JPWO2005093068A1 - Novel proteins and promoters - Google Patents

Abstract

A novel protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4 or 6 involved in energy metabolism in vivo: PGC1αb, the PGC1αb promoter consisting of the base sequence set forth in SEQ ID NO: 33, 34 or 35, and the like; A method for searching for a therapeutic agent for a disease associated with abnormal sugar metabolism using these is provided.

Description

  The present invention relates to a novel gene involved in energy metabolism in a living body: PGC1αb, its promoter, a method for searching for a therapeutic agent for diseases associated with abnormal sugar metabolism, and the like.

The maintenance of homeostasis (homeostasis) is a basic control function for the living body, and particularly the maintenance of the balance between the intake and consumption of energy is essential for maintaining the biological function. When this regulation of energy balance is lost, the living body causes diseases accompanied by metabolic abnormalities such as obesity, diabetes or hyperlipidemia. That is, the energy metabolism abnormality is considered to cause so-called lifestyle-related diseases such as hypertension, arteriosclerosis and diabetes.
Insulin plays the most important role in regulating sugar and lipid metabolism. Insulin is a hormone that has a variety of physiological functions, including metabolic effects of sugars, lipids, and proteins, cell proliferation, cytoskeleton control, muscle or liver cell differentiation, transcriptional regulation, and apoptosis inhibition. In energy metabolism in the living body, energy metabolism is controlled by insulin, and a role such as liver, muscle, or fat plays a large role.
Insulin dysfunction is called insulin resistance and is considered to be a major factor causing metabolic abnormalities together with insulin secretion failure. In other words, insulin resistance is defined as “a state in which more than a normal amount of insulin is required for various actions of insulin at the cell, organ, and individual levels”. Type 2 diabetes is mentioned as a disease accompanied by insulin resistance. Recently, it has become clear that insulin resistance is observed not only in diabetes and impaired glucose tolerance but also in many pathologies such as hypertension, hyperlipidemia and obesity. It is coming. Accordingly, insulin resistance is not only closely related to each disease state, but also easily causes mutual complications, and as a result, synergistically develops and develops arteriosclerosis and the like.
By the way, in type 2 diabetes, it is considered that the organs and tissues responsible for insulin resistance differ between fasting and sugar intake. That is, it is important to improve the insulin resistance in the liver because the gluconeogenesis in the liver increases despite the blood insulin concentration and blood glucose level being higher than those in a normal person on an empty stomach. On the other hand, in insulin resistance at the time of sugar intake, that is, at the time of increasing blood insulin concentration, it is important to improve the decrease in sugar uptake in skeletal muscle and the inhibition of glucose production inhibition in the liver.
Therefore, improvement of insulin resistance in skeletal muscle, that is, anabolic effects such as sugar uptake and glycogen synthesis, energy supply by glycolysis accompanying exercise, and heat production for maintaining body temperature can improve symptoms associated with insulin resistance. Is important to. In fact, De Fronzo et al. Point out that a decrease in glucose uptake via insulin in peripheral tissues (skeletal muscles) is a problem in type 2 diabetes (see Non-Patent Document 1). However, no drug has been found that selectively improves the function of insulin in skeletal muscle.
Recently, a transcriptional co-activator of mouse-derived PPARγ: peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) was found as a new factor involved in the control of heat production (Non-patent Document 2). See). PGC1α is a factor that interacts with the nuclear receptor PPARγ and is cloned as a factor that assists in its transcription activity, and is known to transform white adipocytes into brown adipocytes (see Patent Document 1). . It has also been clarified that PGC1α plays an important regulatory action in the synthesis of a series of proteins (UCP1, mitochondrial enzymes, etc.) involved in the heat production action and in the growth of mitochondria (see Non-Patent Document 3).
For example, in muscle cells, PGC1α induces the expression of UCP2, which is thought to cause energy expenditure in mitochondria. In addition, the forced expression of PGC1α induces the expression of a transcription factor: mtTFA (mitochondrial transcription factor A), which plays an important role in mitochondrial genome replication and transcription reaction, and induces intracellular oxygen consumption and intracellular mitochondria. It has also been clarified that the number increases (see Non-Patent Document 4). PGC1α has also been reported to induce the expression of GLUT4, a sugar transport protein, in muscle cells and increase sugar transport (see Non-Patent Document 5). Furthermore, reports on the relationship between skeletal muscle mitochondrial dysfunction and insulin resistance have been repeated, suggesting a relationship with PGC1α in skeletal muscle (see Non-Patent Documents 6 to 8).
In addition, a method of screening for anti-obesity / anti-diabetic drugs having mitochondrial activation as an action mechanism using PGC1α expression promoting activity as an index is known (see Patent Document 2).
However, PGC1α is involved in the induction mechanism of gluconeogenic genes in the liver. That is, PGC1α is induced by cAMP which is an intracellular second messenger of glucocorticoid and glucagon that induces gluconeogenic genes, and stimulates the transcriptional activity of glucocorticoid receptor and HNF4α to express gluconeogenic genes. Has been reported (see Non-Patent Documents 9 and 10). Therefore, enhancement of PGC1α function promotes gluconeogenic action in the liver, and is not preferable in diseases associated with insulin resistance.
Until now, there has been no known method for selectively improving insulin functions such as heat production in skeletal muscle.
Japanese translation of PCT publication No. 2002-531079 International pamphlet No. 01/090356 DeFronzo et al. , J .; Clin. Invest. 76, 149-155 (1985). Cell, 92, 829-838 (1998). Lowell, et al. , Nature, 404, 652-660 (2000). Cell, 98, 115-124 (1999). Proc. Nat. Am. Soc. 98, 3820-3825 (2001). Mootha, V .; K. et al. Nat. Genet. 34: 267-273. (2003). Patti, M.M. E. et al. Proc. Natl. Acad. Sci. USA. , 100: 8466-8471. (2003). Petersen, K.M. F. et al. Science, 300: 1140-1142. (2003). Yoon, et al. , Nature, 413, 131-138 (2001). Herzig, et al. , Nature, 413, 179-183 (2001).

The problem to be solved by the present invention is a sugar metabolism improving agent, that is, a novel variant of PGC1α that plays an important role in energy metabolism in vivo: PGC1αb, its promoter, and heat production in skeletal muscle using these A method for searching for a sugar metabolism-improving agent that improves the function of skeletal muscle during insulin resistance by improving the action and the like, and a drug containing a substance that promotes the expression of PGC1αb as an active ingredient (especially mitochondrial activation Providing anti-obesity / anti-diabetic drugs).
As a result of intensive studies, the inventors have found PGC1αb, which is a novel variant of PGC1α. Next, when the tissue distribution of PGC1αb was examined, it was surprisingly found that the PGC1αb was hardly expressed in tissues such as the liver and was specifically expressed in skeletal muscle. Furthermore, PGC1αb was induced in skeletal muscle under the conditions of energy consumption by heat and heat production. From the above, it was found that PGC1αb promotes energy metabolism such as heat production specifically for skeletal muscle. Since it is known that exercise therapy that promotes energy metabolism by exercise is effective in insulin resistant patients, PGC1αb is an insulin resistant treatment by enhancing skeletal muscle heat production in insulin resistant patients. Can be a target factor. That is, a substance that promotes the expression or activity of PGC1αb can be a preventive, ameliorating, or therapeutic agent for diseases such as insulin resistance. Further, by promoting energy metabolism in skeletal muscle, it is also an agent for improving diseases associated with abnormal energy metabolism such as sugar metabolism, such as so-called lifestyle-related diseases such as obesity, diabetes, hyperlipidemia, or arteriosclerosis. Can be.
Next, the inventors succeeded in isolating a region having a promoter activity of about 3 kb from human genomic DNA. In this promoter region, a region essential for exhibiting a promoter activity of about 1.3 kb was identified. In addition, a region having a promoter activity of about 3 kb was successfully isolated from mouse genomic DNA. Furthermore, by using an isolated promoter DNA, a method for evaluating the expression control ability of the PGC1αb gene, and a method for searching for a sugar metabolism improving agent, etc., comprising selecting a substance that promotes the promoter activity by the evaluation method I found.
The present invention has been completed based on the above findings.
In the following description, abstract and claims, the above-mentioned PGC1α known before the present invention is referred to as PGC1αa in order to distinguish it from the gene of the present invention.
That is, the present invention
[1] A protein of any one of the following (a) to (f):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6,
(B) consisting of an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence described in SEQ ID NO: 2, 4 or 6, comprising the amino acid sequence described in SEQ ID NO: 8 at the N-terminus; And a protein having mitochondrial activation ability,
(C) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in SEQ ID NO: 2, comprising the amino acid sequence described in SEQ ID NO: 8 at the N-terminus, and having a mitochondrial activation ability;
(D) a protein comprising an amino acid sequence encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3 or 5 and containing the amino acid sequence set forth in SEQ ID NO: 8 at the N-terminus and having the ability to activate mitochondria ,
(E) consisting of an amino acid sequence encoded by DNA consisting of a base sequence having a sequence identity of 80% or more with DNA consisting of the base sequence shown in SEQ ID NO: 1, 3 or 5; A protein having the amino acid sequence of and having the ability to activate mitochondria,
(F) an N-terminal consisting of an amino acid sequence encoded by a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3 or 5 and a DNA that hybridizes under stringent conditions A protein comprising the amino acid sequence of a protein containing the amino acid sequence of SEQ ID NO: 8 and having the ability to activate mitochondria;
[2] A gene comprising a base sequence encoding a protein comprising the amino acid sequence according to [1];
[3] A primer or probe comprising a polynucleotide that specifically recognizes the gene according to [2], or a polynucleotide complementary thereto;
[4] An antibody that specifically recognizes the protein according to [1].
[5] A promoter capable of controlling the expression of the protein according to [1], comprising the gene described in any of (g) to (i) below;
(G) a gene consisting of the base sequence represented by SEQ ID NO: 33, 34 or 35,
(H) a gene comprising a base sequence in which one or more bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 33, 34 or 35, and having transcriptional control ability,
(I) a gene that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 33, 34, or 35 and has a transcriptional control ability;
[6] An expression vector comprising the gene according to [2];
[7] An expression vector comprising the promoter according to [5];
[8] The expression vector according to [7], further comprising a base sequence encoding a gene whose transcription is controlled by the promoter downstream (3 ′ side) of the promoter;
[9] The expression vector according to [8], wherein the gene is the gene according to [2];
[10] A transformed cell into which the expression vector according to any one of [6] to [9] has been introduced;
[11] Evaluation method of expression control ability of PGC1αb gene including the following steps (1), (2) and (3):
(1) contacting a test substance with a cell capable of expressing the PGC1αb gene;
(2) measuring the expression level of the PGC1αb gene in a cell contacted with a test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance;
(3) A step of evaluating the expression control ability of the PGC1αb gene of the test substance using as an index whether or not to change the expression level of the PGC1αb gene based on the comparison result of (2);
[12] Evaluation method of expression control ability of PGC1αb including the following steps (1), (2) and (3):
(1) contacting a test substance with a cell capable of expressing PGC1αb;
(2) measuring the expression level of PGC1αb in a cell contacted with a test substance, and comparing the expression level with the expression level of PGC1αb in a control cell not contacted with the test substance;
(3) Based on the comparison result of (2), a step of evaluating the expression control ability of PGC1αb of the test substance using whether or not the expression level of PGC1αb is varied as an index;
[13] A method for evaluating the ability to control the expression of the PGC1αb gene, comprising the following steps (1) to (3):
(1) A step of contacting a test substance with a cell capable of expressing a reporter gene formed by binding the promoter according to [5],
(2) measuring the expression level of the reporter gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the reporter gene in a control cell not contacted with the test substance, and (3) ( A step of evaluating the expression control ability of the test substance PGC1αb gene using as an index whether or not the expression level of the reporter gene is varied based on the comparison result of 2);
[14] A substance having PGC1αb gene or PGC1αb expression-inducing activity or PGC1αb, using the PGC1αb gene or PGC1αb expression control ability of the test substance evaluated by the evaluation method according to any one of [11] to [13] as an index A method for searching for a PGC1αb expression control substance, which comprises selecting a substance having an expression suppression activity of
[15] A candidate substance having PGC1αb gene or PGC1αb expression-inducing activity, using as an index the PGC1αb gene or PGC1αb expression control ability of the test substance evaluated by the evaluation method according to any one of [11] to [13] A method for searching for a sugar metabolism-improving agent, characterized by screening;
[16] The search method according to [15], which is performed for searching for a candidate substance for a preventive, ameliorating, or therapeutic agent for a disease associated with abnormal sugar metabolism;
[17] The searching method according to [15] or [16], which is carried out for searching for a candidate substance for an agent for preventing, improving or treating an insulin resistant disease;
[18] The search method according to any one of [15] to [17], further comprising selecting a compound using as an index that the test substance does not change the expression of the PGC1αa gene or PGC1αa;
[19] A sugar metabolism improving agent comprising as an active ingredient a compound selected by the search method according to any one of [15] to [18];
[20] An agent for preventing, ameliorating or treating an insulin resistance disease, comprising as an active ingredient a compound selected by the search method according to any one of [15] to [18];
[21] A sugar metabolism-improving agent comprising a PGC1αb or PGC1αb gene expression inducer as an active ingredient;
[22] An insulin resistance ameliorating agent comprising PGC1αb or a PGC1αb gene expression inducer as an active ingredient;
[23] The improving agent according to [21] or [22], which does not change the expression of the PGC1αa or PGC1αa gene;
[24] The improving agent according to [23], which specifically acts on muscle cells in a living body;
[25] The method for searching for a substance that binds to the promoter according to [5], comprising the following steps (1) and (2):
(1) a step of bringing the promoter according to [5] into contact with a test substance, and (2) a step of examining the presence or absence of complex formation between the promoter and the test substance after the step (1);
[26] A method for purifying a substance that binds to the promoter according to [5],
(1) The step of bringing the promoter according to [5] into contact with a sample to form a complex of the promoter and a substance that binds to the promoter contained in the sample, and
(2) A purification method comprising the step of isolating the binding substance from the produced complex after the step (1);
[27] a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8;
[28] The polypeptide of [27], comprising the amino acid sequence of any of SEQ ID NOs: 53 to 55;
[29] The antibody according to [4], which specifically recognizes the peptide according to [27] or [28];
[30] a gene encoding the polypeptide according to [27] or [28];
[31] a polynucleotide comprising the base sequence set forth in SEQ ID NO: 7;
[32] The polynucleotide according to [31], comprising the base sequence according to any of SEQ ID NOs: 50 to 52;
[33] The primer according to [3], comprising at least 5 or more consecutive base sequences of the polynucleotide according to [31] or [32], or the polynucleotide complementary thereto probe;
About.

FIG. 1 is a diagram showing the expression of PGC1αb and PGC1αa in each mouse tissue. The expression level was quantified by RT-PCR, and the value of 36B4 of the control was taken as 1 and expressed as a relative value.
FIG. 2 is a diagram showing exercise-dependent expression induction of PGC1αb in skeletal muscle. The expression level was quantified by RT-PCR, and the value of 36B4 of the control was taken as 1 and expressed as a relative value.
FIG. 3 is a diagram showing the induction of PGC1αb gene expression in cultured muscle cells. The expression level was quantified by RT-PCR, and the value of 36B4 of the control was taken as 1 and expressed as a relative value. Ax represents adenovirus AxCAwt-mPGC1αb, and the amount of virus added is shown in parentheses.
FIG. 4 is a diagram showing the induction of PGC1αb gene expression in cultured muscle cells. The expression level was quantified by RT-PCR, and the value of 36B4 of the control was taken as 1 and expressed as a relative value. Ax represents adenovirus AxCAwt-mPGC1αb, and the amount of virus added is shown in parentheses.
FIG. 5 is a diagram showing the induction of PGC1αb gene expression in mouse striated muscle-derived cultured cells (result of Example 11). The expression level was quantified by RT-PCR, and the value of 36B4 of the control was taken as 1 and expressed as a relative value.
FIG. 6 shows the measurement results of the transcription initiation ability of the human PGC-1αb promoter of the present invention (results of Example 18). The expression level was quantified by RT-PCR, and the value of 36B4 of the control was taken as 1 and expressed as a relative value. Basic is pGL3-Basic, P1 is phPGC1αb-P1 (3.0) -pGL3basic, P2 is phPGC1αb-P2 (1.8) -pGL3basic, P3 is phPGC1αb-P3 (1.3) -pGL3basic, DMSO is control DMSO addition, FSK indicates Forskolin addition.
FIG. 7 shows the measurement results of the transcription initiation ability of the mouse PGC-1αb promoter of the present invention (results of Example 19). The expression level was quantified by RT-PCR, and the value of 36B4 of the control was taken as 1 and expressed as a relative value. Basic represents pGL3-Basic, and mPGC-1αb (1 kb) represents mPGC1αb-P2 (1.0) -pGL3basic. DMSO indicates the addition of DMSO as a control, and Forskolin indicates the addition of Forskolin.

The present invention is described in detail below.
Genetic engineering techniques used in the present invention are described in, for example, “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press and D.C. , M.M. , Glover, DNA Cloning, published by IRL, 1985, and the like.
In this specification, the abbreviations such as amino acids, (poly) peptides, (poly) nucleotides, etc. are defined in the IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. et al. Biochem. , 138: 9 (1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.
In this specification, “gene” or “DNA” is used to include not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. In addition, “gene” or “polynucleotide” is used to include both RNA and DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing DNA (positive strand) and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included. The RNA includes any of total RNA, mRNA, rRNA, and synthetic RNA.
(I) PGC1αb and PGC1αb genes
The present invention relates to PGC1αb, which is a novel variant of PGC1α.
In the present specification, “PGC1αb” represents one type of variant of peroxisome proliferator-activated receptor gamma coactivator-1α, specifically, a protein represented by SEQ ID NO: 2 (human PGC1αb). The PGC1αb of the present invention is characterized in that it includes a partial peptide consisting of the amino acid sequence shown in SEQ ID NO: 8 at about 50 residues at the N-terminus of PGC1αa (Genbank Acc. No. NM — 0316261) that has already been reported. Preferably, PGC1αb is a polypeptide in which the portion corresponding to the N-terminal 17 amino acid residues excluding the first methionine residue of PGC1αa is substituted with the amino acid sequence represented by SEQ ID NO: 8. For example, human PGC1αb is represented by SEQ ID NO: 2 in which the portion corresponding to the N-terminal 17 amino acid residues excluding the first methionine of PGC1αa is replaced with an amino acid sequence consisting of LGLSSMDSILK (SEQ ID NO: 53). Polypeptide.
Further, PGC1αb of the present invention includes homologues (homologues), mutants and the like. For example, as homologues, proteins of other species such as mice and rats corresponding to human proteins can be exemplified, and these are homogenes (http://www.ncbi.nlm.nih.gov/homologene/). Can be a priori identified from the base sequence of the gene identified by. Specifically, mouse PGC1αb has a portion corresponding to the N-terminal 17 amino acid residues excluding the first methionine residue of PGC1αa substituted with an amino acid sequence consisting of LGLSSMDSILK (SEQ ID NO: 54). (Mouse PGC1αb). Rat PGC1αb is represented by SEQ ID NO: 6, in which the portion corresponding to the N-terminal 17 amino acid residues excluding the first methionine of PGC1αa is replaced with an amino acid sequence consisting of SGLSSMDSTLK (SEQ ID NO: 55). Protein (rat PGC1αb).
Examples of the mutant include naturally occurring allelic mutants and non-naturally occurring mutants. Specifically, the proteins shown in the following (b), (c), (e) and (f) are: Can be mentioned.
That is, in the present invention, PGC1αb is not limited to the protein represented by SEQ ID NO: 2, 4 or 6, and represents any one of the following proteins (a) to (f):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6,
(B) consisting of an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence described in SEQ ID NO: 2, 4 or 6, comprising the amino acid sequence described in SEQ ID NO: 8 at the N-terminus; And a protein having mitochondrial activation ability,
(C) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in SEQ ID NO: 2, comprising the amino acid sequence described in SEQ ID NO: 8 at the N-terminus, and having a mitochondrial activation ability;
(D) a protein comprising an amino acid sequence encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3 or 5 and containing the amino acid sequence set forth in SEQ ID NO: 8 at the N-terminus and having the ability to activate mitochondria ,
(E) consisting of an amino acid sequence encoded by DNA consisting of a base sequence having a sequence identity of 80% or more with DNA consisting of the base sequence shown in SEQ ID NO: 1, 3 or 5; A protein having the amino acid sequence of and having the ability to activate mitochondria,
(F) an N-terminal consisting of an amino acid sequence encoded by a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3 or 5 and a DNA that hybridizes under stringent conditions A protein comprising the amino acid sequence of SEQ ID NO: 8 and comprising the amino acid sequence of a protein having the ability to activate mitochondria.
The “mitochondrial activation ability” in the above (b) to (f) can be evaluated by confirming the mitochondrial activation action in the cell under the condition that the protein of the present invention is expressed in the cell. Confirmation of the mitochondrial activation action can be made by measuring the activity of an enzyme present in mitochondria or measuring the expression of a gene related to mitochondrial function and increasing its activity or expression.
Specifically, for example, a reagent WST-1 (DOJINDO, 342-06451) capable of measuring the mitochondrial dehydrogenase activity of living cells by a colorimetric method can be used for examining mitochondrial enzyme activity. Specifically, for example, L6 cells transfected with the expression vector of the protein of the present invention are cultured in a tissue culture dish having a diameter of 10 cm, and when reaching confluence, the cells are transferred to a 96-well tissue culture dish. At this time, the medium is changed to Dulbecco's modified Eagle medium (GIBCO BRL, USA, 21063-029) not containing phenol red. After culturing for 6 hours, the medium is changed to one that does not contain fetal calf serum, and culturing is continued for an appropriate time. After the culture, the medium was once discarded by aspiration, and 100 μl of Dulbecco's modified Eagle medium without phenol red and fetal calf serum was added. 10 μl of PBS containing 5 mM WST-1 and 0.2 mM 1-Methoxy PMS (DOJINDO, 345-040001) Add After reacting at 37 ° C. for 4 hours, mitochondrial enzyme activity can be measured by measuring absorbance at a measurement wavelength of 450 nm.
Or, specifically, for example, the following RT-PCR method can be used as a method for measuring the expression of genes involved in mitochondrial function.
When using the RT-PCR method, after culturing the cell transfected with the expression vector of the protein of the present invention, mitochondria using RNA prepared from the cell or cDNA prepared using the obtained RNA as a template. Design a pair of primers (a normal strand that binds to the cDNA (-strand) and a reverse strand that binds to the + strand) so that the base sequence region encoding the gene involved in the function can be specifically amplified. Synthesize with For example, when studying specifically using mouse-derived cells, UCP2 (Genbank Acc. No. AF111998), which is thought to cause energy consumption in mitochondria, plays an important role in mitochondrial genome replication and transcription reactions. Transcription factors shown: mtTFA (mitochondrial transcription factor A; Genbank Acc. No. NM_009360), transcription factor NRF1 (Genbank Acc. No. AF098077) that binds to the promoters of many mitochondrial genes, oxidative phosphorylation pathway in mitochondria Β ATP synthase (Genbank Acc. No. AF030559) and / or cytochrome c oxidas which are gene groups subunits II; according to the sequence of (COXII Genbank Acc.No.AF378830), synthesized primer may be used. As a template used for the RT-PCR method, specifically, for example, RNA prepared from mouse muscle tissue or mouse striated muscle-derived cultured cell C2C12, or TaqMan Reverse Transcription Reagents (manufactured by ABI) using the obtained RNA as a template. ) Can be used. Specifically, for the RT-PCR reaction, for example, when using the SYBR Green method, an RT-PCR reaction solution is prepared according to the protocol using SYBR Green RT-PCR Reagents (manufactured by Applied Biosystems), and ABI PRIME 7900 Sequence is used. The reaction can be detected and quantified by reacting with Detection System (Applied Biosystems).
Examples of the “deletion, addition or substitution” of amino acids in (b) and “80% or more sequence identity” in (d) include, for example, the amino acid sequence represented by SEQ ID NO: 2, 4, or 6. It includes naturally occurring mutations due to the processing that the protein has in the cell, species differences of individuals from which the protein is derived, individual differences, differences between tissues, and the like, and artificial amino acid mutations.
As a technique for artificially performing the “deletion, addition or substitution” of amino acids in the above (b) (hereinafter sometimes referred to as amino acid modification as a whole), for example, SEQ ID NO: 2, 4 or 6 An example is a method in which conventional site-directed mutagenesis is performed on DNA encoding the amino acid sequence represented, and then this DNA is expressed by a conventional method. Here, as a site-specific mutagenesis method, for example, a method utilizing amber mutation (gapped duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer. Etc.
The number of amino acids modified as described above is at least one residue, specifically one or several, or more. The number of such modifications may be within a range where mitochondrial activation ability and / or immunological activity can be found.
The “sequence identity” in the above (c) and (e) refers to sequence identity and homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or a deletion (for example, a gap) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using the ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. The sequence identity is measured by using sequence analysis software, specifically, an analysis tool provided by Vector NTI, GENETYX-MAC or a public database, for example, the homepage address http Generally available at //www.ddbj.nig.ac.jp.
The sequence identity in the present invention may be 80% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 97%.
Regarding the “stringent conditions” in (f) above, the hybridization used here is, for example, Sambrook J. et al. Frisch E .; F. Maniatis T. It can be carried out according to the usual methods described in the author, Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory press, etc. “Stringent conditions” means, for example, 6 × SSC (a solution containing 1.5 M NaCl and 0.15 M trisodium citrate is set to 10 × SSC) and a hybrid at 45 ° C. in a solution containing 50% formamide. And the like (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6) and the like that are washed with 2 × SSC at 50 ° C. Can do. The salt concentration in the washing step can be selected from, for example, conditions of 2 × SSC at 50 ° C. (low stringency conditions) to 0.2 × SSC at 50 ° C. (high stringency conditions). The temperature in the washing step can be selected, for example, from a temperature from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.
The “complementary base sequence” in the above (f) is a polynucleotide that is in a base-complementary relationship with respect to the base sequence of the PGC1αb gene based on a base pair relationship such as A: T and G: C. Means.
The number of amino acid mutations and mutation sites in the protein are not limited as long as the biological function, that is, the ability to activate mitochondria and / or immunological activity is retained. Indicators that determine how many amino acid residues need to be substituted, inserted or deleted without loss of biological function or immunological activity are computer programs well known to those skilled in the art, For example, it can be found using DNA Star software. For example, the number of mutations is typically within 10% of all amino acids, preferably within 5% of all amino acids, and more preferably within 1% of all amino acids. The amino acid to be substituted is not particularly limited as long as the protein obtained after substitution retains the biological function and / or immunological activity of PGC1αb, but from the viewpoint of maintaining the structure of the protein, the polarity of the residue An amino acid having properties similar to that of the amino acid before substitution, such as charge, solubility, hydrophobicity, hydrophilicity, and amphiphilicity, is preferable. For example, Ala, Val, Leu, Ile, Pro, Met, Phe and Trp are amino acids classified as nonpolar amino acids, and Gly, Ser, Thr, Cys, Tyr, Asn and Gln are mutually non-charged amino acids. Asp and Glu are amino acids that are classified as acidic amino acids, and Lys, Arg, and His are amino acids that are classified as basic amino acids. Therefore, amino acids belonging to the same group can be appropriately selected using these as indices.
The present invention also relates to a gene comprising a base sequence encoding the PGC1αb. The “gene” or “DNA” has not only a “gene” or “DNA” represented by a specific base sequence (SEQ ID NO: 1) but also a biological function equivalent to the protein encoded by them. “Gene” or “DNA” encoding a protein (eg, a homolog) is included. Specifically, the “PGC1αb gene” is not only the human PGC1αb gene represented by SEQ ID NO: 1, but also its homologues or the base having 80% or more sequence identity described in (d) above. "DNA comprising a sequence" and "DNA hybridizing under stringent conditions with the complementary strand of the human PGC1αb gene represented by SEQ ID NO: 1" described in (f) above. That is, as long as the protein comprising the amino acid sequence encoded by these genes (DNA) and containing the amino acid sequence of SEQ ID NO: 8 at the N-terminus has mitochondrial activation ability, the PGC1αb gene of the present invention This is a category.
For example, as a gene (homolog) encoding a homologue of a human-derived protein, genes of other species such as mouse and rat corresponding to the human gene encoding the protein can be exemplified. These genes (homologues) can be identified by HomoMoloGene (http://www.ncbi.nlm.nih.Gov/HomoloGene/). Specifically, the gene (homologue) can be obtained, for example, as follows: The base sequence of a specific human gene is BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). , Http://www.ncbi.nlm.nih.gov/BLAST/), the accession number of the sequence is obtained (Score is the highest, E-value is 0 and Identity is 100%). The UniGene Cluster ID (the number indicated by Hs.) Obtained by inputting the accession number into UniGene (http://www.ncbi.nlm.nih.gov/UniGene/) is input into Homogene. From the list showing the correlation of gene homologues between the other species gene and the human gene obtained as a result, genes of other species such as mice and rats as homologues corresponding to the human gene indicated by a specific base sequence To select. Specifically, the mouse PGC1αb gene represented by SEQ ID NO: 3 and the rat PGC1αb gene represented by SEQ ID NO: 5 can be exemplified.
The gene or DNA does not ask for distinction of the functional region, and can include, for example, an expression control region, a coding region, an exon, or an intron.
The PGC1αb gene of the present invention is a variant of PGC1αa, which is a known gene as described above. Therefore, the PGC1αb gene can be obtained by the same method as the PGC1αa gene for the sequence after the 40th base in the sequence shown in SEQ ID NO: 1. The PGC1αa gene can be found in Genbank Accession No. It is known as NM_013261 and its acquisition method is also known as described in Cell 92 (6), 829-839 (1998). The homologue thereof includes the mouse PGC1αa gene (Genbank Accession No. NM — 008904) and the rat PGC1αb gene described in SEQ ID NO: 5 for the sequence after the 49th base of the mouse PGC1αb gene described in SEQ ID NO: 3. Regarding the sequence after the 1 st base, rat PGC1αa gene (Genbank Accession No. NM — 031347) can be mentioned, and Cell 92 (6), 829-839 (1998), J. Biol. Biol. Chem. 277. (19), 16750-16757 (2002).
The PGC1αb gene can be obtained by a conventional genetic engineering method [for example, Sambrook J. et al. Frisch E .; F. Maniatis T. And the method described in Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory press, etc.].
Specifically, RNA is first prepared from tissues, cells such as humans, mice or rats, or cultured cells derived therefrom. For example, rat muscles are pulverized in a solution containing a strong protein denaturant such as guanidine hydrochloride or guanidine thiocyanate, and the protein is denatured by adding phenol, chloroform or the like to the pulverized product. After the denatured protein is removed by centrifugation or the like, total RNA is extracted from the collected supernatant fraction by a method such as guanidine hydrochloride / phenol method, SDS-phenol method, guanidine thiocyanate / CsCl method or the like. Examples of commercially available reagents based on these methods include ISOGEN (manufactured by Nippon Gene) and Trisol reagent (Gibco BRL).
Using the obtained total RNA as a template, an oligo dT primer is annealed to the poly A sequence of RNA, and reverse transcriptase is allowed to act to synthesize a single-stranded cDNA. Next, a polymerase chain reaction using the single-stranded cDNA as a template and an oligonucleotide designed based on the base sequence of the PGC1αb gene (for example, the base sequence described in SEQ ID NO: 1, 3 or 5) as a primer ( Hereinafter, the PGC1αb gene can be amplified and obtained by performing PCR.
In addition, double-stranded cDNA is synthesized by allowing DNA polymerase to act using the single-stranded cDNA as a template. The obtained double-stranded cDNA is inserted into a vector such as plasmid pUC118 or phage λgt10 to prepare a cDNA library. A DNA having a partial base sequence of the base sequence of the PGC1αb gene (for example, the base sequence represented by SEQ ID NO: 1, 3 or 5) is used as a probe from the cDNA library thus obtained or a commercially available cDNA library. The PGC1αb gene can also be obtained by a hybridization method or PCR using an oligonucleotide designed based on the base sequence of the PGC1αb gene (for example, the base sequence represented by SEQ ID NO: 1, 3 or 5) as a primer.
As a primer used for PCR, for example, a base sequence having a length of about 15 bp to about 50 bp and a G or C base ratio of about 40% to about 60% is encoded with the protein of the present invention as described above. The known nucleotide sequence is selected, and an oligonucleotide is designed and synthesized based on the nucleotide sequence.
The base sequence of the obtained PGC1αb gene was determined using the Maxam Gilbert method (for example, the method described in Maxam, AM & W. Gilbert, Proc. Natl. Acad. Sci. USA, 74, 560, 1977) or the Sanger method. (For example, Sanger, F. & AR Coulson, J. Mol. Biol., 94, 441, 1975, Sanger, F, & Nicklen and AR Coulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977, etc.).
The PGC1αb gene obtained as described above is disclosed in, for example, J. Sambrook, E .; F. Frisch, T .; It can be cloned into a vector according to the genetic engineering method described by Maniatis; published by Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989).
The vector is not particularly limited as long as the PGC1αb gene can be inserted, and a known vector or a commercially available vector can be appropriately used. A vector into which the PGC1αb gene is inserted is also within the scope of the present invention.
Specifically, when Escherichia coli is used as a host cell, for example, plasmid pUC119 (manufactured by Takara Shuzo Co., Ltd.), phagemid pBluescript II (manufactured by Stratagene) and the like can be mentioned. When budding yeast is used as a host cell, plasmid pACT2 (Clontech) and the like can be mentioned. When mammalian cells are used as host cells, plasmids such as pRC / RSV and pRC / CMV (manufactured by Invitrogen), bovine papilloma virus plasmid pBPV (manufactured by Amersham Pharmacia), and EB virus plasmid pCEP4 (manufactured by Invitrogen) And the like, vectors containing an autonomous origin of replication derived from viruses, viruses such as vaccinia virus, and the like. When an insect animal cell (hereinafter referred to as an insect cell) is used as a host cell, an insect virus such as baculovirus can be used.
An expression vector capable of expressing the PGC1αb gene in the host cell is constructed by linking a promoter capable of functioning in the host cell upstream of the PGC1αb gene and incorporating it into the above-described vector. can do.
Here, “to bind in a functional manner” means that when the PGC1αb gene is introduced into the host cell, the promoter and the PGC1αb gene are bound so that they are expressed in the host cell under the control of the promoter. Means that. Examples of the promoter that can function in the host cell include the promoter of the present invention described in [5] above. Further, for example, when the host cell is E. coli, the lactose operon promoter (lacP) of E. coli, the tryptophan operon promoter (trpP), the arginine operon promoter (argP), the galactose operon promoter (galP), the tac promoter Alternatively, synthetic promoters that can function in E. coli such as trc promoter, T7 promoter, T3 promoter, λ phage promoter (λ-pL, λ-pR) and the like can be mentioned. When the host cell is an animal cell or fission yeast, for example, Rous sarcoma virus (RSV) promoter, cytomegalovirus (CMV) promoter, simian virus (SV40) early or late promoter, mouse papilloma virus ( MMTV) promoter and the like. When the host cell is budding yeast, the ADH1 promoter (where the ADH1 promoter is, for example, the yeast expression vector pAAH5 carrying the ADH1 promoter and the terminator available from Washington Research Fundation, Ammerer et al., Method in Enzymology, 101 part. (P.192-201)] can be prepared by an ordinary genetic engineering method.
In general, a DNA in which a promoter that can function in a host cell and a PGC1αb gene are connected in a functional manner is incorporated into a vector that can be used in the host cell, and this is introduced into the host cell. When using a vector having a promoter that can function in the host cell in advance, the PGC1αb gene may be inserted downstream of the promoter so that the promoter possessed by the vector and the PGC1αb gene are linked in a functional manner. . For example, the above-mentioned plasmids pRC / RSV, pRC / CMV, etc. have a cloning site downstream of a promoter that can function in animal cells. By inserting the PGC1αb gene into the cloning site and introducing it into the animal cells, The PGC1αb gene can be expressed. Moreover, the aforementioned yeast plasmid pACT2 has an ADH1 promoter, and if the PGC1αb gene is inserted downstream of the ADH1 promoter of the plasmid or its derivative, the PGC1αb gene can be transformed into a budding yeast such as CG1945 (manufactured by Clontech). An expression vector that can be expressed in can be constructed. When using a vector containing a marker gene (for example, a gene conferring antibiotic resistance such as a kanamycin resistance gene or a neomycin resistance gene), a transformed cell into which the PGC1αb gene has been introduced is selected using the phenotype of the marker gene as an index Useful when doing.
If it is necessary to induce higher expression, a ribosome binding region may be linked upstream of the PGC1αb gene. Examples of the ribosome binding region used include Guarente L. et al. (Cell 20, p543) and Taniguchi et al. (Genetics of Industrial Microorganisms, p202, Kodansha).
By introducing the present vector obtained as described above into a host cell, a transformed cell (hereinafter sometimes referred to as the present transformed cell) can be obtained. The transformed cells are also within the scope of the present invention. The host cell may be any prokaryotic cell such as E. coli or eukaryotic cell such as yeast, animal cell or insect cell, and can be selected according to the expression vector used.
As a method for introducing a vector incorporating the PGC1αb gene (hereinafter sometimes referred to as the present vector) into a host cell, a normal introduction method corresponding to the host cell can be applied.
For example, when Escherichia coli is used as a host cell, conventional methods such as the calcium chloride method and electroporation method described in “Molecular Cloning” (J. Sambrook et al., Cold Spring Harbor, 1989) and the like. This vector can be introduced into a host cell. When mammalian cells or insect cells are used as host cells, the vector is introduced into the cells by a general gene transfer method such as calcium phosphate method, DEAE dextran method, electroporation method or lipofection method. be able to. When yeast is used as a host cell, it can be introduced using, for example, a yeast transformation kit (Clontech) based on the lithium method.
In order to select transformed cells into which this vector has been introduced, for example, the following marker gene is introduced into a host cell at the same time as this vector, and this vector is introduced by a method according to the nature of the introduced marker gene. The cultured host cells may be cultured. For example, when the marker gene is a gene that imparts drug resistance to a selected drug that exhibits lethal activity on host cells (drug resistance-conferring gene), the vector is introduced using a medium containing the drug. The host cells can be cultured. Examples of combinations of a drug resistance-conferring gene and a selective drug include, for example, a combination of a neomycin resistance-conferring gene and neomycin, a combination of a hygromycin resistance-conferring gene and hygromycin, a blasticidin S resistance-conferring gene, and blasticidin S. And the like. In addition, when the marker gene is a gene that complements the auxotrophy of the host cell, the cell into which the present vector has been introduced may be cultured using a minimal medium that does not contain the nutrient.
The PGC1αb gene can be expressed by culturing the transformed cells introduced with the present vector obtained as described above.
The transformed cells can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of Escherichia coli, culturing is performed in a medium appropriately containing an appropriate carbon source, nitrogen source, and micronutrients such as vitamins. The culture method may be any of solid culture and liquid culture, and preferred examples include liquid culture such as aeration and agitation culture.
The PGC1αb gene may be prepared as described above. For example, the PGC1αb gene may be used in the form of a recombinant vector or a recombinant virus containing a nucleic acid comprising the base sequence of the PGC1αb gene. In such a form, for example, viruses such as retrovirus vectors, adenovirus vectors, adeno-associated vectors, herpes simplex virus vectors, SV40 vectors, polyoma virus vectors, papilloma virus vectors, picorna virus vectors and vaccinia virus vectors. You can give a vector. Further, when an adenovirus vector is used, for example, an AdEasy Kit manufactured by QUANTUM is used, the PGC1αb gene is incorporated into the multi-cloning site of Transfer Vector, the resulting recombinant vector is linearized, and then E. coli together with pAdEasy vector. The recombinant virus containing the PGC1αb gene can be produced by incorporating the homologous recombinant DNA into human 293A cells, and this can be recovered and used.
A non-viral vector such as plasmid DNA having a human cytomegavirus promoter region can also be used. In systems that deliver PGC1αb genes locally using non-viral vectors, such as when directly injecting the PGC1αb gene into the fibrotic tissue site, the use of plasmid DNA is extremely beneficial. Any known introduction method can be used by introducing an expression vector into a cell taken out of the body and returning it to the body, that is, using an ex vivo method. For example, a) direct injection, b) transduction via liposome, c) cell transfection by calcium phosphate method, electroporation method, DEAE-dextran method, d) delivery via polybrene, e) protoplast fusion, f) micro Non-viral vectors can be introduced by injection, g) transduction using polylysine, and the like.
PGC1αb can be obtained by combining methods commonly used for isolation and purification of general proteins. For example, the transformed cells obtained by the above culture may be collected by centrifugation or the like, and the transformed cells may be crushed or lysed. If necessary, the protein may be solubilized and purified by singly or in combination with various chromatographic processes such as ion exchange, hydrophobicity, and gel filtration. If necessary, an operation for restoring the higher-order structure of the purified protein may be further performed.
Moreover, PGC1αb is a variant of PGC1αa as described above, and can be obtained by the same method as PGC1αa. Specifically, Biochemical Journal. The method described in Vol367 p413-22 (2002) is mentioned.
(II) Primer or probe
The present invention also relates to a primer or probe comprising a polynucleotide that specifically recognizes the base sequence of the PGC1αb gene, or a polynucleotide complementary thereto.
In the present specification, “polynucleotide” is used to include both RNA and DNA, and the DNA includes any of cDNA, genomic DNA, and synthetic DNA. The RNA includes any of total RNA, mRNA, rRNA, and synthetic RNA.
Here, “specifically recognize” means that, for example, when the Northern blot method is used, the PGC1αb gene or a polynucleotide derived therefrom can be specifically detected, and when the RT-PCR method is used, PGC1αb This means that genes or polynucleotides derived therefrom are specifically produced, but without limitation, those skilled in the art can judge that the above-mentioned detection products or products are derived from these genes. Anything is acceptable. It is preferable that the primer or probe of the present invention can recognize PGC1αb separately from PGC1αa.
PGC1αb is a kind of splicing variant of PGC1αa, and has a different base sequence near the N-terminus of the coding region (ORF). Therefore, PGC1αb can be selectively recognized by using at least one or both of the two primers as a polynucleotide that specifically recognizes the base sequence described in SEQ ID NO: 7.
Here, the “polynucleotide specifically recognizing the base sequence described in SEQ ID NO: 7” means a base sequence of at least 5 bases, preferably 10 bases or more in the base sequence described in SEQ ID NO: 7. Although it will not specifically limit if it is a polynucleotide to have, It is preferable that it is a polynucleotide which consists of a partial sequence of PGC1 (alpha) b gene. That is, the base sequence described in SEQ ID NO: 7 is a polynucleotide containing 15 or more consecutive base sequences among the base sequences represented by SEQ ID NO: 1, 3 or 5, and corresponding to the N-terminal partial sequence Among them, a polynucleotide having a base sequence of at least 5 consecutive bases, preferably 10 bases or more.
More specifically, the “base sequence described in SEQ ID NO: 7” is a partial sequence derived from human PGC1αb represented by SEQ ID NO: 50, a partial sequence derived from mouse PGC1αb represented by SEQ ID NO: 51, and represented by SEQ ID NO: 52. And a partial sequence derived from rat PGC1αb.
On the other hand, another primer used together with “polynucleotide specifically recognizing the base sequence described in SEQ ID NO: 7” should be used together with “polynucleotide specifically recognizing the base sequence described in SEQ ID NO: 7.” The PGC1αb is not particularly limited as long as it can specifically recognize PGC1αb, and may have a length of at least 15 bases continuous in PGC1αb, and the length can be appropriately selected and set. Examples thereof usually have a base length of 15 bp to 100 bp, preferably 15 bp to 50 bp, more preferably 15 bp to 35 bp. When used as a detection probe, those having a base length of usually 15 bp to the total number of sequences, preferably 15 bp to 1 kb, more preferably 100 bp to 1 kb can be exemplified.
Such a primer or probe is based on the base sequence of the PGC1αb gene such as the base sequence represented by SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, for example, primer3 (http: //www.genome.wi. design using mit.edu/cgi-bin/primer/primer3.cgi http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) or vector NTI (Infomax) can do.
Specifically, a candidate primer or probe sequence obtained by applying the PGC1αb gene to the software of primer 3 or vector NTI, or a sequence partially including at least the sequence can be used as a primer or probe.
Here, a complementary polynucleotide (complementary strand, reverse strand) is a full-length sequence of the PGC1αb gene, or a partial sequence having a base sequence of at least 15 bases in the base sequence (here, for convenience, these are “ It is also meant to be a polynucleotide that is in a base-complementary relationship based on a base pair relationship such as A: T and G: C. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions. You may have. It should be noted that the stringent conditions here are taught by a combination of Berger and Kimmel (1987, Guide to Molecular Cloning Techniques in Enzymology, Vol. 152, Academic Press, San Diego CA). It can be determined based on the melting temperature (Tm) of the nucleic acid. For example, as washing conditions after hybridization, conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. The stringent conditions are not particularly limited, but include those described above.
Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the target base strand, and at least 90%, preferably 95% homology with the strand. An example is a chain composed of a base sequence having the same.
In addition, the polynucleotide on the positive strand side includes not only those having the base sequence of the PGC1αb gene or a partial sequence thereof but also a strand comprising a base sequence that is more complementary to the base sequence of the complementary strand. be able to.
Further, the above-mentioned normal and complementary (reverse) polynucleotides may be used in a single-stranded form or in a double-stranded form.
The primer or probe of the present invention can also be used to detect the presence or absence of the expression of PGC1αb gene in mammalian cells such as humans and biological materials (eg, urine, blood, tissue fragments, etc.) or the extent (expression level). it can. That is, the primer of the present invention can accurately determine the activation of the mitochondrial gene group resulting from exercise load in the muscle of mammals such as humans. That is, the primer or probe of the present invention is useful as a tool (sugar metabolism marker) for evaluating the responsiveness of exercise-responsive mitochondrial activation.
In addition, the primer or probe of the present invention is used to detect the presence or absence and expression level of the PGC1αb gene in a method for evaluating the ability of a test substance to improve glucose metabolism such as the ability to promote mitochondrial activity or the ability to induce Glut4 expression, which will be described later. Can be used.
(III) Antibody
The present invention also relates to an antibody that specifically recognizes the protein of the present invention in (I), that is, PGC1αb.
The “antibody” as used herein is not particularly limited in its form, and is an antigen such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a Fab fragment or a fragment generated by a Fab expression library. Some of the above antibodies having binding properties are included. Further, “specifically recognize PGC1αb” preferably recognizes PGC1αb separately from PGC1αa.
The antibody of the present invention may be a polyclonal antibody having a sequence containing the amino acid sequence represented by SEQ ID NO: 8 which is a partial peptide of PGC1αb as an immunizing antigen, or a monoclonal antibody thereof. Furthermore, antigen binding to a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8, which is at least continuous among the amino acid sequences constituting PGC1αb, usually consisting of 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids. It is an antibody having sex.
Specifically, the “polypeptide comprising the amino acid sequence represented by SEQ ID NO: 8” specifically includes a polypeptide comprising a partial sequence specific to human PGC1αb represented by SEQ ID NO: 53, mouse PGC1αb represented by SEQ ID NO: 54 A polypeptide containing a specific partial sequence or a polypeptide containing a rat PGC1αb-specific partial sequence represented by SEQ ID NO: 55 can be exemplified.
Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publ. John Wiley and Sons. Section 11.12-11.13). Specifically, when the antibody of the present invention is a polyclonal antibody, an oligo having a partial amino acid sequence of the protein of the present invention using PGC1αb expressed and purified in Escherichia coli according to a conventional method or according to a conventional method Peptides can be synthesized to immunize non-human animals such as rabbits, and obtained from the sera of the immunized animals according to conventional methods. For example, it can also be obtained by the method described in (Genes Dev, 11: 2052-2065, 1997). On the other hand, in the case of a monoclonal antibody, spleen cells and bone marrow obtained by immunizing non-human animals such as mice with PGC1αb expressed and purified in Escherichia coli according to a conventional method or oligopeptides having partial amino acid sequences of these proteins are obtained. It can be obtained from hybridoma cells prepared by cell fusion with tumor cells (Current protocol in Molecular Biology edit. Ausubel et al. (1987) Publ. John Wiley and Sons. Section 11.1-11.11). .
In addition, proteins used for antibody production are DNA cloning, construction of each plasmid, transfection into a host based on the sequence information (SEQ ID NOs: 1, 3, 5) of genes described in the sequence listing described later. It can be obtained by ordinary genetic engineering methods such as culture of transformed cells and recovery of proteins from the culture. These operations are carried out by methods known to those skilled in the art or methods described in the literature [for example, Sambrook J. et al. Frisch E .; F. Maniatis T. Authors, Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (Methods described in Cold Spring Harbor Laboratory Press; DM Glover et al., DNA Cloning (IRL Press Issue, 1985), etc.], etc. Specifically, a recombinant DNA (expression vector) in which a gene encoding PGC1αb can be expressed in a desired host cell, introduced into the host cell, transformed, The PGC1αb can be obtained by culturing the transformed cells and recovering the target protein from the obtained culture, and the PGC1αb is amino acid sequence information (SEQ ID NO: 2, 4, Accordance), etc., can also be produced by general chemical synthesis method (peptide synthesis).
The antibody of the present invention may be prepared using an oligopeptide having a partial amino acid sequence of PGC1αb. The oligopeptide used for antibody induction does not need to have a functional biological activity, but desirably has the same immunogenic properties as PGC1αb. Preferably, it is a polypeptide having such properties and comprising at least 8 amino acids, preferably 15 amino acids, more preferably 20 amino acids, which is at least continuous in the amino acid sequence of PGC1αb, and contains the amino acid sequence set forth in SEQ ID NO: 8 Polypeptides can be exemplified.
Here, “an oligopeptide having immunogenic properties similar to PGC1αb” refers to an oligopeptide having an immunological activity equivalent to that of PGC1αb, and induces a specific immune response in an appropriate animal or its cells. And a protein having the ability to specifically bind to an antibody against PGC1αb. It is preferable that the antibody can be distinguished from PGC1αb and PGC1αa.
Generation of antibodies against such polypeptides can also be performed by enhancing the immunological response using various adjuvants depending on the host. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels such as aluminum hydroxide, and surfaces such as lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. There are active substances, human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
Since the antibody of the present invention has a property of specifically binding to PGC1αb, the use of the antibody makes it possible to use the antibody in the cells of mammals such as humans and biological products (for example, urine, blood, tissue fragments, etc.) The protein of the present invention can be specifically detected. That is, the antibody is useful for detecting the presence or absence of PGC1αb protein expression in tissues of mammals such as humans, specifically in muscle. For example, in the muscles of mammals such as humans, the activation of mitochondrial genes resulting from exercise load can be accurately determined. Therefore, the antibody of the present invention is useful as a tool (sugar metabolism marker) for evaluating the responsiveness of exercise-responsive mitochondrial activation.
In addition, the antibody of the present invention is used to detect the presence or absence and expression level of PGC1αb in a method for evaluating the ability of a test substance to improve glucose metabolism such as the ability to promote mitochondrial activity or the ability to induce Glut4 expression, which will be described later. be able to.
Here, when the antibody is used for evaluation of sugar metabolism or diagnosis of abnormal sugar metabolism in mammals such as humans, the marker may be an antibody that recognizes the amino acid sequence of human PGC1αb described in SEQ ID NO: 2. preferable.
(IV) PGC1αb promoter, expression vector and transformed cell
The present invention also includes a promoter of PGC1αb, which is a novel variant of PGC1α (peroxisome proliferator-activated receptor gamma coactivator-1α).
The promoter of the present invention comprises (g) DNA consisting of the base sequence represented by SEQ ID NO: 33, 34 or 35, and (h) one or more bases deleted in the base sequence represented by SEQ ID NO: 33, 34 or 35 Hybridizing under stringent conditions with a DNA comprising a substituted or added base sequence and having the ability to control transcription, (i) a DNA comprising the base sequence represented by SEQ ID NO: 33, 34 or 35, And a promoter containing DNA having the ability to control transcription. Here, the DNA consisting of the base sequence represented by SEQ ID NO: 33 represents an about 3 kb PGC1αb promoter region derived from human, and the DNA consisting of the base sequence represented by SEQ ID NO: 34 is about 1.3 kb PGC1αb derived from human. The DNA representing the promoter region and consisting of the base sequence represented by SEQ ID NO: 35 represents a mouse-derived PGC1αb promoter region of about 1.3 kb.
As used herein, the term “promoter” refers to a kind of regulatory gene, a site where RNA polymerase binds and initiates transcription of an operon, and means DNA that contains a sequence necessary to promote transcription. A promoter element under cell-type or tissue-specific control, or sufficient promoter element to cause promoter-dependent gene expression induced by an exogenous signal or factor (eg, a transcriptional activation protein) You may go out.
In the “deletion, addition or substitution” in (h) (hereinafter sometimes referred to as base modification as a whole), for example, the origin of the DNA having the base sequence represented by SEQ ID NO: 33, 34 or 35 It includes naturally occurring mutations due to species differences, individual differences, differences between tissues, etc., and artificial base sequence mutations. Here, as a technique for artificially performing “deletion, addition or substitution” of a base sequence, for example, a conventional site-specific method for DNA comprising the base sequence represented by SEQ ID NO: 33, 34 or 35 is used. There is a technique in which the mutation is introduced and then this DNA is expressed by a conventional method. Examples of the site-specific mutagenesis method include a gapped duplex method (Nucleic Acids Res., 12, 9441-9456 (1984)), a PCR method using a mutagenesis primer, and the like.
The number of bases modified as described above is at least one residue, specifically one or several, or more. The number of such modifications is not particularly limited as long as it retains the ability to control transcription.
The “stringent conditions” in the above (i) are, for example, 6 × SSC (a solution containing 1.5 M NaCl and 0.15 M trisodium citrate is set to 10 × SSC), 45% in a solution containing 50% formamide. Conditions for washing at 50 ° C. with 2 × SSC after forming a hybrid at 0 ° C. (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6) Etc. The salt concentration in the washing step can be selected from, for example, conditions of 2 × SSC at 50 ° C. (low stringency conditions) to 0.2 × SSC at 50 ° C. (high stringency conditions). The temperature in the washing step can be selected, for example, from a temperature from room temperature (low stringency conditions) to 65 ° C. (high stringency conditions). It is also possible to change both the salt concentration and the temperature.
Examples of the method for preparing the promoter of the present invention include chemical synthesis methods, PCR, and hybridization methods.
When preparing using a chemical synthesis method, an automatic DNA synthesizer such as a DNA synthesizer model 380A (manufactured by ABI) or the like can be used.
Next, a method for preparing the promoter of the present invention using PCR will be described. A genomic library used as a template is prepared from a tissue of a mammal such as a human or mouse according to a method described in, for example, “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, etc. can do. Further, commercially available genomic DNA such as human genomic DNA (manufactured by Clontech) or commercially available genomic library such as human genome walker kit (manufactured by Clontech) can be used. Next, when preparing the primer of the present invention containing a primer corresponding to the promoter to be amplified, for example, the promoter of the present invention containing DNA consisting of the base sequence shown in SEQ ID NO: 33, for example, a primer consisting of the base sequence shown in SEQ ID NO: 36 and a sequence PCR is performed using a primer having a base sequence represented by No. 37. The primer can be appropriately designed based on the base sequence represented by SEQ ID NO: 33, and a restriction enzyme recognition sequence or the like may be added to the 5 ′ end side. The DNA amplified as described above can be obtained from "Molecular Cloning: A Laboratory Manual 2nd edition" (1989), Cold Spring Harbor Laboratory Press, "Current Protocols In Molecular Bios. 1987". It can be cloned into a vector according to the usual method described in ISBN0-471-50338-X or the like. Specifically, for example, cloning can be performed using a plasmid vector included in a TA cloning kit manufactured by Invitrogen or a plasmid vector such as pBluescript II manufactured by Stratagene. The base sequence of the cloned DNA is F.I. Sanger, S.M. Nicklen, A.M. R. Coulson, Proceedings of National Academy of Science U. S. A. (1977), 74, 5463-5467, and the like.
Next, a method for preparing the promoter of the present invention using a hybridization method will be described.
First, the DNA used for the probe is labeled. The DNA used for the probe is a DNA having at least a part of the base sequence of the promoter to be prepared, for example, a DNA comprising the base sequence represented by SEQ ID NO: 33 or 35 or a part of the base sequence thereof. DNA having a chain length of 20 to 160 bases, DNA having a base sequence in which one or more bases are deleted, substituted or added in the base sequence of the DNA, under stringent conditions with the DNA DNA which hybridizes to can be mentioned.
Specifically, DNA comprising a base sequence represented by base numbers 1 to 600 of SEQ ID NO: 33, DNA comprising a base sequence represented by base numbers 400 to 1100 of SEQ ID NO: 33, bases of SEQ ID NO: 33 DNA consisting of a base sequence indicated by numbers 900 to 1600, DNA consisting of a base sequence indicated by base numbers 1400 to 2100 of SEQ ID NO: 33, bases indicated by base numbers 1900 to 2600 of SEQ ID NO: 33 Examples thereof include DNA comprising a sequence, DNA comprising a base sequence represented by base numbers 2400 to 3000 of SEQ ID NO: 33, and the like.
Further, DNA consisting of the base sequence indicated by base numbers 1 to 600 of SEQ ID NO: 35, DNA consisting of the base sequence indicated by base numbers 400 to 1100 of SEQ ID NO: 35, base number 900 of SEQ ID NO: 35 DNA consisting of the base sequence shown by the 1600th to 1600, DNA consisting of the base sequence shown by the 1st to 2100th bases of the SEQ ID No. 35, and the base sequence shown by the 1900th to 2600th bases of the SEQ ID No. 35 Examples thereof include DNA, DNA having a base sequence represented by base numbers 2400 to 3000 of SEQ ID NO: 35, and the like.
The DNA used for the probe can be obtained by the usual DNA preparation method described above as the “method for preparing the promoter of the present invention” such as a chemical synthesis method, PCR, or hybridization method. The DNA used for the probe may itself have the ability to control transcription of a gene encoding PGC1αb having the amino acid sequence represented by SEQ ID NO: 2, for example.
In order to label the DNA used for the probe with a radioisotope, for example, Random Labeling Kit manufactured by Boehringer or Takara Shuzo can be used, and dCTP in a normal PCR reaction composition is [α- ³² Instead of P] dCTP, labeling can also be performed by performing a PCR reaction using the DNA used for the probe as a template. In addition, when the DNA used for the probe is labeled with a fluorescent dye, for example, ECL Direct Nucleic Acid Labeling and Detection System (manufactured by Amersham Pharmacia Biotech) or the like can be used. As a DNA library for hybridizing the probe, for example, a genomic DNA library derived from an animal such as a rodent such as a rat can be used. As the DNA library, a commercially available genomic DNA library can be used, and “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press and “Current Protocols In Molecular 7 Biomolecules”. ), John Wiley & Sons, Inc. In accordance with a normal library preparation method described in ISBN0-471-50338-X or the like, for example, using λ vectors such as λ FIX II, λ EMBL3, λ EMBL4, λ DASH II manufactured by Stratagene, Gigapack packaging Extracts (Stratagene) Etc.) can be used for in vitro packaging and a genomic DNA library can be prepared and used.
Examples of the hybridization method include colony hybridization and plaque hybridization, and the method may be selected according to the type of vector used for preparing the library. For example, when the library used is a library constructed with a plasmid vector, colony hybridization can be performed. Specifically, first, DNA of the library is introduced into a host microorganism to obtain transformed cells, the obtained transformed cells are diluted and spread on an agar medium, and cultured at 37 ° C. until colonies appear. Moreover, when the library used is a library constructed with a phage vector, plaque hybridization can be performed. Specifically, first, host microorganisms and library phages are mixed under conditions allowing infection, and further mixed with a soft agar medium, which is then spread on the agar medium. Thereafter, the cells are cultured at 37 ° C. until plaques appear. More specifically, for example, a method described in Molecular Cloning 2nd edition (written by J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press 1989) 2.60 to 2.65, etc. In accordance with NZY agar medium, agar medium 1mm ² About 9.0 x 10 at a density of 0.1 to 1.0 pfu per ⁵ Spread the pfu phage library and incubate at 37 ° C. for 6-10 hours.
Next, in any of the above-described hybridization methods, a membrane filter is placed on the surface of the agar medium on which the above-described culture is performed, and the transformed cells or phages carrying the plasmid are transferred to the membrane filter. The membrane filter is treated with an alkali, then neutralized, and then subjected to a treatment for fixing DNA to the filter. More specifically, for example, in the case of plaque hybridization, the agar medium according to the usual method described in Cloning and Sequence: Plant Biotechnology Experiment Manual (Watanabe, Sugiura Editing, Rural Bunkasha 1989) and the like. Nitrocellulose filter or nylon filter, for example, Hybond-N ⁺ (Amersham Pharmacia Biotech) is placed and allowed to stand for about 1 minute to adsorb phage particles to the membrane filter. Next, the filter is immersed in an alkaline solution (1.5 M sodium chloride, 0.5 N sodium hydroxide) for about 3 minutes to dissolve the phage particles and elute the phage DNA on the filter. .5M sodium chloride, 0.5M Tris-HCl, pH 7.5) for about 5 minutes. The filter is washed with a washing solution (300 mM sodium chloride, 30 mM sodium citrate, 200 mM Tris-HCl) for about 5 minutes, and then, for example, baked at about 80 ° C. for about 90 minutes to fix the phage DNA to the filter. Hybridization is performed using the thus prepared filter and the probe. Reagents and temperature conditions for performing hybridization are, for example, D.I. M.M. Glover, “DNA cloning, a practical approach” IRL PRES (1985) ISBN 0-947946-18-7, Cloning and Sequence: Plant Biotechnology Experiment Manual (Watanabe, Sugiura, Rural Bunkasha 1989), or Molecular Cloning 2nd edition (by J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989) and the like. For example, denatured non-specific DNA containing 450-900 mM sodium chloride, 45-90 mM sodium citrate, sodium dodecyl sulfate (SDS) at a concentration of 0.1-1.0% (W / V) In a concentration of 0 to 200 μg / mL, and in some cases albumin, ficoll, polyvinylpyrrolidone and the like may be included in a concentration of 0 to 0.2% (W / V) respectively, preferably A prehybridization solution containing 900 mM sodium chloride, 90 mM sodium citrate, 1% (W / V) SDS and 100 μg / mL denatured Calf-thymus DNA was prepared as described above. ² The filter is prepared at a rate of 50 to 200 μL per unit, and the filter is immersed in the solution and incubated at 42 to 68 ° C. for 1 to 4 hours, preferably at 45 ° C. for 2 hours. Next, for example, 450 to 900 mM sodium chloride, 45 to 90 mM sodium citrate, SDS at a concentration of 0.1 to 1.0% (W / V), A hybridization solution containing 200 μg / mL and optionally containing albumin, Ficoll, polyvinylpyrrolidone, etc. at a concentration of 0 to 0.2% (W / V), preferably 900 mM sodium chloride A hybridization solution containing 90 mM sodium citrate, 1% (W / V) SDS and 100 μg / mL denatured Calf-thymus DNA, and the probe prepared by the above-described method (filter 1 cm ² 1.0 × 10 per ⁴ ~ 2.0 × 10 ⁶ 1 cm of filter solution mixed with cpm equivalent) ² Prepare a 50-200 μL per solution, soak the filter in the solution, and incubate at 42-68 ° C. for 4-20 hours, preferably at 45 ° C. for 16 hours to carry out the hybridization reaction. After the hybridization reaction, the filter is taken out and contains 42 to 68 ° C. containing 15 to 300 mM sodium chloride, 1.5 to 30 mM sodium citrate, 0.1 to 1.0% (W / V) SDS, and the like. 1 to 4 times of filter washing for 10 to 60 minutes with a washing solution at 55 ° C. containing 300 mM sodium chloride, 30 mM sodium citrate, and 1% (W / V) SDS. Preferably, 15 minutes of washing is performed twice. Further, the filter is lightly rinsed with 2 × SSC solution (containing 300 mM sodium chloride and 30 mM sodium citrate) and then dried. This filter is subjected to, for example, autoradiography to detect the position of the probe on the filter, thereby detecting the position on the filter of the DNA hybridized with the probe used. A clone having the DNA can be isolated by identifying a clone corresponding to the position of the detected DNA on the filter on the original agar medium and catching it. Specifically, for example, the filter is exposed to an imaging plate (Fuji Film) for 4 hours, and then the plate is analyzed using BAS2000 (Fuji Film) to detect a signal. The portion corresponding to the position where the signal was detected in the agar medium used for the production of the filter was cut out to about 5 mm square, and this was cut into about 500 μL of SM buffer (50 mM Tris-HCl pH 7.5, 0.1 M sodium chloride, 7 mM magnesium sulfate and 0.01% (W / V) gelatin) for 2-16 hours, preferably 3 hours to elute phage particles. The obtained phage particle eluate was prepared according to the method described in Molecular Cloning 2nd edition (J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989) 2.60 to 2.65. Spread on agar medium and incubate at 37 ° C. for 6-10 hours. Using this agar medium, phage DNA is immobilized on a filter in the same manner as described above, and hybridization is performed using this filter and the probe described above. The phage particles are eluted from the portion corresponding to the position where the signal is detected in the agar medium used for the preparation of the filter, spread on the agar medium, and the filter is prepared in the same manner as described above. Do. By repeating such identification and purification of the phage clone, a phage clone containing DNA having a base sequence that hybridizes with the probe used is obtained. The DNA possessed by the clone obtained by screening by hybridization as described above is subcloned into a plasmid vector that is easy to prepare and analyze DNA, such as commercially available pUC18, pUC19, pBLUESCRIPT KS +, pBLUESCRIPT KS-, etc. Preparing plasmid DNA; Sanger, S.M. Nicklen, A.M. R. Coulson, Proceedings of National Academy of Science U. S. A. (1977), 74, 5463-5467, etc., and the base sequence can be determined using the dideoxy terminating method. Preparation of a sample used for base sequence analysis is described in, for example, Molecular Cloning 2nd edition (J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989) 13.15. It can be performed according to the primer extension method. In addition, phage clones were prepared according to the method described in Molecular Cloning 2nd edition (J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989) 2.60 to 2.65, etc. Amplified in a liquid medium to prepare a phage solution, from which phage clone DNA is extracted using, for example, Lambda-TRAPPLUS DNA Isolation Kit (manufactured by Clontech), and the DNA is used as a template, for example, by the primer extension method described above. It is also possible to prepare a sample for base sequence analysis and analyze the base sequence. The ability to control the transcription of the DNA thus obtained can also be confirmed as described below. In the present application, “ability to control transcription” means, for example, an activity for initiating transcription of a gene located downstream of a promoter (hereinafter also referred to as promoter activity).
The promoter of the present invention is prepared by introducing a mutation into a promoter or the like containing a DNA consisting of a base sequence represented by base numbers 1 to 3000 of SEQ ID NO: 33 or base numbers 1 to 3022 of SEQ ID NO: 35. Also good. Specifically, for example, A.I. Greener, M.M. Can be obtained by randomly introducing mutations using the method described in Callahan, Strategies (1994) 7, 32-34, and the like. Kramer, et al. Nucleic Acids Research (1984) 12, 9441; Kramer, H .; J. et al. Gapped duplex method described in Frits, Methods in Enzymology (1987) 154, 350, or the like. A. Kunkel, Proc. of Natl. Acad. Sci. U. S. A. (1985) 82,488 or T.W. A. Kunkel, et al. , Methods in Enzymology (1987) 154, 367 and the like, can be obtained by introducing a site-specific mutation using the Kunkel method. Alternatively, it is obtained by preparing a chimeric DNA in which one or several partial base sequences of a promoter containing DNA consisting of the base sequence represented by SEQ ID NO: 33 are replaced with a part of the DNA of another promoter. For example, S.M. Henikoff, et al. Gene (1984) 28, 351, C.I. Yanish-Perron, et al. The method described in Gene (1985) 33, 103 etc. can be used.
In the present specification, the “gene whose transcription is controlled by the promoter downstream (3 ′ side) of the promoter” is not particularly limited as long as it is a gene whose transcription is initiated by the promoter. Specifically, , PGC1αb gene, or a reporter gene used in a reporter gene assay described later.
Here, the PGC1αb gene includes not only the human PGC1αb gene encoding the human PGC1αb represented by SEQ ID NO: 2 and comprising the base sequence represented by SEQ ID NO: 1, but also homologues and mutants thereof. The For example, as homologues, proteins of other species such as mice and rats corresponding to human proteins can be exemplified, and these are homogenes (http://www.ncbi.nlm.nih.gov/homologene/). Can be a priori identified from the base sequence of the gene identified by. Specifically, the mouse PGC1αb gene comprising the nucleotide sequence represented by SEQ ID NO: 3 encoding the mouse PGC1αb represented by SEQ ID NO: 4 or the rat PGC1αb represented by SEQ ID NO: 6 is represented by SEQ ID NO: 5. A rat PGC1αb gene comprising a base sequence can be exemplified.
The promoter of the present invention prepared by the various methods described above can be prepared by a conventional method, for example, “Tamura Takaaki (published by Yodosha), New Transcriptional Control Mechanism (2000)” pages 33-40, “Nomura Shintaro, According to the method described in Toshiki Watanabe (published by Toshiki Watanabe (published by Shujunsha), Deisotope Experiment Protocol (1998)), etc., it is obtained by deleting a part of the base while maintaining the promoter activity (ie, DNA prepared by excision using an appropriate restriction enzyme can also be used as the promoter of the present invention. The ability of the obtained DNA to control the transcription of the PGC1αb gene can be confirmed by the method described later.
Expression vectors containing the promoter of the present invention in a functional form are also within the scope of the present invention. Examples of the expression vector include plasmids described later. That is, the plasmid for inserting the promoter of the present invention may be any plasmid that can function in a desired cell, and a marker gene (for example, a kanamycin resistance gene, a high-gloss gene) for selecting a cell into which the plasmid has been introduced. A mycin resistance gene or a neomycin resistance gene). In addition, in the plasmid, if there is a gene insertion site at a position where the promoter of the present invention and the desired gene are operably linked on the plasmid, for example, downstream of the site where the promoter is inserted, the desired gene Can be preferably used for the construction of a plasmid for the expression in a cell. Here, the gene insertion site is, for example, a base sequence that can be specifically recognized and cleaved by a restriction enzyme that is usually used in genetic engineering techniques, and is a kind of restriction enzyme recognition sequence that exists only on a plasmid having the promoter of the present invention. Is preferred. Specific examples of the plasmid include pUC plasmids [pUC118, pUC119 (manufactured by Takara Shuzo), etc.], pSC101 plasmids, Ti-plasmids [pBI101, pBI121 (manufactured by Clontech), etc.], pBR322 plasmid (manufactured by Boehringer Mannheim), Examples include pcDNA3 plasmid (manufactured by Invitrogen).
The promoter of the present invention can be inserted into the plasmid by a conventional method, for example, “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, “Current Protocols in Biomolecular 7”. Sons, Inc. This can be carried out according to the method described in ISBN0-471-50338-X or the like.
For example, a plasmid containing a reporter gene such as a luciferase gene under the control of the promoter of the present invention among the plasmids can be preferably used when measuring the promoter activity of the promoter of the present invention in the plasmid. Specifically, it can be prepared using a commercially available plasmid containing a luciferase gene, for example, a plasmid such as pGL3-basic (manufactured by Promega) or PicaGene Basic Vector (manufactured by Toyo Ink). Specifically, a plasmid containing the luciferase gene under the control of the promoter of the present invention can be prepared by inserting the promoter of the present invention into a functional form in the gene insertion site of pGL3-basic by a usual method.
A method for preparing the transformed cell of the present invention into which the expression vector of the present invention containing the promoter of the present invention is introduced will be described below.
Examples of the host cell into which the promoter of the present invention or the expression vector of the present invention is introduced include cells such as Escherichia coli, yeast, plant cells and animal cells, and the promoter or the expression vector of the present invention can be amplified in the cell. Any cell that can maintain Preferred examples include animal cells, and particularly preferred are skeletal muscle-derived cells. As a method for introducing the promoter of the present invention or the expression vector of the present invention into a host cell, a method suitable for the cell can be applied. For example, for animal cells, the calcium phosphate method, the electrotransfer method, the DEAE dextran method, the micelle A formation method or the like can be applied. Specifically, for example, as a calcium phosphate method, Grimm, S. et al. et al. , Proc. Natl. Acad. Sci. USA, 93, 10923-10927, and the like. Examples of the electric introduction method and the DEAE dextran method include Ting, A. et al. T. T. et al. , EMBO J. et al. , 15, 6189-6196, and the like. As a micelle formation method, Hawkins, C .; J. et al. et al. , Proc. Natl. Acad. Sci. USA, 93, 13786-13790, and the like. As the micelle formation method, for example, commercially available reagents such as Lipofectamine (manufactured by GibcoBRL) and Fugene (manufactured by Boehringer Mannheim) can be used.
(V) Evaluation method of expression control ability
(V-1) Method using primer or probe
Moreover, this invention includes the evaluation method of the expression control capability of PGC1 (alpha) b gene including the process of the following process (1), (2), and (3).
(1) contacting a test substance with a cell capable of expressing the PGC1αb gene;
(2) measuring the expression level of the PGC1αb gene in a cell contacted with a test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance;
(3) A step of evaluating the expression control ability of the PGC1αb gene of the test substance using as an index whether or not the expression level of the PGC1αb gene is varied based on the comparison result of (2).
The PGC1αb gene used here may be either the endogenous PGC1αb gene of the cell to be used or the PGC1αb gene introduced into the cell as a foreign gene, but the endogenous PGC1αb gene of the cell to be used is preferred. When introduced as a foreign gene, the PGC1αb gene is preferably a PGC1αb gene derived from the cell species used.
The cells used for such screening are not particularly limited as long as they express the PGC1αb gene. Examples thereof include muscle tissue-derived cells and transformed cells into which the aforementioned PGC1αb gene expression vector has been introduced. . Examples of the derived animal species include rodent mammals such as rats, mice and guinea pigs, dogs, monkeys, humans and the like.
In addition to the cells listed above, tissues (eg, muscle tissue fragments) that are aggregates of cells are also included in the category of “cells” used in the screening of the present invention.
Examples of substances that can be used as test substances include, but are not limited to, nucleic acids, peptides, proteins, organic compounds, inorganic compounds, and the like. Specifically, screening includes a sample containing a test substance (test sample) as the tissue / or cell. Can be done in contact. Such test samples are not particularly limited and include, but are not limited to, cell extracts, gene library expression products, synthetic low-molecular compounds, synthetic peptides, natural compounds, and soil extracts.
The “control cell” in the above step (2) represents a cell that can express the PGC1αb gene used in the above step (1) when the test substance is not contacted. “When the test substance is not contacted” includes the case where the same amount of solvent (blank) as the test substance is added instead of the test substance or the case where a negative control substance that does not affect the expression of the PGC1αb gene is added. It is.
In addition, the conditions for bringing the test substance into contact with the cells are not particularly limited, but it is preferable to employ conditions that do not greatly change the culture conditions (temperature, pH, medium composition, etc.) so that the cells do not die.
Detection and quantification of the expression level of such a gene can be carried out by a known method such as Northern blotting or RT-PCR using RNA prepared from the cells or a complementary polynucleotide transcribed therefrom.
When utilizing the Northern blotting method, specifically, a polynucleotide having at least 15 bases continuous in the base sequence of the PGC1αb gene and / or its complementary polynucleotide is used as a primer or a probe. The presence or absence of the expression of the PGC1αb gene and its expression level can be detected and measured.
Here, a complementary polynucleotide (complementary strand, reverse strand) is a full-length sequence of the PGC1αb gene, or a partial sequence having a base sequence of at least 15 bases in the base sequence (here, for convenience, these are “ It is also meant to be a polynucleotide that is in a base-complementary relationship based on a base pair relationship such as A: T and G: C. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions. You may have. Here, the stringent conditions are as described above. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the target base strand, and at least 90%, preferably 95% homology with the strand. An example is a chain composed of a base sequence having the same.
In addition, the polynucleotide on the positive strand side includes not only those having the base sequence of the PGC1αb gene or a partial sequence thereof but also a strand comprising a base sequence that is more complementary to the base sequence of the complementary strand. be able to.
Furthermore, the above-mentioned normal-strand polynucleotide and complementary-strand (reverse-strand) polynucleotide may each be used as a primer or probe in a single-stranded form, or in a double-stranded form.
Specifically, the primer or probe may be a polynucleotide comprising the base sequence (full-length sequence) described in SEQ ID NO: 1 relating to the base sequence of the human PGC1αb gene, or a polynucleotide comprising the complementary sequence thereof. May be. A polynucleotide comprising the base sequence described in SEQ ID NO: 3 relating to the base sequence of the mouse PGC1αb gene (full-length sequence) or the base sequence described in SEQ ID NO: 5 relating to the base sequence of the rat PGC1αb gene (full-length sequence). Alternatively, it may be a polynucleotide consisting of its complementary sequence. Furthermore, if the PGC1αb gene represented by each SEQ ID NO. Or a polynucleotide derived from the gene is selectively (specifically) recognized, a polynucleotide comprising the full-length sequence or a partial sequence of its complementary sequence can be used. There may be. In this case, the primer or probe described in (II) above may be used.
Here, “selectively (specifically)” means that, for example, when Northern blotting is used, the PGC1αb gene or a polynucleotide derived therefrom can be specifically detected, and RT-PCR method. Means that the PGC1αb gene or a polynucleotide derived therefrom is specifically produced, but the present invention is not limited thereto, and those skilled in the art can derive the above detection product or product from these genes. As long as it can be determined that it is to be performed.
The probe or primer is used as a primer for specifically recognizing and amplifying RNA generated from expression of the PGC1αb gene or a polynucleotide derived therefrom, or for specifically detecting the RNA or polynucleotide derived therefrom. Can be used as a probe.
Specifically, the primer or probe is a radioisotope ( ³² P, ³³ P or the like: labeled with RI) or a fluorescent substance, and hybridized with cell-derived RNA transferred to a nylon membrane or the like according to a conventional method, and then the primer or probe (DNA or RNA) and RNA formed A method of detecting and measuring a signal derived from a labeled product (RI or fluorescent substance) of the primer or probe with a radiation detector (BAS-1800II, manufactured by Fuji Film) or a fluorescence detector can do. In addition, using the AlkPhos Direct Labeling and Detection System (manufactured by Amersham Pharmacia Biotech), the probe is labeled according to the protocol, hybridized with RNA derived from the biological tissue of the subject, and then a signal derived from the labeled product of the probe. A method of detecting and measuring with a multi-bioimager STORM860 (manufactured by Amersham Pharmacia Biotech) can also be used.
When the RT-PCR method is used, the presence or absence of expression of the PGC1αb gene in RNA by using a polynucleotide having at least 15 bases continuous in the base sequence of the PGC1αb gene and / or its complementary polynucleotide as a primer And the expression level can be detected and measured. Specifically, a pair of primers prepared based on the sequence of the PGC1αb gene so that a cDNA can be prepared from RNA derived from the biological tissue of a subject according to a conventional method and the target PGC1αb gene region can be amplified using this as a template ( An example of a method for detecting the amplified double-stranded DNA obtained by hybridizing the above-described cDNA (-strand) to the positive strand and the reverse strand bound to the + strand and performing PCR according to a conventional method. can do. In addition, the detection of the amplified double-stranded DNA was performed by a method for detecting the labeled double-stranded DNA produced by performing the PCR using a primer previously labeled with RI or a fluorescent substance. For example, a method may be used in which double-stranded DNA is transferred to a nylon membrane or the like according to a conventional method, and the labeled primer is used as a probe to hybridize with this to detect it. The produced labeled double-stranded DNA product can be measured with an Agilent 2100 Bioanalyzer (manufactured by Yokogawa Analytical Systems). Also, an RT-PCR reaction solution is prepared according to the protocol using SYBR Green RT-PCR Reagents (manufactured by Applied Biosystems), and reacted with ABI PRIME 7900 Sequence Detection System (manufactured by Applied Biosystems). You can also
The gene expression level of the PGC1αb gene can also be detected or quantified using a DNA chip. In this case, the polynucleotide specifically recognizing the PGC1αb gene can be used as a chip probe (for example, in the case of Gene Chip Human Genome U95 A, B, C, D, E of Affymetrix, 25 bp) Used as a polynucleotide probe of length). Such a DNA chip is hybridized with labeled DNA or labeled RNA prepared from the cells used in this evaluation method, and the complex of the probe and labeled DNA or labeled RNA on the chip formed by hybridization is obtained. By detecting the label of labeled DNA or labeled RNA as an indicator, the presence or absence of expression of PGC1αb gene in cells or the expression level (expression level) can be evaluated.
If the expression level of the PGC1αb gene in the cell to which the test substance is added is 1.5 times or more, preferably 2 times or more, more preferably 3 times or more compared to the expression level in the control cell to which no test substance is added, The test substance can be selected as a PGC1αb expression inducer. On the other hand, the expression of the PGC1αb gene in the cells to which the test substance (candidate substance) is added is 3/4 times or less, preferably 1/2 times or less than that in the control cells to which the test substance (candidate substance) is not added. More preferably, if it is 1/3 or less, the test substance can be selected as a substance that suppresses the expression of PGC1αb.
Alternatively, the expression level of the PGC1αb gene in the presence of the test substance (hereinafter referred to as measurement value 1) is compared with the expression level of the gene in the absence of the test substance (hereinafter referred to as measurement value 2). Thus, the expression-inducing activity or the expression-inhibiting activity of the PGC1αb gene of the test substance can be evaluated. In this case, using the measured value, the expression induction rate may be obtained according to the following formula.
Expression induction rate (%) = {(measured value 1−measured value 2) / measured value 2} × 100
A substance whose PGC1αb gene expression induction rate of a test substance shows a statistically significant value, specifically, for example, a substance showing 30% or more, more preferably a substance showing 50% or more is induced to induce PGC1αb expression Select as a substance with ability. In addition, when a measured value shows a negative value, the said test substance is an expression inhibitory substance.
As shown in the Examples, the expression of the PGC1αb gene is specifically and rapidly increased in muscle cells by exercise stimulation. This finding indicates that the expression of the PGC1αb gene is responsive to glucose metabolism.
Therefore, the PGC1αb gene expression promoter obtained by the evaluation method of the present invention is used as a sugar metabolism improving agent described later.
(V-2) Method using antibody
The present invention also relates to a method for evaluating the expression control ability of PGC1αb, comprising the following steps (1), (2) and (3):
(1) contacting a test substance with a cell capable of expressing PGC1αb;
(2) measuring the expression level of PGC1αb in a cell contacted with a test substance, and comparing the expression level with the expression level of PGC1αb in a control cell not contacted with the test substance;
(3) A step of evaluating the PGC1αb expression control ability of the test substance using as an index whether or not the expression level of PGC1αb is varied based on the comparison result of (2).
The cells used in the screening method of the present invention may include all cultured cells having PGC1αb regardless of whether they are endogenous or exogenous. Specifically, the cells described in the above section (V-1) can be mentioned.
The expression of PGC1αb can be easily confirmed by detecting a protein that is a PGC1αb gene expression product.
The expression level of PGC1αb according to the evaluation method of the present invention is the antibody described in (III) above (for example, an antibody that recognizes human PGC1αb represented by SEQ ID NO: 2 or a homolog thereof represented by SEQ ID NO: 4 or 6). Can be quantified according to a known method such as Western blot method using Western blotting uses an antibody of the present invention as a primary antibody and then binds to a primary antibody labeled with a radioisotope such as 125I, a fluorescent substance, an enzyme such as horseradish peroxidase (HRP) as a secondary antibody, etc. And a signal derived from these labeling substances can be measured with a radiation measuring instrument (BAI-1800II: manufactured by Fuji Film Co., Ltd.), a fluorescence detector or the like. In addition, after using the antibody of the present invention as a primary antibody, detection is performed according to the protocol using an ECL Plus Western Blotting Detection System (Amersham Pharmacia Biotech), and measurement is performed with a multibiomea Storm860 (Amersham Pharmacia Biotech) You can also
Examples of the test substance or the control cell include the same ones as in the above (V-1).
If the expression level of PGC1αb in the cells to which the test substance is added is 1.5 times or more, preferably 2 times or more, more preferably 3 times or more compared to the expression level in the control cells to which no test substance is added, The test substance can be selected as a PGC1αb expression inducer. On the other hand, the expression of PGC1αb in the cells to which the test substance (candidate substance) is added is 3/4 or less, preferably 1/2 or less, compared to the expression level in the control cells to which the test substance (candidate substance) is not added. More preferably, if it is 1/3 or less, the test substance can be selected as a substance that suppresses the expression of PGC1αb.
Alternatively, the expression level of PGC1αb in the presence of the test substance (hereinafter referred to as measurement value 1) is compared with the expression level of PGC1αb in the absence of the test substance (hereinafter referred to as measurement value 2). The PGC1αb expression-inducing activity or expression-inhibiting activity of the test substance can be evaluated. In this case, using the measured value, the expression induction rate may be obtained according to the following formula.
Expression induction rate (%) = {(measured value 1−measured value 2) / measured value 2} × 100
A substance whose PGC1αb expression induction rate of a test substance shows a statistically significant value, specifically, for example, a substance showing 30% or more, more preferably a substance showing 50% or more is used as a PGC1αb expression inducing ability. Selected as a substance with In addition, when a measured value shows a negative value, the said test substance is an expression inhibitory substance.
(V-3) Method using promoter activity as an index
The present invention also includes a method for evaluating the expression control ability of the PGC1αb gene, comprising the following steps (1), (2) and (3):
(1) contacting a test substance with a cell capable of expressing a reporter gene to which the promoter of the present invention is bound;
(2) measuring the expression level of the reporter gene in the cell contacted with the test substance, and comparing the expression level with the expression level of the reporter gene in the control cell not contacted with the test substance;
(3) A step of evaluating the expression control ability of the PGC1αb gene of the test substance using as an index whether or not the expression level of the reporter gene is varied based on the comparison result of (2).
The cell used is preferably a transformed cell of the present invention using a mammalian cell as a host, and particularly preferably a transformed cell of the present invention using a mammalian skeletal muscle-derived cell as a host. Examples of the derived animal species include rodent mammals such as rats, mice and guinea pigs, dogs, monkeys, humans and the like. In addition to the cells listed above, tissues (eg, muscle tissue fragments) that are aggregates of cells are also included in the category of “cells” used in the screening of the present invention.
The transformed cell can be prepared as follows.
First, the promoter of the present invention is used as a reporter gene (a gene whose expression can be analyzed) such as glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), β-galactosidase and green fluorescent protein (GFP). By linking in a functional manner, a reporter gene is prepared by ligating the promoter of the present invention in a functional manner. As used herein, “operably linked” means that a gene and one or more regulatory sequences are capable of gene expression when a suitable foreign signal or factor is bound to the regulatory sequence. Means linking in a simple manner. Next, a reporter gene in which the promoter of the present invention is operably linked is inserted into a vector that can be used in a cell into which the reporter gene is introduced, using a conventional genetic engineering technique, thereby preparing a plasmid. To do. The plasmid is then introduced into the cell. Examples of the introduction method into the cell include a calcium phosphate method, an electric introduction method, a DEAE dextran method, and a micelle formation method. As the calcium phosphate method, Grimm, S. et al. et al. , Proc. Natl. Acad. Sci. USA, 93, 10923-10927, etc., as Ting, A. et al. T. T. et al. , EMBO J. et al. , 15, 6189-6196, etc., and micelle formation methods include Hawkins, C .; J. et al. et al. , Proc. Natl. Acad. Sci. USA, 93, 13786-13790, and the like. When using the micelle formation method, commercially available reagents such as Lipofectamine (manufactured by Gibco) and Fugene (manufactured by Boehringer) may be used.
Selection of transformed cells by culturing cells subjected to the plasmid introduction treatment, for example, using a selection marker gene previously contained in the vector and culturing in a medium under selection conditions according to the selection marker gene Can do.
The transformed cell may be prepared from a tissue of a transformed non-human animal described later by a usual method.
Examples of substances that can be used as test substances include, but are not limited to, nucleic acids, peptides, proteins, organic compounds, inorganic compounds, and the like. Specifically, screening includes a sample containing a test substance (test sample) as the tissue / or cell. Can be done in contact. Such test samples are not particularly limited and include, but are not limited to, cell extracts, gene library expression products, synthetic low-molecular compounds, synthetic peptides, natural compounds, and soil extracts.
In the above step (1), the concentration of the test substance to be brought into contact with the cell containing the reporter gene operably linked to the promoter of the present invention may be usually about 0.1 μM to about 100 μM, 1 μM to about 50 μM is preferred, and about 1 μM to about 10 μM is more preferred. The time for contacting the cells with the test substance is usually about 1 hour to about 5 days, preferably about several hours to about 4 days. Preferably, it is about 18 hours or more and about 60 hours, more preferably about 24 hours to 40 hours.
The contact between the cell and the test substance may be performed while culturing the cell under conditions that allow the cell to grow. For example, in the case of the transformed cell of the present invention using a mammalian cell as a host, commercially available D-MEM, OPTI-MEM, RPMI1640 medium (manufactured by Gibco-BRL) and the like supplemented with serum derived from mammals such as fetal bovine serum as appropriate. Can be cultured in the medium. The culture is usually carried out in the presence of about 30 ° C. to about 40 ° C., about 2% (V / V) to 10% (V / V) carbon dioxide, about 35 ° C. to about 37 ° C., about 4% ( More preferably, it is carried out in the presence of (V / V) to about 6% (V / V) carbon dioxide.
For example, when a 24-well plate is used, the number of cells in contact with the test substance is usually about 1 × 10 6. ⁵ Pieces / well to about 1 × 10 ⁶ Individual / well, about 5 × 10 ⁵ Pieces / well to about 8 × 10 ⁵ Pieces / well are preferred.
The “control cell” in the step (2) represents a cell that is not contacted with a test substance in a cell capable of expressing the reporter gene used in the step (1). “When the test substance is not contacted” includes the case where the same amount of solvent (blank) as the test substance is added instead of the test substance or the case where a negative control substance that does not affect the expression of the reporter gene is added. It is.
In addition, the conditions for bringing the test substance into contact with the cells are not particularly limited, but it is preferable to employ conditions that do not greatly change the culture conditions (temperature, pH, medium composition, etc.) so that the cells do not die.
In the above search method, the method of “monitoring the expression level of the reporter gene” is any method that can measure the expression level of the reporter gene in the transformed cells continuously or discontinuously over time. Any method may be used. For example, when an enzyme gene is used as a reporter gene, the enzyme activity measurement is used, and an expression product of the reporter gene, that is, a mRNA corresponding to the gene or a protein corresponding to the gene (specifically, May be used such as Northern blotting and Western blotting.
Examples of reporter genes that can be used in the method of measuring enzyme activity when an enzyme gene is used as a reporter gene include luciferase gene, GFP (Green Fluorescent Protein), chloramphenicol acetyltransferase, β-galactosidase, etc. Preferred examples include those in which the activity of the expressed and produced protein can be easily measured. When a luciferase gene is used as a reporter gene, the following may be performed. (1) For example, after culturing the transformed cell of the present invention into which the plasmid of the present invention containing the luciferase gene under the control of the promoter of the present invention is introduced for several days, an extract of the cell is obtained. (2) Promoter activity can be detected by reacting the extract with luciferin and ATP to cause chemiluminescence and measuring the luminescence intensity. At this time, a commercially available luciferase reaction detection kit such as Picker Gene Dual Kit (manufactured by Toyo Ink) can be used.
As described above, contact between the cell and the test substance and measurement of the promoter activity of the promoter of the present invention in the cell are performed, and when an increase or decrease in the promoter activity is detected, the test substance is added to the promoter. It can be selected as a substance that acts and controls the promoter activity.
That is, if the expression level of the reporter gene in the presence of the test substance is larger than the expression level of the reporter gene that can be present in the absence of the test substance, the test substance induces the expression of the PGC1αb gene that enhances the function of the promoter of the present invention. It can be evaluated as an agent. On the other hand, if the expression level of the reporter gene in the presence of the test substance is smaller than the expression level of the reporter gene in the absence of the test substance, the test substance is an expression inhibitor of the PGC1αb gene that suppresses the function of the promoter of the present invention. It can be evaluated as being.
Specifically, the expression of the reporter gene in the cells to which the test substance is added is 1.5 times or more, preferably 2 times or more, more preferably 3 times or more compared to the expression level in the control cells to which no test substance is added. If so, the test substance can be selected as a PGC1αb gene expression inducer. On the other hand, the expression of the reporter gene in the cells to which the test substance (candidate substance) is added is 3/4 times or less, preferably 1/2 times or less compared to the expression level in the control cells to which no test substance (candidate substance) is added. More preferably, if it is 1/3 or less, the test substance can be selected as a substance that suppresses the expression of PGC1αb.
Alternatively, the expression level of the reporter gene in which the promoter of the present invention is operably linked in the presence of the test substance (hereinafter referred to as measurement value 1) is expressed as the expression level of the reporter gene in the absence of the test substance. (Hereinafter referred to as measured value 2), the PGC1αb gene expression inducing activity or the expression suppressing activity of the test substance can be evaluated. In this case, using the measured value, the expression induction rate may be obtained according to the following formula.
Expression induction rate (%) = {(measured value 1−measured value 2) / measured value 2} × 100
A substance whose PGC1αb gene expression induction rate of a test substance shows a statistically significant value, specifically, for example, a substance showing 30% or more, more preferably a substance showing 50% or more is induced to induce PGC1αb expression Select as a substance with ability. In addition, when a measured value shows a negative value, the said test substance is an expression inhibitory substance.
(VI) Methods for searching for sugar metabolism improvers, etc.
As shown in the Examples of the present invention, the mitochondrial activation action is caused by the enhanced expression of the PGC1αb gene.
Therefore, a substance having mitochondrial activation ability, that is, improved glucose metabolism, using the expression promoting ability of PGC1αb gene or PGC1αb of the test substance evaluated by the evaluation method described in the above (V-1) to (V-3) as an index The method of selecting the agent is also within the scope of the present invention.
That is, the present invention uses the PGC1αb gene or PGC1αb expression-inducing activity of the test substance evaluated by the evaluation method described in (V-1) to (V-3) as an index, and the PGC1αb gene or PGC1αb expression-inducing substance ( There is provided a search method including a step of selecting a substance that increases the expression level as a substance having mitochondrial activation ability, that is, a sugar metabolism improving agent.
Here, the ability to activate mitochondria has the same meaning as described above.
Further, using the PGC1αb gene or PGC1αb expression inducing activity of the test substance evaluated by the evaluation method described in (V-1) to (V-3) as an index, an expression inducer (expression amount of PGC1αb gene or PGC1αb) There is provided a method for searching for a Glut4 expression inducer, comprising a step of selecting a substance to be increased) as a substance having a Glut4 expression induction activity, that is, a Glut4 expression inducer.
Glut4 is a protein that controls the uptake of sugar (ie, glucose) in the cell. When the expression level of Glut4 increases, the sugar is taken from the outside of the cell into the cell via the transporter, and the energy to the cell. Is replenished.
When energy is consumed by exercise, intracellular AMP levels increase and ATP levels decrease. The activity of AMPK, which is a kind of kinase, increases in such an environment. The inventors have found that the expression of PGC1αb is induced by exercise, and the induced PGC1αb induces the expression of Glut4. Furthermore, it was found that the expression of the PGC1αb gene was also induced by a compound known to activate AMPK: 5-aminoimidazole-4-carboxamide rebonucleoside (AICAR) (see Example 9).
As described above, the PGC1αb gene or the PGC1αb expression inducer has an activity of promoting sugar uptake into cells by inducing the expression of Glut4. PGC1αb is expressed specifically in muscle cells, and a PGC1αb gene or a PGC1αb expression inducer selectively promotes sugar uptake in muscle cells.
From the above, the PGC1αb gene or the PGC1αb expression inducer has the ability to improve sugar metabolism. That is, the present invention includes a step of selecting a PGC1αb gene or a PGC1αb expression inducer (a substance that increases the expression level) as a substance having an ability to improve sugar metabolism, that is, a sugar metabolism improving agent. I will provide a.
As shown in Examples of the present invention, PGC1αb is specifically expressed in muscle cells and the like. On the other hand, conventionally known PGC1αa is expressed not only in muscle cells but also in other tissue cells such as liver, kidney and brain. Therefore, a substance that selectively induces the expression of the PGC1αb gene or PGC1αb is very useful as a sugar metabolism improving agent that acts specifically on muscle cells. In addition, a substance that selectively induces or suppresses the expression of the PGC1αb gene or PGC1αb is useful as a medicament for diseases caused by underexpression or overexpression of the translation product of the gene.
Therefore, among the candidate substances for improving sugar metabolism, the expression level of the PGC1αa gene in the cells to which the test substance is added is significantly different from the expression level of the gene in the control cells to which the test substance is not added. It is desirable to select a substance that does not exhibit the expression, that is, a substance that does not change the expression level of the PGC1αa gene or PGC1αa. Whether or not the test substance induces or controls the expression of PGC1αa is the same as the evaluation method described in (V-1) to (V-3) above, in the presence or absence of the test substance. This can be determined by quantifying the expression level of PGC1αa.
The substance that activates the promoter of the present invention, which is selected by the evaluation according to the method described in (V-3) above, is a desired DNA linked under the control of the promoter of the present invention, such as a muscle such as the PGC1αb. It can also be used as a transcriptional regulator when examining effects on cells.
(VII) preventive agent, ameliorating agent and therapeutic agent
The sugar metabolism-improving agent obtained by the search method of the present invention exhibits an action for improving an abnormal decrease in sugar metabolism.
That is, the search method of the present invention provides a candidate substance that becomes an active ingredient of a drug for preventing / ameliorating / treating metabolic diseases such as insulin-resistant diabetes and / or obesity.
The PGC1αb gene or PGC1αb expression-inducing substance or expression-suppressing substance (collectively referred to as expression control substances) as the active ingredient was selected not only those selected using the above screening method, but also selected. It may be industrially produced according to a conventional method based on information on the substance.
Among the candidate substances for the PGC1αb gene or PGC1αb expression inducer selected by the above search method, candidates that are further effective ingredients for ameliorating or treating metabolic glucose metabolism diseases such as insulin-resistant diabetes and / or obesity As a method for selecting a substance, any conventionally known selection method can be used, and typical methods include, for example, an insulin tolerance test and a glucose tolerance test.
The gene / protein expression control substance of the present invention is mixed as it is or with a pharmaceutically acceptable carrier known per se (including excipients, extenders, binders, lubricants, etc.) and conventional additives. And can be prepared as a pharmaceutical composition. The pharmaceutical composition is prepared in a form (oral administration such as tablets, pills, capsules, powders, granules, syrups; parenteral administration such as injections, drops, external preparations, suppositories, etc.), etc. Depending on the dosage, it can be administered orally or parenterally. The dosage varies depending on the type of active ingredient, administration route, administration subject or patient's age, weight, symptoms, etc., and cannot be defined unconditionally, but the daily dosage is several mg to 2 g, preferably about several tens mg. It can be administered once to several times a day.
In addition, if the above substance is encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy. In these cases, the dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but those skilled in the art can appropriately select them.
The gene therapy will be described in detail. In the gene therapy, for example, the PGC1αb gene or a chemically modified form thereof (hereinafter sometimes referred to as the present gene) is directly treated in the same manner as this type of gene therapy. Can be carried out by a method of controlling the expression of the target gene by administering it into the body of the patient, or a method of controlling the expression of the target gene by the cell by introducing these genes into the target cells of the patient.
Here, as the chemical modification, for example, phosphorothioate, phosphorodithioate, alkylphosphotriester, alkylphosphonate, alkylphosphoamidate, etc., derivatives that can enhance the ability to migrate into cells or stability in cells ("Antisense RNA and DNA" published by WILEY-LISS, 1992, pp. 1-50, J. Med. Chem. 36, 1923-1937 (, 1993)). These can be synthesized according to conventional methods.
The gene can be formulated using a stabilizer, buffer, solvent, or the like that is commonly used for administration.
In the method of introducing the present gene into a target cell of a patient, the polynucleotide used preferably has a length of 100 bases or more, more preferably 300 bases or more, and even more preferably 500 bases or more, and has a mitochondrial activation ability. What is necessary is just to express the protein which has. This method also includes an in vivo method for introducing a gene into cells in a living body and an ex vivo method for introducing a gene into a cell once taken out of the body and returning the cell to the body (Nikkei Science, 1994 4). Monthly issue, pages 20-45, monthly pharmacy, 36 (1), 23-48 (1994), experimental medicine special edition, 12 (15), all pages (1994), etc.). Among these, the in vivo method is preferable, and there are a viral introduction method (method using a recombinant virus) and a non-viral introduction method (see the above-mentioned documents).
As a method of using the above recombinant virus, for example, a PGC1αb gene polynucleotide is incorporated into a living body such as a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a poliovirus, or a simbis virus. The method to introduce is mentioned. Of these, methods using retroviruses, adenoviruses, adeno-associated viruses and the like are particularly preferred. Examples of non-viral introduction methods include the liposome method and the lipofectin method, and the liposome method is particularly preferable. Other non-viral introduction methods include, for example, a microinjection method, a calcium phosphate method, an electroporation method, and the like.
The pharmaceutical composition for gene therapy comprises the present gene or a recombinant virus containing these genes and infected cells into which these viruses have been introduced as active ingredients. The dosage form and route of administration of the composition to a patient can be appropriately determined according to the disease or symptom to be treated. For example, it can be administered in an appropriate administration form such as an injection, intravenously, artery, subcutaneously, intramuscularly, etc., or can be directly administered and introduced into a disease target site of a patient. When adopting the in vivo method, the composition for gene therapy includes, for example, a form in which a viral vector containing the gene is embedded in liposomes or membrane-fused liposomes in addition to the administration form such as an injection containing the gene (Sendai). Virus (HVJ) -liposomes). These liposome preparation forms include suspensions, freezing agents, centrifugal concentrated freezing agents, and the like. Moreover, the composition for gene therapy can also be made into the form of the cell culture solution infected with the virus which introduce | transduced the vector containing this gene. The dosage of the active ingredient in these various forms of preparation can be appropriately adjusted depending on the degree of the disease to be treated, the age of the patient, the body weight, and the like. Usually, about 0.0001-100 mg, preferably about 0.001-10 mg per patient adult may be administered once every several days to several months. In the case of a retroviral vector containing this gene, the retroviral titer is about 1 × 10 6 per kg of patient body weight per day. ³ pfu-1x10 ¹⁵ It can be selected from a range of amounts that will be pfu. 1x10 for cells transfected with this gene ⁴ Cells / body-1 × 10 ¹⁵ What is necessary is just to administer a cell / body grade.
(VIII) Method for searching for a substance that binds to the promoter of the present invention
The present invention includes a method for searching for a substance that binds to the promoter of the present invention, which comprises the following steps (1) and (2):
(1) A step of bringing the promoter according to [5] into contact with a test substance,
(2) A step of examining the presence or absence of complex formation between the promoter and the test substance after the step (1).
As the promoter used in the step (1) for bringing the promoter of the present invention into contact with the test substance, a promoter labeled with a radioisotope or a fluorescent dye compound using, for example, a commercially available DNA labeling kit can be used. This is preferable in that a complex with a substance can be easily detected. In order to label the promoter with a radioisotope, for example, a commercially available Random Labeling Kit can be used, and dCTP in a normal PCR reaction composition is [α- ³² Instead of P] dCTP, labeling can also be performed by performing a PCR reaction using the DNA as a template. Further, when the DNA is labeled with a fluorescent dye, for example, ECL Direct Nucleic Acid Labeling and Detection System can be used.
The promoter and test substance are placed in a suitable buffer at about 4 ° C. to about 37 ° C., preferably about 20 ° C. to about 30 ° C., for about 5 minutes to about 60 minutes, preferably about 10 minutes to about 30 minutes. Contact is made in a buffer such as Tris, Hepes, MES, preferably in a Hepes buffer. The concentration of the test substance is usually about 0.1 μM to about 1,000 μM, and preferably 1 μM to 100 μM. The amount of the DNA is usually about 1 fmol to about 10 fmol, and preferably 2 fmol to 7 fmol.
Examples of the method for examining the presence or absence of complex formation between the promoter and the test substance include a gel shift method, a footprint method, and a BIACORE method. When the test substance is a relatively high molecular weight compound (eg, protein, DNA, etc.), a gel shift method, a footprint method, or the like may be used. When the test substance is a compound having a relatively low molecular weight, a BIACORE method, for example, a method described in “Co-authored by Kazuhiro Nagata and Hiroshi Handa, a real-time analysis method of biological substance interaction—mainly BIACORE—Springer Fairlark Tokyo Co., Ltd.” Use it. For example, in the case of the gel shift method, first, the DNA and the test substance mixed solution are subjected to polyacrylamide gel electrophoresis by a usual method. After drying the polyacrylamide gel after electrophoresis, it is possible to examine whether or not the DNA and the test substance are bound by detecting the position of the DNA on the gel by, for example, autoradiography. Specifically, for example, the dried gel is exposed to an imaging plate for about 1 hour to about 1 night, more preferably 6 to 8 hours, and an image image is acquired with BAS2000. When the test substance binds to the promoter, the mobility of the promoter-test substance complex becomes smaller than that of the free promoter, and a band shift on the image image occurs. A test substance when a band shift is detected can be selected as a substance that binds to the promoter of the present invention. Since the binding substance can bind to the promoter of the present invention, it can control the promoter activity of the promoter of the present invention. Furthermore, the expression of a gene under the control of the promoter of the present invention in a cell can be controlled. Specific examples include a transcription factor that binds to the promoter of the present invention. Therefore, the substance is useful as a medicament for diseases caused by overexpression or underexpression of the translation product of the gene. In addition, it is used as a transcriptional regulator when investigating effects on skeletal muscles such as desired DNA linked under the control of the promoter of the present invention, such as DNA that is presumed to be associated with skeletal muscles, and effects on mitochondrial activation. You can also.
There is no limitation in particular as said test substance, For example, the substance as described in said (V-1) can be used.
(IX) Method for purifying a substance that binds to the promoter of the present invention
The present invention also relates to a method for purifying a substance that binds to the promoter of the present invention, comprising the following steps (1) and (2):
(1) The step of bringing the promoter according to [5] into contact with a sample to form a complex of the promoter and a substance that binds to the promoter contained in the sample, and
(2) A purification method comprising the step of isolating the binding substance from the produced complex after the step (1).
The purification method of the present invention will be described below.
The purification method of the present invention comprises a first step of bringing the promoter of the present invention into contact with a sample to produce a complex of the promoter and a substance that binds to the promoter contained in the sample, and the first step Thereafter, a second step of isolating the binding substance from the produced complex is included.
When the promoter of the present invention is brought into contact with the sample, usually, the promoter or the complex of the promoter and the binding substance can be easily recovered by contacting the promoter in a form in which the promoter is bound to a carrier. This is preferable. The type of the carrier to which the promoter is bound is not particularly limited. For example, a commercially available carrier for affinity chromatography, preferably cyanogen bromide activated Sepharose 4B (manufactured by Amersham Pharmacia Biotech) or the like can be used. When the promoter is bound to a carrier, there are a method of directly binding the promoter to the carrier and a method of binding via a spacer. For example, the promoter and cyanogen bromide-activated Sepharose 4B are mixed and stirred at about 1000 ° C. overnight at about 4 ° C. to about 10 ° C. to fix the promoter on Sepharose. Then, in order to eliminate the unreacted cyanogen bromide active group, in a buffer containing an amino group, for example, sodium bicarbonate solution containing 1 M glycine, for example, at about 4 ° C. to about 10 ° C. overnight. put. The obtained gel may be brought into contact with the sample by an ordinary batch method, and an affinity column for a substance that binds to the promoter of the present invention is prepared by filling the gel in a commercially available chromatographic tube. The sample may be brought into contact with the sample by column chromatography.
For example, when contact / isolation is performed by column chromatography, the above-described sample is applied to an affinity column to form a complex of the promoter and a promoter binding substance, followed by washing of the carrier and elution of the binding substance. In this way, the binding substance can be isolated. Specifically, the sample is first loaded, and then washed with a buffer having the same composition as that used for loading to remove components in the sample that have not formed a complex. Thereafter, a substance that binds to the promoter of the present invention can be isolated by performing a gradient that increases the salt concentration contained in the buffer and eluting the substance that binds to the promoter of the present invention. Examples of the salt used for elution include sodium chloride, potassium chloride, and ammonium sulfate.
There are no particular limitations on what is used as the “sample” here, and examples include test substances, synthetic compound libraries, biological samples, and the like listed in (V-1) above. Examples of biological samples include suspensions or extracts derived from any cultured cells, suspensions or extracts derived from animal tissues, and the like. By using a human tissue or cell extract as a sample, an endogenous substance that binds to the PGC1αb promoter can be searched.
By the purification method of the present invention, a substance selected by the method for selecting a substance that controls the promoter activity of the promoter of the present invention or a substance selected by the method for selecting a substance that binds to the promoter of the present invention can be purified.

The present invention will be described in detail in the following examples, but the present invention is not limited thereto.
Example 1
(Preparation of cDNA)
Total RNA was extracted from the tissues using RNeasy Mini Kit (QIAGEN). 11 μl of a mixed solution containing 10 μg of the obtained total RNA and 100 pmol of T7- (dT) 24 primer (manufactured by Amersham) was heated at 70 ° C. for 10 minutes and then cooled on ice. After cooling, 4 μl of 5 × First Strand cDNA Buffer included in SuperScript Choice System for cDNA Synthesis (manufactured by Gibco-BRL), 2 μl of 0.1M DTT included in the kit, and 1 μM included in 0.1 M DTT included in the kit And the mixture was heated at 42 ° C. for 2 minutes. Furthermore, 2 μl (400 U) of Super Script II RT included in the kit was added to the mixed solution, and the mixed solution was heated at 42 ° C. for 1 hour and then cooled on ice. After cooling, 91 μl of DEPC-treated sterilized distilled water, 30 μl of 5 × Second Strand Reaction Buffer included in the kit, 3 μl of 10 mM dNTP Mix, and E. E. coli DNA Ligase 1 μl (10 U), E. coli contained in the kit. 4 μl (40 U) of E. coli DNA Polymerase I and E. coli contained in the kit. 1 μl (2 U) of E. coli RNase H was added and the mixture was reacted at 16 ° C. for 2 hours. Next, 2 μl (10 U) of T4 DNA Polymerase contained in the kit was added to the mixture, and this mixture was reacted at 16 ° C. for 5 minutes, and then 10 μl of 0.5 M EDTA was added to the mixture. Next, 162 μl of a phenol / chloroform / isoamyl alcohol solution (manufactured by Nippon Gene) was added to the mixture and mixed. The mixture was transferred to Phase Lock Gel Light (manufactured by Eppendorf) that had been centrifuged at room temperature and 14,000 rpm for 30 seconds, and this was centrifuged at room temperature and 14,000 rpm for 2 minutes. After centrifugation, 145 μl of the aqueous layer was collected in an Eppendorf tube. The recovered aqueous layer was mixed with 72.5 μl of 7.5 M ammonium acetate solution and 362.5 μl of ethanol, and then the mixture was centrifuged at 14,000 rpm for 20 minutes at 4 ° C. After centrifugation, the supernatant was discarded to obtain a DNA pellet. Thereafter, 0.5 mL of 80% ethanol was added to the DNA pellet. This mixture was centrifuged at 14,000 rpm for 5 minutes at 4 ° C., and the supernatant was discarded to obtain a DNA pellet again. To the obtained DNA pellet, 0.5 mL of 80% ethanol was added again. The mixture was centrifuged at 14,000 rpm for 5 minutes at 4 ° C., and then the supernatant was discarded to obtain a DNA pellet. The obtained DNA pellet was dried and then dissolved in 12 μl of DEPC-treated sterilized distilled water to obtain a cDNA solution.
Example 2
(Cloning of human PGC-1αb gene)
1 μL of human cDNA library (manufactured by Clontech), 20 pmol of primer consisting of the base sequence shown in SEQ ID NO: 9, 20 pmol of primer consisting of the base sequence shown in SEQ ID NO: 10, 1 μL of Ex-taq (Takara Shuzo), attached to Ex-taq A 50 μL reaction solution containing 5 μL of buffer and 4 μL of dNTP was prepared, and PCR was performed. In the PCR, a heat retention cycle comprising 94 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 40 times. The PCR product amplified by PCR was subjected to agarose gel electrophoresis, whereby DNA having a size of about 2.4 kbp was recovered. The recovered DNA was purified using QIAEX II Gel Extraction Kit (QIAGEN). The purified DNA was dissolved in 20 μL of water, 1 μL of the DNA was mixed with pT7-Blue (Takara Shuzo), and the mixture was ligated with ligation kit ver. 1 (Takara Shuzo) was used for ligation reaction at 16 ° C. for 1 hour. 20 μL of the ligation reaction solution thus obtained and E. coli. E. coli JM109 strain competent cell (Toyobo Co., Ltd.) E. coli JM109 transformed cells were obtained. Including the base sequence of the human PGC1αb gene of the present invention shown in SEQ ID NO: 1 using QIAGEN Plasmid Maxi kit (QIAGEN) from cultured cells obtained by culturing the transformed cell in 100 mL of LB medium containing 50 μg / mL ampicillin The recombinant vector was purified and isolated.
Example 3
(Cloning of mouse PGC-1αb gene)
Mouse skeletal muscle cDNA library (Takara Shuzo) 1 μL, primer 20 pmol consisting of the base sequence shown in SEQ ID NO: 11, primer 20 pmol consisting of the base sequence shown in SEQ ID NO: 12, Ex-taq (Takara Shuzo) 1 μL, Ex-taq A 50 μL reaction solution containing 5 μL of the attached buffer and 4 μL of dNTP was prepared, and PCR was performed. In the PCR, a heat retention cycle comprising 94 ° C. for 1 minute, 65 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 40 times. The PCR product amplified by PCR was subjected to agarose gel electrophoresis, whereby DNA having a size of about 2.4 kbp was recovered. The recovered DNA was purified using QIAEX II Gel Extraction Kit (QIAGEN). The purified DNA was dissolved in 20 μL of water, 1 μL of the DNA was mixed with pT7-Blue (Takara Shuzo), and the mixture was ligated with ligation kit ver. 1 (Takara Shuzo) was used for ligation reaction at 16 ° C. for 1 hour. 20 μL of the ligation reaction solution thus obtained and E. coli. E. coli JM109 strain competent cell (Toyobo Co., Ltd.) E. coli JM109 transformed cells were obtained. It contains the nucleotide sequence of the mouse PGC1αb gene of the present invention shown in SEQ ID NO: 3 using QIAGEN Plasmid Maxi kit (QIAGEN) from cultured cells obtained by culturing the transformed cell in 100 mL of LB medium containing 50 μg / mL ampicillin. The recombinant vector was purified and isolated.
Example 4
(Cloning of rat PGC-1αb gene)
Rat cDNA library (manufactured by Clontech) 1 μL, primer 20 pmol consisting of the base sequence shown in SEQ ID NO: 13, primer 20 pmol consisting of the base sequence shown in SEQ ID NO: 14, Ex-taq (Takara Shuzo) 1 μL, attached to Ex-taq A 50 μL reaction solution containing 5 μL of buffer and 4 μL of dNTP was prepared, and PCR was performed. In the PCR, a heat retention cycle comprising 94 ° C. for 1 minute, 65 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 40 times. The PCR product amplified by PCR is subjected to agarose gel electrophoresis to recover DNA of about 2.4 kbp. The recovered DNA is purified using QIAEX II Gel Extraction Kit (QIAGEN). The purified DNA is dissolved in 20 μL of water, 1 μL of the DNA is mixed with pT7-Blue (Takara Shuzo), and the mixture is ligated with ligation kit ver. 1 (Takara Shuzo) is used for ligation reaction at 16 ° C. for 1 hour. 20 μL of the ligation reaction solution obtained in this way and E. coli. E. coli JM109 strain competent cell (Toyobo Co., Ltd.) E. coli JM109 transformed cells are obtained. It contains the nucleotide sequence of the rat PGC1αb gene of the present invention shown in SEQ ID NO: 5 using QIAGEN Plasmid Maxi kit (QIAGEN) from cultured cells obtained by culturing the transformed cell in LB medium containing 50 μg / mL ampicillin. Recombinant vectors can be purified and isolated.
Example 5
(RT-PCR)
When the RT-PCR method is used, RNA prepared from mouse muscle tissue or mouse striated muscle-derived cultured cell C2C12, or prepared using TaqMan Reverse Transfer Reagents (ABI) using the obtained RNA as a template. A pair of primers (a normal strand that binds to the cDNA (-strand) and a reverse strand that binds to the + strand) so that the base sequence region encoding the gene to be quantified can be specifically amplified using the obtained cDNA as a template. Designed and synthesized in the usual way. When quantifying mouse PGC1αb, a primer consisting of the base sequence shown in SEQ ID NO: 15 and a primer consisting of the base sequence shown in SEQ ID NO: 16 were used. Using the synthesized primers, an RT-PCR reaction solution was prepared according to the protocol using SYBR Green RT-PCR Reagents (manufactured by Applied Biosystems), and ABI PRIME 7900 Sequence Detection System (manufactured by Applied Biosystems) was used. The reaction product was detected and quantified. In Examples 6 and 7 below, RT-PCR was performed by the method of this example.
Example 6
(Expression of PGC1αb in each mouse tissue)
CDNA was prepared using RNA prepared from each mouse tissue as a template, and expression of mouse PGC1αb was analyzed by RT-PCR. As an internal standard for expression, 36B4 gene expression quantification was used, and a primer consisting of the base sequence shown in SEQ ID NO: 17 and a primer consisting of the base sequence shown in SEQ ID NO: 18 were used. The relative expression level was determined by dividing the expression quantitative value of the gene to be analyzed in each sample by the 36B4 gene expression quantitative value. For comparison, comparison was made using a primer consisting of the base sequence shown in SEQ ID NO: 19 and a primer which specifically quantifies the mouse PGC1αa gene consisting of the base sequence shown in SEQ ID NO: 20. The results are shown in FIG. PGC1αb was clearly expressed only in muscle and heart, and slightly expressed in brown adipose tissue. In other organs including the liver, no expression was observed under these conditions. On the other hand, PGC1αa known so far was clearly expressed in brown adipose tissue, muscle, kidney, brain and heart, and was also observed in the liver.
Example 7
(Induction of exercise-dependent expression of PGC1αb in skeletal muscle)
47-55 week old male C57BL6j mice (CLEA Japan) were used. The lighting time of the breeding room was from 6:00 to 18:00 in the light period and from 18:00 to 6:00 in the dark period. CE-2 type solid feed (CLEA Japan) was fed as breeding feed, and tap water was given as drinking water.
A treadmill model: MK-680S (rat / mouse treadmill / Muromachi Kikai Co., Ltd.) was used for the exercise load test, and the exercise conditions were 14 m / min x 45 min x 2 (10 min interval). Three hours after the end of exercise, the soleus and long extensor muscles were quickly removed from the lower limbs and rapidly frozen with liquid nitrogen to prevent RNA from being degraded by RNase. RNA was extracted from the extracted soleus and long finger extensor muscles, and RT-PCR was performed.
In 5-aminoimidazole-4-carboxide ribonucleoside (AICAR) treatment test, AICAR (Toronto Research Chemicals Inc.) was administered at a dose of 1 mg / g of body weight (50 mg / ml in selenium). Twelve hours after administration, the soleus and long extensor muscles were removed and immediately frozen in liquid nitrogen to prevent degradation of RNA by RNase. RNA was extracted from the extracted soleus and long finger extensor muscles, and RT-PCR was performed.
As an internal standard for expression, 36B4 gene expression quantification was used, and a primer consisting of the base sequence shown in SEQ ID NO: 17 and a primer consisting of the base sequence shown in SEQ ID NO: 18 were used. The relative expression level was determined by dividing the expression quantitative value of the gene to be analyzed in each sample by the 36B4 gene expression quantitative value. RT-PCR was performed in triplicate, and the average value and standard error were calculated.
The results are shown in FIG. PGC1αb was commonly observed to induce expression in both the soleus and long extensor muscles after exercise and AICAR treatment. On the other hand, in PGC1αa known so far, there was a tendency to be induced by AICAR treatment of the long finger extensor, but in both the soleus and long finger extensor muscles, exercise and induction common to AICAR treatment are I couldn't see it.
Example 8
(Construction of mouse PGC1αb adenovirus vector)
The sense strand of the mouse PGC1αb gene prepared in Example 3 was inserted into the SwaI site of the cosmid vector pAxCAwt (Takara Shuzo) to obtain pAxCAwt-mPGC1αb. The obtained pAxCAwt-mPGC1αb was in vitro packaged using λ packaging kit GigapackXL (Stratagene) and infected with E. coli DH5α. Infected E. coli was cultured in LB medium containing 50 ml of 50 μg / ml ampicillin, and a large amount of cosmid DNA was prepared by Lambda DNA purification Kit (Toyobo).
293 cells (Takara Shuzo) are cultured in 10% FCS-added D-MEM medium (Takara Shuzo) at 37 ° C. under 5% carbon dioxide. 8 μg of pAxCAwt-mPGC1αb prepared as described above and 5 μl of restriction enzyme-treated DNA-TPC (Takara Shuzo) were mixed, and 293 cells were co-transfected by the calcium phosphate method using the mixture. After culturing the cells as they are for 18 hours, the medium is replaced with a D-MEM medium supplemented with 10% FCS (Takara Shuzo) and further cultured for 12 hours. After completion of the culture, the cells were removed from the dish, and the recovered cell suspension and 293 cells were mixed and further cultured in 10% FCS-added D-MEM medium (Takara Shuzo). At the time of death, the culture solution was snap frozen with dry ice. After freezing and thawing 6 times, the supernatant obtained by centrifugation at 5000 rpm for 5 minutes was stored as a primary recombinant adenovirus solution (AxCAwt-mPGC1αb).
After adding 10 μl of the primary recombinant virus solution prepared by the above method to 293 cells (Takara Shuzo), 5% FCS-added D-MEM medium (Takara Shuzo) was added thereto, and then the mixture was incubated at 37 ° C., 5% Cultivation was performed for 1 hour under carbon dioxide. Further, 5% FCS-added D-MEM medium (Takara Shuzo) was added to the mixture, which was then cultured at 37 ° C. under 5% carbon dioxide. After culturing for 3 days, infected 293 cells were collected, a protein fraction was extracted from the cells, and subjected to immunoblotting using a PGC1α antibody (Santa Cruz Biotechnology). The production of recombinant adenovirus was confirmed by confirming the presence or absence of expression of PGC1α protein. Moreover, the culture solution of 293 cells (Takara Shuzo Co., Ltd.) infected with the virus was collected and rapidly frozen with dry ice. After freezing and thawing six times, the supernatant obtained by centrifugation at 5000 rpm for 5 minutes was stored as a secondary recombinant adenovirus solution. Subsequently, this recombinant adenovirus DNA was subcultured by repeating the above procedure. In this way, a high-titer recombinant virus was produced. The titer was measured by an existing method (Japanese Patent Laid-Open No. 7-298877).
Example 9
(Induction of PGC1αb gene expression in cultured muscle cells)
An infection experiment of adenovirus AxCAwt-mPGC1αb to mouse striated muscle-derived cultured cell C2C12 was performed as follows.
C2C12 cells were cultured until they became confluent in D-MEM medium (Takara Shuzo) supplemented with 10% FCS, and then cultured for 5 days in D-MEM medium supplemented with 2% FCS (Takara Shuzo). Differentiation was induced. After differentiation into myotube cells, the cells were infected with AxCAwt-mPGC1αb, and after 48 hours, the cells were collected and RNA was extracted. RT-PCR was performed by the method described in Example 5 to quantify the expression of mouse PGC1αb.
As an internal standard for expression, 36B4 gene expression quantification was used, and a primer consisting of the base sequence shown in SEQ ID NO: 17 and a primer consisting of the base sequence of SEQ ID NO: 18 were used. The relative expression level was determined by dividing the expression quantitative value of the gene to be analyzed in each sample by the 36B4 gene expression quantitative value. For quantification of the mtTFA gene, a primer consisting of the base sequence shown in SEQ ID NO: 21 and a primer consisting of the base sequence shown in SEQ ID NO: 22, and for quantifying the UCP-2 gene, it consists of the base sequence shown in SEQ ID NO: 23 For quantifying the primer and the COXII gene consisting of the base sequence shown in SEQ ID NO: 24, the primer consisting of the base sequence shown in SEQ ID NO: 25, the primer consisting of the base sequence shown in SEQ ID NO: 26, and the NRF gene Is a primer consisting of the base sequence shown in SEQ ID NO: 27, a primer consisting of the base sequence shown in SEQ ID NO: 28, and a primer consisting of the base sequence shown in SEQ ID NO: 29 and SEQ ID NO: 30 for the quantification of the βATP synthase gene. Primer consisting of a base sequence, Glut4 gene Quantitatively, we quantified using primers having the nucleotide sequence represented by primers SEQ ID NO: 32 comprising the nucleotide sequence represented by SEQ ID NO: 31 was compared. The results are shown in FIGS.
The expression of PGC1αb was observed by the infection with adenovirus AxCAwt-mPGC1αb, and the expression was increased by the increase of viral load. UCP2, which is a gene related to mitochondrial function and is considered to cause energy consumption in mitochondria, transcription factor showing an important role in mitochondrial genome replication and transcription reaction: mtTFA (mitochondrial transcription factor A, promoter of many mitochondrial genes In both the transcription factor NRF1 that binds to γ and βATP synthase and cytochrome oxidase subunits II (COXII), which are genes related to the oxidative phosphorylation pathway in mitochondria, an increase in expression corresponding to the increase in the expression of PGC1αb was observed.
Example 10
(Search method)
A PGC1αb gene expression inducer can be searched by determining whether a compound promotes its expression using muscle tissue or cultured cells that express the PGC1αb gene. Mouse striated muscle-derived cultured cell C2C12 was placed in a 10 cm dish (Corning) at 5 × 10 5 per dish. ⁵ Involve cells one by one. The cells were treated with D-MEM medium (high glucose, SIGMA) supplemented with 10% fetal calf serum (JRH life sciences, hereinafter referred to as FCS) and 1 mM sodium pyruvate (Gibco-BRL). Incubate at 37 ° C. under 5% carbon dioxide. On the third day after the start of culture, the medium is exchanged with a medium containing 2% HS (Gibco-BRL) to induce differentiation. For example, a test substance or a culture medium (in the case of a control) and optionally 0.5% dimethyl sulfoxide (hereinafter referred to as DMSO) were added to cultured cell C2C12 derived from mouse striated muscle 7 days after the start of differentiation. The medium can be changed. C2C12 cultured cells cultured after 15 hours are collected, total RNA is prepared from each culture mixture, and cDNA is further prepared. The prepared sample is analyzed by Northern blotting to quantify the expression level of the PGC1αb gene. If the expression of the PGC1αb gene in the cell to which the test substance is added is 1.5 times or more compared to the expression level in the control cell to which no test substance is added, the test substance is a PGC1αb expression inducer, and glucose metabolism It can be selected as a candidate substance for improving substance. At this time, the expression level of the PGC1αa gene is quantified as a control in the same manner. Among the candidate substances for improving sugar metabolism, those that are substances that do not show significant expression induction compared to control cells in which the expression of the PGC1αa gene in the cells to which the test substance is added are not added are selected.
Example 11
(Induction of PGC1αb gene expression in cultured muscle cells)
Mouse striated muscle-derived cultured cell C2C12 was placed in a 10 cm dish (Corning) at 5 × 10 5 per dish. ⁵ I swallowed each cell. The cells were treated with D-MEM medium (high glucose, SIGMA) supplemented with 10% fetal calf serum (JRH life sciences, hereinafter referred to as FCS) and 1 mM sodium pyruvate (Gibco-BRL). Incubated at 37 ° C. under 5% carbon dioxide. On the 3rd day after the start of the culture, the medium was replaced with a medium containing 2% HS (Gibco-BRL) to induce differentiation, and on the 7th day from the start of differentiation, 5-aminoimidazole-4-carbamide ribonucleoside (AICAR; Toronto Research Chemicals Inc.). .) 500 μM, Isoproterenol (SIGMA) 10 μM, Forkolin (DMSO solution SIGMA) 50 μM, Caffeine (SIGMA) 57 mM, CoCl ₂ (SIGMA) The medium was changed to 100 μM and A23187 (SIGMA) 500 nM. C2C12 cultured cells cultured after 15 hours were collected, and RNA was extracted. As an internal standard for expression, expression quantification of 36B4 gene was used, and primers having the sequences of SEQ ID NOs: 40 and 41 were used. The relative expression level was determined by dividing the expression quantitative value of the gene to be analyzed in each sample by the 36B4 gene expression quantitative value. The results are shown in FIG. Apparent induction of PGC1αb gene expression was observed under the conditions of Forkoline, Caffeine, and AICAR.
Example 12
(Cloning of human PGC-1αb promoter of the present invention)
Human Genomic DNA (CLONTECH) 1 μL, Primer 20 pmol consisting of the base sequence shown in SEQ ID NO: 36, Primer 20 pmol consisting of the base sequence shown in SEQ ID NO: 37, Ex-taq (Takara Shuzo) 1 μL, Buffer attached to Ex-taq A 50 μL reaction solution containing 5 μL and 4 μL of dNTP was prepared, and PCR was performed. In this PCR, a heat retention cycle comprising 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 3 minutes was repeated 40 times. The PCR product amplified by PCR was subjected to agarose gel electrophoresis, and DNA having a size of about 3 kbp was recovered.
Similarly, 20 pmol of the primer consisting of the base sequence shown in SEQ ID NO: 44, 20 pmol of the primer consisting of the base sequence shown in SEQ ID NO: 45, and about 1.8 kb of DNA, consisting of the base sequence shown in SEQ ID NO: 46 DNA having a size of about 1.3 kb was recovered using 20 pmol of primer and 20 pmol of a primer consisting of the base sequence represented by SEQ ID NO: 47. The recovered DNA was purified using QIAEX II Gel Extraction Kit (QIAGEN). The purified DNA was dissolved in 20 μL of water, 1 μL of the DNA was mixed with pT7-Blue (Takara Shuzo), and the mixture was ligated with ligation kit ver. 1 (Takara Shuzo) was used for ligation reaction at 16 ° C. for 1 hour. 20 μL of the ligation reaction solution thus obtained and E. coli. E. coli JM109 strain competent cell (Toyobo Co., Ltd.) E. coli JM109 transformed cells were obtained. A recombinant vector comprising the base sequence of the human PGC-1αb promoter of the present invention using QIAGEN Plasmid Maxi kit (QIAGEN) from cultured cells obtained by culturing the transformed cell in 100 mL of LB medium containing 50 μg / mL ampicillin. hPGC1αb-P1 (3.0) -pT7Blue, hPGC1αb-P2 (1.8) -pT7Blue, and hPGC1αb-P3 (1.3) -pT7Blue were purified and isolated.
Example 13
(Determination of the base sequence of the human PGC-1αb promoter of the present invention)
Using the recombinant vector containing the base sequence of the human PGC-1αb promoter of the present invention purified and isolated in Example 5 as a template, Thermo Sequence II dye terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PE Applied Biosystems) using the Sanger method [F. Sanger, S.M. Nicklen, A.M. R. Coulson, Proceedings of National Academy of Science U. S. A. (1977), 74, 5463-5467], the base sequence of the human PGC-1αb promoter of the present invention was determined. The determined base sequence of the fragment of about 3 kbp is shown in SEQ ID NO: 33, and the base sequence of the fragment of about 1.8 kbp in the base sequence shown by SEQ ID NO: 33 is the base sequence from base number 1157 to base number 3000; The base sequence of the 3 kbp fragment is shown in SEQ ID NO: 34. The base sequence shown in SEQ ID NO: 34 corresponds to the base sequence from base number 1648 to base number 3000 in the base sequence shown in SEQ ID NO: 33.
Example 14
(Construction of a plasmid containing the human PGC-1αb promoter and luciferase reporter gene of the present invention)
Recombinant vectors hPGC1αb-P1 (3.0) -pT7Blue, hPGC1αb-P2 (1.8) -pT7Blue, hPGC1αb- containing the base sequence of the human PGC-1αb promoter of the present invention purified and isolated in Example 13 P3 (1.3) -pT7Blue (5 μg) was digested with 70 μL of the reaction solution at 37 ° C. for 1 hour using 5 U each of KpnI and SalI. On the other hand, 5 μg of pGL3-Basic (Promega) was digested for 1 hour at 37 ° C. in a 70 μL reaction solution using 5 U each of KpnI and XhoI. The DNA fragments were collected by subjecting both digestion reaction solutions to agarose gel electrophoresis separately. The recovered DNA fragment was purified using QIAEXII Gel Extraction Kit. Each of the purified DNA fragments was dissolved in 20 μL of water, 1 μL of each of the lysates and ligation kit ver. 16 μL of the A solution contained in 1 and 2 μL of the B solution were mixed. The mixture was subjected to a ligation reaction at 16 ° C. for 1 hour. 20 μL of the ligation reaction solution thus obtained and E. coli. E. coli JM109 strain competent cell. E. coli JM109 transformed cells were obtained. Using the QIAGEN Plasmid Maxi kit from the cultured cells obtained by culturing the transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin, the luciferase reporter gene is downstream (3 ′ side) of the human PGC-1αb promoter of the present invention. Recombinant expression vectors (hereinafter referred to as phPGC1αb-P1 (3.0) -pGL3basic, phPGC1αb-P2 (1.8) -pGL3basic, phPGC1αb-P3 (1.3) -pGL3basic) were purified and isolated. .
Example 15
(Cloning of mouse PGC-1αb promoter of the present invention)
Mouse Genomic DNA (CLONTECH) 1 μL, primer 20 pmol consisting of the base sequence shown in SEQ ID NO: 48, primer 20 pmol consisting of the base sequence shown in SEQ ID NO: 49, Ex-taq 1 μL, buffer attached to Ex-taq 5 μL and dNTP 4 μL A 50 μL reaction solution containing was prepared and subjected to PCR. In the PCR, a heat retention cycle comprising 94 ° C. for 30 seconds, 65 ° C. for 30 seconds, and 72 ° C. for 3 minutes was repeated 40 times. The PCR product amplified by PCR was subjected to agarose gel electrophoresis, and DNA having a size of about 3 kbp was recovered. The recovered DNA was purified using QIAEX II Gel Extraction Kit. The purified DNA is dissolved in 20 μL of water, 1 μL of the DNA is mixed with pT7-Blue, and the mixture is ligated with ligation kit ver. 1 was used for ligation reaction at 16 ° C. for 1 hour. 20 μL of the ligation reaction solution thus obtained and E. coli. E. coli JM109 strain competent cell. E. coli JM109 transformed cells were obtained. A recombinant vector containing the base sequence of the mouse PGC-1αb promoter of the present invention was purified from the cultured cells obtained by culturing the transformed cells in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi kit. Released.
Example 16
(Determination of the base sequence of the mouse PGC-1αb promoter of the present invention)
Using the recombinant vector containing the nucleotide sequence of the mouse PGC-1αb promoter of the present invention purified and isolated in Example 15 as a template, Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and ABI373 DNA sequence reader (PE Applied Biosystems) using the Sanger method [F. Sanger, S.M. Nicklen, A.M. R. Coulson, Proceedings of National Academy of Science U. S. A. (1977), 74, 5463-5467], the base sequence of the mouse PGC-1αb promoter of the present invention was determined. The determined base sequence is shown in SEQ ID NO: 35.
Example 17
(Construction of a plasmid containing the mouse PGC-1αb promoter and luciferase reporter gene of the present invention)
5 μg of the recombinant vector containing the base sequence of the mouse PGC-1αb promoter of the present invention purified and isolated in Example 15 was digested at 37 ° C. for 1 hour in a 70 μL reaction solution using 5 U each of MluI and SmaI. . On the other hand, 5 μg of pGL3-Basic (Promega) was digested with MluI and SmaI in a 70 μL reaction solution at 37 ° C. for 1 hour. The DNA fragments were collected by subjecting both digestion reaction solutions to agarose gel electrophoresis separately. The recovered DNA fragment was purified using QIAEXII Gel Extraction Kit. Each of the purified DNA fragments was dissolved in 20 μL of water, 1 μL of each of the lysates and ligation kit ver. 16 μL of the A solution contained in 1 and 2 μL of the B solution were mixed. The mixture was subjected to a ligation reaction at 16 ° C. for 1 hour. 20 μL of the ligation reaction solution thus obtained and E. coli. E. coli JM109 strain competent cell. E. coli JM109 transformed cells were obtained. Bases from base number 1 to base number 3022 in the base sequence represented by SEQ ID NO: 35 from the cultured cells obtained by culturing the transformed cell in 100 mL of LB medium containing 50 μg / mL ampicillin using QIAGEN Plasmid Maxi kit A recombinant expression vector (hereinafter referred to as mPGC1αb-P1 (3.0) -pGL3basic) containing a luciferase reporter gene downstream (3 ′ side) of the mouse PGC-1αb promoter of the present invention comprising the sequence was purified and isolated. .
In addition, the DNA fragment obtained by cleaving the recombinant vector containing the base sequence of the mouse PGC-1αb promoter of the present invention purified and isolated in Example 15 with KpnI and SmaI was used as the KpnI of pGL3-Basic. Except for introduction into the SmaI site, in accordance with the same method as described above, downstream of the mouse PGC-1αb promoter of the present invention consisting of the base sequence from base number 1631 to base number 3022 in the base sequence represented by SEQ ID NO: 35 (3 A recombinant expression vector containing a luciferase reporter gene on the 'side' (hereinafter referred to as mPGC1αb-P2 (1.0) -pGL3basic) was obtained.
Example 18
(Measurement of transcription initiation ability of human PGC-1αb promoter of the present invention)
Mouse striated muscle-derived cultured cells C2C12 were placed in a 24-well plate (Becton Dickinson) at 5.0 × 10 per well. ⁴ I swallowed each cell. The C2C12 cultured cells were added to D-MEM medium (high glucose, SIGMA) supplemented with 10% fetal calf serum (JRH life sciences, hereinafter referred to as FCS) and 1 mM sodium pyruvate (Gibco-BRL). ) And cultured at 37 ° C. under 5% carbon dioxide overnight. Next, phPGC1αb-P1 (3.0) -pGL3basic, or phPGC1αb-P2 (1.8) -pGL3basic, phPGC1αb-P3 (1.3) -pGL3basic 100 ng, and plasmid pME18s-LacZ 100 ng containing the LacZ gene are mixed. To this, 2 μL of PLUS Reagent (Invitrogen) and 0.5 μL of Lipofectamine Reagent (Invitrogen) were added and mixed, and this was used as a DNA solution for transfection per well of a 24-well plate. This DNA solution for transfection was added to the C2C12 cultured cells, which were further cultured overnight. On the next day, the medium was replaced with a medium containing Forskolin (50 μM) or AICAR (500 μM), and C2C12 cultured cells cultured after 10 to 14 hours were collected. After the cells were washed once with D-PBS, 100 μL per well of a cell lysing agent for Picker Gene Kit (Toyo Ink) diluted with D-PBS containing 1 mg / ml bovine serum albumin was added. This was allowed to stand at room temperature for 15 minutes to obtain a cell lysate. The cell lysate was transferred to a 1.5 ml Eppendorf tube using a pipette and centrifuged (15,000 rpm, 2 minutes, 4 ° C.) to obtain a supernatant. 20 μL of the obtained supernatant was added to 100 μL of the luminescent substrate solution for firefly luciferase attached to the kit, and the amount of luminescence for 10 seconds was quantified with a luminometer (Fluoroskan Ascent FL; Thermo Labsystems). The measured value was taken as a firefly luciferase activity measurement value (measurement value A). Next, 10 μL of a LacZ substrate solution (25 mM o-Nitrophenyl β-D-Galactopyranoside; SIGMA) was added to the supernatant and quantified with a microplate reader (model 3550; Bio-Rad). The measured value was defined as a β-galactosidase activity measurement value (measurement value B). After calculating the value (A / B value) obtained by dividing the measured value A by the measured value B, the A / B value in each C2C12 cultured cell was divided by the A / B value in the control to obtain each C2C12 culture. The relative luciferase activity (relative value) in the cells was calculated. Here, “control” means pGL3-basic instead of recombinant plasmids phPGC1αb-P1 (3.0) -pGL3basic, phPGC1αb-P2 (1.8) -pGL3basic, phPGC1αb-P3 (1.3) -pGL3basic. It means a control striated muscle-derived cell obtained by transfecting only Basic with the same method.
When measurement is performed by inducing differentiation of C2C12 cultured cells, transfection is performed in the same manner as described above, and then the medium is replaced with a medium containing 2% HS on the next day to induce differentiation. Further, the next day, Forskolin (50 μM) Alternatively, stimulation with AICAR (500 μM) was performed. C2C12 cultured cells cultured after 10 to 14 hours were collected, and the activity was measured.
The results are shown in FIG. The phPGC1αb-P1 (3.0) -pGL3basic, phPGC1αb-P2 were used in both the mouse striated muscle-derived cultured cell C2C12 (undifferentiated) and the mouse striated muscle-derived cultured cell C2C12 that had undergone differentiation induction. Significant promoter activity was detected in all of (1.8) -pGL3basic and phPGC1αb-P3 (1.3) -pGL3basic compared to the control, and the activity was enhanced about 5.5 to 8.5 times with Forskolin. It was done.
Example 19
(Measurement of transcription initiation ability of the mouse PGC-1αb promoter of the present invention)
Mouse striated muscle-derived cultured cells C2C12 were placed in a 24-well plate (Becton Dickinson) at 5.0 × 10 per well. ⁴ I swallowed each cell. The cells were used in a D-MEM medium (high glucose, SIGMA) supplemented with 10% fetal calf serum (JRH life sciences, hereinafter referred to as FCS) and 1 mM sodium pyruvate (Gibco-BRL). The cells were cultured overnight at 37 ° C. and 5% carbon dioxide. Next, 100 ng of mPGC1αb-P2 (1.0) -pGL3basic and 100 ng of plasmid pME18s-LacZ containing LacZ gene were mixed, and PLUS Reagent (Invitrogen) 2 μL and Lipofectamine reagent (InvitroLen 0.5 μm) were added. These were mixed to obtain a DNA solution for transfection per well of a 24-well plate. This DNA solution for transfection was added to the C2C12 cultured cells, which were further cultured overnight. On the next day, the medium was replaced with a medium containing Forskolin (50 μM), C2C12 cultured cells cultured after 10 to 14 hours were collected, washed once with D-PBS, and then D containing 1 mg / ml bovine serum albumin. -100 μL per well of a cell lysing agent exclusively for the Picker Gene Kit (Toyo Ink) diluted with PBS was added. This was allowed to stand at room temperature for 15 minutes to obtain a cell lysate. The cell lysate was transferred to a 1.5 ml Eppendorf tube using a pipette and centrifuged (15,000 rpm, 2 minutes, 4 ° C.) to obtain a supernatant. 20 μL of the obtained supernatant was added to 100 μL of the luminescent substrate solution for firefly luciferase attached to the kit, and the amount of luminescence for 10 seconds was quantified with a luminometer (Fluoroskan Ascent FL; Thermo Labsystems). The measured value was taken as a firefly luciferase activity measurement value (measurement value A). Next, 10 μL of a LacZ substrate solution (25 mM o-Nitrophenyl β-D-Galactopyranoside; SIGMA) was added to the supernatant and quantified with a microplate reader (model 3550; Bio-Rad). The measured value was defined as a β-galactosidase activity measurement value (measurement value B). After calculating the value (A / B value) obtained by dividing the measured value A by the measured value B, the A / B value in each C2C12 cultured cell was divided by the A / B value in the control to obtain each C2C12 culture. The relative luciferase activity (relative value) in the cells was calculated. Here, “control” means a control striated muscle-derived cell obtained by transfecting only pGL3-Basic in the same manner instead of the recombinant plasmid mPGC1αb-P2 (1.0) -pGL3basic. To do.
The results are shown in FIG. Significant promoter activity was detected in mPGC1αb-P2 (1.0) -pGL3basic compared to the control, and the activity was enhanced about 1.8 times with Forskolin.
Example 20
(Selection of a substance that controls the promoter activity of the human PGC-1αb promoter of the present invention)
Using the method described in Example 18, phPGC1αb-P1 (3.0) -pGL3basic, or phPGC1αb-P2 (1.8) -pGL3basic, phPGC1αb-P3 (1.3) -pGL3basic were transfected. Mouse striated muscle-derived cultured cell C2C12 was added to a 96-well plate at 1 × 10 6 per well. ⁴ Infuse cells one by one. Then, 1 μM DMSO solution of the test substance is added to the well plate at 0.5 μL per well, and this is incubated overnight. At the same time, as a control, 0.5 μL of DMSO is added instead of 1 μM DMSO solution of the test substance, and this is cultured in the same manner. The firefly luciferase activity measurement value (measurement value A) and the LacZ activity measurement value (measurement value B) are measured using the method described in Example 15. Next, after calculating the value (A / B value) obtained by dividing the measured value A by the measured value B, the A / B value in each cultured cell was divided by the A / B value in the control, and the relative luciferase activity ( Relative value) is calculated. A test substance having a relative luciferase activity (relative value) greater than 1 is selected as a substance having a positive transcriptional regulation ability. Conversely, a test substance having a relative luciferase activity (relative value) smaller than 1 is selected as a substance having a negative transcriptional regulation ability.
Example 21
(Selection of a substance that binds to the human PGC-1αb promoter of the present invention)
5 μg of hPGC1αb-P1 (3.0) -pT7Blue, hPGC1αb-P2 (1.8) -pT7Blue, and hPGC1αb-P3 (1.3) -pT7Blue described in Example 12 were used with 10 U each of KpnI and SalI. Digest for 1 hour at 37 ° C. in 20 μL reaction. By subjecting this digestion reaction solution to agarose gel electrophoresis, DNA fragments are recovered. The recovered DNA fragment is purified using QIAEXII Gel Extraction Kit. 1 μg of purified DNA fragment, [α- ³² 5 μL of P] dCTP (Amersham Pharmacia Biotech), 1 μL each of unlabeled nucleotides (dATP, dTTP, dGTP) (Takara Shuzo), 2 μL of 10 × klenow Buffer (Takara Shuzo) and 1 μL of klenow enzyme (Takara Shuzo). By leaving this mixed solution with distilled water to a total of 20 μL for 1 hour at 37 ° C., a labeled DNA fragment is formed in the reaction system. The reaction solution prepared in this manner was added to unreacted [α- ³² In order to separate P] dCTP and labeled DNA fragment, they were subjected to ProbeQuant G-50 Micro Columns equilibrated with 10 mM Tris-HCl and 1 mM EDTA (hereinafter referred to as TE solution), followed by centrifugation (room temperature, 1 , 200 rpm, 1 minute) to elute the labeled DNA fragment. The radioactivity of the obtained eluate was measured with a scintillator, and 10 ⁴ A labeled DNA fragment solution is prepared by diluting with TE solution so as to have cpm / μL. Subsequently, 5 × binding buffer (50 mM Hepes-potassium hydroxide (pH 7.8), 250 mM potassium chloride, 5 mM EDTA (pH 8.0), 25 mM magnesium chloride, 50% glycerol, 25 mM dithiothreitol, 3.5 mM PMSF, 10 μg / ML Aprotinin, 10 μg / mL Pepstatin, 10 μg / mL Leupeptin and 5 mM sodium orthovanadate.) 5 μL, 2 μg / μL polyIdC 1.5 μL, 10 μM test substance 5 μL and labeled DNA fragment solution 2 μL are mixed. To a total of 25 μL. The mixture is allowed to stand at room temperature for 30 minutes and then subjected to polyacrylamide gel electrophoresis. After electrophoresis, the gel was peeled off from the gel plate and dried under reduced pressure with a vacuum pump at 80 ° C. for 1 hour on Whatman 3MM filter paper. After this is exposed to an imaging plate for 2 hours, an image is acquired with BAS2000. The test substance binds to the labeled DNA fragment and a complex consisting of DNA and the test substance is formed. As a result, the mobility on the gel is smaller than that of the free DNA fragment, and a band shift on the image is detected. In this case, the test substance is selected as a substance that binds to the promoter of the present invention (that is, a binding substance).
Example 22
(Acquisition method of substance binding to human PGC-1αb promoter of the present invention)
(1) Production of an affinity column (affinity column for binding substance) for purifying a substance that binds to the human PGC-1αb promoter of the present invention
200 μg of hPGC1αb-P1 (3.0) -pT7Blue, hPGC1αb-P2 (1.8) -pT7Blue, hPGC1αb-P3 (1.3) -pT7Blue described in Example 12 were used with 50 U each of KpnI and SalI. Digest for 1 hour at 37 ° C. in 200 μL reaction. By subjecting this digestion reaction solution to agarose gel electrophoresis, a DNA fragment existing in the vicinity of about 180 bp was recovered. The recovered DNA fragment is purified using QIAEXII Gel Extraction Kit. 44 mg of the purified DNA fragment and 2 mL of cyanogen bromide activated Sepharose 4B are mixed, and the DNA fragment is immobilized on Sepharose while stirring at 1,000 rpm overnight at 4 ° C. Then, in order to eliminate the unreacted cyanogen bromide active group, 20 mL of sodium hydrogen carbonate solution (pH 9.5) containing 1 M glycine is added to the mixture, which is left at 4 ° C. overnight. The gel thus obtained is packed into a 10 × 300 mm chromatograph tube (Iwaki Glass Co., Ltd.) to produce an affinity column for a binding substance.
(2) Preparation of test substance
Mouse striated muscle-derived cultured cell C2C12 ² 4.5x10 per flask in 40 flasks (Iwaki Glass) ⁶ Inject individual cells. The mouse striated muscle-derived cultured cell C2C12 was added to a D-MEM medium (high-level) supplemented with 10% fetal calf serum (JRH life sciences, hereinafter referred to as FCS) and 1 mM sodium pyruvate (Gibco-BRL). Glucose, SIGMA) and culturing at 37 ° C. under 5% carbon dioxide overnight. After culturing, after removing the medium from the flask, the wall of the flask is washed once with 15 mL of phosphate buffer. Add 1 mL to 2 mL of trypsin-EDTA solution (containing 0.05% trypsin, 0.53 mM EDTA, Gibco) to the flask after washing so that the cells are immersed, and leave it at 37 ° C. for 5 minutes. A cell suspension is obtained by adding about 10 times the amount of the above trypsin-EDTA solution to the FBS-containing medium. The resulting cell suspension is centrifuged (room temperature, 1,300 rpm, 5 minutes), and then the supernatant is removed. The suspension obtained by suspending the remaining cell pellet in 15 mL of phosphate buffer (pH 7.5) is centrifuged (room temperature, 1,300 rpm, 5 minutes), and then the supernatant is removed. The remaining cell pellet is suspended in 10 mL of ice-cooled 10 mM Hepes-potassium hydroxide (pH 7.8), 10 mM potassium chloride and 0.1 mM EDTA (pH 8.0) solution (hereinafter referred to as buffer A). The resulting suspension is cooled on ice for 10 minutes and then centrifuged (4 ° C., 1,300 rpm, 5 minutes). After removing the supernatant, the remaining cell pellet is suspended in 30 ml of buffer A. The cells are completely disrupted while the resulting suspension is cooled on ice using Dounce homogenizer pestle B (Wheaton). The obtained cell lysate is centrifuged (4 ° C., 1,300 rpm, 5 minutes), and then the supernatant is removed. The remaining precipitate is suspended in 2 mL of 50 mM Hepes-KOH (pH 7.8), 420 mM potassium chloride, 0.1 mM EDTA (pH 8.0), 5 mM magnesium chloride and 2% glycerol solution (hereinafter referred to as buffer B). . The resulting suspension is gently rotated on a rotator at 4 ° C. for 30 minutes and then centrifuged (4 ° C., 24,000 g, 30 minutes). A supernatant diluted and 5 times diluted liquid can be prepared as a test substance by adding distilled water to the collected supernatant.
(3) Isolation / recovery process
The test substance is applied to the binding substance affinity column prepared in (1) above. Furthermore, in order to wash | clean the said column, 10 mL of buffer B diluted 5 times is provided. Then, the bound substance is eluted from the column by performing gradient elution in which the concentration of potassium chloride in buffer B is increased to 1M. After recovering the eluate, the binding substance is recovered from the eluate.

  According to the present invention, a novel variant of PGC1α, which plays an important role in energy metabolism in vivo: PGC1αb, its promoter, and a drug that selectively improves insulin functions such as heat production in skeletal muscle using these. It has become possible to provide a method and a therapeutic agent for diseases such as diabetes accompanied by abnormal sugar metabolism, comprising as an active ingredient a substance that promotes the expression of PGC1αb.

SEQ ID NO: 7: Partial sequence of PGC1αb SEQ ID NO: 8: Partial sequence of PGC1αb SEQ ID NO: 9: PCR primer SEQ ID NO: 10: PCR primer SEQ ID NO: 11: PCR primer SEQ ID NO: 12: PCR primer SEQ ID NO: 13: PCR Primer SEQ ID NO: 14: PCR primer SEQ ID NO: 15: PCR primer SEQ ID NO: 16: PCR primer SEQ ID NO: 17: PCR primer SEQ ID NO: 18: PCR primer SEQ ID NO: 19: PCR primer SEQ ID NO: 20: PCR primer SEQ ID NO: 21: PCR primer SEQ ID NO: 22: PCR primer SEQ ID NO: 23: PCR primer SEQ ID NO: 24: PCR primer SEQ ID NO: 25: PCR primer SEQ ID NO: 26: PCR primer SEQ ID NO: 27: PCR primer Immer SEQ ID NO: 28: PCR primer SEQ ID NO: 29: PCR primer SEQ ID NO: 30: PCR primer SEQ ID NO: 31: PCR primer SEQ ID NO: 32: PCR primer SEQ ID NO: 36: PCR primer SEQ ID NO: 37: PCR primer SEQ ID NO: 38: PCR primer SEQ ID NO: 39: PCR primer SEQ ID NO: 40: PCR primer SEQ ID NO: 41: PCR primer SEQ ID NO: 42: PCR primer SEQ ID NO: 43: PCR primer SEQ ID NO: 44: PCR primer sequence No. 45: PCR primer SEQ ID NO: 46: PCR primer SEQ ID NO: 47: PCR primer SEQ ID NO: 48: PCR primer SEQ ID NO: 49: PCR primer SEQ ID NO: 50: Partial sequence of PGC1αb SEQ ID NO: 51: P C1αb partial sequences SEQ ID NO 52: PGC1αb partial sequences SEQ ID NO 53: PGC1αb partial sequences SEQ ID NO 54: PGC1αb partial sequences SEQ ID NO 55: PGC1αb subsequence

Claims (33)

Any one of the following proteins (a) to (f):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6,
(B) consisting of an amino acid sequence in which one or more amino acids are deleted, added or substituted in the amino acid sequence described in SEQ ID NO: 2, 4 or 6, comprising the amino acid sequence described in SEQ ID NO: 8 at the N-terminus; And a protein having mitochondrial activation ability,
(C) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence described in SEQ ID NO: 2, comprising the amino acid sequence described in SEQ ID NO: 8 at the N-terminus, and having a mitochondrial activation ability;
(D) a protein comprising an amino acid sequence encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3 or 5 and containing the amino acid sequence set forth in SEQ ID NO: 8 at the N-terminus and having the ability to activate mitochondria ,
(E) consisting of an amino acid sequence encoded by DNA consisting of a base sequence having a sequence identity of 80% or more with DNA consisting of the base sequence shown in SEQ ID NO: 1, 3 or 5; A protein having the amino acid sequence of and having the ability to activate mitochondria,
(F) an N-terminal consisting of an amino acid sequence encoded by a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3 or 5 and a DNA that hybridizes under stringent conditions A protein comprising the amino acid sequence of SEQ ID NO: 8 and comprising the amino acid sequence of a protein having the ability to activate mitochondria.   A gene comprising a base sequence encoding a protein comprising the amino acid sequence according to claim 1.   A primer or probe comprising a polynucleotide that specifically recognizes the gene according to claim 2, or a polynucleotide complementary thereto.   An antibody that specifically recognizes the protein according to claim 1. A promoter capable of controlling the expression of the protein according to claim 1, comprising a gene described in any of the following (g) to (i);
(G) a gene consisting of the base sequence represented by SEQ ID NO: 33, 34 or 35,
(H) a gene comprising a base sequence in which one or more bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 33, 34 or 35, and having transcriptional control ability,
(I) A gene that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 33, 34, or 35 and has transcription control ability.   An expression vector comprising the gene according to claim 2.   An expression vector comprising the promoter according to claim 5.   The expression vector according to claim 7, further comprising a base sequence encoding a gene whose transcription is controlled by the promoter downstream (3 'side) of the promoter.   The expression vector according to claim 8, wherein the gene is the gene according to claim 2.   A transformed cell into which the expression vector according to any one of claims 6 to 9 has been introduced. Method for evaluating the expression control ability of the PGC1αb gene comprising the following steps (1), (2) and (3):
(1) contacting a test substance with a cell capable of expressing the PGC1αb gene;
(2) measuring the expression level of the PGC1αb gene in a cell contacted with a test substance, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance;
(3) A step of evaluating the expression control ability of the PGC1αb gene of the test substance using as an index whether or not the expression level of the PGC1αb gene is varied based on the comparison result of (2). Evaluation method of expression control ability of PGC1αb including the following steps (1), (2) and (3):
(1) contacting a test substance with a cell capable of expressing PGC1αb;
(2) measuring the expression level of PGC1αb in a cell contacted with a test substance, and comparing the expression level with the expression level of PGC1αb in a control cell not contacted with the test substance;
(3) A step of evaluating the PGC1αb expression control ability of the test substance using as an index whether or not the expression level of PGC1αb is varied based on the comparison result of (2). A method for evaluating the expression control ability of the PGC1αb gene, comprising the following steps (1) to (3):
(1) A step of contacting a test substance with a cell capable of expressing a reporter gene to which the promoter according to claim 5 is bound,
(2) measuring the expression level of the reporter gene in a cell contacted with the test substance, and comparing the expression level with the expression level of the reporter gene in a control cell not contacted with the test substance, and (3) ( A step of evaluating the expression control ability of the PGC1αb gene of the test substance using as an index whether or not the expression level of the reporter gene is varied based on the comparison result of 2).   A substance having PGC1αb gene or PGC1αb expression-inducing activity or PGC1αb expression-inhibiting activity, using as an index the expression control ability of the PGC1αb gene or PGC1αb of the test substance evaluated by the evaluation method according to any one of claims 11 to 13 A method for searching for a PGC1αb expression control substance, comprising:   A candidate substance having a PGC1αb gene or PGC1αb expression-inducing activity is selected using the PGC1αb gene or PGC1αb expression control ability of the test substance evaluated by the evaluation method according to any one of claims 11 to 13 as an index. And a method for searching for a sugar metabolism improving agent.   The search method according to claim 15, wherein the search method is performed for searching for a candidate substance for a preventive, ameliorating or therapeutic agent for a disease associated with abnormal sugar metabolism.   The search method according to claim 15 or 16, which is performed for searching for a candidate substance for an agent for preventing, improving or treating an insulin resistant disease.   The search method according to any one of claims 15 to 17, further comprising selecting a compound using as an index that the test substance does not change the expression of the PGC1αa gene or PGC1αa.   A sugar metabolism-improving agent comprising, as an active ingredient, a compound selected by the search method according to claim 15.   An agent for preventing, ameliorating or treating an insulin resistant disease, comprising as an active ingredient a compound selected by the search method according to any one of claims 15 to 18.   A sugar metabolism-improving agent comprising, as an active ingredient, an expression inducer of PGC1αb or PGC1αb gene.   An insulin resistance improving agent comprising a PGC1αb or PGC1αb gene expression inducer as an active ingredient.   The improving agent according to claim 21 or 22, wherein expression of PGC1αa or PGC1αa gene is not changed.   The improving agent according to claim 23, which specifically acts on muscle cells in a living body. The method for searching for a substance that binds to a promoter according to claim 5, comprising the following steps (1) and (2):
(1) A step of bringing the promoter according to claim 5 into contact with a test substance, and (2) a step of examining the presence or absence of complex formation between the promoter and the test substance after the step (1). A method for purifying a substance that binds to the promoter according to claim 5,
(1) a step of bringing the promoter according to claim 5 into contact with a sample to form a complex of the promoter and a substance that binds to the promoter contained in the sample; and
(2) A purification method comprising the step of isolating the binding substance from the produced complex after the step (1).   A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8.   The polypeptide according to claim 27, comprising the amino acid sequence of any one of SEQ ID NOs: 53 to 55.   The antibody according to claim 4, which specifically recognizes the polypeptide according to claim 27 or 28.   A gene encoding the polypeptide according to claim 27 or 28.   A polynucleotide containing the base sequence set forth in SEQ ID NO: 7.   The polynucleotide according to claim 31, comprising the base sequence according to any one of SEQ ID NOs: 50 to 52.   The primer or probe according to claim 3, comprising at least 5 or more consecutive base sequences of the polynucleotide according to claim 31 or claim 32 or a polynucleotide complementary thereto.

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