JP2008056566A - Hypoxic response promoter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a disease-treating/preventing agent or a food having excellent hypoxic response-promoting activity. <P>SOLUTION: The present invention provides a hypoxic response promoter, a therapeutic or preventive agent for an ischemic disease, a therapeutic agent for a hypoxia-induced dysfunction at high-altitude living and a supplement for high-altitude training, an mTOR-activating agent, an HIF-1α-activating agent or a vascular endothelial growth factor (VEGF) gene and/or an adrenomedullin (ADM) gene expression-inducing agent each containing an amino acid selected from leucine, isoleucine, valine or a mixture thereof. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a hypoxic response promoter and a pharmaceutical composition containing the same, and in particular, the drug can be suitably used in the form of pharmaceuticals and beverages, or in the form contained in foods and drinks.

In general, when cells are exposed to hypoxia, they adapt to hypoxia through changes in intracellular metabolism and gene expression, and prepare for the damage. The play a central in the regulation of such gene expression is transcriptional factor called HIF-1α (H ypoxia- i nducible f actor-1α), the HIF-l [alpha] is efficiently activated, hypoxic stress response Is extremely important (Non-patent Document 1). When the oxygen partial pressure decreases, the stability of the constitutively expressed HIF-1α protein increases, and the amount of HIF-1α protein in the cell increases. HIF-1α translocated to the nucleus binds to the hypoxic response region of the response gene group and induces gene expression at the transcriptional level. Increase in HIF-l [alpha] protein amount is regulated also by promoting translation not only increased stability and its upstream signaling cascades mTOR (m ammalian t arget o f r apamycin) is involved It is considered. For example, growth factors such as insulin, PDGF (platelet-derived growth factor), and FGF (fibroblast growth factor) have no effect on lowering the oxygen partial pressure, and induce an increase in protein amount without proteolysis of HIF-1α (Non-patent literature) 2, Non-Patent Document 3), its action is suppressed by rapamycin, an mTOR activation inhibitor. HIF-1α has been found as a transcriptional activator of erythropoietin that regulates erythropoiesis. However, HIF-1α binding sites in the promoter regions of vascular endothelial growth factor VEGF, enolase 1, transferrin, aldolase A, etc. Was found (Non-Patent Document 4). It is also known to be involved in the control of the expression of genes involved in energy production such as glucose transporters and glycolytic metabolic enzymes. Rapamycin inhibits the activation of HIF-1α by inhibiting mTOR, and by suppressing the expression of VEGF, etc. expressed in certain cancer cells, it stops nutritional supplementation to cancer cells and has an antitumor effect. It has been reported (Non-Patent Document 5). It is widely known that amino acids not only serve as protein substrates but also promote protein synthesis in cells by activating mTOR as a signal (Non-patent Document 6, etc.). However, in experiments at the cell level, it has been reported that the expression of VEGF increases under the condition that the amino acid concentration in the cell culture medium is decreased and the activity of mTOR is decreased (Non-patent Document 7). On the other hand, it is not known whether low molecular weight compounds that activate mTOR such as amino acids enhance the expression of HIF-1α target genes such as VEGF through the activation of intracellular HIF-1α. .

  In an organism, angiogenesis is observed as a result of a physiological hypoxic response in an organ or tissue that has become ischemic due to causes such as arteriosclerosis or thrombus, and attempts to avoid ischemia are common. In many cases, the effect is insufficient, and it is considered that induction of angiogenesis using any drug is a therapeutic method for hypoxic injury such as ischemia. For example, treatment with a protein preparation that induces angiogenesis such as VEGF, and gene therapy of VEGF and HIF-1α have been studied (Non-patent Document 8). In addition, for angiogenesis, there has been a demand for a disease treatment method that regulates HIF-1α. For example, high expression of HIF-1α has been reported to produce more normal vasculature than when VEGF alone or other various factors react to act alone. . At this time, edema, inflammation, ulcers and tumors seen when an angiogenic factor is directly administered alone do not occur, and increasing HIF-1α activation can induce physiological angiogenesis (Non-patent document 9).

  When a person who has lived on a flat ground begins a high-altitude life in a low oxygen state, physiological changes in a low-oxygen environment such as the development of capillaries and an increase in red blood cells occur, and an adaptation phenomenon to the highland life occurs. However, if you climb the highland in a short period of time, or if you become a highland living above 5000m above sea level, various disabilities due to hypoxia will appear. Measures such as high acclimatization and supply of oxygen are taken to avoid physical disabilities in low-oxygen conditions at high altitudes, but groundbreaking solutions such as the need for time and the need for oxygen cylinders There is no situation. In addition, it is often seen that athletes perform high altitude training in athletic competitions, which is training aimed at increasing the amount of oxygen supplied to each body tissue. If the living body can be guided to a state similar to low oxygen in the highland even on flat ground, it is considered that the same effect as highland training and acclimatization can be expected.

  In order to treat ischemic diseases as described above, adapt to high altitude life, and enhance the effects of high altitude training, it is necessary to increase the activity of HIF-1α, which regulates the expression of various genes in response to hypoxia It is done. Under these circumstances, if a drug that can be administered orally and has high safety can increase the activity of the endogenous HIF-1α gene and increase the production of VEGF, etc., there are significant medical benefits. To date, no such drug is known.

Semenza GL.Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology.Trends Mol Med. 2001 Aug; 7 (8): 345-50 Humar R, Kiefer FN, Berns H, Resink TJ, Battegay EJ.Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR) -dependent signaling.FASEB J. 2002 Jun; 16 (8): 771-80 Treins C, Giorgetti-Peraldi S, Murdaca J, Semenza GL, Van Obberghen E. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase / target of rapamycin-dependent signaling pathway.J Biol Chem. 2002 Aug 2; 277 ( 31): 27975-81. Wenger RH, Gassmann M. Oxygen (es) and the hypoxia-inducible factor-1.Biol Chem. 1997 Jul; 378 (7): 609-16 Nat Med. 2004; 10 (6): 594-601 Biochem Biophys Res Commun. 2004 Jan 9; 313 (2): 429-36 Earle KA, Pancholi S, Vernon P, Yudkin JS. Amino acid depletion modulates vascular endothelial growth factor production during the life span of human vascular smooth muscle cells.J Cell Physiol. 1998 Aug; 176 (2): 359-64 Nature Reviews Drug Discovery 2, 863 -872 (2003), Curr Opin Mol Ther. 2004 Apr; 6 (2): 151-9. Elson DA, Thurston G, Huang LE, Ginzinger DG, McDonald DM, Johnson RS, Arbeit JM.Induction of hypervascularity without leakage or inflammation in transgenic mice over expressing hypoxia-inducible factor-1alpha.Genes Dev. 2001 Oct 1; 15 (19 ): 2520-32

An object of the present invention is to provide a disease treating / preventing agent or food having excellent hypoxic response promoting activity.
Another object of the present invention is to provide an agent for treating and / or preventing ischemic disease and anemia, and / or an immunostimulatory agent.
Another object of the present invention is to provide a method for screening an active ingredient of a pharmaceutical or food used for the prevention / treatment of hypoxic injury.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a specific amino acid, preferably a specific branched-chain amino acid has an excellent hypoxic response promoting activity, particularly the transcription factor HIF-1α activating action. Further, the present invention was found by inducing gene expression of VEGF and ADM, which is one of the hypoxia responsive genes and is known to be activated by HIF-1α.
That is, the present invention provides a hypoxic response promoter characterized by containing an amino acid selected from leucine, isoleucine, valine or a mixture thereof.
The present invention also provides a therapeutic and / or prophylactic and / or immunostimulatory agent for ischemic disease, characterized by containing the hypoxic response promoter.

The present invention also provides a therapeutic agent for high altitude life hypoxia-induced disorder or a high altitude training aid, comprising an amino acid selected from leucine, isoleucine, valine or a mixture thereof. .
The present invention also provides a method for screening a hypoxic response promoter characterized by including the following steps.
(1) preparing a cultured cell cultured using a culture solution containing no amino acid;
(2) preparing cultured cells cultured using a culture solution containing amino acids;
(3) Add the test substance to the cultured cells of (1) and (2);
(4) The VEGF gene expression index under normoxic partial pressure and low oxygen partial pressure in the cultured cells of (1) and (2) is measured, and a test substance from which an equivalent or higher VEGF gene expression index is obtained is selected.
The present invention also provides an amino acid-containing drug or food that contains an amino acid selected from leucine, isoleucine, valine, or a mixture thereof and is packaged in a package that describes a high altitude training aid effect.
The present invention also provides an mTOR activator, HIF-1α activator or Vascular Endothelial Growth Factor (VEGF) gene characterized in that it comprises an amino acid selected from leucine, isoleucine, valine or a mixture thereof. An adrenomodullin (ADM) gene expression inducer is provided.

  The hypoxic response promoter of the present invention improves, alleviates, and recovers ischemic diseases and their complications caused by hypoxia through the activation of HIF-1α, which is a major regulator of hypoxic response. be able to. Moreover, although the chemical | medical agent of this invention can be used as a pharmaceutical, it can also be used as a foodstuff. Therefore, the present invention can be widely used particularly in the fields of pharmaceuticals, foods and the like, and therefore the present invention is very useful.

  Treatment for ischemic disease and / or treatment for anemia and / or immunostimulant and / or treatment for high altitude hypoxia-induced disorder and / or high altitude training aid containing hypoxic response promoter of the present invention In the case of other pharmaceutical compositions and food compositions, an mTOR activator per formulation, for example, an amino acid selected from leucine, isoleucine, valine or a mixture thereof, particularly an amino acid typified by leucine, For example, it is preferable to contain about 0.000001-50g. More preferably, it is good to contain about 1-10g. Here, the L-form is preferably used as the amino acid. When the drug or food for disease treatment / prevention of the present invention is used, it can be administered orally, intravenously, subcutaneously, or intramuscularly. However, oral administration is preferred for convenience. The dose varies depending on the symptoms, age and administration method of the patient to be administered, but is usually 0.1 to 30 g / kg / day.

  The therapeutic / preventive pharmaceutical or food of the present invention can be formulated by a conventional method. Examples of the form of the preparation include injections, tablets, granules, fine granules, powders, capsules, creams, suppositories, etc. Examples of the carrier for the preparation include lactose, glucose, D-mannitol, starch, Crystalline cellulose, calcium carbonate, kaolin, starch, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, ethanol, carboxymethylcellulose, carboxymethylcellulose calcium salt, magnesium stearate, talc, acetylcellulose, sucrose, titanium oxide, benzoic acid, P-hydroxybenzoate, sodium dehydroacetate, gum arabic, tragacanth, methylcellulose, egg yolk, surfactant, sucrose, simple syrup, citric acid, distilled water, ethanol, glycerin, propylene glycol , Macrogol, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, glucose, sodium chloride, phenol, thimerosal, p-hydroxybenzoate, sodium bisulfite, etc. Used in admixture with the compounds of the invention. Moreover, it does not necessarily need to be used as a free amino acid, and may be used in the form of an inorganic acid salt, an organic acid salt, an ester that can be hydrolyzed in vivo, or the like. Further, it may be used in the form of two or more leucine or other branched chain amino acids, or peptides in which amino acids are peptide-bonded. Leucine can be used in any of the L-form, D-form, and DL-form, but the L-form is desirable from the viewpoint that it exists in nature. Further, the amino acid in the hypoxic response promoter of the present invention avoids amino acid imbalance in the body at the time of taking, so that leucine, isoleucine, and valine are mixed in an appropriate mixing ratio rather than leucine alone, for example, 100 mass of leucine. A branched chain amino acid in which 10 to 100 parts by mass of isoleucine and 10 to 100 parts by mass of valine are mixed per part is desirable. The hypoxic response promoter of the present invention may be an amino acid alone or may be contained with a general food.

When calculating the dose (intake amount) of the amino acid, which is an active ingredient used in the present invention, the active ingredient of the drug used for the purpose of treatment, prevention, etc. of various disease abnormalities targeted in the present invention Included in the calculation for amino acids that are ingested or administered for other purposes, such as the need for a normal diet or for the treatment of other diseases. There is no need.
For example, it is not necessary to calculate by deducting the amount of amino acids per day taken from the normal diet from the daily dose of the active ingredient in the present invention.
Next, the present invention will be described in more detail by way of examples. It should be noted that the following examples are illustrative of the present invention and are not intended to limit the present invention.

Example 1: Induction of VEGF gene expression by leucine and BCAA <Cell culture and amino acid component modified medium (RPMI-AA)>
Cell line used was H4IIE and cells subcultured with 10% fetal bovine serum and RPMI1640 supplemented with penicillin / streptomycin. As a medium to be acted on during the experiment, a medium (hereinafter referred to as PRMI-AA) in which all amino acids were removed based on RPMI1640 and the glucose concentration was adjusted to 4.5 g / L was used. The PRMI-AA medium was adjusted as follows. 4XInorganic Slats Soln. And 100Xvitamin Soln. Were adjusted and stored as follows, and Other Component and 4X Inorganic Salts Soln. Were added to double-stage distilled water to a final concentration of 1X. Depending on the experimental conditions, a medium in which a final concentration of 800 μM leucine was added to the PRMI-AA medium was used.

4XRPMI base Medium (glucose concentration: 4.5g / l) Created by AA
(components)
Inorganic Salts 4X (500ml)
Ca (NO3) 2 · 4H ₂ O 200mg
KCl 800mg
MgSO ₄ (anhyd.) 97.68mg
NaCl 12g
NaHCO ₃ 4g
Na ₂ HPO ₄ (anhyd.) 1.6g
(Other components) 2L
D-glucose 9g
Glutathione (reduced) 2mg
HEPES 4.76g
Phenol Red 10mg

(vitamins) 100X (500ml)
Biotin 10mg
D-Ca Pantothenate 12.5mg
Choline Chloride 150mg
Folic Acid 50mg
i-Inositol 1.75g
Niaciamide 50mg
Para-aminobenzoic acid 50mg
Pyridoxine HCl 50mg
Riboflavin 10mg
Thiamine HCl 50mg
Vitamine B ₁₂ (100X) 2.5ml
Total 500ml

<Action on cells>
Cultivate H4IIE cells in a 10 cm cell culture dish to 80% Confluent, culture in serum-free RPMI1640 medium for 15 hours before adding the test substance amino acid, and then add amino acid-free from 2 hours before addition of amino acid. The cells were cultured in a RPMI-AA medium. O time, leucine (800 μM: FIG. 1) or BCAA (final concentration 800 μM leucine, 400 μM isoleucine, 480 μM valine: FIG. 2) or essential amino acids other than BCAA (lysine, histidine, arginine, tryptophan, threonine, phenylalanine, methionine) The medium was replaced with RPMI-AA medium supplemented with a final concentration of 800 μM (FIG. 3), and total RNA was extracted after 2 and 8 hours. When rapamycin (Wako Pure Chemical Industries) was added, it was added to the medium so that the final concentration was 100 μM 30 minutes before the replacement with the leucine-containing RPMI-AA medium (FIG. 4). All experiments were performed at N = 4, and VEGF gene expression was examined by the method described below.

<Purification of total RNA>
1 ml of ISOGEN (Nippon Gene) was added to each 10 cm culture dish and homogenized. Next, 200 μl of chloroform was added and lightly stirred. After standing at room temperature for 2 minutes, the mixture was centrifuged at 15000 rpm at 4 ° C. for 10 minutes, and the water tank was collected. A water bath and an equivalent volume of isopropanol were added, and after standing at room temperature for 5 minutes, it was centrifuged at 15000 rpm at 4 ° C. for 10 minutes. The supernatant was discarded, 70% ethanol was added to the precipitated pellet, and the mixture was centrifuged at 15,000 rpm at 4 ° C. for 10 minutes to collect the pellet. The pellet was dried at room temperature for 5 minutes and DEPC (diethyl pyroganate) treated water was added to dissolve the pellet.
<Composition of template>
The cDNA synthesis of the template used for Taqman PCR was performed using SuperScript First-Strand Synthesis System for RT-PCR (GIBCO BRL). 500 ng of total RNA, 0.5 μg / μl Oligo (dT) 12-18 1 μl, and 10 mM dNTPmix 1 μl were dissolved in DEPC-treated water to make a total volume of 10 μl. After reacting at 65 ° C for 5 minutes, cool, add 10XRT Buffer 2μl, 25mM MgCl ₂ 4μl, 0.1M DTT 2μl, RNase inhibitor 1μl, mix, then incubate at 42 ° C for 2 minutes, reverse transcriptase SUPERSCRIPT II RT 1μl (50 units) was added. The reaction was carried out at 42 ° C. for 50 minutes and further at 70 ° C. for 15 minutes.
<Primer design>
Primers were designed for the human VEGF gene and human β-actin as a housekeeping gene as a control. The external database Primer3 shown below was used for the design.
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

Table 1 shows the GenBank numbers, gene names, and primer base sequences of human VEGF gene and human β-actin gene.

Table 1

<Taqman PCR (CYBR Green method) reaction>
The reaction solution having the composition shown in Table 2 was mixed in a PCR tube for Taqman, and PCR reaction was performed in ABI7700 Prism Sequence Dtector. The reaction conditions were as follows.
Reaction conditions: 50 cycles at 50 ° C for 2 minutes → 95 ° C for 10 minutes → (95 ° C for 15 seconds → 60 ° C for 1 minute).
Table 2
PCR reaction solution composition (per tube)
Template cDNA (totel RNA 2.5ng equivalent) 0.1μl
primer soln. (5'-primer) 1μl
primer soln. (3'-primer) 1μl
dH2O
total 10μl

<Data analysis>
Each Taqman PCR reaction was performed at N = 3, and the relative amount of VEGF mRNA in mRNA amount was determined from the fluorescence value. The ratio between the average value and the relative amount of β-actin gene mRNA of the internal standard control obtained in the same sample was compared and shown in FIGS.
<Result>
The gene expression of VEGF was induced by leucine, and an increase in the amount of VEGF mRNA was observed about 3 times in 8 hours (FIG. 1). The same results as with leucine alone were obtained in the amino acid combination of leucine, isoleucine, and valine (FIG. 2). Such an increase in VEGF expression was not observed in the amino acid composition containing no leucine (FIG. 3). In addition, the increase in VEGF expression by leucine was inhibited by treatment with the mTOR inhibitor rapamycin (FIG. 4).
According to this example, it is clear that leucine increases the expression of VEGF mRNA in cells, and that the same effect can be obtained with a mixed amino acid (BCAA) containing leucine, isoleucine and valine. This action was not found in other essential amino acids except BCAA, and was shown to be specific to leucine or BCAA. Furthermore, the increase in the amount of VEGF mRNA by leucine was suppressed by the action of rapamycin, and VEGF expression induction by leucine was considered to be an effect through mTOR activation. From this, it is clear that substances that cause the activation of mTOR such as leucine increase the gene expression of VEGF.

  Attempts to increase VEGF production in vivo by administering VEGF as a protein preparation or gene therapy for the VEGF gene have been attempted in various ischemic diseases such as obstructive arteriosclerosis and heart failure, and have achieved high therapeutic effects. (Nature Reviews Drug Discovery 2, 863 -872 (2003), Curr Opin Mol Ther. 2004 Apr; 6 (2): 151-9.) If an excellent drug can increase endogenous VEGF expression in vivo, it promotes a hypoxic stress response, thereby preventing cell death and improving energy metabolism, thereby achieving therapeutic effects on various diseases. It is clear that

Example 2: Increase in the amount of HIF-1α protein under hypoxic stress by amino acids T cells were isolated and cultured from peripheral blood mononuclear cells (PBMC) in the blood of healthy volunteers, and under normal oxygen concentration and hypoxia The effects of amino acids in the medium on the amount of HIF-1α protein and the expression levels of various mRNAs were examined in culture at a concentration. PBMC was isolated by density gradient centrifugation using Ficoll-Plaque Plus (Amersham Bioscience), suspended in 10% heat-inactivated fetal calf serum (FCS) -RPMI1640 medium, and cultured at 37 ° C for 1 hour. After removing the adherent cells, a cell fraction rich in T cells was obtained using a nylon wool column.
Cultivation at various oxygen concentrations was performed by the method described in J. Immunology, 171: 6534-6540 (2003), and T cell stimulation was performed by adjusting T cells adjusted at a concentration of 2 × 10 ⁶ cells / ml in a 6-well culture dish. After adding 4 ml of each well and allowing to stand for 30 minutes, 10 nM PMA or 5 ng / ml was added by adding an anti-CD3 antibody (UCHT1, BD Pharmingen).

In the experiment examining the amino acid concentration and the expression of HIF-1α, the composition of the 1X medium was 100 mg / L Ca (NO ₃ ) ₂ .4H ₂ O, 400 mg / L, 48.84 mg / L MgSO ₄ , 6000 mg / L NaCl ₂ , Add 800 mg / L NaHPO4, 2000 mg / L D-Glucose, 1 mg / L glutathione, RPMI1640 100Xvitamine solution (Sigma Aldrich) to a final concentration of 1x, and add each amino acid to a final concentration of 0.40 mM Glycine, 0.40 mM L- Alanine, 0.40 mM L-Serine, 0.80 mM L-Threonine, 0.26 mM L-Cystein 2HCL, 0.20 mM L-Methionine, 8 mM L-Glutamine, 0.40 mM L-Asparagine, 0.40 mM L-Glutamic Acid, 0.80 mM L-Aspartic Acid, 0.80 mM L-Valine, 0.8 mM L-leucine, 0.80 mM L-Isoleucine, 0.80 mM L-Phenylalanine, L-Tyrosine ・ 2Na ・ 2H ₂ O 0.08 mM L-Tryptophan, 1.00 mM L-Lysine-HCl, 0.48 Added to mM L-Arginine-HCl, 0.27 mM L-Histidine HCl-H ₂ O, 0.4 mM L-Proline. The amino acid concentration of this medium was defined as 1X, and 1 / 2X, 1 / 4X, 2X, 3X, and 4X media were prepared. The amino acid removal medium (-AA) is a medium excluding all the above amino acids. BCAA-removed medium (-BCAA) is a medium obtained by removing Valine, Leucine, and Isoleucine from the above 1X medium, and leucine-removed medium (-LEU) is a medium obtained by removing Leucine from the above-mentioned 1X medium. The actual experiment was performed by adding 10% heat-inactivated fetal calf serum (FCS), 100 unit / ml penicillin and 100 μg / ml streptomycin to each medium. For amino acid removal stimulation, T cells cultured at a concentration of 2 × 10 ⁶ cells / ml with RPMI 1640 were collected, centrifuged, and the supernatant was removed. The supernatant was further washed once with PBS, and adjusted with a medium having a different amino acid composition.
After culturing the cells for 18 hours under various conditions, the whole cell extract is prepared and antibody specific for HIF-1α (Ab463: Abcam), phosphorylated S6K specific antibody, phosphorylated eIF2α antibody, phosphorylated Akt specific antibody Immunoblotting was performed using various antibodies such as Cell Signaling Technology. In addition, the total RNA extract was prepared, and a primer specific for each mRNA was used to detect by RT-PCR method according to the method described in J. Immunology, 171: 6534-6540 (2003).

FIG. 5 shows the effect of amino acid concentration in the medium on the expression level of HIF-1α protein under low oxygen concentration. As shown in FIG. 5A, when normal oxygen concentration was changed to low oxygen concentration, an increase in HIF-1α protein after 2 hours was observed, but this effect was lost when all amino acids in the medium were removed. At this time, phosphorylation of S6K1 existing downstream of signal transduction by amino acids was lost, and phosphorylation of eIF2α existing downstream of signal transduction in response to amino acid starvation was enhanced. Furthermore, when the amino acid concentration in the medium was changed variously, an increase in the amount of HIF-1α protein was observed depending on the amino acid concentration. This indicates that amino acids are essential for the expression of HIF-1α protein.
Fig. 6 shows the amount of HIF-1α protein after 2 hours when only BCAA is removed (-BCAA) from RPMI1640 medium containing all amino acids under hypoxia (1% oxygen partial pressure). is there. Since removal of only BCAA showed the same effect as removal of all amino acids, it can be seen that BCAA is particularly important among amino acids for the expression of HIF-1α protein. At this time, phosphorylation of S6K1 was also reduced, but Akt phosphorylation was not affected.

FIG. 7 shows the amount of intracellular HIF-1α protein after 2 hours when only leucine was removed from RPMI1640 medium containing all amino acids under low oxygen (1% oxygen partial pressure). It is a thing. Since removal of only BCAA showed the same effect as removal of all amino acids, it is understood that BCAA is important among amino acids, and leucine is most important for the expression of HIF-1α protein. At this time, RNA was extracted 18 hours after hypoxia treatment, and various mRNA levels were examined by RT-PCR. As a result, transcription of the adrenomedullin (ADM) gene known as the target gene of HIF-1α was induced. It was impaired in leucine removal medium (-LEU).
According to this example, leucine or a combination amino acid (BCAA) containing leucine, isoleucine, and valine played an important role in the expression of HIF-1α protein in cells under hypoxia, and the expression of the target gene of HIF-1α It is clear to make adjustments. Moreover, this action was not seen in other amino acids except BCAA, and was shown to be specific to leucine or BCAA. As a target gene of HIF-1α, for example, adrenomedullin (ADM) can be raised, but ADM has a strong vasodilatory action and blood pressure lowering action (Xitamula et a1: Biochem Biophys Res Commun 192,553-560,1993) It is clear that leucine is useful for the treatment of various ischemic diseases and hypertension through increased expression of HIF-1α and subsequent induction of ADM expression.

  In addition, activation of HIF-1α is thought to play an extremely important role in maintaining the survival of activated T cells in peripheral tissues (J. Immunology, 171: 6534-6540 (2003)). In order for T cells to perform biological defense functions such as tumor immunity and immunity against bacterial infection, it is necessary to invade tissues from blood vessels and survive locally for a certain period of time. From this, it is clear that leucine or BCAA is indispensable for the T cell function in vivo, especially for the local function. Therefore, leucine or BCAA activates T cell immunity through activation of HIF-1α and enhances tumor immunity, bacterial infection immunity, parasitic infection immunity, viral infection immunity, and thus various diseases and Can improve the condition.

Figure 2 shows the effect of leucine on VEGF expression. Figure 3 shows the effect of BCAAs on VEGF expression. The influence of the essential amino acid which removed BCAA in VEGF expression is shown. Figure 2 shows the effect of rapamycin on leucine-induced VEGF expression. The influence of the amino acid concentration in the medium on the expression level of HIF-1α protein under low oxygen concentration is shown. When only BCAA is removed (-BCAA) from RPMI1640 medium containing all amino acids under hypoxia (1% oxygen partial pressure), the amount of HIF-1α protein after 2 hours is shown. When only leucine is removed from the RPMI1640 medium containing all amino acids under low oxygen (1% oxygen partial pressure) (-LEU), the amount of intracellular HIF-1α protein after 2 hours is shown.

Claims (16)

  A hypoxic response promoter comprising an amino acid selected from leucine, isoleucine, valine or a mixture thereof.   The hypoxic response promoter according to claim 1, which activates mTOR.   The hypoxic response promoter according to claim 1 or 2, which activates HIF-1α.   The hypoxic response promoter according to any one of claims 1 to 3, which induces expression of Vascular Endothelial Growth Factor (VEGF) gene and / or adrenomodullin (ADM) gene.   A therapeutic / prophylactic agent for ischemic disease, comprising the hypoxic response promoter according to any one of claims 1 to 4.   6. The disease treatment / prevention agent according to claim 5, wherein the ischemic disease is myocardial infarction, heart failure, cerebral infarction, cerebral hyperemia, diabetic vascular disorder, obstructive arteriosclerosis colitis, ulcer, spinal cord disorder, optic neuropathy or neuropathy. .   An immunostimulant comprising the hypoxic response promoter according to any one of claims 1 to 4.   The immunostimulant according to claim 7, wherein the immunity is selected from the group consisting of tumor immunity, bacterial infection immunity, parasitic infection immunity and viral infection immunity.   Use of the hypoxic response promoter according to any one of claims 1 to 4 for preparing an ischemic disease therapeutic agent or an immunostimulant.   A therapeutic agent for highland life hypoxia-induced disorders, comprising an amino acid selected from leucine, isoleucine, valine or a mixture thereof.   A highland training aid comprising an amino acid selected from leucine, isoleucine, valine or a mixture thereof. (1) preparing a cultured cell cultured using a culture solution containing no amino acid;
(2) preparing cultured cells cultured using a culture solution containing amino acids;
(3) Add the test substance to the cultured cells of (1) and (2);
(4) Measure the VEGF gene expression index under normoxic partial pressure and low oxygen partial pressure in the cultured cells of (1) and (2), and select a test substance from which an equivalent or higher VEGF gene expression index is obtained.
A screening method for a hypoxic response promoter, comprising each step.   An amino acid-containing drug or food containing an amino acid selected from leucine, isoleucine, valine, or a mixture thereof, and packaged in a package describing a high-altitude training aid effect.   An activator of mTOR, comprising an amino acid selected from leucine, isoleucine, valine or a mixture thereof.   A HIF-1α activator comprising an amino acid selected from leucine, isoleucine, valine or a mixture thereof.   A Vascular Endothelial Growth Factor (VEGF) gene and / or adrenomodullin (ADM) gene expression inducer, comprising an amino acid selected from leucine, isoleucine, valine or a mixture thereof.