WO2015015818A1 - Composition for ameliorating metabolic disorders caused by diabetes - Google Patents

Abstract

Provided is a composition for ameliorating metabolic disorder(s) caused by diabetes. In the composition for ameliorating one or more kinds of metabolic disorders caused by diabetes, an AMP-activated protein kinase inhibitor is used as an active ingredient.

Description

Composition for improving metabolic disorders due to diabetes

(Cross-reference of related applications)
This application claims priority based on Japanese Patent Application No. 2013-159588 filed on July 31, 2013, the contents of which are hereby incorporated by reference as if set forth herein. Is included.
The present invention relates to a composition for improving metabolic disorders due to diabetes.

Type 1 diabetes mellitus (T1DM) is a disease that causes hyperglycemia, hyperfattyemia, and hyperketonosis due to insulin secretion failure from the pancreas, resulting in decreased skeletal muscle and adipose tissue weight and death. Currently, only insulin is considered a therapeutic agent for T1DM. However, even insulin therapy is known to cause secondary complications such as heart disease, kidney damage, and hypertension in T1DM.

Actually, it is known that long-term insulin administration promotes ectopic lipid deposition in tissues other than adipocytes due to the action of insulin to promote lipid synthesis and cholesterol synthesis, resulting in coronary artery disease. Furthermore, insulin treatment is often life-threatening by causing hypoglycemia. Thus, while insulin therapy is very useful for the treatment of T1DM, additional therapies that reduce the amount used are required.

AMP-activated protein kinase (AMP-activated protein kinase; hereinafter simply referred to as AMPK) detects serine / threonine phosphorylation by detecting an increase in the AMP (ADP) / ATP ratio due to intracellular energy depletion. It is an enzyme. Activation of AMPK promotes catabolic and metabolic effects that lead to ATP production such as mitochondrial biosynthesis, fatty acid oxidation, and sugar utilization, and suppresses anabolic effects that consume ATP such as protein synthesis.

In type 2 diabetes (T2DM), which is independent of insulin, one strategy is to increase the burning of sugar and fat by administering an AMPK activator that activates AMPK. As such AMPK activators, various compounds are known in addition to biguanide oral hypoglycemic agents such as metformin ( Patent Documents 1, 2, and 3).

JP 2013-35867 A Special table 2012-511549 gazette JP 2008-208081 A

To date, no effective drug has yet been found to supplement insulin therapy for T1DM. In addition, AMPK activators are known to be effective as a therapeutic strategy for T2DM. However, AMPK is caused by T1DM and the like in metabolic abnormalities related to sugar, fatty acid, insulin, neutral fat and ketone bodies in blood. The role to play was unknown.

This specification discloses a composition for improving metabolic abnormalities in diabetes.

The present inventors have made various studies on the relationship between metabolic abnormality caused by diabetes and AMPK. Surprisingly, an inhibitor that inhibits AMPK activation is considered to be hyperglycemia, hyperfatty acid, high ketone body in diabetes. It was found that metabolic conditions (metabolic abnormalities) such as blood glucose could be improved. Furthermore, the knowledge that atrophy of skeletal muscle and adipose tissue can be prevented was obtained. This specification provides the following means based on such new findings.

(1) A composition for improving one or more metabolic abnormalities due to diabetes, comprising an AMP-activated protein kinase inhibitor as an active ingredient.
(2) The composition according to (1), wherein the metabolic abnormality is selected from the group consisting of sugar, fatty acid, insulin, neutral fat and ketone body.
(3) The composition according to (1) or (2), wherein the diabetes is insulin-dependent diabetes.
(4) The composition according to any one of (1) to (3), wherein the one or more metabolic abnormalities are selected from the group consisting of fatty acids, neutral fats, and ketone bodies.
(5) The composition according to any one of (1) to (4), wherein the AMP-activated protein kinase inhibitor is represented by the following formula (1).

(6) an AMP-activated protein kinase inhibitor;
A glucagon inhibitor;
A composition for improving one or more types of metabolic abnormalities due to diabetes.
(7) an AMP-activated protein kinase inhibitor;
Insulin,
A composition for improving one or more metabolic abnormalities due to diabetes of insulin-dependent diabetes.
(8) an AMP-activated protein kinase inhibitor;
An IL-6 neutralizing antibody;
A composition for improving one or more metabolic abnormalities selected from the group consisting of sugar, fatty acid, insulin, neutral fat and ketone body in blood due to diabetes of insulin-dependent diabetes .
(9) An AMP-activated protein kinase inhibitor for improving one or more metabolic abnormalities selected from the group consisting of sugar, fatty acid, insulin, neutral fat and ketone body in blood due to diabetes A screening method comprising:
Evaluating the inhibitory activity of a test compound on AMP-activated protein kinase;
A method comprising:

A shows phosphorylated AMPK (Thr172) and total AMPK in soleus and EDL muscles of STZ-treated WT mice. B shows phosphorylated ACC (Ser79), total ACC, α1AMPK, α2AMPK. C, WT and DN-AMPK mouse skeletal muscles (soleus muscle, red and white part of gastrocnemius, EDL muscle), heart, groin (Ing) and epididymis (EpiI) WAT, BAT, pancreas, The results of immunoblotting using an antibody against α1, α2-AMPK in the spleen and the entire brain are shown. A shows the effect of STZ administration on plasma insulin concentration in WT and DN-AMPK mice 2 weeks after the first STZ administration, and the effect of STZ administration on phosphorylation of AKT, an intracellular signal molecule of insulin (Tyr473). Shows the effect, C indicates the effect of STZ administration on blood glucose levels. A shows the effect of STZ administration on body weight in WT and DN-AMPK mice 2 weeks after the first STZ administration, B shows the effect of STZ administration on food intake, and C shows the effect on survival rate. Show. D shows the effect on tissue weight (soleus and gastrocnemius) in WT and DN-AMPK mice 2 weeks after the first STZ administration, and the photograph shows the hind limbs of those mice. E shows the effect on tissue weight [epididymis (EPI) and groin (Ing) WAT and BAT] in similar groups of mice, and the pictures show the intraperitoneal and EPIWAT of these mice (DN-AMPK). Mouse; right, WT mouse: left). A shows the effect of STZ administration on plasma free fatty acid, plasma triglyceride, plasma ketone body concentrations in WT and DN-AMPK mice 2 weeks after the first STZ administration, B shows plasma norepinephrine in similar mice groups, Figure 3 shows the effect of STZ administration on plasma epinephrine, plasma corticosterone and plasma glucagon concentrations. A shows the effect of STZ administration on blood glucose level in WT and DN-AMPK mice after intraperitoneal administration of glucose one week after the first STZ administration, and B shows the effect on plasma insulin concentration. C shows the effect of STZ administration on plasma free fatty acid concentration in WT and DN-AMPK mice in a fat tolerance test (triolein gavage ingestion by gastric ingestion) one week after the first STZ administration. D shows the effect of STZ administration on plasma insulin concentration in WT and DN-AMPK mice in the fat tolerance test (triolein gavage ingestion by gastric ingestion) one week after the first STZ administration. E shows the effect of STZ administration on blood glucose levels in WT and DN-AMPK mice in the pyruvate tolerance test one week after the first STZ administration, and PEPCK (phosphoenols) in the liver two weeks after the first STZ administration. The amount of mRNA of pyruvate carboxykinase) is shown. F shows the amount of PEPCK mRNA in the liver of WT and DN-AMPK mice 2 weeks after the first STZ administration. A shows the effect of STZ administration on the amount of NAMPT and SIRT1 mRNA in the soleus muscle of WT and DN-AMPK mice 2 weeks after the first STZ administration, and B shows the red part of the gastrocnemius muscle of the same group of mice. C shows the effect of STZ administration on the amount of acetylated PGC-1α protein, C shows the effect of STZ administration on the phosphorylation of ULK1 (Ser555) in the soleus muscle of the same group of mice, and D shows the effect of the same group of mice. E shows the effect of STZ administration on the amount of LC-3II protein in the soleus muscle, E shows the effect of STZ administration on TSC2 phosphorylation (Ser1387) in the soleus muscle of the same group of mice, F shows the same group of mice Of STZ administration on S6K phosphorylation (Ser389) in Japanese soleus muscle. A shows the effect of STZ administration on phosphorylation (Thr172) AMPK and total AMPK (α1 and α2) in WAT of the groin area (Ing) of WT and DN-AMPK mice 2 weeks after the first STZ administration. B shows the effect of STZ administration on FAS, LPL, UCP1 and Cidea mRNA levels in the inguinal WAT of a similar group of mice. A shows the effect of STZ administration on the protein level of FNDC5, a precursor of iricin, in the soleus muscle of WT and DN-AMPK mice 2 weeks after the first STZ administration, and B shows the plasma of a similar group of mice. The effect of STZ administration on the concentration of iricin is shown, C shows the effect of STZ administration on the protein level of IL-6, and D shows the effect of STZ administration on plasma IL-6 concentration in the same group of mice. A and B show the effect of STZ administration on plasma leptin and high molecular weight adiponectin concentrations in WT and DN-AMPK mice 2 weeks after the first STZ administration, respectively. A shows the change in plasma glucose concentration after STZ administration in C57BL / 6J mice treated with leptin and IL-6 antibody. The osmotic minipump was implanted subcutaneously on the first day of the experiment and STZ was injected twice on the second and fourth days. The left graph shows the change in plasma glucose concentration for 2 weeks after STZ administration, and the middle and right graphs show the blood glucose level and plasma free fatty acid concentration for 2 weeks after STZ administration. B shows the effect of STZ administration on plasma leptin, plasma IL-6, irisin and insulin concentrations in C57BL / 6J mice treated with leptin and IL-6 antibody 2 weeks after pump implantation and STZ administration. C shows STZ administration on the soleus, EDL muscle, gastrocnemius, Epi and Ing WAT and BAT tissue weights of C57BL / 6J mice treated with leptin and IL-6 antibody 2 weeks after pump implantation and STZ administration. Show the impact. STZ-induced IDDM activates AMPK in skeletal muscle, induces muscle atrophy, and increases plasma concentrations of IL-6 and irisin. IL-6 and / or irisin cause atrophy of adipose tissue and a decrease in plasma leptin levels, and promote UCP1 expression and lipolysis in adipose tissue. Reduced plasma leptin levels suppress glucose and lipid utilization in skeletal muscle, and further cause muscle atrophy. Like insulin, decreased plasma leptin levels increase glucose production in the liver. Inhibition of AMPK activity in skeletal muscle improves metabolic abnormalities and decreases in body weight and tissue weight in IDDM, reduces plasma levels of IL-6 and irisin, and increases plasma levels of leptin and adiponectin. Chronic infusion of IL-6 antibody or leptin improves metabolic abnormalities and tissue weight loss in IDDM, similar to mice expressing muscle-specific DN-AMPK. Four days after the first administration of STZ, an osmotic pump was implanted subcutaneously, whereby when Compound C was continuously administered up to the 14th day, blood glucose level, plasma fatty acid concentration, tissue ( The effect of Compound C on the weight of muscle and fat) is shown. 4 days after the first administration of STZ, an osmotic pump was subcutaneously implanted, whereby the amount of phosphate compound ACC in the soleus muscle on the 14th day when Compound C was continuously administered until the 14th day The influence of Compound C is shown.

The disclosure herein provides one or two selected from the group consisting of blood sugars, fatty acids, insulin, neutral fats and ketone bodies caused by diabetes such as T1DM by inhibition of AMP-activated protein kinase. It relates to the improvement of metabolic abnormalities over species.

The present inventors have found that AMPK is activated in the skeletal muscle of a T1DM model animal, streptozotocin (STZ) -induced insulin-deficient diabetic (IDDM) mouse. Surprisingly, plasma insulin levels remain low in mice that inhibit AMPK activation by expressing dominant negative AMPK that competes with endogenous AMPK in skeletal muscle (STZ-treated DN-AMPK expressing mice). Nevertheless, it was found to improve metabolic abnormalities in this mouse. That is, it was found that the plasma concentrations of glucose, fatty acids, ketone bodies, and gluconeogenic hormones were decreased. It was also found that STZ-treated DN-AMPK-expressing mice increased the amount of adipose tissue, skeletal muscle, and body weight, and then increased survival.

Furthermore, white adipose tissue (WAT) of STZ-induced IDDM mice increased UCP1 (uncoupled protein 1) expression simultaneously with decreased expression of lipid synthase, but they expressed STZ-treated DN-AMPK. The mouse returned to the control level. STZ-treated DN-AMPK expressing mice recovered leptin plasma concentration. Leptin is known to ameliorate abnormalities in systemic glucose and lipid metabolism in IDDM.

Interleukin-6 (IL-6) promotes release from skeletal muscle and lipolysis in adipose tissue, and iricin, a novel myokine, induces the thermogenic protein UCP1 in WAT. In STZ-induced IDDM mice, IL-6 and irisin expression increased in skeletal muscle and their plasma concentrations also increased. However, even if DNZ-AMPK expressing mice were treated with STZ, they were at the control level. Furthermore, administration of IL-6 neutralizing antibody with an osmotic minipump improved metabolic abnormalities in STZ-induced IDDM mice to the same extent as STZ-treated DN-AMPK expressing mice.

Furthermore, when Compound C, which is known as an AMPK inhibitor, was administered to STZ-induced IDDM mice, it was found that the metabolic abnormalities in these mice were improved. That is, it was found that glucose and fatty acids were decreased.

As described above, the present specification clarifies that AMPK plays an important role in metabolic abnormalities in diabetes such as T1DM. Inhibition of AMPK activity in IDDM improved systemic metabolism, prevented muscle and adipose tissue atrophy, and increased survival despite plasma insulin levels remaining low. In addition, the present inventors have revealed that IL-6 and leptin adipocyte hormone are probably mediators involved in abnormal metabolism of IDDM. According to the present specification, it is shown that metabolic abnormalities in diabetes such as T1DM are caused by vicious circles of organ networks including muscles and adipose tissues. This specification shows that inhibition of AMPK activation in skeletal muscle can correct organ network abnormalities related to metabolic regulation and improve vicious circles in diabetes such as T1DM.

Based on the above, this specification indicates that administration of an AMPK inhibitor is one or more selected from the group consisting of blood sugar, fatty acid, insulin, neutral fat and ketone bodies caused by diabetes. Disclose improving metabolic abnormalities. In other words, the AMPK inhibitor improves sugar and fat metabolism abnormalities in such diabetes, and as a result, improves hyperglycemia, hyperfattyemia, and hyperketonosis, and causes skeletal muscle, fat, A reduction in tissue weight can be prevented and treated.

Also, the present specification discloses that administration of IL-6 neutralizing antibody together with inhibition of AMPK activity can improve, prevent and treat metabolic disorders in diabetes and diseases associated with metabolic disorders such as T1DM.

Furthermore, the present specification also provides a finding that there is room for further lowering blood glucose level even when an AMPK inhibitor is administered. For this reason, the improvement of the metabolic abnormality in diabetes by the combination of an AMPK inhibitor and a glucagon inhibitor for lowering blood glucose level is provided. It also provides an improvement in metabolic abnormalities in diabetes by combination with insulin. Hereinafter, various embodiments related to the disclosure of this specification will be described in detail.

Hereinafter, representative and non-limiting specific examples of the present disclosure will be described in detail with reference to the drawings. This detailed description is intended merely to provide those skilled in the art with details to implement preferred examples of the present disclosure and is not intended to limit the scope of the present disclosure. In addition, the additional features disclosed below can be used separately from or in conjunction with other features and inventions to provide a composition for further improving metabolic disorders due to diabetes.

Further, the combinations of features and steps disclosed in the following detailed description are not essential in carrying out the present disclosure in the broadest sense, and are particularly only for explaining representative specific examples of the present disclosure. It is described. Furthermore, various features of the following representative embodiments, as well as various features of those set forth in the independent and dependent claims, are described herein in providing additional and useful embodiments of the present invention. They do not have to be combined in the specific examples or in the order listed.

All features described in this specification and / or claims, apart from the configuration of the features described in the examples and / or claims, are individually disclosed as limitations on the original disclosure and claimed specific matters. And are intended to be disclosed independently of each other. Further, all numerical ranges and group or group descriptions are intended to disclose intermediate configurations thereof as a limitation to the original disclosure and claimed subject matter.

(AMP-activated protein kinase; AMPK)
AMPK is a heterocomplex composed of a catalytic α subunit and regulatory β- and γ-subunits. AMPK plays various roles in vivo.

(1) Activation of AMPK, for example, promotes mitochondrial biosynthesis, fatty acid oxidation and glucose utilization and catabolism processes that produce ATP, and inhibits anabolic pathways such as protein synthesis that consumes ATP (9). AMPK has been shown to promote fatty acid oxidation through direct phosphorylation of acetyl-CoA carboxylase (ACC) (Ser79) and suppression of its activity. Inhibition of ACC activity reduces the production of malonyl CoA, an allosteric inhibitor of carnitine palmitoyltransferase I (CPT1), which transports free fatty acids from the cytosol to the mitochondria, resulting in fatty acid oxidation.

(2) AMPK is also a transcriptional cofactor for expression of mitochondria-related genes, PGC−, via a nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 (sirtuin-1: NAD + dependent deacetylase) dependent pathway. Activates 1α.

(3) AMPK inhibits protein synthesis through direct phosphorylation of TSC2 (tuberous sclerosis protein 2) and thereby suppression of mTOR (mammalian target of rapamamicin) and S6K (P70 S6 kinase) signaling pathway To do.

(4) AMPK directly phosphorylates ULK1 (UNC-51-like kinase 1), induces autophagy, and increases the expression of LC3-II (regulator of autophagy) (17-19).

In the present specification, inhibiting AMPK activity includes any mode of reducing AMPK activity or reducing AMPK activation. Examples of the mode of inhibiting the activity of AMPK include supply (expression) of a protein that competes with AMPK and a substrate, allosteric inhibition of AMPK, inhibition of the pathway by which AMPK is activated, and the like.

(Composition for improving one or more metabolic abnormalities due to diabetes)
This composition contains an AMPK inhibitor as an active ingredient. As described above, the AMPK inhibitor is not particularly limited as long as it selectively reduces AMPK activity. Examples of the AMPK inhibitor include a compound called Compound C shown in the following formula (1) (J Clin Invest. 108, 1167-1174. 2001). This compound is a compound widely used as an inhibitor of AMPK, and selectively inhibits the ATP binding sites of α1 and α2 subunits necessary for the kinase activity of AMPK.

Other AMPK inhibitors include oligosaccharides, which are glucose oligomers having the following isomaltose and isomaltose structures (glucose linked by α1-6 bonds) (Cell Metab. 9, 23-34. 2009). ). They bind to the glycogen binding domain of the β subunit of AMPK and inhibit activity.

In addition, the AMPK inhibitor can also be obtained by a screening method described later.

This composition can improve metabolic abnormalities due to diabetes. Various metabolic abnormalities due to diabetes are known. Among them, one or more metabolic abnormalities selected from the group consisting of sugars, fatty acids, insulin, neutral fats and ketone bodies are mentioned.

The abnormal sugar metabolism means a so-called abnormal blood glucose level, which is the amount of glucose in the blood. Metabolic abnormalities related to blood glucose levels may be determined by commonly adopted standard values, etc., and may be determined by race, gender, age, etc., or by personal standard values. May be. Abnormal blood glucose levels are generally hyperglycemia in diabetes. A method for measuring a blood glucose level is well known.

An abnormality in fatty acid metabolism means an abnormality in the level of free fatty acid in the blood. Metabolic abnormalities related to blood free fatty acids may be determined based on commonly adopted standard values, etc., and are determined based on race, gender, age, etc., or based on personal standard values. It may be. Abnormal metabolism of blood free fatty acids is generally high in diabetes. Methods for measuring blood free fatty acids are well known.

異常 Insulin metabolism abnormality means so-called abnormality of blood insulin level. Metabolic abnormalities related to blood insulin levels may be determined by commonly used reference values, etc., determined by race, gender, age, etc., or by personal reference values It may be a thing. Abnormal blood insulin levels are generally low in diabetes. A method for measuring the blood insulin level is well known.

代謝 Neutral fat metabolism abnormality means abnormal glycerin ester value of fatty acid in blood. Metabolic abnormalities related to blood triglycerides may be determined by commonly used standard values, etc., and determined by race, gender, age, etc., or by personal standard values It may be a thing. Blood triglyceride abnormalities are generally higher than baseline in diabetes. A method for measuring the blood triglyceride level is well known.

The abnormal metabolism of ketone bodies means an abnormality in the total ketone body values of acetoacetic acid, 3-hydroxybutyric acid and acetone in blood. Metabolic abnormalities related to blood ketone bodies may be determined based on commonly used reference values, etc., and determined based on race, gender, age, etc., or based on personal reference values It may be. Blood ketone body abnormalities are generally higher than the reference value in diabetes. The method for measuring blood ketone bodies is well known.

Among these metabolic abnormalities, it is advantageous to use the present composition for abnormal metabolism of fatty acids, neutral fats and ketone bodies. AMPK inhibitors can effectively act and reduce these metabolic abnormalities.

This composition can contain an AMPK inhibitor and a glucagon inhibitor and / or a glucagon receptor inhibitor as active ingredients. There is still room for improvement in the reduction of blood glucose level due to administration of an AMPK inhibitor. The incomplete decrease in blood glucose level is thought to be due to glucagon. Thus, by combining a glucagon inhibitor and / or a glucagon receptor inhibitor with an AMPK inhibitor, it is possible to more effectively reduce the blood glucose level.

Glucagon inhibitors include Glu-001 (Novonordisk, Sorensen H, et al Diabetes 55: 2843-2848, 2006), glucagon receptor antagonists (Eli Lilly, JP 2011-518125), etc., and Yan H, et al. J Pharmacol Exp Ther. 329: 102-111, 2009. Also, known glucagon receptor inhibitors can be mentioned.

This composition may contain an AMPK inhibitor and insulin as active ingredients. As already explained, AMPK inhibitors do not cause hypoglycemia and can be used in combination with insulin. As a result, the dose of insulin can be reduced, the number of injections can be reduced, and the patient's usability can be improved.

This composition may contain an AMPK inhibitor and an IL-6 neutralizing antibody as active ingredients. As already explained, administration of IL-6 neutralizing antibody improves metabolic abnormalities in diabetes such as T1DM. An IL-6 neutralizing antibody can be obtained or obtained by a known method.

The composition disclosed herein may be in the form of a pharmaceutical composition, a food or drink, or a reagent used for research purposes (eg, in vitro or in vivo experiments).

This composition can be suitably used as a pharmaceutical composition administered for the prevention or treatment of diabetes. It can also be suitably used as a food or drink that is taken daily to prevent or improve diabetes.

The active ingredient in the present composition may be in the form of an appropriate salt, if necessary, such as a pharmaceutical form. In the case of using a salt as an active ingredient, acceptable salt forms are well known to those skilled in the art, and those skilled in the art can select an appropriate salt form.

This composition can be formulated by a known pharmaceutical method. For example, various forms such as tablets, powders, granules, capsules, and liquids can be taken orally or parenterally taken into a living body by a method such as intravenous injection.

In these preparations, carriers that are acceptable pharmacologically or as foods and beverages, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, stabilizers, Flavor, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, extender, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, viscous It can be appropriately combined with agents, flavoring agents, solubilizing agents or other additives. In addition, a nutritional supplement can be added and used.

When this composition is used as a pharmaceutical composition, one or more other components effective for improving metabolic abnormality in diabetes, such as T1DM, can be blended. Moreover, you may use together with the other pharmaceutical composition effective in the prevention and treatment of diabetes.

When an AMPK inhibitor is used in combination with other active ingredients, for example, the above-described glucagon inhibitor and / or glucagon receptor inhibitor, insulin, IL-6 neutralizing antibody, etc. A combination of preparations containing two or more active ingredients (kits) so that administration in different dosage forms and different dosages is possible, not limited to a single composition form) Of course, it is possible and appropriate. Such modifications can be made by those skilled in the art within the scope of the design matter as appropriate.

When the composition or the combination is used as a food or drink, the food or drink may be, for example, a health food, a functional food, a food for specified health use, a nutritional supplement, a food for a sick person, or a food additive. The food and drink of the present invention can be ingested as a composition as described above, and can also be ingested as various food and drink. Furthermore, health foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly are also included. Manufacture of the food-drinks in this invention can be implemented with a manufacturing technique well-known in the said technical field. The food or drink may contain one or more other components that can contribute to the improvement of metabolic abnormalities in diabetes. Furthermore, the food and drink may be a multifunctional food and drink by combining with other components that exhibit other functions or other functional foods. For example, a nutritional supplement or the like can be appropriately added and used as a multifunctional food or drink for widely preventing or improving lifestyle-related diseases such as diabetes, hypertension, hyperlipidemia, and arteriosclerosis.

The dose or intake of the composition or the combination is appropriately selected according to age, weight, symptoms, health condition, composition type (pharmaceutical, food and drink, etc.) and the like. The AMPK inhibitor that is the active ingredient of the composition of the present invention is generally used in an effective amount to be able to ameliorate one or more metabolic abnormalities in diabetes including T1DM. Specifically, it is preferable to use once or several times a day so that about 5 mg to 500 mg, preferably about 50 mg to 500 mg per day is administered or ingested in an adult. Usually, it is preferable to take (take) repeatedly for several weeks to several months.

The product (pharmaceutical product, food and drink, reagent) or its instruction relating to the present composition or the present combination or its instructions may be labeled as used for the prevention, treatment or improvement of diabetes. Here, “labeled product or instructions” means that the product body, container, packaging, etc. are marked, or instructions, package inserts, promotional materials, or other printed materials that disclose product information. It means that the display is attached to. Indications may include information on how or by which metabolic abnormalities in diabetes are improved by administration or ingestion of the composition or optionally the combination.

(Methods for improving metabolic abnormalities in diabetes)
The present specification thus provides a method for improving, preventing and treating metabolic abnormalities in diabetes in a subject, characterized in that the subject composition is administered or ingested with the composition or the combination. it can.

(Screening method)
The screening method disclosed in the present specification is an AMPK for improving one or more metabolic abnormalities selected from the group consisting of blood sugar, fatty acids, insulin, neutral fat and ketone bodies due to diabetes. Inhibitor screening method. This screening method can comprise a step of evaluating the AMPK inhibitory activity of the test compound. According to this screening method, it is possible to screen for AMPK inhibition effective in improving diabetes metabolic abnormality.

AMPK inhibitory activity can be evaluated, for example, based on the activity of AMPK already described, using, for example, direct phosphorylation (Ser79) of acetyl-CoA carboxylase (ACC) as an index.

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

First, describe the animals and experimental methods used in the experiment, and then describe the results.

(animal)
Male C57BL / 6J mice (Japan SLC, Hamamatsu, Japan) and mice that express skeletal muscle-specific DN-AMPK (DN-AMPK mice) (29) were used at 10 to 12 weeks after birth. DN-AMPK mice preferentially replace the α1 catalytic subunit of AMPK in which the 157th aspartic acid residue was substituted with alanine (Asp157Ala) preferentially in skeletal muscle under the control of the human α-skeletal actin promoter (29). Mutants were overexpressed. Aspartic acid at position 157 is within the DFG (protein kinase catalytic subunit subdomain VII) motif, where protein kinase is essential for Mg ²⁺ -ATP binding (29). The animals are lit between 06:00 and 18:00 hours and are individually housed in plastic gauges adjusted to 24 ° C ± 1 ° C, which have access to laboratory food (Oriental Yeast, Tokyo, Japan) and water at any time. Maintained as possible.

Mice were injected intraperitoneally (200 mg / kg) with streptozotocin (STZ) (Sigma, Tokyo, Japan) on day 1 and day 3 (STZ-treated WT mice and STZ-treated DN-AMPK mice). . Another group of mice (WT mice and DN-AMPK mice) were injected with vehicle in the same manner as STZ-treated mice. One week after the second injection, a glucose tolerance test (GTT) was performed, followed by a fat tolerance test and a pyruvate tolerance test.

Blood was collected from the tail every day, and the plasma concentration of glucose was measured using a kit (glucose: glucose CII-Test Wako, Osaka, Japan) to obtain a blood glucose level. Two weeks after the second STZ injection, the mice were decapitated at 1 pm and tissue and blood samples were collected.

(DN-AMPK (dominant negative form AMPK))
DN-AMPK is a protein in which the 157th aspartic acid of the AMPKα1 subunit is mutated to alanine. This aspartic acid is essential for ATP binding required for kinase activity, and AMPK having an α1 subunit substituted with alanine loses kinase activity. Moreover, when this is expressed in a cell, normal AMPK, a substrate, and a regulatory subunit are brought together to reduce the intracellular AMPK activity. Furthermore, normal α1 and α2 subunits that could not bind to the regulatory subunit are degraded, resulting in a marked decrease in AMPK activity.

In this experiment, mice that selectively overexpressed DN-AMPK in skeletal muscle using a human actin promoter selective for skeletal muscle were used (provided by Prof. Esaki, National Institute of Nutrition). As shown in the dissertation and the following cited paper, phosphorylation of acetyl-CoA carboxylase (ACC) (a key target protein for AMPK: an important rate-limiting enzyme for fat synthesis) is reduced in skeletal muscle, and AMPK activity It can be seen that is suppressed. In addition, the amount of α2 subunit protein is selectively reduced in skeletal muscle (because the α2 subunit that could not be bound was degraded) (Am J Physiol Endocrinol Metab. 296, E47-E55.).

(Glucose tolerance, pyruvate tolerance and fat tolerance test)
After an overnight fast, animals were injected intraperitoneally with glucose (2 grams / kg) for a glucose tolerance test (GTT). Blood was collected from the animal's tail at 0, 30, 60, 90 and 120 minutes after glucose administration. Plasma glucose concentration and insulin concentration were measured using a kit (mouse insulin ELISA KIT (U type), Shiba goat insulin glucose CII-Test Wako glucose) (Table 1).

The fat tolerance test was performed according to the previous statement (30). That is, after fasting for 4 hours, triolein (Kanto Chemical Co., Inc., Tokyo, Japan) (17 μl / g) was forcibly introduced into the stomach orally in mice, and blood was 0, 30, 60, 90, 10 , Blood was collected from the tail continuously at 180 and 300 minutes. Plasma concentrations of free fatty acids, triglycerides and insulin were measured using kits (free fatty acids: NEFAC-Test Wako, triglycerides: triglycerides E-Test Wako, mouse insulin ELISA KIT (U type), Shibayagi) (Table 1).

(Immunoblot analysis)
Tissue was treated at 50C with 50 mM Tris (pH 7.4), 5 mM EDTA (pH 8.0), 10 mM sodium pyrophosphate, 1% Triton X, 1 mM butyl phosphatase inhibitor (Calbiochem, California, USA) and proteinase inhibitor ( Sigma). The homogenate was centrifuged and the supernatant (20 μg protein) was fractionated by SDS-PAGE. Immunoblot analysis was performed using the specific antibodies shown in Table 2. Immune complexes were visualized with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz, CA, USA) and an ECL chemiluminescent reagent (GE Healthcare, Tokyo, Japan). Protein bands were quantified using ImageJ software (National Institutes of Health, http://rsbweb.nih.gov/ij).

(Measurement of acetyl PGC-1α)
The red part of the gastrocnemius muscle at 4 ° C., 50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM potassium chloride, 1% NP40, 5 mM nicotinamide, 1 mM sodium butyrate phosphatase inhibitor (Calbiochem) and proteinase inhibitor ( Homogenized in lysis buffer containing Sigma). The muscle lysate (2 mg protein) was pre-washed with 40 μl protein G-conjugated Sepharose beads (GE Healthcare, Tokyo, Japan) and 4 μg PGC-1 (H300) antibody (Santa Cruz Biotechnology) and protein A Immunoprecipitated with conjugated Sepharose beads (GE Healthcare). The resulting immunoprecipitate was boiled in 50 μl of sample buffer containing 125 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.02% bromophenol blue, 20% 2-mercaptoethanol. Western blot analysis using antibodies against acetylated lysine (Cell Signaling Technology, Denvers, Mass.) Or total PGC-1α (H300).

(RNA extraction and quantitative real-time PCR)
Total RNA was isolated from tissues using Isogen (Nippon Gene, Wako, Japan), RNA (400 ng) was isolated from oligo (dT) primer (Sigma) and avian myeloblastosis virus reverse transcriptase (Takara, Shiga, Japan) Japan) was used for reverse transcription. The obtained cDNA was quantified by ABI 7500 real-time PCR system (Applied iosystems, Tokyo, Japan) (22) as described above using SYBR green PCR master mix. Data were normalized by the abundance of acidic ribosomal protein for skeletal muscle and by the abundance of 36B4 for adipose tissue samples. The primer sequences are shown in Table 3.

(Measurement of plasma metabolites and hormones)
Plasma concentrations of glucose, corticosterone free fatty acid, triglyceride, ketone body, insulin, glucagon, IL-6 and irisin in blood samples were measured using the kit described in Table 1.

(Chronic infusion of leptin and neutralizing antibody for IL-6)
Recombinant mouse leptin (0.5 mg / ml) (Peprotech Inc., Rocky Hill, NJ) or IL-6 (1 mg / ml) neutralizing antibody (R & D Systems, Minneapolis, MN) (31) A pressure minipump (Alzette, model 2002, Muromachi Kikai Co., Ltd., Tokyo, Japan) was delivered subcutaneously at a rate of 0.5 μL / hr for 14 days. The osmotic minipump was implanted subcutaneously under anesthesia with ketamine (100 mg / kg body weight) and xylazine (10 mg / kg). The control group delivered phosphate buffered saline with the same type of pump. Thereafter, STZ (200 mg / kg) or vehicle was injected intraperitoneally on the second and fourth days after transplantation of the osmotic minipump. Body weight and plasma concentrations of glucose and insulin were monitored daily. Two weeks after transplanting the minipump, the mice were decapitated and blood and tissue samples were taken.

(Statistical analysis)
Data were expressed as mean ± standard error (SEM). Statistical comparisons between groups were tested by analysis of variance (ANOVA) followed by Tukey's HSD post-test. Statistical analysis between the two groups was performed by Student's t test. A P value of <0.05 was considered statistically significant.

(Result 1: STZ-induced IDDM activates AMPK in skeletal muscle)
First, it was examined whether AMPK in skeletal muscle is activated in mice by STZ-induced IDDM. The results are shown in FIGS. 1-1 and 1-2. In STZ-treated WT mice, soleus muscle (red part of skeletal muscle) and extensor digtorum longus (EDL) (mixed muscle including red and white muscle) (A in Fig. 1-1) Thr172 phosphorylation increased. Ser79 phosphorylation of ACC phosphorylated by AMPK was also increased in the soleus muscle of STZ-treated WT mice (FIG. 1-1B).

The expression of the α1 catalytic subunit mutant increased the mutant α1AMPK protein in the soleus, red and white portions of the gastrocnemius muscle, and EDL of DN-AMPK mice. In contrast, α1AMPK protein expression is altered in other tissues such as heart muscle, inguinal (Ing) and epididymis (EPI) WAT, interscapular brown adipose tissue (BAT), pancreas, spleen, and brain. None (B in FIG. 1-1 and C in FIG. 1-2). In contrast, as previously reported, the α2 catalytic subunit of AMPK was decreased in skeletal muscle. This is probably because the subunits of AMPK that are not configured as heterocomplexes are decomposed. In parallel with the activation of AMPK, Ser79 phosphorylation of ACC increased in the soleus muscle of STZ-treated WT mice, but not in DN-AMPK mice (FIG. 1-1B).

STZ treatment decreased plasma insulin concentration (A in FIG. 2) and Serkt phosphorylation of Akt (B in FIG. 2) in the soleus of both WT and DN-AMPK mice. However, plasma glucose concentrations (FIG. 2C) were significantly lower in STZ-treated DN-AMPK mice compared to STZ-treated WT mice. Thus, selective suppression of AMPK activity in skeletal muscle improved hyperglycemia despite plasma insulin levels remaining low and its signaling in skeletal muscle being inhibited.

(Result 2: Selective expression of DN-AMPK in skeletal muscle can reduce weight loss, bulimia, survival in STZ-induced IDDM, and improve tissue weight loss)
WT mice lost weight by STZ treatment (A in Fig. 3-1). However, STZ-treated DN-AMPK mice showed a significant improvement. Overeating caused by STZ administration was also suppressed in STZ-treated DN-AMPK mice (FIG. 3-1 B). Furthermore, all STZ-treated DN-AMPK mice survived 2 weeks after the first injection of STZ, but only 35.7% of STZ-treated WT mice survived (FIG. 3-1). C).

STZ-treated DN-AMPK mice significantly recovered the soleus and gastrocnemius tissue weights compared to STZ-treated WT mice (D in FIG. 3-2). In addition, the tissue weight of epididymis (Epi), inguinal (Ing) WAT, and interscapular BAT decreased by STZ treatment was also recovered in the DN-AMPK mice treated with STZ (E in FIG. 3-2). These data suggest that inhibiting AMPK activation in skeletal muscle improves STZ-induced skeletal muscle and adipose tissue atrophy and weight loss, resulting in increased survival. .

(Result 3: Selective expression of DN-AMPK in skeletal muscle improves systemic glucose and lipid metabolism)
Two weeks after the first STZ injection, STZ-treated WT mice had increased plasma concentrations of free fatty acids, triglycerides and ketone bodies. In DN-AMPK mice treated with STZ, the levels returned to normal levels (A in FIG. 4). Plasma concentrations of norepinephrine, epinephrine and corticosterone were increased in STZ-treated WT mice but not in STZ-treated DN-AMPK mice (FIG. 4B). In contrast, plasma glucagon concentrations remained high in STZ-treated DN-AMPK mice as in STZ-treated WT mice (FIG. 4B). Thus, it was due to hyperglucagonemia in mice (C in FIG. 2) that the blood glucose level remained only partially improved in STZ-treated DN-AMPK mice.

Furthermore, in order to examine the effects of DN-AMPK expressed on skeletal muscles on glucose and lipid metabolism throughout the body, a glucose tolerance test (GTT), a pyruvate tolerance test, and a fat (triolein) tolerance test were performed. STZ-treated WT mice showed a significant increase in blood glucose level in response to GTT (A in FIG. 5-1). However, the blood glucose level (A in FIG. 5-1) improved significantly in STZ-treated DN-AMPK mice. In addition, the triolein intake increased plasma free fatty acid and triglyceride concentrations in STZ-treated WT mice, but significantly improved in STZ-treated DN-AMPK mice (FIG. 5-1C). Also during the experiment, plasma insulin concentrations remained low in STZ-treated DN-AMPK mice as well as in STZ-treated WT mice (FIGS. 5-1 B and D). A pyruvate tolerance test reflecting liver gluconeogenic activity also showed that plasma glucose concentrations increased in response to pyruvate injection were significantly improved in STZ-treated DN-AMPK mice (FIG. 5). -2 E). Phosphoenolpyruvate carboxylase carboxylase (PEPCK) mRNA was decreased in the liver of STZ-treated DN-AMPK mice (F in FIG. 5-2). These data suggest that inhibiting AMPK activity in skeletal muscle improves systemic glucose and lipid metabolism even though plasma insulin levels remain low.

(Result 4: Selective expression of DN-AMPK in skeletal muscle inhibits activation of AMPK-related signals in skeletal muscle in response to STZ-induced IDDM)
FIG. 1-1B shows that IDDM increases ACC phosphorylation in skeletal muscle in parallel with AMPK activation. Therefore, the reaction of other downstream signal molecules related to AMPK in the soleus muscle was examined. In STZ-treated WT mice, the amounts of NAMPT and SIRT1 mRNA increased in the soleus muscle, but these changes were suppressed in STZ-treated DN-AMPK mice (A in FIG. 6). Furthermore, in WT mice treated with STZ, the amount of acetylated PGC-1α decreased in the red part of the gastrocnemius muscle. In contrast, DN-AMPK mice did not decrease (B in FIG. 6).

Autophagy plays an important role in the adaptive response of skeletal muscle in energy crises such as long-term hunger (32-34). AMPK directly phosphorylates ULK1 (Ser555) and consequently regulates autophagy by increasing the amount of LC3-II protein (17-19). STZ-treated WT mice increased ULK1 phosphorylation and LC3-II protein expression in the soleus muscle, but these changes were suppressed in STZ-treated DN-AMPK mice (C and D in FIG. 6).

AMPK inhibits protein synthesis by increasing phosphorylation (Ser1387) of TSC2 (tuberous sclerosis complex) and decreasing mTOR signal (15, 16). In STZ-treated WT mice, phosphorylation of TSC2 (Ser1387) increased, and as a result, phosphorylation of S6K (Thr389) decreased in the soleus muscle. In STZ-treated DN-AMPK mice, they returned to normal levels (E and F in FIG. 6).

(Result 5: Selective expression of DN-AMPK in skeletal muscle normalizes AMPK activation and altered gene expression in STAT-induced IDDM WAT)
In order to investigate the mechanism of WAT atrophy in IDDM mice, activation of AMPK and gene expression were examined in WAT of STZ-treated WT and DN-AMPK mice. Similar to skeletal muscle, AMPK was activated in IngWAT of STZ-treated WT mice but not in STZ-treated DN-AMPK mice (A in FIG. 7). AMPK has been shown to suppress lipid synthesis in adipocytes and inhibit the expression of lipid synthesis-related genes (35). Fatty acid synthase (FAS) and lipoprotein lipase (LPL) mRNA abundance decreased in the inguinal WAT of STZ-treated WT mice, but their expression was significantly restored in STZ-treated DN-AMPK mice (B in FIG. 7). Unexpectedly, STZ-treated WT mice showed increased expression of UCP1 and Cidea (cell-death-inducing DFF45-like effector A) in IngWAT, but were suppressed by STZ-treated DN-AMPK (Fig. 7B). These results suggest that WAT atrophy was induced by suppression of lipogenesis, possibly promoted by increased expression of the thermogenic protein UCP1 in WAT.

(Result 6: STZ-induced IDDM increases the expression of myokines in skeletal muscle in an AMPK-dependent manner)
In order to investigate the mechanism of improving “adipose tissue atrophy” in STZ-induced IDDM by suppressing AMPK activation in skeletal muscle, the expression of myokine that affects adipocyte metabolism was examined. Recent studies have revealed that a novel myokine, ilysin, is generated from FNDC5 (fibrinctin type III domain containing 5) in skeletal muscle in a PGC-1α-dependent manner and induces UCP1 and related gene expression in subcutaneous WAT. (27). Consistent with PGC-1α activation in skeletal muscle, STZ-treated WT mice had increased concentrations of FNDC5 protein and plasma iricin in soleus muscle (A, B in FIG. 8). These returned to normal levels completely in STZ-treated DN-AMPK mice.

IL-6 is another myokine released from skeletal muscle and promotes WAT lipolysis (25, 26). IL-6 has been reported to activate AMPK (26) (26). STZ-treated WT mice had increased levels of IL-6 protein (FIG. 8C) and plasma IL-6 (FIG. 8D) in soleus muscle, but they decreased in STZ-treated DN-AMPK mice . Next, the plasma concentration of IL-15 reported to be released from skeletal muscle (36) was examined. However, IL-15 did not increase in plasma in STZ-treated WT mice (data not shown).

(Result 7: Plasma concentrations of leptin and adiponectin in STZ-treated WT and DN-AMPK mice)
Leptin is secreted from adipocytes and its secretion correlates well with the amount of WAT. Leptin promotes glucose utilization in skeletal muscle through β-adrenergic receptors in sympathetic nerves and tissues (20-22). Furthermore, long-term leptin treatment improves metabolic changes in IDDM, including STZ-induced diabetes, via the central nervous system (23, 24, 37, 38). Another adipocyte secreting hormone, adiponectin, also promotes glucose and lipid utilization in skeletal muscle and suppresses glucose production in the liver (39). Leptin plasma concentration was markedly decreased in STZ-treated WT mice but recovered in STZ-treated DN-AMPK mice (FIG. 9A). Plasma concentrations of high molecular weight adiponectin did not change in STZ-treated WT mice (FIG. 9B), but increased in STZ-treated DN-AMPK mice (FIG. 9B).

(Result 8: Chronic infusion of leptin or IL-6 neutralizing antibody ameliorates metabolic abnormalities in STZ-induced IDDM)
To explore the regulatory effects of leptin and IL-6 on metabolic changes in STZ-induced IDDM, STZ-induced IDDM C57BL / 6J mice were treated with leptin or IL- for 2 weeks using an osmotic minipump. Six neutralizing antibodies were injected. The leptin injection reduced the plasma glucose concentration to a control level that was not treated with STZ (A in FIG. 10-1). Infusion of IL-6 neutralizing antibody also reduced the plasma concentration of glucose (A in FIG. 10-1). Furthermore, plasma free fatty acid concentrations returned to control levels by infusion of leptin and IL-6 antibody (A in FIG. 10-1).

The leptin injection increased the leptin plasma concentration to about 30 ng / ml on the third day after implantation of the minipump, and maintained that concentration until the end of the experiment (2 weeks) (B in FIG. 10-1). Corresponding to the increase in adipose tissue volume, IL-6 antibody infusion significantly increased plasma leptin concentrations relative to STZ treatment alone (STZ + vehicle: 0.12 ± 0.01 ng / ml; STZ + IL-6 Antibody: 4.18 ± 0.57 ng / ml) (B of FIG. 10-1).

Plasma IL-6 and ilysin concentrations were decreased by injection of IL-6 antibody and leptin (B in FIG. 10-1). In contrast, plasma insulin concentrations were low after injection of IL-6 antibody and leptin as well as STZ treatment alone (FIG. 10-1B).

Next, in STZ-induced IDDM, skeletal muscle mass, WAT, and BAT tissue weight after injecting antibodies against leptin or IL-6 were examined. In all injections, the amount of soleus, EDL and gastrocnemius increased (C in Fig. 10-2). Furthermore, the injection of IL-6 antibody increased the amount of EpiWAT, IngWAT and BAT (C in FIG. 10-2). Leptin injection also slightly restored the amount of these adipose tissues (FIG. 10-2C). The reason why the increase in the amount of adipose tissue after leptin injection is smaller than that of IL-6 antibody injection is considered to be because leptin has a catabolic effect on lipid metabolism in adipose tissue (C in FIG. 10-2).

Taken together, these results suggest that STZ-induced IDDM activates AMPK in skeletal muscle and induces muscle atrophy, thereby increasing IL-6 and irisin production (FIG. 11). Increased plasma levels of IL-6 then promote the catabolic process in adipose tissue and reduce leptin production. Ilysin also induces UCP1 expression and reduces the amount of WAT. Decreased plasma leptin levels impair glucose and lipid metabolism in skeletal muscle and induce a vicious cycle in the organ network.

(Result 9: Administration of AMPK inhibitor to STZ-induced IDDM mice improves blood glucose level and blood free fatty acid concentration)
STZ-induced IDDM mice prepared by intraperitoneally administering 200 mg / kg of STZ on day 1 and day 3 were implanted with an osmotic pump subcutaneously on day 4 after STZ administration when diabetes was manifested. Compound C (240 μg / ml, 500 μg / ml), an AMPK inhibitor, was continuously administered subcutaneously until day 14 after STZ was administered at 0.5 μl / hr / week. As a control, vehicle alone was administered subcutaneously. Further, as a control, vehicle and compound C were subcutaneously administered without STZ treatment. The results are shown in FIGS.

As shown in FIG. 12, administration of Compound C significantly decreased blood glucose levels and blood free fatty acid concentrations. Further, as shown in FIG. 13, administration of compound C increased the weights of the soleus, longus extensor and gastrocnemius muscles, and also increased the groin and epididymis and the fat tissue weight of BAT. From these results, it was found that the same improvement effect as that obtained when DN-AMPK was expressed in skeletal muscle can also be obtained by administration of an AMPK inhibitor.

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SEQ ID NOs: 1-18: Primers

Claims (9)

1. A composition for improving one or more metabolic abnormalities caused by diabetes, comprising an AMP-activated protein kinase inhibitor as an active ingredient.
2. The composition according to claim 1, wherein the metabolic abnormality is selected from the group consisting of sugar, fatty acid, insulin, neutral fat and ketone body.
3. The composition according to claim 1 or 2, wherein the diabetes is insulin-dependent diabetes.
4. The composition according to any one of claims 1 to 3, wherein the one or more metabolic abnormalities are selected from the group consisting of fatty acids, neutral fats, and ketone bodies.
5. The composition according to any one of claims 1 to 4, wherein the AMP-activated protein kinase inhibitor is represented by the following formula (1).
   

   
6. An AMP-activated protein kinase inhibitor;
   A glucagon inhibitor and / or a glucagon receptor inhibitor;
   A composition for improving one or more types of metabolic abnormalities due to diabetes.
7. An AMP-activated protein kinase inhibitor;
   Insulin,
   A composition for improving one or more metabolic abnormalities due to diabetes of insulin-dependent diabetes.
8. An AMP-activated protein kinase inhibitor;
   An IL-6 neutralizing antibody;
   A composition for improving one or more metabolic abnormalities selected from the group consisting of sugar, fatty acid, insulin, neutral fat and ketone body in blood due to diabetes of insulin-dependent diabetes .
9. A screening method for an AMP-activated protein kinase inhibitor for improving one or more metabolic abnormalities selected from the group consisting of sugar, fatty acid, insulin, neutral fat and ketone body in blood due to diabetes There,
   Evaluating the inhibitory activity of a test compound on AMP-activated protein kinase;
   A method comprising:

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