JP5717331B2 - Evaluation method and evaluation kit of visceral fat increase inhibitory effect, screening method for substance, and use as marker - Google Patents

Description

  The present invention relates to an evaluation method and an evaluation kit for a visceral fat increase inhibitory effect, a screening method for a substance, and use as a marker. More specifically, the present invention develops visceral fat-type obesity administered with a test substance. Alternatively, the method for evaluating the visceral fat increase inhibitory effect for evaluating the visceral fat increase inhibitory effect of the test substance using the concentration of the marker substance in the body fluid of the animal that can develop as an index, and the visceral fat capable of simply performing the evaluation method The present invention relates to a kit for evaluating an increase inhibitory effect, a method for screening a substance having a desired visceral fat increase inhibitory effect using the evaluation method, and use as a marker.

  In recent years, westernization of eating habits has progressed, and lifestyle-related diseases such as obesity, diabetes, hyperlipidemia, and arteriosclerosis, which are considered to be caused by this, are increasing. These increased incidences are not genetic but mainly due to environmental factors. For example, abnormal lipid metabolism due to ingestion of a high-fat diet or a high-calorie diet causes increased blood lipids, development of insulin resistance, adipocyte hypertrophy, insulin secretion failure, and the like. As a result, diabetes, obesity, arteriosclerosis, etc. occur with high probability, leading to the progress of the disease state.

  In addition, it is becoming clear that visceral fat-type obesity is greatly involved in these lifestyle-related diseases, and so-called “metabolic syndrome” (metabolic syndrome, visceral fat syndrome) has attracted attention. Metabolic syndrome is defined as a condition having two or more of hyperglycemia, hypertension, and lipid abnormality in addition to visceral fat obesity. Metabolic syndrome, which can be said to be a complex disease based on visceral fat obesity, promotes arteriosclerosis and rapidly increases the risk of heart disease and stroke. Therefore, prevention and improvement of metabolic syndrome is important for maintaining a long life and high quality of life.

  In order to prevent and improve metabolic syndrome, it is effective to improve lifestyle habits. For example, it has been proposed to improve exercise habits and eating habits. Regarding eating habits, it is considered that the onset of metabolic syndrome can be prevented and improved by actively taking foods that have the effect of suppressing the increase in visceral fat. Patent Documents 1 and 2 disclose foods and drinks that have the effect of reducing visceral fat. On the other hand, although the screening system for substances having an inhibitory effect on the increase in visceral fat is not clear, Patent Document 3 discloses a screening system related to high cholesterol and high neutral fat.

JP 2008-214253 A JP 2008-169147 A JP 2007-64747 A

  As described above, one measure for preventing and improving metabolic syndrome is to ingest food containing a substance having an inhibitory effect on the increase in visceral fat. For that purpose, it is useful to construct a system for evaluating the visceral fat increase inhibiting effect of a substance and a screening system for substances having an visceral fat increase inhibiting effect. For example, if a biomarker (marker substance) reflecting the state of visceral fat can be identified, it is considered that various evaluation systems and screening systems using the biomarker as an index can be constructed.

  Then, this invention makes it a subject to identify the new biomarker relevant to a visceral fat, and to provide the method etc. which evaluate the visceral fat increase suppression result which a test substance has using this biomarker.

The related invention for solving the above-described problem is at least one of the following marker substances (A01) to (A11) in a body fluid of an animal that has developed or can develop visceral fat-type obesity administered with a test substance: It is a method for evaluating the visceral fat increase inhibitory effect, characterized in that one concentration is compared with a reference value and the visceral fat increase inhibitory effect of the test substance is evaluated.
(A01) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 4610 when subjected to mass spectrometry;
(A02) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 5500 when subjected to mass spectrometry;
(A03) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 7050 when subjected to mass spectrometry;
(A04) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 8330 when subjected to mass spectrometry;
(A05) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 8840 when subjected to mass spectrometry;
(A06) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 8930 when subjected to mass spectrometry;
(A07) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 9070 when subjected to mass spectrometry;
(A08) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 11600 when subjected to mass spectrometry;
(A10) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 13900 when subjected to mass spectrometry;
(A11) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 21400 when subjected to mass spectrometry.

The method for evaluating the inhibitory effect on visceral fat increase according to the present invention is at least one of the marker substances (A01) to (A11) in the body fluid of an animal that develops or can develop visceral fat-type obesity administered with a test substance. One concentration is compared with a reference value, and the visceral fat increase inhibitory effect of the test substance is evaluated. Each of the marker substances (A01) to (A11) is a protein specifically detected in the body fluid of an animal that develops or can develop visceral fat-type obesity. According to the evaluation method of the visceral fat increase inhibitory effect of this invention, the visceral fat increase inhibitory effect of the test substance can be evaluated easily and with high accuracy.

  An “animal that can develop visceral fat-type obesity” refers to an animal that is designed to develop visceral fat-type obesity and has not yet developed visceral fat-type obesity. The “animal that develops or can develop visceral fat-type obesity” refers to “an animal that develops visceral fat-type obesity” or “an animal that can develop visceral fat-type obesity”. In addition to animals that can be bred such as rats, the types of animals include humans.

  Here, the values such as “about 4610”, “about 8930”, “about 21400”, etc. of the mass / charge ratio (hereinafter sometimes abbreviated as “m / z”) in each marker substance are This is a value that takes into account the error range of the measured value, and has a width of approximately ± 0.2%. That is, about 4610 represents about 4610 ± 0.2%, about 8930 represents about 8930 ± 0.2%, and about 21400 represents about 21400 ± 0.2%. The other mass / charge ratios have a width of approximately ± 0.2% in a similar manner. In addition, these marker substances are mainly proteins present in blood. When the test substance has a visceral fat increase inhibitory effect, the concentrations of the marker substances (A01), (A03), (A04), (A05), (A06), (A10), and (A11) in the body fluid of the animal are The lower values are shown, and the concentrations of (A02), (A07), and (A08) show higher values.

The invention according to claim 1 includes the following marker substances (A01), (A02), (A07), and (A07) in the body fluid of an animal that has developed or is capable of developing visceral fat-type obesity administered with a test substance: A method for evaluating the visceral fat increase inhibitory effect, comprising comparing at least one concentration of A08) with a reference value and evaluating the visceral fat increase inhibitory effect of the test substance.
(A01) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 4610 when subjected to mass spectrometry;
(A02) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 5500 when subjected to mass spectrometry;
(A07) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 9070 when subjected to mass spectrometry;
(A08) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 11600 when subjected to mass spectrometry.

The related invention is the above-described evaluation method of the visceral fat increase inhibitory effect characterized by satisfying at least one of the following (a) to (i).
(A) The marker substance (A03) is apolipoprotein C1 or a modified form thereof.
(B) The marker substance (A04) is apolipoprotein C2 or a modified form thereof.
(C) The marker substance (A05) is apolipoprotein A2 or a modified form thereof.
(D) The marker substance (A06) is apolipoprotein C3 or a modified form thereof.
(E) The marker substance (A07) is complement C3 or a modified form thereof.
(F) The marker substance (A08) is β2-microglobulin or a modified form thereof.
(H) The marker substance (A10) is transthyretin or a modified form thereof.
(I) The marker substance (A11) is apolipoprotein M or a modified form thereof.

Apolipoprotein C1, apolipoproteins C2, apolipoprotein A2, apolipoprotein C3, complement C3, beta2-microglobulin, transthyretin, since none of the apolipoprotein M physicochemical properties are well known, the present invention According to the evaluation method of the visceral fat increase inhibitory effect, the analysis of the marker substances (A03) to (A11) is easy.

The invention according to claim 2 is the evaluation method of the visceral fat increase inhibitory effect according to claim 1, characterized in that at least one of the following (e) and (f) is satisfied.
(E) The marker substance (A07) is complement C3 or a modified form thereof.
(F) The marker substance (A08) is β2-microglobulin or a modified product thereof.

  A representative example of “modified protein” is a protein in which at least one of the amino acid residues constituting the protein is modified. “Modification” includes not only addition of a compound or functional group (eg, phosphorylation) but also elimination (eg, dephosphorylation). The “protein or a modified form thereof” includes an isoform of the protein. Further, “protein or a modified form thereof” includes substantially the same protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein. Furthermore, “protein or a modified product thereof” includes a protein fragment derived from the protein that has been cleaved by a protease. In the case of a complex protein, “protein or a modified product thereof” includes its subunits.

A related invention for solving the same problem belongs to any of the following (B1) to (B8) in the body fluid of an animal that has developed or can develop visceral fat-type obesity administered with a test substance It is an evaluation method for the visceral fat increase inhibitory effect characterized by comparing at least one concentration of the marker substance with a reference value and evaluating the visceral fat increase inhibitory effect of the test substance.
(B1) Apolipoprotein C1 or a modified product thereof,
(B2) apolipoprotein C2 or a modified product thereof,
(B3) Apolipoprotein A2 or a modified product thereof,
(B4) Apolipoprotein C3 or a modified product thereof,
(B5) complement C3 or a modified product thereof,
(B6) β2-microglobulin or a modified product thereof,
(B7) glutathioneated transthyretin,
(B8) Apolipoprotein M or a modified product thereof.

The method for evaluating the visceral fat increase inhibitory effect of the present invention belongs to any one of the above (B1) to (B8) in the body fluid of an animal that has developed or can develop visceral fat type obesity administered with a test substance. The concentration of at least one marker substance is compared with a reference value, and the visceral fat increase inhibitory effect of the test substance is evaluated. The marker substances belonging to the above (B1) to (B8) are all proteins that are specifically detected in body fluids of animals that develop or can develop visceral fat-type obesity. According to the evaluation method of the visceral fat increase inhibitory effect of this invention, the visceral fat increase inhibitory effect of the test substance can be evaluated easily and with high accuracy. In particular, apolipoprotein C1, apolipoprotein C2, apolipoprotein A2, apolipoprotein C3, complement C3, β2-microglobulin, transthyretin, and apolipoprotein M are all well known for their physicochemical properties. Is easy.

The invention according to claim 3 is at least one of the marker substances belonging to any of the following (B5) and (B6) in the body fluid of an animal that has developed or is capable of developing visceral fat-type obesity to which a test substance is administered. This is a method for evaluating the visceral fat increase inhibitory effect, wherein one concentration is compared with a reference value and the visceral fat increase inhibitory effect of the test substance is evaluated.
(B5) complement C3 or a modified product thereof,
(B6) β2-microglobulin or a modified product thereof.

  In the present invention, “an animal that can develop visceral fat-type obesity” is an animal that is designed to develop visceral fat-type obesity, and is not an animal that has not yet developed visceral fat-type obesity at present. Point to. The “animal that develops or can develop visceral fat-type obesity” refers to “an animal that develops visceral fat-type obesity” or “an animal that can develop visceral fat-type obesity”. In addition to animals that can be bred such as rats, the types of animals include humans.

  In the present invention, a representative example of a “modified protein” is a protein in which at least one of the amino acid residues constituting the protein is modified. The “modification” includes not only addition of a compound or a functional group. Desorption is also included. In addition, “protein or a modified form thereof” includes an isoform of the protein, a substantially identical protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein, and Protein fragments derived from the protein that have been cleaved by a protease are included. Furthermore, in the case of a complex protein, the “protein or a modified form thereof” includes its subunits.

  The invention according to claim 4 is the viscera according to any one of claims 1 to 3, wherein the animal is an animal that ingests a test substance and a low-fiber high-fat diet. This is a method for evaluating the effect of inhibiting fat increase.

  In the present invention, “an animal that ingests a test substance and ingests a low-fiber high-fat diet” is adopted as “an animal that develops or can develop visceral fat-type obesity administered with a test substance”. That is, in the present invention, an “animal that has developed or can develop visceral fat-type obesity” is set by ingesting a low-fiber high-fat diet, and the test substance is ingested by the animal. In the present invention, for example, an animal can be raised by adding a test substance or a low-fiber high-fat diet to the diet, so that the effect of suppressing visceral fat increase can be more easily evaluated.

  The invention according to claim 5 is the evaluation method of the visceral fat increase inhibitory effect according to any one of claims 1 to 4, wherein the body fluid is blood.

  With such a configuration, a body fluid as a measurement sample can be easily collected, and the visceral fat increase inhibitory effect of the test substance can be evaluated more easily and quickly.

  The invention according to claim 6 is the evaluation method of the visceral fat increase inhibitory effect according to any one of claims 1 to 5, wherein the test substance is a food material.

  With this configuration, the visceral fat increase inhibitory effect of the test substance can be evaluated for the purpose of developing functional foods.

  In the invention according to claim 7, the bodily fluid or bodily fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, and the marker substance in the bodily fluid is captured on the carrier. The method for evaluating the visceral fat increase inhibitory effect according to any one of claims 1 to 6, wherein the concentration of the marker substance in a body fluid is calculated based on the amount of the marker substance.

  In the method for evaluating the visceral fat increase inhibitory effect of the present invention, a carrier on which a substance having affinity for a marker substance is immobilized is used. Then, the body fluid or body fluid component is brought into contact with the carrier, and the marker substance contained in the body fluid or body fluid component is captured on the carrier via a substance having affinity for the marker substance, and the amount of the marker substance captured is increased. Based on this, the concentration of the marker substance in the body fluid is calculated. According to the evaluation method of the visceral fat increase inhibitory effect of the present invention, since the marker substance captured on the carrier is the measurement object, the influence of the contaminant substance contained in the measurement sample can be reduced, and the sensitivity is higher. In addition, the concentration of the marker substance can be measured with high accuracy. Examples of the body fluid component include serum or plasma when the body fluid is blood.

  The invention according to claim 8 is characterized in that the carrier has a flat portion, and the substance having affinity for the marker substance is immobilized on a part of the flat portion. It is the evaluation method of the visceral fat increase inhibitory effect of description.

  In the evaluation method of the visceral fat increase inhibitory effect of the present invention, a carrier having a planar portion is used, and a substance having affinity for the marker substance is immobilized on a part of the planar portion. With this configuration, a substance having affinity for the marker substance can be spot-fixed at a plurality of locations on the carrier. As a result, it is possible to simultaneously process a plurality of measurement samples with one carrier, and simultaneously measure the concentrations of a plurality of marker substances with one carrier, and work efficiency is improved. Furthermore, by reducing the area of each spot, the concentration of the marker substance can be measured even from a very small amount of measurement sample. An example of the carrier having a planar portion is a substrate such as a chip.

  The invention according to claim 9 is the method for evaluating the visceral fat increase inhibitory effect according to claim 7 or 8, wherein the substance having affinity for the marker substance is an ion exchanger or an antibody. .

  In the method for evaluating the visceral fat increase inhibitory effect of the present invention, an ion exchanger or an antibody is used as a substance having affinity for a marker substance, and the marker substance in the measurement sample is captured on the carrier via the ion exchanger or antibody. To do. When the substance is an ion exchanger, various substances are easily available, and a carrier for capturing the marker substance can be easily prepared. Further, when the substance is an antibody, the marker substance can be captured more specifically. Examples of the method for measuring the amount of the captured marker substance include mass spectrometry and immunoassay (in the case of an antibody).

A related invention is a method for screening a substance characterized in that a test substance is evaluated by the above-described evaluation method for suppressing the visceral fat increase, and a substance having a desired visceral fat increase suppressing effect is screened.

The present invention relates to a method for screening a substance, wherein at least one concentration of each of the marker substances (A01) to (A11) and each marker substance belonging to (B1) to (B8) above in a body fluid of an animal is used as a reference value. In comparison, a substance having an evaluation of a desired visceral fat increase inhibitory effect is screened. Each of the marker substances (A01) to (A11) and the marker substances belonging to the above (B1) to (B8) is specific in the body fluid of an animal that has or can develop visceral fat type obesity. It is a protein detected in According to the screening method of the substance of this invention, the substance which has the evaluation of the visceral fat increase inhibitory effect can be easily and accurately screened. In particular, when the test substance is a food material, a food material useful for the development of a functional food having an inhibitory effect on visceral fat increase can be screened.

The invention according to claim 10 evaluates a test substance by the method for evaluating the visceral fat increase inhibitory effect according to any one of claims 1 to 9, and screens a substance having a desired visceral fat increase inhibitory effect. This is a screening method for a substance characterized by the above.

  Invention of Claim 11 is a kit for using for the evaluation method of the visceral fat increase inhibitory effect of any one of Claims 1-9, Comprising: The substance which has affinity with the said marker substance is fixed It is a kit for evaluation of the visceral fat increase inhibitory effect characterized by including the conjugated carrier.

  The kit for evaluating the visceral fat increase inhibitory effect of the present invention includes a carrier on which a substance having affinity for a marker substance is immobilized. With this configuration, it is not necessary to prepare the carrier separately when measuring the concentration of the marker substance, and the concentration of the marker substance can be measured very simply.

  The invention according to claim 12 is the kit for evaluating the visceral fat increase inhibitory effect according to claim 11, wherein the substance having affinity for the marker substance is an ion exchanger.

  With this configuration, the marker substance can be more reliably captured on the carrier.

  A configuration used for screening a substance having a desired inhibitory effect on visceral fat increase is recommended (claim 13).

The invention according to claim 14 is a use of at least one protein of the following (A01) , (A02), (A07), and (A08) present in the body of an animal as a marker reflecting the state of visceral fat. It is.
(A01) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 4610 when subjected to mass spectrometry;
(A02) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 5500 when subjected to mass spectrometry;
(A07) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 9070 when subjected to mass spectrometry;
(A08) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 11600 when subjected to mass spectrometry .

The invention according to claim 15 is the use according to claim 14, characterized in that at least one of the following (e) and (f) is satisfied.
(E) (A07) is complement C3 or a modified form thereof,
(F) (A08) is β2-microglobulin or a modified product thereof .

The invention according to claim 16 is the use of at least one protein of the following (B5) and (B6) present in the body of an animal as a marker reflecting the state of visceral fat.
(B5) complement C3 or a modified product thereof,
(B6) β2-microglobulin or a modified product thereof .

  According to the evaluation method of the visceral fat increase inhibitory effect of the present invention, the evaluation of the visceral fat increase inhibitory effect of the test substance can be easily and highly accurately evaluated.

  According to the screening method for a substance of the present invention, a substance having an evaluation of the visceral fat increase inhibitory effect can be easily and accurately screened.

  According to the kit for evaluating the visceral fat increase inhibitory effect of the present invention, it is not necessary to prepare the carrier separately when measuring the concentration of the marker substance, and the concentration of the marker substance can be measured very simply.

It is the graph which compared the body weight between each group. It is the graph which compared the quantity of the visceral fat between each group. It is the graph which compared the quantity of subcutaneous fat between each group. It is a box diagram about the ion peak whose mass / charge ratio is 4612 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 5501 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 7054 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 8332 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 8836 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 8931 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 9065 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 11463 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 13733 (average value). It is a box diagram about an ion peak whose mass / charge ratio is 13922 (average value). It is a box diagram about the ion peak whose mass / charge ratio is 21383 (average value). 6 is a photograph showing the results of SDS-PAGE performed in Example 2. FIG. 4 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in Example 2. FIG. 6 is a photograph showing the results of SDS-PAGE performed in Example 3. FIG. 6 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in Example 3. FIG. It is an ion peak figure which shows the result of the SELDI-TOF-MS analysis performed in Example 4. 6 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in Example 5. FIG. It is a photograph which shows the result of SDS-PAGE performed in Example 6. It is an ion peak figure which shows the result of the SELDI-TOF-MS analysis performed in Example 6. 6 is an ion peak diagram showing a result of SELDI-TOF-MS analysis performed in Example 7. FIG. 6 is a photograph showing the results of SDS-PAGE performed in Example 8. FIG. 10 is an ion peak diagram showing a result of SELDI-TOF-MS analysis performed in Example 8. FIG. 10 is an ion peak diagram showing the results of SELDI-TOF-MS analysis performed in Example 9. FIG.

  Hereinafter, embodiments for carrying out the present invention will be described in detail.

The evaluation method of the visceral fat increase inhibitory effect of the present invention includes two aspects. In one aspect, the concentration of at least one of the following marker substances (A01) to (A11) in the body fluid of an animal that has developed or can develop visceral fat type obesity administered with the test substance is compared with a reference value. The effect of inhibiting the increase in visceral fat of the test substance is evaluated.
(A01) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 4610 when subjected to mass spectrometry;
(A02) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 5500 when subjected to mass spectrometry;
(A03) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 7050 when subjected to mass spectrometry;
(A04) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 8330 when subjected to mass spectrometry;
(A05) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 8840 when subjected to mass spectrometry;
(A06) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 8930 when subjected to mass spectrometry;
(A07) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 9070 when subjected to mass spectrometry;
(A08) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 11600 when subjected to mass spectrometry;
(A09) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 13700 when subjected to mass spectrometry;
(A10) a protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 13900 when subjected to mass spectrometry;
(A11) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 21400 when subjected to mass spectrometry.

  Each of these marker substances is a protein mainly present in blood, and is specifically detected in the body fluid of an animal that develops or can develop visceral fat-type obesity. In addition, each marker substance of (A01), (A03), (A04), (A05), (A06), (A09), (A10), and (A11) (hereinafter, a group consisting of these marker substances is referred to as “ Group 1 ”may be referred to as a higher value when visceral fat is increased. Therefore, when the test substance has a visceral fat increase inhibitory effect, Indicates a lower value. On the other hand, each marker substance of (A02), (A07), and (A08) (hereinafter, a group consisting of these marker substances may be referred to as “Group 2”) is in a state where visceral fat is increased. Since it shows a low value, when the test substance has a visceral fat increase inhibitory effect, it shows a higher value in the animal administered the test substance.

According to peptide mapping under certain conditions, the marker substance (A03) is apolipoprotein C1, the marker substance (A04) is apolipoprotein C2, the marker substance (A05) is apolipoprotein A2, the marker substance (A06) is apolipoprotein C3, the marker Substance (A07) is complement C3a (des-Arg), marker substance (A08) is β2-microglobulin, marker substance (A09) is transthyretin, marker substance (A10) is transthyretin, marker substance (A11) Is identified as apolipoprotein M. That is, in an embodiment, at least one of the following (a) to (i) is satisfied.
(A) The marker substance (A03) is apolipoprotein C1 or a modified form thereof.
(B) The marker substance (A04) is apolipoprotein C2 or a modified form thereof.
(C) The marker substance (A05) is apolipoprotein A2 or a modified form thereof.
(D) The marker substance (A06) is apolipoprotein C3 or a modified form thereof.
(E) The marker substance (A07) is complement C3 or a modified form thereof.
(F) The marker substance (A08) is β2-microglobulin or a modified form thereof.
(G) The marker substance (A09) is transthyretin or a modified form thereof.
(H) The marker substance (A10) is transthyretin or a modified form thereof.
(I) The marker substance (A11) is apolipoprotein M or a modified form thereof.

In another aspect of the method for evaluating the visceral fat increase inhibitory effect of the present invention, the following (B1) to (B8) in the body fluid of an animal that has developed or can develop visceral fat type obesity administered with a test substance: At least one concentration of the marker substance belonging to any of the above is compared with a reference value, and the visceral fat increase inhibitory effect of the test substance is evaluated.
(B1) Apolipoprotein C1 or a modified product thereof,
(B2) apolipoprotein C2 or a modified product thereof,
(B3) Apolipoprotein A2 or a modified product thereof,
(B4) Apolipoprotein C3 or a modified product thereof,
(B5) complement C3 or a modified product thereof,
(B6) β2-microglobulin or a modified product thereof,
(B7) transthyretin or a modified product thereof,
(B8) Apolipoprotein M or a modified product thereof.

  Examples of protein modifications include methylation, acetylation, adenylylation, myristylation, etc. of N-terminal α-amino group and lysine ε-amino group; addition of sugar or sugar chain to serine / threonine / asparagine; serine / threonine / Examples include phosphorylation of tyrosine, arginine, histidine; cysteine cysteinylation, homocysteinylation, sulfonylation, etc .; γ-carboxylation of glutamic acid; conversion of N-terminal glutamic acid to pyroglutamic acid, and the like. Further, elimination of these modifications (demethylation, elimination of sugars or sugar chains, dephosphorylation, etc.) is also included in “modification”.

  The “protein or a modified form thereof” includes an isoform of the protein. Isoforms include proteins generated by alternative splicing in addition to the various modifications described above. Furthermore, the “protein or a modified form thereof” includes substantially the same protein in which several amino acid residues are deleted, substituted or added in the primary structure of the protein. Furthermore, “protein or a modified product thereof” includes a protein fragment derived from the protein that has been cleaved by a protease. For example, a protein fragment having a length that can be recognized as derived from the protein, for example, a protein fragment composed of 20 or more amino acid residues, a protein fragment having a molecular weight of 2,000 or more, and the like can be mentioned. In the case of a complex protein, the “protein or a modified product thereof” includes its subunits.

  For example, a protein fragment (SEQ ID NO: 2) lacking the 2 amino acid residues (Ala-Pro) on the N-terminal side of apolipoprotein C1 (SEQ ID NO: 1) is included in the “modified product of apolipoprotein C1”.

  For example, complement C3a des Arg (SEQ ID NO: 6), which is a protein fragment of complement C3, is included in “complement C3 or a modification thereof”.

  For transthyretin (SEQ ID NO: 8), various modified transthyretins in which only the Cys residue is modified have been found (Amareth Lim et al., J. Biol. Chem., Vol. 258, No. 50, 2003). For example, “cysteinylated transthyretin” in which cysteine is added to the Cys residue of transthyretin and “glutathionized transthyretin” in which glutathione is added are included in “modified form of transthyretin”. Furthermore, since transthyretin is a complex protein composed of four subunits, the subunit is included in “transthyretin or a modified form thereof”.

  In the evaluation method of the visceral fat increase inhibitory effect of the present invention, only one of the marker substances (A01) to (A11) and each marker substance belonging to (B1) to (B8) may be used, You may use it in combination. There is no particular limitation on the combination method in the case of using a plurality, but for example, the marker substance (s) selected from group 1 and the marker substance (s) selected from group 2 can be combined. .

  In the method for evaluating the inhibitory effect on the increase in visceral fat according to the present invention, “an animal that develops or can develop visceral fat-type obesity administered with a test substance” is used. In a preferred embodiment, “animal fed with a test substance and fed with a low-fiber, high-fat diet” is used as the animal. For example, an animal in which an animal is ingested with a test substance and a low-fiber high-fat diet may be used. A low fiber high fat diet can be prepared, for example, by mixing several percent to several tens of percent of animal fat in a low fiber feed. As other embodiments, it is also possible to use naturally occurring model animals or genetically engineered model animals that develop visceral fat-type obesity. For example, a test substance may be administered to the spontaneous model animal or genetically engineered model animal, and the concentration of each marker substance in the body fluid may be used as an index.

  There are no particular limitations on the type of animal in “animals that develop or can develop visceral fat-type obesity”, and for example, mice, rats, rabbits, pigs and the like can be employed. Furthermore, humans can be employed as animals. In this case, the substance will be evaluated according to the results of clinical trials.

  In the evaluation method of the visceral fat increase inhibitory effect of the present invention, the concentration of the marker substance in the animal body fluid is compared with a reference value. The reference value is not particularly limited and may be set as appropriate according to the purpose. For example, in the case of adopting “an animal that ingests a test substance and a low-fiber high-fat diet”, use the concentration of the marker substance in the body fluid of an animal that ingests only a low-fiber high-fat diet. Can do. An animal fed only with a low-fiber, high-fat diet becomes a model of an animal that has developed or can develop visceral fat-type obesity, and the marker substance concentration in the body fluid becomes an “abnormal value”. And the value (measured value) in the animal (for example, simultaneous intake) which ingested the test substance and ingested the low fiber high fat diet is compared with the reference value (abnormal value), and the measured value is significantly different from the reference value. When there is a difference and it is on the normal side (maintained on the normal side), it can be evaluated that the test substance has an inhibitory effect on visceral fat increase. Specifically, when the marker substance belonging to group 1 is used as an index, when the measured value is significantly lower than the reference value, when the marker substance belonging to group 2 is used as an index, the measured value is the reference. When the value is significantly higher than the value, it can be evaluated that the test substance has an inhibitory effect on visceral fat increase.

Furthermore, there may be a plurality of reference values. For example, in addition to the abnormal value described above, a value (normal value, negative control) in an animal fed a normal diet can be added to the reference value. In particular,
(1) Intake normal diet (normal value),
(2) Ingesting a low-fiber high-fat diet (abnormal value),
(3) Simultaneous intake of test substance and low-fiber high-fat diet,
3 groups are set up and animals are raised. And the said marker substance in the bodily fluid of each animal is measured, and each measured value is compared. At this time, there is a significant difference between (1) and (2), there is a significant difference between (3) and (2), and (3) is closer to the normal side ((1) than (2)) Side) (when maintained on the normal side), the test substance can be evaluated as having a visceral fat increase inhibitory effect.

Furthermore, as a reference value, a value (positive control) in an animal in which a known substance having an inhibitory effect on visceral fat increase and a low-fiber high-fat diet can be simultaneously added. Specifically, in addition to the above (1) to (3),
(4) a group for simultaneously ingesting the known substance and a low fiber high fat diet;
Set up and raise animals. At this time, there is a significant difference between (1) and (2), there is a significant difference between (3) and (2), and (3) is normal ((1) and (2) compared to (2). 4), the test substance can be evaluated as having a visceral fat increase inhibitory effect. That is, such a test substance exhibits the same behavior as the known substance adopted in (4) and can be said to have a similar action.

  When a naturally occurring model animal or genetically engineered model animal that develops visceral fat type obesity is used, instead of the above (2), “a group that ingests a known substance having no inhibitory effect on visceral fat increase”, (3 Instead of (), a “group to ingest the test substance” may be set.

  Blood is preferably used as the body fluid of the animal used in the method for evaluating the visceral fat increase inhibitory effect of the present invention. In particular, it is preferable to use serum or plasma (body fluid component) prepared from blood as a measurement sample. Serum or plasma can be prepared from blood by a known method such as centrifugation.

  Examples of the test substance in the method for evaluating the visceral fat increase inhibitory effect of the present invention include food materials and drug substances. In particular, when food materials are to be evaluated, it can be used for the development of functional foods.

  In the method for evaluating the visceral fat increase inhibitory effect of the present invention, the method for measuring the concentration of a marker substance is a method generally used for protein quantification as long as it can specifically measure the concentration of the marker substance. It can be used as it is. For example, various immunoassays, mass spectrometry (MS), chromatography, electrophoresis and the like can be used.

  According to the immunoassay, the concentration of the marker substance can be accurately measured even with a sample having a lot of contaminants. Examples of immunoassays include classical methods such as precipitation, agglutination, and hemolysis, which directly or indirectly measure antigen-antibody conjugates, and enzyme immunoassays (EIA) with increased detection sensitivity in combination with labeling methods. , Radioimmunoassay (RIA), fluorescent immunoassay (FIA) and the like. The antibody specific for the marker substance used in these immunoassays may be monoclonal or polyclonal.

  According to mass spectrometry, an ion peak derived from each marker substance can be specified, and the amount (concentration) of each marker substance can be measured with the ion peak intensity. As the ionization method for measuring the concentration of the marker substance by mass spectrometry, any of matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI) can be applied. However, MALDI is preferred because it produces less multivalent ions. In particular, according to MALDI-TOF-MS combined with a time-of-flight mass spectrometer (TOF), an ion peak derived from a marker substance can be identified more accurately.

  When measuring the concentration of the marker substance by electrophoresis, for example, the test material is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the target marker substance, and the gel is prepared with an appropriate dye or fluorescent substance. It is only necessary to stain and measure the intensity and fluorescence intensity of the band corresponding to the target marker substance. When the separation of the marker substance is insufficient by SDS-PAGE alone, two-dimensional electrophoresis combined with isoelectric focusing (IEF) can also be used. Furthermore, instead of detecting directly from the gel, Western blotting can also be performed to measure the amount of marker substance on the membrane.

  When measuring the concentration of the marker substance by chromatography, for example, a method by liquid high performance chromatography (HPLC) can be used. That is, the concentration of the marker substance in the sample can be measured by subjecting the sample to HPLC to separate the target marker substance and measuring the peak area of the chromatogram.

  In a preferred embodiment, a marker substance is captured on a carrier, and the captured marker substance is a measurement target. That is, a substance having affinity for the marker substance is immobilized on the carrier, and the marker substance is captured on the carrier via the substance having the affinity. Then, the concentration of the marker substance in the body fluid is calculated based on the amount of the captured marker substance. According to this embodiment, it is possible to reduce the influence of contaminants contained in the sample, and to measure the concentration of the marker substance with higher sensitivity and higher accuracy. As an example of the carrier that can be used in the present embodiment, a carrier having a flat portion such as a substrate can be used in addition to a general one such as beads, metal, glass, resin, and the like. In the case of using a substrate, it is preferable to immobilize a substance having affinity for the marker substance on a part of the planar portion. As an example, there may be mentioned a carrier in which a chip is used as a substrate and a substance having affinity for a marker substance is spot-fixed at a plurality of spots on the surface. Examples of “affinity” include ion binding, affinity between metal chelate and histidine residue in protein, antigen and antibody, enzyme and substrate, bioaffinity such as hormone and receptor, and hydrophobicity. Chemical interactions such as interactions.

  When capturing the marker substance on the carrier by ionic bonding, the ion exchanger is immobilized on the carrier. In this case, either a cation exchanger or an anion exchanger can be used as the ion exchanger, and further, a strong cation exchanger, a weak cation exchanger, a strong anion exchanger, and a weak anion exchanger. Either of these can be used, but a strong anion exchanger and a weak cation exchanger are preferably used. Examples of strong anion exchangers include quaternary ammonium (trimethylaminomethyl) (QA), quaternary aminoethyl (diethyl, mono-2-hydroxybutylaminoethyl) (QAE), quaternary ammonium (trimethylammonium) ( And those having a strong anion exchange group such as QMA). Examples of weak cation exchangers include those having weak cation exchange groups such as carboxymethyl (CM). Examples of strong cation exchangers include those having a strong cation exchange group such as sulfopropyl (SP). Furthermore, examples of the weak anion exchanger include those having a weak anion exchange group such as dimethylaminoethyl (DE) and diethylaminoethyl (DEAE).

When capturing a marker substance via a metal chelate, for example, a metal chelate such as Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Al ³⁺ , Fe ³⁺ , Ga ³⁺ is fixed. A fluorinated carrier can be used.

  When a marker substance is captured on a carrier by an antibody, an antibody specific for the marker substance may be immobilized on the carrier.

  When a marker substance is captured on a carrier by hydrophobic interaction, a substance having a hydrophobic group is immobilized on the carrier. Examples of hydrophobic groups include C4-C20 alkyl groups, phenyl groups, and the like.

  In this embodiment, when an immunoassay is used as a method for measuring a marker substance, it is preferable to use a carrier on which an antibody is immobilized. In this way, an immunoassay system using the antibody immobilized on the carrier as the primary antibody can be easily constructed. For example, two types of antibodies specific to a marker substance and having different epitopes are prepared, one is immobilized on a carrier as a primary antibody, and the other is enzyme-labeled as a secondary antibody to construct a sandwich EIA system. . In addition, immunoassay systems based on binding inhibition methods and competitive methods can be constructed. Further, when a substrate is used as a carrier, immunoassay using an antibody chip is possible. According to the antibody chip, the concentration of a plurality of marker substances can be measured simultaneously, and rapid measurement is possible.

  On the other hand, when mass spectrometry is used in the present embodiment, for example, in addition to antibodies, ion exchangers, metal chelates, or carriers on which hydrophobic groups are immobilized can be used. Since binding by these substances is not as specific as bioaffinity such as antigens and antibodies, when using a carrier on which these substances are immobilized, substances other than the marker substance can also be captured on the carrier. According to the analysis, there is no problem because it is quantified by the mass spectrometer spectrum reflecting the molecular weight. In particular, using a substrate as a carrier, surface-enhanced laser desorption / ionization-time-of-flight mass spectrometry (hereinafter referred to as "SELDI-TOF-MS") ), The concentration of the marker substance can be measured more accurately. As the types of substrates that can be used, cation exchange substrates, anion exchange substrates, normal phase substrates, reverse phase substrates, metal ion substrates, antibody substrates, etc. can be used, but cation exchange substrates, particularly weak cation exchanges. A substrate and an anion exchange substrate, particularly a strong anion exchange substrate are preferably used.

  The method for screening a substance of the present invention comprises evaluating a test substance by the method for evaluating the visceral fat increase inhibitory effect of the present invention, and screening for a substance having a desired visceral fat increase inhibitory effect. In the method for screening a substance of the present invention, the same embodiment as the above-described method for evaluating the visceral fat increase inhibitory effect of the present invention can be employed.

  In order to simply perform the evaluation method of the visceral fat increase inhibitory effect of the present invention, an evaluation kit can be constructed by collecting necessary reagents. Examples of the evaluation kit include those containing a carrier on which a substance having affinity for a marker substance is immobilized. In particular, according to the evaluation kit including a substrate on which a weak cation exchanger such as CM or a strong anion exchanger such as QA or QAE is immobilized as a carrier, SELDI-TOF-MS or the like is easily performed. be able to. The kit may contain other reagents such as standard substances and various pretreatment buffers. The kit can also be used as a kit for screening for a substance having an inhibitory effect on visceral fat increase. Examples of the configuration of the kit of the present invention are given below.

[Example of kit configuration]
(1) Weak cation exchange substrate: 1 sheet (2) Strong anion exchange substrate: 1 sheet (3) Substrate cleaning buffer A (pH 3.0): appropriate amount (4) Substrate cleaning buffer B (pH 9.0) : Appropriate amount (5) Standard product of each marker substance:

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

  Six to eight 4-week-old SD rats were prepared per group. The first group of rats was fed with CE-2 (Claire Japan) as a normal diet. The second group of rats was fed a low fiber, high fat diet (low fiber diet mixed with 30% beef tallow). The third group was fed with a mixed feed of the low-fiber high-fat diet and a known material having a visceral fat increase inhibitory effect. The fourth group was fed with a mixed feed of a normal feed and a known material having a visceral fat increase inhibitory effect. Each feed was freely consumed and was bred for 3 weeks (21 days). After completion of the breeding, the body weight of each rat, the amount of visceral fat per body weight (mesentery, kidney, around the testis) and the amount of subcutaneous fat per body weight (scapula, around the lower limbs) were measured and compared to 4 groups. . Furthermore, whole blood was collected from each rat to prepare plasma.

  FIG. 1 shows the measurement results of body weight, FIG. 2 shows the measurement results of visceral fat, and FIG. 3 shows the measurement results of subcutaneous fat. 1-3, symbol * indicates a significant difference with respect to the first group, symbol # indicates a significant difference with respect to the second group, “*” and “#” are 5% significant, and “**” and “##” are 1%. Significance, “***” and “##” mean 0.1% significance. First, as shown in FIG. 1, the second group and the third group were significantly heavier than the first group and the fourth group (normal feed group). Moreover, there was no significant difference between the third group and the fourth group. That is, no influence on the body weight by the known feed was observed. Further, as shown in FIG. 2, the amount of visceral fat was significantly higher in the second group and the third group than in the first group and the fourth group (normal feed group). Furthermore, the amount of visceral fat was significantly smaller in the third group than in the second group. Thereby, the visceral fat suppression effect by the said known feed was supported. In addition, as shown in FIG. 3, the tendency similar to visceral fat was shown also about subcutaneous fat. From the above, the validity of this animal experiment was supported. In this experiment, the breeding was completed in a short period of 3 weeks (21 days), and it is considered that the rats of the second group at the end of the breeding were in visceral fat-type obesity at the beginning of the onset.

2. Protein chip analysis and ion peak selection To 20 μL of each plasma sample, 30 μL of denaturation buffer (9 M urea, 2% CHAPS, 50 mM Tris-HCl (pH 9.0)) was added for pretreatment to denature the protein. Next, each pretreated body fluid sample was applied to a strong anion exchange resin column (Q-Sepharose, GE Healthcare). Next, a pH 9.0 buffer solution (50 mM Tris-HCl (pH 9.0), 0.1% (w / v) 1-o-N-octyl-β-D-glucopyranoside (hereinafter referred to as “OGP”). )), PH 5.0 buffer (100 mM sodium acetate (pH 5.0), 0.1% (w / v) OGP), pH 3.0 buffer (50 mM sodium citrate (pH 3.0), 0. 1% (w / v) OGP) and organic solvent (mixed solution consisting of 0.1% trifluoroacetic acid, 50.0% acetonitrile) were eluted in order with 200 μL each, and fraction 1 (eluted at pH 9.0, passed) ), Fraction 2 (eluted at pH 5.0), fraction 3 (eluted at pH 3.0), and fraction 4 (eluted with an organic solvent).

  10 μL of each of the obtained fractions was diluted 10-fold with a pH 3.0 protein chip binding buffer (50 mM sodium citrate), and then added to a weak cation exchange chip CM10 (BioRad). Similarly, 10 μL of each obtained fraction was diluted 10-fold with a pH 9.0 protein chip binding buffer (50 mM Tris-HCl (pH 9.0)) and then added to a strong anion exchange chip Q10 (Bio-Rad). did. Each chip was washed 3 times with each binding buffer, then once with deionized water and dried. Next, sinapinic acid (SPA-H, SPA-L) or α-cyano-4-hydroxycinnamic acid (CHCA), which is an energy absorbing molecule, is added and a protein chip reader Model PBS IIc (Bio-Rad) is used. Then, SELDI-TOF-MS was performed. In addition, the measurement molecular weight range (m / z) was performed in the range of 3000-200000. Moreover, the measurement was performed in duplicate and the average value of m / z was calculated. Data analysis was performed using Protein Chip Software, Ciphergen Express Data Manager, and Biomarker Patterns Software (all from Bio-Rad). Specifically, after performing baseline correction, molecular weight calibration, and spectrum normalization processing, single marker analysis and multiflow analysis combining several markers were performed. As a result, a large number of peaks were detected depending on the combination of the type of crude fraction, the type of protein chip, the washing conditions of the chip, and the like. For each peak, a p-value (Mann-Whitney test method), ROC area, and ion peak intensity were calculated, and 11 candidate peaks were selected using the following conditions (1) to (4) as indices.

(1) Candidate peak search (basic)
There is a significant difference in ionic strength between the first group and the second group (p <0.05).
(2) Effect verification of the known substance There is a significant difference in ionic strength between the second group and the third group (p <0.05 or p <0.1), and the value of the third group is more It is closer to the value of the first group than the value of the second group (the value of the third group has returned to the first group side).
(3) Increase / decrease pattern analysis Only the second group shows a high value or a low value, and there is not much difference among the first group, the third group, and the fourth group. In addition, even if there is a difference between the third group and the fourth group, if the value of the fourth group is farther from the second group than the value of the third group, this condition is satisfied. handle.

3. Identification of marker substance (A01) Fraction 4 (organic solvent) was brought into contact with a weak cation exchange chip CM10, washed with a pH 3.0 protein chip binding buffer and subjected to SELDI-TOF-MS (EAM: CHCA). In some cases, an ion peak with a mass / charge ratio of 4612 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 4 shows a box diagram when the peak intensity of this peak is plotted for each group. In the figure, the upper and lower edges of the bag are the maximum and minimum values, respectively, the upper and lower sides of the box are the third quartile (75th percentile) and the first quartile (25th percentile), respectively, and the line in the box is the center Value (the same applies to FIG. 5 and subsequent figures). As a result, the ROC area (first group vs second group) of this peak is 0.750, the p value (first group vs second group) is 0.018, and the p value (second group vs third group) is It was 0.014.

  From the above, a protein (marker substance (A01)) that produces a peak with a mass / charge ratio of about 4610 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A01) in the body fluid of an animal that develops or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 4610 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

4). Identification of Marker Substance (A02) Fraction 1 (pH 9.0) is contacted with a weak cation exchange chip CM10, washed with a pH 3.0 protein chip binding buffer, and SELDI-TOF-MS (EAM: SPA-H) , An ion peak with a mass / charge ratio of 5501 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 5 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.833, the p value (first group vs second group) is 0.002, and the p value (second group vs third group) is 0.004.

  From the above, a protein (marker substance (A02)) that produces a peak with a mass / charge ratio of about 5500 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A02) in the body fluid of an animal that has developed or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed by the same procedure, a protein that produces a peak with a mass / charge ratio of about 5500 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

5. Identification of marker substance (A03) Fraction 3 (pH 3.0) is brought into contact with a weak cation exchange chip CM10, washed with a protein chip binding buffer of pH 3.0, and then SELDI-TOF-MS (EAM: SPA-L) , An ion peak with a mass / charge ratio of 7055 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 6 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.750, the p value (first group vs second group) is 0.029, and the p value (second group vs third group) is 0.018.

  From the above, a protein (marker substance (A03)) that produces a peak with a mass / charge ratio of about 7050 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A03) in the body fluid of an animal that has developed or can develop visceral fat-type obesity to which the test substance is administered as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 7050 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

6). Identification of marker substance (A04) Fraction 4 (organic solvent) is brought into contact with a weak cation exchange chip CM10 and washed with a protein chip binding buffer having a pH of 3.0 to obtain SELDI-TOF-MS (EAM: SPA-H). When performed, an ion peak with a mass / charge ratio of 8333 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 7 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.984, the p value (first group vs second group) is 0.00002, and the p value (second group vs third group) is It was 0.060.

  From the above, a protein (marker substance (A04)) that produces a peak with a mass / charge ratio of about 8330 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A04) in the body fluid of an animal that develops or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 8330 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

7). Identification of Marker Substance (A05) Fraction 1 (pH 9.0) is contacted with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-L) , An ion peak with a mass / charge ratio of 8337 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 8 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.802, the p value (first group vs second group) is 0.004, and the p value (second group vs third group) is 0.090.

  From the above, a protein (marker substance (A05)) that produces a peak with a mass / charge ratio of about 8340 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A05) in the body fluid of an animal that has developed or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 8340 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

8). Identification of Marker Substance (A06) Fraction 4 (organic solvent) is brought into contact with the weak cation exchange chip CM10, washed with a pH 3.0 protein chip binding buffer, and SELDI-TOF-MS (EAM: SPA-H) is obtained. When performed, an ion peak with a mass / charge ratio of 8931 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 9 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.984, the p value (first group vs second group) is 0.00001, and the p value (second group vs third group) is 0.001.

  From the above, a protein (marker substance (A06)) that produces a peak with a mass / charge ratio of about 8930 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A06) in the body fluid of an animal that has developed or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 8930 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

9. Identification of marker substance (A07) Fraction 1 (pH 9.0) is contacted with weak cation exchange chip CM10, washed with pH 3.0 protein chip binding buffer, and SELDI-TOF-MS (EAM: CHCA) is performed. In this case, an ion peak having a mass / charge ratio of 9065 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 10 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.714, the p value (first group vs second group) is 0.046, and the p value (second group vs third group) is 0.018.

  From the above, a protein (marker substance (A07)) that produces a peak with a mass / charge ratio of about 9070 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A07) in the body fluid of an animal that develops or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 9070 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

10. Identification of Marker Substance (A08) Fraction 1 (pH 9.0) is contacted with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-H) , An ion peak with a mass / charge ratio of 11643 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a high value in the first group, the third group, and the fourth group, and a low value in the second group. FIG. 11 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.729, the p value (first group vs second group) is 0.033, and the p value (second group vs third group) is 0.090.

  From the above, a protein (marker substance (A08)) that produces a peak with a mass / charge ratio of about 11600 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A08) in the body fluid of an animal that develops or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, a protein that produces a peak with a mass / charge ratio of about 11600 when a body fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure. When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

11. Identification of marker substance (A09) Fraction 4 (organic solvent) is brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-L) is obtained. When performed, an ion peak with a mass / charge ratio of 13733 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 12 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.703, the p value (first group vs second group) is 0.046, and the p value (second group vs third group) is 0.055.

  From the above, a protein (marker substance (A09)) that produces a peak with a mass / charge ratio of about 13700 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A09) in the body fluid of an animal that develops or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 13700 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

12 Identification of marker substance (A10) Fraction 2 (pH 5.0) is brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-H) , An ion peak with a mass / charge ratio of 13922 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 13 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.755, the p value (first group vs second group) is 0.020, and the p value (second group vs third group) is It was 0.042.

  From the above, the protein (marker substance (A10)) that produces a peak with a mass / charge ratio of about 13900 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A10) in the body fluid of an animal that has developed or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, a protein that produces a peak with a mass / charge ratio of about 13900 when a bodily fluid sample is prepared by performing the same animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure. When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

13. Identification of marker substance (A11) Fraction 4 (organic solvent) is brought into contact with strong anion exchange chip Q10, washed with a protein chip binding buffer of pH 9.0, and SELDI-TOF-MS (EAM: SPA-H) is obtained. When performed, an ion peak with a mass / charge ratio of 21384 (average value) was detected. As a result of the increase / decrease pattern analysis, this peak showed a low value in the first group, the third group, and the fourth group, and a high value in the second group. FIG. 14 shows a box diagram when the peak intensity of this peak is plotted for each group. As a result, the ROC area (first group vs second group) of this peak is 0.750, the p value (first group vs second group) is 0.011, and the p value (second group vs third group) is 0.083.

  From the above, a protein (marker substance (A11)) that produces a peak with a mass / charge ratio of about 21400 when subjected to SELDI-TOF-MS is specific to animals that develop or can develop visceral fat-type obesity. It has been found that it is a substance and can be a marker reflecting the state of visceral fat. Thereby, using the concentration of the marker substance (A11) in the body fluid of an animal that develops or can develop visceral fat-type obesity administered with the test substance as an index, the evaluation of the visceral fat increase inhibitory effect of the test substance, and It has been shown that such substances can be screened. For example, when a body fluid sample is prepared by performing a similar animal experiment using a desired test substance and SELDI-TOF-MS is performed in the same procedure, a protein that produces a peak with a mass / charge ratio of about 21400 When the concentration of is maintained at a normal value, the test substance can be evaluated as having an inhibitory effect on the increase in visceral fat.

  In addition, when a protein equivalent to the marker substances (A01) to (A11) is also present in human body fluid, there is a possibility that visceral fat-type obesity can be diagnosed using the concentration of the protein in human body fluid as an index. Also suggested.

Purification and identification of marker substance (A03) A column (5 mL) packed with strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl pH 9.0, 1 M urea, 0.22). % CHAPS). Denaturation buffer (50 mM Tris-HCl pH 9.0, 9 M urea, 2% CHAPS) was added to 450 mL of rat standard serum (Chemicon) 1.5 mL, followed by addition to the equilibrated column. The non-adsorbed fraction was collected. The collected fraction was subjected to SELDI-TOF-MS analysis, and a peak showing a mass / charge ratio equivalent to the mass / charge ratio (average value: 7054) of the marker substance (A03) was confirmed.

  A part of the collected fractions were subjected to acetone precipitation treatment, and then subjected to SDS-PAGE using a polypeptide separation gel (15-20%) NTH-5AOT gel (DRC) (FIG. 15). In FIG. 15, lanes 1 to 6 are all collected fractions, and M is a molecular weight marker. The separated band of about 7.0 kDa (arrow a in FIG. 15) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and a peak showing a mass / charge ratio equivalent to that of the marker substance (A03) is confirmed. (Arrow in FIG. 16).

  The same SDS-PAGE was performed again to cut out a band of about 7.0 kDa, and after reduction alkylation treatment, 0.01 μg / μL trypsin solution (dissolved in 50 mM ammonium bicarbonate (pH 8.0)) was allowed to act. Digested in the gel. 1 μL of the digested sample was dropped on a metal plate, 0.4 μL of saturated CHCA solution was further dropped and dried, and then measured with a mass spectrometer Proteomics Analyzer 4700 (Applied Biosystems). At least 5 peaks were observed. And their accurate masses were calculated as “129.66”, “1052.49”, “1279.62”, “781.45”, and “1138.54”. Based on these data, a known protein was searched by Mascot database (Matrix Science), and peptide mass fingerprinting was performed. These peptides were consistent with peptides having amino acid sequences represented by SEQ ID NOs: 10 to 14, respectively. The target protein was identified as “apolipoprotein C1” with a probability of 99% or more. Table 1 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported rat apolipoprotein C1 (mature type) is shown in SEQ ID NO: 1.

  Here, in humans, mice and the like, those in which 2 amino acid residues on the N-terminal side of apolipoprotein C1 are deleted have been found. The mass of the full length rat apolipoprotein C1 (SEQ ID NO: 1) is 7187 Da, while the mass of the rat apolipoprotein C1 fragment (SEQ ID NO: 2) lacking the N-terminal two amino acid residues (Ala-Pro) is 7019 Da. Therefore, the marker substance (A03) was finally identified as a fragment of apolipoprotein C1 (SEQ ID NO: 2).

Purification and identification of marker substance (A04) Strong anion exchange resin HiTrap Q HP (5 mL) (GE Healthcare) was equilibrated with 50 mM sodium acetate buffer (pH 6.0). 1 mL of rat standard serum (Chemicon) was diluted 10-fold with 50 mM sodium acetate buffer (pH 6.0), filtered through 0.45 μL of Millex, and added to the equilibrated column. The column was washed sequentially with 5 column volumes (CV) of 50 mM sodium acetate buffer (pH 6.0), 12 CV of 50 mM sodium acetate buffer (pH 6.0) / 200 mM NaCl, and 5 CV of 50 mM sodium acetate buffer (pH 6.0). Thereafter, elution was performed with 50 mM sodium acetate buffer (pH 3.0), and a fraction having a high absorbance at 280 nm was collected (about 1 mL). The collected fraction was subjected to SELDI-TOF-MS analysis, and a peak having a mass / charge ratio equivalent to the mass / charge ratio (average value: 8333) of the marker substance (A04) was confirmed.

  A portion of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT gel (DRC) (FIG. 17). In FIG. 17, lanes 1 to 6 are all collected fractions, and M is a molecular weight marker. The separated band of about 8.3 kDa (arrow a in FIG. 17) was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and the mass / charge ratio (average value: 8333) of the marker substance (A04) A peak showing an equivalent mass / charge ratio was confirmed (arrow in FIG. 18).

  The same SDS-PAGE was performed again to cut out a band of about 8.3 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2. As a result, at least 7 peaks were detected, and their exact masses were It was calculated as “950.49”, “1055.54”, “1324.74”, “196.98”, “928.46”, “2261.18”, and “2447.26”. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides coincided with the peptides of the amino acid sequences represented by SEQ ID NOs: 15 to 21, respectively, and the target protein was 99% or more. Was identified as “apolipoprotein C2”. Table 2 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported rat apolipoprotein C2 is shown in SEQ ID NO: 3.

Purification and identification of marker substance (A05) In the SELDI-TOF-MS analysis for the column non-adsorbed fraction performed in Example 2, the mass / charge ratio (average value: 8837) equivalent to the mass / charge ratio of the marker substance (A05) A peak indicating the charge ratio was confirmed.

  A band of about 8.8 kDa (arrow b in FIG. 15) separated by SDS-PAGE performed in Example 2 was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and it was equivalent to the marker substance (A05) A peak indicating the mass / charge ratio was confirmed (FIG. 19).

  A similar SDS-PAGE was performed again to cut out a band of about 8.8 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2. As a result, at least three peaks were detected, and their accurate masses were “126.54”, “860.50”, and “1270.63” were calculated. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides coincided with the peptides of the amino acid sequences represented by SEQ ID NOs: 22 to 24, respectively, and the target protein was 99% or more. Was identified as “apolipoprotein A2”. Table 3 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported rat apolipoprotein A2 is shown in SEQ ID NO: 4.

Purification and identification of marker substance (A06) In the SELDI-TOF-MS analysis on the non-adsorbed fraction performed in Example 3, the mass / charge ratio (average value: 8931) equivalent to the mass / charge ratio of the marker substance (A06) A peak indicating the charge ratio was confirmed.

  A band of about 8.9 kDa (arrow b in FIG. 17) separated by SDS-PAGE performed in Example 3 was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and it was equivalent to the marker substance (A06) A peak indicating the mass / charge ratio of was confirmed (FIG. 20).

  The same SDS-PAGE was performed again to cut out a band of about 8.9 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2. As a result, at least two peaks were detected, and their accurate masses were “2262.00” and “968.53” were calculated. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides matched the peptides of the amino acid sequences represented by SEQ ID NOs: 25 and 26, respectively, and the target protein was 99% or more. Was identified as “apolipoprotein C3”. Table 4 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported rat apolipoprotein C3 is shown in SEQ ID NO: 5.

Purification and identification of marker substance (A07) A column (1 mL) packed with strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl pH 9.0, 1 M urea, 0.22). % CHAPS). 750 μL of denaturation buffer (50 mM Tris-HCl pH 9.0, 9 M urea, 2% CHAPS) was added to 500 μL of rat standard serum (Chemicon), and denaturation treatment was performed at 4 ° C. for 20 minutes. The denatured sample was centrifuged at 12,000 G for 10 minutes at 4 ° C., filtered through a 0.45 μm filter, added to the equilibrated column, and the non-adsorbed fraction was collected. The collected fraction was subjected to SELDI-TOF-MS analysis, and a peak showing a mass / charge ratio equivalent to the mass / charge ratio (average value: 9065) of the marker substance (A07) was confirmed.

  Weak cation exchange resin HiTrap CM FF 1 mL (GE Healthcare) was equilibrated with 50 mM HEPES-NaOH (pH 7.0). 3 mL of the collected fraction and 2.3 mL of 100 mM sodium acetate buffer (pH 5.0) were mixed, filtered through a 0.45 μL filter, and then added to the equilibrated column. After washing the column with 50 mM HEPES-NaOH (pH 7.0), 5 CV 50 mM HEPES-NaOH (pH 7.0) / 50 mM NaCl, 5 CV 50 mM HEPES-NaOH (pH 7.0) / 100 mM NaCl, and 5 CV 50 mM This was washed successively with HEPES-NaOH (pH 7.0) / 150 mM NaCl, and eluted with 5 CV of 50 mM HEPES-NaOH (pH 7.0) / 1 M NaCl. SELDI-TOF-MS analysis was performed on the eluted fraction, and a peak showing a mass / charge ratio equivalent to the mass / charge ratio (average value: 9065) of the marker substance (A07) was confirmed.

  A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separation gel (15-20%) NTH-5AOT gel (DRC) (FIG. 21). In FIG. 21, lanes 1 to 6 are all collected fractions, and M is a molecular weight marker. The separated band of about 9.1 kDa (arrow in FIG. 21) is cut out, protein is extracted, SELDI-TOF-MS analysis is performed, and it is equivalent to the mass / charge ratio (average value: 9065) of the marker substance (A07) A peak indicating the mass / charge ratio was confirmed (arrow in FIG. 22).

  When the same SDS-PAGE was performed again to cut out a band of about 9.1 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2, at least three peaks were detected. It was calculated as “1148.43”, “1438.38”, and “838.34”. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides coincided with the peptides of the amino acid sequences represented by SEQ ID NOs: 27 to 29, respectively, and the target protein was 99% or more. Was identified as “complement C3a (des-Arg)”. Table 5 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported rat complement C3a (des-Arg) is shown in SEQ ID NO: 6.

Purification and identification of marker substance (A08) In the SELDI-TOF-MS analysis for the non-adsorbed fraction performed in Example 2, the mass / charge ratio (average value: 11642) equivalent to the mass / charge ratio of the marker substance (A08) A peak indicating the charge ratio was confirmed.

  The approximately 11.6 kDa band (arrow c in FIG. 15) separated by SDS-PAGE performed in Example 2 was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and it was equivalent to the marker substance (A08) A peak indicating the mass / charge ratio of was confirmed (FIG. 23).

  The same SDS-PAGE was performed again to cut out a band of about 11.6 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2. As a result, at least 5 peaks were detected, and their accurate masses were “1091.59”, “3445.48”, “1496.73”, “2822.17”, and “777.39” were calculated. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides coincided with the peptides having the amino acid sequences represented by SEQ ID NOs: 30 to 34, respectively, and the target protein was 99% or more. Was identified as “β2-macroglobulin”. Table 6 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported rat β2-macroglobulin is shown in SEQ ID NO: 7.

Purification and identification of marker substances (A09) and (A10) A column (5 mL) packed with strong anion exchange resin Q-Sepharose HP (GE Healthcare) was equilibrated with buffer (50 mM Tris-HCl pH 9.0, 1 M urea). , 0.22% CHAPS). Denaturation buffer (50 mM Tris-HCl pH 9.0, 9 M urea, 2% CHAPS) was added to 450 mL of rat standard serum (Chemicon) 1.5 mL, followed by addition to the equilibrated column. After sequentially washing with 5 CV of 50 mM Tris-HCl (pH 9.0), 5 CV of 50 mM sodium acetate buffer (pH 5.0), and 5 CV of 50 mM sodium citrate buffer (pH 3.0), 33.3% isopropanol / 16 Elution was performed with 7% acetonitrile / 0.1% trifluoroacetic acid, and the eluted fraction was collected.

  A part of the collected fraction was subjected to acetone precipitation, and then subjected to SDS-PAGE using a polypeptide separating gel (15-20%) NTH-5AOT gel (DRC) (FIG. 24). In FIG. 24, lanes 1 to 6 are all collected fractions, and M is a molecular weight marker. The separated band around 13.7 kDa (arrow a in FIG. 24) was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and the mass / charge ratio (average of marker substances (A09) and (A10)) A peak containing a mass / charge ratio equivalent to the value: 13733, 13922) was confirmed (arrow in FIG. 25).

  The same SDS-PAGE was performed again to cut out a band around about 13.7 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2. As a result, at least 7 peaks were detected, and their accurate masses were detected. Were calculated as “1352.41”, “1587.52”, “2439.83”, “3125.12”, “871.30”, “2516.88”, and “2599.66”. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides coincided with the peptides having the amino acid sequences represented by SEQ ID NOs: 35 to 41, respectively, and the target protein was 99% or more. Was identified as “transthyretin”. Table 7 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported rat transthyretin is shown in SEQ ID NO: 8.

  As described above, various modified transthyretins in which only one Cys residue is modified have been found for transthyretin. From each charge / mass ratio (average value: 13733, 13922) of the marker substances (A09) and (A10), the marker substance (A09) is “cysteinylated transthyretin” and the marker substance (A10) is “glutathionized transthysine”. Retin "was identified.

Purification and identification of marker substance (A11) In the SELDI-TOF-MS analysis for the column non-adsorbed fraction performed in Example 8, the mass / charge ratio (average value: 21383) equivalent to the mass / charge ratio of the marker substance (A11) A peak indicating the charge ratio was confirmed.

  The approximately 21.4 kDa band (arrow b in FIG. 24) separated by SDS-PAGE performed in Example 8 was cut out, protein was extracted, SELDI-TOF-MS analysis was performed, and it was equivalent to the marker substance (A11) A peak indicating the mass / charge ratio was confirmed (FIG. 26).

  The same SDS-PAGE was performed again to cut out a band of about 21.4 kDa, and in-gel digestion and mass spectrometry were performed in the same manner as in Example 2. As a result, at least 6 peaks were detected, and their accurate masses were “1262.49”, “1654.61”, “938.30”, “825.35”, “263.86”, and “803.37” were calculated. Based on these data, peptide mass fingerprinting was performed in the same manner as in Example 2. As a result, these peptides coincided with the peptides of the amino acid sequences represented by SEQ ID NOs: 42 to 47, respectively, and the target protein was 99% or more. Was identified as “apolipoprotein M”. Table 8 shows the correspondence between the exact mass, amino acid sequence, and SEQ ID NO of each peptide. The amino acid sequence of the previously reported rat apolipoprotein M is shown in SEQ ID NO: 9.

Claims (16)

Based on the concentration of at least one of the following marker substances (A01) , (A02), (A07), and (A08) in the body fluid of an animal that has developed or is likely to develop visceral fat-type obesity administered with the test substance The evaluation method of the visceral fat increase inhibitory effect characterized by evaluating the visceral fat increase inhibitory effect which a test substance has compared with a value.
(A01) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 4610 when subjected to mass spectrometry;
(A02) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 5500 when subjected to mass spectrometry;
(A07) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 9070 when subjected to mass spectrometry;
(A08) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 11600 when subjected to mass spectrometry . The evaluation method of the visceral fat increase inhibitory effect according to claim 1, wherein at least one of the following (e) and (f) is satisfied.
(E) The marker substance (A07) is complement C3 or a modified form thereof.
(F) The marker substance (A08) is β2-microglobulin or a modified product thereof . Comparing at least one concentration of a marker substance belonging to any one of the following (B5) and (B6) in a body fluid of an animal that has developed or is capable of developing visceral fat-type obesity administered with a test substance with a reference value; The evaluation method of the visceral fat increase inhibitory effect characterized by evaluating the visceral fat increase inhibitory effect which a test substance has.
(B5) complement C3 or a modified product thereof,
(B6) β2-microglobulin or a modified product thereof .   The said animal is an animal which ingested the test substance and ingested the low fiber high fat diet, The evaluation method of the visceral fat increase suppression effect of any one of Claims 1-3 characterized by the above-mentioned.   The said body fluid is blood, The evaluation method of the visceral fat increase inhibitory effect of any one of Claims 1-4 characterized by the above-mentioned.   The said test substance is a food material, The evaluation method of the visceral fat increase inhibitory effect of any one of Claims 1-5 characterized by the above-mentioned.   The bodily fluid or bodily fluid component is brought into contact with a carrier on which a substance having affinity for the marker substance is immobilized, the marker substance in the bodily fluid is captured on the carrier, and based on the amount of the captured marker substance The method for evaluating the visceral fat increase inhibitory effect according to any one of claims 1 to 6, wherein the concentration of the marker substance in a body fluid is calculated.   The evaluation of the visceral fat increase inhibitory effect according to claim 7, wherein the carrier has a planar portion, and the substance having affinity for the marker substance is immobilized on a part of the planar portion. Method.   The method for evaluating the visceral fat increase inhibitory effect according to claim 7 or 8, wherein the substance having affinity for the marker substance is an ion exchanger or an antibody.   10. A method for screening a substance comprising: evaluating a test substance by the method for evaluating the visceral fat increase inhibitory effect according to any one of claims 1 to 9; and screening a substance having a desired visceral fat increase inhibitory effect. .   A kit for use in the method for evaluating the visceral fat increase inhibitory effect according to any one of claims 1 to 9, comprising a carrier on which a substance having affinity for the marker substance is immobilized. Kit for evaluating the inhibitory effect on visceral fat increase.   12. The kit for evaluating the visceral fat increase inhibitory effect according to claim 11, wherein the substance having affinity for the marker substance is an ion exchanger.   The kit for evaluating the visceral fat increase inhibitory effect according to claim 11 or 12, which is used for screening a substance having a desired visceral fat increase inhibitory effect. Use of at least one protein of the following (A01) , (A02), (A07), and (A08) present in an animal body as a marker reflecting the state of visceral fat.
(A01) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 4610 when subjected to mass spectrometry;
(A02) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 5500 when subjected to mass spectrometry;
(A07) a protein that binds to a weak cation exchanger at pH 3.0 and produces an ion peak with a mass / charge ratio of about 9070 when subjected to mass spectrometry;
(A08) A protein that binds to a strong anion exchanger at pH 9.0 and produces an ion peak with a mass / charge ratio of about 11600 when subjected to mass spectrometry . Use according to claim 14, characterized in that at least one of the following (e) and (f) is fulfilled:
(E) (A07) is complement C3 or a modified form thereof,
(F) (A08) is β2-microglobulin or a modified product thereof . Use of at least one protein of the following (B5) and (B6) present in an animal body as a marker reflecting the state of visceral fat.
(B5) complement C3 or a modified product thereof,
(B6) β2-microglobulin or a modified product thereof .