JP2011116753A - FAT DIFFERENTIATION REGULATOR USING BOND OF PROHIBITIN 2 AND PGC1alpha - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To newly identify a protein in which PHB2 is bonded to clarify a fat differentiation regulation mechanism, to provide a new method of screening a fat differentiation regulator based on the result of the clarification, and to provide a new fat differentiation regulator. <P>SOLUTION: A method of screening a regulator in formation of a complex of prohibitin 2 (PHB2) and a peroxisome proliferator-activated receptor γ/coactivator (PGC1α), including: selecting a material to be tested which promotes or inhibits formation of the complex of the PHB2 and the PGC1α. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a fat differentiation regulator comprising a substance that regulates the formation of a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ / coactivator (PGC1α). The present invention further relates to a method for screening a drug based on the binding of prohibitin 2 (PHB2) to peroxisome proliferator-activated receptor γ · coactivator (PGC1α).

  Prohibitin (hereinafter also referred to as PHB) was first isolated in mammals as a cytostatic factor. PHB is a highly conserved protein from yeast to mammals. It is known that PHB protein has two proteins PHB1 and PHB2 having similar primary structures of amino acids, form a complex with each other, and localize in the inner mitochondrial membrane. The yeast PHB protein has been shown to perform a chaperone-like function responsible for cell cycle control and stabilization of newly synthesized mitochondrial proteins. In nematodes, it has been reported that PHB1 is involved in aging and early development. On the other hand, in mammals, various functions such as transcription control by PHB1 and PHB2 have been suggested.

  In Patent Document 1, repressor of estrogen receptor activity (REA), which is a homologous protein of PHB2, was identified as a substance that acts as a repressor of estrogen receptor (ER) expression, and the interaction between estrogen receptor (ER) and REA is identified. Methods for screening for substances that manipulate expression regulation are described.

  In Patent Document 2, a method of screening for a PHB2 gene regulator in a cell in which PHB2 moves from the mitochondria into the nucleus in the presence of an estrogen receptor (ER) in an estradiol (E2) -dependent manner and expresses a nuclear receptor such as ERα. Is described. Example 13 of Patent Document 2 describes the study of the transcriptional repression activity of PHB2 protein. Specifically, the ERE-Luc gene, the ERα gene, the PHB-2 gene, and the PGC-1α gene inserted into the respective expression vectors were introduced into HeLa cells, and a Renilla luciferase having a CMV promoter was simultaneously introduced as a control. The luciferase activity was measured using this as an internal control. FIG. 14 of Patent Document 2 shows the result of measurement of relative luciferase activity in HeLa cells into which each gene was introduced. In Example 13 of Patent Document 2, when the ERα gene and the PGC-1α gene, which is a coactivator of ERα, are introduced into HeLa cells, the transcriptional activity is enhanced, particularly in the presence of E2, that the transcriptional activity is further enhanced. When co-expressing PHB2 protein, the effect of suppressing transcriptional activity was detected, and it is described that PHB2 protein acts to suppress transcriptional regulation of PGC-1α protein, which is a coactivator of ERα. . As described above, Patent Document 2 teaches that PHB2 protein acts to suppress the transcriptional regulation of PGC-1α protein. However, PHB2 protein regulates the transcription of PGC-1α protein by any action mechanism. There is no teaching about working in a restraint. That is, Patent Document 2 does not suggest any interaction or binding between PHB2 and PGC1α. In addition, since transcription is generally controlled by a very complex mechanism, even if it becomes clear that one protein acts to suppress transcriptional regulation of another protein, such transcriptional regulation It is necessary to conduct further analysis as to what kind of mechanism of action the suppression of is performed. Therefore, even if the fact that transcriptional regulation is suppressed is found, it does not predict that the proteins directly interact with each other or are directly bound to each other.

  Non-patent document 1 shows that REA is related to obesity because the expression level of REA was halved in REA knockout mice and showed significant weight gain. On the other hand, Non-Patent Document 2 describes that in PPARγ knockout mice, an increase in adipocytes is suppressed by a decrease in the expression of a gene transcribed by PPARγ. Non-Patent Document 3 describes that obesity can be improved by suppressing the activity with an antagonist of PPARγ.

International Publication WO00 / 26232 International Publication No. WO2007 / 061022

Molecular Cellular Biology, Vol.25, No.5, pp1989-1999 (2005) J Biol Chem, Vol.276, No.44, pp41245-41254 (2001) J Clin Invest, Vol.108, No.7, pp1001-1013 (2001)

  It has been reported that the physiological effects of estrogen are related to sex, metabolic syndrome, and osteoporosis. In particular, obesity in postmenopausal women with reduced estrogen secretion is evident. In addition, metabolic syndrome such as fatty liver is prominent in male patients who cannot synthesize estrogen. However, their mechanism of action is not clear. It is also known that PHB2 shuttles mitochondria and nucleus in the presence of estrogen receptor in an estradiol-dependent manner and suppresses estrogen receptor activity in the nucleus, but the interacting protein in the nucleus of PHB2 is other than estrogen receptor It is not very clear. Furthermore, obesity suppression cannot be explained only by the transcriptional regulatory action of estrogen receptor.

  The present invention elucidates the regulation mechanism of adipose differentiation by newly identifying a protein to which PHB2 binds, and based on this, a novel adipose differentiation regulator and a novel screening method for adipose differentiation regulator are disclosed. It was set as a problem to be solved.

  The present inventors diligently studied to solve the above problems, and identified that the interacting protein in the nucleus of PHB2 is a peroxisome proliferator-activated receptor γ · coactivator (PGC1α). And, as a result of examining whether or not the activity of the transcription factor PPARγ having an action of promoting fat differentiation is suppressed through the binding of PHB2 and PGC1α, PHB2 directly binds to PGC1α, As a result, it was clarified that the transcription activity of PPARγ was suppressed. Furthermore, the present inventors have clarified that estradiol moves PHB2 existing in mitochondria in the adipocyte to the nucleus, and that estradiol suppresses adipogenesis of the adipocyte. Furthermore, the present inventors have clarified that a PHB2 mutant localized in the nucleus suppresses adipogenesis of preadipocytes even in the absence of estradiol. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) A fat differentiation regulator comprising a substance that regulates the formation of a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ / coactivator (PGC1α).
(2) Peroxisome proliferator-activated receptor γ · coactivator (PGC1α) activity regulator comprising a partial fragment or mutant of prohibitin 2 (PHB2).
(3) A fat differentiation regulator comprising a fragment or mutant of prohibitin 2 (PHB2).
(4) A complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ / coactivator (PGC1α).
(5) PHB2 and PGC1α comprising selecting a test substance that promotes or inhibits the formation of a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ · coactivator (PGC1α) Screening method for modulators of complex formation.
(6) An agent for regulating fat differentiation, comprising selecting a test substance that modulates inhibition of coactivator activity of peroxisome proliferator-activated receptor γ · coactivator (PGC1α) by prohibitin 2 (PHB2) Screening method.
(7) A screening method for an agent for regulating fat differentiation, comprising selecting a test substance that regulates inhibition of transcription activity of peroxisome proliferator-activated receptor γ (PPARγ) by prohibitin 2 (PHB2).

  ADVANTAGE OF THE INVENTION According to this invention, the screening method of a fat differentiation regulator can be provided. Furthermore, according to the present invention, it is possible to provide a fat differentiation regulator and a peroxisome growth factor-activated receptor γ · coactivator (PGC1α) activity regulator.

FIG. 1A shows a medium for differentiating 3T3-L1 cells into adipocytes. FIG. 1B shows cells on day 0 and day 7 after differentiation induction. FIG. 1C shows the results of RT-PCR examining the gene expression of differentiation markers PPARγ and aP2 (FABP4) in cells at 0, 2, 4, 7, and 9 days after differentiation induction. FIG. 1D shows the results of RT-PCR using RNA extracted from 3T3-L1 cells that 3T3-L1 cells endogenously express estrogen receptor α (ERα) and PPARγ coactivator 1α (PGC1α). Show. FIG. 1E shows the results of addition of 17β estradiol (E2) and E2 inhibitor ICI 182,780 (ICI) to the differentiation medium of 3T3-L1 cells, and the influence on differentiation was measured by adipogenesis assay. FIG. 1F shows the results of cell immunostaining using an anti-PHB2 antibody capable of immunostaining. Figure 2A shows the suppression of PPARγ transcriptional activity by PHB2 using a luciferase reporter gene (PPRE-TK-Luc) that has a PPAR-responsive element (PPRE) upstream of the thymidine kinase (TK) promoter. Results are shown. Figure 2B shows a fusion protein of GAL4-DBD and PGC1α (DBD-PGC1α), and a luciferase reporter gene (GAL4-Luc) that has a UAS (upstream activator sequence) sequence upstream of GAL4 recognition sequence. The result of having performed transcriptional activity analysis is shown. Fig. 2C shows that GST-PHB2 and PGC1α-myc were mixed in a test tube, GST-PHB2 complex was purified with glutathione beads, and Western blotting was performed using anti-myc antibody. The result of having examined whether PGC1 (alpha) -myc exists in it is shown. Fig. 2D shows an immunoprecipitation experiment using an anti-DBD antibody prepared by preparing a cell extract in which HeLa cells co-expressed a fusion protein (PHB2-FLAG) with a FLAG tag added to the carboxyl terminus of DBD-PGC1α and PHB2. The results are shown. FIG. 2E shows the result of an immunoprecipitation experiment using a cell extract obtained by co-expressing DBD-PGC1α (1-600) and PHB2-FLAG in HeLa cells with an anti-FLAG antibody. FIG. 3A shows the results of examining whether or not the transfer of endogenous PHB2 from the mitochondria to the nucleus by estrogen depends on ERα. FIG. 3B shows the results of examining whether translocation of endogenous PHB2 to the nucleus by E2 administration regulates the activities of PPARγ and PGC1α. FIG. 3C shows the results of examining the suppression of the transcriptional activity of DBD-PGC1α in the presence of E2 in an experiment in which ERα was co-expressed with DBD-PGC1α and GAL4-Luc. Fig. 4A shows the introduction of PHB2 wild type and FLAG tag fusion (PHB2-FLAG) and PHB2C and FLAG tag fusion (PHB2C-FLAG) into 3T3-L1 cells, followed by cell immunostaining with anti-FLAG antibody. The results are shown. FIG. 4B shows the results of performing adipogenesis assay by inducing fat differentiation using 3T3-L1 cells introduced with GFP, PHB2, PHB2C, and PGC1α by lentivirus. FIG. 4C shows the results of extracting RNA from cells on day 7 after differentiation and measuring the gene expression level by quantitative PCR. FIG. 5A shows the results of comparison of changes over time in body weights of C57BL6J female mice that were ovariectomized 10 weeks after birth on an E2-containing diet or an E2-free diet. FIG. 5B shows the result of measuring the weight of the uterus after dissection of the mouse after 60 days of breeding. FIG. 5C shows the results of measuring the cancellous bone density of the tibia using X-ray CT of mice after 60 days of breeding. FIG. 5D shows the results of measuring visceral fat (VAT) and subcutaneous fat (SAT) amounts by CT. FIG. 5E shows the results of comparison of fat cell diameters after dissecting the mice in each group and preparing tissue sections of periovar fat. FIG. 5F shows the results of confirming the intracellular localization of endogenous PHB2 in adipose tissue by tissue immunostaining. FIG. 6 shows a model of the molecular mechanism in obesity suppression of PHB2. FIG. 7A shows the results of measurement of suppression of transcriptional activity of PPARα using PPRE-TK-Luc as a reporter gene by PHB2 in the presence of PPARα and PGC1α. FIG. 7B shows the results of measurement of suppression of transcriptional activity of ERα in the reporter gene ERE-Luc having an estrogen responsive element (ERE) by PHB2 in the presence of ERα and PGC1α. FIG. 7C shows the results of preparing a plurality of deletion mutants of PHB2 and examining their activities in a system using GAL4-DBD-PGC1α and GAL4-Luc. Fig. 8 shows the results of examining the transcriptional activity of various coactivators against the transcriptional activity of PPARγ, a transcription factor that promotes adipose differentiation, and whether PHB2 suppresses the transcriptional activity of PPARγ in the presence of these coactivators. The result of having examined is shown.

Embodiments of the present invention will be described below.
(1) Screening method for adipose differentiation regulator In the present invention, it was identified that the interacting protein in the nucleus of PHB2 is a peroxisome proliferator-activated receptor γ · coactivator (PGC1α). And it was clarified that PHB2 directly binds to PGC1α and suppresses the transcriptional activity of PPARγ. Furthermore, it has been clarified that PHB2 existing in mitochondria in adipose precursor cells is transferred to the nucleus by estradiol, and that estradiol suppresses adipose differentiation of adipose precursor cells. Furthermore, it was also clarified that PHB2 mutants localized in the nucleus suppress fat differentiation of adipose precursor cells even in the absence of estradiol.

  The human PHB2 gene is registered in NCBI as NCBI registration number NM_007273. The human PHB2 protein is registered with NCBI as NCBI registration number NP_009204. The base sequence of the human PHB2 gene is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the human PHB2 protein is shown in SEQ ID NO: 2 in the sequence listing. Examples of PHB2 genes or PHB2 proteins derived from organisms other than humans include, for example, Saccharomyces cerevisiae (PHB2_YEAST (swissprot: accession P50085)), Nematode (Caenorhabditis elegasis Esp2) , Derived from Arabidopsis thaliana (ATPHB2 (PROHIBITIN 2) [Arabidopsis thaliana] (NCBI registration number NP_973755)), derived from Xenopus tropicalis (prohibitin 2X icalis] (NCBI Registration No. NP_001016551)), mouse-derived (PHB2_MOUSE (swissprot: accession 035129)) and rat (PHB2_RAT (swissprot: accession Q5XIH7)) include those of. In the present invention, the PHB2 gene is not limited to DNA consisting of the base sequence described in the above-mentioned registration number, and in these DNAs, one or more (eg, 1 to 10, preferably 1 to 5) bases are present. A DNA encoding a protein consisting of a base sequence deleted, substituted or added and having a PHB2 protein function is also included. The PHB2 gene has 90% or more, preferably 95% or more, 97% or more, more preferably 98% or more, particularly preferably 99% or more identity to the DNA comprising the base sequence described in the above registration number. And a DNA encoding a protein having a PHB2 protein function. Or not only DNA which codes the amino acid sequence as described in the registration number mentioned above, In these amino acid sequences, 1 or multiple (for example, 1-10, preferably 1-5) amino acids are deleted, substituted or A DNA encoding a protein consisting of an added amino acid sequence and having a PHB2 protein function is also included in the PHB2 gene.

The human peroxisome proliferator-activated receptor γ · coactivator (PGC1α) gene is registered in NCBI as NCBI registration number NM — 031261. The human PGC1α protein is registered with NCBI as NCBI registration number NP — 037393.
The peroxisome proliferator-activated receptor γ (PPARγ) gene is registered with NCBI as NCBI accession number NM — 015869. Human PPARγ protein is registered with NCBI as NCBI registration number NP_056853.

  The present invention will be described based on the functions newly found for the above-described PHB2.

  In the present invention, it was first identified that prohibitin 2 (PHB2) binds to peroxisome proliferator-activated receptor γ · coactivator (PGC1α). That is, a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ · coactivator (PGC1α) was first identified by the present invention and is included within the scope of the present invention.

  In the present invention, PHB2 and PGC1α are selected by selecting a test substance that promotes or inhibits the formation of a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ · coactivator (PGC1α). Can be screened for modulators of complex formation. That is, in the present invention, the formation of a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ / coactivator (PGC1α) in the presence of the test substance is analyzed. A test substance that promotes or inhibits formation can be selected as a regulator of the formation of a complex of PHB2 and PGC1α.

  Furthermore, according to the present invention, in the presence of a test substance, (a) binding of prohibitin 2 (PHB2) to peroxisome proliferator-activated receptor γ · coactivator (PGC1α), (b) prohibitin 2 ( Inhibition of coactivator activity of peroxisome proliferator-activated receptor γ · coactivator (PGC1α) by PHB2), (c) Peroxisome proliferator-activated receptor γ (PPARγ) by prohibitin 2 (PHB2) By measuring either the suppression of transcriptional activity of (d) or estradiol-induced migration of prohibitin 2 (PHB2) to the nucleus, and selecting a test substance that changes any of the above (a) to (d) A screening method for a fat differentiation regulator can be used.

  In one example of the present invention, a fat differentiation regulator by selecting a test substance that regulates inhibition of coactivator activity of peroxisome proliferator-activated receptor γ · coactivator (PGC1α) by prohibitin 2 (PHB2) Or screening for a fat differentiation regulator by selecting a test substance that regulates inhibition of transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) by prohibitin 2 (PHB2) Also good.

  In the present invention, (a) binding of PHB2 and PGC1α, (b) suppression of PGC1α coactivator activity by PHB2, (c) suppression of PPARγ transcription activity by PHB2, or (d) nucleus of PHB2 by estradiol A substance that promotes any one or more of the transition to can be selected as a candidate substance for a fat differentiation inhibitor.

  Conversely, (a) binding of PHB2 and PGC1α, (b) suppression of PGC1α coactivator activity by PHB2, (c) suppression of PPARγ transcriptional activity by PHB2, or (d) PHB2 nuclear activation by estradiol A substance that suppresses any one or more of the migration can be selected as a candidate substance for a fat differentiation promoter.

  The screening method can be performed in either a cell-free system or a cell system. When performed in a cell-free system, for example, in the presence and absence of the test substance, PHB2 and PGC1α are allowed to interact to form a complex, compared to the case in the absence of the test substance, A test substance that causes a difference in the amount of the complex formed in the presence of the test substance can be selected as a substance that promotes or inhibits the formation of the complex. Moreover, when performing by a cell system, a PHB2 gene expression cell can be used, for example. Any cell may be used as long as it expresses the PHB2 gene. For example, HeLa cells (derived from human cervical cancer), MCF-7 cells (derived from human breast cancer), U-2OS cells (derived from human osteosarcoma), and human fibers Examples include human-derived cell lines such as blasts, B6.1 cells (derived from mouse myeloma), mouse embryonic stem cells, and SWISS 3T3 cells (derived from mouse fibroblasts). Alternatively, cells into which the PHB2 gene has been introduced (or overexpressed) are also included in the PHB2 gene expressing cells. For example, the PHB2 gene contained in the PCR product or vector can be introduced into cells by electroporation, calcium phosphate, lipofection, or the like. In the present invention, cells in which PGC1α is further expressed can be used as the above-mentioned PHB2 gene-expressing cells. For example, the PGC1α gene contained in the PCR product or vector can be introduced into cells by electroporation method, calcium phosphate method, lipofection method, virus vector method or the like. As a method for confirming that the PHB2 gene and the PGC1α gene are expressed in the above cells, at the mRNA level, for example, RT-PCR using a primer or probe specific to the PHB2 gene and the PGC1α gene, quantitative PCR, or Northern blotting The method of confirming by is mentioned. In addition, at the protein level, for example, the expression of PHB2 gene and PGC1α gene should be confirmed using immunological methods such as ELISA, flow cytometry, Western blotting using antibodies specific for PHB2 protein and PGC1α protein. Can do.

  Examples of test substances that can be used in the present invention include nucleic acids, peptides, proteins, synthetic compounds, microbial culture supernatants, natural components derived from plants and marine organisms, plant extracts, animal tissue extracts, and the like. . The test substance may be an individual low-molecular synthetic compound, a compound present in a natural product extract, a compound library, a phage display library, or a combinatorial library. The test substance is preferably a low molecular compound, and a compound library of low molecular compounds can also be used. The construction of a compound library is known to those skilled in the art, and a commercially available compound library can also be used.

  In the screening method of the present invention, for example, (a) binding of prohibitin 2 (PHB2) to peroxisome proliferator-activated receptor γ / coactivator (PGC1α), (b) parsing with prohibitin 2 (PHB2). Inhibition of coactivator activity of oxysome growth factor-activated receptor γ-coactivator (PGC1α), (c) transcription activity of peroxisome growth factor-activated receptor γ (PPARγ) by prohibitin 2 (PHB2) The test substance can be screened using as an index either suppression or (d) migration of prohibitin 2 (PHB2) to the nucleus by estradiol.

(A):
When binding of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ / coactivator (PGC1α) is used as an index, as described above, prohibitin is present in the presence and absence of the test substance. 2 (PHB2) and peroxisome proliferator-activated receptor γ · coactivator (PGC1α) may be analyzed to select a test substance that promotes or inhibits the formation of the complex.

(B):
When inhibition of the coactivator activity of peroxisome proliferator-activated receptor γ · coactivator (PGC1α) by indicator 2 (PHB2) is used as an indicator, prohibitin is present in the presence and absence of the test substance. 2 (PHB2) and peroxisome proliferator-activated receptor γ · coactivator (PGC1α) are brought into contact, and this system further includes a measurement system capable of measuring the coactivator activity of PGC1α. As a measurement system capable of measuring the coactivator activity of PGC1α, a luciferase reporter gene (PPRE-TK-Luc) having a PPAR-responsive element (PPRE) upstream of a thymidine kinase (TK) promoter, PPARγ gene, PHB2 A luciferase reporter gene (GAL4-Luc) that has a gene co-expressed with PGC1α, or a fusion protein of GAL4-DBD and PGC1α (DBD-PGC1α) and a UAS (upstream activator sequence) sequence that is a recognition sequence for GAL4 upstream ) And the PHB2 gene are co-expressed. In the system described above, inhibition of PGC1α coactivator activity can be measured using the expression level of the luciferase reporter gene as an index.

(C):
When inhibition of the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) by prohibitin 2 (PHB2) is used as an indicator, prohibitin 2 (PHB2) and peroxidase in the presence and absence of the test substance are used. An oxysome growth factor-activated receptor γ · coactivator (PGC1α) is contacted, and a measurement system capable of measuring suppression of PPARγ transcription activity is further included in this system. As a measurement system capable of measuring the suppression of the transcriptional activity of PPARγ, a luciferase reporter gene (PPRE-TK-Luc) having a PPAR responsive element (PPRE) upstream of the thymidine kinase (TK) promoter, PPARγ gene, PHB2 Examples include a system in which a gene and PGC1α are co-expressed. In the system described above, suppression of the transcriptional activity of PPARγ can be measured using the expression level of the luciferase reporter gene as an index.

(D):
In the case of using estradiol as a marker for the transition of prohibitin 2 (PHB2) to the nucleus, in the presence and absence of the test substance, prohibitin 2 (PHB2) and peroxisome proliferator activated receptor A γ coactivator (PGC1α) is brought into contact. Thereafter, screening can be performed by analyzing the localization of prohibitin 2 (PHB2) in cells by, for example, cell immunostaining.

  If the above index is significantly changed in the presence of the test substance compared to the absence of the test substance, the test substance is determined to be a PHB2 activity regulator or a fat differentiation regulator. be able to.

  The PHB2 activity regulator or fat differentiation regulator selected by the screening method of the present invention described above is also included in the scope of the present invention.

(2) Fat differentiation regulator and PGC1α activity regulator According to the present invention, the formation of a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ / coactivator (PGC1α) is regulated. A fat differentiation regulator comprising the substance to be provided is provided. For example, a substance that promotes the formation of a complex of PHB2 and PGC1α is useful as a fat differentiation inhibitor, and a substance that suppresses the formation of a complex of PHB2 and PGC1α is useful as a fat differentiation promoter. A substance that regulates the formation of a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ / coactivator (PGC1α) used in the present invention is selected by the screening method of the present invention described above. Can be used.

  Furthermore, according to the present invention, a peroxisome proliferator-activated receptor γ · coactivator (PGC1α) activity modulator comprising a partial fragment or mutant of prohibitin 2 (PHB2), and prohibitin 2 (PHB2) A fat differentiation regulator is provided comprising a partial fragment or variant.

  As a partial fragment or variant of prohibitin 2 (PHB2), a protein consisting of a part of the amino acid sequence of prohibitin 2 (PHB2) and having a PGC1α activity regulating action or a fat differentiation regulating action, “PHB2” or In the amino acid sequence of “PHB2 partial fragment”, one or more (for example, 1 to 30, preferably 1 to 20, more preferably 1 to 10, particularly preferably 1 to 5) amino acids are deleted. And a protein having a substituted and / or added amino acid sequence and having a PGC1α activity regulating action or fat differentiation regulating action. The partial fragment or mutant of prohibitin 2 (PHB2) used in the present invention is preferably a protein that is localized in the nucleus even in the absence of estradiol. In addition, a method for introducing a desired amino acid mutation into a predetermined amino acid sequence is known to those skilled in the art. Proteins with mutations can be prepared.

  Peroxisome proliferator-activated receptor γ · coactivator (PGC1α) activity inhibitor and adipose differentiation inhibitor include a partial fragment of PHB2 consisting of an amino acid sequence excluding the micotondoria localization sequence from the amino acid sequence of PHB2. Can be used. As an example, the peroxisome proliferator-activated receptor γ · coactivator (PGC1α) activity inhibitor and the adipose differentiation inhibitor include amino acid numbers 51-299, amino acid numbers 51-150, or amino acid numbers 201 of PHB2. A partial fragment of PHB2 consisting of -299 can be used. Moreover, as a PGC1 (alpha) activity promoter and a fat differentiation promoter, the partial fragment of PHB2 which consists of amino acid numbers 51-250, amino acid numbers 51-200, or amino acid numbers 101-250 of PHB2 can be used.

  The PGC1α activity regulator and fat differentiation regulator of the present invention can be administered to any mammal including humans, but are preferably administered to humans. The PGC1α activity regulator and the fat differentiation regulator of the present invention can be used, for example, for improving or treating metabolic syndrome, improving or treating obesity, and for controlling aging by regulating PGC1α activity. Or can be used to improve or treat fatty liver.

  The preparation form of the PGC1α activity modulator and the fat differentiation regulator of the present invention is not particularly limited, and an appropriate form most suitable for the purpose of treatment or prevention is selected from the preparation forms for oral administration or parenteral administration. Is possible. Examples of the dosage form suitable for oral administration include tablets, capsules, powders, granules, fine granules, syrups, solutions, emulsions, suspensions, chewables and the like. Suitable dosage forms include, for example, transdermal absorption in the form of injections (subcutaneous injection, intramuscular injection, intravenous injection, etc.), drops, inhalants, sprays, suppositories, gels or ointments. Examples include, but are not limited to, transmucosal absorbents, transdermal absorbents in the form of patches, tapes and the like.

  Liquid preparations suitable for oral administration, such as solutions, emulsions or syrups, are water, sorbits, sugars such as fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil. , P-hydroxybenzoic acid esters and other preservatives, strawberry flavor, peppermint and other flavors. For the production of solid preparations such as capsules, tablets, powders or granules, excipients such as lactose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc In addition, binders such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, surfactants such as fatty acid esters, plasticizers such as glycerin, and the like can be used.

  An injectable or infusion preparation suitable for parenteral administration preferably contains the above-mentioned substances as active ingredients dissolved or suspended in a sterile aqueous medium isotonic with the blood of the recipient. For example, in the case of an injection, a solution can be prepared using a salt solution or an aqueous medium composed of a mixture of salt water and another solution. For preparations for parenteral administration, one or two selected from glycols, oils, flavors, preservatives, excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc. The above auxiliary components can also be added.

  The dosage and frequency of administration of the PGC1α activity regulator and the fat differentiation regulator of the present invention depend on various factors such as the type and severity of symptoms, the dosage form, conditions such as patient age and weight, and the presence or absence of complications. Generally, the dose of the active ingredient is about 10 μg / kg to 10 mg / kg per day.

  EXAMPLES Hereinafter, although an Example is given and this invention is further demonstrated, this invention is not limited to these Examples.

Example 1
Adipose precursor cells (3T3-L1 cells) are cell lines suitable for adipocyte differentiation studies. By using a differentiation medium supplemented with drugs such as dexamethasone, IBMX, and insulin, 3T3-L1 cells differentiate into adipocytes (FIG. 1A). Seven days after induction, many lipid droplets containing neutral fat that are stained red with oil red O appear in the cytoplasm (FIG. 1B).

After differentiation induction, cells were collected on 0, 2, 4, 7, and 9 days, and RNA was extracted and subjected to RT-PCR. The experimental conditions for RT-PCR are as follows.
In RT-PCR, cDNA was first synthesized from 1 μg of total RNA by reverse transcription using Superscript II. Next, PCR was performed using this cDNA as a template and rTaq polymerase (TAKARA). PCR primers and conditions are as shown below.

  For the mouse PPARγ gene, primers of 5′-AAG AGC TGA CCC AAT GGT TG-3 ′ (SEQ ID NO: 3) and 5′-TCC ATA GTG GAA GCC TGA TGC-3 ′ (SEQ ID NO: 4) are used. The reaction of 94 ° C., 30 sec, 55 ° C., 30 sec, 74 ° C. and 1 min was performed 30 times.

  For the adipocyte-specific fatty acid binding protein (aP2) gene, 5'-ATG TGT GAT GCC TTT GTG GGA-3 '(SEQ ID NO: 5) and 5'-TGC CCT TTC ATA AAC TCT TGT-3' (sequence Using the primer No. 6), the reaction at 94 ° C., 30 sec, 55 ° C., 30 sec, 74 ° C. and 1 min was performed 20 times.

  For amplification of the mouse GAPDH gene, primers 5'-GCC CAT CAC CAT CTT CCA G-3 '(SEQ ID NO: 7) and 5'-TGA GCC CTT CCA CAA TGC C-3' (SEQ ID NO: 8) are used. The reaction of 94 ° C., 30 sec, 55 ° C., 30 sec, 74 ° C. and 1 min was performed 25 times.

  For amplification of mouse ERα gene, primers of 5'-ACC GTG TCC TGG ACA AGA TC-3 '(SEQ ID NO: 9) and 5'-GTC ATA GAG GGG CAC AAC GT-3' (SEQ ID NO: 10) are used. The reaction at 94 ° C., 30 sec, 55 ° C., 30 sec, 74 ° C. and 1 min was carried out 40 times.

  Furthermore, for amplification of the mouse PGC1α gene, primers of 5'-gaa agg gcc aaa cag aga ga -3 '(SEQ ID NO: 11) and 5'- gta aat cac acg gcg ctc tt -3' (SEQ ID NO: 12) are used. The reaction at 94 ° C., 30 sec, 55 ° C., 30 sec, 74 ° C. and 1 min was carried out 40 times.

  These reaction products were electrophoresed on a 3% agarose gel to analyze the products. As differentiation progressed, gene expression of differentiation markers PPARγ and aP2 (FABP4) increased (FIG. 1C). GAPDH is a control. It was confirmed by RT-PCR using RNA extracted from 3T3-L1 cells that 3T3-L1 cells endogenously express estrogen receptor α (ERα) and PPARγ coactivator 1α (PGC1α) (FIG. 1D). .

  3T3-L1 cells that have differentiated into adipocytes can be extracted and quantified after staining with oil red O to measure the degree of adipogenesis (adipogenesis assay). 17β estradiol (E2) and E2 inhibitor ICI 182,780 (ICI) were added to the differentiation medium of 3T3-L1 cells, and the influence on differentiation was examined.

  Adipogenesis assay was performed according to the protocol of Adipogenesis Assay Kit (Chemicon). 3T3-L1 cells are seeded in a 24-well plate, cultured for 7 days in adipose differentiation medium, washed with PBS, stained with 0.36% oil red O staining solution for 15 minutes, washed 3 times with the washing solution, and then with the extract. Oil red O stained with neutral fat was extracted. The absorbance was measured at 520 nm, and the amount of neutral fat accumulated in the cells was quantified (n = 4).

  As a result, fat differentiation was inhibited in a concentration-dependent manner by the addition of E2. On the other hand, addition of ICI did not inhibit differentiation, and addition of ICI suppressed differentiation inhibition by E2 (FIG. 1E). From the above, it was shown that estrogen has an adipocyte differentiation inhibitory action. Statistical differences were examined by T-test.

Cell immunostaining was performed using an anti-PHB2 antibody capable of immunostaining. The anti-PHB2 antibody capable of immunostaining was prepared by immunizing rabbits with GST-PHB2 protein prepared in E. coli as an antigen. Cellular immunostaining was performed as follows. 3T3-L1 preadipocytes are seeded in 35-mm poly-L-lysine-coated glass-bottomed dishes (Matsunami Glass Ind.) And added to cell culture medium supplemented with 10 ^-8 M ICI or 10 ^-7 M E2 for 2 hours After incubation, the cells were treated with a fixative (4% PFA and 0.4% Triton X-100 in PBS) at room temperature for 20 minutes. After washing with PBS, cell staining was performed using anti-GST-PHB2 antibody (diluted 500 times) as the primary antibody and Alexa 488-conjugated anti-rabbit IgG (Molecular Probes) (diluted 1000 times) as the secondary antibody. . The fluorescence image was taken and analyzed using a TCS SP5 (Leica) confocal laser microscope.

As a result, the endogenous PHB2 of 3T3-L1 cells was localized mainly in mitochondria when ICI was added, but when E2 was added, a part of the endogenous PHB2 was transferred to the nucleus. (FIG. 1F).
From the above, 3T3-L1 cells have endogenous expression of ERα and PGC1α genes, estrogen suppresses 3T3-L1 cell fat differentiation, and 3T3-L1 cells have endogenous PHB2 by estrogen stimulation. It was shown that a part of migrating from the mitochondria to the nucleus.

Example 2
The nuclear action of PHB2 was examined using a dual luciferase assay with renifer reniformis-derived luciferase (RLuc) as an internal standard.

  Dual luciferase assay was performed according to the protocol using Dual-luciferase Reporter Assay System (Promega). Specifically, HeLa cells have a Renilla luciferase reporter gene (TK-RLuc) upstream of the thymidine kinase (TK) promoter and a firefly luciferase reporter gene (PPRE) upstream of the TK promoter with a PPAR responsive element (PPRE). -TK-Luc), PPARγ gene, PGC1α gene, PHB2 gene and the like were mixed in the indicated combinations, and DNA was introduced into the cells using Lipofectamine 2000 (Invitrogen). After 5 hours, the medium was changed, and after 22 hours in total, the cells were collected with a cell lysate and then measured with a luminometer. Using the activity of Renilla luciferase as an internal standard, the transcriptional activity was compared with firefly luciferase (n = 3-4). In addition, PPARγ agonists were added at a final concentration of 10 μM, Pioglitazone (Pio) and 10 μM Troglitazone (Tro), respectively, to the medium at the time of medium replacement.

  In addition, GAL4 assay uses TK-RLuc as an internal standard and firefly luciferase reporter gene (GAL4-Luc) having UAS (upstream activator sequence) sequence upstream of GAL4 recognition sequence as a reporter gene. Display binding domain (GAL4-DBD), GAL4-DBD and PGC1α fusion protein (DBD-PGC1α) gene, PHB2 (PHB2-wt) gene, PHB2 N-terminal deletion mutant (PHB2 (51-299)) gene The gene was introduced in the combinations shown in (1). The luciferase activity was measured according to the aforementioned dual luciferase assay.

  First, using the luciferase reporter gene (PPRE-TK-Luc) having a PPAR responsive element (PPRE) upstream of the thymidine kinase (TK) promoter, the PPARγ gene and the PHB2 gene were coexpressed. Inhibition of the transcriptional activity of PPARγ was not significant. However, when PGC1α, which is a coactivator of PPARγ, was coexpressed, PHB2 showed a remarkable transcription repressing activity (FIG. 2A). Next, in order to examine the possibility that PHB2 directly acts on PGC1α, a fusion protein of GAL4-DBD and PGC1α (DBD-PGC1α) was prepared, and the upstream activator sequence (UAS) sequence, which is a recognition sequence for GAL4, was upstream. When the transcriptional activity analysis was performed using the luciferase reporter gene (GAL4-Luc) contained in PHB2, PHB2 significantly suppressed the transcriptional activity of DBD-PGC1α (FIG. 2B).

  Whether or not PHB2 directly binds to PGC1α protein is expressed in E. coli and purified glutathione S transferase (GST) and PHB2 fusion protein (GST-PHB2), and the myc tag at the carboxyl terminus of PGC1α expressed in HeLa cells It was examined by GST-pulldown assay using a protein (PGC1α-myc) fused with.

  The GST-pulldown assay was performed as follows. First, PGC1α-Myc was transfected into HeLa cells using Lipofectamine 2000. After culturing for 18 hours, the cells were collected, and the cell extract was prepared with TNE buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA) supplemented with 0.1% NP-40. 2 μg each of GST or GST-PHB2 protein expressed in E. coli and the above extract were mixed together with GST beads in TNE (0.1% NP-40) overnight. After washing three times with TNE (1% NP-40), SDS-PAGE was performed using these precipitates, transferred to an ECL-nitrocellulose membrane (Amersham), and then mouse monoclonal anti-Myc antibody (BD Biosciences) was added. A 1000-fold dilution was used as a primary antibody, and a secondary antibody was an HRP-conjugated anti-mouse Ig antibody (GE Healthcare) used at a 2000-fold dilution. Detection was performed using an ECL detection kit (GE Healthcare). For HeLa (PGC1α-myc) input, a 1/40 cell extract was used. As GST-PHB2 protein, full-length GST-PHB2 (1-299) and C-terminal deletion mutants GST-PHB2 (1-179) and GST-PHB2 (1-100) were used.

  As described above, after mixing GST-PHB2 and PGC1α-myc in a test tube, purify the GST-PHB2 complex with glutathione beads, and perform Western blotting using an anti-myc antibody, this GST-PHB2 complex When PGC1α-myc was present, it was shown that PHB2 binds to PGC1α (FIG. 2C). Analysis of deletion mutants of PHB2 showed that the binding site for PGC1α was present at the 179-299 amino acid site of the PHB2 protein (FIG. 2C).

  Next, immunoprecipitation experiments were performed. First, DBD-PGC1α or DBD-fused PGC1α C-terminal deletion mutant and PHB2 carboxyl-terminal fusion protein (PHB2-FLAG) were co-expressed in HeLa cells, and each cell was recovered and RIPA Suspend in buffer (10 mM Tris HCl (pH 8.0), 1% DOC, 1% Triton X-100, 0.1% SDS, 150 mM NaCl). Qing was extracted. First, Protein G Sepharose was added to the extract, and the centrifuged supernatant was recovered to remove non-specific binding, and then 1 μg of rabbit polyclonal anti-DBD antibody (Santa Cruz) and Protein G Sepharose were added to the supernatant. Then, after binding at 4 ° C., the complex binding to the DBD fusion protein was purified by centrifugation and washing. Using this, SDS-PAGE was performed, transferred to a nitrocellulose membrane, and then Western blotting was performed at a 30,000-fold dilution of anti-FLAG antibody (M2, Sigma) to detect whether the FLAG fusion protein was precipitated together with the DBD fusion protein. As a reverse experiment, as described above, immunoprecipitation was performed with an anti-FLAG antibody, and then Western blotting was performed with an anti-DBD antibody to detect whether the DBD fusion protein was precipitated together with the FLAG fusion protein.

  When a cell extract was prepared by co-expressing DBD-PGC1α and PHB2-carboxylated FLAG-tagged fusion protein (PHB2-FLAG) in HeLa cells, and immunoprecipitation experiments were performed using anti-DBD antibodies, PHB2 Binding to PGC1α and experiments using PGC1α deletion mutants indicated that the PHB2 binding site was present at the 401-600 amino acid site of the PGC1α protein (FIG. 2D). On the other hand, when a cell extract in which DBD-PGC1α (1-600) and PHB2-FLAG were co-expressed in HeLa cells was subjected to an immunoprecipitation experiment with anti-FLAG antibody, PGC1α was coprecipitated with PHB2 (Fig. 2E). Further, detailed analysis of deletion mutants of PHB2 showed that the binding site of PGC1α exists at the 151-200 amino acid site of PHB2 protein (FIG. 2F). Considering the result of FIG. 2C, it was considered that the PGC1α binding site in the PHB2 protein exists in the region of 171 to 200 amino acids. These results indicate that PHB2 binds directly to PGC1α, the coactivator of PPARγ, PHB2 suppresses PGC1α activity, and as a result, PHB2 suppresses PPARγ transcriptional activity through PGC1α activity regulation. It was.

Example 3
HeLa cells are cultured in a glass bottom dish, and transient transfection of mutant ERα (ERα241G) with deletion of 256-303 amino acids, including wild-type ERα and nuclear translocation signal, using Lipofectamine 2000 did. The next day, the cells were replaced with a cell culture solution supplemented with 10 ⁻⁸ M ICI or 10 ⁻⁷ M E2 and cultured for 2 hours, and then cell immunostaining was performed using the anti-GST-PHB2 antibody by the method described above. The fluorescence image was observed using a TCS SP5 (Leica) confocal laser microscope.

  We first examined whether the transfer of endogenous PHB2 from the mitochondria to the nucleus by estrogen depends on ERα. In HeLa cells, endogenous PHB2 was shown to migrate from the mitochondria to the nucleus in the presence of E2 only when wild-type ERα was expressed. On the other hand, when ERα was not introduced, or when mutated ERα (ERα241G) in which the 256-303 amino acid site including the nuclear translocation signal was deleted was introduced, the translocation to the nucleus was observed even in the presence of E2. It could not be observed (FIG. 3A). In addition, even when wild-type ERα was introduced, transfer to the nucleus could not be observed in the absence of E2 (FIG. 3A). That is, it was revealed that both E2 and ERα are required for the transfer of endogenous PHB2 from the mitochondria to the nucleus.

  Next, it was examined whether the transfer of endogenous PHB2 to the nucleus by E2 administration regulates the activities of PPARγ and PGC1α.

Dual luciferase assay was performed as described above (n = 3-4). However, ERα was added to the gene to be coexpressed. In addition, an experimental system to which 10 ⁻⁷ M E2 was added at the time of medium exchange 5 hours after gene introduction was added. In addition, the GAL4 assay was also carried out according to the above description, and when ERα was added to the gene to be co-expressed, or an experimental system in which 10 ⁻⁷ M E2 was added at the time of medium change 5 hours after gene introduction was used. added.

  As shown in FIG. 3B, when PPARγ and PGC1α are co-expressed in PPRE-TK-Luc, the transcriptional activity of PPARγ-PGC1α is maximized, and even if ERα is co-expressed, transcription is performed in the absence of E2. The activity remains relatively high (FIG. 3B, E2 (−)). However, when ERα was co-expressed, it was revealed that the transcriptional activity was remarkably suppressed under the condition where E2 was present (FIG. 3B, E2 (+)). Similarly, in an experiment where ERα was co-expressed with DBD-PGC1α and GAL4-Luc, the transcriptional activity of DBD-PGC1α was suppressed in the presence of E2 (FIG. 3C). From the above, in the presence of E2-ERα, endogenous PHB2 is transferred from the mitochondria to the nucleus. As a result, PHB2 transferred to the nucleus suppresses the activity of PGC1α, and through this inhibition, the transcriptional activity of PPARγ It was strongly suggested to suppress

Example 4
PHB2 mutation lacking the mitochondrial translocation signal located at the amino terminus, which translocates to the nucleus even in the absence of E2, for the purpose of investigating whether PHB2 nuclear translocation directly has an adipogenic inhibitory effect The body (PHB2C: 51-299AA) was prepared with a lentivirus and introduced into 3T3-L1 cells to examine the presence or absence of a PHB2 adipogenesis-inhibiting action not involving E2.

  3T3-L1 preadipocytes transfected with lentivirus were seeded in 35-mm poly-L-lysine-coated glass-bottomed dishes (Matsunami Glass Ind.) And subjected to cell immunostaining as described above . The primary antibody used was a rabbit polyclonal anti-FLAG antibody (Sigma) diluted 1,000-fold as the primary antibody, and Alexa 488-conjugated anti-rabbit IgG (Molecular Probes) as the secondary antibody diluted 1000-fold. The fluorescence image was taken and analyzed using a TCS SP5 (Leica) confocal laser microscope.

  When a fusion of PHB2 wild type and FLAG tag (PHB2-FLAG) and a fusion of PHB2C and FLAG tag (PHB2C-FLAG) were introduced into 3T3-L1 cells, PHB2C-FLAG was not mitochondria, but cytoplasm and nucleus It was shown by cell immunostaining with an anti-FLAG antibody that it was localized in (FIG. 4A).

  The Adipogenesis assay was performed as described above using 3T3-L1 cells into which lentivirus was introduced, and the culture time was measured 5 days and 7 days after the start of induction (n = 4).

  When adipogenesis was induced using 3T3-L1 cells transfected with GFP, PHB2, PHB2C, and PGC1α with lentivirus, adipogenesis assay markedly suppressed fat differentiation in 3T3-L1 cells introduced with PHB2C (FIG. 4B).

Total RNA was extracted from 3T3-L1 cells into which lentivirus had been introduced, and 1 μg of this was used to synthesize cDNA using reverse transcriptase. Quantitative PCR was performed using LightCycler 1.5 (Roche Applied Science) using the LightCycler ^™ FastStart DNA Master ^PLUS SYBR Green I kit (Roche Applied Science) according to a standard method. PCR primers are as shown below.

  For the mouse PPARγ gene, primers 5′-AAG AGC TGA CCC AAT GGT TG-3 ′ (SEQ ID NO: 13) and 5′-TCC ATA GTG GAA GCC TGA TGC-3 ′ (SEQ ID NO: 14) were used. For the aP2 gene, 5'-ATG TGT GAT GCC TTT GTG GGA-3 '(SEQ ID NO: 15) and 5'-TGC CCT TTC ATA AAC TCT TGT-3' (SEQ ID NO: 16) were used, and Adiponectin For the gene, 5′-GCA CTG GCA AGT TCT ACT GCA A-3 ′ (SEQ ID NO: 17) and 5′-GTA GGT GAA GAG AAC GGC CTT GT-3 ′ (SEQ ID NO: 18) were used. As a control, 5'-GCC CAT CAC CAT CTT CCA G-3 '(SEQ ID NO: 19) and 5'-TGA GCC CTT CCA CAA TGC C-3' (SEQ ID NO: 20) primers are used for amplification of the GAPDH gene. It was. Using the amount of GAPDH gene as an internal standard, the ratio of other gene expression levels was examined (n = 3).

  RNA was extracted from each cell on the 7th day after differentiation, and the gene expression level was examined by quantitative PCR.The gene expression levels of adipose differentiation marker genes PPARγ, aP2 (FABP4), and adiponectin were PHB2C. Decreased significantly in 3T3-L1 cells transfected with (Fig. 4C). From the above, it was shown that PHB2 translocation into the nucleus directly suppresses adipose differentiation without involving E2.

Example 5
It was verified in vivo whether obesity suppression by E2 is accompanied by changes in the intracellular localization of PHB2.
First, a mouse subjected to ovariectomy (ovarectomy; OVX) was prepared and verified as a model mouse for postmenopausal obesity. 10 weeks old C57BL6J female mice were bred with 0.1 mg / kg E2-containing diet or E2-free diet one week after OVX treatment, and the changes in body weight over time were compared. Although the body weight of the reared mouse (E2 non-administered group) increased, no weight increase was observed in the E2-containing group (FIG. 5A). In order to confirm the effect of E2 on mice, uterine weights were measured after dissection of mice reared for 60 days, and uterine weights were reduced in the E2 non-administered group (FIG. 5B). However, the uterine weight was increased in the E2 administration group (FIG. 5B). On the other hand, when the mice after 60 days were measured for cancellous bone density of the tibia using X-ray CT, bone density decreased in the E2 non-administered group, but increased in the E2 treated group. (FIG. 5C). Similarly, when the visceral fat (VAT) and subcutaneous fat (SAT) levels were measured by CT, the fat level increased in the E2 non-administered group, but decreased in the E2 group (Fig. 5D). From the above, it was shown that OVX mice become a model of postmenopausal obesity, and that hormone replacement therapy by E2 administration is effective for mice and suppresses obesity.

  After dissecting these groups of mice, tissue sections of periovar fat were prepared, stained with hematoxylin and eosin (HE), and the diameters of these fat cells were compared. However, the diameter decreased when E2 was administered (FIG. 5E).

  Each group of mice was reflux fixed with 4% paraformaldehyde. Thereafter, frozen sections were prepared from periovar white adipose tissue. Tissue immunostaining is performed using rabbit polyclonal anti-GST-PHB2 antibody (diluted 500 times) as the primary antibody and Cy5-conjugated anti-rabbit IgG (GE Healthcare) (diluted 500 times) as the secondary antibody. went. The fluorescence image was taken and analyzed using a TCS SP5 (Leica) confocal laser microscope.

  In the above-mentioned adipose tissue, the intracellular localization of endogenous PHB2 was confirmed by tissue immunostaining, and it was found that PHB2 was localized in the nucleus in the E2 administration group compared to the E2 non-administration group. (FIG. 5F). From the above, it has been clarified that PHB2 is transferred from mitochondria to the nucleus by E2 in the mouse body, and that E2 administration suppresses the increase in white adipocyte volume.

Example 6
From the above, (1) PHB2 localized in mitochondria moves to the nucleus in the presence of ERα and E2, and (2) PHB2 is a coactivator of transcription factor PPARγ that promotes fat differentiation in the nucleus, PGC1α By binding directly to the protein, it suppresses its activity and suppresses the transcriptional activity of PPARγ through this suppression. (3) Even in the absence of E2, PHB2 nuclear localization suppresses adipose differentiation As a direct action of PHB2, it was shown that it has an activity of inhibiting fat differentiation. Furthermore, (4) E2 induces the nuclear localization of endogenous PHB2 in the adipose tissue of OVX mice, which is a model of postmenopausal obesity, and further suppresses adipocyte obesity. In FIG. 6, the model of the molecular mechanism in obesity suppression of PHB2 is shown.

Example 7
Dual luciferase assay was performed as described above (n = 3). TK-RLuc as an internal standard, firefly luciferase reporter gene (ERE-Luc) having an estrogen responsive element upstream of the TK promoter as a reporter gene, ERα, mouse PGC1α gene, PHB2 gene, etc. as co-expressed genes Introduced. An experiment in which 10 ⁻⁷ M E2 was added as a ligand at the time of medium exchange 5 hours after gene introduction was added.

On the other hand, regarding the transcriptional activity regulation of PPARα, TK-RLuc, PPRE-Luc, PPARα, human PGC1α gene, PHB2 gene and the like were introduced in the indicated combinations. An experiment was conducted in which 10 ⁻⁵ M Fenofibrate (FFB-10) or 10 ⁻⁴ M WY14643 (WY) was added as a ligand when the medium was changed 5 hours after gene introduction.

  In addition, the GAL4 assay was also carried out according to the above description, and gene transfer was carried out with combinations of GAL4-DBD and PGC1α fusion protein (DBD-PGC1α) gene, PHB2 gene, and various deletion mutant genes of PHB2 as shown.

  Although PHB2 was shown to suppress the coactivator activity of PGC1α, the effect on transcription factors bound by PGC1α other than PPARγ was examined by dual luciferase assay. First, in the presence of PPARα and PGC1α, PHB2 suppressed the transcriptional activity of PPARα using PPRE-TK-Luc as a reporter gene (FIG. 7A). Similarly, in the presence of ERα and PGC1α, PHB2 suppressed the transcriptional activity of ERα in the reporter gene ERE-Luc having an estrogen responsive element (ERE) (FIG. 7B). From the above, it was shown that PHB2 also regulates the transcriptional activity of PGC1α other than PPARγ by regulating the coactivator activity of PGC1α.

  On the other hand, in order to determine the domain that contributes to the regulation of PHB2 PGC1α activity, several deletion mutants were prepared and their activities were examined in a system using GAL4-DBD-PGC1α and GAL4-Luc. Multiple sites that regulate the activity of PGC1α were found (FIG. 7C). These deletion mutants propose prototypes of PGC1α activity modulators.

  From the above, it was shown that PHB2 regulates the activity of various transcription factors regulated by PGC1α through the regulation of PGC1α activity (FIG. 7D). Peptides derived from PHB2 analogs and mutants, and substances that substitute their activities are considered to be drugs that regulate these functions.

Example 8
We examined which coactivators promoted transcriptional activity by adding various coactivators to the transcriptional activity of PPARγ, a transcription factor that promotes adipose differentiation, and further examined the transcriptional activity of PPARγ in the presence of these coactivators. We investigated whether PHB2 suppresses.

  Dual luciferase assay was performed according to the protocol using Dual-luciferase Reporter Assay System (Promega). Specifically, HeLa cells have a Renilla luciferase reporter gene (TK-RLuc) upstream of the thymidine kinase (TK) promoter and a firefly luciferase reporter gene (PPRE) upstream of the TK promoter with a PPAR responsive element (PPRE). -TK-Luc), PPARγ gene, PGC-1α gene, PGC-1β gene (NCBI registration number NM_133249), SRC-1 gene (NCBI registration number NP_003734), SRC-2 gene (NCBI registration number NP_006531), SRC- Three genes (NCBI registration number NP_858045), PHB2 gene, and the like were mixed in the indicated combinations, and DNA was introduced into the cells using Lipofectamine 2000 (Invitrogen). After 5 hours, the medium was changed, and after 22 hours in total, the cells were collected with a cell lysate and then measured with a luminometer. Using the activity of Renilla luciferase as an internal standard, the transcriptional activity was compared with firefly luciferase (n = 3).

  Using the dual luciferase assay, first, using the luciferase reporter gene (PPRE-TK-Luc) that has a PPAR responsive element (PPRE) upstream of the thymidine kinase (TK) promoter, the PPARγ gene and PGC-1α And other coactivator genes (PGC-1β, SRC-1, SRC-2, SRC-3) were co-expressed, and the combination of PPARγ gene and PGC-1α was more PPARγ than other coactivators. Markedly increased the transcriptional activity. When PHB2 gene was added to these combinations, PHB2 remarkably suppressed the transcriptional activity of the combination of PPARγ gene and PGC-1α (FIG. 8).

Claims (7)

A fat differentiation regulator comprising a substance that regulates the formation of a complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ · coactivator (PGC1α). A peroxisome proliferator-activated receptor γ · coactivator (PGC1α) activity regulator comprising a partial fragment or mutant of prohibitin 2 (PHB2). A fat differentiation regulator comprising a partial fragment or mutant of prohibitin 2 (PHB2). A complex of prohibitin 2 (PHB2) and peroxisome proliferator-activated receptor γ · coactivator (PGC1α). A complex of PHB2 and PGC1α comprising selecting a test substance that promotes or inhibits the formation of a complex of prohibitin 2 (PHB2) and peroxisome growth factor-activated receptor γ · coactivator (PGC1α) For screening for regulators of the formation of sucrose. A method for screening a fat differentiation regulator, comprising selecting a test substance that regulates inhibition of coactivator activity of peroxisome proliferator-activated receptor γ · coactivator (PGC1α) by prohibitin 2 (PHB2). A method for screening a fat differentiation regulator, comprising selecting a test substance that regulates inhibition of transcription activity of peroxisome proliferator-activated receptor γ (PPARγ) by prohibitin 2 (PHB2).