JP5044786B2 - Method for measuring metabolic rate of 2-deoxyglucose - Google Patents

Description

  The present invention relates to a method for measuring the metabolic rate of 2-deoxyglucose (2DG), and more particularly to a method for estimating the glucose metabolic rate in body tissues or cells using non-radioactive 2DG.

Lifestyle-related diseases such as obesity and diabetes have become a major social problem, and the development of therapeutic methods and drugs is urgent. Techniques for measuring glucose metabolism rates in various organs and tissues using experimental animals are used not only for elucidating the causes of obesity and diabetes, but also for determining the effects of anti-diabetic and metabolic improving effects of new drugs.
Similarly to glucose, 2-deoxyglucose (2DG) is taken into cells by a glucose transporter on the cell membrane and metabolized to 2-deoxyglucose-6-phosphate (2DG-6-P) by hexokinase (HK). However, it is not metabolized thereafter and accumulates in the cell. Therefore, the amount of 2DG-6-P accumulated in the cell is the best indicator of the glucose uptake rate (metabolic rate). Therefore, the measurement method using 2DG labeled with a radioactive compound is the glucose metabolism in each tissue in the body. It is generally used as a method for measuring speed (Non-patent Document 1).
On the other hand, a method for selectively quantifying 2DG-6-P accumulated in cells using non-radioactive 2DG using an enzyme has been devised (Non-patent Document 2). However, the methods that have been published so far were developed mainly to measure the glucose metabolism rate of a single nerve cell, so that they are commonly used in obesity and diabetes research fields, such as skeletal muscle and adipose tissue that are rich in glucose metabolites. It was difficult to measure the rate of glucose metabolism in typical tissues. In addition, a method for easily calculating a glucose metabolism rate by administering a large amount of non-radioactive 2DG to an animal has been reported (Non-Patent Document 3), but this method has a concern about an influence on glucose metabolism itself. Therefore, the measurement method using non-radioactive 2DG is hardly used despite its convenience.

Sokoloff, LM, et al., J Neurochem 28: 897-916, 1977. Akabayashi, A et al., Biomedical Research 10: 173-177, 1989. Ueyama, A, et al., Biol Signals Recept 9: 267-274, 2000.

In order to estimate the glucose metabolism rate in body tissues and cells using non-radioactive 2DG, it is necessary to metabolize endogenous glucose metabolite (G6P) to the extent that it does not affect the measurement of 2DG. Conventionally, no suitable method has been proposed. On the other hand, when a large amount of 2DG is administered to an animal in order to reduce the influence on the measurement of glucose metabolite (G6P) (Non-patent Document 3), there is a problem that the large amount of 2DG disturbs glucose metabolism in the animal body.
Therefore, the present invention provides a method for estimating the glucose metabolic rate in each tissue or cell in the body using a small amount of non-radioactive 2DG.

  In order to measure the rate of glucose metabolism using a small amount of 2DG, the present inventors have a low concentration of endogenous glucose metabolites (glucose-6-phosphate (G-6-P)) that affect the measurement. Glucose-6-phosphate dehydrogenase (G6PDH) and nicotinamide adenine dinucleotide (NAD) are removed to increase 2-DG metabolite 2-deoxyglucose-6-phosphate (2DG-6-P). To solve the problems of conventional methods by measuring the amount of reduced nicotinamide adenine dinucleotide phosphate (NADPH) produced by treatment with concentrations of G6PDH and nicotinamide adenine dinucleotide phosphate (NADP) The present invention has been completed. The amount of NADPH may be measured after amplification.

That is, the present invention is a method for measuring the metabolic rate of 2-deoxyglucose (2DG) comprising the following steps.
(1) G6PDH and a concentration of 50 to 300 μM in a tissue collected from a non-human animal to which 2DG has been administered or a cultured cell of a mammal to which 2DG has been administered so that the concentration is 0.5 to 1 u / ml. Adding NAD and further treating with acid (step 3),
(2) Further, G6PDH is added so that the concentration is 30 u / ml or more, NADP is added so that the concentration is 5 to 50 μM, and further, the step of treatment with alkali is performed (step 4), and (3) and (2). Measuring the amount of NADPH produced (step 6)

Furthermore, the present invention is a method for measuring the metabolic rate of 2-deoxyglucose (2DG) comprising the following steps.
(1) administering 2DG into a vein of a non-human mammal (step 1);
(2) collecting a tissue from the mammal, homogenizing the collected tissue, performing a heat treatment, and collecting the extract (step 2);
(3) A step of adding G6PDH so that the concentration becomes 0.5 to 1 u / ml and NAD so that the concentration becomes 50 to 300 μM and further treating with an acid (step 3),
(4) A step of adding G6PDH so that the concentration becomes 30 u / ml or more and NADP so that the concentration becomes 5 to 50 μM and further treating with alkali (Step 4),
(5) Further, a step of adding a mixed solution containing glycol-6-phosphate, glutathione reductase, G6PDH, glutathione, and 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) (step 5), and ( 6) Measuring the amount of DTNB reaction (step 6)

The method may further include the following steps.
(7) collecting blood from the non-human mammal;
(8) G6PDH to a concentration of 0.5 to 1 u / ml, NAD to a concentration of 50 to 300 μM, hexokinase to a concentration of 1 to 10 u / ml, Adding ATP and MgCl ₂ to ₁ to 3 mM and further treating with acid (step 3),
(9) A step of adding G6PDH so that the concentration becomes 30 u / ml or more and NADP so that the concentration becomes 5 to 50 μM, and further treating with alkali (step 4),
(10) Further, a step of adding a mixed solution containing glycol-6-phosphate, glutathione reductase, G6PDH, glutathione, and 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) (step 5), and ( 11) A step of measuring the reaction amount of DTNB (step 6), and (12) From the obtained reaction amount of each DTNB, the disappearance rate of 2DG in blood and 2-deoxyglucose-6-phosphate (2DG in tissue) -6-P) calculating the amount and calculating the glucose metabolism rate (step 6)

Furthermore, the present invention is a method for measuring the metabolic rate of 2-deoxyglucose (2DG) comprising the following steps.
(1) adding 2DG to a culture solution of mammalian cells (step 1),
(2) Further, G6PDH is added to the cell extract to a concentration of 0.5 to 1 u / ml, and NAD is added to a concentration of 50 to 300 μM, followed by further treatment with acid (step 3).
(3) Further, a step of adding G6PDH to a concentration of 30 u / ml or more and NADP to a concentration of 5 to 50 μM, and further treating with alkali (step 4)
(4) Further, a step of adding a mixed solution containing glycol-6-phosphate, glutathione reductase, G6PDH, glutathione, and 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) (step 5), and ( 5) Measuring the amount of DTNB reaction (step 6)

The method of the present invention can easily and easily measure glucose metabolism rate even in organs containing a large amount of glucose metabolites such as skeletal muscle and adipose tissue. This method can also be performed using cultured cells.
Since the method of the present invention uses only a small amount of 2DG, it does not cause the problem of disturbing glucose metabolism in the animal body. On the other hand, endogenous glucose metabolites (G-6-P), nicotinamide adenine dinucleotide (NAD, NADH), and nicotinamide adenine dinucleotide phosphate (NADP, NADPH) are degraded before measurement. Does not affect the measurement of NADPH produced in proportion to the amount of 2DG-6-P.
Furthermore, since the method of the present invention comprises only a step of adding a reagent to the object to be measured, it can be automated by using a single 96- or 384-well plate and using a robot.

In vivo, glucose is converted into glucose-6-phosphate (G-6-P) by the action of hexokinase and glucokinase in muscle and adipose tissue, and then metabolized by actions of glycogen synthase and the like. Diabetes is due to this impaired glucose metabolism due to lack of insulin.
In the method of the present invention, 2-deoxyglucose (2DG) is administered instead of glucose, and the accumulated amount of 2-deoxyglucose-6-phosphate (2DG-6-P), which is a metabolite of 2DG, is measured. . In order to know the amount of 2DG-6-P, glucose-6-phosphate dehydrogenase (G6PDH) was added, and the reaction product of 2DG-6-P and G6PDH, nicotinamide adenine dinucleotide phosphate (NADPH) Measure the amount of Thereby, the metabolic rate of 2DG in each tissue or cell in the body can be known, and this corresponds to the glucose metabolic rate, so that the glucose metabolic rate in each tissue or cell in the body can be estimated.

Specifically, the present invention includes the following three methods.
(A) Administer 2DG to non-human animals, collect tissue after a certain period of time, decompose G-6-P in the tissue extract with low concentrations of G6PDH and NAD, and then add high concentrations of G6PDH and NADP Measure the amount of NADPH produced. This amount of NADPH corresponds to the amount of 2DG-6-P accumulated in cells in the tissue.
(B) In the method of (a), blood is further collected after a certain time, and the disappearance rate of 2DG is measured by hexokinase and the method of (a). From the disappearance rate of 2DG and the amount of 2DG-6-P, Calculate the glucose metabolism rate in the tissue.
(C) 2DG is administered to a cell culture solution of a mammal, and cultured cells are collected after a certain time. The amount of 2DG-6-P in the cell extract is measured by the same method as in (a).

Hereinafter, the method for measuring the metabolic rate of 2-deoxyglucose (2DG) of the present invention will be described step by step.
(1) Step 1: First, 2DG is administered to a non-human animal. As the non-human animal, any mammal may be used, but usually a mouse or a rat is used. Usually, 2DG is injected into a vein or artery. The dose of 2DG may be an amount that does not disturb glucose metabolism in the animal body and is sufficient for the measurement of the present invention, and 2 to 10 μmol for mice and 10 to 30 μmol for rats with a body weight of 300 g are appropriate amounts. Conceivable.
Further, cultured cells may be used, and the cells may be human or non-human as long as they are mammals. The appropriate amount of 2DG administered to the cell culture medium is generally considered to be 0.5 to 1.5 mM.

(2) Step 2: In order to measure the amount of 2DG-6-P accumulated in the tissue after a certain time after administration of 2DG, the tissue is collected in the case of a non-human animal. In addition, blood is collected when calculating the 2DG metabolic rate of the tissue. The collected tissue is homogenized by adding a buffer solution, subjected to heat treatment, and the extract is collected by centrifugation. The heat treatment may be performed under any conditions that denature proteins, and is usually performed at 95 ° C. for 15 minutes. The blood is centrifuged to collect serum or plasma, heat-treated, centrifuged again, and the supernatant collected. In the case of cultured cells, the cells are homogenized with a buffer, then heat-treated and centrifuged, and the supernatant (extract) is collected.

(3) Step 3: Nicotinamide adenine dinucleotide (NAD) and a low concentration of glucose-6-phosphate dehydrogenase (G6PDH) are added to the extract of tissue or cultured cells to which 2DG has been administered.
These are added to decompose in advance glucose or glucose metabolite (G-6-P) already present in the living body or cells. NAD is present in a small amount in living organisms or cells, but is added so that the final concentration (that is, the concentration at the time of reaction) is 50 to 300 μM, preferably 75 to 200 μM. G6PDH is added so that the final concentration is 0.5 to 1 u / ml. Furthermore, it is preferable to add a buffer, preferably 5 to 15 mM Tris-HCl buffer so that the pH is 7 to 9.
When these are added, and preferably allowed to stand for 5 hours, nicotinamide adenine dinucleotide (NADH) is produced as shown in the following formula, and this is treated with an acid. At this time, the pH is preferably 3 or less. As the acid, hydrochloric acid or the like is used, and the concentration after the addition is suitably 40 to 100 mN. After treatment with acid, neutralize with alkali. As the alkali, NaOH can be used, and its concentration is suitably 40 mM to 100 mM.

By this series of operations, the influence of endogenous glucose metabolite (G-6-P), NADPH and NADH can be eliminated when NADPH is measured later. Therefore, it is possible to quantitatively measure NADPH at a low concentration, and thus a small amount of 2DG can be administered.
In addition, G6PDH added here functions only in the degradation of glucose metabolite (G-6-P) due to its low concentration, and the degradation of 2-deoxyglucose-6-phosphate (2DG-6-P) in the latter stage Does not affect. This is due to the low affinity of 2DG-6-P for G6PDH compared to G-6-P.
Furthermore, by using NAD, the reactivity of G6PDH, which degrades 2DG-6-P at the latter stage, is higher than when using NADP, and a smaller amount of 2DG-6-P can be measured. Although NADP can be used, NAD is suitable for measuring a small amount of 2DG-6-P.
G6PDH usually uses an enzyme derived from Leuconostoc meseteroides that reacts not only with NADP but also with NAD and has an activity that is stable for a long time, but other G6PDH that reacts with NAD and has an activity that is stable for a long time may be used.

そこで、2DG-6-Pを分解するためにNADPと高濃度のG6PDHが加えられる。NADPは最終濃度（即ち、反応時の濃度）が５〜５０μＭ、好ましくは５〜１５μＭとなるように加えられ、G6PDHは、最終濃度が３０u/ml以上となるように加えられる。この上限は、特に限定はないが、６０u/mlであれば十分である。G6PDHは、活性が長時間安定であるLeuconostoc
meseteroides由来の酵素が通常用いられる。更に、ｐHが７〜９となるように緩衝液、好ましくは５〜１５ｍMのTris-HCl緩衝液を加えることが好ましい。その後３０分以上静置することが好ましい。 (4) Step 4: Nicotinamide adenine dinucleotide phosphate (NADP) and a high concentration of G6PDH are added to the object to be treated obtained in the previous step.
When 2DG is administered to a living body or a cell, it is taken into the cell by the same glucose transporter as glucose and becomes 2-deoxyglucose-6-phosphate (2DG-6-P). However, since it does not react with intracellular metabolic enzymes, it is not metabolized and accumulates in the cell.

Therefore, NADP and high concentration of G6PDH are added to decompose 2DG-6-P. NADP is added so that the final concentration (that is, the concentration at the time of reaction) is 5 to 50 μM, preferably 5 to 15 μM, and G6PDH is added so that the final concentration is 30 u / ml or more. The upper limit is not particularly limited, but 60 u / ml is sufficient. G6PDH is a Leuconostoc whose activity is stable for a long time
Enzymes from meseteroides are usually used. Furthermore, it is preferable to add a buffer, preferably 5 to 15 mM Tris-HCl buffer so that the pH is 7 to 9. It is preferable to leave still for 30 minutes after that.

なお、血液中の２ＤＧの消失速度を測定するためには、ヘキソキナーゼ（ＨＫ）、ＡＴＰ及びＭｇＣｌ_２を作用させて２ＤＧを全量２ＤＧ−６−Ｐに変換した後、同様に測定するが、この操作は前段階で行なわれる。用いるヘキソキナーゼの量は３〜１０ｕ／ｍｌ、ＡＴＰ及びＭｇＣｌ_２の濃度はそれぞれ１〜３ｍＭである。
残存するNADPはアルカリで分解する。ｐH１１以上として５０℃以上で処理することが好ましい。アルカリとしては、NaOHを用いることができ、その濃度は40mM〜100mＭが適当である。 By this operation, nicotinamide adenine dinucleotide phosphate (NADPH) is generated as shown in the following formula. This amount of NADPH corresponds to the amount of accumulated 2DG-6-P.
It is also possible to use NAD instead of NADP. However, since the production rate of NADH is slow, it is necessary to lengthen the reaction time. In addition, in order to measure a small amount of NADH, it is necessary to use an enzyme cycling method described later in (5) (iii).

In addition, in order to measure the disappearance rate of 2DG in blood, hexokinase (HK), ATP and MgCl ₂ are allowed to act to convert 2DG into a total amount of 2DG-6-P and then measured in the same manner. Is performed in the previous stage. The amount of hexokinase used is 3 to 10 u / ml, and the concentrations of ATP and MgCl ₂ are 1 to 3 mM, respectively.
The remaining NADP decomposes with alkali. It is preferable to treat at 50 ° C. or higher as pH 11 or higher. As the alkali, NaOH can be used, and its concentration is suitably 40 mM to 100 mM.

(5) Step 5: The produced NADPH (or NADH) may be measured as it is because G-6-P, endogenous NADH and NADPH have already been removed, but the added 2DG is small. Therefore, since the amount of NADPH (or NADH) produced is small, it is preferable to measure after amplification.
As a method for amplifying NADPH, any known method may be used, and examples thereof include the following methods.

(i) Enzyme cycling method (Anal Chem 99: 297-303, 1979):
A solution containing NADPH was added to glucose-6-phosphate (G-6-P) (concentration of 10 mM or more during reaction), glutathione reductase (GR) (concentration of 1 to 10 u / ml during reaction), G6PDH (during reaction) Concentration of 20 to 100 u / ml), glutathione (GSSG) (concentration at reaction 5 to 20 μM), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) (concentration at reaction 1 to 5 mM) By treating with a liquid at a temperature from room temperature to 37 ° C., reaction amplification and color development can be performed simultaneously. Mix 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) just before use. The mixing ratio is not particularly limited, but the concentration during the reaction is adjusted to 1 to 5 mM. G6PDH uses an enzyme derived from Leuconostoc meseteroides, which normally has a stable activity for a long time and is not easily inhibited by anions and the like.

This reaction is shown by the following formula. The reaction amount of DTNB can be measured by the absorbance of the produced 5-mercapto-2-nitrobenzoic acid at 420 nm. The reaction amount of DTNB (that is, the absorbance) corresponds to the amount of NADPH and the amount of 2DG-6-P.

(ii) Alternative method for enzyme cycling (Anal Chem 128: 186-190, 1983):
G6PDH, GDH (glutamate dehydrogenase), NH ₄ ⁺ (ammonium ion), α-keto-glutarate, and G-6-P are added to the NADPH-containing solution. Utilizing a reaction in which 6-P-gluconate is produced in a large amount according to the amount of NADPH. The amount of 6-P-gluconate produced is converted back to NADPH by 6-P-gluconate dehydrogenase, and the amount produced can be known by measuring the absorbance at 340 nm.

(iii) Alternative method of enzyme cycling (Anal Chem 53: 86-97, 1973.):
In the reaction represented by Chemical Formula 3 (Chemical Formula 3), NAD is used instead of NADP, and a small amount of produced NADH is measured. Add ADH (alcohol dehydrogenase), ethanol, oxaloacetate, MDH (malate dehydrogenase) to the solution containing NADH, and malate (malate) according to the amount of NAD based on the reaction of the following formula. ) Is used in a reaction that produces a large amount. The produced malic acid is converted back to NADH by MDH, and the amount of production can be known by measuring the absorbance at 340 nm. GOT (glutamate oxaloacetate transaminase) and glutamate are added to promote the reaction to reconvert to NADH.

(iv) Alternative method of enzyme cycling (Nature 193: 454-456, 1962):
Add G6PDH, ADH (alcohol dehydrogenase), glucose-6-P (G-6-P), PMS (oxidized methylphenazenium methylsulfate), DCPI (dichlorophenolindophenol) to the solution containing NADPH, Based on the reaction of the following formula, a reaction in which a large amount of leucoDCPI is generated according to the amount of NADPH is used. Since DCPI has an absorbance at 600 nm, the amount of leucoDCP produced can be determined from the decrease in absorbance at 600 nm. There is also a modification using ethylphenazinium ethyl sulfate instead of PMS and a modification using thiazolyl blue instead of DCPI.

  Among these methods, method (ii) is the most common method for quantifying trace amounts of NADPH, but in (ii) and (iii), amplification reaction by color cycling and color reaction must be performed separately. Moreover, a long reaction time is required to measure a small amount of NADPH and NADH. In addition, the method (iv) can carry out the proliferation reaction and the color development reaction at the same time, but is not stable because the reaction rate changes with time. On the other hand, in the method (i), the enzyme cycling and the color reaction by DTNB can be performed at the same time, so the reaction time is greatly shortened and all reactions can be performed in one 96-well plate, and the reaction is also stable. ing. Therefore, the method (i) is preferable as the amplification method of the present invention.

(6) Step 6: The amount of NADPH (or NADH) is measured using the method described in (5). Any method may be used for measuring the amount of NADPH and NADH. By measuring this amount, the accumulated amount of 2DG-6-P can be known, and the change in the accumulated amount of 2DG-6-P is an indicator of the metabolic rate of 2DG. The obtained 2DG metabolic rate is used as an estimate of glucose metabolic rate.
Alternatively, the glucose metabolic rate constant (Ki, min ⁻¹ ) may be calculated from the accumulated amount of 2DG-6-P and the 2DG amount in blood based on Sokoloff's formula (Equation 1). Since 2DG-6-P accumulated in the tissue depends on the blood 2DG amount and blood glucose concentration, the glucose metabolism rate constant (Ki) that is independent of the 2DG dose, blood glucose concentration, and circulating blood volume is calculated. It is desirable to do. In order to measure the glucose metabolism rate constant, it is necessary to calculate the 2DG elimination rate (Kp) in the blood as in the Sokoloff equation. On the other hand, the rate of 2DG disappearance in the blood changes depending on the glucose utilization rate throughout the body, but at the same time, it is also affected by the circulating blood volume and cardiac output, so it can be used as an indicator of the glucose utilization rate throughout the body. Can not.

The solution used in the method described above can be used as a kit for measuring the metabolic rate of 2DG. The kit consists of the following five solutions.
(1) Solution 1: a buffer solution containing G6PDH and NAD, wherein the NAD content is 50 to 600 nmol per unit of G6PDH This solution 1 is used in Step 3 above. A characteristic is that the concentration of G6PDH during the reaction is low, and this mixing ratio makes it possible to reduce the concentration of G6PDH during the reaction.
(2) Solution 2: hexokinase (HK), a buffer solution containing ATP and MgCl _2, the content of ATP is 0.3μmol or more per HK1unit, the content of MgCl ₂ is a 0.1~0.5μmol per HK1unit Buffer Solution 2 is used to measure the disappearance rate of 2DG in the blood in Step 3 above, and is added to Solution 1 for use.
(3) Solution 3: A buffer solution containing G6PDH and NADP, wherein the NADP content is 0.1 to 0.5 nmol per G6PDH unit. This solution 3 is used in Step 4 above. A characteristic is that the concentration of G6PDH during the reaction is high, and this mixing ratio makes it possible to increase the concentration of G6PDH during the reaction.
(4) Solution 4: A buffer solution containing G6PDH, glycol-6-phosphate, glutathione and glutathione reductase, wherein the content of glycol-6-phosphate is 0.2 μmol or more per G6PDH unit, and the content of glutathione is 1 unit of G6PDH Buffer solution containing 0.2 to 0.5 nmol and glutathione reductase content of 0.03 unit or more per unit of G6PDH (5) Buffer solution containing 5: 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) The solution 4 and the solution 5 are used in the above step 5 ((i) enzyme cycling method) and mixed immediately before use.
The buffer used here is preferably a buffer having a pH of 7 to 9, particularly a Tris-HCl buffer.

The following examples illustrate the invention but are not intended to limit the invention.
In the measurement of this example, a solution having the following composition was used.
1. Reaction mixture A1 (for tissue): 10 mM Tris-HCl (pH 8.1), 0.03% bovine serum albumin (BSA), 195 μM NAD (oriental yeast), 1.3 mM EDTA, 1.1 unit / ml G6PDH (Leuconostoc meseteroides-derived enzyme, Oriental yeast)
2. Reaction mixture A2 (for blood): 10 mM Tris-HCl (pH 8.1), 0.03% bovine serum albumin (BSA), 195 μM NAD, 1.1 unit / ml G6PDH (Leuconostoc meseteroides-derived enzyme, oriental yeast), 7.5 unit / ml Hexokinase (Sigma), 2.6 mM ATP, 1.3 mM MgCl ₂
3. Reaction mixture B: 10 mM Tris-HCl (pH 8.1), 200 μM NADP (Roche Applied Science), 40 mM EDTA, 860 unit / ml G6PDH
4). Amplification / coloring solution (mix solution C and solution D in a volume ratio of 7: 2 immediately before use)
(1) Solution C: 10 mM Tris-HCl (pH 8.1), 29 mM G-6-P (oriental yeast), 2.9 mM reduced glutathione (GSH) (Sigma), 2.9 mM EDTA, 124.7 unit / ml G6PDH (Leuconostoc meseteroides-derived enzyme, oriental yeast) (reaction concentration 43unit / ml), 8.7unit / ml glutathione reductase (GR) (oriental yeast) (reaction concentration 3unit / ml)
(2) Solution D: 10 mM Tris-HCl (pH 8.1), 20 mM 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma)

Test example 1
In this test example, a constant concentration of 2DG (Sigma) or 2DG-6-P was added to the blood and skeletal muscle lysate of mice not administered with 2DG, and after the amplification / coloring solution was allowed to act, Absorbance at 420 nm was measured, and calibration curves were prepared for 2DG-6-P and 2DG, respectively. The calibration curve created in this way is shown in FIG.

Example 1
After the silicone catheter was placed in the jugular vein of the mouse and the body weight recovered, 5 μmol of 2DG (Sigma) (100 μl of physiological saline) was administered from the catheter in an unanesthetized and unconstrained state.
Twenty minutes later, Nembutal was administered from the catheter to euthanize the mice, and each tissue was collected and stored at -80 ° C. 500 μl of Tris-HCl buffer (10 mM Tris-HCl, pH 8.1) was added to the tissue weight (˜10 mg), homogenized (POLYTRON PT 1200), and heat-treated at 95 ° C. for 15 minutes. Thereafter, the mixture was centrifuged at 14,000 rpm and 4 ° C. for 15 minutes, and the supernatant was collected and stored at −80 ° C.
From the supernatant obtained above, the amount of 2DG-6-P was measured by the following procedure.
1. The supernatant obtained from each tissue is diluted with 10 mM Tris-HCl buffer (soleus, long extensor: x2-4 times; gastrocnemius: x2 times; adipose tissue: x2 times; brain: x8 Double; heart: x8 times).
2. Place 20 μl of each sample in a 96-well plate.
3. To this, 65 μl of the reaction mixture A1 is added and allowed to stand overnight at room temperature. After the reaction mixture A1 was added (that is, during the reaction), the concentration of G6PDH was 0.86 unit / ml, and the concentration of NAD was 150 μM.
4). To this is added 5 μl of 1N HCl and left at 38 ° C. for 15 minutes.
5. To this, 5 μl of 1N NaOH / 50 mM Tris-HCl (pH 8.1) is added for neutralization.
6). To this, 5 μl of reaction mixture B is added and left at 38 ° C. for 1 hour. After adding the reaction mixture B, the concentration of G6PDH was 43 units / ml, and the concentration of NADP was 10 μM.
7). Add 5 μl of 1.5N NaOH and leave it at 70 ° C. for 1 hour.
8). To this, 5 μl of 1.5N HCl / 50 mM Tris-HCl (pH 8.1) is added for neutralization.
9. Add 90 μl of amplification / development solution. The concentration of G6PDH after adding the amplification / coloring solution was 43 units / ml, the concentration of glutathione reductase was 3 units / ml, and the concentration of DTNB was 2 mM.
10. Measure absorbance at 420 nm at 30 ° C. using a microplate reader.
11. The amount of tissue 2DG-6-P is calculated from the calibration curve prepared in Test Example 1.
The results are shown in Table 1.

Example 2
Before the mouse of Example 1 was sacrificed, blood (20 μl) was collected from the catheter every 3 minutes, 6 minutes, 10 minutes, 15 minutes, and 20 minutes after DG administration. The obtained blood was centrifuged at 3000 rpm, 4 ° C. for 15 minutes, serum was collected, this serum was diluted 30 times with buffer (10 mM Tris-HCl, PH8.1), and allowed to stand at 95 ° C. for 15 minutes. . Thereafter, the mixture was centrifuged at 14,000 rpm and 4 ° C. for 15 minutes, and the supernatant was used as a sample and dispensed and stored at −80 ° C.
The amount of 2DG was measured from the obtained sample by the following procedure.
1. Place 5 μl of sample in a 96-well plate.
2. To this is added 15 μl of buffer (10 mM Tris-HCl, pH 8.1).
3. Add 65 μl of the reaction mixture A2 and leave it at room temperature overnight. After adding the reaction mixture A2, the concentration of G6PDH was 0.86 unit / ml, the concentration of NAD was 150 μM, and the concentration of hexokinase was 5.8 unit / ml.
4). To this is added 5 μl of 1N HCl and left at 38 ° C. for 15 minutes.
5. To this, 5 μl of 1N NaOH / 50 mM Tris-HCl (PH 8.1) is added for neutralization.
6). To this, 5 μl of reaction mixture B is added and left at 38 ° C. for 1 hour. After adding the reaction mixture B, the concentration of G6PDH was 43 units / ml, and the concentration of NADP was 10 μM.
7). Add 5 μl of 1.5N NaOH and leave at 70 ° C. for 1 hour.
8). To this, 5 μl of 1.5N HCl / 50 mM Tris-HCl (pH 8.1) is added for neutralization.
9. Add 90 μl of amplification / development solution. The concentration of G6PDH after adding the amplification / coloring solution was 43 units / ml, the concentration of glutathione reductase was 3 units / ml, and the concentration of DTNB was 2 mM.
10. Measure absorbance at 420 nm at 30 ° C. using a microplate reader.
11. The blood 2DG amount is calculated from the calibration curve prepared in Test Example 1.

血液中2DGは時間とともに指数関数的に減少した。血糖値に比べて血液中2DG濃度は、30分の1以下であり、2DGを投与しても血糖値には全く影響しなかった。その結果、2DGの消失速度(Fractional turn over rate, Kp x 100)は6.8±1.5 %であった。
The results are shown in Table 2 and FIG.

2DG in the blood decreased exponentially with time. Compared with blood glucose level, blood 2DG concentration was 1/30 or less, and administration of 2DG did not affect blood glucose level at all. As a result, the disappearance rate of 2DG (Fractional turn over rate, Kp x 100) was 6.8 ± 1.5%.

本測定法で算出した各組織のグルコース代謝速度定数（Ki）は、2[³H]DGを用いた方法（比較例１）とほぼ同じ値であった。 The glucose metabolic rate constant (Ki) was calculated from the tissue 2DG-6-P amount obtained in Examples 1 and 2 and the blood 2DG amount based on the Sokoloff equation. The results are shown in Table 3 and FIG.

The glucose metabolism rate constant (Ki) of each tissue calculated by this measurement method was almost the same value as the method using 2 [ ³ H] DG (Comparative Example 1).

骨格筋、脂肪組織、心臓などいわゆるインスリン感受性組織においてグルコース代謝速度定数が亢進した（表５）。既に報告されているように、大脳皮質のグルコース代謝速度定数は変化しなかった。骨格筋などインスリン感受性組織でのグルコースの利用亢進に伴い、血液中2DG量の減少も促進した（表４）。2DGの消失速度(Fractional turnover rate, Kp x 100)は7.5±0.4 %であった。
Example 3
A silicone catheter was placed in the jugular vein of a mouse, and after weight recovery, insulin (0.75 unit / kg body weight) was administered from the catheter in an unanesthetized and unrestrained state, and 15 μm later, 5 μmol of 2DG (saline) 100 μl) was administered. In order to maintain the blood glucose level, a glucose solution was administered from the jugular vein catheter after insulin administration. Blood (20 μl) was collected from the catheter at 3, 6, 10, 15, and 20 minutes. In addition, after collecting the final blood, Nembutal was immediately administered from the catheter to euthanize the mouse, the tissue was collected, and the tissue was stored at -80 ° C.
The amount of 2DG in blood and the amount of 2DG-6-P in tissue were measured in the same manner as in Examples 1 and 2, and the glucose metabolic rate constant (Ki) was calculated. The results are shown in Tables 4 and 5.

Glucose metabolic rate constants increased in so-called insulin-sensitive tissues such as skeletal muscle, adipose tissue, and heart (Table 5). As already reported, the glucose metabolic rate constant of the cerebral cortex did not change. With the increase of glucose utilization in insulin-sensitive tissues such as skeletal muscle, the decrease of 2DG in blood was also promoted (Table 4). The disappearance rate of 2DG (Fractional turnover rate, Kp x 100) was 7.5 ± 0.4%.

Comparative Example 1
In this comparative example, the glucose metabolism rate was measured using 2DG labeled with a radioactive substance.
A mixed solution of 2- [ ³ H] DG and [ ¹⁴ C] sucrose was administered from an intravenous catheter (2- [ ³ H] DG 5 μCi, [ ¹⁴ C] sucrose 1 μCi per mouse). Subsequently, blood (20 μl) was collected 3 minutes, 6 minutes, 10 minutes, 15 minutes and 20 minutes later. Immediately after collecting the last blood, Nembutal anesthetic is administered from the catheter, the mouse is euthanized, and the tissue is collected. And ³ H, ¹⁴ C radioactivity in blood at each time point, a ³ H, ¹⁴ C radioactivity of the tissue was measured by a liquid scintillation counter. Tissue radioactivity was measured by dissolving in organic base. Based on Sokoloff's equation, the metabolic rate of glucose (constant Ki) was calculated from 2- [ ³ H] DG (dpm / mg tissue) incorporated into the cells and the ³ H radioactivity of blood at each time point. . The results are shown in Table 6 and FIG.

Example 4
3T3-L1 cultured cells are cultured with Dulbecco's Modified A Gourmetium (DMEM) (including 20 mM glucose) and 10% fetal bovine serum in a 6-well culture dish. After becoming confluent, 2x dexamethasone and isobutylmethylxanthine The cells were allowed to act for a day, and then insulin (0.1 μM) was added to differentiate into adipocytes.
One week after culturing after adding insulin, in order to remove the influence of physiologically active substances contained in fetal calf serum, the culture medium was replaced with DMEM (glucose 20 mM) not containing fetal calf serum and cultured for 4 hours. Thereafter, the cells were further washed with PBS (phosphate-buffered saline), and then cultured at 37 ° C. for 15 minutes in PBS containing insulin (0.1 μM) and 5 mM glucose. After 15 minutes, 2DG (1 mM) was added to this culture solution, and further cultured for 15 minutes. After 15 minutes, after washing the cells with PBS, the cells were collected, and a cell lysate was prepared with 500 μl of 10 mM Tris-HCl buffer (pH 8.1). This was heat-treated at 95 ° C. for 15 minutes, centrifuged at 14,000 rpm at 4 ° C. for 15 minutes, and the supernatant was collected and stored at −80 ° C.
Using the supernatant obtained above as a sample, the amount of 2DG-6-P was measured by the following procedure.
1. Place 20 μl of each sample in a 96-well plate.
3. To this, 65 μl of the reaction mixture A1 is added and allowed to stand overnight at room temperature. After the reaction mixture A1 was added (that is, during the reaction), the concentration of G6PDH was 0.86 unit / ml, and the concentration of NAD was 150 μM.
4). To this is added 5 μl of 1N HCl and left at 38 ° C. for 15 minutes.
5. To this, 5 μl of 1N NaOH / 50 mM Tris-HCl (pH 8.1) is added for neutralization.
6). To this, 5 μl of reaction mixture B is added and left at 38 ° C. for 1 hour. After adding the reaction mixture B, the concentration of G6PDH was 43 units / ml, and the concentration of NADP was 10 μM.
7). Add 5 μl of 1.5N NaOH and leave it at 70 ° C. for 1 hour.
8). To this, 5 μl of 1.5N HCl / 50 mM Tris-HCl (pH 8.1) is added for neutralization.
9. Add 90 μl of amplification / development solution. The concentration of G6PDH after adding the amplification / coloring solution was 43 units / ml, the concentration of glutathione reductase was 3 units / ml, and the concentration of DTNB was 2 mM.
10. Measure absorbance at 420 nm at 30 ° C. using a microplate reader.
11. As in Test Example 1, a certain amount of 2DG-6-P is added to the lysate of cultured cells to which 2DG has not been added to create a calibration curve, and the amount of intracellular 2DG-6-P is calculated.
The results are shown in Table 7. Insulin increased the amount of 2DG-6-P in cultured adipocytes. This indicates that the metabolic rate of 2DG has increased.

It is a figure which shows the calibration curve (left figure) of 2DG amount in blood, and the calibration curve (right figure) of 2DG-6-P amount in a structure | tissue. It is a figure which shows 2DG amount and blood glucose level in the blood with respect to elapsed time. The elapsed time indicates the time after 2DG was administered into the vein of the mouse. It is a figure which shows the glucose metabolism rate constant (Ki) in each structure | tissue.

Claims (5)

A method for measuring the metabolic rate of 2-deoxyglucose (2DG) comprising the following steps.
(1) G6PDH and a concentration of 50 to 300 μM in a tissue collected from a non-human animal to which 2DG has been administered or a cultured cell of a mammal to which 2DG has been administered so that the concentration is 0.5 to 1 u / ml. Adding NAD and further treating with acid,
(2) Further, a step of adding G6PDH to a concentration of 30 u / ml or more and NADP to a concentration of 5 to 50 μM and further treating with alkali, and (3) NADPH produced in (2) To measure the amount of A method for measuring the metabolic rate of 2-deoxyglucose (2DG) comprising the following steps.
(1) administering 2DG into a vein of a non-human mammal;
(2) collecting tissue from the mammal, homogenizing the collected tissue, performing heat treatment, and collecting the extract;
(3) A step of adding G6PDH so that the concentration becomes 0.5 to 1 u / ml and NAD so that the concentration becomes 50 to 300 μM and further treating with an acid.
(4) A step of adding G6PDH so that the concentration becomes 30 u / ml or more and NADP so that the concentration becomes 5 to 50 μM, and further treating with alkali.
(5) further adding a mixture comprising glycol-6-phosphate, glutathione reductase, G6PDH, glutathione, and 5,5-dithiobis (2-nitrobenzoic acid) (DTNB); and (6) DTNB Measuring reaction volume The method for measuring the metabolic rate of 2-deoxyglucose (2DG) according to claim 2, further comprising the following steps.
(7) collecting blood from the non-human mammal;
(8) G6PDH is added to the blood so that the concentration is 0.5 to 1 u / ml, NAD is added so that the concentration is 50 to 300 μM, and hexokinase is used so that the concentration is 1 to 10 u / ml. Adding ATP and MgCl ₂ so that each becomes ₁ to 3 mM, and further treating with acid,
(9) adding G6PDH so that the concentration becomes 30 u / ml or more, and NADP so that the concentration becomes 5 to 50 μM, and further treating with alkali;
(10) a step of adding a mixture further comprising glycol-6-phosphate, glutathione reductase, G6PDH, glutathione, and 5,5-dithiobis (2-nitrobenzoic acid) (DTNB); and (11) of DTNB The stage of measuring the reaction amount, and (12) The disappearance rate of 2DG in blood and the amount of 2-deoxyglucose-6-phosphate (2DG-6-P) in the tissue are calculated from the obtained reaction amount of each DTNB And calculating the glucose metabolism rate A method for measuring the metabolic rate of 2-deoxyglucose (2DG) comprising the following steps.
(1) adding 2DG to a culture of mammalian cells;
(2) Furthermore, G6PDH is added to the cell extract to a concentration of 0.5 to 1 u / ml, and NAD is added to a concentration of 50 to 300 μM.
(3) Further, G6PDH is added so that the concentration becomes 30 u / ml or more, and NADP is added so that the concentration becomes 5 to 50 μM, and further treated with alkali (4) Further, glycol-6-phosphate, glutathione Adding a mixture containing reductase, G6PDH, glutathione, and 5,5-dithiobis (2-nitrobenzoic acid) (DTNB); and (5) measuring a reaction amount of DTNB. A kit for measuring the metabolic rate of 2-deoxyglucose (2DG) comprising the following five solutions.
(1) A G6PDH and buffer containing NAD, buffer content of NAD is 50~600nmol per G6PDH1unit (2) hexokinase (HK), a buffer solution containing ATP and MgCl _2, the content of ATP Is a buffer solution containing 0.3 μmol or more per HK1 unit and a MgCl ₂ content of 0.1 to 0.5 μmol per HK1unit (3) A buffer solution containing G6PDH and NADP, wherein the NADP content is 0.1 to 0.1 per G6PDH unit 0.5 nmol of buffer (4) A buffer containing G6PDH, glycol-6-phosphate, glutathione and glutathione reductase, wherein the content of glycol-6-phosphate is 0.2 μmol or more per G6PDH unit, Content per G6PDH 1 unit .2～0.5Nmol, buffer content of glutathione reductase is 0.03unit or more per G6PDH1unit (5) 5,5- dithiobis (2-nitrobenzoic acid) buffer containing (DTNB)

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