JP2002148267A - Method for detecting insufficient working of insulin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple method for detecting insufficient working of insulin which imposes little burden upon a patient. SOLUTION: By this method, the insufficient working of insulin can be detected easily, by using the secular change of the measured concentration value of mannose in the blood of the patient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting insufficient insulin action required for diagnosing arteriosclerosis associated with diabetes or obesity.

[0002]

2. Description of the Related Art Insulin is the only hormone that promotes glucose utilization in the body, but when this hormone is deficient or no longer works effectively, the blood glucose level rises abnormally, causing diabetes. Become. Therefore, from the analysis of urinary glucose, fasting blood glucose, and fluctuation pattern of blood glucose due to glucose tolerance test, etc., the state of hyperglycemia is grasped, and diabetes is diagnosed by indirectly judging "insufficient insulin action". I've been.

[0003] This lack of insulin action is associated not only with "decrease in the absolute amount of insulin secretion" from the pancreas but also with a decrease in glucose utilization efficiency due to "insulin resistance". For example, mild hyperglycemia associated with obesity (IG
In T), the sensitivity to insulin is reduced, resulting in hypersecretion of insulin. This hyperinsulinemia is considered to be an important factor in arteriosclerosis associated with obesity. Thus, the lack of insulin action is closely related to obesity and diabetes.

[0004] Conventional methods for detecting insufficient insulin action have been performed simply by analyzing the fasting blood glucose level, the blood glucose level after oral glucose loading, and changes over time. More strictly, in order to ascertain the amount of insulin secretion and the decrease in sensitivity to insulin (insulin resistance), both the fasting blood glucose level and the blood insulin level are measured to determine the relationship between the two (the HOMA index). ) Is analyzed. Most strictly, glucose and insulin are loaded intravenously, and the dynamic relationship between the blood glucose level and the blood insulin level is analyzed.

On the other hand, regarding the clinical significance of the measurement of mannose concentration, (1) blood sugar level and mannose concentration are correlated (J.
Clin. Endocrinol. Metab., 62, p984-989 (1986), Sca
nd.J. Clin. Lab. Invest., 59, p607-612 (1999)),
(2) When the mannose / glucose concentration ratio is non-insulin-dependent diabetes (Clin. Chim. Acta, 251, p91-103 (199
6)), dyslipidemia, increased urinary protein excretion and BMI (Body M
ass Index (Body Mass Index) increased (Scand. J. Clin. Lab.
Invest., 59, p607-612 (1999)).

[0006]

However, the method for detecting insufficient insulin action using only the blood glucose level has a problem that the blood glucose level fluctuates greatly due to the consumption of glucose in the tissue. In addition, the method of detecting insufficient insulin action by combining the blood insulin level and the blood glucose level is more rigorous, but has a problem that the burden on the patient due to the insulin load is large. Even if it is a simple method using only fasting blood sampling, there is also a problem that measuring the insulin immunologically is more expensive than the biochemical measuring method. In addition, the single-point measurement of the mannose value to date could not show any clinical usefulness exceeding the conventional test method.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that blood mannose levels decrease in response to insulin. As a result, the present invention has been accomplished based on the fact that the insulin action can be easily grasped.

That is, the present invention provides: (1) a method for detecting insufficient insulin action, which comprises using a time-dependent change in the measured value of blood mannose concentration; (2) insufficient insulin action is caused by a decrease in suppression of glucose release. (3) The detection method according to (1) or (2), wherein a time-dependent change in the measured value of the blood mannose concentration during a glucose load is used. (4) Insulin The use of blood mannose concentrations over time to detect deficits.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The present invention is a method characterized by using a time-dependent change in the measured value of blood mannose concentration in the detection of insulin action deficiency, further using a time-dependent change in the measured value of blood mannose concentration during glucose loading. Is the way. The method for measuring blood mannose concentration used in the present invention may be any method that can be measured with clinically significant accuracy, and is not particularly limited. For example, gas chromatography (Pflugers Arch., 420, p367-375 (1992), Biol.
Mass Spectrom., 23, p590-595 (1994)) and liquid chromatography (Am. J. Clin. Pathol., 53, p793-802 (19)
70)) and a method of measuring with a general-purpose biochemical analyzer recently used in clinical examination sites (Japanese Patent Laid-Open No. 11-26689).
No. 6), but a biochemical analysis method suitable for routine measurement at a clinical site is preferable. As described above, the insulin action in the present invention means a decrease in blood glucose concentration due to insulin, and reflects insulin secretion (blood concentration) and insulin effectiveness (= glucose utilization efficiency). It is. Decreased glucose utilization efficiency is referred to as insulin resistance, which further reduces glucose utilization in peripheral tissues such as muscle and adipose tissue and decreases suppression of glucose release in liver and kidney. It is explained by both sides. As described later, glucose release is promoted by the degradation of glycogen in the liver, and mannose is released together with glucose. Since the inhibitory effect of insulin on hepatic glucose release is known, changes over time in blood mannose measurements can be used to test whether insulin resistance is due to reduced glucose utilization or reduced glucose release. And The method of testing for insufficient insulin action in organs such as the liver is also included in the present invention.

[0010] The blood used in the present invention is whole blood, plasma or serum, preferably plasma or serum. The measurement of blood mannose concentration in the present invention is preferably performed in a situation where a change in blood insulin concentration is expected.
For example, before and after a glucose tolerance test on an empty stomach, before and after a meal in daily life, before and after a drug load that causes insulin fluctuation, before and after exercise load, and before and after falling asleep, blood levels and changes thereof are measured, and diabetes and The analysis may be performed by a method convenient for diagnosing an insulin-resistant patient due to obesity and for grasping a more detailed condition.

[0011] The glucose load in the present invention may be any method as long as it can load efficiently, and is not particularly limited. There are a load test by daily meal, an oral load test (OGTT) of glucose and maltose, etc., which are frequently used as a diagnostic method of diabetes, a glucose load test by intravenous injection, and the like, but preferably OGTT, and OGTT.
Can be performed simultaneously with the diabetes test.

Furthermore, the measurement of blood mannose concentration in a glucose load is based on the fact that insulin is secreted in response to glucose, and mannose is considered to fluctuate due to the secreted insulin. It is appropriate to perform the measurement at a later point in time from the vicinity of the time to reach. As will be described later, the minimum value of the mannose concentration in the glucose tolerance test in normal rats is 20 minutes later, but in humans (healthy subjects), it gradually decreases after glucose loading and goes to the minimum value 120 minutes after glucose loading (before glucose loading). (About 50 to 80% of the value of the above). The optimal observation point for analyzing the variation of the mannose value varies depending on the species difference of the animal. In the case of humans, the measurement is preferably performed 10 minutes to 3 hours after loading, more preferably 30 minutes to 2 hours after loading.

[0013]

EXAMPLES The present invention will be described more specifically with reference to Experimental Examples and Examples, but the present invention is not limited to these.

Example 1 Insulin tolerance test Cannulation Wistar rats (♂, 240-270) as normal rats
g, Clea Japan) under anesthesia, an operation was performed in which a cannula was placed in the right atrium in order to continuously collect blood from the same individual without stress. Specifically, under Nembutal anesthesia, the rat was held in a dorsal position on a holding table, the periphery of the upper part of the right jugular vein was shaved, and the skin where the jugular vein was beating was incised. The connective tissue was bluntly dissected using forceps to expose the jugular vein, and a suture (No. 2) was applied to the rostral and caudal sides of the jugular vein, and the rostral thread was tightly tied. The tail thread was left loose. The blood flow from the heart side was stopped with a clip, a small cut was made in the blood vessel with a Wecker scissor, and a cannula filled with a physiological saline solution containing 10 U / mL of heparin (heparin supplemented diet) was inserted. The pectoral muscle was sewn with the thread previously hung on the stopper of the cannula, and the cannula was fixed. Further, a small incision was made on the skin on the back of the neck, forceps were inserted through the cut, the cannula was pulled out, and passed through the center hole of the cannula case body.
The case was secured to the dorsal skin with sutures (No. 4). An appropriate amount of a heparinized diet was injected so that blood did not remain in the cannula, and the filling solution was filled in the cannula.

Administration and blood collection 0.25 U / mL insulin was administered from the cannula, 5 minutes before administration, immediately before administration, and 5, 10, 20, and 20 minutes after administration.
80 μL of blood was collected from the cannula for a total of 8 times at 30, 45, and 60 minutes.

Blood Glucose Measurement Using 20 μL of the collected blood, the blood glucose level was measured by Antosense II (Bayer Sankyo).

Measurement of Mannose To 50 μL of the collected whole blood, 50 μL of heparinized diet was added, and the mixture was centrifuged at 7,500 × g for 15 minutes. To 70 μL of the supernatant, 10 μL of 1.5 M perchloric acid was added. After standing for 10 minutes under cooling, the protein was removed by centrifugation at 7,500 × g for 15 minutes. The concentration of mannose in the supernatant was
Using a liquid chromatography analyzer (HPLC) in which a column for sugar analysis was set, quantification was performed under the following conditions.

HPLC conditions Column: Finepak gel SA-121 Column temperature: 80 ° C. Column flow rate: 0.5 mL / min Mobile phase: Buffer A (0.25 M boric acid solution), Buffer B
(0.60 M boric acid solution) Mobile phase composition: A / B = 8/2 (0 min) → 6/4 (17
Min) Linear gradient Reaction reagent: 20% acetonitrile aqueous solution containing 100 mM boric acid, 50 mM guanidine hydrochloride and 0.5 mM sodium periodate Reaction temperature: 160 ° C. Reagent flow rate: 0.5 mL / min Fluorescence detection: excitation wavelength 310 nm, detection Wavelength 415nm

Experimental Results The results are shown in FIG. 1 and Table 1. This suggests that blood mannose was reduced in response to insulin.

Table 1. Fluctuation in blood mannose level during inulin loading (μM) Before loading After loading Decrease (0 min) (20 min) (pre-loading-post-loading) Normal rats 99.9 67.8 32.1

Example 2 Glucose Tolerance Test In place of the insulin of Example 1, 1 mL of a 50% glucose solution was replaced with a normal Wistar rat and a GK rat as a type 2 diabetes model (♂, 180 to 200 g, CLEA Japan).
, And the same test was performed. These results are shown in FIG.
(Normal Wistar rats) and FIG. 3 (diabetic GK rats). In both cases, the blood glucose level peaked after 5 minutes, but the mannose value was about 20 minutes later, and was 20 to 3 minutes.
The lowest value was reached after 0 minutes. Normally, insulin secretion in normal rats is almost parallel to glucose, and thus tends to be delayed by 5 to 10 minutes from the time of insulin loading by the time required for insulin secretion. further,
In order to compare the changes in the blood mannose concentration between normal rats and diabetic rats, the amount of decrease in the mannose value at 20 minutes after glucose loading and the ratio of the decrease to the pre-loading value (% ) Are shown in Table 2. The ratio (%) of the decrease amount of the mannose value to the pre-load value in the diabetic rat was remarkably reduced to about 1/3 and 1/4 of the normal rat, respectively. This may reflect a lack of insulin action in diabetic rats.

Table 2. Fluctuations in blood mannose level during glucose loading (μM) Before loading After loading Reduction (0 min) (20 min) Percentage of reduction relative to pre-load (%) Normal rats 96.0 61.9 34.1 35.5 GK rats (diabetes) 164.5 150.2 14.3 8.7

Example 3 Glucose Tolerance Test 2 Cannulation Wistar rats (♂, 7 weeks old, CLEA Japan) as normal rats and GK rats of type 2 diabetes model (の, 9)
(Kure Nihon, Japan) was cannulated in the same manner as in Example 1.

Administration and blood collection 1 mL of 50% glucose solution (2 g / k
g) immediately before administration (0 minutes), 2, 5, 7,
150 μL for 10, 15, 30 minutes and a total of 7 times
Blood was drawn from the cannula.

Blood glucose measurement was measured in the same manner as in Example 1. Mannose measurement Heparin 10 U / mL fresh food 9 in 90 μL of collected whole blood 9
0 μL was added, and the mixture was centrifuged at 7,500 × g for 15 minutes. 1.5 μL of 1.5M perchloric acid was added to 120 μL of the supernatant.
Add 10 μL, leave under ice-cooling for 10 minutes,
For 15 minutes to remove proteins. The concentration of mannose in the supernatant was determined using a liquid chromatography analyzer (H
PLC) under the same conditions as in Example 1.

Insulin Measurement Whole blood was centrifuged at 7,500 × g for 10 minutes, and 5 μL of the supernatant was used to measure the insulin concentration using an insulin measurement kit by ELISA (Morinaga Biochemical Laboratory).

The results are shown in FIG. 4 (normal Wistar rats) and FIG. 5 (diabetic GK rats). Blood sugar level
Both peaked after 2 minutes. In normal rats,
A large amount of insulin was released in response to blood sugar.
In rats, insulin was present but changed very little. Reflecting this, the mannose value of the normal rat gradually decreased, and decreased by 34 μM after 30 minutes. On the other hand, in GK rats, the concentration was only slightly reduced by 1.7 μM. These values are summarized in Table 3 to further clarify the relationship between insulin and mannose. These results revealed that mannose levels fluctuated not with blood glucose but with insulin.

Table 3. Fluctuations in blood insulin and mannose levels during glucose loading Insulin level (pg / mL) Mannose level (μM) Before loading After loading Before loading After loading Decrease (0 min) (2 min) (30 min) (0 Min) (30 min) Normal rat 1310 11000 6200 91.2 57.2 34.0 GK rat 857 1760 1143 126.7 125.0 1.7

Example 4 Glucose Tolerance Test in Humans A 75 g oral glucose tolerance test (7
5g OGTT) was performed to track changes in plasma glucose and mannose concentrations, and the results are shown in FIG. As is clear from this figure, the mannose value in humans gradually decreased after the sugar load and reached the minimum value after 120 minutes. As mentioned above, the minimum value in rats is 20
Since it is minutes later, it can be seen that the optimal observation point for analyzing the fluctuation of the mannose value changes depending on the species difference of the animal.

Example 5 Perfusion experiment in rat liver (mannose release from liver) Preparation of rat perfused liver (Miller type) Wistar rat (♂, 310-400 g, CLEA Japan)
Under pentobarbital anesthesia (0.1 mL / 100 g)
(Body weight), and an operation for perfused liver separation was performed. That is,
Loosely thread two threads on the portal vein (one with the hepatic artery and the other on the small intestine, portal vein only) and a cannula (diameter 1.
1mm), and fix it tightly with the small intestinal thread.
0.6 mL of heparin diluted to U / mL was injected with a syringe. The perfusate from the perfusion device was pumped into the liver through the cannula at a pump flow rate of 30 mL / min, and the peritoneal vena cava was cut immediately below the right kidney. The cannula was tied with a thread on the liver side and fixed, and at the same time the hepatic artery was ligated. At this stage, the blood in the liver was almost completely removed, and the liver became pale yellow. The chest was opened and the descending vena cava was cut. Immediately above the right kidney, the peritoneal vena cava was ligated with a thread, and the perfusate was allowed to flow in from the portal vein side, pass through the liver, and flow out of the descending vena cava. Finally, it was separated from other organs without damaging the liver.

Hepatic perfusion and drug administration A perfusate previously bubbled with a mixed gas of 95% oxygen and 5% carbon dioxide is inhaled using a peristaltic pump, and small bubbles are removed with an air trap. While being warmed to 37 ° C., into the liver via a portal cannula. The liver was placed on a mesh and covered with a plastic case to keep humidity and temperature. The upper part of the liver was irradiated with light (100 W) so that the temperature became constant at 37 ° C. In the separated liver,
The perfusate was allowed to stand on the mesh for about 15 minutes while sending the perfusate at a flow rate of 30 mL / min, and then the perfusate was collected six times every minute for 6 minutes. At the end of 2 minutes from the beginning of this collection, the valve of the perfusion line was switched to 0.2 μg
The perfusate was flushed with epinephrine at a concentration of / mL.

The perfusate was 5 mM glucose, 11
8 mM sodium chloride, 4.7 mM potassium chloride, 2.
A glucose-added Krebs-Ringer solution consisting of 5 mM calcium chloride, 1.2 mM monobasic dihydrogen phosphate, 1.2 mM magnesium sulfate, 24.2 mM sodium bicarbonate (pH 7.4) was used.

Relationship between Glucose Release and Mannose Release Glucose and mannose concentrations in the collected perfusate were measured, and the results of comparing the kinetics of both with the release of hepatic glucose are shown in FIG. Epinephrine is a drug that promotes glycogen breakdown in the liver and promotes glucose release,
Reflux of epinephrine also enhanced mannose release almost parallel to glucose release. From this, fluctuations in mannose levels in blood reflect the situation of glucose release from organs such as the liver, and therefore, a decrease in mannose levels due to insulin reflects a decrease in glucose release from organs such as the liver. It is considered something.

As described above, the degree of insulin action deficiency can be easily ascertained by measuring the blood mannose level and analyzing the fluctuation over time, and particularly, the level of glucose release from organs such as the liver due to insulin. The state of inhibition can be grasped, for example, by using in combination with other markers such as a blood glucose-related marker, the disease state associated with diabetes or obesity, for example, insufficient insulin action in diabetes decreases the ability to inhibit glucose release, or decreases the ability to utilize glucose. It is expected that more detailed discrimination as to whether or not the decrease will be possible, and that it will be a marker for knowing the effect of insulin on suppressing hepatic glucose release.

[Brief description of the drawings]

FIG. 1 shows the results of an insulin tolerance test on normal rats.

FIG. 2 shows the results of a glucose tolerance test on normal rats.

FIG. 3 shows the results of a glucose tolerance test on diabetic rats.

FIG. 4 shows the results of a glucose tolerance test on normal rats to which the measured values of insulin were added.

FIG. 5 shows the results of a glucose tolerance test on diabetic rats to which the measured values of insulin were added.

FIG. 6: Dynamics of glucose and mannose in a healthy person (human) in a glucose tolerance test

FIG. 7: Dynamics of glucose and mannose in rat perfused liver

Claims (4)

[Claims] 1. A method for detecting insulin action deficiency, comprising using a time-dependent change in a measured value of a blood mannose concentration. 2. The insulin deficiency is an insulin deficiency caused by a decrease in suppression of glucose release.
The detection method described 3. The detection method according to claim 1, wherein a time-dependent change in the measured value of blood mannose concentration during glucose load is used. 4. Use of time-dependent measurements of blood mannose concentration to detect insulin deficiency.