptide
synthesized using an ordinary method and including a partial amino
acid sequence of the protein (Current protocols in Molecular
Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons.
Section 11.4 to 11.11).
[0120] The CCDC3 protein used as an immunogen for the production of
the antibody can be obtained according to procedures such as DNA
cloning, construction of plasmids, host transfection, culturing the
transformant, and collection of the protein from the culture, based
on the gene sequence information (for example, SEQ ID NO:1 or 2)
provided by the present invention. These procedures can be
performed according to methods known to skilled artisan, or
previous methods (for example, Molecular Cloning, T. Maniatis et
al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS
(1985)).
[0121] Specifically, the protein as an immunogen for the production
of the antibody of the present invention can be obtained by
producing a recombinant vector (expression vector) that allows the
CCDC3 protein coding gene to be expressed in a desired host cell.
The recombinant vector is then introduced into the host cell to
obtain a transformant. The transformant is cultured, and the target
protein is collected from the resulting culture. The partial
peptide of CCDC3 protein can be produced using a common chemical
synthesis (peptide synthesis) technique based on the amino acid
sequence information (SEQ ID NO:3, 4) provided by the present
invention.
[0122] Note that the CCDC3 protein of the present invention
encompasses not only the proteins concerning the amino acid
sequence of SEQ ID NO:3 or NO:4, but homologues thereof. An example
of such homologues is a protein that has the amino acid sequence of
SEQ ID NO:3 or NO:4 with the deletion, substitution, or addition of
one or more amino acids, and that has immunologically the same
activity as the protein represented by SEQ ID NO:3 or NO:4.
[0123] Examples of the protein with the same immunological activity
include proteins that induce specific immune reaction in a suitable
animal or in cells thereof, and that are capable of specifically
binding to the antibody against the CCDC3 protein.
[0124] The number of amino acid mutations, and the mutation sites
in the protein are not limited, as long as the immunological
activity is maintained. The index as to which and how many amino
acid residues should be substituted, inserted, or deleted without
losing the immunological activity can be found by using a computer
program known to skilled artisan, for example, such as DNA Star
software. For example, the number of mutations is typically within
10%, preferably within 5%, further preferably within 1% of the all
amino acids. Substituted amino acids are not particularly limited,
as long as the protein resulting from the substitution maintains
the immunological activity of the CCDC3 protein. From the
standpoint of maintaining the protein structure, the substituted
amino acids are preferably those similar in properties, such as the
reside polarity, charge, solubility, hydrophobicity,
hydrophilicity, and amphipathicity, to the amino acids before the
substitution. For example, amino acids can be classified into
nonpolar amino acids (Ala, Val, Leu, Ile, Pro, Met, Phe, Trp),
uncharged amino acids (Gly, Ser, Thr, Cys, Tyr, Asn, Gln), acidic
amino acids (Asp, Glu), and basic amino acids (Lys, Arg, His).
Amino acids belonging to the same group can thus be appropriately
selected using this classification as an index.
[0125] The antibody of the present invention may be one prepared by
using an oligopeptide having a partial amino acid sequence of CCDC3
protein. The oligo(poly)peptide used to produce the antibody does
not need to have a functional biological activity, but should
desirably have immunogen properties similar to those of the CCDC3
protein. A preferred example is an oligo(poly)peptide that has such
immunogen properties, and that includes at least 8 contiguous amino
acids, preferably 15 contiguous amino acids, more preferably 20
contiguous amino acids of the CCDC3 protein amino acid
sequence.
[0126] The antibody against the oligo(poly)peptide also may be
produced by enhancing the immunological reaction with various
adjuvants selected according to the type of host. Non-limiting
examples of the adjuvants include Freund's adjuvant, mineral gel
such as aluminum hydroxide, surface active materials such as
lysolecithin, pluronic polyol, polyanion, peptide, oil emulsion,
keyhole lympet hemocyanin, and dinitrophenol, and human adjuvants
such as BCG (Bacille de Calmette et Guerin) and corynebacterium
parvum.
[0127] The antibody that recognizes the CCDC3 protein concerned
with the present invention includes, but is not limited to, the
following antibodies (i) to (iii).
(i) Antibody having the amino acid sequences of SEQ ID NOS:31, 32,
and 33 for CDR1, CDR2, and CDR3, respectively, of the heavy-chain
variable domain, and the amino acid sequences of SEQ ID NOS:35, 36,
and 37 for CDR1, CDR2, and CDR3, respectively, of the light-chain
variable domain.
[0128] Non-limiting examples of the antibody include preferably an
antibody that has a heavy-chain variable domain with the amino acid
sequence represented by SEQ ID NO:30, and a variable light domain
with the amino acid sequence represented by SEQ ID NO:34.
(ii) Antibody having the amino acid sequences of SEQ ID NOS:39, 40,
and 41 for CDR1, CDR2, and CDR3, respectively, of the heavy-chain
variable domain, and the amino acid sequences of SEQ ID NOS:43, 44,
and 45, respectively, of the light-chain variable domain.
[0129] Non-limiting examples of the antibody include preferably an
antibody that has a heavy-chain variable domain with the amino acid
sequence of SEQ ID NO:38, and a variable light domain with the
amino acid sequence of SEQ ID NO:42.
(iii) Antibody having the amino acid sequences of SEQ ID NOS:47,
48, and 49 for CDR1, CDR2, and CDR3, respectively, of the
heavy-chain variable domain, and the amino acid sequences of SEQ ID
NOS:51, 52, and 53 for CDR1, CDR2, and CDR3, respectively, of the
light-chain variable domain.
[0130] Non-limiting examples of the antibody include preferably an
antibody that has a heavy-chain variable domain with the amino acid
sequence of SEQ ID NO:46, and a variable light domain with the
amino acid sequence of SEQ ID NO:50.
[0131] The antibody that recognizes the CCDC3 protein concerned
with the present invention also includes antibodies that bind to
the epitope bound to the antibodies (i) to (iii).
[0132] The antibody of the present invention has the property to
specifically bind to the CCDC3 protein, and thus can be used to
specifically detect the CCDC3 protein expressed in the tissue of a
subject, or the CCDC3 protein secreted into the blood. That is, the
antibody is useful as a probe for detecting the presence or absence
of CCDC3 protein, or the extent of CCDC3 protein in the biological
tissue or biological sample (including blood) of a subject.
[0133] Specifically, the antibody of the present invention can be
used as a probe, using an ordinary method, for the detection of
CCDC3 protein according to a known detection method, for example,
such as western blotting and ELISA, using a tissue extract of
protein prepared according to an ordinary method from the collected
specimen, which may be blood collected from a subject, or a part of
the subject's visceral adipose tissue obtained by biopsy or some
other procedure.
[0134] Visceral obesity can be diagnosed by determining the
difference in the amount of CCDC3 protein between the biological
tissue or biological sample of a subject and the biological tissue
or biological sample of a normal individual (non-visceral obesity).
Specifically, because the CCDC3 gene has higher expression in the
visceral adipose tissue of a visceral obesity individual than in
the visceral adipose tissue of a normal individual (non-visceral
obesity), visceral obesity is suspected when the amount of CCDC3
protein in the biological tissue of a subject, or the amount of
CCDC3 protein secreted into a biological sample (such as blood) is
higher than in the biological tissue or biological sample of a
normal individual (non-visceral obesity) by 50% or more, preferably
100% or more, more preferably 200% or more.
(3) Test Drug Kit
[0135] The present invention provides a test drug kit that includes
the test drug and is used for the detection or diagnosis of
visceral obesity. The detection kit includes at least one of the
oligo or polynucleotide used as a probe or a primer (may be
labeled, or may be immobilized on a solid phase), and the antibody
above. In addition to the probe and primer, the test drug kit of
the present invention may appropriately include reagents,
instruments, and other components required for performing the
method of the present invention, including hybridization reagents,
probe labels, label detectors, and buffers, as required.
3. Visceral Obesity Detection Method (Diagnosis Method)
[0136] The present invention provides a visceral obesity detection
method (diagnosis method) that uses the test drug of the present
invention.
[0137] Specifically, the detection method (diagnosis method) of the
present invention detects the gene (mRNA) expression levels of the
visceral obesity-associated CCDC3 gene contained in the biological
sample collected from a subject, and the protein (CCDC3 protein)
derived from the gene, and measures the mRNA expression amount or
the amount of the product protein to detect (diagnose) the presence
or absence of visceral obesity in the subject, or the extent of
visceral obesity.
[0138] The detection method of the present invention includes the
following steps (1) and (2).
[0139] (1) The step of measuring the CCDC3 mRNA expression amount
or the amount of the product CCDC3 protein in a biological sample
(test sample) collected from a subject.
[0140] (2) The step of comparing the measured mRNA expression
amount (subject expression amount) or the measured amount of the
product CCDC3 protein (subject production amount) of CCDC3 with the
mRNA expression amount (control expression amount) or the amount of
the product CCDC3 protein (control production amount) of CCDC3 in a
biological sample (control sample) collected from a non-visceral
obesity individual.
[0141] The subject can be determined as having visceral obesity
when the subject expression amount or subject production amount is
higher than the control expression amount or control production
amount as measured by the method.
[0142] Examples of the biological sample include samples prepared
from the biological tissue (e.g., visceral adipose tissue)
collected from a subject, and samples prepared from blood. Specific
examples include RNA-containing samples prepared from such tissues,
samples containing polynucleotides prepared from RNA-containing
samples, and samples containing proteins prepared from the tissues.
The RNA-, polynucleotide-, and protein-containing samples can be
prepared according to an ordinary method from the collected
specimen, which may be blood, or a part of the subject's visceral
adipose tissue collected by methods such as biopsy.
[0143] Specifically, the detection method of the present invention
is performed using the following procedures, according to the type
of the biological sample used as the measurement target.
(1) Use of RNA as Measured Biological Sample
[0144] When RNA is used as the measurement target, visceral obesity
can be detected specifically by a method that includes the
following steps (a), (b), and (c).
[0145] (a) The step of binding the test drug (polynucleotide) of
the present invention to the RNA prepared from a biological sample
(test sample) of a subject, or to a complementary polynucleotide
transcribed from the RNA.
[0146] (b) The step of measuring the test sample-derived RNA or the
complementary polynucleotide transcribed from the RNA by using the
test drug as an index after binding the test drug to the RNA or the
complementary polynucleotide.
[0147] (c) The step of comparing the measurement result (subject
result) from (b) with the result (control result) obtained in the
same manner in steps (a) and (b) for a biological sample (control
sample) derived from a non-visceral obesity individual.
[0148] The detection method (diagnosis method) is performed by
detecting and measuring the CCDC3 gene expression level in the RNA.
Specifically, the method can be performed using a known method such
as northern blotting, RT-PCR method, DNA chip analysis, and in situ
hybridization analysis, using the polynucleotide test drug
(polynucleotide) of the present invention as a primer or a
probe.
[0149] When using northern blotting, the test drug of the present
invention can be used as a probe to detect and measure the presence
or absence of CCDC3 gene expression in the RNA, or the expression
level of CCDC3. As a specific example, the test drug of the present
invention (complementary strand) is labeled with a radioisotope
(.sup.32P, .sup.33P:RI) or a fluorescence substance, and hybridized
with the biological tissue-derived RNA of a subject after
transferring the RNA to a nylon membrane or the like using an
ordinary method. The resulting test drug (DNA) and RNA double
strand is then used for the detection and measurement of signals
from the test drug labels (labeling substance such as RI or
fluorescence substance) using a detector such as a radiation
detector (BAS-1800II, Fuji Film) or a fluorescence detector. In
another exemplary method, the test drug (probe DNA) is labeled
according to the protocol of the AlkPhos Direct Labelling and
Detection System (Amersham Pharmacia Biotech), and hybridized with
the biological tissue-derived RNA of a subject. Signals from the
test drug labels can then be detected and measured using a multi
bio-imager STORM 860 (Amersham Pharmacia Biotech).
[0150] When using a RT-PCR method, the test drug of the present
invention can be used as a primer for the detection and measurement
of the presence or absence of CCDC3 gene expression in the RNA, or
the expression level of CCDC3. As a specific example, cDNA is
prepared from the biological tissue-derived RNA of a subject using
an ordinary method. The cDNA is then hybridized with a pair of
primers (a plus strand that binds to the cDNA (-strand), and an
opposite strand that bind to the +strand) prepared from the test
drug of the present invention, in a manner allowing the target
CCDC3 gene region to be amplified using the cDNA as the template. A
PCR method is then performed according to an ordinary method, and
the resulting amplified double-stranded DNA is detected. Note that
the amplified double-stranded DNA may be detected, for example, by
detecting the labeled double-stranded DNA produced by PCR with
primers labeled beforehand with RI or fluorescence substance, or by
detecting the hybridization product of a labeled test drug (probe)
and the product double-stranded DNA transferred to a nylon membrane
or the like using an ordinary method. Note that the labeled
double-stranded DNA product may be measured using devices such as
Agilent 2100 bioanalyzer (Yokogawa Analytical Systems). Further, a
RT-PCR reaction mixture may be prepared according to the protocol
of the SYBR Green RT-PCR Reagents (Applied Biosystems), and the
product of the reaction performed with ABI PRISM 7700 Sequence
Detection System (Applied Biosystems) may be detected.
[0151] When using DNA chip analysis, detection may be performed by
a method in which a DNA chip carrying the test drug of the present
invention as a DNA probe (single-stranded or double-stranded) is
prepared and hybridized with the cRNA prepared from the biological
tissue-derived RNA of a subject using an ordinary method, and in
which the resulting DNA and cRNA double-strand is bound to the
labeled probe prepared from the test drug of the present invention.
The DNA chip may be one capable of detecting and measuring the
CCDC3 gene expression level.
(2) Use of Protein as Measured Biological Sample
[0152] When protein is used as the measurement target, the visceral
obesity detection method (diagnosis method) of the present
invention is performed by detecting the CCDC3 protein in a
biological sample, and measuring the amount of the detected
protein. Specifically, the method can be performed by using a
method that includes the following steps (a), (b), and (c).
[0153] (a) The step of binding the CCDC3 protein in a biological
sample (test sample) of a subject to the test drug of the present
invention (antibody that recognizes the CCDC3 protein) concerning
the antibody.
[0154] (b) The step of measuring the CCDC3 protein bound to the
test drug in the test sample, using the test drug as an index.
[0155] (c) The step of comparing the measured result (subject
result) from (b) with the result (control result) obtained in the
same manner in steps (a) and (b) for a biological sample (control
sample) derived from a non-visceral obesity individual.
[0156] More specifically, the detection method may be performed by
using a method that detects and quantifies the CCDC3 protein using
a known method such as western blotting, using the antibody
(antibody that recognizes the CCDC3 protein) as the test drug of
the present invention.
[0157] Western blotting may be performed using the primary antibody
and the secondary antibody, for which the test drug of the present
invention, and a labeled antibody (antibody that binds to the
primary antibody) labeled with a radioisotope (such as .sup.125I),
a fluorescence substance, or an enzyme (such as horseradish
peroxidase (HRP)) are used, respectively. Signals from the
radioisotope, fluorescence substance, or other labeling substances
for the labeled compound can then be detected and measured using
devices such as a radiation measurement device (for example,
BAS-1800II; Fuji Film), and a fluorescence detector. It is also
possible to use the test drug of the present invention as the
primary antibody, and then perform detection according to the
protocol of the ECL Plus Western Blotting Detection System
(Amersham Pharmacia Biotech), followed by measurement with a multi
bio-imager STORM 860 (Amersham Pharmacia Biotech).
[0158] Note that the measured CCDC3 protein has a glucose output
effect in liver cells, and the effect to increase the expression of
a rate-limiting enzyme gene in gluconeogenesis, as will be
described later in Example 5. There is a certain correlation
between protein amount and protein function and activity. Thus, the
detection (diagnosis) of visceral obesity according to the present
invention also can be performed by measuring the protein function
or activity, instead of measuring the amount of the product CCDC3
protein. Specifically, the detection method of the present
invention also encompasses a visceral obesity detection (diagnosis)
method that involves measurement and detection according to the
measurement method disclosed in the present application, using the
function or activity of the CCDC3 protein as an index.
(3) Determination of the Presence or Absence of Visceral Obesity
(Diagnosis)
[0159] Visceral obesity can be diagnosed by comparing and
determining the difference between the gene (mRNA) expression level
of the CCDC3 gene in a biological tissue (such as visceral adipose
tissue and blood) of a subject, or the production amount or the
function or activity of the gene expression product protein (CCDC3
protein) (hereinafter, also collectively referred to as "protein
levels"), and the gene expression level (mRNA expression amount) or
the protein level (amount of the product CCDC3 protein) in a
biological tissue (such as visceral adipose tissue and blood) of a
normal individual (non-visceral obesity individual).
[0160] The procedure requires a biological sample (RNA or protein)
collected from the biological tissue of a normal individual
(non-visceral obesity individual). Such samples can be collected
from the biological tissue (such as visceral adipose tissue and
blood) of a non-visceral obesity individual by using methods such
as biopsy and blood collection. As used herein, "non-visceral
obesity individual" means an individual diagnosed as not having
visceral obesity as measured by at least conventional test methods,
for example, such as visceral fat area measurement by computed
tomography (CT), and abdominal circumference measurement. In the
specification, "non-visceral obesity individual" is also referred
to as simply "normal individual" or "non-visceral obesity
individual".
[0161] The comparison of the gene expression level (mRNA expression
amount) or protein amount (amount of the product CCDC3 protein)
between a subject's biological tissue and a normal individual's
biological tissue may be performed by concurrently performing
measurement for the subject's biological sample and the normal
individual's biological sample. When measurement is not performed
concurrently, the mean value or statistical intermediate value of
the gene expression levels (mRNA expression amount) of the CCDC3
gene, or of the amounts of the gene expression product protein
(amounts or levels of the product CCDC3 protein) measured under
uniform measurement conditions using a plurality of (at least two,
preferably 3 or more, more preferably 5 or more) normal biological
tissues may be used for comparison as the gene expression level
(mRNA expression amount) or protein amount (amount of the product
CCDC3 protein) of the normal individual.
[0162] In determining whether the subject has visceral obesity,
visceral obesity is determined or suspected in the subject when the
gene expression level of the CCDC3 gene, or the protein level of
the expression product CCDC3 protein in the biological tissue of
the subject is significantly higher than the levels in the normal
individual. Specifically, an increase from the normal individual
levels by 50% or more, preferably 100% or more, more preferably
200% or more can be used as an index.
4. Method for Screening Components Effective for Ameliorating
Visceral Obesity or Diabetes
[0163] As will be described later in Examples, the present
inventors found strong involvement of CCDC3 protein in visceral
obesity or in the progression of visceral obesity, based on the
findings that CCDC3 gene expression increases specific to the
visceral fat of visceral obesity individuals, and that the CCDC3
protein increases the liver glucose release, and the expression of
the rate-limiting enzyme in gluconeogenesis. Knowing that the CCDC3
protein is involved in the onset or progression of visceral obesity
and diabetes, it is considered that the substance that can suppress
the CCDC3 gene expression (mRNA expression) or CCDC3 protein
function and can thus suppress the liver glucose release or
gluconeogenesis is useful as a component that effectively
ameliorates visceral obesity or diabetes, or as an active
ingredient that prevents MS, cardiovascular disease, or diabetes
complications caused by progression of visceral obesity.
[0164] The screening method of the present invention described
below screens test substances for a substance that has any of the
following properties, using these as an index.
[0165] (3-1) suppression of CCDC3 gene expression (mRNA
expression)
[0166] (3-2) decrease in the amount of CCDC3 protein production
[0167] (3-3) decrease in CCDC3 protein activity
[0168] By searching for such a substance, the method intends to
obtain an active ingredient of visceral obesity ameliorating
agents, or an active ingredient of preventing agents (anti-visceral
obesity agents) for metabolic disease and cardiovascular disease,
such as MS, caused by progression of visceral obesity. The method
also intends to obtain an active ingredient of diabetes
ameliorating agents, or an active ingredient of preventing agents
(antidiabetic agents) for diabetes complications caused by
progression of diabetes.
[0169] Examples of the candidate substance for the active
ingredient of anti-visceral obesity agents or antidiabetic agents
include nucleic acids, peptides, proteins, organic compounds
(including low-molecular compounds and high-molecular compounds),
and inorganic compounds. The screening method of the present
invention can be performed for samples that contain such candidate
substances (will be collectively referred to as "test substances").
Examples of samples that contain candidate substances include cell
extracts, gene library expression products, microorganism culture
supernatants, bacterial components, and fungal components.
(1) Screening Method for Substance Having CCDC3 Gene Expression
Suppressing Activity
[0170] The method screens test substances for a substance that has
CCDC3 gene expression suppressing effect, using the CCDC3 mRNA
expression amount as an index, and obtains the substance as an
active ingredient of anti-visceral obesity agents or antidiabetic
agents.
[0171] Specifically, the method can be performed by performing the
following steps (1) to (3).
[0172] (1) The step of contacting test substances to cells capable
of expressing CCDC3 gene.
[0173] (2) The step of measuring the CCDC3 gene expression amount
in the cells contacted to the test substances.
[0174] (3) The step of selecting a test substance that makes the
CCDC3 gene expression amount lower than in control cells (control
expression amount) not brought into contact with the test
substances.
[0175] Any cells, endogenous or exogenous, may be used for the
screening, as long as the CCDC3 gene can be expressed. The origin
of CCDC3 gene is not particularly limited either, and the CCDC3
gene may originate in humans (SEQ ID NO:1), or in other biological
species, including non-human mammals such as mice. Preferably,
human-derived CCDC3 gene is used. Specific examples of the cells
include CCDC3 gene-containing visceral adipose tissue cells
(including cells from humans and other biological species), and
primary culture cells of isolated visceral adipose tissue cells. It
is also possible to use transformed cells prepared to be capable of
CCDC3 mRNA expression by introducing a CCDC3 gene cDNA-containing
expression vector according to an ordinary method. Tissue, as a
collection of cells, also falls within the definition of the cells
used for screening.
[0176] In step (1) of the screening method of the present
invention, the contact conditions for the test substances and the
CCDC3 gene expressible cells are not particularly limited.
Preferably, culture conditions (such as temperature, pH, and medium
composition) that do not cause cell death and that allow for
expression of the CCDC3 gene are selected.
[0177] Screening of the candidate substances may be performed by
contacting the test substances and the CCDC3 gene expressible
cells, for example, under the foregoing conditions, in order to
search for a substance that suppresses CCDC3 gene expression and
lowers the mRNA expression amount. Specifically, the CCDC3 mRNA
expression amount in the CCDC3 gene expressible cells cultured in
the presence of the test substances is used as an index when it is
lower than the CCDC3 mRNA expression amount (control expression
amount) in the corresponding CCDC3 gene expressible cells cultured
in the absence of the test substances. The test substances
contacted to the cells can then be screened for a substance that
has a CCDC3 gene expression suppressing effect, or for a candidate
substance of an active ingredient of anti-visceral obesity agents
or antidiabetic agents.
[0178] The measurement (detection, quantification) of the CCDC3
mRNA expression amount can be performed by measuring the CCDC3 mRNA
expression amount in the CCDC3 gene expressible cells with, for
example, an oligonucleotide (the test drug of the present
invention) having a complementary sequence of the CCDC3 mRNA base
sequence, using known methods such as northern blotting, RT-PCR
method, and real-time quantitative PCR method. The measurement of
the CCDC3 mRNA expression amount also can be performed by using a
method that uses a DNA array.
[0179] The detection and quantification of the CCDC3 gene
expression level also can be performed by measuring the activity of
a marker gene-derived protein, using a cell line obtained by
introducing a fused gene prepared by attaching a marker gene, for
example, such as a luciferase gene to the gene region (expression
regulatory region) that regulates CCDC3 gene expression. The
screening method for a CCDC3 gene expression control substance
according to the present invention encompasses a method that
searches for a target substance using the expression amount of the
marker gene as an index. In this regard, the concept of the "CCDC3
gene" used in the screening method of the present invention
includes a fused gene of the CCDC3 gene expression regulatory
region and the marker gene.
[0180] Preferably, the marker gene is a structural gene of an
enzyme that catalyzes luminescent reaction or color reaction. Aside
from the luciferase gene, other specific examples include reporter
genes such as alkaliphosphatase gene, chloramphenicol
acetyltransferase gene, .beta. glucuronidase gene, .beta.
galactosidase gene, and aequorin gene. Production of the fused
gene, and the measurement of the marker gene-derived activity may
be performed by using known methods.
[0181] The substance selected by the screening method of the
present invention can position itself as a gene expression
suppressing agent for the CCDC3 gene. The substance suppressing the
CCDC3 gene (mRNA) expression becomes a strong candidate substance
for visceral obesity ameliorating medicaments, or for medicaments
that suppress disease progression and thus prevent the progression
of the disease into metabolic disease and cardiovascular disease
such as MS. Further, the substance suppressing the CCDC3 gene
(mRNA) expression becomes a strong candidate substance for
medicaments that ameliorate diabetes through amelioration of the
hyperglycemia state, or for medicaments that suppress disease
progression and thus prevent the progression of the disease into
diabetes complications.
(2) Substance Screening Method Using CCDC3 Production Lowering
Activity as Index
[0182] The method screens test substances for a substance having a
CCDC3 protein production lowering effect, using a decrease in the
amount of CCDC3 protein production as an index, and obtains the
substance as an active ingredient of anti-visceral obesity agents
or antidiabetic agents.
[0183] Specifically, the method can be performed by performing the
following steps (1') to (3').
[0184] (1') The step of contacting test substances to cells capable
of producing CCDC3 protein.
[0185] (2') The step of measuring the amount of the product CCDC3
protein in the cells contacted to the test substances.
[0186] (3') The step of selecting a test substance that makes the
CCDC3 production amount lower than in control cells (cells capable
of CCDC3 production; control production amount) not brought into
contact with the test substances.
[0187] Any cells, endogenous or exogenous, may be used for the
screening (including control cells), as long as the CCDC3 gene can
be expressed and CCDC3 protein is produced. The origin of CCDC3
gene is not particularly limited either, and the CCDC3 gene may
originate in humans, or in other biological species, including
non-human mammals such as mice. Preferably, human-derived CCDC3
gene is used. Specific examples of the cells include CCDC3
gene-containing visceral adipose tissue cells (including cells from
humans and other biological species), and primary culture cells of
isolated visceral adipose tissue cells.
[0188] It is also possible to use transformed cells prepared to be
capable of CCDC3 protein production by introducing a CCDC3 gene
cDNA-containing expression vector according to an ordinary method.
Tissue, as a collection of cells, also falls within the definition
of the cells used for screening.
[0189] In step (1') of the screening method (3-2) of the present
invention, the contact conditions for the test substances and the
cells capable of CCDC3 protein production are not particularly
limited. Preferably, culture conditions (such as temperature, pH,
and medium composition) that do not cause cell death and that allow
for production of the CCDC3 protein are selected.
[0190] Screening of the candidate substances may be performed by
contacting the test substances and the cells capable of CCDC3
protein production, for example, under the foregoing conditions, in
order to search for a substance that lowers the amount of the CCDC3
protein produced. Specifically, the amount of the CCDC3 protein
produced in the cells capable of CCDC3 protein production cultured
in the presence of the test substances is used as an index when it
is lower than the CCDC3 production amount (control production
amount) in the corresponding cells capable of CCDC3 protein
production cultured in the absence of the test substances. The test
substances contacted to the cells can then be screened for a
substance that has a CCDC3 protein production lowering effect, or
for a candidate substance of an active ingredient of anti-visceral
obesity agents or antidiabetic agents.
[0191] The measurement (detection, quantification) of the CCDC3
protein production amount can be performed by measuring the CCDC3
protein amount from the cells capable of producing CCDC3 protein,
using the antibody (anti-CCDC3 antibody; the test drug of the
present invention) for the CCDC3 protein according to a known
method such as western blotting, immunoprecipitation, and ELISA.
Specifically, western blotting may be performed using the primary
antibody and the secondary antibody, for which the test drug of the
present invention, and a labeled antibody (antibody that binds to
the primary antibody) labeled with a radioisotope (such as
.sup.125I), a fluorescence substance, or an enzyme (such as
horseradish peroxidase (HRP)) are used, respectively. Signals from
these labeling substances can then be measured using devices such
as a radiation measurement device (for example, BAS-1800II; Fuji
Film), and a fluorescence detector. It is also possible to use the
test drug of the present invention as the primary antibody, and
then perform detection according to the protocol of the ECL Plus
Western Blotting Detection System (Amersham Pharmacia Biotech),
followed by measurement with a multi bio-imager STORM 860 (Amersham
Pharmacia Biotech).
[0192] The substance selected by the screening method of the
present invention can position itself as a CCDC3 protein production
suppressing agent. The substance suppressing the CCDC3 protein
production becomes a strong candidate substance for visceral
obesity ameliorating medicaments, or for medicaments that suppress
disease progression and thus prevent the progression of the disease
into metabolic disease and cardiovascular disease such as MS.
(3) Substance Screening Method Using CCDC3 Protein Function or
Activity Lowering Effect as Index
[0193] The method screens test substances for a substance having a
CCDC3 protein function or activity lowering effect, using a
decrease in the CCDC3 protein effect as an index, and obtains the
substance as an active ingredient of anti-visceral obesity agents
or antidiabetic agents.
[0194] Specifically, the method can be performed by performing the
following steps (1'') to (3'').
[0195] (1'') The step of contacting test substances, cells that
react with CCDC3 protein, and CCDC3 protein to one another.
[0196] (2'') The step of detecting CCDC3 protein function or
activity in the cells.
[0197] (3'') The step of selecting a test substance that makes the
detected function or activity lower than the CCDC3 protein function
or activity of the control cells (cells that react with CCDC3
protein) contacted to CCDC3 protein but not to the test
substances.
[0198] The cells used for screening that react with CCDC3 protein
are cells that show some biological activity in contact with CCDC3
protein. Specific examples of such cells include liver-derived cell
lines, and isolated primary cultured cells of the liver. Tissue, as
a collection of cells, also falls within the definition of the
cells used for screening.
[0199] Screening of the candidate substances may be performed by
contacting the test substances to the cells that react with CCDC3
protein, or to the cell fractions thereof, for example, under the
foregoing conditions, in order to search for a substance that
lowers the CCDC3 protein effect. Specifically, the CCDC3 protein
function or activity in the CCDC3 protein reacting cells cultured
with CCDC3 protein in the presence of the test substances is used
as an index when it is lower than the CCDC3 protein function or
activity (control function or activity) in the corresponding the
CCDC3 protein reacting cells cultured with CCDC3 protein in the
absence of the test substances. The test substances can then be
screened for a substance that has a CCDC3 protein function or
activity lowering effect, or for a candidate substance of an active
ingredient of anti-visceral obesity agents or antidiabetic
agents.
[0200] Examples of the CCDC3 protein function or activity include
the glucose output promoting effect from the liver cells in
response to the CCDC3 protein added to the liver cells, and the
effect of increasing the mRNA expression amounts of the
gluconeogenesis rate-limiting enzymes glucose-6-phosphatase
(G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). These
functions and activities can be measured using the methods
described in Example 5.
(4) Screening Method Using CCDC3 Transgenic Mice
[0201] The method screens for test substances for a substance
(candidate substance) that can be used as an active ingredient of
anti-visceral obesity agents or antidiabetic agents, using, as an
index, a decrease in the body weight or glucose level of CCDC3
transgenic mice (CCDC3 Tg mice) transformed to express CCDC3 mRNA
and produce CCDC3 protein at high levels.
[0202] Specifically, the method can be performed by performing the
following steps (a) to (c).
[0203] (a) The step of administering a test substance to CCDC3 Tg
mice.
[0204] (b) Measuring the body weight (subject weight) or glucose
level (subject glucose level) of the test substance-administered
CCDC3 Tg mice over a time course, and comparing the result with the
time-course body weight (control body weight) or glucose level
(control glucose level) of CCDC3 Tg mice that did not receive the
test substance.
[0205] (c) The step of selecting a test substance that makes the
subject weight or subject glucose level lower than the control body
weight or control glucose level, the test substance being selected
as an active ingredient that ameliorates visceral obesity or
diabetes.
[0206] The CCDC3 Tg mice used for screening are mice artificially
transformed to express CCDC3 mRNA at high levels, and can be
produced using various methods, including a method involving DNA
microinjection into the pronucleus of a fertilized egg, and a
method that produces chimeric mice after introduction of DNA into
an embryonic stem cell (Mouse Laboratory Manual, 2nd ed., Tokyo
Metropolitan Institute of Medical Science, Test Animal Research
Department, ed., Springer Japan (2005)). The methods described in
Example 7 below also may be used to produce the mice. The mice are
characterized by their significantly increased body weights and
high glucose levels compared to normal mice even on the same diet
(FIGS. 7 and 8).
[0207] Screening of the candidate substances can be performed by
serially measuring the body weights and glucose levels of the test
substance-administered CCDC3 Tg mice (subject mice) and the test
substance-free CCDC3 Tg mice (control mice) grown in the same
environment. The test substance that significantly lowers the body
weights of the subject mice compared with the control mice can then
be selected as an active ingredient that ameliorates visceral
obesity. The test substance that significantly lowers the glucose
levels of the subject mice compared to the control mice can be
selected as an active ingredient that ameliorates diabetes.
[0208] The substance screened for by the screening methods (1) to
(4) has the effect to suppress increased (enhanced) expression of
CCDC3 gene in visceral adipose tissue cells and thus suppress liver
glucose release or gluconeogenesis. The substance can thus be used
as an active ingredient of compositions (anti-visceral obesity
agents) that ameliorate visceral obesity or prevent metabolic
disease cardiovascular disease, such as MS, caused by progression
of the disease. The substance also can be used as an active
ingredient of compositions (antidiabetic agents) that ameliorate
diabetes or prevent progression of the disease (including onset of
diabetes complications).
[0209] The candidate substance screened for by the screening method
can be further screened using pathological non-human animals having
visceral obesity, or pathological non-human animals having
metabolic disease or cardiovascular disease such as MS, or
diabetes. The candidate substance so screened also can be used in
an efficacy test or safety test using pathological non-human
animals having visceral obesity or diabetes, or pathological
non-human animals having metabolic disease or cardiovascular
disease such as MS. It is also possible to use the candidate
substance in a clinical test for visceral obesity individuals
(humans), or for individuals (humans) in the stage prior to disease
onset or individuals suffering from metabolic disease or
cardiovascular disease, such as MS, caused by the progression of
the disease. More practical active ingredients for anti-visceral
obesity agents and antidiabetic agents can be screened and obtained
through these tests.
[0210] The substance so screened may be industrially produced by
using chemical synthesis, biological synthesis (including
fermentation), and genetic procedures, depending on the type of
substance, after performing structure analysis, as required. The
product substance can then be used for the preparation of
anti-visceral obesity agents and antidiabetic agents.
5. Anti-Visceral Obesity Agent, Antidiabetic Agent
[0211] CCDC3 gene siRNA can suppress CCDC3 mRNA expression in CCDC3
gene-introduced cells, as will be described later in Example 6. The
siRNA can be used as an active ingredient of visceral obesity
ameliorating agents, or an active ingredient of medicinal agents
(anti-visceral obesity agents) for preventing progression of
metabolic disease and cardiovascular disease, such as MS, caused by
the progression of the disease. The siRNA also has use as an active
ingredient of diabetes ameliorating agents, or an active ingredient
of medicinal agents (antidiabetic agents) for preventing
progression of diabetes complications caused by the progression of
the disease.
[0212] The present invention thus provides an anti-visceral obesity
agent that includes siRNA as an active ingredient for suppressing
CCDC3 gene expression. The present invention also provides an
antidiabetic agent that includes siRNA as an active ingredient for
suppressing CCDC3 gene expression.
[0213] The siRNA concerned with the present invention is
double-stranded, and is formed by hybridization of a sense strand
(a sequence complementary to the target sequence of CCDC3 gene) and
an antisense strand complementary to the sense strand. The double
strand may be a siRNA (shRNA) molecule of a closed-end structure,
for example, a hairpin structure. The siRNA is not particularly
limited, as long as it can suppress CCDC3 gene expression. Examples
of mouse CCDC3 gene siRNA include:
[0214] siRNA #1 that targets the 2236-2256 region of a mouse CCDC3
gene base sequence (SEQ ID NO:2): sense strand r
(gacacaugauacugauaaa)dTdT (SEQ ID NO:5), antisense strand r
(uuuaucaguaucauguguc)dTdG (SEQ ID NO:6),
[0215] siRNA #2 that targets the 1506-1526 region: sense strand r
(gaaagauguuaagacuuaa)dTdT (SEQ ID NO:7), antisense strand r
(uuaagucuuaacaucuuuc)dAdG (SEQ ID NO:8), and
[0216] siRNA #3 that targets the 2011-2131 region: sense strand r
(gaaguauuugcauaacaaa)dTdT (SEQ ID NO:9), antisense strand r
(uuuguuaugcaaauacuuc)dTdA (SEQ ID NO:10).
[0217] Human CCDC3 gene siRNA can be constructed in the same
manner.
[0218] The active ingredient of anti-visceral obesity agents or
antidiabetic agents of the present invention is not limited to the
siRNA that suppresses CCDC3 gene expression, and a nucleic acid
that suppresses CCDC3 gene also can be used as the active
ingredient of anti-visceral obesity agents or antidiabetic agents.
Examples of nucleic acid include miRNA, antisense
poly(oligo)nucleotides, ribozymes, aptamers, and decoys for the
CCDC3 gene. The antisense poly(oligo)nucleotide has a base sequence
complementary to or substantially complementary to the CCDC3 gene
base sequence, or has a part of such a sequence, and hybridizes
with the CCDC3 gene RNA to inhibit the synthesis or function of the
RNA, or modulate or regulate CCDC3 gene expression through
interaction with the RNA. Any antisense poly(oligo)nucleotide,
including, for example, antisense RNA and antisense DNA, may be
used, as long as it has the foregoing effects.
[0219] The antisense poly(oligo)nucleotide is configured from
typically about 10 to 1,000 bases, preferably about 15 to 500
bases, further preferably about 16 to 30 bases. In order to prevent
degradation due to hydrolase such as nuclease, the phosphate
residues (phosphates) of the nucleotides forming the antisense DNA
may be substituted with chemically modified phosphate residues, for
example, such as phosphorothioate, methylphosphonate, and
phosphorothioate. The antisense poly(oligo)nucleotide can be
produced by using a known device such as a DNA synthesizer.
[0220] The active ingredient of anti-visceral obesity agents or
antidiabetic agents according to the present invention is not
limited to the nucleic acid that suppresses CCDC3 gene, and
substances having the effect to suppress the CCDC3 protein function
also can be used as the active ingredient of anti-visceral obesity
agents or antidiabetic agents. Examples of such substances include
antibodies against CCDC3 protein, particularly neutralizing
antibodies.
[0221] As used herein, neutralizing antibodies mean antibodies
having the properties of inhibiting the intrinsic functions or
activities of antigens upon binding to the antigens. Neutralizing
antibodies against CCDC3 protein mean antibodies having the
properties of inhibiting the functions or activities of CCDC3
protein upon binding to the CCDC3 protein.
[0222] In addition to the active ingredient (including siRNA,
antisense poly(oligo)nucleotide, and antibody) that suppresses
CCDC3 gene expression or CCDC3 functions, the anti-visceral obesity
agent or antidiabetic agent of the present invention may also
include any types of carriers and additives, for example,
pharmaceutically acceptable carriers and additives.
[0223] Non-limiting examples of pharmaceutically acceptable
carriers and additives include excipients such as sucrose and
starch; binders such as cellulose and methylcellulose;
disintegrants such as starch and carboxymethylcellulose; lubricants
such as magnesium stearate and aerosil; aromatizers such as citric
acid and menthol; preservatives such as sodium benzoate and sodium
bisulfite; stabilizers such as citric acid and sodium citrate;
suspensions such as methylcellulose and polyvinylpyrrolidone;
dispersants such as surfactants; diluents such as water and
physiological saline; and base wax.
[0224] Examples of preparations suited for oral administration
include liquid, capsule formulations, sachets, tablets, suspension
agents, and emulsions. Examples of preparations suited for
parenteral administration (for example, subcutaneous injection,
intramuscular injection, local injection, intraperitoneal
administration) include aqueous and non-aqueous isotonic aseptic
injections, which may contain an antioxidant agent, a buffer, an
antibacterial agent, a tonicity agent, or other such components.
Other examples of parenteral administration preparations include
aqueous and non-aqueous aseptic suspension agents, which may
contain a suspension, a solubilizer, a thickener, a stabilizer, an
antiseptic, or other such components.
[0225] The dose of the preventative or therapeutic agent of the
present invention varies depending on factors such as the body
weight and age of the subject, and the seriousness of disease, and
cannot be set definitively. For example, the agent may be given in
several milligrams to several ten milligrams/kg body weight/day for
adults in terms of an active ingredient amount, once a day, or in
divided portions several times a day.
[0226] When the active ingredient is encoded by DNA, it is
considered possible to perform a gene therapy by incorporating the
DNA into a vector for gene therapy. Further, when the active
ingredient is an antisense polynucleotide, a gene therapy can be
performed by incorporating the antisense polynucleotide either
directly or via a vector for gene therapy. As above, the dose of
the gene therapy composition, and the administration method vary
depending on factors such as the body weight, age, and symptom of a
patient, and may be appropriately selected by a skilled
artisan.
EXAMPLES
[0227] The present invention is described below based on Examples.
Note, however, that the present invention is not limited to the
following Examples. Further, in the Examples below, procedures such
as gene modifications and cell cultures followed the methods
described in, for example, Molecular Cloning: A Laboratory Manual,
Second Edition (1989) (Cold Spring Harbor Laboratory Press),
Current Protocols in Molecular Biology (Greene Publishing
Associates and Wiley-Interscience), unless otherwise stated.
Example 1
Microarray Analysis Using Human Adipose Tissue
(1) Obesity Patients Adipose Tissue Used in DNA Microarray
Analysis
[0228] Abdominal visceral adipose tissue and abdominal subcutaneous
adipose tissue were donated by patients arranged to undergo
surgical operation at Shiga University of Medical Science Hospital,
after explaining the content of the research and obtaining their
consent. A total of 10 patients, male, from age 55 to 75 were
categorized into two groups: non-visceral obesity individuals
(normal glucose tolerance) with abdominal circumferences of less
than 85 cm (5 cases, average abdominal circumference of 78.5
cm.+-.2.5 cm), and visceral obesity individuals (normal glucose
tolerance) with abdominal circumferences of 85 cm and higher (5
cases, average abdominal circumference of 90.7 cm.+-.3.6 cm).
(2) Search for Genes that Show Varying Expression Specific to
Obesity Patients Visceral Adipose Tissue
[0229] The frozen visceral adipose tissue and frozen subcutaneous
adipose tissue of the five non-visceral obesity individuals and the
five visceral obesity individuals were sliced into about a 5
mm.times.5 mm size, and homogenized in QIAzol Lysis Reagent
(Qiagen) using TissueLyser (Qiagen). Chloroform was added and mixed
with the resulting homogenate, and the aqueous layer was collected
by centrifugation. Total RNA was isolated and purified from the
aqueous layer using RNeasy Lipid Tissue Mini Kit (Qiagen),
according to the protocol attached to the Kit. The purified total
RNA was quantified at 260 nm absorbance, and checked for the
absence of RNA degradation using an Agilent 2100 Bioanalyzer
(Agilent Technologies).
[0230] This was followed by labeled cRNA synthesis, which was
performed using a Low RNA Input Linear Amplification Kit (Agilent
Technologies) according to the single-dye method experiment
protocol attached to the Kit. cDNA was synthesized with 500 ng of
the purified total RNA by reverse transcription reaction, using
oligo-dT primers that included T7 promoter added to the 5' end. By
using the cDNA as a template, labeled cRNA was synthesized by in
vitro transcription performed with T7 RNA Polymerase in the
presence of Cy3-labeled UTP (PerkinElmer). The resulting labeled
cRNA was purified with RNeasy Mini Kit (Qiagen), and the yield was
quantified at 260 nm absorbance.
[0231] After fragmenting the Cy3-labeled cRNA, hybridization was
performed at 65.degree. C. for 17 hours using Whole Human Genome
Oligo Microarray (Agilent Technologies). The microarray was washed
and dried, and a Cy3 wavelength fluorescence image was obtained
using a DNA microarray scanner (Agilent Technologies). The
fluorescence signal of each probe was then quantified using Feature
Extraction Software (Agilent Technologies). Data were analyzed
using GeneSpring version 7.0 (Agilent Technologies). The
microarrays were normalized by setting the same median value for
the signals of the all probes on the array.
[0232] The analysis revealed the presence of the coiled-coil domain
containing protein 3 (CCDC3) gene, whose expression in the visceral
adipose tissue of the visceral obesity individuals was higher than
in the visceral adipose tissue of the non-visceral obesity
individuals by a factor of 3.2 (left-hand side in FIG. 1). In
contrast, in the subcutaneous adipose tissue, CCDC3 gene expression
was only slightly higher in the visceral obesity individuals than
in the non-visceral obesity individuals (right-hand side in FIG.
1). There results demonstrated that the increased gene expression
was a phenomenon specific to the visceral adipose tissue.
Example 2
CCDC3 Expression Analysis in Obesity Model db/db Mice
[0233] (1) Real-time PCR was performed to examine whether the
increased CCDC3 gene expression in the visceral fat of the obesity
patients changes in the adipose tissue of obesity pathological
model mice (db/db mice). For the analysis, db/db male mice (CLEA
Japan), 15 weeks old were used, and db/m mice (CLEA Japan) were
used as a control group.
[0234] The mesenteric adipose tissue (visceral adipose tissue), and
the subcutaneous adipose tissue and periepididymal adipose tissue
(both for comparison) were collected from five mice in each group,
and RNA was prepared. The RNAs of the five mice in each group were
pooled as a total RNA sample for microarray analysis. The methods
of Example 1(2) were used for the RNA preparation, cDNA synthesis,
microarray analysis, and detection. Note, however, that a Whole
Mouse Genome Oligo Microarray for mice (Agilent Technologies) was
used as the microarray. The analysis confirmed a 2.1-fold increase
in CCDC3 gene expression in the mesenteric fat of the obesity model
mice (db/db mice) over the control groups (FIG. 2).
[0235] The results of the microarray analysis of the humans and the
obesity model mice suggested that a CCDC3 gene expression amount
could be used as an index to determine obesity, particularly
visceral obesity.
[0236] (2) The obesity pathological model mice DIO (Diet-Induced
Obese) mice, 20 weeks old, were used for experiment that compared
high expression genes (2-fold or more, vs CE-2 diet mice) and low
expression genes (0.5-fold or more, vs CE-2 diet mice) in the
mesenteric fat and periepididymal fat. Note that the DIO mice were
produced from C57BL/6J Jcl male mice (CLEA Japan), 7 weeks old,
that had the high-fat diet (HFD) 58Y1DIO P.D. 60% Energy From
Fat-Blue (Japan SLC) for 13 weeks. As a control group, mice
produced from C57BL/6J Jcl male mice (CLEA Japan), 7 weeks old,
that had CLEA Rodent Diet CE-2 (CLEA Japan) for 13 weeks were used.
An investigation found that the number of genes that showed
expression changes under a high-fat diet load was 1,241
(338+229=567 high expression genes, 360+314=674 low expression
genes) in the mesenteric fat, and 3,205 (1,349+229=1,578 high
expression genes, 1,313+314=1,627 low expression genes) in the
periepididymal fat (Table 1), demonstrating that expression varies
in greater numbers of genes in the periepididymal fat. However,
only a few genes showed expression changes in the both mesenteric
and periepididymal adipose tissues (229 high expression genes, 314
low expression genes), revealing that these adipose tissues show
different gene expression changes, even though the both are white
adipose tissues (see Table 1).
TABLE-US-00001 TABLE 1 Number of high Number of low expression
genes expression genes Mesenteric fat only 338 360 Epididymal fat
only 1349 1313 Mesenteric fat and 229 314 epididymal fat
Example 3
Confirmation for Secretory Protein
[0237] The amino acid sequence of CCDC3 protein was analyzed by
using two algorithms, Neural Network (NN) and Hidden Markov Model
(HMM) of SignalP 3.0, a webtool for predicting the signal sequence
site and its cleavage site in an amino acid sequence. The both
algorithms suggested the possibility that the CCDC3 protein is a
protein with a signal sequence, and it was inferred that its
cleavage site was between amino acid residues 21 and 22.
[0238] To confirm this, human CCDC3 overexpression vector
pReceiver-CCDC3 was introduced into HEK293T cells, and the CCDC3
protein secreted into a medium was detected by western blotting.
Specifically, CCDC3 expression vector pReceiver-CCDC3 was
introduced into HEK293T cells cultured to about 80% confluence in a
10-cm petri dish, according to the manual attached to Lipofectamine
2000 (Invitrogen). After 40 hours from the introduction, the
culture and a cell extract dissolved in 1-mL Lysis buffer (25
mmol/L Tris-HCl (pH 8) containing 0.1 mol/L NaCl, 1% Triton X-100,
and protein inhibitor) were collected, and subjected to western
blotting in 10 .mu.L and 0.8 .mu.L, respectively (reduced state).
For the detection, anti-FLAG M2-HRP antibodies (SIGMA) and ECL plus
(GE Healthcare Biosciences) were used. The results are presented in
FIG. 3.
[0239] The analysis results confirmed the CCDC3 protein signal in
the culture (medium), suggesting that the CCDC3 protein is a
secretory protein.
Example 4
Purification of Human CCDC3 Recombinant Protein
[0240] A human CCDC3 expression vector pReceiver-M14-humam CCDC3
with a 3.times. Flag tag fused on the C-terminus side was purchased
from GeneCopoeia. The human CCDC3 expression vector was used as a
template, and a plasmid with an inserted base sequence that encodes
a 3.times. Flag tag cutting, thrombin-specific cut amino acid
recognition sequence was constructed, and used for protein
expression purification. The human CCDC3 expression vector was
transfected into Freestyle293 cells using 293fectin (Invitorogen),
according to an ordinary method. The cells were shake cultured in a
Freestyle293 expression medium (Gibco) at 37.degree. C. for 2 days,
and the culture supernatant was collected by centrifugation. The
culture supernatant was then applied to an ANTI-FLAG M2 Affinity
Gel (Sigma) column equilibrated beforehand with a 0.5 M
NaCl-containing 20 mM HEPES-Na buffer (pH 7.5) to adsorb the
3.times. Flag tag-fused CCDC3 protein. After being washed with the
same buffer equivalent of the volume of five columns, the CCDC3
protein was eluted with the same buffer containing 0.1 mg/ml
3.times. FLAG Peptide (Sigma). The elution fraction was
electrophoresed by SDS-PAGE, and a 3.times. Flag tag-fused CCDC3
protein with a molecular weight of 34 kDa was confirmed. The CCDC3
protein-containing fraction was concentrated with Amicon Ultra-15
Centrifugal Filter 10k MWCO (Millipore), and 1 unit of thrombin
(Sigma, T7513-100u) was added per about 10 .mu.g of the CCDC3
protein to cut the 3.times. Flag tag in a 15-hour treatment at room
temperature. After the cutting treatment, the liquid was applied to
Benzamidine Sepharose 6B resin to remove the thrombin. As the final
purification, the sample was applied to a Superdex 200 16/60 column
(GE healthcare) equilibrated with the buffer, and the elution
fraction was electrophoresed by SDS-PAGE to collect a CCDC3 protein
with a molecular weight 31 kDa. The protein was obtained as a
purified sample of the human CCDC3 recombinant protein.
Example 5
Influence on Human CCDC3 Recombinant Protein Stimulation on Liver
Glucose Release
(1) Isolation of Rat Liver Cells
[0241] Wistar rats (10 weeks old, male) were anesthetized with
pentobarbital, and the liver was perfused by cannulation with an
about 350 mL of Liver Perfusion Medium (Invitrogen 17701-038) that
contained 2% penicillinstreptomycin. Perfusion was continued
further for about 10 min with Liver Digest Medium (Invitrogen
17703-034). The perfused liver was transferred to a 15-cm petri
dish, and shaken in Hepatocyte Wash Medium (Invitrogen 17704-024)
supplemented with 1% FBS (GIBCO) and 1% penicillinstreptomycin
(GIBCO) to liberate the cells. The liver cells suspended in
Hepatocyte Wash Medium was filtered through a gauze (4 layers), and
collected into a 50-ml Falcon tube. After centrifugation at 400 rpm
for 1 min at 4.degree. C., the supernatant was discarded, and the
precipitated liver cells were suspended in a new Hepatocyte Wash
Medium, and centrifuged at 400 rpm for 1 min at 4.degree. C. After
removing the supernatant, the collected liver cells were suspended
in an ice-cooled Hepatocyte Wash Medium supplemented with 10% FBS
and 1% penicillinstreptomycin, and centrifuged (400 rpm, 2 min,
4.degree. C.). The washing procedure was repeated twice. The
resulting liver cells were suspended in an about 10-mL 10% FBS
Hepatocyte Wash Medium, and counted after trypan blue staining. The
cells were appropriately diluted with 10% FBS William's E Medium
(SIGMA-ALDRICH, W4128), and used for the experiments below.
(2) Glucose Output Measurement in Liver Cells
[0242] The liver cells isolated as above were inoculated on a
24-well collagen plate (BIOCOAT) in 2.5.times.10.sup.5/well, and
cultured for 4 hours. The cells were then cultured overnight in a
William's medium E supplemented with 0.5% BSA (SIGMA) and 100 nM
dexamethasone (SIGMA). On the next day, the medium was exchanged
with a medium prepared by adding gluconeogenesis substrate (2 mM
lactate (SIGMA-ALDRICH, L-1750) and 0.2 mM pyruvate (Nacalai
tesque)) to glucose free DMEM (SIGMA, D5030), and assessment was
made whether the human recombinant CCDC3 protein (100 nM) prepared
in Example 4 has any influence on glucose output in the presence or
absence of protein stimulation.
[0243] As a positive control of liver glucose release increase, 3
nM glucagon (Peptide Institute, 4098-s) was used. After medium
exchange, the cells were cultured at 37.degree. C. for 4 hours, and
the supernatant was collected. The liver cells were washed with
PBS(-), and dissolved in 0.25 N NaOH (250 .mu.l). The glucose
concentration in the supernatant, and the protein amount were
quantified using a glucose C-Il-test wako (WAKO, 437-90902), and a
BCA protein assay kit (PIERCE 23225), respectively. The glucose
concentration was corrected with the liver cell protein amount. The
results are presented in FIG. 4. As shown in FIG. 4, it was
confirmed that the glucose output increased by stimulation with 100
nM CCDC3 protein (CCDC3 protein 0 nM vs 100 nM, 1.2-fold increase,
n=6, p<0.01).
(3) Measurement of Gluconeogenesis System Gene Expression
[0244] RNA was prepared from the rat liver cells cultured for 4
hours, using an RNeasy mini kit (QIAGEN) according to the attached
manual. cDNA was synthesized from the total RNA (500 ng) using a
High Capacity RNA-to-cDNA Kit (Applied Biosystems) according to the
attached manual. By using the cDNA as a template, real-time
quantitative PCR was performed to examine the mRNA expression
amounts of the gluconeogenesis rate-limiting enzymes
glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate
carboxykinase (PEPCK). Ribosomal protein S18 (Rps18) was used as a
correction gene. Quantification of mRNA amounts was performed by
real-time quantitative PCR with SYBR Green I, using rat G6Pase and
rat Rps18 real-time quantitative PCR primers synthesized by Takara
Bio.
TABLE-US-00002 G6Pase: Forward primer: (SEQ ID NO: 8)
5'-TGCGTGCCATAGGACTCATCA-3', Reverse primer: (SEQ ID NO: 9)
5'-AGCAAACAATTGCCCACCGTA-3' Rps18: Forward primer: (SEQ ID NO: 10)
5'-AAGTTTCAGCACATCCTGCGAGTA-3', Reverse primer: (SEQ ID NO: 11)
5'-TTGGTGAGGTCAATGTCTGCTTTC-3'
[0245] POWER SYBR GREEN PCR MASTER MIX (Applied Biosystems) was
used as the reaction reagent, and the quantification was performed
by a reaction at 50.degree. C. for 2 min, and then at 95.degree. C.
for 10 min, followed by 40 cycles of reaction at 95.degree. C. for
15 sec and at 60.degree. C. for 60 sec. PCR and fluorescence
measurement were performed using 7500 Real time PCR System (Applied
Biosystems). For the rat PEPCK, the real-time quantitative PCR was
performed using Taqman probe Rn01529009_g1 (Applied Biosystems).
Taqman Universal PCR Master Mix (Applied Biosystems) was used as
the reaction reagent, and the quantification was performed by a
reaction at 50.degree. C. for 2 min, and then at 95.degree. C. for
10 min, followed by 40 cycles of reaction at 95.degree. C. for 15
sec and at 60.degree. C. for 1 min. PCR and fluorescence
measurement were performed using 7500 Real time PCR System (Applied
Biosystems).
[0246] The results are presented in FIG. 5. As shown in FIG. 5, the
analysis results confirmed that stimulation by 100 nM CCDC3 protein
significantly increased the mRNA expression of the gluconeogenesis
rate-limiting enzymes G6Pase and PEPCK (CCDC3 protein 0 nM vs 100
nM: 2.6-fold increase in G6Pase, 2.2-fold increase in PEPCK, n=5,
p<0.01 for each enzyme). The results thus suggested that the
CCDC3 protein increases liver glucose release by accelerating the
gluconeogenesis system.
Example 6
CCDC3 Expression Suppression by Introduced siRNA
(1) 3T3-L1 (Mouse Pre-Adipocyte) Culture
[0247] 3T3-L1 cells (mouse pre-adipocyte) were cultured to
confluence in a 10-cm petri dish (Biocoat CollagenI Cellware 100 mm
Dish, BD Biosciences), and induced to differentiate in an exchanged
differentiation medium D-MEM (+10% FBS, +1% Penicillin
G/Streptomycin, 5 .mu.g/ml insulin, 0.5 mM
3-isobutyl-1-methylxanthin, 1 .mu.M dexamethasone). On day 2 of the
induced differentiation, the medium was exchanged with D-MEM (+10%
FBS, +1% Penicillin G/Streptomycin), and the cells were cultured
until day 5 of the induced differentiation.
(2) CCDC3 siRNA Introduction Using Nucleofector (AMAXA) The
differentiated 3T3-L1 cells were detached from the petri dish with
trypsin (GIBCO), suspended in D-MEM medium, and collected into a
tube. After 5-min centrifugation at 500 rpm, the D-MEM medium was
removed, and gently suspended in a Nucleofector Solution (200
.mu.l/100-mm petri dish) attached to Cell Line Nucleofector Kit L
(AMAXA). The prepared 100 .mu.l of cell suspension was dispensed in
a 1.5-ml tube, and 200 pmol CCDC3 siRNA (QIAGEN, HP GenomeWide
siRNA (siRNA #1: sense strand r (gacacaugauacugauaaa)dTdT (SEQ ID
NO:5), antisense strand r (uuuaucaguaucauguguc)dTdG (SEQ ID NO:6);
siRNA #2: sense strand r (gaaagauguuaagacuuaa)dTdT (SEQ ID NO:7),
antisense strand r (uuaagucuuaacaucuuuc)dAdG (SEQ ID NO:8); siRNA
#3: sense strand r (gaaguauuugcauaacaaa)dTdT (SEQ ID NO:9),
antisense strand r (uuuguuaugcaaauacuuc)dTdA (SEQ ID NO:10)), or
control siRNA (QIAGEN, AllStars RNAi Controls) was added. The
mixture was gently suspended, and transferred to a cuvette attached
to the kit. Note that the CCDC3 siRNA #1, #2, and #3 are siRNAs
that target the 2236-2256, 1506-1526, and 2011-2131 regions,
respectively, of the mouse CCDC3 gene base sequence. The cuvette
was set on the main unit of Nucleofector, and electroporation was
performed. Then, 500 .mu.l of D-MEM (+10% FBS, +1% Penicillin
G/Streptomycin) was added to the cuvette, and gently suspended
using the attached dropper. The cell suspension was inoculated to
the preincubated D-MEM (1.5 ml; +10% FBS, +1% Penicillin
G/Streptomycin) on a 6-well plate (Biocoat CollagenI Cellware
6-well plate, BD Biosciences), and RNA was prepared after culturing
the cells for 2 days.
(3) RNA Preparation and CCDC3 Expression Analysis
[0248] RNA was prepared from the CCDC3 siRNA-introduced cells,
using an RNeasy mini kit (QIAGEN) according to the attached manual.
cDNA was synthesized from the total RNA using a SuperScript
First-strand Synthesis System for RT-PCR (Invitrogen) according to
the attached manual. By using the cDNA as a template, real-time
quantitative PCR was performed to examine the CCDC3 mRNA expression
amounts. 36B4 was used as the correction gene. Quantification of
mRNA amounts was performed by real-time quantitative PCR with SYBR
Green I, using the mouse CCDC3 real-time quantitative PCR primers
synthesized by Takara Bio (Forward primer:
5'-TGGTTCATCGCCTCTAATGGTG-3' (SEQ ID NO:15), Reverse primer:
5'-TGGAATGGGTTCTAGGTGGTCAA-3' (SEQ ID NO:16)), and the mouse 36B4
real-time quantitative PCR primers (Forward primer:
5'-CAACGGCAGCATTTATAACCC-3' (SEQ ID NO:17), Reverse primer:
5'-CCCATTGATGATGGAGTGTGG-3' (SEQ ID NO:18)). POWER SYBR GREEN PCR
MASTER MIX (Applied Biosystems) was used as the reaction reagent,
and the quantification was performed by a reaction at 50.degree. C.
for 2 min, and then at 95.degree. C. for 10 min, followed by 40
cycles of reaction at 95.degree. C. for 15 sec and at 60.degree. C.
for 60 sec. PCR and fluorescence measurement were performed using
7500 Real time PCR System (Applied Biosystems).
[0249] The results are presented in FIG. 6. As shown in FIG. 6, it
was confirmed that the introduction of the three types of CCDC3
siRNA suppressed CCDC3 mRNA expression by 80% or more.
Example 7
Production and Analysis of Transgenic (Tg) Mice Using
Lentivirus
(1) Construction of Plasmid for Producing CCDC3 Lentivirus
Vector
[0250] Total RNA was purified from the adipose tissue collected
from wild-type C57BL/6 male mice, using TRIzol Reagent
(Invitrogen), and cDNA was synthesized with oligo dT primers using
SuperScript III Reverse transcriptase (Invitrogen). By using the
cDNA as a template, PCR was performed with specific primers of the
base sequences below using KOD FX (Toyobo). As a result,
amplification fragments were obtained that had EcoRI and BamHI
restriction enzyme sites introduced therein.
TABLE-US-00003 CCDC3 EcoRI-F3 primer: (SEQ ID NO: 19)
gcgaattctagccgtgcacccagctctccggag CCDC3 BamHI-R3 primer: (SEQ ID
NO: 20) gcggatcctagagcttgcggttgttctcagtcagc
[0251] The DNA amplification fragments were treated with EcoRI and
BamHI restriction enzymes, and inserted and ligated at the EcoRI
and BamHI cleavage sites of a pBluescript KS (Stratagene) to obtain
a plasmid, which was then sequenced to confirm sequence. By using
the plasmid as a template, PCR was performed with specific primers
of the base sequences below to obtain a CCDC3 gene ORF sequence
that had a kozak consensus sequence and a STOP codon.
TABLE-US-00004 CCDC3 Kozak-F primer: (SEQ ID NO: 21)
gcgaattccaccatgccgctcccgctgctgctc CCDC3 STOP-R primer: (SEQ ID NO:
22) gcggatccctacccatgcagatagggggc
[0252] The DNA amplification fragments were treated with EcoRI and
BamHI restriction enzymes, and inserted to the EcoRI and BamHI
sites of pCDH1-MCS1 (System Biosciences) to produce a virus
producing plasmid pCDH1-CMV-CCDC3. The virus producing plasmid
pCDH1-CMV-CCDC3 was sequenced to confirm sequence.
(2) Preparation of CCDC3 Lentivirus Vector
[0253] 293FT cells (Invitrogen) were cultured under 5% CO.sub.2
37.degree. C. conditions using DMEM (high glucose) that contained
10% FCS (fetal bovine serum). The cells were transfected using
FuGENE6 (Roche) according to the attached protocol, and the
pCDH1-CMV-CCDC3 plasmid was introduced together with the packaging
plasmids (pPACKH1-GAG, pPACKH1-REV, pVSV-G) of the pPACKH1
Lentivector Packaging Kit (System Biosciences). After 72 hours from
the transfection, the culture supernatant was collected, filtered
through a 0.45-.mu.m filter (Millipore), and concentrated by
ultracentrifugation (Beckman SW32Ti rotor; 19,400 rpm, 4.degree.
C., 2 hours). The supernatant was carefully discarded, and
suspended in PBS. This was followed by purification using a sucrose
density gradient method (Beckman SW55Ti rotor; 21,000 rpm,
4.degree. C., 2 hours), and finally by suspension in PBS to prepare
a concentrated lentivirus vector for transgenic mice production.
The physical titer (number of particles) of the virus vector was
measured using a p24 Antigen ELISA kit (ZeptoMetrix).
(3) Production of Transgenic Mice Using Lentivirus Vector
[0254] BDF1 female mice were administered with 51 U of PMSG
(pregnant mare serum gonadotropin), and, 48 hours later,
intraperitoneally with 51 U of hCG (chorionic gonadotrophin) using
an ordinary method to induce multiple ovulation. The mice were then
crossed with BDF1 male mice. On the next day of the crossing, the
oviduct of a female mouse with a confirmed vaginal plug was
collected after about 40 hours from the crossing, and perfused with
0.4% BSA-containing physiological saline. The collected two-cell
embryo was cultured briefly in a KSOM medium (Ark Resource), and
treated with acidic Tyrode (Ark Resource) for about 1 to 2 min to
melt and remove the embryo transparent body. The embryo with
successfully removed zona pellucida was washed in a KSOM medium,
and cultured in the same medium until virus infection. The embryo
after the removal of zona pellucida was virally infected by being
cultured for 2 days with drops (6 .mu.l/spot) of KSOM medium
diluted to contain about 100 pg/.mu.l, 333 pg/.mu.l, or 1,000
pg/.mu.l of virus particles. All embryos were cultured under 5%
CO.sub.2 37.degree. C. conditions. Following the virus infection,
the embryo developed to the blastocyst or morula stage was
transplanted into the uterus of a pseudopregnant mouse 2.5 days
after checking the vaginal plug. After 17 days from the transplant,
baby mice were obtained by natural birth or Caesarean section.
(4) Screening of CCDC3 Transgenic Mice
[0255] Genomic DNA was purified from the tail end of the baby mice
collected after 3 weeks from birth, using a DNeasy Blood &
Tissue Kit (QIAGEN). By using the purified DNA as a template, PCR
was performed with the primer set of the following base sequences
for genotyping.
TABLE-US-00005 CCDC3-F1 primer: (SEQ ID NO: 23)
ggagagggagggctcttcta CCDC3-R1 primer: (SEQ ID NO: 24)
gttctcctgggtgtctggaa
[0256] Total RNA was purified from the tail end of the transgenic
mice screened by PCR after 4 to 5 weeks from birth, using an RNeasy
Plus Mini Kit (QIAGEN). By using the purified RNA as a template,
CCDC3 gene expression analysis was performed with the primer set of
the base sequences below, using a Quantitect SYBR Green RT-PCR Kit
(QIAGEN). mGAPDH gene was used as an endogenous control gene in the
expression analysis, and transgenic mice (CCDC3 Tg mice) with the
CCDC3 gene expression amount four times or greater than that in the
control group were screened for phenotyping.
TABLE-US-00006 CCDC3-Q PCR-F primer: (SEQ ID NO: 25)
gctcaaccttactggtctgggcta CCDC3-Q PCR-R primer: (SEQ ID NO: 26)
catcttggaaattgactccgtgtg mGAPDH-Q PCR-F primer: (SEQ ID NO: 27)
aaatggtgaaggtcggtgtg mGAPDH-Q PCR-R primer: (SEQ ID NO: 28)
tgaaggggtcgttgatgg
(5) Phenotyping of CCDC3 Transgenic Mice
[0257] CCDC3 Tg mice and a virus infection control group, 8 weeks
old, were given the high-fat diet (HFD) 58Y1 DIO P.D. 60% Energy
From Fat-Blue (Japan SLC), and the body weights were measured over
time (FIG. 7). On day 52 post HFD feeding, casual glucose levels
were measured (FIG. 8). The casual glucose levels were measured
with a Glucocard DIA meter a (Arkray), using trace amounts of blood
collected by cutting the tail vein with a razor. For the virus
infection control group, a lentivirus vector was used produced in
the same manner as for the pCDH1-CMV-CCDC3, using a
pCDH1-CMV-loxP-stop-loxP-EGFP plasmid.
[0258] The analysis confirmed increased body weights in the HFD
CCDC3 Tg mice over the control group, as shown in FIG. 7. Further,
as shown in FIG. 8, the CCDC3 Tg mice had increased casual glucose
levels over the control group, as measured on day 52 post HFD
feeding. These results suggested the possibility that CCDC3 has the
effect to influence body weight and glucose level.
Example 8
Preparation of Monoclonal Antibodies that Recognize Human CCDC3
Protein
[0259] Human CCDC3 monoclonal antibodies were prepared as
antibodies that recognize CCDC3, according to the method described
in Immunochemistry in Practice (Blackwell Scientific
Publications).
(1) Selection of Antigens
[0260] In order to acquire CCDC3-recognizing antibodies, the human
recombinant CCDC3 prepared in the foregoing Examples, and its
conjugates with KLH (keyhole limpet hemocyanin) were used as
immunogens. Because the steric structure of human CCDC3 has not
been elucidated, the C-terminus peptide of the amino acid sequence
from position 259 to 270 of human CCDC3, having a high possibility
of being exposed on the surface of the steric structure was
selected as the immunogen.
(2) Immunogen Preparation
[0261] A human CCDC3 protein C-terminus partial peptide (259-270,
12 amino acid residues, ARGPVRPPYLRG: SEQ ID NO:29) was
synthesized, and a sulfo-SMCC aqueous solution with 10 times the
molar quantity was added to a 0.1 M phosphate buffer (pH 7.1) that
contained 1.5 mg of the partial peptide. This was followed by
reversed-phase HPLC (column: YMC-pack ODS-A, 6.0.times.150 mm,
mobile phase: acetonitrile/0.1% trifluoroacetate aqueous solution)
to separate 1.4 mg of a maleimidized peptide fraction. The
maleimidized peptide fraction was freeze dried, and used in the
conjugation described below. Separately, a Sulfo-SPDP (Thermo
scientific) aqueous solution with 700 times the molar quantity was
added to 2 mL of 0.1 M phosphate buffered saline (pH 7.2) that
contained 20 mg of mariculture keyhole limpet hemocyanin (KLH,
Thermo scientific). The mixture was left unattended at 4.degree. C.
for 16 hours, and then at room temperature for 30 min after adding
0.2 mL of a 0.1 M mercaptoethylamine aqueous solution. This was
followed by gel filtration to separate SH group-introduced KLH,
using a PD-10 column (GE Healthcare) equilibrated with 5 mM
EDTA-containing 0.1 M phosphate buffer (pH 6.0). A freeze-dried
powder (1.2 mg) of the maleimidized C-terminus peptide was added to
the 5 mM EDTA-containing 0.1 M phosphate buffer (pH 6.0) that
contained the SH group-introduced KLH (10 mg), and the mixture was
allowed to react at room temperature for 1 hour, and at 4.degree.
C. overnight. The reaction mixture was dialyzed against purified
water, freeze dried, and used as the immunogen (antigen 1).
[0262] Separately, a Sulfo-EMCS (Thermo scientific) was added to
the human recombinant CCDC3 (1.1 mg) purified in Example 4 at a
molar ratio of 5:1, and the mixture was allowed to react at room
temperature for 3 hours to prepare maleimide human CCDC3. Then,
3.75 .mu.M sulfo-LC-SPDS (Thermo scientific) was added to 20 mg of
mariculture keyhole limpet hemocyanin (KLH, Thermo scientific). The
mixture was allowed to react at room temperature for 3 hours to
prepare a SH group-introduced KLH. The maleimide human CCDC3 (1 mg)
and the SH group-introduced KLH (8 mg) were mixed, and reacted at
4.degree. C. overnight. The reaction mixture was dialyzed against
10 mM PB (pH 7.5) and 0.5 M NaCl, preserved at -80.degree. C., and
used as the immunogen (antigen 2).
[0263] The three immunogens, including the two immunogens prepared
above and the human recombinant CCDC3 (antigen3), were
intraperitoneally administered (100 .mu.g each) to Balb/c female
mice, 4 weeks old, with a Freund's complete adjuvant (initial
immunization). After 21, 42, 63, and 84 days, the immunogen (100
pg) was administered with a Freund's incomplete adjuvant for
boosting. After 104 days, the immunogen (100 .mu.g) suspended in 10
mM PB (pH 7.5) and 0.5 M NaCl (0.1 mL) was intraperitoneally
administered for final immunization.
(3) Hybridoma Production
[0264] Spleen was removed after 3 days from the final immunization,
and spleen cells were collected. The spleen cells were fused with
mouse myeloma cells (p3.times.63-Ag8.U1, Tokyo Shuryu Kenkyusho)
using 50% polyethyleneglycol 4000, and selected in a medium that
contained hypoxanthine, aminopterin, and thymidine.
(4) Biotin Labeling of Peptide in Human CCDC3 Protein C-Terminus
Region
[0265] An aqueous solution (32 .mu.l) containing an equimolar (160
.mu.g) PEO-maleimide activated biotin (PIERCE) was added to 5 mM
EDTA-containing 0.1 M phosphate buffer (pH 6.0) that contained a 13
mer peptide (0.4 mg) obtained by adding a Cys residue to the
N-terminus of the peptide (259-270, 12 amino acid residues) in the
C-terminus region of human CCDC3 protein. The mixture was left
unattended at room temperature for 2 hours, and the peptide of the
biotin-labeled human CCDC3 protein C-terminus region was purified
by reversed-phase HPLC (column: YMC-pack ODS-A, 6.0.times.150 mm,
mobile phase: acetonitrile/0.1% trifluoroacetate aqueous
solution).
(5) Biotin Labeling of Recombinant Human CCDC3 Protein
[0266] Human recombinant CCDC3 protein (0.31 mg) dissolved in 0.31
ml of 10 mM phosphate buffer (pH 7.2), 0.5 M NaCl, and 0.01% Tween
20 was added to 4.1 .mu.l of a 20 mM PEO-maleimide activated biotin
(PIERCE) aqueous solution, and the mixture was allowed to react at
4.degree. C. overnight. This was followed by gel filtration using a
Superdex 75 (GE Healthcare Biosciences) equilibrated with 10 mM
phosphate buffer (pH 7.2) and 0.5 M NaCl to purify the labeled
human CCDC3.
(6) Selection of Human CCDC3 Monoclonal Antibodies
[0267] Specific antibody producing cells were screened 10 days
after the cells were fused. Screening was performed by ELISA, as
follows. A Tris buffer (50 mM Tris-HCl, pH 7.5, 35 .mu.l)
containing anti-mouse IgG antibodies (0.35 .mu.g; Shibayagi Co.,
Ltd.) was added to each well of a 384-well microtiter plate (Nunc),
and the antibodies were immobilized at 4.degree. C. for 16 hours.
The wells were washed once with 90 .mu.l of washing liquid
(physiological saline containing 0.01% Tween 20), and left
unattended at room temperature for 2 hours for blocking after
adding 200 .mu.l of Block Ace (Dainippon Pharma) (anti-mouse IgG
antibody-anchored plate). After washing each well once with washing
liquid (90 .mu.l), a hybridoma culture supernatant (15 .mu.l) and
buffer A (10 .mu.l; 50 mM Tris buffer containing 0.5% bovine serum
albumin, 0.01% Tween 80, 0.05% Proclin 150, and 0.5 M NaCl; pH 7.4)
were added. When the immunogen was the peptide-KLH conjugate,
buffer A (10 .mu.l) containing 0.02 ng of the biotin-human CCDC3
protein C-terminus region peptide and 2 ng of Streptavidin-HRP
(PIERCE) was added. When the immunogen was the human recombinant
CCDC3 protein or its KLH-conjugate, buffer A (10 .mu.l) containing
1 ng of the biotin-human recombinant CCDC3 protein and 4 ng of
Streptavidin-HRP (PIERCE) was added. In either case, reaction was
performed at 4.degree. C. for 16 hours. After washing each well
three times with washing liquid (90 .mu.l), 25 .mu.l of
TMB+Substrate-Chromogen (DAKO) was added to develop color at room
temperature for 30 min. The reaction was stopped by adding 25 .mu.l
of 0.5 M sulfuric acid, and absorbance at 450 nm was measured.
[0268] After the screening, three clones (3H6, 6G12, 11E10) with
strong affinity to the human recombinant CCDC3 protein were
obtained. Note that the 3H6, 6G12, and 11E10 antibodies were
obtained from the mouse-derived hybridomas immunized with antigen
1, antigen 3, and antigen 2, respectively. The subclass of each
antibody was determined using a mouse monoclonal antibody isotyping
ELISA kit (BD biosciences). The antibodies 3H6 and 11E10 were of
isotype IgG2b, and the antibody 6G12 was of IgG1.
(7) Antibody Affinity Evaluation by ELISA
[0269] A buffer A containing 10 .mu.l of CCDC3 antibody was added
to the anti-mouse IgG antibody-anchored plate, and 10 .mu.l of 0.01
to 100 nM human recombinant CCDC3 was added. The mixture was
allowed to react at 4.degree. C. for 16 hours after adding buffer A
(10 .mu.l) that contained 1 ng of biotin-human recombinant CCDC3
protein and 4 ng of Streptavidin-HRP (PIERCE). Each well was washed
three times with washing liquid (90 .mu.l), and 25 .mu.l of
TMB+Substrate-Chromogen (DAKO) was added to develop color at room
temperature for 30 min. The reaction was stopped by adding 25 .mu.l
of 0.5 M sulfuric acid, and absorbance at 450 nm was measured. The
results are presented in FIG. 9.
[0270] As can be seen from the results, the IC.sub.50 values of the
antibodies against human CCDC3 protein were 9.4 nM, 3.2 nM, and
0.10 nM for the 3H6, 6G12, and 11E10 antibodies, respectively.
Example 9
Evaluation of Antibody Binding to Human CCDC3 Protein by Western
Blotting
[0271] Western blotting was performed using the 3H6, 6G12, and
11E10 antibodies. Human recombinant CCDC3 protein (50 ng) was
subjected to SDS-PAGE (reduced state). For detection, anti-mouse
IgG-HRP antibodies (GE Healthcare Biosciences) and ECL advance (GE
Healthcare Biosciences) were used. The results are presented in
FIG. 10. The results revealed that the all antibodies were positive
in western blotting, and bound to the human recombinant CCDC3
protein.
Example 10
Determination of Antibody Sequence
[0272] The sequences of the heavy-chain variable domain and
light-chain variable domain of the 3H6, 6G12, and 11E10 antibodies
obtained in Example 8 were determined using an ordinary method.
(1) 3H6 Antibody
TABLE-US-00007 [0273] Heavy-chain variable domain (SEQ ID NO: 30)
QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMIWVKQAPGKDLKWMAWINTYSGEPTCADDFK
GRFAFSLETSASTAYLQINNLKNEDMATYFCARDDGYYGYFDYWGQGTTLTVSS
[0274] Note that the underlined regions in the heavy-chain variable
domain correspond to CDR1 (SEQ ID NO:31), CDR2 (SEQ ID NO:32), and
CDR3 (SEQ ID NO:33) from the N-terminus side (left).
TABLE-US-00008 Light-chain variable domain (SEQ ID NO: 34)
DVVMTQTPLSLPVSLGDQASISCRSSQTLVHSDGNTYLHWYLQKPGQSPKLLIYKISNRFSGVPD
RFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPTFGGGTKLEIKRA
[0275] Note that the underlined regions in the light-chain variable
domain correspond to CDR1 (SEQ ID NO:35), CDR2 (SEQ ID NO:36), and
CDR3 (SEQ ID NO:37) from the N-terminus side (left).
(2) 6G12 Antibody
TABLE-US-00009 [0276] Heavy-chain variable domain (SEQ ID NO: 38)
QVQLQQPGAELVRPGASVKMSCKASGYTFSSYLMDWVKQRPGQGFECIGNINPNTGSTIYNERFK
GKAKLTVDKSSSTAYMQLISLTSEDSAVYYCAADGYYAPFTYWGQGTLVTVSA
[0277] Note that the underlined regions in the heavy-chain variable
domain correspond to CDR1 (SEQ ID NO:39), CDR2 (SEQ ID NO:40), and
CDR3 (SEQ ID NO:41) from the N-terminus side (left).
TABLE-US-00010 Light-chain variable domain (SEQ ID NO: 42)
DIVMTQAAPSVPVTPGESVSISCRSSKSLLHNNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPD
RFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGAGTKLELKRA
[0278] Note that the underlined regions in the light-chain variable
domain correspond to CDR1 (SEQ ID NO:43), CDR2 (SEQ ID NO:44), and
CDR3 (SEQ ID NO:45) from the N-terminus side (left).
(3) 11E10 Antibody
TABLE-US-00011 [0279] Heavy-chain variable domain (SEQ ID NO: 46)
QVQLQQSGPELVKPGASVKISCKASGYSFRSYYIHWMKQSPGQGLEWIAWIYPGGGKIYYNDNEK
FKDKVTLTADTSSSTAYMQLTGLTSEDSAVYFCARWGGYYVGSILDYWGQGTSVTVSS
[0280] Note that the underlined regions in the heavy-chain variable
domain correspond to CDR1 (SEQ ID NO:47), CDR2 (SEQ ID NO:48), and
CDR3 (SEQ ID NO:49) from the N-terminus side.
TABLE-US-00012 Light-chain variable domain (SEQ ID NO: 50)
DGDIVMTQSQKFLSTSVGDRVSVTCKASQNVDNNVAWYQQRPGQSPKPLIYLTSSRYSGVPDRFT
GSGSGTDFTLTITNVQSEDLAEYFCQQYKTYPLTFSVGTKLELKRA
[0281] Note that the underlined regions in the light-chain variable
domain correspond to CDR1 (SEQ ID NO:51), CDR2 (SEQ ID NO:52), and
CDR3 (SEQ ID NO:53) from the N-terminus side (left).
Example 11
Preparation of Antibodies that Recognize Mouse CCDC3 Protein
(1) Selection of Antigens
[0282] A KLH conjugate of mouse recombinant CCDC3 protein prepared
in the same manner as for the human recombinant CCDC3 protein
prepared above was used as the immunogen. The peptide in the
C-terminus region with the amino acid sequence from position 262 to
273 of mouse CCDC3 was selected as the immunogen.
(2) Immunogen Preparation
[0283] A peptide (13 amino acids) with a cysteine introduced to the
C-terminus of the peptide (positions 262 to 273) in the C-terminus
region of mouse CCDC3 protein was synthesized (Scrum Inc.). The
synthetic peptide (2.9 mg) was dissolved in a 0.1 M phosphate
buffer (PH 6.0) that contained 5 mM EDTA (0.29 mL), and an aqueous
solution (1 mL) containing 10 mg of Imject Maleimide activated
mariculture keyhole limpet hemocyanin (Thermo scientic) was added.
The mixture was then allowed to react at room temperature for 1
hour, and then at 4.degree. C. overnight. The reaction mixture was
dialyzed against purified water, freeze dried, and used as the
immunogen.
[0284] Separately, Sulfo-EMCS (Thermo scientic) was added at a
molar ratio of 5:1 to 1.1 mg of mouse recombinant CCDC3 purified in
the same manner as for the human CCDC3 protein. The mixture was
allowed to react at room temperature for 3 hours to prepare
maleimide mouse CCDC3. Then, 3.75 .mu.M sulfo-LC-SPDS (Thermo
scientic) was added to 20 mg of mariculture keyhole limpet
hemocyanin (Thermo scientic), and reacted at room temperature for 3
hours to prepare SH group-introduced KLH. The maleimide mouse CCDC3
(1 mg) and the SH group-introduced KLH (8 mg) were mixed, and
reacted at 4.degree. C. overnight. The reaction mixture was
dialyzed against 10 mM PB (PH 7.5) and 0.15 M NaCl, preserved at
-80.degree. C., and used as the immunogen.
[0285] The two immunogens prepared as above (100 .mu.g each) were
intraperitoneally administered to Balb/c female mice, 4 weeks old,
together with a Freund's complete adjuvant (initial immunization).
After 21, 42, 63, and 84 days, the immunogen (100 .mu.g) was
administered with a Freund's incomplete adjuvant for boosting.
After 104 days, the immunogen (100 .mu.g) suspended in
physiological saline (0.1 mL) was intraperitoneally administered
for final immunization.
(3) Hybridoma Production
[0286] Spleen was removed after 3 days from the final immunization,
and spleen cells were collected. The spleen cells were fused with
mouse myeloma cells (p3.times.63-Ag8.U1, Tokyo Shuryu Kenkyusho)
using 50% polyethyleneglycol 4000, and selected in a medium that
contained hypoxanthine, aminopterin, and thymidine.
(4) Biotin Labeling of Partial Peptide in Mouse CCDC3 Protein
C-Terminus Region
[0287] An aqueous solution (40 .mu.l) containing an equimolar (200
.mu.g) PEO-maleimide activated biotin (PIERCE) was added to 5 mM
EDTA-containing 0.1 M phosphate buffer (pH 6.0) that contained a
peptide (13 amino acids; 0.5 mg) obtained by introducing a Cys
residue to the C-terminus of the partial peptide (amino acid
sequence from position 262 to 273) in the C-terminus region of
mouse CCDC3 protein. The mixture was left unattended at room
temperature for 2 hours, and the partial peptide of the
biotin-labeled mouse CCDC3 protein C-terminus region was purified
by reversed-phase HPLC (column: YMC-pack ODS-A, 6.0.times.150 mm,
mobile phase: acetonitrile/0.1% trifluoroacetate aqueous
solution).
(5) Biotin labeling of Mouse Recombinant CCDC3 Protein
[0288] Mouse recombinant CCDC3 protein (0.22 mg) dissolved in 0.20
ml of 10 mM phosphate buffer (pH 7.2), 0.5 M NaCl, and 0.01% Tween
20 was added to 3.2 .mu.l of a 20 mM PEO-maleimide activated biotin
(PIERCE) aqueous solution, and the mixture was allowed to react at
4.degree. C. overnight. This was followed by gel filtration using a
Superdex 75 (GE Healthcare Biosciences) equilibrated with 10 mM
phosphate buffer (pH 7.2) and 0.5 M NaCl to purify the labeled
mouse CCDC3.
(6) Selection of Anti-CCCD3 Antibodies
[0289] Specific antibody producing cells were screened 10 days
after the cells were fused. Screening was performed by ELISA, as
follows. A Tris buffer (50 mM Tris-HCl, pH 7.5, 35 .mu.l)
containing anti-mouse IgG antibodies (0.35 .mu.g; Shibayagi Co.,
Ltd.) was added to each well of a 384-well microtiter plate (Nunc),
and the antibodies were immobilized at 4.degree. C. for 16 hours.
The wells were washed once with 90 .mu.l of washing liquid
(physiological saline containing 0.01% Tween 20), and left
unattended at room temperature for 2 hours for blocking after
adding 200 .mu.l of Block Ace (Dainippon Pharma) (anti-mouse IgG
antibody-anchored plate). After washing each well once with washing
liquid (90 .mu.l), a hybridoma culture supernatant (15 .mu.l) and
buffer B (10 .mu.l; 50 mM Tris buffer containing 0.5% bovine serum
albumin, 0.01% Tween 80, 0.05% Proclin 150, and 0.15 M NaCl; pH
7.4) were added. When the immunogen was the peptide-KLH conjugate,
buffer B (10 .mu.l) containing 0.02 ng of the biotin-mouse CCDC3
protein C-terminus region partial peptide and 2 ng of
Streptavidin-HRP (PIERCE) was added. When the immunogen was the
mouse recombinant CCDC3 protein-KLH-conjugate, buffer B (10 .mu.l)
containing 1 ng of the biotin-mouse recombinant CCDC3 protein and 4
ng of Streptavidin-HRP (PIERCE) was added. In either case, reaction
was performed at 4.degree. C. for 16 hours. After washing each well
three times with washing liquid (90 .mu.l), 25 .mu.l of
TMB+Substrate-Chromogen (DAKO) was added to develop color at room
temperature for 30 min. The reaction was stopped by adding 25 .mu.l
of 0.05 M sulfuric acid, and absorbance at 450 nm was measured.
[0290] After the screening, clones (13A8, 24F6) with strong
affinity to the mouse recombinant CCDC3 protein were obtained. The
subclass of each antibody was determined using a mouse monoclonal
antibody isotyping ELISA kit (BD biosciences). The antibodies 13A8,
13D4, and 24F6 were of IgG1, IgG2a, and IgM, respectively.
(7) Antibody Affinity Evaluation by ELISA
[0291] A buffer A containing 10 .mu.l of the selected anti-CCDC3
antibody was added to the anti-mouse IgG antibody-anchored plate,
and 10 .mu.l of 0.01 to 100 nM mouse recombinant CCDC3 protein was
added. For the 13A8 antibody, a buffer B (10 .mu.l) containing 1 ng
of the C-terminus region partial peptide of biotin-mouse
recombinant CCDC3 protein and 4 ng of Streptavidin-HRP (PIERCE) was
added. For the 13D4 and 24F6 antibodies, a buffer B (10 .mu.l)
containing 0.02 ng of the biotin-mouse recombinant CCDC3 protein
and 2 ng of Streptavidin-HRP (PIERCE) was added. Each was allowed
to react at 4.degree. C. for 16 hours. Each well was washed three
times with washing liquid (90 .mu.l), and 25 .mu.l of
TMB+Substrate-Chromogen (DAKO) was added to develop color at room
temperature for 30 min. The reaction was stopped by adding 25 .mu.l
of 0.05 M sulfuric acid, and absorbance at 450 nm was measured. The
results are presented in FIG. 11. As can be seen from the figure,
the IC.sub.50 values of the antibodies against the mouse CCDC3
protein were 7.4 nM, 17 nM, and 2.6 nM for the 13A8, 13D4, and 24F6
antibodies, respectively.
Example 12
Evaluation of Antibody Binding to Mouse CCDC3 Protein by Western
Blotting
[0292] Western blotting was performed using the 13A8 antibodies.
Mouse recombinant CCDC3 protein (50 ng) was subjected to SDS-PAGE
(reduced state). For detection, anti-mouse IgG-HRP antibodies (GE
Healthcare Biosciences) and ECL advance (GE Healthcare Biosciences)
were used. The results are presented in FIG. 12. The results
revealed that the all antibodies were positive in western blotting,
and bound to the mouse recombinant CCDC3 protein (FIG. 12).
Example 13
Setting CCDC3 Concentration Measurement Method by Chemiluminescence
Detection Sandwich ELISA in Human Blood Plasma
[0293] Two sandwich ELISA methods were set using 6G12 antibodies
against human CCDC3 protein, and 3H6 antibodies against the human
CCDC3 protein C-terminus partial peptide (anchored antibodies), and
using 11E10 antibodies against human CCDC3 protein (detection
antibodies).
(1) Preparation of Biotin Labeled 6G12 and 3H6
[0294] The IgG fraction of the 6G12 or 3H6 antibodies was reacted
with NHS-PEO-biotin (PIERCE) at a molar ratio of 1:10 in 0.1 M
phosphate buffer. This was followed by gel filtration performed
with a PD-10 column (GE Healthcare) equilibrated with 50 mM
tris-HCL buffer (pH 7.5) to separate the biotinylated 6G12 or 3H6
fraction. In the sandwich ELISA, the biotinylated 6G12 or 3H6
antibodies were used by being immobilized on a 96-well microtiter
plate that had been coated with goat anti-biotin antibodies.
(2) Preparation of Alkaliphosphatase-Labeled 11E10
Fab'Antibodies
[0295] The detection antibodies were obtained by labeling the 11E10
antibody Fab' fraction with alkaliphosphatase (ALP, Roche,
recombinant), and were prepared according to the following
procedure. Pepsin (Roche, derived from pig gastric mucosa) was
added to a 0.1 M sodium acetate buffer (pH 4.2) that contained the
IgG fraction of the 11E10 antibodies (added in 40 .mu.g per
milligram of IgG1), and incubated at 37.degree. C. for 16 hours.
This was followed by high speed gel filtration (LC-6A system
(Shimadzu Corporation) equipped with a TSK-GEL G3000 column (Tosoh)
equilibrated with 5 mM EDTA-containing 0.1 M phosphate buffer, pH
6.0) to separate the F(ab').sub.2 fraction. Then, a 0.1 M
mercaptoethylamine aqueous solution was added to the F(ab').sub.2
fraction in 1/10 the volume of the F(ab').sub.2 solution. The
mixture was allowed to react at 37.degree. C. for 1.5 hours, and
the Fab' fraction was separated by high speed gel filtration.
Separately, sulfo-SMCC (PIERCE) was added to 0.2 mL of a 0.05 M
borate buffer (pH 7.6, containing 1 mM MgCl.sub.2 and 0.1 mM
ZnCl.sub.2 with ALP) in 5 times the molar quantity of ALP, and
reacted at 30.degree. C. for 30 min. This was followed by gel
filtration with a PD-10 column (GE Healthcare) equilibrated with a
1.0 mM MgCl.sub.2- and 0.1 mM ZnCl.sub.2-containing 0.1 M tris-HCL
buffer (pH 6.8) to collect the maleimidized ALP fraction. The
maleimidized ALP fraction and the 11E10 Fab' fraction were mixed at
a molar ratio of 1:3, and allowed to react at 4.degree. C.
overnight. This was followed by high speed gel filtration to
separate the target ALP-labeled 11E10 Fab' fraction.
(3) Chemiluminescence Detection Sandwich ELISA
[0296] As a standard substance, the human recombinant CCDC3 protein
was used by being diluted with buffer A (50 mM Tris buffer
containing 1.0% bovine serum albumin, 0.1% Tween 80, 0.05% sodium
azide, 0.5 M NaCl, 1 mM MgCl.sub.2, and 0.1 mM ZnCl.sub.2, pH 7.5).
A Tris buffer (50 mM Tris-HCl, pH 7.5, 100 .mu.L) containing goat
anti-biotin antibodies (1.0 .mu.g; Bethyl) was added to each well
of a 96-well white microtiter plate (NUNC, Maxi-Sorp), and the
mixture was left unattended at 4.degree. C. for 16 hours to
immobilize the antibodies. The wells were washed once with 250
.mu.L of washing liquid (physiological saline containing 0.01%
Tween 20), and left unattended at room temperature for 2 hours for
blocking after adding 150 .mu.L of Block Ace (Dainippon Pharma).
After washing each well once with 250 .mu.L of washing liquid, a
buffer A containing 100 ng of the biotinylated 6G12 antibodies or
3H6 antibodies was added, and the mixture was left unattended at
room temperature for 1 to 2 hours. After washing each well three
times with washing liquid, a 1 to 500 pM standard human recombinant
CCDC3 protein solution or a biological solution sample (50 .mu.L)
and buffer A (50 .mu.L) were added and mixed. The mixture was left
unattended at 4.degree. C. for 16 to 24 hours. After washing each
well three times with washing liquid, a buffer A (100 .mu.L)
containing 10 ng of the ALP-labeled 11E10 Fab' fraction was added,
and the mixture was left unattended at room temperature for 2 to 3
hours. After washing each well three times with washing liquid, a
luminescent substrate CDP-Star.RTM./Emerald II (100 .mu.L; Applied
Biosystems) was added, and the mixture was left unattended at room
temperature. After 20 minutes from adding the substrate,
luminescence intensity was measured with a multilabel counter
ARVO.RTM. (PerkinElmer).
[0297] As a result, a desirable standard curve was obtained in the
standard human CCDC3 protein in the 1 to 500 pM range, with the
minimum detection limit of 1 pM, as shown in FIG. 13. It is
therefore possible to specifically and sensitively measure the
CCDC3 protein in a human biological solution such as a human blood
plasma sample.
Example 14
Analysis of Correlation Between CCDC3 Concentration and the Extent
of Obesity in Human Blood Plasma
(1) Preparation of Human Blood Plasma
[0298] Blood was collected from 85 donors after obtaining consent
for the experiment.
[0299] In all blood samples, hemoglobin A1c (HbA1c) was also
measured during the measurement of CCDC3 protein concentration in
the blood plasma. The collected blood was transferred to a blood
plasma separation tube (EDTA.2Na blood collection tube), and
centrifuged at 3,000 rpm for 30 min at 4.degree. C. The supernatant
was then used as blood plasma for testing.
[0300] The measurement of CCDC3 protein concentration in the blood
plasma was performed using ELISA with a combination of 3H6 and
11E10 antibodies, according to the chemiluminescence detection
sandwich ELISA method described in Example 13(3).
[0301] 25 out of the 85 subjects were also measured for the
visceral fat area (visceral fat area in the abdominal cross section
at navel), using CT (computed tomography). Among the 25 subjects,
eight classified as non-visceral obesity with the visceral fat area
of less than 100 cm.sup.2, and seventeen as visceral obesity with
the visceral fat area of 100 cm.sup.2 or more.
(2) Measurement of CCDC3 Protein Concentration in Human Blood
Plasma and Correlation Analysis with the Extent of Obesity
[0302] Measurements of CCDC3 protein concentration in the blood
plasma revealed that the blood plasma CCDC3 protein concentration
was higher in the visceral obesity group (17 subjects) with the
visceral fat area of 100 cm.sup.2 or more than in the non-visceral
obesity group (8 subjects) with the visceral fat area of less than
100 cm.sup.2 (FIG. 14). Further, by comparing the blood plasma
CCDC3 protein concentration between a subject group (64 subjects)
with the normal HbA1c values of 5.8% or less and a subject group
(21 subjects) with the HbA1c values above 5.8% (HbA1c is an index
that reflects long-term glucose levels), it was found that the
CCDC3 protein blood plasma concentration was higher in the
HbA1c>5.8% subject group than in the HbA1c 5.8% subject group,
as shown in FIG. 15.
Example 15
CCDC3 Expression Analysis in Obesity Model DIO mouse Adipose Tissue
by Immunostaining
(1) Mouse Adipose Tissue Perfusion Fixation
[0303] DIO mice were produced by giving the high-fat diet (HFD)
58Y1 DIO P.D. 60% Energy From Fat-Blue (Japan SLC) to C57BL/6J Jcl
male mice (CLEA Japan), 7 weeks old, for 31 weeks. Mice that had
the CLEA Rodent Diet CE-2 (CLEA Japan) for 43 weeks were used as
control group mice. Each mouse was subjected to laparotomy under
halothane anesthesia, and a syringe was inserted near the aorta
from the left ventricle of the heart. After cutting the tip of the
right auricle of the right atrial appendage, the tissue was washed
by a reflux of phosphate buffer saline (Lonza). The tissue was then
fixed by perfusion with 4% paraformaldehyde (TAAB). Thereafter,
mesenteric adipose tissue (visceral adipose tissue) and
subcutaneous adipose tissue were collected, and fixed in a
paraformaldehyde solution.
(2) Immunostaining
[0304] Frozen thin slices (10 .mu.m thick) were produced from the
collected adipose tissues under -35 to -40.degree. C. conditions.
These were dipped in a phosphate buffer to accommodate the tissues
to the wet state, and washed for 3 min in phosphate buffer after
being dipped in Avidin blocking solution (Avidin/Biotin Blocking
Kit (VECTOR)) for 15 min. The tissue slices were further washed for
3 min in phosphate buffer after being dipped in Biotin blocking
solution (Avidin/Biotin Blocking Kit (VECTOR)) for 15 min. The
samples were then placed in a slice slide moist chamber, and
reacted for 60 min in a M.O.M Mouse IgG Blocking Reagent (M.O.M.
Immunodetection Kit (VECTOR)) solution. Then, primary antibodies
(mouse monoclonal 13D4) diluted to a 10 .mu.g/mL concentration with
an M.O.M. diluting solution were dipped in the same moist chamber
at ordinary temperature for 30 min. After being washed three times
with phosphate buffer for 5 min, the samples were treated for 10
min with M.O.M. Biotinylated Anti-Mouse (horse) IgG Reagent used as
the secondary antibodies. The samples were then washed three times
with phosphate buffer for 5 min, and reacted with Vectastain ABC-AP
Reagent (ABC-AP Universal Kit (VECTOR)) for 5 min. After being
washed three times with phosphate buffer for 5 min, the samples
were treated with a levamisol-added Alkaline Phosphatase substrate
solution (BCIP/NBT solution (VECTOR); pH 9.5) to develop color for
about 5 min. The samples were gently washed with phosphate buffer,
and prepared as specimens after aqueous mounting with Aqua
Poly/Mount (BioSciences).
[0305] FIG. 16 represents the result of the observation of the
specimens under a microscope. It was found that, compared to the
mesenteric fat of the control group mice, the DIO mouse mesenteric
fat had clearly more cells stained dark blue, an indicator of CCDC3
expression and production. The mesenteric fat of the DIO mice was
thus found to have more enhanced CCDC3 protein expression and
production over the control group. On the other hand, in the
subcutaneous fat, the immunostained image did not show any
difference between the control group mice and the DIO mice,
suggesting that there was no difference in the amount of CCDC3
protein expression and production. The results therefore
demonstrated that an increase in CCDC3 protein expression and
production in the adipose tissue of obesity mice was specific to
the visceral adipose tissue (mesenteric adipose tissue).
Sequence Listing Free Text
[0306] SEQ ID NOS:5 to 10 are the bases sequences of the siRNA for
the CCDC3 gene concerned with the present invention
[0307] SEQ ID NOS:11 and 12 are the base sequences of rat G6Pase
real-time quantitative PCR primers (forward primer, reverse
primer)
[0308] SEQ ID NOS:13 and 14 are the base sequences of rat Rps18
real-time quantitative PCR primers (forward primer, reverse
primer)
[0309] SEQ ID NOS:15 and 16 are the base sequences of mouse CCDC3
real-time quantitative PCR primers (forward primer, reverse
primer)
[0310] SEQ ID NOS:17 and 18 are the base sequences of mouse 36B4
real-time quantitative PCR primers (forward primer, reverse primer)
base sequence
[0311] SEQ ID NO:19 is the base sequence of CCDC3 EcoRI-F3
primer
[0312] SEQ ID NO:20 is the base sequence of CCDC3 BamHI-R3
primer
[0313] SEQ ID NO:21 is the base sequence of CCDC3 Kozak-F
primer
[0314] SEQ ID NO:22 is the base sequence of CCDC3 STOP-R primer
[0315] SEQ ID NO:23 is the base sequence of CCDC3-F1 primer
[0316] SEQ ID NO:24 is the base sequence of CCDC3-R1 primer
[0317] SEQ ID NO:25 is the base sequence of CCDC3-QPCR-F primer
[0318] SEQ ID NO:26 is the base sequence of CCDC3-QPCR-R primer
[0319] SEQ ID NO:27 is the base sequence of mGAPDH-QPCR-F
primer
[0320] SEQ ID NO:28 is the base sequence of mGAPDH-QPCR-R
primer
[0321] SEQ ID NO:29 is the amino acid sequence of the C-terminus
partial peptide (259-270 region) of human CCDC3 protein
[0322] SEQ ID NO:30 is the amino acid sequence of the heavy-chain
variable domain of 3H6 antibody
[0323] SEQ ID NO:31 is the amino acid sequence of the CDR1 of the
heavy-chain variable domain of 3H6 antibody
[0324] SEQ ID NO:32 is the amino acid sequence of the CDR2 of the
heavy-chain variable domain of 3H6 antibody
[0325] SEQ ID NO:33 is the amino acid sequence of the CDR3 of the
heavy-chain variable domain of 3H6 antibody
[0326] SEQ ID NO:34 is the amino acid sequence of the light-chain
variable domain of 3H6 antibody
[0327] SEQ ID NO:35 is the amino acid sequence of the CDR1 of the
light-chain variable domain of 3H6 antibody
[0328] SEQ ID NO:36 is the amino acid sequence of the CDR2 of the
light-chain variable domain of 3H6 antibody
[0329] SEQ ID NO:37 is the amino acid sequence of the CDR3 of the
light-chain variable domain of 3H6 antibody
[0330] SEQ ID NO:38 is the amino acid sequence of the heavy-chain
variable domain of 6G12 antibody
[0331] SEQ ID NO:39 is the amino acid sequence of the CDR1 of the
heavy-chain variable domain of 6G12 antibody
[0332] SEQ ID NO:40 is the amino acid sequence of the CDR2 of the
heavy-chain variable domain of 6G12 antibody
[0333] SEQ ID NO:41 is the amino acid sequence of the CDR3 of the
heavy-chain variable domain of 6G12 antibody
[0334] SEQ ID NO:42 is the amino acid sequence of the light-chain
variable domain of 6G12 antibody
[0335] SEQ ID NO:43 is the amino acid sequence of the CDR1 of the
light-chain variable domain of 6G12 antibody
[0336] SEQ ID NO:44 is the amino acid sequence of the CDR2 of the
light-chain variable domain of 6G12 antibody
[0337] SEQ ID NO:45 is the amino acid sequence of the CDR3 of the
light-chain variable domain of 6G12 antibody
[0338] SEQ ID NO:46 is the amino acid sequence of the heavy-chain
variable domain of 11E10 antibody
[0339] SEQ ID NO:47 is the amino acid sequence of the CDR1 of the
heavy-chain variable domain of 11E10 antibody
[0340] SEQ ID NO:48 is the amino acid sequence of the CDR2 of the
heavy-chain variable domain of 11E10 antibody
[0341] SEQ ID NO:49 is the amino acid sequence of the CDR3 of the
heavy-chain variable domain of 11E10 antibody
[0342] SEQ ID NO:50 is the amino acid sequence of the light-chain
variable domain of 11E10 antibody
[0343] SEQ ID NO:51 is the amino acid sequence of the CDR1 of the
light-chain variable domain of 11E10 antibody
[0344] SEQ ID NO:52 is the amino acid sequence of the CDR2 of the
light-chain variable domain of 11E10 antibody
[0345] SEQ ID NO:53 is the amino acid sequence of the CDR3 of the
light-chain variable domain of 11E10 antibody
SEQUENCE LISTING
[0346] PCT_visceral obesity
test.sub.--20100512.sub.--204601.sub.--1.txt
Sequence CWU 1
1
5312753DNAHomo sapiens 1cccgtggcgg agacagcggt gcgctcagct cccgggagcg
gcccgagcag ccgagcgccc 60agggctgccc ttcccgggcc ggcgggctcc ccgggctccc
cgccgccgcc ccgtgcgccc 120cgggagggcc cggcatgctg cgccagctgc
tgctcgccgc gctctgcctg gcgggtcccc 180cagcgcccgc gcgcgcctgc
cagctgccct ccgagtggag gcccctgagc gagggctgcc 240gcgccgagct
ggccgagacc atcgtgtacg ccagggtgct ggcgctgcac cccgaggcgc
300ccggcctcta caaccacctg ccctggcagt accacgccgg ccaggggggc
ctcttctact 360cggccgaggt cgagatgctg tgcgaccagg cgtggggcag
catgctggag gtgcccgccg 420gctccaggct caacctcacc ggcctgggct
acttctcgtg ccactcccac accgtggtcc 480aggactactc ctatttcttc
ttcctcagga tggatgaaaa ttataacctc ttgcctcacg 540gagtcaattt
ccaagatgcc atcttcccag acactcaaga gaacagaagg atgttttcta
600gccttttcca gttttcaaac tgttcgcaag ggcagcagct ggcgactttc
tccagtgact 660gggaaatcca ggaagacagt aggctcatgt gctcctcggt
gcagaaggcc ttgtttgagg 720aggaggacca cgtcaagaaa ctgcagcaga
aagtggccac cctggagaag cgcaaccggc 780agctccggga gcgagtgaag
aaggtcaaga ggtccttgcg gcaggcgcgt aagaagggcc 840gccacctgga
gctggcgaac cagaaactca gtgagaagct ggcggcgggc gcgctgccgc
900acatcaacgc ccgggggccc gtgcgccccc cctacctgcg ggggtaacgg
gcctgggggc 960tgccaggtgt gcagggccaa tcctggcggt aattgagaat
gagtgaggtt tcgtacatgc 1020agctatttca agggttgtaa gagtttttgt
ttttaatcac gcatttggta gagtctaaat 1080ggataaaatg caaggcttgc
tttccccttg ggtgctggcc tcaatgtcag accccacgcg 1140ctgccccttc
ctggcctgac cccagacgca gtgcctggca gtccagaggc agtgggatcc
1200ctgagtgctg aatgctcgcc tgcagagcag cccagaaaga gccctgactg
gggagagaac 1260attttagaat ctctagtgta aaagacatca acgtgcttag
cctttatttc agaaaaaaat 1320cagggtggtt cccagctccc cagtccagga
caaccattag tcctgatgag tgagctgacg 1380ctggtgctgg aacctgctgg
cacctcactg gccacatctt tggaagggga tggtggcctt 1440gcatccaaga
tgcctgaaaa tcagcacgtg cagggcctcc ctatccagcc agcattttcc
1500ttccagctga ggcaggtgaa gacttcataa gctcatcaca ggggagggaa
ttaggagcag 1560ggcagcaggt aattaaacaa gataaattat acctgatttc
caacaccagc tacaaagagt 1620tgaagatgat acctatgggt cgcgttaaca
cagggggcaa ctgccttgat cggcctgcca 1680tgggtcatca gactgcttcc
taaattgaga gaaactgagc aatctctcag ccactgctat 1740agtctaactt
cttgtttgct gagtaattgt ttctaatgtc tctgaactca aagtgaggtg
1800ctccaagacg ctgtgaactt ctgcaaagac acctccttac ctactgggat
cacgtgacct 1860gacctcactc ccagccaggc tcccaaaggg ctcattccag
ccattccaat ctcttcttct 1920ttatgcaaac acttttcccc cacaacaagc
cttgtttgtt ccgataggaa tacgtgtacg 1980tcagtgcact tgtccttacg
tcagttcctt acaccaccaa agcacttcac ctttctggaa 2040ataaaacttt
taagacacta ctataagtaa aaatgagagt attcactaga cttattgctc
2100aggcacattt gagtgggtcc cagctgtgtg attaagaagt caactgggtg
gccttttctg 2160ggttatcttc tgatcatggc ctttcaaccc aacaagggcc
cttccctgct cttccaccag 2220taaaggctcc tggcctctca tcaggatctg
ccccccagag acccccccag acactgcagg 2280gcctggtgat gctgtcctct
gtaccggaaa tggcaggcac tgtcagattt ccactcttct 2340gcctttagga
aggctgggtg cttcttgctc tgacagccag tctggggaga tgactcttac
2400gttgcttgag tcttggtggc aggctgctgt ccacggggga gaagtctctg
ctctggactg 2460gacagaagag agacttttac cctggggcac tcacacggcc
aagcttctgc caccacttca 2520ttagctgtat tctccatagt atggtgaaat
agcaggtgcg tcttctagtt tattcctcct 2580ggggacattt cctcaaagca
gttttgcgcc cccgcaaggg aatggtcagc ctaagggtaa 2640tgtacagccc
gtgcttggag aaccatggaa gctacacccc tacaggtgca tactgttctg
2700cttttccaat aaatacgagc ggcgatttca accacaaaaa aaaaaaaaaa aaa
275322769DNAMus musculus 2gccgaagagc tagccgtgca cccagctctc
cggagcgcgt gcaggcgagc cgagcgcccc 60gtccgcggtt ctcgggcagg cgctgcgggc
tccccggctc cccgccgtcc cgggcacccg 120ggcgggccat gcgcccgggc
tagagcgtag ccgccggcat gccgctcccg ctgctgctcg 180ccgcgctctg
cctcgccgcc tccccggcgc ccgcgcgcgc ctgccagctg ccgtcggagt
240ggagaccctt gagcgaaggc tgccgcgccg agctagccga gaccatcgtg
tatgccaagg 300tgctggcgct gcaccccgag gtgcctggcc tctacaacta
cctgccgtgg cagtaccaag 360ctggagaggg agggctcttc tactccgccg
aggtggagat gctgtgtgac caggcgtggg 420gcagtatgtt ggaggtgccc
gccggctccc ggctcaacct tactggtctg ggctacttct 480cctgccactc
ccacacggtg gtccaggact actcttattt cttctttgta aggatggatg
540agaattacaa tctcttgcca cacggagtca atttccaaga tgccatcttt
ccagacaccc 600aggagaacag aaggatgttt tccagccttt tccagtttgc
taactgttcc caagggcagc 660agctgacgac tttctcgagt gactgggagg
tccaggaaga caacaggctc atgtgctcct 720cggtgcagaa ggccctgttt
gaggaagagg accatgtcaa gaagctgcag cagaaggtgg 780ccaccctgga
gaagcggaac aggcagctcc gagagcgggt caagaaggtc aagaggtctc
840tgaggcaggc acggaagaac agccgccacc tggagctggt gaaccaaaaa
ctcaatgaga 900agctaggggc ctccagtgct cagcagcaca ttaacgctct
gggccgggag cccgtgcgtg 960ccccctatct gcatgggtag caggggcagg
tggatgctgg cctgccttgc ctgtgccggc 1020tgactgagaa caaccgcaag
ctctatatat gcacttactt caaacattat agtagctatc 1080catttggcat
ttggtagagt ctaactgggt aaattgtcag gttttgcttt ctttcccatt
1140gggtcctggc cttactggcc atttgacttc agatcccatg tgccacatgt
cctctttctg 1200tcctggtgcc acacaagagc cttaaaaccg gaaggcaata
gtatcttgag tctggccctc 1260agcctctaga gagttccttc gctgggggga
gaacattcta gaacctcggt atacatgaag 1320atatcacagg actctcttgt
ttcagaaggg agtcggtgtt gaggctcagc aaccagggtt 1380ggcacacgca
tgcgtgttct aaaatgttct ggaatgttct gccttttctc gtgtttttag
1440aaatggtgtc tacgtgtaaa tgtgcagggc acccacccgt gcttcagtca
ccatttctcc 1500caaagctgaa agatgttaag acttaatgag ttcattacag
gtaagggaac tgggcgagga 1560gctgaggcag ctagtaattg gtaaagataa
attatatctg ttttccaacc ccagctcctt 1620ggggctgaca tgattcccta
caggatgttt taatctaaag cgtaattgct tttatagaca 1680taccatattt
catcactcat gtttccaaac tggaattaag ctcaacctaa ctttgtggag
1740tcttaacttt gtgttagcgc agcttgccat ggctccgagt ttaaggtcac
tgacttgcac 1800acactgagac tttctactct gcagaaacag ctccctgcct
gaaggggttc aataagctct 1860atgctagggt cccaaagggc tttgtccagg
gattctaacc caagcttcta tgcaacctcc 1920aagcctctga cccaacacac
acacacacac acacacacac acacacacac acactgctca 1980ggttctcata
aaaggtctac gtgtacccca tagaagtatt tgcataacaa acactttctt
2040ttaataagag tgctttcact atccttccca tggccatgtt tgatcatgtc
ccatttgtgt 2100tactaagaag tcaatggagt ggctgctggt tcatcgcctc
taatggtggc cctccagatc 2160taatgccgtg ccacactgtt ccaccaatag
aaagaatcct tcagctctac accattgacc 2220acctagaacc cattccagac
acatgatact gataaaagtg gtcattactg tcacactgcc 2280gcctttctgc
ttgcaggaag tccaggcctc tgtgagaagt ccctcctgtc acatgagtat
2340tggtaggggt ggggataagt ttgtgccctt ggctggtcag ggcatgatgg
gaaggaactt 2400ctattctggg accaatcttc atccactctc ccccactcag
cctctttaag cagtgctatt 2460cataatatgc tacagaggtg tgccttctca
ttaattctcc ctgggagcat taagttaaag 2520cgcttttgtt acctcccaag
ggaatgaatg gcaagcttag gggtgatgga gtcaggactc 2580ggagaactgt
ggaagccgaa gatgtcttat gttctgcttt tccaataaac atgagcagtg
2640cttccaatgg ctgtggaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 2700aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2760aaaaaaaaa 27693270PRTHomo sapiens 3Met
Leu Arg Gln Leu Leu Leu Ala Ala Leu Cys Leu Ala Gly Pro Pro 1 5 10
15 Ala Pro Ala Arg Ala Cys Gln Leu Pro Ser Glu Trp Arg Pro Leu Ser
20 25 30 Glu Gly Cys Arg Ala Glu Leu Ala Glu Thr Ile Val Tyr Ala
Arg Val 35 40 45 Leu Ala Leu His Pro Glu Ala Pro Gly Leu Tyr Asn
His Leu Pro Trp 50 55 60 Gln Tyr His Ala Gly Gln Gly Gly Leu Phe
Tyr Ser Ala Glu Val Glu 65 70 75 80 Met Leu Cys Asp Gln Ala Trp Gly
Ser Met Leu Glu Val Pro Ala Gly 85 90 95 Ser Arg Leu Asn Leu Thr
Gly Leu Gly Tyr Phe Ser Cys His Ser His 100 105 110 Thr Val Val Gln
Asp Tyr Ser Tyr Phe Phe Phe Leu Arg Met Asp Glu 115 120 125 Asn Tyr
Asn Leu Leu Pro His Gly Val Asn Phe Gln Asp Ala Ile Phe 130 135 140
Pro Asp Thr Gln Glu Asn Arg Arg Met Phe Ser Ser Leu Phe Gln Phe 145
150 155 160 Ser Asn Cys Ser Gln Gly Gln Gln Leu Ala Thr Phe Ser Ser
Asp Trp 165 170 175 Glu Ile Gln Glu Asp Ser Arg Leu Met Cys Ser Ser
Val Gln Lys Ala 180 185 190 Leu Phe Glu Glu Glu Asp His Val Lys Lys
Leu Gln Gln Lys Val Ala 195 200 205 Thr Leu Glu Lys Arg Asn Arg Gln
Leu Arg Glu Arg Val Lys Lys Val 210 215 220 Lys Arg Ser Leu Arg Gln
Ala Arg Lys Lys Gly Arg His Leu Glu Leu 225 230 235 240 Ala Asn Gln
Lys Leu Ser Glu Lys Leu Ala Ala Gly Ala Leu Pro His 245 250 255 Ile
Asn Ala Arg Gly Pro Val Arg Pro Pro Tyr Leu Arg Gly 260 265 270
4273PRTMus musculus 4Met Pro Leu Pro Leu Leu Leu Ala Ala Leu Cys
Leu Ala Ala Ser Pro 1 5 10 15 Ala Pro Ala Arg Ala Cys Gln Leu Pro
Ser Glu Trp Arg Pro Leu Ser 20 25 30 Glu Gly Cys Arg Ala Glu Leu
Ala Glu Thr Ile Val Tyr Ala Lys Val 35 40 45 Leu Ala Leu His Pro
Glu Val Pro Gly Leu Tyr Asn Tyr Leu Pro Trp 50 55 60 Gln Tyr Gln
Ala Gly Glu Gly Gly Leu Phe Tyr Ser Ala Glu Val Glu 65 70 75 80 Met
Leu Cys Asp Gln Ala Trp Gly Ser Met Leu Glu Val Pro Ala Gly 85 90
95 Ser Arg Leu Asn Leu Thr Gly Leu Gly Tyr Phe Ser Cys His Ser His
100 105 110 Thr Val Val Gln Asp Tyr Ser Tyr Phe Phe Phe Val Arg Met
Asp Glu 115 120 125 Asn Tyr Asn Leu Leu Pro His Gly Val Asn Phe Gln
Asp Ala Ile Phe 130 135 140 Pro Asp Thr Gln Glu Asn Arg Arg Met Phe
Ser Ser Leu Phe Gln Phe 145 150 155 160 Ala Asn Cys Ser Gln Gly Gln
Gln Leu Thr Thr Phe Ser Ser Asp Trp 165 170 175 Glu Val Gln Glu Asp
Asn Arg Leu Met Cys Ser Ser Val Gln Lys Ala 180 185 190 Leu Phe Glu
Glu Glu Asp His Val Lys Lys Leu Gln Gln Lys Val Ala 195 200 205 Thr
Leu Glu Lys Arg Asn Arg Gln Leu Arg Glu Arg Val Lys Lys Val 210 215
220 Lys Arg Ser Leu Arg Gln Ala Arg Lys Asn Ser Arg His Leu Glu Leu
225 230 235 240 Val Asn Gln Lys Leu Asn Glu Lys Leu Gly Ala Ser Ser
Ala Gln Gln 245 250 255 His Ile Asn Ala Leu Gly Arg Glu Pro Val Arg
Ala Pro Tyr Leu His 260 265 270 Gly 519RNAArtificial SequencesiRNA
#1, sense strand r 5gacacaugau acugauaaa 19619RNAArtificial
SequencesiRNA #1, antisense strand r 6uuuaucagua ucauguguc
19719RNAArtificial SequencesiRNA #2, sense strand r 7gaaagauguu
aagacuuaa 19819RNAArtificial SequencesiRNA #2, antisense strand r
8uuaagucuua acaucuuuc 19919RNAArtificial SequencesiRNA #3, sense
strand r 9gaaguauuug cauaacaaa 191019RNAArtificial SequencesiRNA
#3, antisense strand r 10uuuguuaugc aaauacuuc 191121DNAArtificial
SequenceForward primer for amplifying G6Pase 11tgcgtgccat
aggactcatc a 211221DNAArtificial SequenceReverse primer for
amplifying G6Pase 12agcaaacaat tgcccaccgt a 211324DNAArtificial
SequenceForward primer for amplifying Rps18 13aagtttcagc acatcctgcg
agta 241424DNAArtificial SequenceReverse primer for amplifying
Rps18 14ttggtgaggt caatgtctgc tttc 241522DNAArtificial
SequenceForward primer for amplifying mouse CCDC3 gene in real-time
quantitative PCR 15tggttcatcg cctctaatgg tg 221623DNAArtificial
SequenceReverse primer for amplifying mouse CCDC3 gene in real-time
quantitative PCR 16tggaatgggt tctaggtggt caa 231721DNAArtificial
SequenceForward primer for amplifying mouse 36B4 gene in real-time
quantitative PCR 17caacggcagc atttataacc c 211821DNAArtificial
SequenceReverse primer for amplifying mouse 36B4 gene in real-time
quantitative PCR 18cccattgatg atggagtgtg g 211933DNAArtificial
SequenceCCDC3 EcoRI-F3 primer 19gcgaattcta gccgtgcacc cagctctccg
gag 332035DNAArtificial SequenceCCDC3 BamHI-R3 primer 20gcggatccta
gagcttgcgg ttgttctcag tcagc 352133DNAArtificial SequenceCCDC3
Kozak-F primer 21gcgaattcca ccatgccgct cccgctgctg ctc
332229DNAArtificial SequenceCCDC3 STOP-R primer 22gcggatccct
acccatgcag atagggggc 292320DNAArtificial SequenceCCDC3-F1 primer
23ggagagggag ggctcttcta 202420DNAArtificial SequenceCCDC3-R1 primer
24gttctcctgg gtgtctggaa 202524DNAArtificial SequenceCCDC3-QPCR-F
primer 25gctcaacctt actggtctgg gcta 242624DNAArtificial
SequenceCCDC3-QPCR-R primer 26catcttggaa attgactccg tgtg
242720DNAArtificial SequencemGAPDH-QPCR-F primer 27aaatggtgaa
ggtcggtgtg 202818DNAArtificial SequencemGAPDH-QPCR-R primer
28tgaaggggtc gttgatgg 182912PRTHomo sapiens 29Ala Arg Gly Pro Val
Arg Pro Pro Tyr Leu Arg Gly 1 5 10 30119PRTHomo sapiens 30Gln Ile
Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15
Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Gly Met Ile Trp Val Lys Gln Ala Pro Gly Lys Asp Leu Lys Trp
Met 35 40 45 Ala Trp Ile Asn Thr Tyr Ser Gly Glu Pro Thr Cys Ala
Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser
Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu
Asp Met Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Asp Asp Gly Tyr Tyr
Gly Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val
Ser Ser 115 315PRTHomo sapiens 31Asn Tyr Gly Met Ile 1 5
3217PRTHomo sapiens 32Trp Ile Asn Thr Tyr Ser Gly Glu Pro Thr Cys
Ala Asp Asp Phe Lys 1 5 10 15 Gly 3310PRTHomo sapiens 33Asp Asp Gly
Tyr Tyr Gly Tyr Phe Asp Tyr 1 5 10 34114PRTHomo sapiens 34Asp Val
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Thr Leu Val His Ser 20
25 30 Asp Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly
Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Ala 3516PRTHomo
sapiens 35Arg Ser Ser Gln Thr Leu Val His Ser Asp Gly Asn Thr Tyr
Leu His 1 5 10 15 367PRTHomo sapiens 36Lys Ile Ser Asn Arg Phe Ser
1 5 3710PRTHomo sapiens 37Ser Gln Ser Thr His Val Pro Pro Thr Phe 1
5 10 38118PRTHomo sapiens 38Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Ser Ser Tyr 20 25 30 Leu Met Asp Trp Val Lys
Gln Arg Pro Gly Gln Gly Phe Glu Cys Ile 35 40 45 Gly Asn Ile Asn
Pro Asn Thr Gly Ser Thr Ile Tyr Asn Glu Arg Phe 50 55 60 Lys Gly
Lys Ala Lys Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ile Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Ala Asp Gly Tyr Tyr Ala Pro Phe Thr Tyr Trp Gly Gln Gly
Thr 100 105 110 Leu Val Thr Val Ser Ala 115 395PRTHomo sapiens
39Ser Tyr Leu Met Asp 1 5 4017PRTHomo sapiens 40Asn Ile Asn Pro Asn
Thr Gly Ser Thr Ile Tyr Asn Glu Arg Phe Lys 1
5 10 15 Gly 419PRTHomo sapiens 41Asp Gly Tyr Tyr Ala Pro Phe Thr
Tyr 1 5 42114PRTHomo sapiens 42Asp Ile Val Met Thr Gln Ala Ala Pro
Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys
Arg Ser Ser Lys Ser Leu Leu His Asn 20 25 30 Asn Gly Asn Thr Tyr
Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu
Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
His 85 90 95 Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu
Glu Leu Lys 100 105 110 Arg Ala 4316PRTHomo sapiens 43Arg Ser Ser
Lys Ser Leu Leu His Asn Asn Gly Asn Thr Tyr Leu Tyr 1 5 10 15
447PRTHomo sapiens 44Arg Met Ser Asn Leu Ala Ser 1 5 4510PRTHomo
sapiens 45Met Gln His Leu Glu Tyr Pro Phe Thr Phe 1 5 10
46123PRTHomo sapiens 46Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Arg Ser Tyr 20 25 30 Tyr Ile His Trp Met Lys Gln
Ser Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Trp Ile Tyr Pro
Gly Gly Gly Lys Ile Tyr Tyr Asn Asp Asn Glu 50 55 60 Lys Phe Lys
Asp Lys Val Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr 65 70 75 80 Ala
Tyr Met Gln Leu Thr Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90
95 Phe Cys Ala Arg Trp Gly Gly Tyr Tyr Val Gly Ser Ile Leu Asp Tyr
100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120
475PRTHomo sapiens 47Ser Tyr Tyr Ile His 1 5 4819PRTHomo sapiens
48Trp Ile Tyr Pro Gly Gly Gly Lys Ile Tyr Tyr Asn Asp Asn Glu Lys 1
5 10 15 Phe Lys Asp 4912PRTHomo sapiens 49Trp Gly Gly Tyr Tyr Val
Gly Ser Ile Leu Asp Tyr 1 5 10 50111PRTHomo sapiens 50Asp Gly Asp
Ile Val Met Thr Gln Ser Gln Lys Phe Leu Ser Thr Ser 1 5 10 15 Val
Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp 20 25
30 Asn Asn Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ser Pro Lys Pro
35 40 45 Leu Ile Tyr Leu Thr Ser Ser Arg Tyr Ser Gly Val Pro Asp
Arg Phe 50 55 60 Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Thr Asn Val 65 70 75 80 Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys
Gln Gln Tyr Lys Thr Tyr 85 90 95 Pro Leu Thr Phe Ser Val Gly Thr
Lys Leu Glu Leu Lys Arg Ala 100 105 110 5111PRTHomo sapiens 51Lys
Ala Ser Gln Asn Val Asp Asn Asn Val Ala 1 5 10 527PRTHomo sapiens
52Leu Thr Ser Ser Arg Tyr Ser 1 5 5312PRTHomo sapiens 53Gln Gln Tyr
Lys Thr Tyr Pro Leu Thr Phe Ser Val 1 5 10
* * * * *
References