Glucagon:

Chapter 16

Glucagon: MOLECU LAR BIOLOGY AN D STRUCTURE-ACTiViTY VICTOR J. HRUBY

Introduction Discovery of Glucagon Glucagon Structure Glucagon Actions In Vivo Glucagon Mechanisms of Action Glucagon Biosynthesis and Secretion Glucagon" Biomedical Mechanisms of Action Glucagon/Glucagon Receptor: Structure-Activity Approaches Used to Study Glucagon/Receptor Actions Glucagon Structure-Activity Relationships Summary

Principles of Medical Biology, Volume 10B Molecular and Cellular Endocrinology, pages 387-401. Copyright 9 1997 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 1-55938-815-3

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INTRODUCTION Discovery of Glucagon Glucose homeostasis is of critical importance to human health due to the central importance of glucose as a source of energy, and the fact that brain tissues do not synthesize it. Thus maintaining adequate glucose levels in the blood are necessary for survival. On the other hand, inappropriate levels of glucose in the blood are a primary symptom of diabetes, a major degenerative disease in society. Normal glucose homeostasis is primarily maintained by glucagon and insulin. Following the discovery of insulin in the pancreas by Banting and Best (1921) and its ability to lower blood glucose levels in normal and in diabetic states, a second factor was discovered by Kimball and Murlin (1923) in the pancreas that could raise glucose levels in animals and it was given the name glucagon. Glucagon thus has a counterregulatory effect on glucose levels in the blood relative to insulin; the interrelated bioactivities of these two hormones are critical to understanding glucose homeostasis in normal and diabetic states.

Glucagon Structure Glucagon from the pancreas was purified to eventually give a crystalline compound (Staub et al., 1955) and shortly thereafter its sequence (I) was determined (Bromer et al., 1956) as shown in Figure 1. About 20 years later, an X-ray crystal structure was determined (Sasaki et al., 1975). The availability of crystalline glucagon at an early date and the determination of its primary structure has made glucagon an important hormone in the development of our ideas of the mechanisms of action of peptide hormones.

Glucagon Actions In Vivo Given our current understanding, it is interesting to note that despite its near co-discovery with insulin, and its clear counterregulatory hormonal activities (relative to insulin), and the discovery by Sutherland of the mechanism of its action via cAMP (discussed by Sutherland, 1972), the acceptance of glucagon as the key counterregulatory hormone to insulin required more time before it became generally accepted. However, the metabolic studies of Exton and colleagues (e.g. Exton and Park, 1969), studies of plasma glucagon levels in vivo (e.g. Unger et al., 1963),

H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyrl0-Ser-Lys-Tyr-Leu Asp-Ser-Arg-Arg-Ala-Gln 20 -Asp-Phe- Val - Gin -T rp . Leu . Met . . Asn . Thr OH

Figure 1. The structure of glucagon.

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and numerous related studies, eventually led to the conclusion that glucagon was a second peptide hormone of the pancreas whose primary action was the prevention of hypoglycemia and of cerebral neuroglycopenia (e.g. Unger and Orci, 1981). The secretion of glucagon is primarily controlled by blood glucose levels, and during the hypoglycemic state (approximately 80 mg% glucose), glucagon is released into the blood and via circulation delivered to its target organs, especially the liver, where it stimulates glucose production via the metabolic processes of glycogenolysis and gluconeogenesis (e.g. Johnson et al., 1972). Inaddition, glucagon stimulates lipolysis in fat cells (Harris et al., 1979; Witters et al., 1979), and also has been reported to exert effects on heart and kidney tissue (Glick et al., 1968; Bailly et al., 1980). All of these studies and many others have provided the clear understanding that in the normal metabolic state there is a close coordination of anatomically linked glucagon- and insulin-secreting islet cells (Orci et al., 1975) which maintain blood glucose levels at a very narrow range, and that this control constitutes one of the most important homeostatic systems for the maintenance of good health. On the other hand, in all forms of diabetes, this normal relationship between glucagon and insulin is disrupted in various Ways (e.g. MUller et al., 1970, 1971; Dobbs et al., 1975) which in turn led to the suggestion that glucagon may be involved in the pathogenesis of diabetes mellitus (Unger and Orci, 1975, 1983). The precise manner in which glucagon participates in the diabetic state is still a matter of considerable controversy. In summary, in the normal man, glucagon plays two critical physiological roles. In its first role, glucagon plays a homeostatic function. In concert with insulin, it participates in the maintenance of normal glucose levels. In its second physiological role, glucagon produces hyperglycemia during stress, providing the fuel needed by the brain, muscle, and other tissues to respond to stress.

GLUCAGON MECHANISMS OF ACTION Glucagon Biosynthesisand Secretion Glucagon is produced and processed from preproglucagon in pancreatic ct cells, in small intestine L cells, and in certain hypothalamic cells (Schroeder et al., 1984; Tricoli et al., 1984). Preproglucagon consists of a signal peptide, glicitin-related polypeptide, glucagon(1-29), glucagon-like peptide-1 (GLP-I), and glucagon-like peptide-2 (GLP-2). Though several of these peptides other than glucagon(1-29) have received increased attention in recent years, we will only discuss here the biosynthesis of glucagon(1-29). Glucagon itself is produced primarily by processing the preproglucagon to proglucagon and then by a series of specific enzymes to glucagon. This complete processing occurs only in the pancreatic ot cells (Hellerstr/Sm et al., 1974; Noe and Bauer, 1975: Patzelt et al., 1979). Higher molecular weight forms are released from the gut and can be found in circulation. It appears

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that only glucagon(1-29, Figure 1) is released from the normally functioning ct cell of the pancreas. Ordinarily, release of glucagon is under inhibitory control by insulin (Samok et al., 1965), glucose, and somatostatin (Koerker et al., 1974). Secretion is controlled by a number of factors. The major stimulus of glucagon secretion is hypoglycemia, and this appears to be the case whether the origins of hypoglycemia are starvation, alcohol infusion, insulin administration, or whatever (Iversen and Hermansen, 1977). Glucagon secretion also is stimulated by various amino acids (Assan et al., 1977) such as arginine, omithine, and many others including glycine, alanine, serine, glutamic acid, valine, tyrosine and asparagine. Both raising blood glucose levels and free fatty acids can exert inhibitory effects on glucagon secretion (Lefevre, 1972). Glucagon increases glucose production and lipolysis and decreases the level of circulating amino acids, thus ensuring that secretion of glucagon occurs only under conditions requiting fuel mobilization. In diabetes, hyperglucagonemia is often present despite the elevated levels of glucose. Current evidence (Starke et al., 1985) suggests that the elevated levels of glucagon in diabetes are entirely due to the insulin deficiency, at least in the insulin-deficient form of diabetes, since bringing insulin levels to normal in insulin-deficient diabetes can completely reverse hyperglucagonemia.

Glucagon: Biomedical Mechanisms of Action The biochemical actions of glucagon are thought to occur via the glucagon receptor. The human glucagon receptor has been cloned recently (Lok et al., 1994; MacNeil et al., 1994; Buggy et al., 1995) along with glucagon genes from other species (e.g. Svoboda et al., 1993; Jelinek et al., 1993; Carruthers et al., 1994; Burcelin et al., 1995). This will provide a new tool for examining the mechanisms of action of glucagon. In the meantime, a good deal has been learned in the past 40 years regarding the biochemical mechanism of glucagon action. In fact, glucagon has served as a paradigm for obtaining insights into the mechanisms of hormonal signal transduction, and in some ways we now know more about the mechanisms of glucagon action than that of insulin. As a result of the classical work of Suthefland and coworkers (Sutherland and Rail, 1960; Sutherland et al., 1968), it has been thought that most, if not all, of the metabolic actions of glucagon are mediated via the formation of 3',5'-cyclic adenosine monophosphate (cAMP). Glucagon initiates its action in the liver by binding to the specific glucagon receptors on the plasma membrane (Rodbell et al., 1971a) which subsequently enhances intracellular cAMP levels by activation of adenylyl cyclase (Rodbell et al., 1971b,c). When glucagon is bound to its liver plasma membrane receptor the complex interacts with the stimulatory G protein, Gs (Gilman, 1984). The a-subunit from the Gs protein (which has three subunits) is freed and interacts with adenylyl cyclase to activate it, and the enzyme in turn now can catalyze the conversion of ATP to cAMP (Figure 2). This causes a rapid

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increase in intracellular levels of cAMP (the intracellular levels of cAMP are lowered by a phosphodiesterase). The increase in cAMP levels causes various changes in cellular metabolism by phosphorylating numerous cellular proteins (Garrison, 1978; Garrison et al., 1979). Of particular importance to glucose production is a direct activation of the protein cAMP-dependent protein kinase, dissociating its regulatory dimeric subunits as a result of nucleotide binding to the regulatory subunit. This results in activation of the two catalytic subunits which occurs upon dissociation from the complex (Rubin and Rosen, 1975; Ciudad et al., 1987). These subunits catalyze phosphorylation of a number of intracellular enzymes, which mediates all known actions of glucagon. In particular, phosphorylase b is activated (Figure 2) (Friedmann, 1976), which in turn can lead to enhanced glycogenolysis and release of glucose into the blood stream. Insulin is thought to oppose glucagon action at these latter sites of interaction by reducing the dissociation of the regulatory subunits of cAMP-dependent protein kinase and thus decreasing its activity (Gabbay and Lardy, 1984). Glucagon also increases glucose output by inhibiting glucagon synthesis. This is accomplished because cAMP-dependent protein kinase phosphorylates the active form of glucagon synthetase converting it to the inactive b form (Jett and Soderling, 1979), thus preventing glucagon formation (Exton, 1987).

Glucagon

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Glucagon.Glucagon Receptor

Adenyl Cyclase

/ ATP

~

= cAMP

cAMP-Dependent Protein Kinase

Protein Kinase (Inactive)

DP Phosphorylase b, (Inactive)

= Phosphorylase b (Active)

ATP Glycogen

ADP

- Glucose

Figure 2. Cascade of reactions that leads to the formation of glucose.

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Moreover, the actions of glucagon are more complex than simple regulation of glycogen metabolism to glucose. In addition, glucagon stimulates hepatic gluconeogenesis and ketogenesis. The former occurs because glucagon increases the rate of phosphoenolpyruvate production and decreases the rate of its disposal by the enzyme pyruvate kinase. It does this by inhibiting pyruvate kinase activity directly via a cAMP-dependent protein kinase phosphorylation (Riou et al., 1976; Engstrom, 1978; Pilkis et al., 1982). Glucagon also modifies pyruvate kinase activity by its effect on the levels of fructose-1,6-diphosphate, an allosteric activation of pyruvate kinase, the rate-limiting enzyme of gluconeogenesis (Claus and Pilkis, 1981; Van Schaftingen et al., 1981; E1-Maghrabi et al., 1982). After feeding, fructose-2,6-biphosphate levels are high, which promotes glycolysis and inhibits gluconeogenesis. In the fasted state or in the diabetic state, this enzyme is phosphorylated, as a result of the high levels of glucagon and low levels of insulin. The lowering of fructose-2,6-biphosphate results in inactivation of the rate-limiting enzyme of glycolysis, 6-phosphofructo-l-kinase, and stimulates the rate-limiting enzyme of gluconeogenesis, fructose-l,6-biphosphate. Glucagon also plays an additional role in "activating" the gluconeogenic enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxykinase. However, this activation does not occur via phosphorylation (Leiter et al., 1978), but by changes in the levels of their substrates and effectors (Siess et al., 1977; MacDonald et al., 1978) (Figure 3).

l

l

cAMP-DependentProtein Kinase

6oPhosphofructo-2-kinaselfructose-2,6-biphosphatase

t cAMP

t

Fructose-2,6-Biphosphate Glycogen Synthetasea Phosphorylasea

cAMP

Pyruvate Kinase I Glycogenolysis

l

~ Glycolysis

l

t Gluconeogenesis Glycogenesis

l

Figure 3. Effects of CAMP on various enzymes involved in glycolysis and gluconeogenesis.

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The antagonism by insulin of the above effects of glucagon is thought to be exerted by enhancing phosphodiesterase activity which reduces cAMP levels, and by opposing the glucagon-mediated activation of cAMP-dependent protein kinase (Gabbay and Lardy, 1984) which reduces the overall phosphorylation state of the enzymes of glycogenesis, glycogenolysis, the bifunctional enzyme, and the L-type pyruvate kinase (Figure 3). As for ketone production, glucagon stimulates hepatic ketone production by its effects on glucose metabolism. In the fed state, when insulin levels are high relative to glucagon, the bifunctional enzyme is not phosphorylated. Acting as a kinase, it increases the flow of three-carbon fragments down the glycolytic pathway of the liver providing the substrates needed for lipogenesis in the liver. The resulting fatty acids form triglycerides and are secreted by the liver as very-low-density lipoproteins which are transported to the adipocytes where they are stored. In the normal state, these newly formed fatty acids do not form ketones because malonyl CoA, a powerful inhibitor of carnitine palmitoyl transferase-1, is blocked. The high levels of malonyl CoA that form during lipogenesis block this conversion. In the diabetic and fasting states when glucagon is high relative to insulin, the bifunctional enzyme is not phosphorylated and acts as a kinase providing the three carbon fragments which act as substrates for lipogenesis. In these circumstances, hepatic malonyl CoA levels become very low and carnitine palmitoyl transferase-1 is released from inhibition. This allows transesterification to fatty acyl carnitine and entry into the mitochondrial membrane. The activity level determines the amount of ketones produced and released into the blood. As the glucagon-to-insulin ratio increases, increased fatty acid oxidation and ketogenesis occurs. This is particularly a problem in diabetes (McGarry and Foster, 1977, 1980). Clearly a potent glucagon antagonist should have a dramatic effect on the regulatory mechanisms of ketogenesis by effectively decreasing the glucagon/insulin signal ratio by blocking the glucagon receptor. Used in conjunction with insulin, it may be even more effective. It is generally accepted that, in addition to the well-established cAMP (G-protein/adenylyl cyclase) signal elicited by glucagon, a second Ca 2§ signal, is activated by glucagon. Less clear are the biochemical physiological origins of this signal, though several hypothesis have been put forward based on the available evidence (Wakelam et al., 1986; Pitmer and Fain, 1991; Tang and Houslay, 1992). One current thought is that in addition to the cAMP-mediated pathway, glucagon may be acting through the inositol triphosphate(IP3)-mediated pathway, and that its effect on various metabolic changes are the result of phosphorylation of key enzymes. More work is needed to further evaluate the possible importance of non-cAMP-dependent pathways in the action of glucagon in the normal and the diabetic state. Potent, clean glucagon receptor antagonists will be needed to properly evaluate the significance of these possible responses.

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GLUCAGON/GLUCAGON RECEPTOR: STRUCTURE-ACTIVITY Approaches Used to Study Glucagon/Receptor Actions As previously discussed, all of the physiological actions of glucagon are thought to be mediated by the interactions of glucagon with its receptors on plasma membranes. Classically, the interactions of glucagon with its receptor have been studied by binding in vitro and in vivo assays. Among the most widely used are the following: 1. Receptor binding assays using purified rat liver plasma membranes (Pohl et al., 1971) and [125I]glucagon in competitive binding experiments (Lin et al., 1975). 2. In vitro cAMP levels have been measured by the method of Lin et al. (1975) and Solomon et al. (1974). In view of the recent cloning of the human glucagon receptor (vid~ supra), it is likely that binding and in vitro cAMP assays with rat membranes will be supplemented or replaced using membranes containing the stably transfected human receptor. 3. Whole cell rat hepatocytes prepared by the methods of Berry and Friend (1969) as modified by Heyworth and Houslay (1983) also have been widely used as an in vitro assay to examine the effects of glucagon-glucagon receptor interactions, and the methods continue to be improved to provide long-lived viable hepatic cells with minimal damage. Recently, the classical cAMP accumulation assay of Brown et al. (1972) has been substantially improved for much greater sensitivity in measuring cAMP accumulation in response to glucagon agonists (Van Tine et al., 1996). 4. It also is possible to use perfused liver slices to examine the various biological activities related to glucagon (and insulin) action (Smith et al., 1986; McKee et al., 1988). These methods have greatly aided studies of the early events of receptor transduction following glucagon agonist- and antagonist-glucagon receptor interactions. Of course, it also is possible to study the activity of the various enzymes in the cascade since they might be of interest to some specific downstream function one is examining. 5. Finally, in vivo it is important to examine in normal and diabetic animals such critical parameters as glucose levels, ketone bodies, blood pH, and so forth, and well-developed methods are available in most clinical laboratories for such studies.

Glucagon Structure-Activity Relationships Extensive structure-activity studies have been done on glucagon during the past 25 years (most recently reviewed by Hruby et al., 1986). A brief examination of

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what has been discovered can provide insights into our present knowledge and where new insights are needed. As a starting point, it is interesting to note that the hormone responsible for most if not all of glucagon's activity for interactions with hepatic glucagon receptors is the 29 amino acid structure shown in Figure 1, and we will concentrate all of our discussion of this product of the proglucagon gene. The receptor for this hormone (vid~ supra) is a seven-transmembrane G-linked receptor. Interestingly, though several earlier reports suggested that smaller fragments of glucagon could interact with the glucagon receptor to give agonist or partial antagonist activity (Hruby et al., 1986a), none of these earlier reports have held up to more recent critical scrutiny. Thus far, though a few residues can be removed from the N- or C-terminal with maintenance of some agonist activity, in general the potency is greatly reduced, and even [des-Hisl]glucagon was shown to be a weak partial agonist/antagonist (Lin et al., 1975: Hruby et al., 1976). It appears that essentially the entire glucagon(1-29) sequence is necessary for potent agonist activity at the glucagon receptor (Frandsen et al., 1981; Hruby et al., 1986). Even for antagonist activity at the glucagon receptor, essentially the entire glucagon sequence appears to be required (Hruby et al., 1986a,b). Some time ago, Collins et al. (1992) reported a weak organic antagonist CP-99, 711 for the glucagon receptor, but this result has not been confirmed and no subsequent work has appeared. Thus, it appears that the complete or near complete glucagon sequence is needed for agonist and possibly for antagonist activities for glucagon. This is rather unusual, since for many peptide ligands for the seven-transmembrane G-linked receptor superfamily, it has been possible to find potent peptide agonists much reduced in size from the native peptide, and for antagonists, it often has been possible to find quite small peptide and non-peptide ligands. Many attempts have been made to prepare glucagon super-agonist analogues, but so far the only reported success has been the analogue [Lys 17'18, Glu21]glucagon (Krstenansky et al., 1986a), which has increased helical structure in the C-terminus of glucagon. Other substitutions in the C-terminal region (Hruby et al., 1986a), in the 1-5 N-terminal region (Hruby et al., 1986b; Unson et al., 1987; Zechel et al., 1991), or in the central region of glucagon (Hruby et al., 1986b; Krstenansky et al., 1986b) have been unsuccessful. Indeed, as key residues have been modified, the more usual result has been a change from glucagon agonist activity to partial agonist or even antagonist activity. Indeed, this feature of the structure-activity studies of glucagon was one of the earliest observations when it was found that [des-Hisl]glu cagon was a partial agonist/antagonist (Lin et al., 1975; Hruby et al., 1976). Shortly thereafter, the first potent glucagon antagonist, THG-glucagon, was reported (Bregman et al., 1980) which was shown to lower blood glucagon levels in diabetic rats (Johnson et al., 1982), presumably by blocking the effect of endogenous glucagon. Though this analogue was useful in examining the possibility of two separate signal-transduction systems for glucagon (Wakelam et al., 1986), it subsequently was shown to be a weak partial agonist in some systems (Hruby et al., 1981; Corvera

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et al., 1984; Garcia-Saint et al., 1986; Hruby et al., 1996). Since the need for obtaining clean, stable antagonists for the glucagon receptor will be critical for sorting out the roles of glucagon in normal and especially diabetic states, we will limit the rest of our remarks to glucagon antagonist studies which have been the major focus of structure-activity relationships during the past decade. The early work clearly established the importance of the His I and Lys 12residues for developing antagonist activity in glucagon. Modifications by total synthesis of glucagon analogues have further demonstrated that appropriate modifications or deletions in positions 1, 4, 5, and especially 6, 9, and 16 of glucagon can lead to potent antagonists (e.g. Unson et al., 1987; Unson et al., 1989; Zechel et al., 1991; Dharanipragada et al., 1993; Unson, 1994; Azizeh et al., 1995; 1996; Sturm et al., 1997). Of critical importance was the discovery of the importance of the Asp 9 residue to glucagon activity when it was found that [des-His ~, Glu9]glucagon and several related analogues (Unson et al., 1987, 1989) were antagonists, and that des-Phe 6 analogues also were antagonists (Zechel et al., 1991). Subsequently, it was discovered by Unson and Merrifield (1994) that the Ser 16 residue in glucagon (Figure 1) also was critical for receptor transduction, and that suitable modifications could lead to antagonists. Taken together, these results clearly demonstrated the critical importance of the His l, Phe 6, Asp 9, and Ser 16residues in receptor transduction and the important role also played by the residues Gly4, Thr 5, Tyr l~ Lys 12, and Tyr 13 in glucagon for agonist/antagonist activity. Despite these important insights, it was recently demonstrated that many of these antagonists, perhaps most, still have minor residual partial agonist activity (Van Tine et al., 1996). However, Azizeh et al. have developed a pure, clean glucagon antagonist, [des-His 1, des-Phe 6, Glu9]glu cagon (Azizeh et al., 1995; Van Tine et al., 1996), and subsequently other related analogues that are pure antagonists (Azizeh et al., 1997). The way is now open to obtain pure, potent glucagon receptor antagonists for investigating in detail the role of glucagon alone and in conjunction with insulin in normal and diabetic states in more detail and with more clarity than has hitherto been possible. SUMMARY

The actions of glucagon at hepatic and fat cell receptors is critical for the control of glucose levels and glucose homeostasis in normal and diabetic states. Its interactions with insulin in maintaining homeostasis in normal animals is understood in considerable detail and tools are now available to obtain an even deeper understanding. In the diabetic state, the interplay of glucagon and insulin (and other factors) in hyperglycemia, ketoses, and other manifestations of diabetes is less clear, and provides a continuing challenge for biochemists, physiologists, endocrinologists, and molecular biologists. Fortunately, new tools in the form of potent receptor agonists and antagonists, cloned receptors, assays with enhanced sensitivity, and increased understanding of receptor transduction mechanism should greatly aid in obtaining these needed new insights and understanding.

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ACKNOWLEDGMENTS The partial support of the U. S. Public Health Service Grant DK-21085 is gratefully acknowledged. I am particularly grateful for the outstanding collaborations of the excellent undergraduate and graduate students, postdoctoral associates, and fellow colleagues who have worked with me in our studies. The contents of this paper are solely the responsibility of the author and do not necessarily represent the views of the USPHS.

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RECOMMENDED READING Burcelin, R., Katz, E.B., & Charron, M.J. (1996). Molecular and cellular aspects of the glucagon receptor-Role in diabetes and metabolism. Diabetes Metab. (Paris) 22, 373-396.