RESEARCH

There are eight interdependent, projects with the main goal of understanding aspects of the pathogenesis of diabetes, including exercise, stress and hypoglycemia.

1) Extrapancreatic Glucagon

Since the discovery of insulin it was clear that diabetes is related to insulin deficiency. What was not known was whether glucagon, the second hormone of the endocrine pancreas, also played an important role in physiology and diabetes. There were two reasons for this: 1) Glucagon can increase glucose in plasma but only when given at very high concentrations; 2) Diabetes can be induced in animals either by selective chemical destruction of the insulin-producing B-cells or by total pancreatectomy, which removes the B-cells and the glucagon-producing A-cells. If glucagon were the hormone to counteract insulin, one would have expected that total pancreatectomy would induce milder diabetes than the selective destruction of the B-cells, but that was not the case. Dr. Unger from Dallas, Texas developed an immunoassay of glucagon and at least three laboratories, including his own, claimed that pancreatectomy in dogs results in the total disappearance of glucagon from blood. Mladen's first post-doctoral fellow, Dr. Kawamori, came from Japan. He is presently Chairman of the Department of Medicine at the Tokyo Juntendo Medical School and many of his medical residents trained in Mladen's laboratory. In this way, the contact and collaboration with Dr. Kawamori has continued over many years. When the pancreas was removed from dogs and they were not treated with insulin, glucagon in blood did not disappear (measured by Dr. S. Pek from the University of Michigan), but instead increased to very high levels. These results created a lot of excitement because it was contrary to the dogma. Mladen was so active discussing his views that a friend remarked that it seemed he was speaking in two conference rooms at the same time. It was his first major scientific battle won. This led to the discovery, stunning at the time that a pancreatic hormone can be produced in large amounts outside its original endocrine gland. Over the next ten years using tracer methods to measure glucose fluxes, biochemical, histological, electron microscopy, immunological methods and purifying stomach glucagon to homogeneity, they determined beyond a doubt that the parietal mucosa of the dog stomach can synthesize and secrete true glucagon while the remaining gastrointestinal tract does not produce glucagon but glucagon-like peptides instead. In Toronto he collaborated with Dr. C. Yip and in Geneva with Drs. Jeanrenaud, Miller, Girardier, Prentki and Renold (3,4).  This explained why, following pancreatectomy, diabetes is as severe as with selective destruction of the B-cells:- because the stomach can produce the same amount of glucagon as the pancreas. Interestingly, in one dog that was sacrificed 5 years after pancreatectomy, he showed in collaboration with Dr. Orci in Geneva that there was an incredibly large hyperplasia of the A-cells in its stomach. By electron microscopy the parietal mucosa of the stomach looked like a glucagon-producing endocrine gland. These findings changed some classical views of endocrinology and provided further proof that one hormone is not necessarily produced in only one endocrine gland.

The most exciting symposium that he attended was a 1974 Conference on Glucagon held at the Santa Ynez ranch in California. This property belonged to Mr. Kroc, founder and owner of McDonald's. The scientific program was administered by his brother, Dr. Robert L. Kroc. A few years before the conference there were major breakthroughs in the field of glucagon research and 12 scientists, including Mladen, lectured on these breakthroughs. Guilleman discovered somatostatin and some other hypothalamic-releasing hormones and received the Nobel Prize for this work shortly thereafter. Somatostatin suppresses secretion of glucagon and insulin and therefore it became possible, with the replacement of one pancreatic hormone to precisely determine the distinct functions of insulin and glucagon. Roger Unger provided the evidence that diabetes is not only caused by insulin deficiency but also by glucagon excess. Mladen's discovery of extrapancreatic glucagon in dogs was essential to confirm Unger's suggestion that diabetic hyperglycemia requires no only hypoinsulinemia, but also the presence of glucagon. Marty Rodbell suggested that G-proteins act as transducers in cell signaling by glucagon and other hormones. He received the Nobel Prize for this discovery 20 years later. The highest awards given in North America for diabetes research are the Lilly Award for Young Researchers, and the Banting Medal and Lectureship for Life Achievement. The participants at this particular Kroc Symposium would in the future be recipients of 7 Banting Medals, 5 Lilly Awards, 3 Claude Bernard Medals (the highest European Award for Diabetes Research) and 2 Nobel prizes.

2) Tracer Methods to Measure Glucose Fluxes

Tracer methods are a necessary tool because a change in glucose turnover can occur independently of the changes of glucose concentration. For example, if the pancreas releases insulin and glucagon at the same time, glucagon will increase glucose production by the liver while insulin will increase glucose uptake to the same degree. Using tracer methodology, Mladen showed glucagon's exquisite potency with only physiological amounts given. This explained why previous workers not using tracer methods believed that glucagon could exert effects only in very high amounts and in many organs, which could not explain its physiological role. Mladen showed that in physiological doses glucagon is very selective, acting only on the liver.

It is easy to measure glucose turnover in steady state. However, if one wishes to learn how various control systems work it is necessary to drive the system out of steady state. Drs. Steele, Altszuler and De Bodo in Brookhaven developed a mathematical model to do just this. Their model was based on certain assumptions regarding the distribution of glucose in extracellular fluid. The problem with their model was that it was not experimentally validated. It had been used only in dogs with uncertainty about the precision of the measurements. The first validations, performed in Toronto by Hetenyi and Cowan, provided information about averaged data over an entire experimental period. Dr. Geza Hetenyi came to the Department of Physiology at the C.H. Best Institute during the Hungarian revolution and played a key role as Mladen's mentor in both science and music. Mladen argued that the question was not whether such tracer methods were right or wrong, but that what is important is to determine the error throughout an experiment or clinical trial. Dr. J. Radziuk, a graduate student of Dr. K. Norwich and Mladen (5), compared the results of one tracer whose infusion rate was known, against the results of a different tracer whose infusion rate was unknown. In this way they could check the accuracy of the methodology on a minute-to-minute basis throughout the period of change. They concluded that the methodology, which Hetenyi and Cowan modified from the original proposal of Steele, had an error of only 10%. It also implied, however, that comparisons of two experimental conditions are only valid if the difference is much larger than the error of the method. The validation studies convinced clinical investigators that this methodology could be used with confidence. Application of non-steady state glucose turnover enabled him and his colleagues to study the selective effects of metabolic hormones (glucagon, insulin, catecholamines, IGF-1, glucocorticoids) and their interactions. This is a cornerstone for understanding the physiology of metabolic hormone actions as related to pathogenesis of diabetes, stress and exercise. With a close friend and colleague, George Steiner, they conducted the first clinical studies, using the validated tracer infusion method. This is now a standard method for measuring glucose turnover in man. They used the non-steady state approach for the first time to demonstrate that insulin resistance is present not only in obesity but also in lean, hypertriglyceridemics (6). It was known that obesity leads to insulin resistance but what was amazing was that the same degree of insulin resistance could be demonstrated in lean subjects with lipid disorders. With a double tracer technique they also quantified the role of the Cori cycle (recycling of glucose from liver to periphery and back) in metabolic adaptations. An important metabolic adaptation is that during fasting glucose production decreases, which minimizes protein degradation in muscle, the main source of gluconeogenesis. However, the most important mechanism for reducing gluconeogenesis is to recycle glucose through the Cori cycle, thus minimizing the need for carbon from amino acids to produce glucose. They determined for the first time that during fasting, the absolute rate of glucose recycling is unchanged, but since glucose production decreased, the proportion of glucose production, which was represented by recycling increased.  The collaboration with George prompted his interest in clinical research and stimulated him to strengthen the ties between physiology and medicine. Many basic scientists were apprehensive about breaking the barriers between themselves and clinical researchers. This has greatly changed since each large teaching hospital now has its own research Institute and since cross-appointments between various departments are flourishing.

The Federation of American Societies of Experimental Biology (FASEB) holds a Symposium every year to consider problems and advances of tracer methodology. he became Chairman and organizer of the Tracer Methodology Study Group in 1972 and continued until 1975. This was a very exciting task, especially when he became the first person asked to edit and publish the Proceedings from this Symposium in Federation Proceedings, a very prestigious and widely read journal.

The main method used to assess insulin's effects in vivo is the euglycemic, hyperinsulinemic clamp developed by Dr. Andres at National Institutes of Health. This is a critical method in diabetes because it can assess the magnitude of insulin resistance. With the clamp combined with tracers, one can assess hepatic glucose production separately from overall glucose utilization. The problem observed in every laboratory was that glucose production measurements were frequently negative, an artefact that invalidated the measurements. With a post-doctoral fellow, Dr. Diane Finegood, and in collaboration with Dr. Richard Bergman of the University of Southern California, they established a new methodology whereby the tracer infusion was not constant. They were able to predict variations in tracer infusion such that specific activity of glucose could be maintained constant and thereby the measurement of glucose production became very precise (7). Bergman and he developed a close friendship with an active exchange of ideas well beyond their scientific interests. This tracer method is now generally accepted as state-of-the-art methodology. However, when a glucose clamp is not used, one cannot predict precisely the changes in specific activity that will occur. Using different models, he nevertheless showed that it is possible to minimize the changes in specific activity and demonstrated that accurate data could be obtained if the change in specific activity is under 25%.  This new method was used in collaboration with Errol Marliss, from McGill University, for the first time during strenuous exercise, when rapid increments of glucose turnover occur within minutes and therefore the new tracer method is particularly essential (24).

Advances in tracer methodology opened the door for very precise investigation of the hormonal interactions and teleology. With Alan Cherrington, then a graduate student, he demonstrated how precisely glucagon and insulin need to interact to increase glucose turnover but maintain glucose homeostasis (8). With Friedrich Kemmer, a postdoctoral fellow from Germany, and colleagues O. and A. Sirek they solved an old problem. The question was why hypophysectomy can under certain conditions normalize plasma glucose in diabetes. They demonstrated that this is due to a decrease of glucose production, which is itself in part due to normalization of glucagon secretion. Contrary to earlier views, hypophysectomy deteriorated peripheral glucose uptake (9). With postdoctoral fellows, Kamil El-Tayeb and Pat Brubaker, and surgeon, Dr. Lavina Lickley they demonstrated that beta-endorphins, which are known to have opiate-like effects during stress, also have important effects on glucose metabolism. They potentiate epinephrine's hyperglycemic effect both by increasing glucose production and decreasing peripheral glucose clearance.

3) Exercise and Diabetes

Mladen's mentor in Toronto, Gerry Wrenshall, was a Type 1 diabetic. It was general knowledge that exercise improves metabolic control in diabetes and therefore he would go for a fast walk after each meal. They began exercise experiments in depancreatized dogs that were either infused with or totally deprived of insulin. The surprise was that in uncontrolled diabetic dogs during exercise, blood sugar did not decrease but instead it sharply increased due to an excessive increase in hepatic glucose production. The main beneficial effect of exercise in normal man and animals is an increase of glucose metabolic clearance. Glucose clearance reflects the efficiency of tissues to extract glucose independently of glucose concentration. This parameter is very important for the study of regulation of glucose transport because glucose concentration by itself promotes glucose utilization. They were the first to promote this concept in their first published exercise paper (10). In contrast to normal dogs, in diabetic animals glucose clearance did not increase. It was even more disturbing that an intraportal insulin infusion that decreased plasma glucose from 350 to 140 mg% still resulted in an increase of plasma glucose without the increase of glucose clearance during exercise. Clearly an acute basal intraportal insulin infusion did not improve insulin resistance. These experiments in dogs consisted of a 100m/min run on a flat treadmill, reflecting light exercise. In later experiments they increased the incline by 12% to reflect more intense exercise. It was interesting that under those conditions a basal insulin infusion could indeed normalize glucose turnover, illustrating the importance of the level of difficulty of exercise.

When Berson and Yallow developed the immunoassay for insulin in the sixties, it became evident that during exercise in non-diabetic subjects, insulin in plasma decreases up to 50%. Mladen hypothesized that since blood flow in muscle during exercise can increase by 20-fold or more despite a decrease in plasma insulin, the perfusion of muscle with insulin is maintained. Other laboratories indicated that muscle contraction increases glucose uptake independently of insulin. Mladen concluded that insulin during exercise in vivo is necessary to counterbalance factors that would decrease glucose uptake such as increased fatty acids and catecholamines (11). They then examined the mechanism of exercise-induced hypoglycemia in depancreatized dogs maintained on long-acting insulin. With Kawamori, he was very surprised to find that during exercise, plasma insulin increased dramatically. It was interesting that nobody had made this observation before, although the insulin assay had been available for quite a few years. A friend remarked at the time that what he observed was highly unlikely since it would surely have been noticed before. Nevertheless, he could now for the first time explain the mechanism of the development of hypoglycemia during exercise in insulin-treated patients (12). With increased insulin levels during exercise, glucose production did not increase to match the increased glucose uptake by the muscle. Even when insulin does not increase, the fact that exogenous insulin does not decrease may also inhibit the liver, but to a lesser extent. During his sabbatical leave in Geneva working with Michael Berger, Philippe Halban, (now President of the European Association for the Study of Diabetes) and Albert Renold he injected tritiated insulin, which was synthesized for the first time by Halban. This gave them the opportunity to study the pharmacokinetics of insulin absorption in rats running on a treadmill. Mobilization of insulin from its subcutaneous depot increased dramatically and most of the radioactive insulin was found in skeletal muscle. This also confirmed their hypothesis that during exercise perfusion of muscle with insulin can increase, presumably due to increased blood flow in the muscle (13). In collaboration with Drs. Zinman and Marliss in Toronto, they were then able to demonstrate the same observations in Type 1 diabetics. The diabetic subjects in which insulin infusion established near-normal plasma glucose had normal turnover rates in exercise. With injection of insulin, however, plasma insulin increased in the same fashion as in dogs and rats and again the increment of glucose production was prevented (14). He thus developed a hypothesis that explained why exercise in diabetics can result in unchanged glucose levels, in hyper- or hypoglycemia and this hypothesis was widely accepted. With insulin deficiency or resistance, there is overproduction and underutilization of glucose, and hyperglycemia ensues. On the other hand, with excessive insulin due to enhanced mobilization from its subcutaneous depot, glucose production is inhibited and its utilization is enhanced.

In Oxford, U.K., he worked in the laboratory of Eric Newsholme. Phosphorous nuclear magnetic resonance spectroscopy offered an opportunity to study intracellular pH and phosphocreatine continuously and non-invasively. This was done in collaboration with John Challis and George Rada and was the first NMR spectroscopy of muscle performed in diabetic rats (15). During muscular contractions in diabetic rats that were not insulin-treated, the bioenergetics studied were deficient.  Insulin-treated rats were normal. The big surprise was that if insulin treatment was discontinued for three days all the parameters studied remained normal. This clearly indicated that the continued presence of normal insulin values is not needed for glucose metabolism during exercise. The deficiency of diabetic rats with respect to muscular contraction was thus a consequence of chronic effects of the diabetic state. However, an effect of acute insulin withdrawal was observed in the post-contractile state. The rate of resynthesis of glycogen was greatly diminished. It is now well known that this defect is observed not only in diabetes, but also in non-diabetic offspring of diabetic parents.

Guilleman's discovery of somatostatin permitted him to study with Dr. B. Issekutz from Halifax, for the first time, the role of glucagon in regulation of glucose production during exercise in normal dogs (16). Dr. Issekutz left his native Hungary during the revolution, and was a pioneer is studying the effects of exercise on glucose and FFA turnover. A suppression of glucagon greatly decreased the increment of glucose production, resulting in hypoglycemia during exercise. This greatly changed the type of fuel utilized by the muscle. Hypoglycemia resulted in an increased release of catecholamines, which suppressed muscle glucose clearance but increased lipolysis. They demonstrated, and it is now widely accepted, that the main regulator of glucose production during moderate exercise is the ratio of glucagon to insulin. By preventing the drop in plasma glucose he was able to determine, with Ph.D. student D. Wasserman and in collaboration with Dr. L. Lickley, that glucagon controls most of the glucose production increment (17). Dr. Lickley is a surgeon who divided her main interest between breast cancer and experimental diabetes. She is a close friend and Head of Surgery in Women's College Hospital in Toronto. This collaboration continued for 20 years. The conclusion was that an important role of glucagon during exercise is to spare muscle glycogen by stimulating glucose production by the liver. This is of particular importance for repetitive or endurance exercise. Glucagon is even more important in exercising, diabetic dogs where suppression of glucagon completely prevents increments in glucose production (18). They also demonstrated that in diabetes defective glucose clearance during exercise can be fully normalized by the beta-blockade of catecholamines but only if some insulin is present. This clearly demonstrated that the main role of insulin during exercise is not its direct effect on the muscle but rather to counteract the negative effects of catecholamines on glucose utilization. The field of metabolic control and exercise and diabetes was reviewed with the late Michael Berger, former President of EASD (19).

He had a very productive and enjoyable collaboration with Dr. Amira Klip. It was known at the time that glucose is transported into various tissues by specialized proteins-glucose transporters. A stunning discovery by Cushman and Kono was that in the fat cell, by far the largest number of transporters is found in cell plasma and that insulin translocates the transporters to the plasma membrane where they exert their function. It was much more difficult to determine glucose transporters in the muscle and Amira was one of the first to develop the methodology required for the muscle. They demonstrated, together with a graduate student D. Dimitrakoudis, that in mild diabetic rats, glucose transporter number is decreased both in plasma membrane and inside the cell and that this was due not to insulin deficiency, but to hyperglycemia. They explored this problem using phlorizin. Since the time of Minkowski, more than 100 years ago, it had been known that phlorizin prevents reabsorption of glucose in the kidney. Therefore, it is possible to normalize glucose in diabetic animals by phlorizin treatment. Plasma glucose is normalized because excessive amounts of glucose are secreted in the urine. When they treated diabetic rats with phlorizin for a few days, glucose concentration normalized and they could normalize not only the number of glucose transporters but also the genetic expression of the gene for glucose transporters in the muscle (20).

This work with Amira continued with a post-doctoral fellow, Dr. Marette. They showed for the first time that most of the glucose transporters are translocated not to the plasma membrane, but to the transverse tubules of the skeletal muscle. This is physiologically very important because transverse tubules represent a larger surface area than the plasma membrane, are exposed to the extracellular milieu and play an important role in transporting ions and nutrients into the muscle fibre. A Ph.D. student in their laboratories, Theos Tsakiridis, demonstrated that the actin network, an important part of the cytoskeleton, is essential for the insulin stimulation of glucose transport and transporters (21). However, they further concluded that this network might be part of the signaling process rather than only a simple vehicle for transport of the vesicles. This was particularly evident when the stimulation of glucose transport was caused by mitochondrial uncoupling of the oxidative chain. This was achieved by dinitrolphenol, which mimics the effect of hypoxia and perhaps exercise. This signaling process was completely different from that with insulin. The key signals required for insulin stimulation, phosphatidylinositol 3-kinase and the actin network were not required when the oxidation chain was uncoupled (22). This observation, with observations from other laboratories explains why the effects of insulin and exercise are additive and why muscular contraction per se can stimulate glucose transport without insulin.

An ongoing collaboration with Errol Marliss from McGill University examines the effect of strenuous exercise in normal and Type 1 diabetic subjects. In contrast to his previous work in man and animals during moderate exercise, the changes in glucagon and insulin during strenuous exercise are not important in hepatic regulation of glucose production. This was demonstrated by using somatostatin infusions to clamp insulin, glucagon and growth hormone so that the secretion of those hormones could not be affected by exercise. Because in strenuous exercise, concentration of plasma catecholamines is excessive (16x), they postulated that in strenuous exercise control of glucose production shifts from the glucagon/insulin ratio to the effect of catecholamines (23). This would also imply that during strenuous exercise, mobilization of glycogen in muscle could play a bigger role than mobilization of glucose from the liver, since catecholamines are potent stimulators for both muscular and hepatic glycogenolysis. Interestingly, in contrast to moderate exercise, feeding did not shut down endogenous glucose production, an important mechanism to maintain glucose supply at the highest possible rate (23).

Insulin deficiency results in increased FFA levels, which according to Randle's hypothesis could inhibit peripheral glucose uptake. Mladen investigated this effect during moderate exercise in dogs with a post-doctoral fellow, Dr. Q. Shi and Dr. K. Yamatani from Japan. The main effect of inhibition of FFA oxidation was to decrease glucose production by the liver rather than a direct effect on glucose uptake in the muscle as proposed by Randle. Normalization of glucose production during exercise improved plasma glucose concentration in diabetic dogs, which in turn increased metabolic glucose clearance (MCR) in the muscle. These observations led to the hypothesis about the relationship between glucose concentration and glucose uptake in the muscle, protecting the muscle against excessive hyperglycemia. This will be described in more detail later.

His new early concepts regarding metabolic regulations in physiology and diabetes during exercise attracted wide attention. He was invited to organize the first Symposium on “Exercise and Diabetes” sponsored by the Kroc Foundation in 1978, and held in the same place where he had attended the Glucagon Symposium four years earlier (see books edited). This initiated a number of meetings on this topic, held every three years, in both Europe and the United States. It is generally considered that this conference greatly stimulated research in the field of diabetes and exercise, which is of paramount importance with respect to the disease. More recently, epidemiologists concluded that exercise not only can improve but may also prevent type 2 diabetes. Thus, exercise is considered a cornerstone in the treatment and prevention of type 2 diabetes. With Michael Berger, he wrote a review (24) that has been updated with different collaborators many times in a variety of books, of which the most important are 3, 4, 5, 6^th Edition of Ellenberg and Rifkin's Diabetes Mellitus (25).

The last six years we explored the mechanism of preventing hyperglycemia in an animal model of obese diabetic rats (ZDF). These animals have a deficient leptin receptor, and therefore, they eat excessively. With development of obesity, they also develop Type 2 diabetes. In a paper resulting from my D.Sc. in Zagreb, I indicated that new islet cells can originate either from hyperplasia of the islet cells, or from the cells of pancreatic ducts (1). Methodology of that time was very simple: now we have the availability of molecular methods to study the growth of insulin producing beta cells. What is amazing is that with only 1 hour of swimming per day, the rats, although obese, did not develop diabetes. The reason for this is that obese animals and humans are insulin resistant, but they only develop diabetes if the pancreas is unable to release huge amounts of insulin. Exercise stimulated the growth of the beta cells and we could demonstrate marked increase of new beta cells. Increased beta cell mass was also accompanied by an increased function. In diabetes a number of proteins are not processed properly and they accumulate in the beta cells. The prevention of hyperglycemia also prevented accumulation of such proteins and it prevented oxidative stress. We concluded that exercise first, improves uptake of glucose in the muscle (increased insulin sensitivity). High blood sugar is toxic for the beta cells and therefore, their mass and function deteriorates. However, prevention of hyperglycemia also prevents toxic effects on the beta cells. Improvement of the function of beta cells helps to prevent diabetes. Swimming as a modality of exercise is accompanied by stress. We then investigated the effect of volitional exercise where the rats have an opportunity to volunteer exercising in their cages. We observed the same effect. A very hot area of research right now is the fact that low grade infection occurs in obesity. That is an important factor that decreases action of insulin (insulin resistance). We investigated a number of markers of inflammation and could show that despite obesity, exercise attenuates the inflammation. Thus, such work in animals indicates the surprising fact that just one hour of exercise per day can prevent diabetes and strengthen the notion that exercise, and of course food control, are the key factors that can prevent onset of  Type 2 diabetes. When the rats stopped exercising, diabetes developed. This strengthened the notion that in order to prevent diabetes, a continuous, uninterrupted exercise program is needed (41).

It is well known that continuous stress deteriorates diabetes and probably can also accelerate the onset of diabetes. Very surprisingly, that is not the case in all types of stress. When we exposed the obese rats to neurogenic stress (rats are put into plastic tubing which restrains their movements), the onset of diabetes was also prevented. Physiological and molecular analysis indicated that this type of stress can decrease body weight and decrease the activity of the Hypothalamic-Pituitary-Adrenal Axis, which both have a beneficial effect. This is in the same line with work of the father of stress, Hans Selye. We indicated that certain types of stress improve, rather than deteriorate the health. Importantly, our work contrasts with common views that all stressors are deleterious for diabetes and illustrates that intermittent exposure to mild stressors and the ensuing adaptations may instead be important for normal physiological functioning by preparing the body to deal with threats to homeostasis (42).

4) The Relative Importance of Portal and Peripheral Insulin in Regulating Glucose Production:

One such example is the control of glucose production. Together with Dr. Adria Giacca, a post-doctoral fellow who came from Italy and who is now an Associate Professor in the Department of Physiology, University of Toronto, they demonstrated that in diabetic dogs a peripheral infusion of insulin is more effective in suppressing glucose production by the liver than a portal insulin infusion (26). This was very surprising since the general belief was that increased glucose production in diabetes is at least in part due to the fact that diabetics are treated with peripheral insulin injections. However, some 30 years ago Rachmiel Levine had already indicated this possibility, since insulin has very little effect on isolated liver cells. We thus confirmed an earlier key paper of Richard Bergman that arrived at the same conclusion working in normal dogs. The question arose as to how a peripheral metabolic effect of insulin can send signals to the liver. They investigated this in diabetic dogs and in normal and diabetic human subjects. Insulin cannot be delivered intraportally in humans, so in collaboration with Dr. Gary Lewis they infused tolbutamide to stimulate endogenous insulin secretion. Two weeks later they infused insulin peripherally and were able to mimic insulin secretion rates produced by tolbutamide. They concluded that in normal man, both peripheral signals and direct hepatic insulin secretion are of importance. We speculated that the direct hepatic effect of insulin is initially to inhibit glycogenolysis while the indirect effect of insulin eventually and predominantly suppresses gluconeogenesis. The main peripheral signal is insulin suppression of glucagon and free fatty acids (FFAs) (27). The residual direct effect of insulin is demonstrated when both glucagon and FFA are replaced so that a peripheral signal is abolished. They also concluded that in Type 2 diabetes, the sustained suppression of glucose production is due exclusively to peripheral signals (28).

5) Stress and Diabetes:

The effects of acute and chronic stress are wide-ranging and it is well known that they can markedly offset metabolic control in diabetes. Together with graduate students, P. Miles and M. Lekas, and post-doctoral fellows, Z.Q. Shi and K. Yamatani, Mladen induced acute stress in dogs by inserting a chronic cannula into the third ventricle (29,30). Carbachol injections mimic the muscarinic action of acetylcholine, which is a major neurotransmitter of the brain. Such an injection induces a release of all counterregulatory hormones (vasopressin, cortisol, catecholamines and glucagon). It is known that some types of stress can selectively suppress insulin secretion but in this model, insulin is not affected. In normal dogs, this stress induces a large increase in glucose turnover with only a 5% change in glucose concentration. Glucose homeostasis is maintained because stress induces an increase in glucose utilization that is independent of insulin. This was the first demonstration of a neuroendocrine pathway that can increase peripheral glucose utilization independently of insulin. In contrast, stress induced a major increase in glucose concentration because glucose uptake did not increase, as shown by graduate student Shirya Rashid. Resistance to stress in diabetes reflects a chronic defect that cannot be improved by acute hyperinsulinemic euglycemic clamps. Similarly to exercise, beta-blockade can in part restore the response of glucose uptake to stress (31). Thus they discovered the mechanism whereby stress in diabetes can offset glucose homeostasis.

6) Protection Against Excessive Hyperglycemia of Muscle and Liver, but not Pancreas

The next important invitation was to deliver the 1995 Solomon Berson Distinguished Lectureship of the American Physiological Society - Endocrinology and Metabolism Section at the Federation of American Societies of Experimental Biology in Atlanta entitled “The Yin-Yang of Carbohydrate Metabolism”. He is the only Canadian to have given that lecture.

The ancient Chinese philosophy of the Yin and the Yang can be used as a metaphor for the balance between active and passive interrelationships. In this philosophy, yin is dark and passive, while yang is light and active. In harmony the two are symbolized as the light and dark halves of a circle, and literally mean the dark and sunny sides of a hill. What is particularly useful with respect to glucose homeostasis is the yin-yang implication of an entire series of opposites, whose interplay (as one increases, the other decreases) defines the actual dynamic process. The thesis of his Berson Lecture was the proposition that in some organs, decreased glucose efficiency is not a defect, but rather a protective mechanism against diabetic complications. Glucose utilization is a result of opposing forces related to the effect of glucose itself, and to the interaction between the effects of glucose and insulin. These interactions are dynamic and reflect a continuum of synergistic and contrasting processes. Most of the complications of diabetes are due to chronic elevation of plasma glucose. Through mass effect, an excessive amount of glucose enters a variety of tissues resulting either in glycosylation of many proteins or in augmentation of otherwise insignificant metabolic pathways. This does not occur in either the muscle or the liver.

In diabetes, glucose uptake in the muscle is not decreased. The defect can be observed by measuring metabolic glucose clearance (MCR), which represents the ratio of glucose utilization to plasma glucose concentration and reflects the efficiency of glucose extraction by the tissues. Glucose clearance is significantly decreased in diabetes, which is one reason for development of hyperglycemia. This was attributed to a toxic effect of hyperglycemia on glucose uptake in peripheral tissues. He suggested that this is mainly an adaptive effect. Together with Dr. Geza Hetenyi, a former Chairman of the Department of Physiology at the University of Ottawa, they infused phlorizin to diabetic dogs either for a few days or acutely. The only effect of phlorizin is the prevention of glucose reabsorption by the kidney, causing glycosuria. With chronic or acute glucose normalization the defective glucose clearance substantially increased, providing the evidence that most, if not all of the diabetic defect was adaptive and could be fully normalized in dogs after many months. In his recent experiments (32) performed with an MD/PhD student, S. Fisher, he demonstrated that during exercise the severe defect of MCR in diabetic dogs was fully restored by an acute normalization of glucose by phlorizin and this was independent of FFA turnover and insulin levels. This demonstrates that regulation of glucose uptake during exercise is mainly dependent on muscular contraction and not insulin or FFAs. However, the energy balance in the muscle was maintained because when the FFA turnover was high, glucose oxidation decreased. In contrast to MCR, glucose uptake was near normal during rest and exercise in both normoglycemic and hyperglycemic dogs. However, because of decreased glucose transporters as described earlier, glucose clearance also decreases so that the net result is that the changes of glucose uptake are minimized. With Marliss, he demonstrated the same phenomenon in Type-1 diabetic patients whose plasma glucose was maintained overnight with insulin infusion at moderately hyperglycemic levels. During rest and strenuous exercise glucose utilization was normal. However, metabolic clearance rate (MCR) was decreased during rest and exercise (33). Again, this illustrates a perfect balance between glucose concentration and glucose MCR. However, because of low MCR blood sugar did not decrease following exercise, as it did in non-diabetics. This implies that diabetic subjects who require less insulin during and after moderate exercise, may require additional insulin injections following strenuous exercise (23). With Dr. Shi and graduate student, J. Mathoo, He demonstrated that the same mechanisms also work in the isolated hindquarter of the rat. In the total absence of insulin by appropriate infusions of glucose they kept plasma glucose concentrations normal, hypo- or hyperglycemic. With hyperglycemia, the number of glucose transporters in plasma membrane decreased while with hypoglycemia it increased. This was reflected with increased or decreased rates of MCR. Thus, during rest and exercise the muscle is protected against hypo- or hyperglycemia. Other laboratories have investigated the reason for muscle resistance against insulin, which also diminishes glucose uptake. However, both mechanisms contribute to diabetic hyperglycemia, which causes diabetic complications in other organs.

7) Glucose Cycling:

Glucose uptake in the liver is modified by three non-equilibrium reactions:- the glucose cycle, the fructose-6-phosphate cycle and the phosphoenol pyruvate cycle. Suad and Mladen were particularly interested in the glucose cycle in which entry of glucose is accelerated by the enzyme glucokinase, which yields glucose-6 phosphate. Some of the glucose-6 phosphate is then cycled back into glucose through the enzyme glucose-6 phosphatase. Thus through this mechanism a small amount of glucose taken up by the liver is recycled into the blood stream. They found that the activity of glucose cycling was increased in depancreatized and alloxan-induced diabetic dogs, in lean and obese Type-2 diabetic subjects as well as in acromegaly and hyperthyroidism (34,35). The percent increase in post-absorptive glucose cycling is more marked than the increase in glucose production. They suggested, therefore, that measurement of glucose cycling in addition to glucose production could be a valuable tool to assess the early metabolic derangements of glucose intolerance. The mechanism of increased glucose cycling in depancreatized dogs was then investigated (36). With Drs. Shi, Giacca and van der Werve, Chairman of the Department of Nutrition at the University of Montreal they measured liver enzymes by biopsy and glucose cycling by the double tracer method during anaesthesia. They concluded that increased hepatic glucose cycling from diabetes is mainly due to the increase of substrates for glucokinase and glucose-6 phosphatase rather than from changes in the total amount of enzymes. This has some similarity to regulations in the muscle because again glucose concentration by itself can regulate part of its rate of entry into the liver. It is not known how effective this mechanism is in reducing excessive metabolism of glucose into the liver.

8) Hypoglycemia:

A major acute complication of diabetes is a defective response of glucagon, catecholamines and glucocorticoids to insulin-induced hypoglycemia. The threat of hypoglycemia has increased since the treatment for diabetes has aimed for tight blood glucose control to decrease the risk of diabetic complications. Therefore, it is very important to develop a treatment strategy that would decrease the risk of hypoglycemia. The defect of glucagons and epinephrine response to hypoglycemia is puzzling because both counterregulatory responses are normal or even excessive during some stresses, such as moderate and strenuous exercise (23). Mladen tried to answer this question in dogs and rats by inducing diabetes by alloxan and streptozotocin, respectively. With this treatment, not only are most of the beta-cells destroyed but also the number of islets is markedly reduced. With Drs. Sudha Rastogi and Suad Efendic he showed that although in each islet the number of glucagon cells is greatly increased the total amount of glucagon in pancreas remains unchanged because of the reduction in the number of islet cells. It is well known that the release of glucagon by the pancreas is inhibited by both insulin and somatostatin. Since most beta-cells have been destroyed, somatostatin is the main paracrine inhibitor of the A-cell in diabetes. That is why it is of particular interest that in diabetic islets the ratio of somatostatin to glucagon is markedly increased. An acute insulin injection increased this ratio further. Theirs was the first demonstration that part of the defective mechanism in hypoglycemia may reflect alterations of this ratio in diabetes. Since somatostatin inhibits glucagon release, this could explain why glucagon-producing alpha cells are less sensitive to hypoglycemia, while they remain normally responsive to other stresses (37). The other factor that affects alpha cells is chronic hyperglycemia. With Dr. Shi (38) he then demonstrated that the defective glucagon responses are in part due to chronic hyperglycemia and hyperinsulinemia. Normalization of hypoglycemia without, but not with insulin, restored in part, glucagon's responsiveness in diabetic rats. This occurs because hyperinsulinemia offsets the beneficial effect of normalization of glucose.

Presently, he is investigating with Dr. Steve Matthews, a molecular neuroendocrinologist in the department, and graduate student Owen Chan, the gene expression of stress hormones and their receptors in the brain to find out how diabetes affects the function of the hypothalamic-pituitary-adrenal (HPA) axis and its relationship to sympathoadrenal responses in hypoglycemia. They demonstrated that diabetes increases the activity of the HPA axis, which is evidenced by increased expression of stress hormones and their receptors in the brain and by an increase of peripheral glucocorticoids. The impaired stress responses involved also decreased pituitary and adrenal sensitivity, and the basal hyperactivation of diabetic HPA axis is due to decreased glucocorticoid negative feedback sensitivity. With insulin treatment, glucocorticoid concentration normalizes but central components of the HPA axis remain increased. Since the central HPA axis is also associated with sympathetic activity, this could explain why insulin-treated diabetics retain a defect of the sympathoadrenal response to hypoglycemia. Response of the HPA axis to hypoglycemia is greatly reduced in diabetic rats. Normally during hyperglycemia expression of corticotrophin-releasing hormones increases and the expression of mineralocorticoid hormones decreases. That does not occur in diabetic rats. They also demonstrated that normalization of plasma glucose with or without insulin treatment can normalize the responses of the HPA axis to hypoglycemia. Thus, hyperglycemia and not hypoinsulinemia plays a key role in the fine turning of the HPA axis (39). It is well known that repetitive (antecedent) episodes of hypoglycemia increase the threat of further hypoglycemia episodes. They investigated with graduate student, Karen Inouye, the reason why counterregulatory resonses of epinephrine are deficient (40). They demonstrated that there is a defect of enzyme expression in the adrenal medulla that controls epinephrine and norepinephrine synthesis, which is further jeopardized with antecedent hypoglycemia. This could explain the main defect, which is diminished epinephrine response. This indicates the importance of the regulation of synthesis of those enzymes and opens new possibilities for pharmacological intervention.

When we treated the rats with insulin we could normalize glucagon and epinephrine defects, but the defect of the HPA axis persisted. Our insulin treatment normalized fasting blood sugar, but a moderate defect of fed blood sugar persisted. We therefore concluded that full restoration of counterregulation would only be possible if the control of blood sugar is fully normalized, which at the present time cannot be achieved.

We were then interested whether recurrent restraint stress also increases the threat of hypoglycemia similarly to the effect of episodes of antecedent hypoglycemia. Indeed in diabetic rats, these two effects (antecedent hypoglycemia and antecedent stress) were comparable with respect to the HPA axis. The defect correlated with decreased basal gene expression of PVN AVP and the anterior pituitary POMC mRNA. In these rats, there was practically no glucagon response to hypoglycemia and therefore, recurrent restraint stress could not further jeopardize glucagon responses. However, in contrast to antecedent hypoglycemia, recurrent restraint stress did not impair catecholamines counterregulation.

In this biographical sketch, a long journey has been taken to describe Mladen's research endeavours, which are all related to the physiology and pathophysiology of carbohydrate metabolism with a special emphasis on diabetes. To bring it all together, it would be useful to quote the citation that was read during his induction into the Royal Society of Canada: “He pioneered tracer methods for nonsteady-state glucose turnover, providing a cornerstone for quantifying hormonal interactions in glucoregulation and pathogenesis of diabetes. He established the significance of glucagon-insulin interaction in health and diabetes. His hypothesis concerning factors that determine beneficial or deleterious glucoregulatory effects of exercise in diabetes is universally accepted. He demonstrated by tracer, cellular and molecular methods how muscle, liver and pancreatic a-cells adapt to hyperglycemia: a critical concept in diabetes. By purifying and determining biological activity of stomach glucagon, he provided the first evidence of glucagon's extrapancreatic site, changing prevailing concepts that one hormone is synthesized in one gland.”

Mladen has received several awards and honours for his work:

• Albert Renold Award of the American Diabetes Association for distinguished career in the training of diabetes research scientists and facilitation of research (the only Canadian awarded) (2005),
• Keynote speaker on Endocrinology and Diabetes;
• The society of Chinese Bioscientists of North America (2006),
• Canadian Diabetes Association Inaugural Life - Time Achievement Award for leadership in diabetes research and contribution to the Canadian diabetes community (2007),
• Fellow, Royal Society of Canada (FRSC);
• Corresponding Member, Croatian Academy of Arts and Sciences;
• Poll Visiting Scholar, University of Washington, Seattle, Washington;
• Novo Nordisk Lecture, Karolinska Institute, Stockholm;
• Solomon A. Berson Distinguished Lectureship of American Physiological Society - Endocrinology and Metabolism Section, FASEB, Atlanta;
• Foreign Adjunct Professor, Karolinska Institute, Stockholm; Mizuno Inaugural Lectureship and Award, Fourth International Symposium on Exercise and Diabetes - Osaka University, Japan;
• Honorary Degree of Doctor of Medicine - Karolinska Institute Medical Faculty (one of the two Canadians in the whole history of Karolinska institute);
• Banting Medal and Lectureship For Distinguished Scientific Achievement (American Diabetes Association – the only Canadian working in Canada);
• R. Kroc Lectureship, University of Southern California School of Medicine, Los Angeles;
• Peter J. Laurie Memorial Lecture of the Juvenile Diabetes Foundation Canada, Toronto; Canada Council Killam Scholar;
• Elected Fellow of the Royal College of Physicians and Surgeons of Canada, Medical Scientist Category;
• MRC Visiting Scientist Award during sabbatical leave at University of Oxford, England;
• Visiting Research Fellowship at Merton College, University of Oxford, England;
• Inaugural Banting and Best Memorial Lecture and Canadian Diabetes Association Award: 12th Congress of the International Diabetes Federation, Madrid, Spain;
• Pfizer Lecturer and Travelling Fellow of the Clinical Research Institute, University of Montreal;
• Upjohn Lecturer of the Faculty of Health Sciences, University of Ottawa;
• Vuk Vrhovac Memorial Lecture, University of Zagreb, Croatia (to commemorate the 50th Anniversary of the Foundation of the Institute for Diabetes);
• Faculty Scholar of the Josiah Macy Foundation during sabbatical leave at the University of Geneva;
• Elected Professor, Department of Medicine, University of Zagreb;
• Honorary Executive Member of the Croatian World Congress Physicians;
• Diploma for contributions to Medical Science and Health in Croatia, on the occasion of the 125^th anniversary of the Medical Council of Croatia;
• Honorary member of the Turkish Diabetes Association.