Hello, my name is Jens Holst I'm a professor of medical physiology at the University of Copenhagen. And I'm here to talk about glucose regulation. Let us begin by considering the normal concentrations of glucose in the circulating body fluids. What should we chose, blood or plasma? We will choose plasma, because the concentrations in the red blood cells is low. What matters is the amount of glucose that can diffuse freely and reach target cells, serving as fuel or exerting regulatory functions. The concentration in plasma is around 5 millimoles per liter, corresponding to 90 milligrams per deciliter, and is remarkably constant. In healthy subjects, it rarely falls below 4 mmol/l, and rarely increases beyond 7 mmol/l. With this tight control, one could imagine that glucose control is important, and this is indeed the case. Numerous tissues depend on glucose for energy supply. To support vital functions. Particularly the central nervous system cannot operate without adequate supplies of glucose, and the same is true for the red blood cells. If glucose falls too much, this is what we call hypoglycaemia, we may experience confusion, convulsions, loss of consciousness. And eventually death. Conversely acute elevations of plasma glucose are associated with impaired neural functions. Especially cognitive functions. But the real danger is the damage to a large number of proteins caused by prolonged elevations of plasma glucose. This is what causes the devastating diabetic complications. Neuropathy, retinopathy, nephropathy the so called microvascular complications. Chronic hyperglycemia is also associated with macrovascular damage, which may lead to myocardial infarction, stroke and amputations. But how is it possible to maintain this constant glucose level? To answer this question we must consider the glucose fluxes inside the body that occur during daily life. And that will reveal that the consumption and production are so closely matched that deviations from five millimole are very rare in spite of very large flux differences depending on the physiological state. The glucose and plasma is either derived from the diet, [COUGH] and absorbed from the gut into the bloodstream, after digestion of dietary carbohydrates. Alternatively, it is produced in tissues capable of producing glucose for export. The amount of glucose from the diet varies, of course, but may amount to as much as 3000 millimoles. Or some 500 grammes, or 8 to 9 kilojoules per 24 hours. Where does all this glucose go? It enters the volume of distribution for glucose and that is roughly equivalent to the extracellular space. This is because glucose cannot cross the cell membranes and enter the cells unless these are equipped with special transporters. The extracellular space roughly corresponds to about 20% of the body weight. That is 14 liters in a person with a body weight of 70 kilograms. 3,000 millimoles in 14 litres? This would amount to more than 200 millimoles per liter which would be absolutely lethal. But glucose is also removed from the circulation. The brain and the rest of the central nervous system needs a constant supply of some 35 millimoles per hour. That's about half of the pool. What is the pool? So it's these 14 liters of extracellular volume with a concentration of 5 millimoles per liter which equals 70 millimoles per liter. And then we have the muscles. During maximum muscular work the muscles may take up several hundred of millimoles per hour, which would of course rapidly empty the pool. It's clear that we need very efficient mechanisms to ensure that the glucose concentration stays normal in both of these situations. So, what can the body do with the absorbed glucose? There are two possibilities. One is that it can be metabolized in the tissue. But this process is of course limited to the energy requirements of the tissues. Once these are satisfied no more glucose is disposed of in that way. The second is to deposit the glucose, this occurs in several tissues. The most important ones being the liver In the skeletal muscular tissues. Here glucose units are combined into large molecular weight glycogen molecules facilitated by the glycogen synthese pathway. The glucose stored as glycogen can be mobilized again. The liver can split the glycogen molecules again. And actually re export the individual glucose units. The latter is due to the fact that the liver cells express an enzyme called glucose six phosphatase which allows exit of glucose. The muscles can also split glycogen but only for internal use, that cannot very well export glucose. Once the glycogen deposits are filled, the organism cannot store anymore glucose, but may instead convert the glucose into fat. Both the liver and the fat cells of the adipose tissues are capable of synthesizing fatty acids. And eventually triglycerides from glucose. It's well recognized that storage in this way is almost unlimited. Can fat be mobilized to bring back glucose to the bloodstream? Not readily. The triglycerides may undergo lipolysis whereby the fatty acids are liberated. The fatty acids may then be exported and transported to tissues in need of energy, particularly the muscles, for combustion. The backbone of the triglycerides, the glycerol moiety, may also be exported and transported to the liver. Where it can serve as substrate for the process designated gluconeogenesis, whereby new molecules of glucose are synthesized. Clearly, the liver plays a central role in the regulation of the plasma glucose concentration it does so, not only because of its ability to take up considerable amounts of glucose, but also to produce glucose if needed. To summarize, the liver takes up glucose and stores it as glycogen in the process named glycogenolysis. But it can also produce glucose for export to the circulation either by mobilizing the stores of glycogen, so called glycogenolysis Or by production of new glucose from various substrates, so-called gluconeogenesis. We mentioned glycerol as a substrate, but there are other important substrates. These include lactate, derived from anaerobic metabolism of glucose in the tissues, as well as amino acids liberated from peripheral tissues, for instance, during fasting. It is possible to measure the fluxes of glucose in humans with minimal invasion by infusing at a constant rate, isotopically-labeled glucose, which allows one to follow the fate of the molecules in the body. By measuring the varying dilution of the tracer, the radioactive glucose, in the glucose pool, it is possible to determine both the formation of glucose in the body, the so-called rate of appearance, and the total glucose disposal, the rate of disappearance. We should mention that also the kidneys are capable of producing small amounts of glucose by gluconeogenesis. The kidneys may also help to dispose of glucose at very high concentrations. The maximum capacity for re-absorption of glucose in the kidneys is reached at a plasma concentration around ten millimoles per liter. And at higher concentrations, glucose is, therefore, lost in the urine. Clearly, this is not important for healthy individuals, but occurs frequently in diabetes where we talk about glucose urea. Since the excretion of glucose is accompanied by a considerable loss of water, so-called Osmotic Diuresis. Glycose urea may lead to serious losses of fluid and electrolytes in patients with dysregulated diabetes. Thus, in a person in nutritional balance, dietary carbohydrates are either combusted or rapidly deposited as glycogen and muscles and liver. In the interdigestive periods, when glucose uptake from the gut has ceased, the liver starts to export glucose, and is capable of maintaining a constant plasma glucose concentration for lengthy periods. In the beginning, the predominating mechanism will be glycogenolysis. But the liver can only store glycogen enough to support the bodily needs for about 24 hours. However, long before the stores are exhausted, the liver starts to produce glucose by gluconeogenesis. And this pathway is sufficient to maintain plasma glucose levels for many days, as is evident from studies of people subjected to starvation which does normally not cause hypoglycemia. This situation is obviously extremely demanding with respect to supplies of substrates, which eventually will result in a catabolic state where the gluconeogenic substrate is amino acids from body proteins. The question then arises, where are the sensors and regulatory mechanisms in glucose homeostasis? What makes the liver switch its functions according to the metabolic demands? What makes skeletal muscles switch from carbohydrate to fatty acid oxidation? The answer is, of course, the pancreatic endocrine Islets of Langerhans, albeit several other mechanisms may play a roll as well. Our approximately 2 million pancreatic islets, which make up 1 to 2% of the pancreatic volume, contain five endocrine cell types. The two most important ones, making up more than 90% of the cells, are the insulin-producing beta cells and the glucagon-producing alpha cells. In total, a couple of thousandths per islet. The remaining cell types are firstly, the somatostatin-producing delta cells, which probably exert important paracrine regulatory functions within the islets. Secondly cells producing pancreatic polypeptide with no known function. And finally, ghrelin-producing cells, the importance of which is also unclear. The arrangement of the cells in the islet varies somewhat between the species and appears somewhat irregular in humans. The arrangement is probably very important for intra-islet regulatory processes. For example, somatostatin powerfully inhibits the secretion of both insulin and glucagon. And insulin is thought to inhibit glucagon secretion, while glucagon stimulates insulin secretion. For two such cells lying next to each other, it is easy to imagine that they might influence each other's functions. But these interrelationships are not very well worked out. Nevertheless, the main function of the endocrine pancreas is well-established. It reacts by increasing instant secretion as the concentration of glucose in the plasma that profuses it rises, and with increasing secretion of glucagon, if the glucose concentration falls. One way of studying this is to isolate surgically the pancreas and keep it alive with artificial media for which one can control glucose concentrations. The mechanism of glucose stimulation of the beta cells has been worked out in considerable detail. The beta cell is a glucose sensor and controller in one. It reacts to changes in plasma glucose concentrations by producing appropriate amounts of insulin. So what is the glucose sensor? Like in other cells, glucose cannot directly pass into the beta cells, but it is quipped with a glucose transporter, a transmembrane protein that facilitates passage of glucose. This particular transporter is called Glut 2 and it's characterized by having a Km close to the normal plasma glucose concentration. This causes the transporter to operate at 1-order kinetics for glucose, and therefore ensures the plasma glucose transport into the beta cell is proportional to the exterior glucose concentration. Once in the beta cells, the glucose is phosphorlyated by a specific enzyme, glucokinase, with a similar Km as the transporter, so that the phosphorylation rate is roughly proportional to the plasma concentrations. These two molecules, the transporter and the glucokinase, constitute the glucose sensor of the beta cells. Since together, they allow formation of glucose-6-phosphate at a rate that is proportional to plasma glucose. Phosphorylated glucose then enters glycolysis, with ensuing formation of ATP. This cytosolic ATP interacts with certain ATP-sensitive potassium channels in the beta cell membrane, the KATP channels, where increased ATP reduces the opening probability of the channel. Again, to an extent that is proportional to plasma glucose. The reduction diminishes the efflux of potassium ions from the cell. Since the membrane potential of the beta cells to a large extent is generated by efflux of positively charged potassium ions, this means that the cell will become depolarized. The depolarization, in turn, will increase the opening probability of voltage gated calcium channels. And because of the steep gradient for calcium, with 10,000 times higher concentrations outside compared to inside the cells, calcium will enter the cell. An elevated intracellular level of free ionized calcium is exactly what is needed to initiate the process of exocytosis, whereby intracellular insulin containing granules are transported to the cell membranes, where they open and release their contents to the exterior. Clearly, any other process that causes depolarization or elevated intracellular calcium levels may also influence secretion. Thus, other fuels including lipids and amino acids may also generate ATP and influence secretion. Certain hormone and neurotransmitter receptors are coupled to intracellular signaling pathways that may elevate intracellular calcium from intracellular stores. Some hormones, notably the incretin hormones, which we will talk about later, activate receptors coupled to the membrane associated adenylate cyclase, leading to the formation of cyclic AMP, which may both directly and indirectly influence both the kATP channels and exocytosis. Interestingly, the sulfonylureas, among the most widely used anti-diabetic drugs, exert their action by binding to and blocking the kATP channels, and in this way activating the machinery for insulin release. By acting on the kATP channels they bypass glucose metabolism and therefore will cause insulin secretion regardless of the glucose levels surrounding the beta cells. This explains that they may produce inappropriate amounts of insulin and therefore cause unintended hypoglycemia. The alpha cells share some of the biochemical features of the beta cells but react, as mentioned, with decreasing secretion in response to increasing glucose concentrations, as nicely illustrated in these experiments in healthy volunteers exposed to both high and low plasma concentrations of glucose. The mechanisms responsible for this have still not been completely worked out, undoubtedly because pure isolated alpha cells are not easy to get hold of. There is good evidence that one of the functions of the somatostatin secreting delta cells is to regulate glucagon secretion. As mentioned, the main regulatory mechanisms for the Isles is their ability to react to change in concentrations of glucose in plasma with appropriate alterations in the secretion of insulin and glucagon. But is this enough to keep plasma glucose constant? Indeed, the islets will respond to several other important stimuli. For instance, what is the role of other nutrients? Lipids have limited effects on both insulin and glucagon secretion. But recent evidence suggests that beta cells are equipped with a number of receptors for both the long- and short-chain fatty acids, which may play a role in its maintenance of insulin secretion. Otherwise the effects of lipids are most often discussed into the context of lipotoxicity, deleterious effects on beta cell function of high lipid levels as seen in obesity. However, as briefly mentioned, several amino acids provide a powerful stimulus to both insulin and glucagon secretion. Indeed, both alpha and beta cell function may be evaluated with arginine tests, where arginine is injected intravenously and insulin and glucagon responses are measured in plasma shortly after. Protein-rich meals likewise produce a strong stimulus. The combined action on both hormones makes sense. The insulin response serves to enhance peripheral uptake of amino acids and their incorporation into tissue proteins in agreement with insulin's general anabolic activity. However, if the meal does not contain an equivalent amount of carbohydrate, one could fear that the insulin response might result in hypoglycemia. But this is prevented by the simultaneous stimulation of glucagon secretion. That this actually happens has been demonstrated in simulation experiments. The presence of cholinergic and other neurotransmitter receptors on the beta and alpha cells suggests that the autonomic intervention of the islets also plays a role. Thus vagal stimulation provides a powerful stimulus to both insulin and glucagon secretion, suggesting that, for instance, meal stimulation, in addition to the effects of absorbed nutrients, also engages a neurocomponent. Indeed, there are numerous reports on the existence of a cephalic phase for insulin secretion. But the sympathetic division of the autonomic nervous system may be even more important. Stimulation of the sympathetic nerve supply to the pancreas will strongly inhibit insulin secretion and enhance glucagon secretion. This is one of the mechanisms engaged during muscular work, and as we shall see later, these changes are essential for the maintenance of plasma glucose levels in this situation. But are hormones from outside the pancreas also important for islet function? The answer is yes. In fact, it turns out that up to 70% of the postprandial insulin response is caused by the actions of hormones secreted from the gut, the so-called incretin hormones. The amplification of insulin secretion by gut hormones is called the incretin effect. The incretin effect is normally evaluated by comparing the insulin responses to an oral glucose load and to an intravenous infusion of glucose adjusted to result in similar glucose concentrations. Thus, as shown in the figure, it is clear that the oral route causes much higher insulin secretion. The incretin effect is very important for keeping down postprandial glucose levels. Have a look at these experiments in healthy individuals. Here, glucose in amounts ranging from 25 to 100 grams were given orally, and the resulting excretions were copied by intravenous infusions. The most surprising observation is that the glucose excretions are virtually identical, in spite of up to four-fold differences in glucose loads. It is the incretin effect. In the next figure we see the insulin responses to the various glucose loads. And it's clear that insulin secretion is dramatically and dose-dependently increased in response to the increasing oral loads. In other words, the incretin effect ensures that plasma glucose excretions after carbohydrate loading are kept at a low and relatively constant level regardless of the amount of carbohydrate ingested. Which are the hormones responsible for the incretin effect? The two most important ones are glucose-dependent insulinotropic polypeptide, in short, GIP, and the glucagon-like peptide-1, GLP-1. Both have remarkable effects on the beta cells. The incretin effect is of particular clinical interest because it is almost completely lost in patients with type 2 diabetes and this loss contributes considerably to the inability of these patients to secrete sufficient amounts of insulin. Fortunately one of the hormones, GLP-1, is nevertheless capable of stimulating insulin secretion in supraphysiological doses. And because of this, it is possible to treat type 2 diabetes with GLP-1 agonists. [MUSIC]
