Role of the Endocrine Pancreas in Glucose Homeostasis During Exercise

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appear that the presence of hypoglycemia somehow sensitizes the liver to glucagon action. Recently, we studied this phenomenon and found that even in the face of a 25-fold rise in hepatic insulin, a 70 pg/mL rise in glucagon was almost 3 times as effective at stimulating glucose production under hypoglycemic as opposed to euglycemic conditions (9). It is this ability to overcome insulin’s inhibitory signal that allows glucagon to fulfill its role as the primary defender of blood glucose (BG). It is now evident that plasma glucagon levels also change in response to nutrient intake. Amino acids stimulate glucagon secretion, while glucose inhibits it. We thus wondered whether changes in plasma glucagon associated with meal ingestion could alter the liver’s ability to take up and store glucose. To examine this question, we carried out a hyperglycemic (2 times basal) hyperinsulinemic (4 times basal) clamp in the conscious dog while either raising (≈20 pg/mL) or lowering (≈14 pg/mL) plasma glucagon (10). This difference in glucagon was associated with a marked difference in net hepatic glucose uptake (≈2.1 vs. 4.3 mg/kg/min). Clearly, therefore, glucagon also plays a role in the postprandial response of the liver. From work carried out by ourselves and others, it is now clear that glucagon levels are absolutely or relatively elevated in individuals with diabetes and that the hormone contributes significantly to the metabolic abnormalities associated with the disease (11). Likewise, the abnormal response of glucagon to a meal in patients with type 2 diabetes is now known to be one of the causes for postprandial hyperglycemia (12). Finally, it is now also well recognized that the alpha cell quickly loses its ability to respond to low BG levels in patients with type 1 diabetes, in part explaining the high frequency of hypoglycemia in such patients (7). Thus, after nearly 50 years of study, both the physiologic and pathophysiologic relevance of glucagon is unquestioned. The hormone that Mladen introduced me to in 1968 has proven to be important indeed. Nevertheless, issues related to this interesting hormone persist. Why does the alpha cell fail in people with type 1 diabetes? What are the downstream mediators of glucagon action (adenosine monophosphate-activated protein kinase [AMPK], steroid receptor coactivator-1 [SRC-1], etc.)? How important is glucagon’s effect on hepatic fat metabolism? How important are the decreases in plasma glucagon associated with incretin therapy? Hopefully, over the next few years, the answers to these questions will be forthcoming.

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Unger RH, Eisentraut AM, McCall MS, et al. Glucagon antibodies and an immunoassay for glucagon. J Clin Invest. 1961;40:1280-1289. Cherrington A, Vranic M, Fono P, et al. Effect of glucagon on glucose turnover and plasma free fatty acids in depancreatized dogs maintained on matched insulin infusions. Can J Physiol Pharmacol. 1972;50:946-954.

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Cherrington AD, Williams PE, Shulman GI, et al. Differential time course of glucagon’s effect on glycogenolysis and gluconeogenesis in the conscious dog. Diabetes. 1981;30:180-187. 4. Cherrington AD, Liljenquist JE, Shulman GI, et al. Importance of hypoglycemia-induced glucose production during isolated glucagon deficiency. Am J Physiol. 1979;236:E263-E271. 5. Cherrington AD. Control of glucose production in vivo by insulin and glucagon. In: Jefferson LS, Cherrington AD, eds. The Handbook of Physiology: The Endocrine Pancreas and Metabolic Regulation. New York, NY: Oxford Press; 2001:759-785. 6. Wasserman DH, Spalding JA, Lacy DB, et al. Glucagon is a primary controller of hepatic glycogenolysis and gluconeogenesis during muscular work. Am J Physiol. 1989;257(1 pt 1):E108-E117. 7. Cryer PE. Glucose counterregulation in man. Diabetes. 1981;30:261-264. 8. Dobbins RL, Connolly CC, Neal DW, et al. Role of glucagon in countering the hypoglycemia induced by insulin infusion in dogs. Am J Physiol. 1991;261(6 pt 1):E773-E781. 9. Rivera N, Everett CA, Stettler K, et al. Hypoglycemia increases the sensitivity of the liver to glucagon. Diabetes. 2006;55(suppl. 1):A47. 10. Holste LC, Connolly CC, Moore MC, et al. Physiological changes in circulating glucagon alter hepatic glucose disposition during portal glucose delivery. Am J Physiol. 1997;273(3 pt 1):E488-E496. 11. Dobbs R, Sakurai H, Sasaki H, et al. Glucagon: role in the hyperglycemia of diabetes mellitus. Science. 1975;187:544-547. 12. Shah P, Vella A, Basu A, et al. Lack of suppression of glucagon contributes to postprandial hyperglycemia in subjects with type 2 diabetes mellitus. J Clin Endocrinol Metab. 2000;85:4053-4059.

ROLE OF THE ENDOCRINE PANCREAS IN GLUCOSE HOMEOSTASIS DURING EXERCISE David H. Wasserman PhD Some years ago—around 1980—I was living on the streets of Santa Monica, California, not but a stone’s throw from the Pacific Ocean. I had a degree from UCLA in kinesiology, the study of movement. It occurred to me that I should take that degree and do something with my life. It had already become clear that diabetes and metabolic disease were on track to be public health problems on an immense scale. With my interest in exercise and the knowledge that exercise plays an important role in metabolic disease, I thought that the study of exercise and diabetes would provide a foundation upon which I could build a career and, in the process, work toward resolving an important public health problem. I spent days in the library and talked to numerous experts about how to best accomplish this goal. The consensus was clear: Dr. Mladen Vranic at the University of Toronto was the best person with whom to begin my studies of exercise and diabetes. I therefore asked Professor Vranic to consider my application to work as a doctoral student in his laboratory. He agreed to take a chance on a poor kid with sketchy credentials, and this made all the difference for me. In autumn of 1981, I moved 5000 km to Canada. That winter, I saw snow fall for the first time. Mladen summoned me into his office upon my arrival in Toronto and assigned me with the task of defining the roles of glucagon and catecholamines during exercise in health and diabetes. To do this, I undertook studies using dog models and sophisticated techniques for manipulating

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hormone action and measuring glucose fluxes using isotopes. Mladen had pioneered the development of these techniques a decade before, and this was to be my dissertation project. This work showed that in the absence of glucagon, blood glucose (BG) fell during exercise. However, a highly sensitive insulincounterregulatory response protected the body from severe hypoglycemia (1). We further showed that cortisol and the catecholamines rose during exercise with decrements in BG as small as 10 mg/dL; thus, the mechanisms for protecting BG are highly sensitized during exercise. The role of the presence of glucagon was quantified by clamping BG at euglycemia, thereby preventing sensitive compensatory mechanisms; this enabled us to calculate that 60% of the glucose released by the liver during exercise is due to the presence of glucagon. Consistent with these findings, glucagon suppression during exercise in alloxan-diabetic dogs resulted in a marked fall in BG due, again, to a reduction in the rate that glucose was released by the liver (2). We further demonstrated that betaadrenergic blockade lowers BG during exercise (3). However, its mechanism of action was much different: in contrast to glucagon, which acted entirely through the liver, there was no effect of beta-adrenergic blockade on the liver. Instead, its glucose-lowering effect was mediated by increasing the peripheral utilization of glucose in the insulin-deficient diabetic dog. Nonetheless, despite the compelling studies documenting the importance of glucagon during exercise, the question of what was regulating the rate of glucose release during exercise was still shrouded in controversy. Upon my graduation from the University of Toronto, my student visa expired and I was required to leave Canada. I moved to Nashville, where I continued my training with another “Vranic alumnus”, Alan Cherrington at Vanderbilt University School of Medicine. I remain at Vanderbilt to this day. I continued the work I had begun in Toronto by quantifying the role of the exercise-induced increase in glucagon on specific pathways in the liver, by combining hepatic arteriovenous difference techniques and isotopic methods during treadmill exercise in the dog. My laboratory showed that the rise in glucagon is necessary for exerciseinduced stimulation of glycogenolysis (4), gluconeogenesis (4), pathways for nitrogen disposal (5) and fatty acid oxidation (6). We also demonstrated that, within the exercise environment, glucagon is 6-fold more effective at stimulating glucose production than it is under sedentary conditions (7). More recently, my laboratory showed in the mouse that glucagon action during exercise and other metabolic stresses is associated with a marked discharge in hepatic energy stores (8). The resulting increase in the ratio of adenosine monophosphate (AMP) to adenosine triphosphate (ATP) leads to activation of AMP-activated protein kinase and, we believe, activates allosterically a number of other liver enzymes important to the exercise response. Nonetheless,

in contrast to the sensitivity of the liver to changes in pancreatic hormone secretion, we have also demonstrated that glucose uptake and oxidation by working muscles of healthy humans (9) and animals (10) are insulin independent. A corollary to the ability to mobilize glucose during exercise is that the body must have the capacity to replenish liver glycogen afterwards. My laboratory at this point turned its attention to this sensitive process by showing that prior exercise significantly accelerates the repletion of liver glycogen by increasing the rate of glucose absorption from the gut after ingestion (11,12). This occurs through increased extraction of glucose by the liver (13) and by direction of more of the glucose taken up after exercise to liver glycogen (14). We also demonstrated that different mechanisms appear to govern the accelerated rates of hepatic glucose uptake and glycogen deposition. Hence, the post-exercise increase in hepatic glucose uptake during a glucose load is closely linked to increased hepatic insulin sensitivity (14). On the other hand, the fate of glucose taken up by the liver is independent of insulin action, but is more closely linked to glycogen mass (15). Hence, preventing the decrease in glycogen mass during exercise by preventing the glucagon and insulin responses to exercise with somatostatin did not diminish the accelerated hepatic glucose uptake (16). However, it did alter the metabolic fate of the glucose, as the percentage of glucose diverted to glycogen was greatly reduced (16). In recent years, my laboratory has delineated the physiology of glucose metabolism during exercise and insulin stimulation by using genetically altered mouse models. As Director of the Vanderbilt–National Institutes of Health (NIH) Mouse Metabolic Phenotyping Center (MMPC), I have transferred procedures developed for studying large animals (which I learned with Mladen) to the mouse. We have published key methodology papers on how to glucoseclamp mice (17,18). Using the methodology developed in the Vanderbilt–NIH MMPC, my laboratory has also redefined the paradigm for the control of muscle glucose uptake. Previously, muscle glucose uptake had been defined as rate-limited by glucose membrane transport. However, we have shown in transgenic mice that muscle glucose uptake is under distributed control during both exercise and insulin stimulation and in insulin resistance. Thus, the rate of glucose uptake by muscle is determined by the rate of glucose delivery to muscle, membrane glucose transport and the rate of glucose phosphorylation within muscle (19-26). The lessons I learned from my PhD mentor, Mladen Vranic, still resonate with me today. I greatly appreciate the roles that Mladen and his longtime colleague, Lavina Lickley, played in my scientific development. The tools and concepts I learned in Toronto to study metabolism in vivo serve me to this day. I am grateful to have overlapped with such quality individuals as Kamal El Tayeb, Tom Hatton, CANADIAN JOURNAL OF DIABETES. 2010;34(3):186-202.

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Clement Gautier, Ola Bjorkman, Patricia Brubaker and Diane Finegood. The latter 2 were participants in the symposium honouring Mladen, and continue to be productive figures in the scientific community. Finally, I will always be grateful to the University of Toronto and the province of Ontario for making it possible for a kid from the States to come to Canada and receive such exceptional training.

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Wasserman DH, Lickley HL, Vranic M. Interactions between glucagon and other counterregulatory hormones during normoglycemic and hypoglycemic exercise in dogs. J Clin Invest. 1984;74:1404-1413. Wasserman DH, Lickley HL, Vranic M. Important role of glucagon during exercise in diabetic dogs. J Appl Physiol. 1985;59:1272-1281. Wasserman DH, Lickley HLA, Vranic M. Role of beta-adrenergic mechanisms during exercise in poorly controlled diabetes. J Appl Physiol. 1985;59:1282-1289. Wasserman DH, Spalding JA, Lacy DB, et al. Glucagon is a primary controller of the increments in hepatic glycogenolysis and gluconeogenesis during exercise. Am J Physiol. 1989;257(1 pt 1):E108-E117. Krishna MG, Coker RH, Lacy DB, et al. Glucagon response to exercise is critical for accelerated hepatic glutamine metabolism and nitrogen disposal. Am J Physiol Endocrinol Metab. 2000;279:E638-E645. Wasserman DH, Spalding JA, Bracy D, et al. Exercise-induced rise in glucagon and the increase in ketogenesis during prolonged muscular work. Diabetes. 1989;38:799-807. Wasserman DH. Four grams of glucose. Am J Physiol Endocrinol Metab. 2008;296:E11-21. Berglund ED, Lee-Young RS, Lustig DG, et al. Hepatic energy state is regulated by glucagon receptor signaling in mice. J Clin Invest. 2009;119:2412-2422. Wasserman DH, Geer RJ, Rice DE, et al. Interaction of exercise and insulin action in humans. Am J Physiol. 1991;260(1 pt 1):E37-E45. Wasserman DH, Mohr T, Kelly P, et al. The impact of insulin deficiency on glucose fluxes and muscle glucose metabolism during exercise. Diabetes. 1992;41:1229-1238. Hamilton KS, Gibbons FK, Bracy DP, et al. Effect of prior exercise on the partitioning of an intestinal glucose load between splanchnic bed and skeletal muscle. J Clin Invest. 1996;98:125-135. Pencek RR, Koyama Y, Lacy DB, et al. Prior exercise enhances passive absorption of intraduodenal glucose. J Appl Physiol. 2003;95:1132-1138. Galassetti P, Coker RH, Lacy DB, et al. Prior exercise increases net hepatic glucose uptake during a glucose load. Am J Physiol. 1999;276 (6 pt 1):E1022-E1029. Pencek RR, James FD, Lacy DB, et al. Interaction of insulin and prior exercise in control of hepatic metabolism of a glucose load. Diabetes. 2003;52:1897-1903. Galassetti P, Hamilton KS, Gibbons FK, et al. Effect of prior fast duration on disposition of an intraduodenal glucose load in the conscious dog. Am J Physiol. 1999;276(3 pt 1):E543-E552. Pencek RR, James FD, Lacy DB, et al. Exercise-induced changes in insulin and glucagon are not required for enhanced glucose uptake by the liver following exercise but influence the fate of glucose within the liver. Diabetes. 2004;53:3041-3047. Ayala JE, Bracy DP, McGuinness OP, et al. Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes. 2006;55:390-397. Berglund ED, Li CY, Poffenberger G, et al. Glucose metabolism in vivo in four commonly used inbred mouse strains. Diabetes. 2008;57:1790-1799. Ayala JE, Bracy DP, Julien BM, et al. Chronic treatment with sildenafil improves energy balance and insulin action in high fat-fed conscious mice. Diabetes. 2007;56:1025-1033. Fueger PT, Bracy DP, Malabanan CM, et al. Hexokinase II overexpression improves exercise-stimulated but not insulin-stimulated muscle glucose uptake in high fat fed C57BL/6J mice. Diabetes. 2004;53:306-314.

21. Fueger PT, Bracy DP, Malabanan CM, et al. Distributed control of glucose uptake by working muscles of conscious mice: roles of transport and phosphorylation. Am J Physiol Endocrinol Metab. 2004;286:E77-E84. 22. Fueger PT, Heikkinen S, Bracy DP, et al. Hexokinase II partial knockout impairs exercise-stimulated muscle glucose uptake in oxidative muscles of mice. Am J Physiol Endocrinol Metab. 2003;285:E958-E963. 23. Fueger PT, Hess HS, Bracy DP, et al. Regulation of insulin-stimulated muscle glucose uptake in the conscious mouse: role of glucose transport is dependent on glucose phosphorylation capacity. Endocrinology. 2004;145:4912-4916. 24. Fueger PT, Hess HS, Posey KA, et al. Control of exercise-stimulated muscle glucose uptake by GLUT4 is dependent on glucose phosphorylation capacity in the conscious mouse. J Biol Chem. 2004;279:50956-50961. 25. Fueger PT, Shearer J, Bracy DP, et al. Control of muscle glucose uptake: test of the rate-limiting step paradigm in conscious, unrestrained mice. J Physiol. 2005;562:925-935. 26. Halseth AE, Bracy DP, Wasserman DH. Overexpression of hexokinase II increases insulin-and exercise-stimulated muscle glucose uptake in vivo. Am J Physiol. 1999;276(1 pt 1):E70-E77.

FROM ENTEROGLUCAGON TO THE GLUCAGON-LIKE PEPTIDES, GLP-1 AND GLP-2 Patricia L. Brubaker PhD Having completed my PhD studies at McGill University in the area of adrenocorticotrophic hormone processing and biological activities, I had a strong desire to extend my expertise in biochemistry into a new field, namely that of peptide hormones in diabetes. I quickly discovered that Mladen Vranic at the University of Toronto had conducted studies of extrapancreatic glucagon, also known as intestinal- or enteroglucagon, dating back to 1974 (1-3). Although the proglucagon gene had not yet been cloned, it was well established at that time that enteroglucagon was composed of 2 peptides, both lacking hyperglycemic activity despite the fact that they contained the sequence of glucagon within their longer sequences; hence, it was presumed that proglucagon was posttranslationally processed in the intestine to release enteroglucagon rather than glucagon. I therefore decided to join Mladen’s laboratory as a postdoctoral fellow to study these peptides, arriving on September 1, 1982. On first blush, my expertise in peptide biochemistry seemed to be a good match for Mladen’s laboratory and this particular project. Nonetheless, Mladen took a big chance in allowing me the opportunity to work with him, as his main interest at that time lay in in vivo studies of glucose homeostasis, primarily using the conscious dog as a model, whereas my studies were to focus on the development of novel in vitro models to study proglucagon processing as well enteroglucagon secretion, using the rat as a model. Thus it was that I, a peptide biochemist, began to learn about tracer approaches and in vivo physiology from Mladen and his graduate students, Kamal El-Tayeb in particular, while Mladen began a crash course in cell biology. Together, we developed a novel approach to placing rat intestinal cells into culture, enabling studies on the posttranslational processing of the newly cloned (in 1983) proglucagon molecule, as well