John James Richard Macleod – Nobel Lecture

Nobel Lecture, May 26, 1925

The physiology of insulin and its source in the animal body

The knowledge that the isles of Langerhans of the pancreas have the function of secreting into the blood a hormone which plays an essential role in the regulation of the metabolism of the carbohydrates, is the outcome of numerous investigations extending over many years, and to the development of this knowledge workers in various fields of medical science have contributed.

In 1889, when Minkowski and von Mering discovered that complete extirpation of the pancreas leads to fatal diabetes, practically nothing was known concerning the significance of the ductless glands, and few conceived that it would be possible to extract, from various of them, substances capable of replacing the lost function, when administered to animals from which some particular gland had been removed. Although, at this time, it was known that the thyroid gland is atrophied in myxoedema and in cretinism, it was not until 1892 that Murray discovered that administration of the gland removes the symptoms, and it was only later that the doctrine of internal secretions, first enunciated by Claude Bernard in 1856 in connection with the production of sugar by the liver, came to take its place in physiological teaching. Minkowski in the complete account of his researches, published in 1893, considered the antidiabetic function of the pancreas to be dependent upon its acting as a ductless gland, and no doubt he had in mind that it performed this through an internal secretion, although the positive statement that such exists was first made by Lepine, who thought that it took the form of a glycolytic enzyme. But, so far, there was no hint as to the actual structure within the pancreas upon which the antidiabetic influence of the gland depends and it is primarily to the anatomists, Laguesse and Diamare, that we owe the hypothesis that this must be the collection of cells, named after their discoverer, the isles of Langerhans. By careful studies of the cytological characteristics of the cells of these islets, as distinguished from those of the much more numerous secreting acini among which they lie, and by painstaking examination of the anatomical relationships of the two kinds of cells in different classes of vertebrates, Laguesse and Diamare concluded that the islets must be responsible for the antidiabetic influence.

As this anatomical work was in progress, the potent action of extracts of the suprarenal gland on the blood pressure and other physiological functions was discovered, in 1894, by Oliver and Schafer, thus adding strong support to the hypothesis that the ductless glands function by producing internal secretions. The hypothesis, that the islets of Langerhans of the pancreas must act in a similar manner, gained a firm hold among physiologists and clinical workers, with the result that many attempted to alleviate the symptoms of diabetes by administration of pancreas, or of extracts of the gland, to patients suffering from the disease. No success attended these attempts partly, we believe, because the antidiabetic principle was destroyed, either during the preparation of the extracts or by the action of the digestice juices, and partly, because of imperfect knowledge of the clinical course of the disease, particularly with regard to the relationship of diet to it. Notwithstanding the failure of these attempts, the hypothesis that the isles of Langerhans are the structures to which the pancreas owes its antidiabetic function was still maintained, and indeed strengthened, by the supporting evidence furnished by the graft experiments of Minkowski and Hédon. These workers showed that no diabetic symptoms supervene in dogs when a portion of the pancreas is transplanted into the wall of the abdomen prior to, or at the same time as, removal of the remainder of the gland, but immediately do so in full intensity when this graft is subsequently excised. Moreover, it was known that ligation of the ducts of the pancreas, or their injection by oil or paraffin, is not followed by diabetes. Since, in neither of these types of experiment, can any of the digestive secretion gain the intestine it was clear that the anti-diabetic function of the pancreas must be independent of its digestive function. It may be well to point out also that the graft experiments once and for all disproved the view held by some (by Pflüger, for example), that damage to the nerve structures adjacent to the pancreas, or in the duodenal wall, is responsible for the diabetic symptoms.

A distinct step forward was taken in 1900 when Schulze and Ssobolev discovered that the degenerative changes which follow ligation of the ducts affect the cells of the acini much more markedly than those of the islets, and although among those who repeated these researches, there were some who failed to corroborate the findings, the conclusions of Schulze and Ssobolev were generally accepted. It was not long after this that the first, though unsuccessful, attempt was made to see whether an extract of the degenerated residue of duct-ligated pancreas might not relieve the symptoms of diabetes. About this time also (1906) - as was revealed in 1922 by the opening of a sealed package deposited with the Société de Biologie - Gley had found similar extracts to diminish the symptoms in diabetic dogs, and in the same year, Miss Dewitt had tried their effects on glycolysis.

The insular hypothesis of diabetes was meanwhile strongly supported by the careful histological studies of the pancreas of patients who had died from the disease (Opie) for, although it had been known, even prior to the experiments of Minkowski and von Mering, that the gland is often the seat of morbid change, it was not realized that the islets are the structures which are chiefly affected.

In 1903-1904, Rennie, by anatomical studies in certain Teleostei, gave strong support to the view of Diamare, that the islet cells in these fishes exist as separate glands of relatively large size and more or less independent of the pancreatic acini. Both workers attempted to demonstrate an effect of extracts of these glands on sugar or starch solutions, but without success. They administered them by mouth to diabetic patients with no favourable results, although in one case, in which an extract was given subcutaneously, there was decided alleviation of the diabetic symptoms (Rennie and Fraser).

About this time the significance of internal secretions in the control of animal functions was clearly demonstrated by the discovery of secretin by Bayhss and Starling (1902), and the term "hormone" came into use to designate their active principles. Many believed that the antidiabetic function of the pancreas must depend on a hormone secreted by the isles of Langerhans, but neither the graft experiments already referred to, nor the transfusion experiments of Hédon - in which it was found that when the blood of a normal dog was transfused in a diabetic one the symptoms were alleviated - could prove the hypothesis. To do this it was necessary to- show that extracts of the islets, or at least of the pancreas, are capable of removing the symptoms of diabetes. In 1907 Zuelzer published results which must be considered, in the light of what we now know, as really demonstrating the presence of the antidiabetic hormone in alcoholic extracts of pancreas. But unfortunately, even although several diabetic patients were benefited by administration of the extracts, the investigations were not sufficiently completed to convince others, and, apparently, Zuelzer himself was discouraged in continuing them because of toxic reactions in the treated patients.

To describe, even in mere outline, the further attempts to prepare active antidiabetic extracts of the pancreas would far exceed the limits of this essay. To Knowlton and Starling, Meltzer and Kleiner, E. L. Scott, Murlin and Cramer, and to Clark, we owe much, for although none of these investigators succeeded in demonstrating beyond doubt that an extract having antidiabetic properties could be prepared from the pancreas, they all obtained results which were sufficiently positive to keep alive the hope that some day this would be possible. Special reference must also be made to the more recent work of Paulesco who prepared extracts having very decided effects on the sugar and the urea of the blood of diabetic animals.

Believing that the want of success to prepare extracts of uniform potency as due to the destruction of the antidiabetic hormone by the digestive enzymes also present in the gland, F. G. Banting suggested preparing them from duct-ligated pancreas, and with the aid of C. H. Best, and under my direction, he succeeded in 1922 in showing that such extracts reduced the hyperglycaemia and glycosuria in depancreatized dogs. The general symptoms of diabetes were also found to be alleviated and the duration of life of the depancreatized animal prolonged, by the repeated injection of alcoholic extracts of foetal, as well as of adultox pancreas. Later it was shown, in collaboration with Collip, that other symptoms of diabetes, namely the ketonuria and the absence of glycogen from the liver, were favourably influenced by the extracts and, with Hepburn, that the respiratory quotient became raised. These results on depancreatized dogs showed beyond doubt that the antidiabetic hormone was present in potent form in the extracts, and the time seemed ripe to investigate their action on the clinical forms of diabetes. This was done by Banting in a severe case under the care of W. R. Campbell, with the result that the hyperglycaemia and glycosuria were diminished. At the same time, however, it was found that it would be necessary to rid the extracts of irritating substances before the value of their repeated injection in the treatment of diabetes in man could be adequately put to the test. This was accomplished by Collip, and the name insulin was decided upon for the purified extract. This name had previously been suggested by Sir E. Sharpey Schafer (1916), who had been one of the first to support the hypothesis of the insular derivation of the antidiabetic hormone. I need not here detail the rapid progress which it was now possible to make in studying the therapeutic value of insulin in the treatment of diabetes in man; for it is with experimental aspects of the subject that this essay is concerned.

The invariable lowering of the blood sugar which was observed to result from the administration of insulin in animals rendered diabetic by pancreatectomy, raised the question as to whether such would also occur in those forms of hyperglycaemia which can be induced by other experimental procedures, such as the injection of epinephrin, piqûre, or asphyxia. As the first step in the investigation of this question, Collip injected insulin into normal rabbits and found the blood sugar to become lowered, thus furnishing a valuable method for testing the potency of various preparations and, therefore, for affording a basis for their physiological assay. At the same time it was found that neither piqûre, nor epinephrin, nor asphyxia caused any hyperglycaemia in rabbits in which, as a result of injection with insulin, the blood sugar was at a low level to start with.

Peculiar symptoms (convulsions and coma) were observed in many of the injected animals, and it was soon possible to show that these were related to the lowering of the blood sugar and that they usually supervened when this was about 0.045 per cent. Sometimes the animals recovered spontaneously from these symptoms, but more frequently the coma became so profound, with marked fall of body temperature, that death occurred. That the lowering of blood sugar is closely related to the occurrence of the symptoms, was proved by finding that the subcutaneous injection of a solution of glucose was followed, almost immediately, by complete recovery, even in cases in which death was imminent from deep coma. It has been found, in collaboration with Noble, that glucose is remarkably specific in this regard, the only other sugar which approaches it being mannose and, in certain animals, such as the mouse, maltose. Laevulose and galactose are decidedly inferior in their antidoting action, the pentoses are entirely inactive and none of the disaccharides, other than maltose, has any effect. It is evident that this specificity in the action of glucose, in combating the hypoglycaemic symptoms, offers an opportunity to determine, not only what related substances are readily converted into glucose in the animal body, but also what groupings in the glucose molecule itself are significant for the effects. By substituting various side chains in the molecule, as for example, by methyl groups, it has been found, in collaboration with Herring and Irvine, that none of these substitution products is effective, even such compounds as the mono-methylglucosides being entirely inactive.

The fall in blood sugar is dependent upon increased diffusion of sugar into the tissues and not to its more rapid destruction in the blood itself. Thus, Eadie and I could detect no change in the rate of glycolysis by adding insulin to blood incubated under sterile conditions outside the body, or in blood withdrawn from animals injected some minutes before death with insulin. Hepburn and Latchford, on the other hand, demonstrated that the addition of insulin to the fluid perfused through the excised mammalian heart markedly increased the rate at which the percentage of sugar became diminished in it.

The striking relationship between the concentration of glucose in the blood and the normal functioning of the nervous system, which is revealed by these observations, had already been noted by Mann and Magath in their experiments on hepatectomized dogs. They observed that when the blood sugar fell to about 0.045 per cent, characteristic symptoms supervened which could be antidoted by glucose, and to a less extent, by laevulose and mannose. We must conclude that when the tension of glucose in the tissue cells falls below a certain level (glucatonia), a condition of irritability becomes developed; but little is known as to what the underlying cause for this may be. Olmsted and Logan have advanced some evidence that it may depend on interference with the process of oxidation in the nerve cells, or that these are irritated by substances produced elsewhere in the body by faulty oxidation. More recent experiments by Argyll Campbell on the tension of oxygen in the tissues lend support to this view.

These observations emphasize the great importance of a certain tension of glucose within the tissue cells. They help us to understand why it is that the concentration of this sugar in the circulating fluids of animals of every order and species in which it has been determined, varies only within narrow limits, even after prolonged periods of starvation, or following muscular exercise.

We must imagine that it is by lowering the tension of glucose within the tissue cells that insulin primarily acts, so that the glucose of the blood plasma, with which the tissue glucose is in equilibrium, diffuses into the cells to maintain the tension. With Eadie we have found that the free glucose extractable from the muscles by warm alcohol is reduced following the injecting of insulin, but we know nothing of the fate of the glucose which disappears. It is not converted into glycogen (McCormick, Noble and Macleod, Dudley and Marrian, Cori, etc.) nor is it immediately oxidized, since the respiratory metabolism (intake 0, and respiratory quotient) does not become increased at the time when the blood sugar is falling (Eadie, Dickson, Macleod, and Pember; Trevan and Boock; Krogh; Boothby and Wilder, etc.)¹.

Although the intake of oxygen may become greater in certain animals such as dogs, cats, and man when hypoglycaemic symptoms make their appearance, this does not occur when sugar is also administered. In the light of these results we have concluded that the glucose which disappears must become converted into some hitherto unidentified substance, but we have been unable to obtain any clue as to what this substance may be. Large amounts of it must be formed to account for the enormous quantities of glucose which may vanish from the blood, as when glucose is injected along with insulin. We have, for example, injected into rabbits, in the course of eight hours, as much as IO grams of glucose per kilo body weight along with insulin, without finding, at the end, any increase in blood sugar, or in the free or the combined sugar of the muscles or liver. Burn and Dale have also shown that very large quantities of glucose can be injected along with insulin into eviscerated animals without increasing the percentage of the blood sugar. It is conceivable that between glucose and the material which is finally oxidized in the tissues there exists, not one, but a group of substances constantly changing from one into another in an equilibrated system, and that no one of them ever accumulates in sufficient quantity to make its identification possible by available chemical methods.

Be this as it may, it is significant that the percentage of inorganic phosphoric acid in the blood declines at the same rate as the sugar, although, in the recovery process, the phosphoric acid begins to rise decidedly before the sugar in animals injected with insulin. Accompanying this fall in the phosphates of the blood, those of the urine entirely disappear for several hours and then return to considerably above the normal level so that, in urine collected throughout the 24 hours, an excess is excreted, as compared with the amount on days during which no insulin is given. (Winter and Smith, Allan and Sokhey, etc.). These facts would seem to indicate that in the process responsible for the disappearance of glucose in the tissues there is a stage when compounds of phosphoric acid with sugar or its immediate breakdown products are formed. One immediately thinks of the possibility that an increase in the amount of the substance, described by Embden and his school, in muscle, and named lactacidogen, might be responsible, but we have been unable to demonstrate that this is the case (Eadie, Macleod, and Noble). At the present time we are entirely at a loss to account for the disappearing glucose. When this problem is solved it may be anticipated that a great advance will become possible in our knowledge of the intermediary metabolism of the carbohydrates.

Having outlined the known facts with regard to its physiological action, we may now turn to the interesting question of the source of insulin. The observations of Banting and Best, that simple extracts of the residue of pancreas remaining several weeks after the ducts are tied possess antidiabetic properties, does not necessarily prove that insulin is derived from the islets. As Bensley and others have shown there may still remain, at this period after duct-ligation, a considerable amount of more or less normal acinar tissue. Even were the gland allowed to degenerate for a sufficient time so that all acinar tissue had disappeared - which is considerably over a year in the rabbit - it would be difficult, in the event that extracts of the residue still contained insulin, to be certain that this insulin is not of the type which it is possible to extract from various materials, including even the tissues of depancreatized animals, as Best and Scott have shown.

Further investigation of the problem was therefore undertaken by continuing the work of Diamare and Rennie on certain of the Teleostei, such as Lophius and Myoxocephalus, in which the islet cells exist apart from the acinar tissue, as the so-called "principal islets". Extracts were made by alcohol from these structures, as well as from the acinar tissue, and it was found that, whereas very large yields of insulin are readily obtainable from the islets, little or none at all can usually be prepared from the pancreas itself. Indeed, extracts of the latter sometimes cause the blood sugar of rabbits to become raised, instead of lowered. The lowering of the blood sugar by acinar extracts when it occurred, may have been due to the presence of a few scattered microscopic islets, such as have been observed by Slater Jackson to exist in the pancreatic bands of Myoxocephalus. That the principal islet may have some acinar tissue associated with it does not detract from the value of the foregoing observations as evidence supporting the insular hypothesis, since it has been shown that extracts of the acinar tissue are comparatively impotent. In this connection it is of interest to note that insulin can be readily prepared from the pancreas of the Elasmobranchi (Raja and Squalus), which occurs as a compact gland with the islet tissue included in it, much in the same manner as in the mammalian pancreas.

But in view of the fact that insulin, or at least extracts capable of lowering the blood sugar in normal rabbits, can be prepared from other tissues than the pancreas or the principal islets, there still remains the possibility that it might be secreted internally from some of these. It is indeed possible that although this did not occur in the normal animal, in which a sufficient amount was coming from the pancreas, it might occur when the normal. secretion was cut off, as in diabetes. By such a vicarious functioning of extra-pancreatic potential sources of insulin, the tolerance of the diabetic organism for carbohydrate might become raised. It seemed important, therefore, to see whether diabetes would result from excision of the principal islets alone, an operation which is possible in Myoxocephalus, the two principal islets being readily removable without exposure of the fish to air for more than 15 minutes.

In a large number of fish in which this operation was performed, it was found, in collaboration with McCormick, that the blood sugar became raised, often to ten times the normal value. At this high level it remained so long as the fish were kept alive - 11 days in one case, 5-10 days in others. The hyperglycaemia in itself was not sufficient to prove that the fish had become diabetic as a result of the isletectomy, for it was found, in other fish that were exposed to air for a period equal to that required for the operation (about 15 minutes), that the blood sugar rose, sometimes almost as much as in the operated ones. This asphyxial hyperglycaemia, however, was found to disappear within four days², nor while it lasted was it so pronounced as in the isletectomized fish. There is no doubt that removal of the islets in Myoxocephalus causes pronounced diabetes, as judged by the behaviour of the blood sugar, and, it is of interest to add that there was, on an average, considerably more fat and less glycogen present in the liver of the operated fish than in those of the controls. It remains to determine whether, by giving insulin to the isletectomized fish, the blood sugar can be brought down to the normal level. So far we have been unable to demonstrate any very potent influence of insulin on the blood sugar of normal fish, although there is some indication that it can retard the development of asphyxial hyperglycaemia.

Taking all the evidence into consideration the conclusion seems justified that the only source from which physiologically effective insulin can be secreted within the animal body is the islet tissue.

And finally permit me to say something concerning the behaviour of depancreatized animals kept alive by means of insulin. By such studies it is possible that we may be able to determine whether the lost power to utilize carbohydrate can be reacquired in any measure, and also whether the secretion of pancreatic juice and of insulin include all the functions of the gland. With the collaboration of Frank N. Allan, I. L. Chaikoff, J. Markowitz, and W. W. Simpson, several completely depancreatized dogs have now been kept alive, by daily injections of insulin, for many months, the operation on * one of them, which is at present under observation, having been performed over eighteen months ago. But this result was not immediately achieved.

In the earlier observations it was observed that, notwithstanding the fact that the animals ate large amounts of meat, the body weight steadily fell, no doubt because of inadequate intestinal digestion and absorption. The addition of cane sugar, in amounts sufficient to cause a mild degree of glycosuria (50 to 100 g daily), had the immediate effect of preventing the loss of body weight, and in most animals, of causing it to become increased, especially when large amounts were given. Four of these animals lived in excellent nutritive condition for periods varying between one and seven months, when each in turn developed symptoms of acute jaundice (bile pigment in urine, yellowing of sclera and skin) accompanied by rise in rectal temperature, anuria and progressive bodily weakness, ending fatally in from two to three days after the onset. The post mortem examination revealed, in each case, an extremely fatty liver with no significant pathological changes elsewhere in the body. Under the microscope it was difficult to see any liver cells that were not completely filled with fat, except for a few towards the centres of the lobules which were only moderately invaded. In some way or other, absence of the pancreas leads to a fatal breakdown of the hepatic function. Two possibilities may be considered: the one, that the pancreas secretes internally, besides insulin, some other hormone which is necessary for the functional integrity of the liver, perhaps a hormone having to do with its action on fat metabolism; and the other, that in the absence of the pancreatic ferments, the process of intestinal digestion becomes of such a (bacterial) type that substances having a toxic effect on the liver cells are absorbed into the portal blood. It was therefore decided to add raw ox pancreas (50 g) to the daily diet, and it is as an outcome of this addition that the animals have thrived without showing any symptoms of hepatic breakdown. This favourable result may be dependent either on the restoration of the pancreatic enzymes, thus preventing the development of toxic substances, or because some hormone which withstands digestive action is absorbed from the ingested gland. We are at present observing the effect of adding trypsin to the food, instead of raw pancreas, but although the animal thus treated is in excellent nutritive condition we cannot as yet say whether it may not ultimately develop the hepatic symptoms. It may be added that the toxic theory is supported by the observation that in the absence of raw pancreas, or trypsin, not more than fifty per cent of the ingested meat is assimilated, whereas over eighty per cent is assimilated when either of these is present.

The carbohydrate balance is being determined at intervals in several diabetic animals, in order to see whether any of the lost power to secrete insulin may be reacquired. This is done by determining the proportion of the ingested sugar which reappears in the urine daily while the animals are under the same dose of insulin, but so far no change has been detected. While it is certain that any considerable reacquirement of the power to secrete insulin would be revealed by this method, it is possible that a very scanty secretion might be masked on account of the relatively large amounts administered daily from without, for it has been shown, by Frank N. Allan, that the glucose equivalent of each unit of insulin is very much higher when the total number of units administered is small than when it is large. Another method for investigating this problem remains available, namely to observe whether the diabetic symptoms which supervene when insulin is discontinued are less severe after several months treatment than they are soon after the removal of the pancreas. Our attempts to make this observation have, so far, been frustrated by the very rapid downward progress of the animals after discontinuing insulin. Unless they are given large quantities of meat they die in a few days of symptoms not unlike those of diabetic coma.

It has been stated by Carlson and Drennan that the diabetic symptoms are very much less than usual when pancreatectomy is performed on pregnant animals near full time, and this they have attributed to the secretion of insulin from the foetal pancreas. We could obtain no evidence in support of this hypothesis in the present investigations. Thus, one of the depancreatized dogs gave birth to five pups without any change whatsoever in her sugar balance throughout the pregnancy, although, on the day after the pups were born, severe symptoms of hypoglycaemia developed, no doubt because of the removal of glucose from the body to form the lactose of the milk. There was therefore no evidence that the developing foetuses contributed any significant amount of insulin to the maternal organism. In the face of the relatively large amounts of insulin injected into the mother, however, it is possible, in view of Allan's results, that the small contribution from the foetuses could have no measurable influence on the maternal sugar balance.

I have attempted to review but a small part of the work relating to insulin and have only cursorily referred to the perplexing problem of the mechanism of its action in the animal body. Facts of importance in this regard come almost daily to light and it is to be anticipated that, as these accumulate, a great advance will become possible in our knowledge of the history of carbohydrates in the animal body.

1. Practically all observers have confirmed the observation first made by Dickson and Pember that the R. Q. (respiratory quotient) rises somewhat in normal animals injected with insulin but the extent of this rise is not sufficient to indicate that increased combustio of glucose can be the significant cause for the rapid reduction in blood sugar.

2. There was one fish in which the blood sugar remained at a high level even eight days after asphyxia.

From Nobel Lectures, Physiology or Medicine 1922-1941.