IXDUCED OXIDATION O F GLUCOSE I K PRESEKCE OF INSULIN ACTISG AS AX ISDUCTOR BY HIRA LAL DUBE AND N. R. DHAR
In previous papers' we have advanced the view that insulin acts as a promoter in the oxidation of glucose in the animal body. The experimental results recorded in this paper shows that in oitro insulin acts as an inductor in the oxidation of glucose in presence of phosphates by passing air, although Spoehr and Smith? did not observe any increased oxidation of glucose in presence of insulin. The experimental details are not available in the paper of Spoehr and Smith. All our experiments were carried out in diffused daylight inside the room at a temperature of about 25'. In these experiments a slow current of air was passed through a series of bottles, containing 2 0 7 ~sodium hydroxide solution, baryta and concentrated sulphuric acid to free the air from carbon dioxide gas and moisture. This carbon dioxide free air was passed through the solution of glucose containing insulin and other substances, such as ferrous hydroxide, sodium phosphate, etc. X measured volume of air was passed. The insulin used was obtained from British Drug House, containing 5 cc. in the tube and I cc. contained 2 0 units. In every experiment a fresh solution of the insulin was taken, as it was observed during the experiments that the solutions putrifies on keeping. I t is practically neutral and does not reduce Fehling's solution. 5 cc. of the insulin ( 2 0 units) in the tube was made up to j o cc. by adding distilled water and this aqueous insulin was used in the following experiments. Extra pure glucose of Merck was used for the experiments. The volume of the solution to be oxidized was always made up to I O O cc. by adding distilled water. 36.5 litres of air were passed in 15 hours. Experiments with Insulin and Sodium Phosphate In each of these experiments I O cc. of 1VC glucose solution was taken and it was estimated by the reduction of Fehling's solution and finally weighing the precipitate as cupric oxide. I O cc. glucose = 0.2310 gram CuO. In the following experiment 0.348 N solution of disodium hydrogen phosphate was used: No. of Litres of Insulin Sodium experi- air passed in cc. Phosphate ment in cc.
I
36.5
IO
IO
Actual Amount weight of of glucose glucose left after in I O cc. experiof the ment solution In grm. taken in grm. (Blank) O.IOOO 0.0982
Amount of glucose oxidised in grm.
0.0018
Percentage amount of glucose oxidised
1.8
'Dhar: Chemie der Zelle und Gewebe, 12, 217 (1925); J. Phys. Chem., 29, 376 (I9Zj). * J. Am. Chem. SOC.,48, 236 (1926).
INDUCED OXIDATION O F GLGCOSE IN PRESENCE O F INSULIN
445
I n the followingexperiments 0.41j N solution of sodium phosphate was used: 36.5 36.5 73 36.5
2
3 4 5
IO
IO
0.1000
20
IO
0.1000
25 25
IO
O.IOOO
30
O.IOOO
0.0987 0.0916 0.0761 0.0678
0.0013 0.0084 0.0239 0.0322
1.3 8.4 23.9 32.2
I n the following experiments no insulin was used: Actual Amount of weight of glucose left Litres of Sodium glucose in after experiexperi- air passed phosphate I O cc. of ment in ment (0.348N) the soln. grm. in cc. taken in grm. (Blank)
No. of
I
36.5
IO
O.IOOO
0,0997
Amount of glucose oxidised in grm.
Percentage amount of glucose oxidised
0.0003
0.3
In the following experiments 0.415 X disodium hydrogen phosphate was used: 2
73
IO
O.IOOO
0.0844
0.0156
15.6
3
36.5
30
0.1000
0.0961
0.0039
3.9
The above tables clearly show that there is more oxidation of glucose in presence of insulin and phosphate than in phosphate alone. This leaves no doubt that insulin acts as an inductor in the oxidation of glucose in presence of sodium phosphate. It is well known that the part which phosphate plays in the animal metabolism is unique. We have carried on experiments with freshly precipitated cerous and ferrous hydroxides also and the results obtained conclusively prove that they also help insulin in the oxidation of glucose. The same amount of cerous hydroxide was used in each of these experiments: Actual Amount of weight of glucose left glucose in after experiI O cc. of ment in the solution grm. taken in grm. (Blank)
No. of experiment
Litres of air passed
Insulin in cc.
I
36.5 36.5 36.5 73
IO
0.1000
20
0.1000
25
0.1000
25
O.IOOO
2
3 4
0.0830 0.0789 0.0789 0.0675
Amount of glucose oxidised in grm.
Percentage amount of glucose oxidised
0.0170
17.0
0.0211
21.1
0.0~12
21.2
0.0325
32.5
I n the following experiments also the same amount of cerous hydroxide but no insulin was used: I 2
36.5 73
-
0.1000
0.0845
0.1000
O.OjO1
o.015j 0,0299
15.5 29.9
I n the following experiments ferrous hydroxide was used instead of cerous hydroxide:
446
HIRA LAL DUBE AND N . R . DHAR
No. of experiment I 2
Insulin in cc.
Litres. of air Dassed
73 73
25
Actual weight of glucose in I O cc. of the solution taken in grm. (Blank)
0.0806 0.0923
0.1000
&in
Amount of glucose left after experiment in grm.
0.1000
Amount of glucose oxidised in grm.
Percentage amount of glucose oxidised
0.0194
19.4 7.7
0.0077
We have also carried on some experiments with glucose and insulin alone and we find that there is very slight oxidation. The following results were obtained : 0.0992 0.0008 0.8 I 36.5 IO 0.1000 0.0010 1.0 25 0.1000 0.0990 2 36.5 By these results we are led to believe that there is some oxidation of glucose in presence of insulin alone in vitro, but by adding cerous or ferrous hydroxides, which act as surfaces the oxidation is facilitated. We have observed that insulin by itself is oxidized by passing air a t 25' and carbon dioxide is evolved, but when glucose is added the oxidation of insulin is great,ly retarded. This led us to believe that insulin acts as an inductor in the oxidation of carbohydrates. In several publications' we have shown that the slow oxidation of substances can be retarded by another reducing agent, which is slowly oxidized along with the primary reaction. The oxidation of insulin induces the oxidation of glucose. We have tried the oxidation of glucose in presence of insulin and sodium bi-carbonate and sodium carbonate, but in the case of sodium bicarbonate we find that the oxidation is practically the same in presence or absence of insulin. In the case of sodium carbonate the result,s are curious. There is more oxidation in presence of sodium carbonate alone than in presence of sodium carbonate and insulin. We have obtained the following results. In the following experiments IO cc of I.ooj hT sodium bicarbonate solution was used: No. of
experirnent
Litres of air passed
I
36.5
2
73
3
73
Insulin in cc.
IO
~
%
Actual weight of glucose in I O cc. of the solution taken in grm. (Blank)
Amount of glucose left after experiment in grm.
Amount of glucose oxidised in grm.
Percentage amount of glucose oxidised
0.1000
0.0983 0.0987 0.0982 i ~
o.oo17 0.0013 o.oo18
1.3 1.8
0.1000
~
o~. 1000 ~
i
I n the following experiments 30 cc. solution was used: I 36.5 25 0.1000 2 36.5 0.1000 3 36.5 Insulin 0.1000
1.7
of the normal sodium carbonate
0.0786 0.0858 o.ojo3
0.0214 0.0142 0.0297
21.6 14.2 29.2
'Dhar: Roc. Akad. Wet. Amsterdam, 29, 1023 (1921);Z. anorg. allgem. Chem., 144, 289 (1925).
INDUCED OXIDATION OF GLUCOSE IN PRESENCE OF INSULIN
447
It is difficult to explain satisfactorily these results. Since 1922 much experimental investigation has been carried on with insulin and its influence on glucose metabolism in the animal body and it is generally believed that it helps glucose metabolism. From our experiments it is clear that insulin plays an important part in the carbohydrates metabolism zn vztro and it acts as an inductor. It is easily oxidised by passing air and when mixed with glucose solution the oxidation of insulin is retarded, while it helps the oxidation of glucose. On addition of sodium phosphate or cerous or ferrous hydroxide the oxidation of insulin is accelerated and there is a large amount of carbon dioxide liberated and a t the same time the oxidation of glucose is also increased. Sodium phosphate and cerous and ferrous hydroxides facilitate the oxidation of glucose. In the animal body also phosphate is present which must be helping the oxidation of glucose by the secretion of the pancreas. Moreover, surfaces are also present in the animal system. The behaviour of insulin resembles that of glutathione. Glutathione is an auto-oxidisable substance. It oxidises itself by the oxygen of the atmosphere and at the same time induces the oxidation of the cell constituents. Harrison’ has shown that traces of iron cause a marked acceleration in the auto-oxidation of glutathione; and hence the oxidation of tissue components induced by glutathione w ill also be accelerated by iron. The same behaviour is seen in the case of insulin also and our experiments show that insulin, like glutathione is our auto-oxidisable substance, which induces the oxidation of glucose in the animal system. It is probable that insulin is a polypeptid, a group of substances known to participate in inportant ways in the metabolism of the body. When analysed by the method of Folin and Looney, there is found 17.9% tyrosin, 7.1% cystin, 0.87, tryptophan and 8.5% histidin in insulin. Insulin is not digested by pepsin nor by trypsin, but in an alkaline medium, in which trypsin is present, insulin becomes inactivated. It may be reactivated, however, showing that it is not destroyed. It appears from our experimental results that in presence of sodium carbonate, insulin is inactivated and does not increase the oxidation of glucose by air in vitro. It will be interesting to note here that Bertrand and MBcheboeuf2 have found that insulin contains very small amounts of nickel and cobalt salts (the amount is never greater than a fraction of a milligram per kilo of tissue). The amount of sugar metabolised under the action of insulin is increased when nickel or cobalt compound is given simultaneously. The recent results obtained by Svedberg3 seem to demonstrate that insulin is a well-defined protein belonging to the same class as egg albumin and Bence Jones protein. As pointed out by Dr. H. Jensen of the Johns Hopkins University, Baltimore, this fact makes it very improbable that the synthesis of insulin will ever become possible. Biochem. J., 18, 1009 (1924). *Compt. rend., 182, 1305,1506;183, 5, 257, 326 (1926). Nature, 127,438 (1931).
HIRA LAL DUBE AND N , R. DHAR
448
summary Glucose is appreciably oxidised by passing air through solutions of glucose in presence of insulin at 25'. Phosphates, cerous and ferrous hydrooxides markedly accelerate this induced oxidation of glucose by air in presence of insulin. ( 2 ) Insulin is oxidised by passing air and in presence of glucose, the oxidation of insulin is retarded but the oxidation of insulin induces the oxidation of glucose. This is the probable mechanism of the increased oxidation of glucose in presence of insulin in the animal body. (3) Sodium carbonate appears to inactivate insulin and in presence of sodium carbonate, there is no increase in the oxidation of glucose due to insulin. (I)
Chemical Laboratory, Alluhabad University, Alluhabad, India, M a y 24, 1951.
