JP2007217368A - PGC-1alpha EXPRESSION PROMOTER, PGC-1alpha EXPRESSION INHIBITOR AND METHOD FOR USING THEM - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a PGC-1α (Peroxisome proliferator activated receptor γ coactivator 1α) expression promoter through a selective β<SB>2</SB>receptor and a PGC-1α expression inhibitor through a β receptor and to provide a method for using them. <P>SOLUTION: The expression of PGC-1α mRNA in skeletal muscle is promoted by administration of a medicine (clenbuterol) stimulating a β<SB>2</SB>receptor. The expression increase of PGC-1α mRNA in skeletal muscle by locomotor stimulation is inhibited by administration of a medicine (propranolol) blocking a β receptor. In this manner, a medicine containing a β<SB>2</SB>stimulator promotes PGC-1α expression and increases the number of mitochondria in skeletal muscle. Also, a medicine containing a β blocker controls the expression increase of PGC-1α and increases the number of mitochondria in skeletal muscle. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a PGC-1α expression promoter, a PGC-1α expression inhibitor, and methods for using them.

  PGC-1α (Peroxisome proliferator activated receptor γ coactivator 1α), a nuclear receptor coactivator, is expressed in brown adipose tissue, skeletal muscle, heart, kidney and brain and is involved in mitochondrial biosynthesis (eg, non- (See Patent Documents 1 and 2).

  In L6 myotube cells, when the intracellular calcium ion concentration is increased by caffeine or ionomycin, the expression level of PGC-1α increases. Moreover, when AICAR (5-Aminoimidazole-4-Carboxamide Ribonucleoside) known as an AMPK activator is administered, the expression level of PGC-1α increases. Thus, when a Ca-releasing agent or an AMPK activator is added to myocytes, the expression level of PGC-1α increases, but at the same time, the concentrations of mitochondrial enzymes and GLUT4 increase, and DNA of NRF-1 and NRF-2 Binding activity is also increased. These series of events are very similar to those observed when muscle cells adapt to loading exercise, and L6 myotubes are a good model for studying the mechanism of adaptive responses that exercise occurs in muscle mitochondria. (For example, refer nonpatent literature 3).

  On the other hand, it has also been known that AMPK activity is enhanced in skeletal muscle during exercise, and that the expression level of PGC-1α increases when AICAR is administered to cultured skeletal muscle (for example, Non-Patent Document 4). reference).

  From these results, it was considered that the increase in the expression of PGC-1 during exercise was mediated by an increase in AMPK activity (see, for example, Non-Patent Document 4).

However, in the skeletal muscles of AMPKα1 knockout mice or AMPKα2 knockout mice, the amount of PGC-1α increased after exercise (see, for example, Non-Patent Document 5), as in wild-type mice. This suggests that the expression of PGC-1α can be enhanced at least by pathways other than the AMPK pathway.
Puigsever P, Wu Z, Park CW, Graves R, Wright M, and Spiegelman BM. A cold-unducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92: 829-839, 1998. Wu Z, Puigserver P, Andersson U, Zhang C, Adelmant G, Mootha V, Troy A, Cinti S, Lowell B, Scarpulla RC, and Spiegelman BM. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. : 115-124.1999. Ojuka EO, Proc Nutr Soc 63 (2), 275-278 (2004). Terada S et al, Biochem Biophys Res Commun 296 (2), 350-4 (2002). Jorgensen et al. FASEB J 19, 1146-1148 (2005).

  Therefore, the present invention has found a novel PGC-1α expression promoter and a novel PGC-1α expression inhibitor by elucidating the mechanism of increase in the expression level of PGC-1α by exercise in muscle cells, and a method for using them. The purpose is to provide.

In view of the above object, the present inventors have made intensive efforts to elucidate the mechanism of increase in the expression level of PGC-1α by exercise in muscle cells. As a result, as shown in the following examples, selective β ₂ stimulators And β-blocker administration revealed that the expression level of PGC-1α in skeletal muscle was changed, and a new β-receptor signaling system was clarified.

That is, the PGC-1α expression promoter according to the present invention contains a β ₂ stimulator as an active ingredient. Here, the β ₂ stimulator is preferably, for example, clenbuterol or a clenbuterol compound.

  In addition, the promoter is characterized by promoting the expression of PGC-1α in skeletal muscle.

  Furthermore, the promoter is used for prevention / treatment of a disease caused by a low expression level of PGC-1α. Here, the disease is, for example, obesity or diabetes.

  Moreover, the PGC-1α expression inhibitor according to the present invention contains a β-blocker as an active ingredient. Here, it is preferable that the β blocker is, for example, propranolol or a propranolol compound.

  Moreover, the said inhibitor suppresses the expression of PGC-1 (alpha) in a skeletal muscle, It is characterized by the above-mentioned.

  Furthermore, the said inhibitor is used for the prevention and treatment of the disease resulting from the high expression level of PGC-1 (alpha).

The method of promoting the expression of PCG-1α according to the present invention is a method of promoting the expression of PCG-1α in a cultured cell, cultured tissue, or cultured organ having a β ₂ receptor-PGC-1α signal transduction system. The β ₂ receptor in the cultured cell, the cultured tissue, or the cultured organ is stimulated. Here, the cultured cell, the cultured tissue, or the cultured organ is preferably derived from skeletal muscle.

Furthermore, the method for promoting the expression of PCG-1α according to the present invention is a method for promoting the expression of PCG-1α in a human or non-human vertebrate, which stimulates β ₂ receptor in the vertebrate. It is characterized by that.

Here, the method preferably stimulates the β ₂ receptor, for example, by administering clenbuterol or a clenbuterol compound.

  The method is characterized by promoting the expression of PGC-1α in skeletal muscle.

  Furthermore, the method is characterized in that it is used for prevention / treatment of diseases caused by low expression levels of PGC-1α. Here, the disease is, for example, obesity or diabetes.

  Moreover, the method for suppressing the expression of PCG-1α according to the present invention is a method for suppressing the expression of PCG-1α in a cultured cell, tissue or organ having a β receptor-PGC-1α signal transduction system. The β-receptor in the cultured cell, the cultured tissue, or the cultured organ is blocked. Here, the cultured cell, the cultured tissue, or the cultured organ is preferably derived from skeletal muscle.

  Furthermore, the method for suppressing the expression of PCG-1α according to the present invention is a method for suppressing the expression of PCG-1α in a human or non-human vertebrate, wherein the β receptor in the vertebrate is blocked. It is characterized by.

  Here, it is preferable that the method blocks the β receptor by administering, for example, propranolol or a propranolol compound.

  The method is characterized by suppressing the expression of PCG-1α in skeletal muscle.

  Furthermore, the method is characterized in that it is used for prevention / treatment of diseases caused by high expression levels of PGC-1α.

The present invention can provide a PGC-1α expression promoter via a selective β ₂ receptor, a PGC-1α expression inhibitor via a β receptor, and methods for using them.

  Hereinafter, the present invention will be described specifically and in detail with reference to examples, but the present invention is not limited thereto.

  Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

=== Pharmacological action ===
When the β receptor is stimulated by administration of a drug or the like, the GTP binding protein (Gs) is activated, this activation activates adenylate cyclase, and this activation activates protein kinase A, This activation activates cAMP response element binding protein (CREB), resulting in the expression of β action (Gonzalez GA, and Montminy MR. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59: 675-680, 1989.).

As shown in the following examples, administration of a drug that stimulates β ₂ receptor (clenbuterol) promotes the expression of PGC-1α mRNA in skeletal muscle, while a drug that blocks β receptor (propranolol). When administered, the expression of PGC-1α mRNA in skeletal muscle, which was increased by exercise stimulation, was suppressed. In addition, mitochondrial increase and muscle redness were observed in PGC-1α transgenic mice (Lin J et al., Nature 418, 797-801, 2002, Miura S et al., J. Biol. Chem. , 278, 31385-31390, 2003). Here, as described above, PGC-1α is a transcription factor involved in mitochondrial biosynthesis. Therefore, a drug containing a β ₂ stimulator promotes the expression of PGC-1α (referred to herein as the β ₂ receptor-PGC-1α signal transduction system) and increases the number of skeletal muscle mitochondria, , A drug containing a β-blocker can suppress an increase in the expression of PGC-1α (herein referred to as a β receptor-PGC-1α signal transduction system) and suppress an increase in the number of mitochondria in skeletal muscle. it can.

=== Usefulness of Drugs Containing β ₂ Stimulants ===
Generally, lack of exercise (insufficient energy consumption) is known as one of the causes of obesity. Therefore, the drug containing the β ₂ stimulator of the present invention can increase the number of mitochondrial skeletal muscles, increase the amount of energy consumed in the body, and prevent and treat obesity.

In addition, there is exercise therapy as one of the prevention / treatment methods for diabetes (mainly non-insulin-dependent daiabetes mellitus (NIDDM)). Continuing physical training is said to improve insulin sensitivity in peripheral tissues, particularly muscles that have been reduced by type 2 diabetes. Therefore, the drug containing β ₂ stimulator of the present invention prevents diabetes by increasing the number of mitochondria in skeletal muscle, increasing the energy consumption in the body, and improving the insulin sensitivity of peripheral tissues.・ Can be treated. Moreover, hypertension, arteriosclerosis, even in lifestyle-related diseases such as hyperlipidemia, since the physical training of mild to moderate is needed, drugs containing beta ₂ stimulants of the present invention, these diseases It is also useful in the prevention and treatment of cancer.

Furthermore, β receptors are ciliary muscle (β ₂ ), sinoatrial node (β ₁ ), atrial muscle (β ₁ ), atrioventricular node (β ₁ ), His bundle and Purkinje fiber (β ₁ ), ventricular muscle. (Β ₁ ), coronary blood vessels (α <β ₂ ), skeletal muscle blood vessels (α ₁ <β ₂ ), pulmonary blood vessels (α ₁ <β ₂ ), visceral blood vessels (α> β ₂ ), renal blood vessels (α ₁ > Β ₁ , β ₂ ), systemic blood vessels (α ₁ , β ₂ ), bronchial muscle (β ₂ ), bronchial gland (α ₁ , β ₂ ), gastric movement / tension (α, β ₂ ), intestine Exercise / tension (α ₁ , β ₁ , β ₂ ), bile duct (β ₂ ), detrusor muscle (β ₂ ), pregnancy uterus (only when relaxed) (β ₂ ), non-pregnant uterus (β ₂ ), spleen capsule It is also expressed in (α ₁ > β), liver (β ₂ ), pancreatic β cells (α ₂ , β ₂ ), adipocytes (α ₁ , β ₁ ), salivary glands (α ₁ , β), and the like. Therefore, the drug containing the β ₂ stimulator of the present invention can increase the number of mitochondria and produce energy even in an organ expressing the β ₂ receptor. For example, mitochondrial diseases (e.g., chronic progressive extraocular palsy syndrome, Melas, Murf, Leigh (Lee) encephalopathy) are not only symptoms of skeletal muscle such as muscle weakness, muscle atrophy, but also intelligence decline, convulsions, myoclonus, In diseases with neurological symptoms such as cerebellar ataxia, hearing loss, and extraocular muscle paralysis, mitochondrial DNA mutation is considered to be one of the causes of the onset. However, if the normal mitochondria are present, by administering an agent containing beta ₂ stimulants of the present invention, it is possible to increase the number of mitochondria, this drug in patients with mitochondrial disease Is also considered useful.

When a drug containing a β ₂ stimulant is administered, relaxation of the ciliary muscle (β ₂ ), which is β ₂ action, expansion of coronary blood vessels (α <β ₂ ), blood vessels of skeletal muscles (α ₁ < β ₂ ) dilation, pulmonary blood vessel (α ₁ <β ₂ ) dilation, visceral blood vessel (α> β ₂ ) contraction, renal blood vessel (α ₁ > β ₁ , β ₂ ) contraction, systemic blood vessel (α ₁ , Β ₂ ) contraction / expansion, bronchial muscle (β ₂ ) relaxation, bronchial gland (α ₁ , β ₂ ) secretion inhibition / secretion promotion, reduction of stomach movement / tension (α, β ₂ ), intestinal movement・ Reduction of tension (α ₁ , β ₁ , β ₂ ), relaxation of bile duct (β ₂ ), relaxation of detrusor muscle (β ₂ ), relaxation of pregnancy uterus (only during relaxation) (β ₂ ), non-pregnancy uterus ( β ₂ ) relaxation, contraction of spleen capsule (α ₁ > β), promotion of glycogenolysis / gluconeogenesis in liver (β ₂ ), decreased secretion / secretion in pancreatic β cells (α ₂ , β ₂ ) It is also expected to have effects such as increase and viscosity promotion in salivary glands (α ₁ , β).

In addition, since salbutamol, terbutaline, trimethoquinol, procaterol and the like, which are known as β ₂ stimulants, have the same mechanism of action as clenbuterol, even when these drugs are administered, the same effects as the drug of the present invention Can be obtained.

=== Usefulness of drugs containing β-blockers ===
As above-mentioned, the chemical | medical agent containing (beta) blocker can suppress the expression of PGC-1 (alpha), and can suppress the increase in the number of mitochondria of the skeletal muscle by sympathetic nervous system stimulation, such as an exercise | movement. Therefore, the medicine containing the β-blocker of the present invention is useful for prevention / treatment of diseases caused by the amount of PGC-1α or diseases in which energy consumption in skeletal muscles is to be reduced. Note, when administered a drug containing a beta blocker of the present invention, the ciliary muscle (beta ₂₎ contraction, heart rate decreased in the sinus node (beta _1), decreased contraction force in the atrium muscle (beta _1), Automaticity and conduction velocity of atrioventricular node (β ₁ ), automaticity and reduction of conduction velocity of His bundle and Purkinje fiber (β ₁ ), decrease in contraction force of ventricular muscle (β ₁ ), coronary blood vessels (α < β ₂ ) contraction, skeletal muscle blood vessel (α ₁ <β ₂ ) contraction, pulmonary blood vessel (α ₁ <β ₂ ) contraction, visceral blood vessel (α> β ₂ ) expansion, renal blood vessel (α ₁ > β ₁ , β ₂ ), systemic blood vessels (α ₁ , β ₂ ) contraction / expansion, bronchial muscle (β ₂ ) contraction, secretion promotion / secretion suppression in bronchial gland (α ₁ , β ₂ ), stomach increased exercise and contraction (alpha, beta _2), motion and contraction of the intestinal _{(α 1, β 1, β} 2) increased, biliary (beta ₂₎ contraction, yield detrusor (beta ₂₎ Shrinkage, contraction of the pregnancy uterus (only when relaxed) (β ₂ ), contraction of the non-pregnant uterus (β ₂ ), relaxation of the spleen capsule (α ₁ > β), inhibition of glycogenolysis and glucose formation in the liver (β ₂ ) Secretion increase / decrease in pancreatic β cells (α ₂ , β ₂ ), inhibition of lipolysis in fat cells (α ₁ , β ₁ ), inhibition of fatty acid release into blood, viscosity in salivary glands (α ₁ , β) It is also expected to have effects such as thickening suppression.

  In addition, since alprenolol, oxprenolol, pindolol, bufetrol, bupranolol, indenolol, carteolol, bucmolol, timolol, nadolol, etc., known as β-blockers have the same mechanism of action as propranolol, these Even when the drug is administered, the same effect as that of the drug of the present invention can be obtained.

=== Manufacturing and Administration Method for the above Drugs ===
The drug containing the β ₂ stimulator of the present invention is not limited as long as it contains a compound capable of stimulating the β ₂ receptor. In addition, the drug containing the β-blocker of the present invention is not limited as long as it contains a compound capable of blocking the β-receptor.

Pharmaceutically acceptable carriers that may be used in the drug containing drug and beta blockers containing beta ₂ stimulants, may be used various organic or inorganic carrier substances conventionally used as preparation materials, for example, solid preparation May contain a solvent, a solubilizing agent, a suspending agent, a tonicity agent, a buffering agent, a soothing agent, and the like in a liquid preparation. Further, if necessary, additives such as usual preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like may be appropriately contained. As the dosage form, oral preparations include, for example, tablets, capsules, granules, powders, fine granules, syrups, sustained-release tablets / capsules / granules, cashews, chewable tablets or drops. Examples of injections include solution injections, emulsion injections, and solid injections.

  The dose of the drug of the present invention varies depending on age, weight, indication, or administration / intake route, but is not particularly limited as long as the above-described effects can be exhibited and the side effects to be generated are within an acceptable range.

As an administration method, for example, in humans or non-human vertebrates, the expression level of PGC-1α in cells, tissues, or organs caused by the target disease is measured, and the expression level of PGC-1α is below a predetermined value. case, administering an agent containing beta ₂ stimulants of the present invention as an active ingredient. On the other hand, when the expression level of PGC-1α is a predetermined value or more, a drug containing the β-blocker of the present invention as an active ingredient is administered. Here, the “predetermined value” may be, for example, a value that is generally determined that the expression level of PGC-1α is higher than that of a healthy person, or a reference value that is determined by a medical worker. PGC-1α can be measured by collecting cells, tissues, or organs and performing PCR, Southern blotting, Northern blotting, or the like. In addition, the agent may be administered by measuring the number of mitochondria in cells, tissues, or organs considered to be the cause of the target disease, and measuring this number.

Any cell, tissue, or organ to be administered may be used as long as it has a β ₂ receptor-PGC-1α signal transduction system or a β receptor-PGC-1α signal transduction system. Such cells with signal transduction system beta ₂ receptor-PGC-l [alpha], tissue, or to increase the energy production of organs, cultured cells with signal transduction system beta ₂ receptor-PGC-l [alpha] , cultured tissue, or drugs may be administered which comprises, as an active ingredient a beta ₂ stimulants of the present invention in a culture organ, or the like. On the other hand, in order to reduce the energy production amount of cells, tissues, or organs having a β receptor-PGC-1α signal transmission system, cultured cells and culture tissues having a β receptor-PGC-1α signal transmission system Alternatively, a drug containing the β-blocker of the present invention as an active ingredient may be administered to a cultured organ or the like. Whether a cell, tissue, or organ has a β ₂ receptor-PGC-1α signal transduction system can be easily identified by administering a β ₂ stimulator and measuring the amount of PGC-1α. Whether a cell, tissue, or organ has a β receptor-PGC-1α signal transduction system can be easily identified by administering a β-blocker and measuring the amount of PGC-1α.

  Hereinafter, the present invention will be specifically described by way of examples. These examples are for explaining the present invention, and do not limit the technical scope of the present invention.

<Example 1: Inhibition of expression of PGC-1α mRNA by propranolol>
(1) Preparation of sample In order to examine whether the expression of PGC-1α increased by exercise is inhibited by propranolol (β receptor antagonist), the following experiment was performed. In the experiment, 10-week-old C57BL / 6J female mice (Japan SLC, Inc.) were used. These mice have a light / dark cycle of 12 hours, cages (temperature 22 ° C.), guidelines (National Institute of Health and Nutrition Animal Experiment Guidelines and NIH Animal Experiment Guidelines (NIH Publication Number 85-23,1985: http: / /grants1.nih.gov/grants/onlaw/references/phspol.htm)).

  First, propranolol (Sigma Chemical, st. Louis, MO) was subcutaneously administered to mice (10 mg per mouse body weight (kg)). One hour after the administration, the mice were run for 45 minutes using a treadmill (15 m / min). These mice were sacrificed over time (0 hours, 3 hours, 6 hours, and 9 hours after the end of running the treadmill), and the gastrocnemius muscles were removed. The excised gastrocnemius muscle was used for analysis by the “Northern blot method” described below. In addition, the mouse | mouth which did not administer propranolol but administered subcutaneously the same amount physiological saline as propranolol was used as control.

(2) Preparation of PGC-1α cDNA In order to carry out the “Northern blot method” described below, PGC-1α cDNA was prepared using the gastrocnemius muscle extracted by the above method.
Total RNA was extracted from the gastrocnemius muscle extracted by the above method using TRIZOL (Invitrogen). Next, using the Advantage One-Step RT-PCR kit (Clontech Lab., Palo Alto, Calif.), The forward primer (SEQ ID NO: 5′-ATGGCTTGGACAGTGC-3 ′) and reverse primer (SEQ ID NO: 5′-TTACCTGCGCCAAGCTTCCTCT -3 ′) was used to amplify full-length PGC-1α cDNA from 1 μg of extracted total RNA.

(3) Quantification of PGC-1α using Northern blotting To quantitate the expression level of PGC-1α in the gastrocnemius muscle extracted by the above method, Northern blotting was performed. Statistical analysis was performed by Fisher's statistical analysis method (Statview 5.0, Abacus Concepts, Inc., Berkeley, Calif.) For comparison with each group, and Student's T test for comparison between the two groups. Note that p <0.05 was considered significant.

  First, total RNA was isolated from the gastrocnemius muscle extracted by the above method using TRIzol reagent (Invitrogen). This RNA was denatured using glyoxal and dimethyl sulfoxide, and electrophoresis (10 μg / lane) was performed using a 1% agarose gel. After electrophoresis, it was transferred to a nylon membrane (NEN, Boston, MA) (12 hours) and cross-linked by UV irradiation.

As the probe, the PGC-1α cDNA described in “(2) Preparation of PGC-1α cDNA” described above was used. Hybridization was performed using a probe labeled with ³² P-dCTP (Amersham Biosciences, Tokyo, Japan) using a random prime labeling kit (Amersham Biosciences). After washing the membrane, each signal was quantified using an image analyzer (BAS 1800-II, Fuji Film, Tokyo, Japan).

  The results of examining the expression of PGC-1α are shown in FIG. 1A. In the group not receiving propranolol, there was an increase in endogenous PGC-1α transcripts of 6.5 kb, 5 kb, and 3 kb requiring polyadenylation signals at 3 hours, 6 hours, and 9 hours after exercise. It was. In particular, it was revealed that the expression of PGC-1α was most increased after 3 hours of exercise (FIG. 1B). Further, immediately after exercise, no increase in PGC-1α transcript was observed. On the other hand, in the group administered with propranolol, the increase in the expression level of PGC-1α due to exercise stimulation was all suppressed.

  From the above, it was revealed that the amount of PGC-1α mRNA in skeletal muscle increases with exercise, but the increase is suppressed by administering propranolol.

<Example 2: Increase in expression of PGC-1α mRNA by clenbuterol>
(1) Preparation of sample In order to investigate whether the expression of PGC-1α is enhanced by a β receptor agonist, the following experiment was performed using clenbuterol, which is a β ₂ receptor agonist. In the following experiment, the same mouse as in Example 1 was used.
Clenbuterol (Sigma Chemical, st. Louis, MO) was subcutaneously administered to mice at 0.01 mg, 0.1 mg, 1 mg, or 5 mg per mouse body weight (kg). These mice were sacrificed 4 hours later and the gastrocnemius muscle was removed. The excised gastrocnemius muscle was used for analysis by the “Northern blot method” described below. In addition, the mouse | mouth which did not administer clenbuterol and subcutaneously administered the same amount physiological saline as clenbuterol was used as control.

(2) Preparation of PGC-1α cDNA In order to carry out the “Northern blot method” described below, PGC-1α cDNA was prepared using the gastrocnemius muscle extracted by the above method. The manufacturing method is the same as that in Example 1.

(3) Quantification of PGC-1α using Northern blotting To quantitate the expression level of PGC-1α in the gastrocnemius muscle extracted by the above method, Northern blotting was performed. Northern blotting is as described in Example 1. Each group was compared by Fisher's statistical analysis method (Statview 5.0, Abacus Concepts, Inc., Berkeley, CA). Note that p <0.05 was considered significant.

  The result of examining the expression of PGC-1α is shown in FIG. 2A. When Clenbuterol was administered, an increase in endogenous PGC-1α transcript was observed. In particular, it was found that the expression of PGC-1α was most increased in the group administered with 1 mg of clenbuterol per body weight of the mouse (FIG. 2B). On the other hand, the expression level of PGC-1α did not increase in the group administered with 0.01 mg of clenbuterol per body weight of the mouse or in the group not administered with clenbuterol.

  From the above results, it was revealed that the amount of PGC-1α mRNA in skeletal muscle increases when Clenbuterol at a certain concentration or higher is administered.

<Example 3: Inhibition of expression of CREB phosphorylation by propranolol>
In order to examine whether the intracellular signal transduction mechanism via β receptor is functioning in skeletal muscle, the phosphorylation of cAMP response element binding protein (CREB) was examined using propranolol (β receptor antagonist). . In addition, norepinephrine (NE) acts directly on the β receptor to produce an excitatory effect on the target organ. As a result, the intracellular cAMP concentration is increased and protein kinase A is activated. As a result, serine at position 133 of CREB is phosphorylated (Gonzalez GA, and Montminy MR. Cyclic AMP stimulated somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59: 675-680, 1989.). In the following experiment, the same mouse as in Example 1 was used.

(1) Preparation of sample First, propranolol (Sigma Chemical, st. Louis, MO) was subcutaneously administered to mice (10 mg per mouse body weight (kg)). From 1 hour after administration, the mice were run for 45 minutes using a treadmill (15 m / min). Three hours after the running of the treadmill, the mouse was sacrificed and the gastrocnemius muscle was removed. The excised gastrocnemius muscle was used for analysis by the “Western blot method” described below. In addition, the mouse | mouth which did not administer propranolol but administered subcutaneously the same amount of physiological saline as propranolol was used as control.

(2) Western blotting method In order to quantify phosphorylated CREB and total CREB in gastrocnemius muscle extracted by the above method, Western blot analysis was performed. Statistical analysis was performed by Fisher's statistical analysis method (Statview 5.0, Abacus Concepts, Inc., Berkeley, Calif.) For comparison with each group, and Student's T test for comparison between the two groups. p <0.05 was considered significant.

  First, the gastrocnemius muscle extracted by the above method was homogenized under ice cooling, the crushed material was centrifuged for 60 minutes (175,000 × g), and the supernatant was used for electrophoresis described below. The protein concentration was measured using Micro BCA Protein Assay Kit (PIERCE, IL, USA).

  Next, 7.5 μg of crude protein was separated on a 10% polyacrylamide slab gel (Bio-Rad Laboratories) and transferred to a nitrocellulose membrane (Bio-Rad Laboratories) by electroblotting.

  After blocking this membrane with 3% BSA, primary antibodies (an antibody recognizing a phosphorylated protein at position 133 serine of CRDB (Cell Signaling Technology, Beverly, MA, catalog number 9191)) and a CREB antibody (Cell Signaling Technology, Beverly , MA, catalog number 9192)) and incubated at 4 ° C. for 12 hours. The membrane was washed and then incubated with a secondary antibody (Rabbit IgG, Horseradish Peroxidase-linked whole antibody (from donkey) (Amersham Biosciences, UK)) for 1 hour at room temperature. ECL Western Blotting Detection Reagents (Amersham Biosciences, UK) was used for color development. The results are shown in FIG.

  As a result, it was found that the amount of phosphorylated CREB in which serine at position 133 was phosphorylated in the exercised mouse was increased 7.7 times compared to the non-exercise mouse. On the other hand, it was found that the amount of CREB that was not phosphorylated did not change in any mouse (FIGS. 3A and B). It was also found that in mice that had been exercised after propranolol administration, the amount of phosphorylated CREB increased only 2.7 times compared to mice that had not been exercised after propranolol administration.

  From the above, it has been clarified that the intracellular signal transduction mechanism via β receptor is involved in the increase of PGC-1α expression by exercise stimulation in skeletal muscle.

In one Example of this invention, it is the figure which showed the result of having detected PGC-1 (alpha) mRNA of the gastrocnemius muscle by propranol administration by the Northern blot method. Gastrocnemius muscles removed from individual mice are used for each lane. 18S was used as a control. In one Example of this invention, it is the figure which showed the result of having quantified the PGC-1 (alpha) mRNA of the gastrocnemius muscle by propranol administration by the Northern blot method. The vertical axis represents the amount of PGC-1α mRNA in% compared to the control (group administered with physiological saline). The amount of PGC-1α mRNA is shown as the sum of three PGC-1α transcripts (6.5 kb, 5 kb, and 3 kb). A white column indicates a non-moved mouse, and a black column indicates a moved mouse. Each value shows the mean ± SE of the amount of PGC-1α mRNA of gastrocnemius muscle extracted from 3 mice. The results of statistical analysis with the control were shown as ++ when p <0.001 and ++ when p <0.01. In one Example of this invention, it is the figure which showed the result of having detected PGC-1 (alpha) mRNA of the gastrocnemius muscle by Clenbuterol administration by the Northern blot method. Gastrocnemius muscles removed from individual mice are used for each lane. 18S was used as a control. In one Example of this invention, it is the figure which showed the result of having quantified the PGC-1 (alpha) mRNA of the gastrocnemius muscle by Clenbuterol administration by the Northern blot method. The vertical axis represents the amount of PGC-1α mRNA in% compared to the control (group administered with physiological saline). The amount of PGC-1α mRNA is shown as the sum of three PGC-1α transcripts (6.5 kb, 5 kb, and 3 kb). Each value shows the mean ± SE of the amount of PGC-1α mRNA of gastrocnemius muscle extracted from 3 mice. As a result of statistical analysis with the control, p <0.001 is represented by +++. In one Example of this invention, it is the figure which showed the result of having detected the phosphorylated CREB of the gastrocnemius muscle by propranolol administration by Western blotting. The gastrocnemius muscle was extracted from the exercised and non-exercised mice. In one Example of this invention, it is the figure which showed the result of having detected CREB of the gastrocnemius muscle by propranolol administration by the western blotting method. The gastrocnemius muscle was extracted from the exercised and non-exercised mice. In one Example of this invention, it is the figure which showed the result of having quantified the phosphorylated CREB of the gastrocnemius muscle by propranolol administration by the Western blot method. The vertical axis represents the amount of phosphorylated CREB compared to the control (non-exercising group) in%. Each value represents the mean ± SE of the amount of phosphorylated CREB of gastrocnemius muscles extracted from 3 mice. The result of statistical analysis with the control was indicated by + when p <0.005.

Claims (22)

PGC-l [alpha] expression promoting agent containing beta ₂ stimulants as an active ingredient. The accelerator according to claim 1, wherein the β ₂ stimulant is clenbuterol or a clenbuterol compound.   The promoter according to claim 1 or 2, which promotes the expression of PGC-1α in skeletal muscle.   The promoter according to any one of claims 1 to 3, which is used for prevention / treatment of a disease caused by a low expression level of PGC-1α.   The promoter according to claim 4, wherein the disease is obesity or diabetes.   A PGC-1α expression inhibitor containing a β-blocker as an active ingredient.   The inhibitor according to claim 6, wherein the β-blocker is propranolol or a propranolol compound.   The inhibitor according to claim 6 or 7, which suppresses the expression of PGC-1α in skeletal muscle.   The inhibitor according to any one of claims 6 to 8, which is used for prevention / treatment of a disease caused by a high expression level of PGC-1α. A method of promoting the expression of PCG-1α in a cultured cell, tissue, or organ having a β ₂ receptor-PGC-1α signaling system,
A method of stimulating β ₂ receptor in the cultured cell, the cultured tissue, or the cultured organ.   The method according to claim 10, wherein the cultured cell, the cultured tissue, or the cultured organ is derived from skeletal muscle. A method for promoting the expression of PCG-1α in vertebrates other than humans, comprising:
Wherein the stimulating beta ₂ receptors in said vertebrate. The method according to claim 10, wherein β ₂ receptor is stimulated by administering clenbuterol or a clenbuterol compound.   The method according to claim 12 or 13, which promotes the expression of PGC-1α in skeletal muscle.   The method according to any one of claims 12 to 14, wherein the method is used for prevention / treatment of a disease caused by a low expression level of PGC-1α.   The method according to claim 15, wherein the disease is obesity or diabetes. A method of suppressing the expression of PCG-1α in a cultured cell, tissue, or organ having a β receptor-PGC-1α signal transduction system,
A method comprising blocking β receptor in the cultured cell, the cultured tissue, or the cultured organ.   The method according to claim 17, wherein the cultured cell, the cultured tissue, or the cultured organ is derived from skeletal muscle. A method of suppressing the expression of PCG-1α in vertebrates other than humans,
Blocking the β-receptor in said vertebrate.   The method according to any one of claims 17 to 19, wherein β receptor is blocked by administering propranolol or a propranolol compound.   21. The method according to claim 19 or 20, wherein the expression of PCG-1α in skeletal muscle is suppressed. The method according to any one of claims 19 to 21, which is used for prevention / treatment of a disease caused by a high expression level of PGC-1α.