JP5797051B2 - Method for searching for adipocyte differentiation / maturation inhibitor and / or fat accumulation inhibitor - Google Patents

Description

  The present invention relates to a method for searching for an adipocyte differentiation / maturation inhibitor and / or a fat accumulation inhibitor.

  In recent years, the obese population has been increasing due to westernization of eating habits, lack of exercise and stress. Obesity has been reported to cause various lifestyle-related diseases such as insulin resistance and arteriosclerosis, and is a major social problem in terms of maintaining and promoting the health of the people.

  Obesity refers to a state in which body fat has accumulated excessively, and is caused by the amount of energy consumed exceeding the amount of energy consumed. Therefore, reducing the amount of energy intake or increasing the amount of energy consumed is thought to lead to the prevention and improvement of obesity. Examples of methods for preventing and improving obesity and various lifestyle-related diseases associated with obesity include exercise, calorie restriction, and improvement of lifestyle habits. These are generally considered effective for the prevention and amelioration of obesity, but in many cases sufficient effects cannot be obtained in many cases.

  So far, extensive research has been conducted with the aim of developing obesity-improving agents and lipid metabolism promoters. For example, for the purpose of promoting energy metabolism, PPAR (Peroxisome Proliferator Activated Receptor: Peroxisome proliferator activated receptor) activator (Non-patent Documents 1 to 3) or inhibitor (Non-patent Document 4), AMPK (AMP-activated protein kinase) activator (Non-patent Documents 5 and 6) and the like. In addition, ACC-1 (acetyl-CoA carboxylase) inhibitor (Non-patent Document 7), FAS (Fatty Acid Synthase) inhibitor (Patent Document 1), and the like can be cited as the purpose of suppressing fat synthesis.

  Obesity is also closely linked to adipocyte hypertrophy and an increase in cell number, and therefore suppresses the differentiation of preadipocytes into mature adipocytes, or suppresses fat accumulation in adipocytes. Is considered effective in the treatment and prevention of obesity. Insulin, dexamethasone and isobutylmethylxanthine are generally used as induction stimuli for differentiating fat cells into mature fat cells. With these differentiation-inducing stimuli, the expression of transcription factors, PPARs and C / EBPs, which are master regulators of adipocyte differentiation, increased, followed by ACC-1, FAS and DGAT (diacylglycerol acyltransferase) and lipid synthesis involved in lipid synthesis. Furthermore, the expression of various lipid metabolism-related genes specifically expressed in mature adipocytes such as GLUT4 (glucose transporter 4) and LPL (lipoprotein lipase) involved in sugar / lipid uptake increases. In addition, Fabp4 (fatty acid binding protein 4), which is a fatty acid transport carrier, is also expressed specifically in differentiated adipocytes and is used as a marker for differentiation and maturation of adipocytes.

  As with other cell types, adipocytes are surrounded by an extracellular matrix (ECM) around the cells. It has been reported that ECM not only provides a scaffold for cell adhesion, but also contributes to many physiological activities such as cell proliferation, differentiation, and migration via receptors present on the cell membrane surface. The molecular composition of ECM varies from cell to cell, and cells are thought to express and maintain cell functions in an appropriate state by forming an environment called ECM having a specific molecular composition.

Laminin is a protein known as a major component of the basement membrane, which is an ECM that exists between the epithelial cell layer and the stromal cell layer. Laminin forms a heterotrimer composed of α, β, and γ chains, and plays a central role in cell adhesion to the basement membrane. Laminin is also known to have many biological activities such as cell proliferation, migration and neurite outgrowth.
The activity of laminin is thought to be exerted through the laminin signal system through the binding of laminin to a cell surface receptor and intracellular signaling coupled thereto. Integrins α6β1, α3β1, α6β4, α7β1, dystroglycan, syndecan and the like are known as main receptors for receiving laminin (Non-patent Document 8). Integrins are heterodimeric proteins composed of α-subunits and β-subunits. Currently, 24 integrins have been reported in mammals by combining 18 kinds of α-subunits and 8 kinds of β-subunits, and each integrin specifically recognizes an ECM ligand (Non-patent Document 9). There are three main information transmission pathways downstream of the integrin. First, pathways that control cytoskeletal systems such as actin via Rho, second, pathways that transmit signals related to survival such as apoptosis suppression via phosphorylation of PI3K / Akt, and third, phosphorylation of MAPK (Erk) It is a pathway that transmits signals related to cell growth through oxidation.

  Laminin is present in various tissues and is also expressed in adipose tissue. It has been reported that laminin-8 (α4β1γ1) is particularly expressed in adipocytes, and that gene expression increases with differentiation and maturation (Non-patent Document 10). However, the expression level change and physiological significance of laminin in adipose tissue have not yet been fully clarified. In addition, the effect of laminin on adipocyte properties such as differentiation maturation and fat accumulation has not been elucidated.

JP 2007-63156 A

Curr. Opin. Lipidol., 1999, 10: 151-159 BMC Pharmacol., 2004, 4 (23) Cell 113, 2003: 159-170 Folia. Pharmacol. Jpn., 2003, 122: 294-300 Molecular Medicine., 2002, 39 (4): 398-407 Diabetes., 2001, 50 (5): 1076-1082 Nat.Med., 2000, 6 (10): 1115-1120 Matrix Biology, 2006, 25 (3): 189-197 J. Biol. Chem., 2000, 275 (29): 21785-21788 Matrix Biology, 1997, 16 (4): 223-230

  The present invention relates to a method for searching for an adipocyte differentiation / maturation inhibitor or fat accumulation inhibitor that is effective in reducing systemic or local body fat, using laminin signal inhibitory activity as an index.

  The present inventors investigated the effect of the extracellular environment containing the extracellular matrix on adipocyte characteristics. As a result, it was found that culturing adipocytes in the presence of laminin enhances differentiation and maturation of cells and fat accumulation, and suppression of laminin signal is effective in suppressing differentiation and maturation of adipocytes and fat accumulation I found out.

That is, the present invention provides the following.
(1) A method for searching for an adipocyte differentiation / maturation inhibitor and / or a fat accumulation inhibitor, characterized by evaluating laminin signal inhibitory activity of a test substance in an adipocyte.
(2) The method according to (1), wherein the laminin signal suppressing activity is an activity of inhibiting the binding between laminin and a laminin receptor.
(3) The method according to (1), wherein the laminin signal inhibitory activity is an activity that suppresses the expression of laminin or a laminin receptor.
(4) The method according to (1), wherein the laminin signal suppressing activity is an activity of suppressing intracellular signal transduction caused by laminin.

  ADVANTAGE OF THE INVENTION According to this invention, the differentiation maturation or fat accumulation of an adipocyte can be suppressed, and the material effective in the reduction | decrease of whole body or local body fat can be provided.

Effect of laminin on differentiation and maturation of adipocytes: Influence of laminin on the expression level of genes specifically expressed in differentiated adipocytes. LM; laminin, *; P <0.05, **; P <0.01, ***; P <0.001 (Student's t-test), error bar = standard error, n = 3. Laminin promotes fat accumulation. A: Oil-red O stained adipocytes, B: Cell staining (relative absorbance of isopropanol extract of Oil-red O stained cells). LM; laminin, *; P <0.05 (Student's t-test), error bar = standard error, n = 3. Insulin stimulation responsive enhancement effect by laminin: (A) Effect of laminin on phosphorylation of signaling molecules (Akt, Erk, IRS-1) downstream of insulin receptor. (B) Effect of laminin on signaling molecule Akt in the absence of insulin. Inhibition of adipocyte differentiation and maturation by laminin partial peptide. LM; laminin, *; P <0.05 (Student's t-test), error bar = standard error, n = 3.

As shown in Examples described later, laminin has an action of promoting the differentiation and maturation of fat cells and an action of promoting fat accumulation in fat cells. Moreover, as shown in the below-mentioned Example, the differentiation and maturation of an adipocyte is prevented by inhibiting the coupling | bonding of a laminin and a receptor. That is, a substance capable of suppressing the transmission of a series of laminin signals caused by laminin to cells is useful as a substance having an effect of suppressing differentiation and maturation of adipocytes and suppressing fat accumulation in adipocytes.
Accordingly, the present invention provides a method for searching for an adipocyte differentiation / maturation inhibitor and / or a fat accumulation inhibitor characterized by evaluating laminin signal inhibitory activity of a test substance in adipocytes.

  In this specification, a laminin signal refers to a series of processes in which a signal is transmitted into a cell via laminin receptors present on the surface of a cell membrane from laminin, which is a kind of extracellular matrix (ECM). That is, the laminin signal can be measured using, for example, laminin expression level or laminin receptor expression level by cells, binding of laminin receptor and laminin on the cell surface, intracellular signal transduction caused by laminin, etc. as indicators. it can. These indicators may be measured either alone or in combination.

Examples of laminin include subtypes such as α1, α2, α3, α4, α5, β1, β2, β3, γ1, γ2, and γ3.
Examples of laminin receptors include integrins such as integrin α6β1, α3β1, α6β4, α7β1, dystroglycan, syndecan and the like.

  The expression level of laminin molecules and laminin receptor molecules may be determined by any method known in the art as a method for measuring proteins. For example, the expression level of the molecule may be determined by directly measuring the protein expression level, or the subtype, polymorphism, proteoglycan fused with a sugar, polymer with other proteins, fragmentation or release. Expression levels of mature peptides, immature forms and degradation forms of messenger RNA (mRNA) encoding them; and activities of transcriptional regulatory regions that control their expression, etc. The expression level may be calculated based on this.

  For example, the expression level of a laminin molecule is the expression level of α1, α2, α3, α4, α5, β1, β2, β3, γ1, γ2, γ3, Alternatively, the expression level of the mRNA may be evaluated by measuring the expression level of the laminin receptor molecule integrin, such as α-subunits such as α3, α4, α7 or β1, β4, etc. constituting the integrin protein. You may evaluate by measuring the expression level of (beta) -subunit, or the expression level of those mRNA.

  When measuring the protein expression level, for example, a protein extracted from adipose tissue may be quantified by a conventional method. Further, for example, the target molecule may be specifically quantified by an ELISA method or subjected to electrophoresis such as SDS-PAGE and then separated, and then the target molecule may be detected and quantified. When measuring the RNA expression level, cDNA is synthesized from RNA extracted from adipose tissue by a conventional method, and this is subjected to the PCR method to detect and quantify RNA corresponding to the target molecule.

  Intracellular signal transduction caused by laminin can be evaluated based on, for example, the activity of molecules in the intracellular signal transduction system downstream of the laminin receptor. As the intracellular signal transduction system downstream of the laminin receptor, for example, when the receptor is an integrin, there are mainly the following three. First, pathways that control cytoskeletal systems such as actin via Rho, second, pathways that transmit signals related to survival, such as apoptosis suppression, through phosphorylation of PI3K / Akt, and third, phosphorus of MAPK (Erk) It is a pathway that transmits signals related to cell growth through oxidation. Therefore, examples of the molecules of the intracellular signal transduction system include Rho, PI3K / Akt, MAPK (Erk1 / 2), IRS-1, and molecules that control their activities. The activity of a molecule can be evaluated using, for example, the expression level of the protein or mRNA of the molecule or phosphorylation of the molecule as an index.

  The method for measuring the expression level of a protein or its mRNA is as described above. Is molecular phosphorylation specifically determined by an ELISA method for a protein extracted from adipose tissue using a conventional measurement method performed in the art, for example, a specific phosphorylated antibody against a target molecule? Alternatively, it can be measured by a method of detecting and quantifying the target molecule after separation by electrophoresis such as SDS-PAGE.

  The binding between laminin receptor on the cell surface and laminin is performed by a conventional measurement method used in the art, for example, laminin labeled with fluorescence or RI is incubated with laminin receptor-expressing cells, and the amount of laminin binding is measured. It can be measured by the method. In addition, a molecular interaction measuring instrument such as Biacore can also be used.

  The test substance used in the method of the present invention is not particularly limited as long as it is a substance desired to be used as a fat accumulation regulator. The test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, etc., and may be a compound, a composition or a mixture. Good.

  The adipocytes used in the method of the present invention may be adipose tissue-derived adipocytes collected from a living body or cultured adipocytes.

  Preferably, in the method of the present invention, adipocytes are cultured in the presence of laminin and a test substance. At this time, a substance capable of promoting differentiation induction of adipocytes such as insulin, dexamethasone, isobutylmethylxanthine may be further added. It is desirable to culture the adipocytes for a time sufficient for the cells to differentiate into mature adipocytes (for example, about 10 days after induction of differentiation). Thereafter, the laminin signal in the cultured adipocytes is measured. The method for measuring the laminin signal is as described above. Subsequently, the laminin signal inhibitory activity of the cell by a test substance is evaluated based on the measured laminin signal. Evaluation of laminin signal inhibitory activity is, for example, between a test substance addition group with a higher concentration and a test substance addition group with a lower concentration; between a test substance addition group and a non-addition group; a test substance addition group and a control substance This can be done by comparing the laminin signal of cells between the addition group; or before and after the addition of the test substance. A test substance that suppresses cellular laminin signal can be selected as an adipocyte differentiation / maturation inhibitor and / or a fat accumulation inhibitor.

An example of a more specific procedure of the method of the present invention will be described below.
(A) Coat the bottom of the cell culture vessel with the purified laminin. Separately, prepare a culture container without laminin coating.
(B) Adipose-derived cells are respectively seeded in laminin-coated and uncoated culture containers (test group and control group, respectively). Usually, the medium is high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. The seeded cells are cultured under conditions of 37 ° C. and 5% CO ₂ .
(C) The cells are normally cultured until they become 100% confluent, and then the medium is replaced with a differentiation-inducing medium. As the differentiation induction medium, a normal medium supplemented with 5 μg / ml insulin, 1 μM dexamethasone and 0.115 mg / ml isobutylmethylxanthine is used. The day when the medium is changed to the differentiation-inducing medium is referred to as day1, and the medium is changed again to the normal medium at day3. Thereafter, the medium is changed every two days.
(D) In any of the above (b) to (c) or in all steps, a test substance is contained in the culture medium of the test group.
(E) The laminin signal of the cell is measured at day 10, the measured value is compared between the control group and the test group, and the influence of the test substance on the laminin signal is evaluated. A test substance in which laminin signal inhibitory activity is observed is selected as an adipocyte differentiation / maturation inhibitor and / or a fat accumulation inhibitor.

  For example, in the method of the present invention, when the laminin signal is measured according to the above procedures (a) to (e), the laminin signal of the adipocytes is significantly compared with the control group to which the test substance was not added. A test substance preferably suppressed to 20% or less, more preferably 50% or less, can be selected as an adipocyte differentiation / maturation inhibitor and / or a fat accumulation inhibitor.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Example 1 Effect of Laminin Promoting Adipocyte Differentiation and Maturation For the experiment, mouse preadipocyte-derived cell line 3T3-L1 cells (ATCC) were used. As a normal medium, high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum was used, and cultured under conditions of 37 ° C. and 5% CO ₂ .
3T3-L1 cells were seeded in a 6-well dish (BD) coated with laminin, normally cultured until 100% confluent, and then the medium was replaced with a differentiation-inducing medium. The differentiation induction medium used was a medium supplemented with 5 μg / ml insulin, 1 μM dexamethasone, and 0.115 mg / ml isobutylmethylxanthine. The day when the medium was changed to the differentiation-inducing medium was defined as day1, and the medium was replaced with the normal medium again on day3. Thereafter, the medium was changed every two days, and the cells were collected on day 10 (laminin group). As a control, cells cultured under the same conditions using a dish without laminin coat were collected (control group).

Total RNA was prepared from the obtained cells, and cDNA was obtained by reverse transcription reaction. Using the obtained cDNA as a template, each of Fabp4, PPARγ, GLUT4, LPL, ACC-1, FAS, DGAT, HSL, and ATGL by quantitative PCR using 7500 Fast Real-Time PCR System (Applied Biosystems) The expression level of the gene was measured.
Fabp4 is known as a differentiation / maturation marker for adipocytes. PPARγ is known as a master regulator of adipocyte differentiation. GLUT4 and LPL are genes related to sugar and lipid uptake, ACC-1, FAS and DGAT are lipid synthesis-related genes, HSL and ATGL are lipolysis-related genes, both of which are involved in energy metabolism. Known as the major gene involved.
The measured expression level of each gene was corrected by an internal standard (36B4 gene expression level). The obtained values were graphed as an average value ± standard error (n = 3). The significant difference test was performed using Student's t-test.

  The results are shown in FIG. The level of expression of Fabp4, an adipocyte differentiation / maturation marker, was significantly increased by laminin and reached 2.4 times that of the control group. In addition, the mRNA expression level of the transcription factor PPARγ, which is a master regulator of adipocyte differentiation, was significantly increased by laminin and reached 3.5 times that of the control group. Furthermore, the expression of major genes involved in energy metabolism (GLUT4, LPL, ACC-1, FAS and DGAT) was also significantly increased by laminin. From these results, it was shown that laminin promotes differentiation and maturation of adipocytes. Furthermore, GLUT4, LPL, ACC-1, FAS and DGAT are genes involved in sugar / lipid uptake and lipid synthesis, indicating that laminin promotes fat accumulation in adipocytes. Therefore, suppression of laminin signal leads to suppression of adipocyte differentiation and maturation and fat accumulation, and laminin signal is effective as an index for searching for adipocyte differentiation maturation inhibitor or fat accumulation inhibitor. .

Example 2 Effect of promoting fat accumulation by laminin After fixing fat cells collected by the same method as in Example 1 with 10% formalin, lipid droplets in the cells were stained with Oil-red O staining solution, and examined with a stereomicroscope. Observed and photographed. The Oil-red O staining solution used was an Oil red-O stock solution (0.5% Oil red-O / isopropanol) and DDW mixed at 3: 2 and filtered through a 0.5 mm filter. In order to quantify the degree of staining (fat content) of the cells, the pigment was extracted from the cells with isopropanol, the absorbance OD450 was measured, and the relative absorbance with the absorbance of the control group being 1 was determined. The obtained absorbance was graphed as an average value ± standard error (n = 3). The significant difference test was performed using Student's t-test.

  The results are shown in FIG. The oil-red O staining degree of the laminin group was stronger than that of the control group (FIG. 2A), and a significant difference was also observed in the quantitative analysis of the staining degree (FIG. 2B). These results indicated that laminin promoted fat cell fat accumulation. Accordingly, suppression of laminin signal leads to suppression of fat accumulation in adipocytes, and laminin signal is effective as an index for searching for a fat cell fat accumulation inhibitor.

Example 3 Insulin signal responsive enhancement effect by laminin In this example, Akt, Erk and IRS-1 and their activated (phosphorylated) forms p-Akt, p-Erk and p-IRS-1 were Western blotted. The effect of laminin on molecular activation was examined, and the effect of laminin on the intracellular signal transduction system was evaluated. Akt, Erk, and insulin receptor substrate (IRS) -1 are insulin signaling molecules that exist downstream of the insulin receptor and transmit a signal due to insulin stimulation through phosphorylation thereof.

3T3-L1 cells were seeded in a 6-well dish (BD) coated with laminin and cultured normally until 100% confluent. After overnight culture in serum-free medium, the medium was replaced with a medium supplemented with insulin at a final concentration of 5 μg / ml, and the cells were collected at predetermined time intervals (5 min, 15 min, 30 min, 60 min) (laminin). group). As a control, cells cultured under the same conditions using a dish without laminin coat were collected (control group).
Lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM Sodium Chloride, 1% Triton X-100) was added to the collected cells and well homogenized with a 25G needle. After leaving still on ice for 15 minutes, it centrifuged at 12000 rpm and 4 degreeC for 10 minutes. The supernatant was obtained as a protein solution.
Using the obtained protein solution as a sample, Western blotting using the following antibodies was performed to detect each of the above molecules.
anti-Akt (cell signaling; # 9272)
anti-phospho-Akt Ser473 (cell signaling; # 9271)
anti-Erk1 / 2 (cell signaling; # 4695)
anti-phospho-Erk1 / 2 Thr202 / Tyr204 (cell signaling; # 9101)
anti-IRS-1 (upstate biotechnology; # 06-248)
anti-phospho-IRS-1 Tyr895 (cell signaling; # 3070)

  In the same procedure, but without adding insulin to the medium, the cells are cultured and collected for a predetermined time (5 minutes, 10 minutes, 20 minutes, 30 minutes, and 60 minutes) to prepare a protein solution, Akt and its activated form p-Akt were detected by Western blotting.

The results are shown in FIGS. 3A and B. All of Akt, Erk and IRS-1 were expressed to the same extent in the laminin group and the control group. Expression of activated forms of molecules p-Akt and p-IRS-1 increased in the laminin group compared to the control group, indicating that laminin promoted activation of these molecules. (FIG. 3A). Therefore, laminin enhances the response to insulin stimulation, particularly the response through the PI3K / Akt mediated pathway. From these results, it is possible that laminin enhances the activation of the insulin signal transduction system, which may contribute to the effect of promoting the differentiation and maturation of fat cells and the effect of promoting fat accumulation. It was suggested that differentiation and maturation of fat cells and fat accumulation can be evaluated using activity as an index.
On the other hand, the activation promotion effect of the molecule by laminin was not observed in the absence of insulin (FIG. 3B). Therefore, laminin does not directly induce phosphorylation of these molecules, but is thought to support the phosphorylation of molecules by insulin stimulation. The presence of intracellular signaling molecules was suggested. In addition to the above-mentioned insulin signaling molecules, the activity of other intracellular signaling molecules activated by laminin can also be used as an index for evaluating differentiation and maturation of fat cells and fat accumulation. .

Example 4 Inhibition of Adipocyte Differentiation and Maturation by Suppressing Laminin Signal Laminin partial peptide YIGSR (Tyr-Ile-Gly-Ser-Arg: SEQ ID NO: 1, Graf) that competitively inhibits the binding of laminin to the corresponding receptor et al, Biochemistry, 1987, 26 (22): 6896-6900 and Nomizu Motoi, Protein, Nucleic Acid, Enzyme, 2000, 45 (14): 2475-2482) The effect of suppressing differentiation / maturation of adipocytes by signal suppression was examined.
Rat primary cultured cells derived from subcutaneous fat were suspended in a normal medium supplemented with the C-terminal amidated form of the peptide shown in SEQ ID NO: 1 (final concentration 100 μg / ml), incubated at 37 ° C. for 30 minutes, and then laminin. It seed | inoculated to the coated 6-hole dish (BD company). Thereafter, cells were cultured in the same manner as in Example 1 except that the C-terminal amidated form of the above peptide (final concentration 100 μg / ml) was added to all the media, and adipocytes were collected (laminin + inhibition) Peptide group). As a comparative control, cells cultured under the same conditions without adding the peptide were collected (laminin group). As a negative control, cells cultured under the same conditions using a dish without laminin coat were collected (control group).

  Total RNA was prepared from the obtained cells, and quantitative PCR was performed on Fabp4 expression using cDNA obtained by reverse transcription reaction as a template. The Fabp4 gene expression level was corrected by an internal standard (36B4 gene expression level). The obtained values were graphed as an average value ± standard error (n = 3). Significance test was performed using Student's t-test.

  The results are shown in FIG. The expression of Fabp4, a marker for differentiation and maturation of adipocytes, increases in the presence of laminin (laminin group), but its expression level is determined by competitively inhibiting the binding of laminin and its receptor with a C-terminal amidated form of laminin partial peptide. Decreased significantly (laminin + inhibitory peptide group). The action of laminin to promote differentiation and maturation of adipocytes can be suppressed by suppressing laminin signal, for example, by inhibiting the binding between laminin and its receptor.

Claims (5)

A method for searching for an adipocyte differentiation / maturation inhibitor and / or a fat accumulation inhibitor characterized by evaluating laminin signal inhibitory activity of a test substance in an adipocyte ,
The method wherein the laminin signal is expression of one or more genes selected from the group consisting of Fabp4, PPARγ, GLUT4, LPL, ACC-1, FAS and DGAT . The method according to claim 1, wherein an increase in the expression level of the gene represents activation of a laminin signal. The method according to claim 1 or 2, wherein the laminin signal suppressing activity is an activity of inhibiting the binding between laminin and a laminin receptor. The laminin signaling inhibitory activity The method of claim 1 or 2, wherein the activity of suppressing the expression of laminin or laminin receptor. The method according to claim 1 or 2 , wherein the laminin signal inhibitory activity is an activity to suppress intracellular signal transduction caused by laminin.