Novel diagnostic marker and novel therapeutic composition for diabetes, obesity and / or fatty liver

Abstract

To provide a novel diagnosis marker and a novel treatment composition for diabetes, obesity, and / or fatty liver.SOLUTION: The present inventors have reached the present invention by finding out that inputting αMG into a living body increases the concentration of glucagon in blood in the living body and that using the action of accelerating secretion of glucagon by the αMG enables the diagnosis and the treatment of diabetes, obesity, and / or fatty liver.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel diagnostic markers and novel therapeutic compositions for diabetes, obesity and / or fatty liver. [Background technology]

[0002] The increasing number of diabetes patients in developed countries, including Japan, is becoming a serious problem. It has been known that diabetes is caused not only by insufficient secretion of insulin, a blood glucose-lowering hormone secreted by pancreatic beta cells, or insulin resistance, but also by increased endogenous glucose production due to excessive glucagon secretion from pancreatic alpha cells. However, the mechanism of glucagon secretion has not been fully elucidated, and the only diagnostic indicators for diabetes are blood glucose level and insulin. Furthermore, it has been difficult to sensitively diagnose abnormal glucagon secretion using conventional glucose tolerance tests and meal tolerance tests.

[0003] It was previously thought that glucose uptake in pancreatic α cells was solely mediated by the glucose transporter GLUT-1 (glucose transporter-1). However, the present inventors recently discovered that pancreatic α cells also contain Na + They found that SGLT-1 (sodium / glucose cotransporter-1), which cotransports glucose into cells together with α-D-glucose, is expressed in the cells (Non-Patent Document 1). In addition, in an in vitro experiment using a cultured pancreatic α-cell line, αMG (Methyl α-D-Glucoside), a non-metabolizable glucose analogue and SGLT-specific substrate, increased intracellular Ca2+ levels. 2+ We also found that SGLT-1 regulates glucagon secretion by increasing the concentration of SGLT-1 (Non-patent Document 1). + These results suggest that there may be a novel mechanism for controlling glucagon secretion that is independent of glucose metabolism, through the cotransport of SGLT-1 and the substrate (glucose). However, it was unclear whether SGLT-1 in pancreatic α cells affects endogenous glucagon secretion in vivo.

[0004] In addition, current diabetes medications have limited effectiveness, resulting in many inadequately treated cases. Effective and safe treatments for obesity and fatty liver have yet to be developed, and treatments currently rely on dietary therapy. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Suga et al. Mol Metab., 19:1-12, 2019 Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide novel diagnostic markers and novel therapeutic compositions for diabetes, obesity and / or fatty liver. [Means for solving the problem]

[0007] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that administering αMG to a living body increases blood glucagon levels in the body, and that this glucagon secretion-promoting effect of αMG can be utilized to diagnose and treat diabetes, obesity, and / or fatty liver, thereby arriving at the present invention.

[0008] That is, the present invention is as follows. [1] Glucagon in samples from living organisms administered αMG (Methyl α-D-Glucoside) data collection for the diagnosis of diabetes, obesity and / or fatty liver, including measuring the amount of How to get it. [2] The method according to [1], wherein the sample is plasma or serum. [3] In the above [1] or [2], the measurement is performed by enzyme immunoassay (EIA). The method described. [4] The method according to any one of [1] to [3], wherein the diagnosis is based on the criteria that the patient is diagnosed with diabetes, obesity, and / or fatty liver if the amount of glucagon in a sample derived from a living organism administered αMG is higher than the reference value, or if the amount of glucagon in a sample derived from a living organism administered αMG is higher than the amount of glucagon in a sample derived from a healthy individual administered αMG. [5] A diagnostic kit for diabetes, obesity, and / or fatty liver, comprising a reagent for measuring the amount of glucagon in a sample derived from a living body administered with αMG (Methyl α-D-Glucoside). ,kit. [6] The kit according to [5], wherein the reagent comprises an anti-glucagon antibody. [7] Contains αMG (Methyl α-D-Glucoside), which promotes weight loss, insulin resistance, and glucose tolerance. Agents for improving performance and / or increasing basal metabolism. [8] A pharmaceutical composition for treating diabetes, obesity and / or fatty liver, comprising the agent according to [7]. [9] The pharmaceutical composition described in [8], which is administered continuously. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a novel diagnostic marker and a novel therapeutic composition for diabetes, obesity, and / or fatty liver by utilizing the glucagon secretion-promoting action of αMG, an SGLT-specific substrate. [Brief explanation of the drawings]

[0010] [Figure 1] This shows the results of measuring blood glucagon levels after administration of αMG in mice fed a normal diet. Results after administration of PBS as a negative control are also shown, as well as the results after administration of αMG and an SGLT inhibitor (phlorizin). [Figure 2]These figures show the results of measuring blood glucagon levels (left), blood glucose levels (middle), and hepatic gluconeogenesis (right) after administration of αMG to mice fed a high-fat, high-sucrose diet. PBS was administered as a negative control. Furthermore, for the measurement of blood glucagon levels and blood glucose levels, αMG was administered at low or high concentrations. [Figure 3] The following photographs show the results of body weight and food intake (upper left), casual blood glucose levels (lower left), fasting blood glucose levels and postprandial blood glucose levels (upper right) as indicators of insulin resistance and glucose tolerance, and H&E staining of the liver as an indicator of fatty liver in mice fed a high-fat, high-sucrose diet following continuous administration of αMG. PBS was administered as a negative control. [Figure 4] The graphs show the results of measuring the respiratory quotient (top) and energy expenditure (bottom) following continuous administration of αMG in mice fed a high-fat, high-sucrose diet. PBS was administered as a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0011] One embodiment of the present invention is a method for producing a medicament for the treatment of inflammatory bowel disease derived from a living body administered with αMG (Methyl α-D-Glucoside). A data acquisition method for diagnosing diabetes, obesity and / or fatty liver, comprising measuring the amount of glucagon in a sample.

[0012] The data acquisition method of this embodiment is intended to aid in the diagnosis of diabetes, obesity and / or fatty liver.

[0013] The diabetes is not particularly limited, but examples include type 1 diabetes and type 2 diabetes, with type 2 diabetes being preferred. Obesity is not particularly limited, but for example, BMI (Body Mass Index) is an index of BMI of 25.0 or more. It may refer to a state in which the body fat percentage is 20% or more for men and 25% or more for women, using body fat percentage as an indicator. Fatty liver is a condition in which fatty tissue occupies more than 30% of the liver, although this is not limited to this condition. It may refer to either alcoholic fatty liver or non-alcoholic fatty liver.

[0014] The animal from which the specimen is derived is preferably a mammal such as a human or a mouse, and more preferably a human. The specimen is preferably a body fluid, more preferably blood, and particularly preferably plasma or serum. Collection, preparation and storage of body fluids can be carried out according to conventional methods.

[0015] In this embodiment, the living body to which αMG is administered is not particularly limited, but in the case of an adult human, it is a living body to which an agent containing αMG at a concentration of 0.55 to 1.1 M is administered in an amount of 66 to 132 g / 60 kg body weight. The time from administration of αMG to the living body to collection of the sample is preferably 5 to 10 minutes.

[0016] The method for measuring the amount of glucagon is not particularly limited, but for example, enzyme immunoassay (EIA) ) or enzyme-linked immunosorbent assay (ELISA), An example is the sandwich ELISA method described in Non-Patent Document 1.

[0017] In the present invention, the diagnostic criteria for determining whether a subject has diabetes, obesity, and / or fatty liver are not particularly limited, but may be, for example, that the subject has diabetes, obesity, and / or fatty liver when the amount of glucagon in a sample derived from a living organism administered αMG is higher than the reference value, or when the amount of glucagon in a sample derived from a living organism administered αMG is higher than the amount of glucagon in a sample derived from a healthy subject administered αMG. The reference value may be a cutoff value predetermined based on the measured values ​​of healthy individuals by a method known to those skilled in the art. For example, the cutoff value may be the mean value of the measured values ​​of healthy individuals + 3 × standard deviation (SD). In the present invention, a healthy subject means a subject who does not have a disease accompanied by diabetes, obesity and / or fatty liver.

[0018] Another embodiment of the present invention is a kit for diagnosing diabetes, obesity, and / or fatty liver, which comprises a reagent for measuring the amount of glucagon in a sample derived from a living body administered αMG (Methyl α-D-Glucoside).

[0019] In this embodiment, the reagent for measuring the amount of glucagon is not particularly limited, but if the kit is a kit using an immunological assay such as EIA or ELISA, the reagent for measuring the amount of glucagon preferably contains an anti-glucagon antibody. These immunological assays can be performed by conventional methods, and the reaction temperature, reaction time, types of reagents such as antibodies, labeling reagents, buffer solutions, and blocking solutions, concentrations of these reagents, and methods for detecting fluorescence or color development can all be determined in accordance with conventional methods.

[0020] The kit is a kit for diagnosing diabetes, obesity, and / or fatty liver, and the diagnostic criteria for determining whether or not a subject has diabetes, obesity, and / or fatty liver can be the same as those described in the data acquisition method of the present invention. The kit may further include an instruction manual or the like that describes a method for measuring the amount of glucagon included in the data acquisition method according to the present invention.

[0021] Another embodiment of the present invention is an agent for weight loss, improvement of insulin resistance, improvement of glucose tolerance and / or enhancement of basal metabolism, comprising αMG.

[0022] Weight loss refers to a decrease in body weight after administration of the agent according to this embodiment compared to before administration of the agent according to this embodiment. Improvement of insulin resistance means improving a state in which insulin is less effective. For example, it can be determined that insulin resistance has been improved by the fasting blood glucose level being lower after administration of the agent of this embodiment compared to before administration of the agent of this embodiment. Improvement of glucose tolerance means improving the decline in the ability to lower blood glucose levels that have increased due to meals, etc., to normal levels. For example, it can be determined that glucose tolerance has been improved if postprandial blood glucose levels are lower after administration of the agent of this embodiment compared to before administration of the agent of this embodiment. Increased basal metabolism can be determined, for example, by a decrease in respiratory quotient or an increase in energy consumption after administration of the agent of this embodiment compared to before administration of the agent of this embodiment, indicating that basal metabolism has been updated.

[0023] The αMG content in this preparation is not particularly limited as long as the preparation is capable of reducing body weight, improving insulin resistance, improving glucose tolerance and / or increasing basal metabolism, but when the preparation is a liquid, it is preferably 0.10 to 1.50M, more preferably 0.55 to 1.30M, more preferably 1.00 to 1.20M, and particularly preferably 1.1M.

[0024] The dosage of the agent to be administered to a living body is not particularly limited, as long as the agent is capable of reducing body weight, improving insulin resistance, improving glucose tolerance, and / or increasing basal metabolism, and is appropriately selected depending on the patient's age, sex, body weight, symptoms, type of cancer, etc. For example, for an adult, the dosage may be 10 to 200 g / 60 kg body weight per day, or 50 to 150 g / 60 kg body weight per day, with 132 g / 60 kg body weight being particularly preferred.

[0025] The method of administration of the present agent is not particularly limited as long as the agent can reduce body weight, improve insulin resistance, improve glucose tolerance and / or increase basal metabolism, and may be administered orally or parenterally.

[0026] The agent can be formulated into a desired dosage form depending on the administration method. For example, for oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. For parenteral administration, it can be formulated into injections, liquid preparations, suspensions, and the like.

[0027] This agent may be used in combination with other agents or medicines, or may be used in combination with other treatment methods.

[0028] Another embodiment of the present invention is a pharmaceutical composition for treating diabetes, obesity, and / or fatty liver, comprising an agent for weight loss, improvement of insulin resistance, improvement of glucose tolerance, and / or enhancement of basal metabolism, comprising the aforementioned αMG.

[0029] The content of αMG in this pharmaceutical composition, the dosage of this pharmaceutical composition to a living body, the administration method, the dosage form, etc., can be determined from the information described in the above section on pharmaceuticals. This pharmaceutical composition may be used in combination with other agents or medicines, or may be used in combination with other therapies.

[0030] The pharmaceutical composition may be administered continuously, and by continuing to administer it continuously over a long period of time, the therapeutic effect on diabetes, obesity and / or fatty liver can be enhanced. [Example]

[0031] The present invention will be described below using examples, but the present invention is not limited to these examples.

[0032] Example 1 [αMG, a specific substrate for SGLT, increases blood glucagon levels in mice via SGLT] C57BL / 6J mice (Charles River Laboratories) aged 14 to 17 weeks and fed a normal diet were intraperitoneally injected with 10 μl / g body weight of 1.1 M αMG (Fujifilm Wako Pure Chemical Industries, Ltd.) solution (n = 10 to 14). Mice fed a normal diet were given CE-2 (purchased from Claire) as food. Blood samples were collected from the mice, and blood glucagon concentrations were measured using an ELISA method known to those skilled in the art (Glucagon ELISA kit, Mercodia, Sweden). The results confirmed that blood glucagon concentrations were elevated in αMG-treated mice compared with control mice intraperitoneally injected with PBS. Furthermore, pretreatment with an SGLT inhibitor (Phlorizin, Cayman Chemical Company) using a method known to those skilled in the art inhibited αMG-induced endogenous glucagon secretion, reducing glucagon concentrations to levels comparable to those in control mice (PBS-treated mice) (Figure 1). These results demonstrate that αMG increases blood glucagon concentrations in mice via SGLT.

[0033] <Example 2> [αMG induces glucagon secretion more strongly in diabetic model mice] The present inventors have previously investigated the effects of 22-week-old C57BL / 6J mice (Charles River Laboratories) and 16-week-old db / db mice (Charles River Laboratories) on a high-fat, high-sucrose diet. We found that the expression level of SGLT-1 in pancreatic α-cells was increased in diabetic model mice compared to control mice (Non-Patent Document 1). The high-fat, high-sucrose diet-fed mice were fed a high-fat, high-sucrose diet (HFHSD) containing 20% ​​sucrose. The HFHSD contained protein (17.2%), fat (54.5%), and carbohydrates (28.3%) (Oriental Yeast Co., Ltd.). Based on the results described in Non-Patent Document 1, the present inventors investigated whether αMG-induced glucagon secretion was more potently induced in diabetic model mice. C57BL / 6J mice (n = 8-12) fed a high-fat, high-sucrose diet were given 0.55M (Low) and 1.1M 10 μl / g body weight of αMG solution (High) was intraperitoneally injected. After 10 minutes, blood was collected from the mice and The blood glucagon concentration was measured in the same manner as in Example 1, and the blood glucose level was measured over time (0, 15, 30, 60, 90) using a blood glucose meter (Sanwa Kagaku Kenkyusho Co., Ltd.) by a method known to those skilled in the art. , 120 minutes later). The results showed that αMG-administered mice showed dose-dependent increases in blood glucagon and blood glucose levels compared to control mice that received an intraperitoneal injection of PBS, and that blood glucagon and blood glucose levels were particularly markedly elevated in the high-dose αMG group (indicated as High αMG or αMG(high) in the figure) (Figure 2, left and center). Furthermore, control mice and αMG-treated mice (n = 3–5) were treated with 10 μl of 1.1 M αMG solution per gram of body weight. After 60 minutes, liver samples were collected from the mice, and mRNA expression of the hepatic gluconeogenic enzyme (G6pc) was measured by a method known to those skilled in the art. In the livers of mice, the expression of hepatic gluconeogenic enzyme (G6pc) mRNA was significantly increased compared to the livers of control mice. An increase was observed (right panel of Figure 2). Therefore, it was shown that evaluation of endogenous glucagon secretion in living organisms administered with αMG can be applied as a new diagnostic method for understanding the pathology of diabetes.

[0034] Example 3 [SGLT-specific substrate (αMG) is a potential therapeutic agent for diabetes, obesity, and fatty liver] Glucagon is known to have a transient blood glucose increase effect by promoting hepatic gluconeogenesis, while also suppressing appetite, increasing metabolism, and reducing body weight through lipolysis. Therefore, the inventors investigated whether promoting endogenous glucagon secretion by chronic administration of αMG to the body would have an ameliorative effect on diabetes, obesity, and fatty liver. C57BL / 6J mice (n = 8–9) fed a high-fat, high-sucrose diet were intraperitoneally injected with 10 μl / g body weight of 1.1 M αMG solution three times daily. Food intake, body weight, and random blood glucose levels were monitored over time using methods known to those skilled in the art. Compared with control mice given PBS intraperitoneally, the αMG-treated mice exhibited significant appetite suppression (reduced food intake), weight loss (upper left panel in Figure 3 ), and reduced random blood glucose levels (lower left panel in Figure 3 ). Furthermore, fasting and postprandial blood glucose levels were measured in each mouse using methods known to those skilled in the art. Compared with control mice given PBS intraperitoneally, fasting and postprandial blood glucose levels were reduced in the αMG-treated mice, confirming the improvement of insulin resistance and glucose tolerance due to αMG administration (upper right panel in Figure 3 ). Livers were also collected from each mouse and stained with hematoxylin and eosin (H&E) using methods known to those skilled in the art. As a result, the αMG-treated mice had reduced adipose tissue compared to the control mice that received an intraperitoneal injection of PBS, and the improvement in fatty liver was confirmed histologically (lower right panel of Figure 3). Furthermore, the effect of αMG administration on energy expenditure was evaluated using a respiratory metabolic monitoring system (Oxymax, Columbus Instruments) by methods known to those skilled in the art. As a result, the αMG-administered mice showed a decreased respiratory quotient and a decreased pulmonary function compared to the control mice intraperitoneally injected with PBS. The results showed an increase in basal metabolic rate and energy expenditure (Figure 4). In other words, it was demonstrated that αMG administration increases basal metabolic rate. These results confirm that the promotion of endogenous glucagon secretion by chronic administration of αMG to the body has an ameliorative effect on diabetes, obesity, and fatty liver.

Claims

1. Contains αMG (Methyl α-D-Glucoside) for weight loss and / or increased basal metabolism Agent.

2. A pharmaceutical composition for treating obesity and / or fatty liver, comprising the agent according to claim 1.

3. The pharmaceutical composition of claim 2, which is administered continuously.

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