Cellular Cancer Therapy, part 4

Chapter 3      General Facts About Insulin

    The normal quantity of glucose in the blood, determined by the glucose oxidase method, is 60—80 mg./100 ml. In arterial blood the concentration of glucose is 15—30 mg/100 ml more than in venous blood. The concentration of the blood glucose is maintained approximately constant (homeostasis) independently of the intake of carbohydrates through the ingestion of food. Homeostasis of the glucose is determined by various regulatory hormones: some elevate its concentration in the blood, and others lower it. The chart below shows these two types of hormonal mechanisms:

    Apart from these chemical messengers that regulate the level of glycemia, the autonomic nervous system (sympathetic and parasympathetic) and the CNS (picadura puncture of the fourth ventricle, which Claude Bernard called "diabetic" in 1855) also participate in this regulation, as well as the liver, in that it is this organ that stores glucose In the form of glucogen.

    Bernard supposed that the increase in hepatic glucogen after a meal was a direct consequence of the glucose ingested and was transformed into glucogen in the liver. This is only partially true.

    Nowadays, it is known that a large part of the glucose that is ingested and penetrates into the hepatic circulation passes through the liver to eventually be metabolized in other places. However, some of it passes through the hepatic cells by membrane transport and is converted there into glucogen through glucogenesis. This process has to be facilitated by the energy stored in the ATP, which phosphorylates glucose in the presence of the enzyme hexocinase. In this way, glucose-6—phosphate is formed and then, by phosphoglucomutase, transformed into glucogen through the intermediary stage of uridindiphosphoglucose.

    However, these means can only produce a small part of the total amount of hepatic glucogen, since this polymer can also be formed from certain amino acids that, like glucose, are products of digestion. It can also be formed starting from the lactic acid (produced in the muscles and which the blood passes on to the liver), and to a smaller degree, from fats. The formation of glucogen from compounds that are not carbohydrates is called gluconeogenesis. The amino acids that form glucogen (glycine, alanine, serine, cysteine) are known as glucogenic amino acids. The administration of these to diabetic animals causes the appearance of sugar in the urine. For example, alanine is broken down and transformed into pyruvate by transamination. Pyruvate can be oxidized by the activity of the citric acid cycle or transformed, by way of fructose phosphate and glucose phosphate into glucogen.

    The metabolism of the lactate derived from muscle tissue follows the same steps. The course of the metabolism of fats is less clear, but for the moment it suffices to say that the glucogen stored in the liver comes from several sources.

    The other important function of the liver within this framework, and also discovered by Bernard, is the enzymatic breakdown of glucogen into glucose. This process, called glucogenolysis, is carried out in the first place by phosphorylation which transforms the glucogen into glucose-1—phosphate. This, in turn, converted into glucose—6—phosphate, and further into glucose and inorganic phosphates by glucose—6—phosphate. Thus, the liver contributes in three important ways to the metabolism of carbohydrates and, in doing so, provides everything necessary for the controlled storage of macroenergetic molecules and their release into the blood.

    The role played by the muscles is no less important, but their contribution is different from that of the liver, for they have to do with the oxidation release of energy and its manifestation as muscular work. The glucose released in the liver and a large portion of that obtained directly by the absorption after meals passes into the muscle cells by active transport. In the interior of the cell, it is phosphorylated by the transfer of the terminal phosphate group of the ATP and the glucose—6—phosphate formed in this way enters into one of the metabolic cycles of the liver. A part of the glucose-6—phosphate is reconstituted into glucogen; and part is metabolized directly with a release of energy. At the right time, the stored glucogen is despolymerized and used in this way. We can separate the breakdown of glucogen into two phases: one, called glucolysis, is the anaerobic breakdown of glucose—6—phosphate forming pyruvate, which is transformed quickly into lactate and acetylcoenzyme A. The other is aerobic and depends, as a result, on an adequate sup 17 of oxygen to the muscle tissue; this phase includes the oxidation breakdown of the acetyl group of the acetylcoenzyme A to CO2 and H2O by way of the citric acid cycle or the Krebs cycle. These processes also occur in the liver, but the difference between the hepatic cells and the muscle cells is that the latter do not have glucose—6—phosphates which break down glucose—6— phosphate, or fructose—1, 6—diphosphatase which converts glucose—1, 6—diphosphate into fructose—6—phosphate. Therefore, the muscle cells, differently from the liver cells, cannot transform glucogen into glucose nor carry out gluconeogenesis. Figure 3.1 provides a summary of these processes.

    As has already been said, the existence of glucose in the blood is called glycemia. The homeostatic regulation of its concentration can he easily shown by studying, in man, ‘the course that it follows after the ingestion of carbohydrates. An immediate result is the elevation of the level of blood sugar, which is called hyperglycemia and subsequent recovery constitute what is known as the glucose tolerance Lest, and it is clinically used to investigate abnormalities in the metabolism of carbohydrates. The person to he investigated fasts during some eight hours and drinks a glucose solution. Samples of blood are taken before the test and every 30 minutes from then on, and the glucose content of the samples is determined by any of the known methods, preferably the glucose oxidase method. The glucose level, initially observed to he about 70—80 ng/100 ml, reaches a maximum in 3O—45 minutes, and soon begins to re— turn to normal which it reaches after about two hours. The initial elevation is due to the flooding of the blood with glucose before the regulating mechanism can control it. The subsequent fall is due, on the one hand, to the oxidation of the glucose, and on the other, to its conversion into glucogen in the tissues, particularly in the liver and the muscles.

    The description above refers to the regulation of the level of blood glucose in normal individuals; it shows the action of a system of flux equilibrium when the homeostatic regulating mechanisms are functioning normally. However, they are not always functioning normally and the study of what happens in these circumstances is what has led to the under— standing of how the metabolism of carbohydrates is regulated. In patients with a slight alternation of glucose metabolism, the tolerance test for glucose shows that the glycemia in fasting is within normal limits but its increment after the ingestion of glucose is greater and more prolonged than normal, the effects of which can be discovered in the urine. In normal people, the glucose contained in the blood is filtered to the proximal renal tubules through the glomerules and absorbed actively in the distal tubules, which is why the urine is glucose—free. If, in spite of this, the level of glucose in the blood goes beyond a critical point, the tubules are incapable of absorbing all of t}Le glucose that passes in the urine.

    As a consequence, a little of it will be eliminated in the urine, with the result that it will become sweet. The critical point is reached by a concentration of approximately 160 mg/100 ml.

    In 1969, a peculiar characteristic of the microanatomy of the mammalian pancreas was discovered. This organ is formed preponderantly of cells that secrete the digestive enzymes of the pancreatic juices, but Paul Langerhans demonstrated the existence, of many snail islands or groups of cells that can easily be distinguished in the zymogenous tissue. Other investigators proved later the great importance that these cells have in the metabolism of carbohydrates. These groups of cells are now called the Isles of Langerhans, and produce an internal secretion which in 1901 was given the name ‘insulin.’ The importance of the discovery of insulin was that it opened the way for the preparation of pancreatic extracts to be administered to human patients.

    The production of insulin in the ‘isles’ of pancreatic tissue is a characteristic of the vertebrates that includes everything from fish to mammals, and now we know the real origin of insulin. It has been known for a long time that there are two types of cells in the isles of Langerhans in mammals, that is, A (or alpha) cells and B (or beta) cells. The B cells are already found in animals such as lampreys, which are vertebrates that do not have mandibles, and are situated at a lower evolutionary level than fish. However, both kinds of cells, A and B, exist in the pancreatic tissue of all other vertebrates from fish to man. There is also frequently a third type of cell called D or A1. Of the three types of cells, which can be distinguished by their different reactions to staining, the only one which produces insulin is type B. One of the things that support this conclusion is that these cells contain granules which give a positive reaction to the histochemical tests for the sulfahydrile groups which are found in the insulin molecule.

    In 1926, J. J. Abel isolated insulin in crystal form. At that time it was known that this hormone was of a proteinic nature but the determination of its chemical composition presented formidable ‘difficulties. It was known that protein molecules were very complex, but the fundamental properties of their structure were not understood, though it seemed possible that their properties could be determined by the specific order or the position of the residues of the amino acids along the polypeptide chain; but the idea could not be proven, since methods for determining the order of amino acids in proteins still had not been developed.

    Until 1945 the knowledge in this field progressed little; but during the next ten years, the work of Sanger at the University of Cambridge established the complete chemical structure of the insulin molecule. The insulin molecule has a molecular weight of 6,000 and is composed of two polypeptide chains, A and B, where the first is shorter.

    The two chains are united in two places by disulfide bridges of cysteine residue, and the two points of A are connected by a third disulfide bridge. This description refers to the structure of the insulin of the bovine pancreas.

    It was said above that the B cells and the insulin they secrete are a basic characteristic of physiological organization. However, thanks to Sanger’s work, we now know that while the insulin molecule possesses the characteristic property of making tine glucose level of the blood lower and achieving other effects associated with this action, its chemical structure is not uniform, for there are even some variations in it among mammals, as can be seen in the chart in Fig. 3.2.

    In spite of these variations, they do not seem to correspond to important correlative variations in the biological power of the molecules. Many of these variations are found in the A chain, which is protected by the disulfide bridge within it, but other variations occur in other parts of this chain, and in the B chain as well. It is clear that these variations consist in the substitution of one amino acid for another and it is supposed that these substitutions are the result of genetic mutations that modify the programming of the secretion of the hormone in such a way that they do not alter its final ability to influence the metabolism of carbohydrates.

    Even more prominent differences exist in the composition of the amino acids of the teleostic fish——the codfish (Gadus callarias) and the bonito (Symnosarda alleterata)—— as between those and bovine insulin. Even so, just as is the case with the differences among mammalian insulins, when these products are tested on rats, little difference in their biological activity can be noticed.

    The insulins of different species, however, differ in terms of their antigenicity. Insulin, since it is a protein, can act as an antigen, that is, injecting insulin from one species into another the latter can produce antibodies to combat the insulin of the former. This means that, for example, bovine insulin can cause the production of antibodies in the horse, and in certain circumstances, the serum of the horse that contains these antibodies can neutralize the biological activity of the insulin in the bovine serum. Based on this fact, it can be demonstrated that the concentration of bovine—insulin antibodies in the horse necessary to neutralize one biological unit of codfish insulin is forty times greater than that necessary for the neutralization of one unit of homologous insulin. From those results and other data we can draw some conclusions not only about insulin, but also about other proteins. The order of amino acids in a protein is known as the primary structure of the molecule. However, the chains that make it up can be arranged not as a linear chain. but in the form of a spiral, giving it a secondary structure, and, in turn, the spirals themselves can ho bent and intertwined to yield a tertiary structure. We might accept that all of the properties of the molecule arc determined by the overall. structure or configuration; and if this wore so, then it would also be conceivable that only a small portion of the molecule could be responsible o for the specific activity that is manifested in a particular regulating response. This small portion could be considered an ‘active site,’ and the rest of the molecule would have other properties such as the antigenetic ones we just mentioned.

    As far as we know, this idea is purely speculative, though it gives a possible explanation as to why some amino acids can be substituted without altering the molecule’s fundamental biological activity, and, consequently, we can consider these substitutions as occurring in parts of the molecule that do not have an active site. However, in the particular case of insulin, we arc forced to conclude that the portions of the molecule that determine its immunological properties should differ from those that condition its metabolic effects. This is the result of considering the immunological differences between the different hinds of insulin, which all have similar metabolic effects.

    When the relationship between the molecular structure of a protein hormone and its biological activity is known, hopefully we will be able to understand the way in which it carries out this activity. This problem leads us to the fundamental question about the nature of the relation between a biologically active molecule and the cells which it acts upon.

    At this time , this is undoubtedly the fundamental question to be asked.

    One of the most important things is to distinguish clearly between the physiological effects of a regulating agent and the means by which these effects are initiated within the white cells. With respect to the former, the most evident effects of the administration of insulin to an animal are the lessening of hyperglycemia, and the increased content of glucogen in the striated. muscles. Many other effects can be found, but in general the majority of them arc the consequence of the reciprocal relations among the relevant metabolic pathways. It is thought that insulin has direct influence on the metabolism of proteins and the retention of nitrogen, as well.

    We still have given no information about how insulin produces these effects, since to understand this means that an ample analysis at the level of the coil would he necessary. To this end one should begin with the fact that the muscles of normal animals treated with insulin can take in more glucose from the blood. For example, if a rabbit is given injections of glucose in doses of 1.5 g/kg/hour, during six hours, its glycemia is considerably raised. If during this period insulin is administered as well, then there is a considerable. increment in the glucogen in the muscles, but the glucogen in the liver decreases constantly during the treatment with insulin. Experiments with rat diaphragms in vitro are even more convincing. By adding glucose tagged with radioactive carbon to the medium in which the diaphragm is kept, its glucose uptake can be studied, since the uptake is indexed by the increasing radioactivity of the tissue. Thus it can be demonstrated that insulin favors glucose uptake as well as glucogenesis in the diaphragm tissue.

    If one wants to explain the way in which insulin produces these effects, it is necessary to isolate a stage in the uptake or metabolism of glucose that is specifically stimulated by insulin. Many of the steps in Fig. 3.1 can be excluded. For example, the complete removal of the pancreas does not effect glucolysis nor does it have any effect on the oxidation of pyruvate or citrate in the Citric Acid cycle. Consequently it is logical to infer that insulin influences one of the first stages of the metabolism of carbohydrates in muscle cells, before the metabolic pathways divide.

    One point that has been investigated in depth is that insulin might favor the formation of glucose—6—phosphate by stimulating the activity of hexocinase, the enzyme that catalyzes these processes. This hypothesis, however, has not found support in experimental results, and is no longer considered to be correct. One possible alternative is that insulin facilitates the transport of glucose through the fiber membrane, so that there is more of it available for glucogenesis. This opinion, defended especially by Levine, finds substantial evidential support, and also, in conjunction with what is now known, offers at least an operational hypothesis for the explanation of the effect of insulin on carbohydrate metabolism (see studies by Donato P. Senior). Of course it is less clear whether it explains as well the effect of the hormone on the synthesis of proteins, since there is no proof that insulin favors the passage of amino acids into the cell. In this case, it may well be that the hormone acts directly on the ribosomes; but it would be a bit premature to suppose that the way which insulin acts has finally been established, since even in the particular case of the metabolism of carbohydrates it is doubtful that it influences only membrane transport, as there is proof that it also stimulates glucogenesis directly.

Figure 3.3. The possibility of a reaction at the cell surface (1) which precedes the first phosphorylation of glucose (2). The reaction at the cell surface would be responsible for the transport of the carbohydrate to the interior of the cell.

    Up to here only the secretion of the B cells, which are found in all of the vertebrates, has been considered, and their function is well known. The same cannot be said of the A cells, which exist in fish and on up the phylogenetic scale, as they have not been identified in lampreys and such. For a long time these cells were considered unimportant, but now it is known that they produce glucagon which has a hyperglycemic effect and counteracts the hypoglycemic effect of insulin. Moreover, it is presumed that the transient hyperglycemic effect induced by tile intravenous injection of commercial insulin before subsequent hypoglycemia, is due to the fact that these insulins are ordinarily contaminated with glucagon.

    Glucagon is a crystalline polypeptide whose molecular weight is 3,485. The molecule consists of a single chain of 29 amino acids, thereby being smaller titan insulin. This chain contains tryptophan, which does not exist in insulin; but it does not possess the cysteine that insulin has. The proof that A cells secrete glucagon is partially based on the fact that even when the zymogen—secreting tissue has atrophied by tying the pancreatic conduit and the B cells have disappeared because of the action of aloxane, glucagon can still be extracted from the pancreas. On the other hand, the histochemical tests provide further support, since the A cells have a positive reaction to tryptophan, but negative for the sulfahydrate groups present in cysteine.

    If one wants to understand how glucagon acts, it is necessary to keep in mind that, in the liver, the despolymerization of glucogen into glucose is carried out in three steps:

1. Glucogen+ Pi —-> glucose—1—phosphate

2. Glucose—1—phosphate —-> glucose—6—phosphate

3. Glucose—6—phosphate —-> glucose+ Pi

(Pi represents inorganic phosphates.)

    The first of these reactions is the slowest, being catalyzed by phosphorylation. Thus the velocity of glucose release in the liver will depend on the activity of the phosphorylase present in the liver cells. In this stage is where we believe that glucagon has its effect on the increasing activity Of the enzyme, since, part of the cell’s content of’ the enzyme ±s in inactive form, becoming active, though, through phosphorylation, which is favored by the action of glucagon. This is how the effect of glucagon makes the quantity of glucogen in the liver decrease and the glycemia level increase. Moreover, it seems to favor gluconeogenesis, thereby increasing the total amount of carbohydrate available to be put into circulation.

    Some investigators believe, based on these facts, that glucagon is secreted like a hormone in response to the lowering of the glycemia level. However, its real behavior and significance continue to be obscure and it is still not possible to say whether it is a true hormone, just as there is no evidence for interpreting the role of the D cells and their secretions. One can only say that, as in the case of other components of the endocrine system, progress brings with it unsuspected complexity.

RECIPROCAL HORMONE ACTION IN THE REGULATION OF METABOLISM

    Experiments with glucagon have produced data that indicate that insulin does not act in isolation and its action has been described abstractly as in a mammal of unspecified size and age. Therefore, it is necessary to consider, from a more real point of view, both insulin and the mammalian organism.

    Insulin has reciprocal effects, which regulate metabolism, with hormones that originate in endocrine glands that have no anatomical relation with the isles of Langerhans. As an example one can mention the somatotropin of the hypophysis (the growth hormone).

    It is now known that somatotropin is a very complex protein that has differences from species to species as to its molecular composition which are comparable to the differences found in the insulin molecule in different species. It is possible that these differences influence the fact that mammalian somatotropins can foster growth in lower orders such as the teleosteos, while this hormone, when taken from the latter has no effect on the former. Our knowledge of this hormone has been greatly advanced by the studies of C. H. Li and his collaborators at Berkeley; they have determined the order of the amino acids in the human hormone molecule. This was a notable achievement because with a molecular weight of 21,500 it, is much bigger than insulin.

    Growth, as is well known, is a regulated phenomenon in which anabolism predominates and includes the synthesis of proteins for the permanent structure of the animal, involving the retention of nitrogen, which is influenced by insulin, which is why it is not surprising that there is a certain reciprocal effect of the two hormones. Bernardo A. Houssay demonstrated this reciprocal action for the first time in Buenos Aires in 1920. He demonstrated that the excision of the hypophysis in diabetic dogs (caused by removal of the pancreas) caused the intensity of the diabetic manifestations to decrease. If these animals were given pituitary extracts, the symptoms of diabetes increased as if they were simply pancreatectomized. These and other experiments demonstrated without a doubt that the hypophysis (or more correctly, the pars distalis) secretes a substance with effects that are the opposite of those of insulin. In tile beginning, this substance was called the diabetogenic factor of the hypophysis, but afterwards it was demonstrated that it was somatotropin.

    We know that somatotropin acts differently on the different metabolic pathways. It stimulates anabolism of proteins and increases the oxidation of fats. It also ro4uccs the velocity of glucose uptake in the muscle tissue and consequently limits the use of carbohydrates. By decreasing the carbohydrate metabolism, the effect of somatotropin tends to elevate the level of glycemia, which makes it diabetogenic under these conditions.

    Another example of its diabetogenic action is this: if somatotropin is continuously injected into intact animals, big insulin der4and is created which occasions the depletion of the Isles of Langerhans. The animals are diabetic at this time and suffer from a lack of insulin. However, this diabetes is different from that produced by pancreatectomy, because the excess somatotropin favors the retention of nitrogen while the pancreatectomy destroys the protein reserves causing a subsequent increase in the release of nitrogen. In any case, these are not the only effects of the hormone on metabolism. Thus it is, thought that it acts to stimulate the A and B cells, which results in an increment in the insulin in the pancreas, and a very complicated situation arises once more which is very poorly understood and continues to be the object of intensive study.

    Two other components of the endocrine system of vertebrates act reciprocally with insulin in the regulation of the metabolic pathways. One is the adrenal cortex which secretes various hormones (adrenocortical hormones) which have different effects’ on the metabolism of carbohydrates, and of water and the electrolytes that are essential to life. Some of these hormones, for example cortisol, are similar to somatotropin in that they constrain the metabolism of carbohydrates in the muscle tissue and this is the reason they are called glucocorticoids. Their actions differ from that of somatotropin in that the latter provokes a reduction of the absorption of amino acids by the cells. It is thought that this difference is due to the fact that glucocorticoids favor gluconeogenesis in the liver, spurring the transport of the proteins to it from other places in the body, and to which is due at least in part the tremendous protein consumption characteristic of the diabetic animal.

    The other constituent of the endocrine system that acts together with insulin is the suprarenal medulla. This tissue is totally different from a functional point of view, from the adrenocortical tissue around it. The suprarenal medulla secretes two hormones, adrenaline and noradrenaline, which are often called catecholamines, since their nucleus is of catechol. In general, it can be said that they have the effect of mobilizing the reserves that the organism has when it is under great demands.. For example, both hormones provoke an .increase in blood pressure, while adrenaline in particular increases the blood flow through the heart and stimulates glucogenolysis in the liver, by which the glycemia level is raised.

    Due to this, the action of adrenaline is similar to that of glucagon, which is mediated by increased phosphorylase activity. Ultimately, an increment in the release of lactic, acid from the muscles occurs. In this way adrenaline contributes to the activity of the animal by fomenting glucolysis in the muscle cells.

    This whole group of events can be considered as an emergency response fostered by a temporal distortion of the metabolism of carbohydrates’ where the animal has to depend on the regulatory mechanisms cited above to reestablish its normal situation and preserve homeostasis in the metabolism. There are other hormones that have not yet been mentioned whose actions are important for growth and metabolism. Among them we find thyroxin (T4) arid tri—iodo—thyronine (T3). The effects of both arc fundamentally similar, though not necessarily identical. A lack of thyroid bodies or of their hormones produces subnormal growth which is manifested in cretinism. Cretins will be dwarves unless they are treated with thyroid extract, and they differ from hypophysary dwarves in the defective development of their brains.

    One impressive fact is that Man and other vertebrates are capable of maintaining a constant or almost constant composition of organic liquids in an environment where there are incessant variations in the availability of water. This consistency, an essential factor in the homeostasis of mammals, depends to a large extent on two internal secretions that are entirely different from each other. One is produced by the hypophysis and the other is secreted by the suprarenal glands.

    The hypophysis is made up of two components: the adenohypophysis (which includes the pars tuberalis, pars distalis and pars intermedia) , and the neurohypophysis which, in higher vertebrates, is made up of the medial eminence, the infundibulum and the posterior lobe. The pars intermedia and neural lobe together form the posterior lobe. These two components come from separate embryonic origins, though they become closely related in one of the first stages of development. This close relationship is manefested in the intense reciprocal nature of their functioning.

    The adenohypophysis develops as an evagination of the so—called Rathke’s bag, which is bulging out of the embryonic bucal cavity and is made up of numerous cell types that secrete at least seven hormones. The peculiar role of the adenophypophysis in the endocrine system is the result of its appearance as the development of the inferior part (infundibulun) of the diencephalic floor, that is, the posterior part of the prosencephalon. The diencephalon is mainly in charge of the functioning of the regulatory mechanisms in vertebrates. The reason for this is that its floor and the.. inferior parts of its walls fern the territory called the hypothalamus, which is the encephalic center of the sympathetic and parasympathetic constituents of the autonomic nervous system. Through the application of electric stimuli to the hypothalamus we can cot proof of this. Thus, it is possible to provoke responses like an increase in blood pressure and pupil dilation, which normally are produced by the sympathetic component. The stimulation of other parts of the diencephalon produces reactions of the parasympathetic system.

part 5