Glucagon and Cyclic AMP: Time to Turn the Page?

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Current Diabetes Reviews, 2012, 8, 362-381

Glucagon and Cyclic AMP: Time to Turn the Page? Robert L. Rodgers* Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI, 02881, USA Abstract: It is well established that glucagon can stimulate adipose lipolysis, myocardial contractility, and hepatic glucose output by activating a GPCR and adenylate cyclase (AC) and increasing cAMP production. It is also widely reported that activation of AC in all three tissues requires pharmacological levels of the hormone, exceeding 0.1 nM. Extensive evidence is presented here supporting the view that cAMP does not mediate metabolic actions of glucagon on adipose, heart, or liver in vivo. Only pharmacological levels stimulate AC, adipose lipolysis, or cardiac contractility. Physiological concentrations of glucagon (below 0.1 nM) duplicate metabolic effects of insulin on the heart by activating a PI3K- dependent signal without stimulating AC. In the liver, glucagon can enhance gluconeogenesis and glucose output - by increasing the expression of PEPCK or inhibiting the activity of PK - at pharmacological concentrations by activating AC coupled to a low-affinity GPCR, but also at physiological concentrations by activating a high affinity receptor without generating cAMP. Plausible AC/cAMP-independent signals mediating the increase in gluconeogenesis include p38 MAPK (PEPCK expression) and IP3/DAG/Ca 2+ (PK activity). None of glucagon’s physiological effects can be explained by activation of spare receptors or amplification of the AC/cAMP signal. In a new model proposed here, glucagon antagonizes insulin on the liver but mimics insulin on the heart without activating AC. Confirmation of the model would have broad implications, applicable not only to the general field of metabolic endocrinology but also to the specific role of glucagon in the pathogenesis and treatment of diabetes.

Key Words: Glucagon, cyclic AMP, adipose, heart, liver, phosphoenolpyruvate carboxykinase, pyruvate kinase. INTRODUCTION The peptide hormone glucagon is an important endocrine regulator of carbohydrate metabolism [1-3]. Together with epinephrine, glucagon was central to the discovery of cyclic adenosine monophosphate (cAMP) as the first known “second messenger”, by Sutherland, Rall, and coworkers, more than 50 years ago [4-7]. In the intervening decades, all four of “Sutherland’s criteria” [8] have been repeatedly fulfilled for cAMP acting as the “second messenger” for either epinephrine or glucagon: 1) The agonist must be shown to stimulate adenylate cyclase (AC); 2) The tissue levels of cAMP (and, presumably, the activities of cAMP-associated signal components), should change appropriately with the response (e.g., an increase in the signal should precede or coincide with the onset of the response and the directions of the signal and response should be functionally appropriate); 3) Inhibition of cAMP breakdown (i.e., inhibition of cAMP phosphodiesterase) should elicit responses that are similar to those of AC activators; and 4) exogenous cAMP or lipophilic derivatives should mimic cAMP-dependent agonist effects. We now know that glucagon and epinephrine activate cognate G protein-coupled receptors (GPCR) linked to isoforms of AC, and subsequently activate cAMP-dependent protein kinase (PKA) and other protein kinases that ultimately phosphorylate and alter the activities of downstream enzymes and terminal intracellular effectors [9]. The collective evidence has established beyond doubt (Fig. 1) that activation of

*Address correspondence to this author at the Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI, 02881, USA; Tel: 401-874-5033; Fax: 401-874-2646; E-mail: [email protected] 1573-3998/12 $58.00+.00

AC - and production of cAMP - by glucagon stimulates lipolysis in adipose tissue, inotropy and chronotropy in heart, and glucose output in liver [10-19]. Most if not all of the cAMP/PKA-dependent hepatic actions of glucagon make sense physiologically [20-22]. Briefly, in the postprandial state the ratios of insulin to glucagon levels in the blood increase. Dietary glucose then becomes a predominant source of carbohydrate utilization by extrahepatic tissues, with insulin more actively stimulating glucose uptake and utilization in heart, skeletal muscle, and other insulin-dependent tissues. Glucose derived from hepatic gluconeogenesis or glycogenolysis contributes less to total glycemia because inhibitory actions of insulin dominate stimulatory effects of glucagon. During fasting, the ratios of insulin to glucagon decline. The inhibitory effects of insulin on hepatic glucose output are at least partially lifted, while the stimulatory effects of glucagon become more evident. Normoglycemia is thus maintained by reciprocal fluctuations in glucagon and insulin levels in response to variations in dietary glucose intake and blood glucose levels. The prevailing view is that glucagon produces these and other physiological effects in vivo predominantly through activation of the AC/cAMP/PKA pathway in liver. Whether glucagon also influences adipose lipolysis or cardiac contractility in vivo is a bit more controversial. It is important to emphasize here that presumed physiological actions of glucagon, and the role of cAMP in mediating them, are often inferred from results of studies carried out ex vivo or in vitro. For the most part, evidence supporting the current model depicted in Fig. (1) has been collected from isolated organs, cells, or cell-free systems, mainly from the laboratory rat, but also occasionally from other mammalian species and from biopsies of human © 2012 Bentham Science Publishers

Glucagon and Cyclic AMP

Current Diabetes Reviews, 2012, Vol. 8, No. 5

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Fig. (1). Current model of the actions of glucagon on its three predominant target tissues: adipose, heart, and liver. In this model, all three major responses, adipose lipolysis, myocardial contractility, and hepatic glucose output, are mediated by activation of the glucagon GPCR associated with adenylate cyclase and the production of cyclic AMP. A second GPCR, associated with increases in intracellular calcium, is also depicted (adapted from [1, 3, 245]).

Fig. (2). Comparisons of concentration-effect curves for the actions of glucagon on adenylate cyclase (AC) activation or cyclic AMP generation and adipose lipolysis (A), myocardial contractility (B), and hepatic gluconeogenesis or glucose output (C). All curves are composites compiled from multiple reports: A) [13, 23-27]; B) [63-72]; and C) [24, 69, 88-103]. The shaded area is the average glucagon blood range in the rat, from references [30-39].

tissues. In vivo studies that are applicable to the involvement of cAMP in the actions of endogenous glucagon are less common but have been pursued more actively in recent years. ELEPHANT IN THE ROOM One fundamental and seemingly obvious principle not specifically addressed by Sutherland’s four criteria can be stated as follows: In order for an intracellular signal and its attendant response to be physiologically relevant, both must be influenced by physiological concentrations of the hormone. Good presumptive evidence to test whether this “fifth criterion” is fulfilled can be obtained experimentally by comparing the effective concentrations required to generate both the intracellular signal and the overt response, and then comparing those concentrations to each other and to the

range of concentrations found in blood (Fig. 2). Three possible outcomes apply here: 1). The concentration-effect (doseresponse) curve for generating the signal overlaps, or is very close to, the curve for the overt response, and the lower and upper extremes of the two curves bracket the hormone’s physiological concentration range. Such a finding would be compelling evidence that the signal of interest mediates the response and that the response is physiologically relevant; 2) The curves for the response and the signal of interest completely or nearly overlap but the concentrations required to generate either of them substantially or totally exceed peak blood levels. This would be strong evidence that the signal mediates the response but that neither is physiologically relevant; and 3) The curve for the response of interest overlaps the physiological concentration range, but the curve for the signal presumed to generate that response does not. The latter is instead displaced substantially or completely to the right,

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so that the threshold concentration of the signal curve nearly or completely exceeds peak physiological blood concentrations. In that case, the most plausible interpretation would be that the response is physiologically relevant but is not mediated by the signal of interest. Amassing and analyzing the ex vivo data this way allows the formulation of rational hypotheses concerning the potential role of cAMP in the actions of endogenous glucagon in experimental animals or humans. A survey of the literature compiled over the last few decades confirms that, specifically for glucagon as the agonist and AC/cAMP as the signal, outcome 1) does not apply to any of that hormone’s presumed cAMP-dependent actions on adipose tissue, heart, or liver. Instead, outcome 2) applies to adipose lipolysis and myocardial contractility (Fig. 2 A and B), while outcome 3) applies to hepatic glucose output (Fig. 2C). That is the 50-year-old elephant in the room. Adipose Over the last four decades or so, a variety of reports [13, 23-27] have consistently demonstrated that glucagon increases lipolysis in isolated adipocytes at concentrations between a threshold of close to 0.10 nanomolar (nM) and a maximum of 1,000 nM (Fig. 2A). The response is dependent on activation of AC. The curve for increasing tissue cAMP or activating AC overlaps the lipolysis curve completely, so that both effects are manifested in a concentrationdependent manner over an identical concentration range. The intracellular signal events and molecular targets responsible for cAMP-dependent activation of adipose lipolysis have been well characterized [28, 29]. Both curves, however, lie well to the right of the normal blood concentration range. Blood glucagon concentrations in the rat (mean ± sem) vary between a statistical minimum of 0.030 ± 0.004 nM (10-10.5 M) postprandially to a maximum of 0.085 ± 0.032 nM (10-10.1 M) during starvation-, exercise-, or insulin-induced hypoglycemia [average from refs. 30-39]. The blood concentration range in the human is similar: 0.035 ± 0.005 – 0.064 ± 0.012 nM [average from refs. 40-47]. The collective data summarized in Fig. (2A) are thus consistent with outcome 2 described above. The full range of blood glucagon concentrations is almost entirely below the threshold of the composite curve describing AC activation or lipolysis. The summary evidence, compiled over decades of research from many sources, clearly shows that cAMP mediates the lipolytic response in rat hepatocytes, but that supraphysiological concentrations of glucagon are required to generate both the signal and the effect [48-50]. The apparent EC 50 for the single composite curve representing the stimulation of either AC or lipolysis is around 4.5 nM (pD 2 8.4), approximately 50 fold greater than maximal blood levels. Ligand binding analysis is consistent with the positions of the dose-response curves. The affinity constant (K d ) for specific glucagon binding to isolated rat adipocytes of 6.4 ± 2.4 nM (pKd 8.2) [average from refs. 13, 14, 26, 27, 32] is not markedly different from the EC 50 value of 4.5 nM. In one study of human adipocyte membranes, however, the Kd for glucagon was reported to be 0.5 nM [51]. The binding curves in the rat appear to be monophasic, with no evidence of a second, higher-affinity binding site. For a while, there were suspicions that endogenous adenosine may have artificially suppressed glucagon potency in isolated adipocytes

Robert L. Rodgers

[49], but this hypothesis did not seem to survive subsequent scrutiny [51]. Human adipose tissue is less responsive than the rat’s. The density of glucagon receptors in human adipocyte membranes is much lower than that of glucagon-likepeptide receptors [52]. The majority of studies of human adipose tissue ex vivo indicate that glucagon weakly stimulates lipolysis, but only at concentrations above 10 nM, and binds with a Kd of around 6 nM [53-56]. Thus, in humans, the threshold concentration for activating AC and lipolysis is around 100 times maximal blood levels. Clearly, experimental and clinical studies show that, under normal conditions, blood glucagon would rarely if ever reach the threshold concentrations necessary to activate adipocyte AC, especially in humans. Studies of the lipolytic actions of glucagon in vivo tend to bear this out. Of course, interpretation of in vivo studies can be problematic because of unavoidable indirect or uncontrolled effects that might obscure or confound the results. For example, infusion of somatostatin into human volunteers, to suppress endogenous glucagon secretion, decreases free fatty acid (FFA) levels without altering blood concentrations of insulin or other hormones [57]. But somatostatin can also directly suppress lipolysis in isolated adipocytes [58]. As Fig. (2A) would predict, however, infusion of glucagon into depancreatized dogs, in which insulin and glucose levels are controlled, yielded a blood glucagon concentration of 0.16 nM but did not affect plasma free fatty acid levels [59]. Further, in glucagon receptor (GR -/-) knockout mice, circulating levels of free fatty acids are not different from those in the wild type after feeding or 5 hours of fasting, but are unexpectedly elevated after 16 hours of fasting [60, 61]. The paradoxical effect of prolonged fasting despite the absence of functional glucagon GPCRs could not be attributed to hypoinsulinemia. Instead, it was tentatively explained as a lifting of endogenous glucagon’s chronic induction of hepatic lipid oxidation enzyme expression. In summary, both ex vivo and in vivo evidence, collected over decades, uniformly support previous suggestions [1, 28] that the activation of AC and promotion of lipolysis in adipose tissue is a pharmacological action of glucagon without physiological relevance. Heart Similar questions were raised early on about whether glucagon activates AC and increases contractility of the heart in vivo. In 1964, Kreisberg and Williamson [62] were among the first to report cAMP-generating and inotropic actions of glucagon, on the isolated perfused rat heart, that were appropriately correlated in time and magnitude. But they needed to administer pharmacological concentrations to generate both responses. In their discussion, they speculated that it is unlikely that the effects of glucagon on cardiac contractility (or on hepatic metabolism - see following section) occurs in vivo, because of the wide disparity between the concentrations required to activate AC in those tissues and the levels found in the blood. It seems that the substantial number of investigations of glucagon’s cardiac effects since that time did not dispel those concerns. On the one hand, the collective evidence presented in Fig. (2B) [63-72] verifies that the concentration-effect curves for increases in AC/cAMP and contractile force overlap, supporting the long-held and wellestablished view that the signal mediating mechanical actions


Fig. (3). Concentration-effect curves for glucagon on glycolysis and left ventricular pressure in perfused working rat hearts. The apparent EC50 values for the glycolytic and inotropic dose-response curves are 0.03 and 1.6 nM, respectively. The shaded area represents the average concentration range in rat or human blood [31, 35, 40, 42]. The figure is from reference [75].

of the hormone on the heart is indeed cAMP/PKAdependent. As in adipose tissue, the roles of cAMP and downstream intracellular events responsible for the contractile response in heart have been thoroughly described [73]. On the other hand, the totality of information, amassed over roughly 4 decades, also shows that the overlapping curves for AC activation and contractility lie completely outside the physiological concentration range (Fig. 2B). The distance between the apparent EC 50 of the composite curve – describing the inotropic and cyclic AMP-dependent effects – and the average maximum blood concentration is about 500 fold, ten times higher than the analogous disparity in adipose tissue described above. Again, as in adipose tissue, half-maximal concentrations for response generation and receptor binding are similar. The apparent EC 50 of 38 nM (pD 2 7.4) for stimulating inotropy or AC (Fig. 2B) is fairly close to a reported Kd of 52 nM (pKd 7.3) for specific glucagon binding to myocardial membranes in vitro [69]. Therefore, the results in heart, as in adipose, are consistent with outcome 2 above. As is true for its effects on lipolysis and AC activation in adipose, glucagon does not increase myocardial contractility unless it also activates AC and elevates tissue cAMP levels at pharmacological concentrations [15, 71]. Thus, it has been well established by many reports that the AC/cAMP signal pathway does indeed mediate the inotropic effects of glucagon. However, there is apparently no evidence that endogenous glucagon affects or regulates myocardial contractility in vivo, even under extreme fasting conditions. For instance, this author is not aware of any studies showing that inhibition of glucagon receptors in vivo rapidly reduces myocardial contractility. Thus, neither the activation of AC nor the attendant stimulation of contractile force represents physiological actions of the hormone on the myocardium because the concentrations required to produce those responses are too high. Apparently, Kreisberg and Williamson were right, at least about whether glucagon activates the AC/cAMP pathway and alters myocardial contractile force in vivo.


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Fig. (4). Enhancement of akt phosphorylation by physiological concentrations of either glucagon or insulin in perfused working rat hearts. In both cases, the effect is blocked by the presence of the PI3K/akt inhibitor LY 294002 in the perfusate. The figure is from reference [75].

If glucagon does not influence myocardial contractility in vivo, then perhaps instead it regulates cardiac fuel metabolism. It is well established that glucagon and insulin exert opposing actions on fuel metabolism in the liver (see below), and that insulin regulates utilization of glucose and fatty acids in the heart. But apparently no studies had been reported showing that glucagon might also influence myocardial glucose utilization at physiological concentrations [74]. Glucagon can enhance glycogenolysis in heart by activating glycogen phosphorylase [64]. But that effect is at least partially dependent on stimulation of AC by pharmacological levels of the hormone. Recently, however, we reported a previously unknown metabolic effect of glucagon on the heart that was elicited by physiologically relevant hormone concentrations without activating AC (Fig. 3). We showed that glucagon robustly stimulated myocardial glycolysis in a concentrationdependent manner [75]. Notably, the concentrations of glucagon sufficient to generate the glycolytic response completely overlapped blood levels, did not affect contractility, and appeared to actually decrease tissue cAMP levels. The apparent EC 50 for the glycolytic response was 0.03 nM, over 1200 times lower than the EC 50 for the cAMP/inotropic response shown in Fig. (2A). The signal mediating the glycolytic effect was dependent on the activation of phosphoinositol-3-kinase/akt (PI3K/akt), the same signal that mediates rapid metabolic effects of insulin on heart tissue (Fig. 4). Metabolic responses to peak physiological concentrations of glucagon and insulin were found to be statistically identical [75, 76]. Both hormones stimulated glycolysis and glucose oxidation, and inhibited palmitate oxidation; all three effects produced by the two hormones were quantitatively indistinguishable (Fig. 5). Based on what is known of the insulin effect [77], the enhancement of glycolytic flux by glucagon probably involves both a stimulation of glucose uptake and


Robert L. Rodgers

Fig. (5). Effects of physiological concentrations of glucagon or insulin on glycolysis, glucose oxidation, and palmitate oxidation in perfused working rat hearts. The figure is from references [75 and 76].

alterations in the activities of the glycolytic enzymes phosphofructokinase 1 and 2. Not surprisingly, subinotropic concentrations of glucagon have been reported to produce insulin-like cardioprotective effects in the hypoxic myocardium [78]. Thus, a previously unrecognized, cAMP- independent physiological role of glucagon in the regulation of myocardial fuel metabolism has now been revealed. Unexpectedly, it seems that glucagon and insulin do not oppose each other on heart as they do on liver, but instead mimic each other on myocardial fuel metabolism by activating a PI3K/aktdependent signal. These observations are consistent with outcome 3 above, indicating that the insulin-like enhancement of myocardial glucose utilization is a physiological effect that is independent of the AC/cAMP pathway. Whether the promotion of myocardial glucose utilization by glucagon is mediated by a high-affinity isoform of the cloned glucagon GPCR or by a receptor belonging to a different class is not known. GPCRs for other ligands can be coupled to the activation of PI3K and akt [79]. These include the muscarinic M 2 receptor [80], angiotensin II receptors, alpha adrenoceptors [81], and chemokine receptors [82]. Both subclasses of PI3K in heart, IA and IB, consist of catalytic (p110) and regulatory (p85) subunits. Subclass IA is generally associated with tyrosine kinase receptors, including the insulin receptor, while subclass 1B is more often associated with GPCRs [83-85]. Both Gq and Gi isoforms have been linked to class 1B PI3K [17, 81]. It should be noted here, however, that the potency of glucagon in enhancing myocardial glycolysis (Fig. 3) is considerably higher than it is in promoting hepatic glucose output (Fig. 2C), the latter of which is presumably mediated by a high-affinity isoform of the glucagon GPCR. The apparent EC 50 value for the cardiac glycolytic response is 0.03 nM, nearly 5 times lower than the EC 50 value of 0.14 nM for the hepatic metabolic response, leaving open the possibility that the cardiac insulin-like effect is mediated by a non-GPCR. A recent report is consistent with that hypothesis [86]. In pre-glucose clamped diet-

induced obese mice, administration of anti-glucagon GPCR antibodies markedly elevated plasma glucagon concentrations, decreased plasma glucose and insulin levels, and reduced hepatic glucose output. In spite of producing hypoinsulinemia and hypoglycemia, however, antibody administration nearly doubled the rate of cardiac glucose uptake. The last effect was likely related to the hyperglucagonemia, but would not be expected to occur if the high-affinity cardiac glucagon receptor were an isoform of the cloned GPCR, because it, like the hepatic receptor, would also presumably have been blocked by the antibody. Liver The answer to the central question of this review as it applies to the liver is more complex than it is with regard to adipose or heart. Recall that in adipose tissue, glucagon does not activate lipolysis without also activating AC at pharmacological concentrations. In heart, by contrast, the hormone produces physiological and pharmacological responses that are clearly distinguishable. The first is a PI3K-dependent promotion of glucose utilization at physiological concentrations, and the second is a cAMP-dependent stimulation of inotropy at pharmacological concentrations. The threshold concentration required to activate AC in adipose is approximately 0.1 nM, and even higher than that in heart (Figs. 2A and 2B). The same critical concentration, dividing physiological and pharmacological actions of the hormone, seems to apply to liver as well (Fig. 2C). But the hepatic responses appear to be unique in at least one important respect: physiological and pharmacological effects of glucagon on the liver, elicited by concentrations below and above 0.1 nM, are coincidentally similar or identical but likely mediated by different cellular signals. Often, hepatic processes such as gluconeogenesis, observed to be influenced or regulated by glucagon in vivo, are presumed to be mediated by cAMP because activation of AC by higher concentrations affect the same metabolic processes, in the same direction, in isolated



Table 1. Two glucagon binding affinity constants in liver Preparation

Kd1 (nM)

Kd2 (nM)

Reference

DHM

0.19

2.6

RH

0.14

1.3

95

PHM

0.08

----

108

RH

-----

2.9

114

RHM

-----

1.2

100

RHM

-----

3.0

24

RHM

-----

2.0

69

RHM

-----

3.8

39

X (± sem)

0.14 ± 0.03

107

2.4 ± 0.3

Abbreviations: DHM: dog hepatocyte membranes; RH: rat hepatocytes; PHM: pig hepatocyte membranes; RHM: rat hepatocyte membranes.

hepatocytes or perfused livers ex vivo. Thus, glucagon appears to produce only pharmacological effects on adipose tissue, different physiological and pharmacological effects on heart that are mediated by different signals, and the same physiological and pharmacological effects in liver that are mediated by different signals. The dual action of glucagon on the liver is illustrated in Fig. (2C). As in heart, the hormone produces two distinct concentration-effect curves over a broad concentration range. This was anticipated by an early report from Pilkis and coworkers [87]. They showed that 0.1 nM glucagon increased glucose output from perfused rat liver by about 50% of the maximum, but increased the release of cAMP into the medium by only about 1% of the maximum produced by 4 nM. A number of subsequent studies on hepatocytes verifies that the lower half of the left-hand curve in liver - between 0 and approximately 0.1 nM (peak physiological concentrations) – depicts a significant increase in glucose output (app. 45% max) with little or no activation of AC or increases in tissue levels of cAMP [average of 11 curves from ref.s 88-98]. The right-hand curve - generated by concentrations above 0.1 nM - shows that activation of AC by pharmacological concentrations can further increase glucose output to a higher maximum [average of 10 curves from ref.s 24, 69, 90-92, 99-103]. The curve depicting AC/cAMP activation in Fig. (2C) is very close to, if not identical with, the analogous curve generated in adipose tissue shown in Fig. (2B). In adipocytes, the curves for AC activation and lipolysis overlap, but in hepatocytes it is evident that the curves for AC activation and glucose output are widely separated. Recall that in adipose, the apparent EC 50 value is the same for both AC activation and lipolysis: approximately 4.5 nM (pD 2 8.4). In rat liver, the EC 50 value for AC activation and related intracellular events is similar, approximately 3.5 nM (pD 2 8.5). This is fairly close to an EC 50 of 6 nM reported for activating AC in human liver [24]. By contrast, the apparent EC 50 for the lefthand glucose output curve in Fig. (2C) is 0.14 nM (pD 2 9.8), approximately 25 fold lower than the EC 50 value for AC activation. The metabolic EC 50 concentration is even lower (0.05 nM) if the hormone is administered in a pulsatile manner [104] to minimize tachyphylaxis [105]. Interestingly, 0.05 nM is very close to the EC 50 value of 0.03 nM for the myocardial glycolysis curve in Fig. (3). The separate and distinct hepatic dose-response curves, compiled from multi-

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ple sources, are consistent with outcome 3 above. The curve produced by physiological concentrations, up to 0.1 nM, does not seem to involve activation of AC. Only the upper portion of the glucose output curve, representing extended increases in glucose output produced by concentrations exceeding 0.1 nM, appears to be dependent at least in part on AC activation. The ex vivo data thus predict that endogenous glucagon is able to chronically regulate hepatic glucose output in vivo without activating AC. This hypothesis is supported by the observation that a small molecule glucagon receptor antagonist significantly suppresses hepatic glucose output during fasting [106]. The physiological and pharmacological actions of glucagon in liver, as in heart, may be mediated either by different receptors or by functionally distinct isoforms of the glucagon GPCR. A high affinity glucagon binding site has apparently not yet been found in heart, but it has in liver. Evidence For Two Glucagon Binding Sites in Liver Most studies of glucagon binding reveal a low-affinity, high capacity site in intact hepatocytes or membrane preparations (Table 1). The low-affinity binding site represents the cloned glucagon GPCR associated with AC. The average experimental Kd value of 2.4 nM (Kd 2 in Table 1) for that receptor is very close to the apparent EC 50 of 3.5 nM for the composite right-hand curve describing AC or PKA activation (Fig. 2C). It is also nearly identical to a reported affinity constant of 2.5 nM in human liver [24]. However, at least three experimental studies have also demonstrated the presence of a higher affinity, lower capacity site in liver [95, 107, 108]. The average Kd for the higher affinity site is approximately 0.14 nM (Kd 1 in Table 1), which is identical to the apparent EC 50 of 0.14 nM for the composite left-hand curve in Fig. (2C) representing physiological effects of the hormone. The high- and low-affinity receptors in liver have been designated as G1 and G2 respectively [109]. Both appear to be isoforms of the glucagon GPCR. Expression of the cloned glucagon GPCR in cultured kidney cells results in both high- and low-affinity binding, with K d values of 0.7 and 37 nM [110]. The high-affinity binding site seems to be associated with a Ca2+-dependent signal (see below), while the lower-affinity site is linked to AC activation. Metabolic enzymes and other intracellular effectors whose activities or expressions can be influenced by activation of the lowaffinity GPCR associated with AC have been well characterized [1, 11-114]. An important question here is whether any or all of those intracellular effector enzymes can be similarly regulated by physiological concentrations of the hormone through activation of the high-affinity hepatic receptor in vivo. Dual Actions of Glucagon in Liver: Candidate Physiological Target Enzymes In the fasted state, glucagon stimulates hepatic glucose output by promoting gluconeogenesis with little or no stimulation of glycogenolysis [115]. Table 2 consists of a list of intracellular effector enzymes that are involved in the regulation of hepatic gluconeogenesis and glucose output, and whose activities or expressions can be influenced by glucagon [3, 21, 116 -118]. Depending on the target enzyme, the nature of the regulation can be either transcriptional (delayed


Robert L. Rodgers

Table 2. Hepatic metabolic enzymes influenced or regulated by glucagon at concentrations above and below 0.1 nM. Effective Concentrations 0.1 nM

Enzyme

> 0.1 nM

References

Transcriptional regulation Pyruvate kinase expression

+

248 – 250

Glucose-6-phosphatase expression

+

143, 252

PEPCK expression

+

+

121, 124, 132

F-1,6-BP activity

+

+

174, 259

Glycogen synthase activity

+

+

101, 107, 253 – 255,260

Glycogen phosphorylase activity

+

+

101, 207, 233,

Post-translational regulation

254, 256- 258, 261

6-PF-2K/F-2,6-BP activity

+

+

220, 259, 262 - 264

Pyruvate kinase activity

+

+

174, 175, 177, 265, 266

Abbreviations: 6-PF-2K, 6-phosphofructo-2-kinase; F-1,6-BP, fructose-1,6-bisphosphatase; F-2,6-BP, fructose-2,6-bisphosphatase; PEPCK, phosphoenolpyruvate carboxykinase.

alterations in enzyme expression), posttranslational (relatively rapid alterations in activity), or both. Rapid actions on enzyme activity can be elicited directly, most often by protein kinase-mediated phosphorylation, or indirectly by alterations in the intracellular levels of allosteric modulators. All of the effector enzymes listed in Table 2 can be affected by concentrations of glucagon exceeding 0.1 nM. Thus, they are all subject to regulation by AC/cAMP/PKA- dependent mechanisms through activation of the low-affinity glucagon receptor. Accordingly, they can be mimicked or duplicated by PKA itself or lipid-soluble analogs of cAMP. Note also from Table 2 that at least all but two of the listed processes - the expressions of glucose-6-phosphatase and pyruvate kinase - can also be influenced by glucagon at concentrations at or below 0.1 nM, and thus are candidates for cAMP-independent regulation by the hormone. It is important to emphasize that the directions of the changes - in either expression or activity - induced by glucagon are the same on all targets regardless of whether the hormone produces them at physiological or pharmacological concentrations, i.e., above or below 0.1 nM. Accordingly, they may be dually and redundantly regulated by glucagon via cAMPdependent and cAMP-independent signal pathways. We can focus here on phophoenolpyruvate carboxykinase and pyruvate kinase. Those two enzymes specifically are important regulators of hepatic gluconeogenesis and are wellcharacterized glucagon targets [119]. Delayed increases in phosphoenolpyruvate carboxykinase expression and rapid inhibition of pyruvate kinase activity are two dominant effects of glucagon in promoting hepatic gluconeogenesis and glucose output [119]. Transcriptional Carboxykinase

Regulation

of

Phosphoenolpyruvate

Phosphoenolpyruvate carboxykinase (PEPCK) is a ratelimiting enzyme in the hepatic gluconeogenic pathway [21]. Its expression, and thus its influence on gluconeogenesis, is

regulated reciprocally by glucagon and insulin [21, 120-124] (Fig. 6A and B). Peak physiological concentrations of insulin are sufficient to inhibit PEPCK expression by about 80%. The effect of insulin (Fig. 6B) to suppress the expression of PEPCK - or of glucose-6-phosphatase - is largely attributable to activation of a PI3K/akt-dependent signal pathway and downstream phosphorylation and inhibition of the forkhead box protein O1/A2 transcription factor (Fox01) and the transducer of regulated cAMP response element binding protein (CREB) activity 2 (TORC2) [125-130]. Recent evidence also implicates the high-mobility AT-hook 1 (HMGA1) nuclear factor in hepatic PI3K-mediated effects of insulin [131]. Glucagon elicits opposite effects on the expressions of many of the same enzymes and cofactors. At concentrations exceeding 0.1 nM, glucagon activates AC and increases the synthesis and production of mRNA for PEPCK as well as for glucose-6-phosphatase [132, 133]. The mechanism involves PKA-induced phosphorylation of CREB and dephosphorylation of TORC2. Subsequent association of TORC2 with CREB increases the expression of the coactivator peroxisome proliferator activated receptor –-coactivator 1 (PGC-1) which, in association with transcription factors Fox01, HNF4, and others, promotes the transcription of PEPCK and glucose-6-phosphatase [22, 118, 133 – 145]. Notably, fasting can similarly influence several nuclear regulatory proteins involved in the expression of gluconeogenic genes and hepatic glucose output, including CREB, PGC-1, C/EBP, and TORC2 [127, 146 – 148]. As noted above, however, fasting rarely increases blood glucagon levels above 0.085 nM, which is not sufficient to activate AC. Further, the absence of hypoglycemia in TORC2 mutants is consistent with the view that influencing hepatic TORC2 may not be necessary for glycemic control by endogenous glucagon in vivo [149]. Accordingly, activation of the AC/cAMP pathway by glucagon apparently accounts only for the upper half - beyond 0.1 nM - of the dose-response curve for PEPCK expression shown in Fig. (6A).



369

Fig. (6). Concentration-effect curves for glucagon (A) and insulin (B) on the expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat hepatocytes. Shaded areas represent physiological blood concentration ranges for glucagon (see legend Fig. 2A) or insulin [179]. The curves are from [120] and [124].

In contrast, regulation of PEPCK by physiological concentrations, below 0.1 nM, is presumably via a cAMP/TORC2 - independent mechanism, or at least a pathway that bypasses the PKA-dependent component [121, 124, 150, 151]. The peak physiological concentration of glucagon (0.1 nM) strongly stimulates transcription rate, mRNA abundance, and PEPCK activity in rat hepatocytes [124], with little or no activation of AC (Figs. 2C and 6A). Physiological validity of the cAMP/PKA-independent response is further supported by studies of exogenous glucagon actions in humans [44, 152]. Glucagon infusion in normal subjects, sufficient to raise blood glucagon levels only from 0.04 to 0.08 nM, increased the rate of hepatic glucose output and gluconeogenesis from glutamine by approximately 1.5 to 2 fold over 60 minutes, in spite of a parallel increase in plasma insulin levels [44]. Recent transgenic mouse studies provide additional insight. If regulation of PEPCK in vivo requires continual activation of AC and related signal pathways, regardless of hormone concentration, then PEPCK expression should be suppressed by any of three interventions: 1) removal of critical hepatic AC/PKA-related signal components, 2) elimination or inhibition of hepatic glucagon receptors, or 3) imposition of aglucagonemia. Alternatively, if cAMPdependent alterations of PEPCK expression were produced only by concentrations exceeding maximum blood levels, then interventions 2) and 3) would still inhibit PEPCK expression in vivo, but intervention 1) would not. This is indeed what happens. As discussed above, TORC2 is involved in the regulation of PEPCK expression and is dephosphorylated and activated only by high concentrations of glucagon as well as by exogenous cAMP [118, 127, 138, 149, 151-155]. Accordingly, the absence of the TORC2 regulator [149] inhibits the ability of exogenous glucagon, at a pharmacological concentration of 50 nM, to enhance the expression of PEPCK above baseline. However, TORC2-null mice have normal expressions of PEPCK, and normal blood glucagon, insulin, and glucose levels as well, when compared to the wild type [149]. By contrast, glucagon receptor knockout mice are characterized by suppressed hepatic PEPCK expression but normal phospho-CREB levels and blood insulin

concentrations in spite of hyperglucagonemia [61]. Aglucagonemic mice, whose alpha cells had been transgenically ablated, display pronounced (70%) suppression of basal hepatic PEPCK expression and moderately decreased glucose output, also with normal insulin levels [154]. In the dietinduced obese mouse study mentioned above, administration of anti-glucagon GPCR antibodies markedly decreased hepatic PEPCK expression without altering the expression of pyruvate kinase [86], as predicted for these enzymes in Table 2. Thus, a substantial body of evidence, both ex vivo and in vivo, is consistent with the hypothesis that endogenous glucagon regulates the hepatic expression of PEPCK and glucose output by activating a high- affinity GPCR and a signal that does not involve activating the PKA-dependent TORC2/CREB pathway [155]. Which signal or signals do mediate the physiological regulation of PEPCK expression by glucagon remains an open question. Other signals that have been linked to glucagon include NR4A, LKP1, AMPK, mTOR, and JNK1, and ERK1/2 [156 – 166]. Activation of most of them, however, requires PKA activation by either cAMP or high hormone concentrations. In addition, none of their intracellular effectors is clearly associated with increases in hepatic PEPCK expression or stimulation of gluconeogenesis produced by physiological glucagon concentrations. For example, NR4A (NUR77), an orphan nuclear receptor, has been implicated in the regulation of hepatic glucose metabolism [160]. Either dibutyryl cAMP ex vivo or a very high dose of glucagon in vivo profoundly (30 – 1,000 fold) increases the activity of hepatic NR4A as well as expressions of several gluconeogenic enzymes including glucose-6-phosphatase (see Table 2) but - notably - not PEPCK [160]. Glucagon is capable of phosphorylating AMP kinase (AMPK), but only at high concentrations required to activate PKA [142, 167]. In any case, phosphorylation and activation of AMPK leads to decreased PEPCK expression and diminished rates of gluconeogenesis [142, 167]. AMPK may, however, be involved in the modulation by endogenous glucagon of hepatic lipid metabolism [168, 169]. Apparently, the PLC/DAG/IP3 signal (Fig. 1) is not a candidate for PEPCK upregulation either.


Activators of this calcium-dependent signal, such as vasopressin or phenylephrine, inhibit the expression of PEPCK in hepatocytes [170, 171]. However, a PLC-dependent signal does seem to be implicated in the rapid, posttranscriptional actions of glucagon on pyruvate kinase activity (see following section). One intriguing possibility for a mediator of PEPCK regulation by endogenous glucagon in vivo is p38 MAPK, but the evidence is not completely consistent. This signal component has been implicated in glucagon-induced inhibition of hepatic lipogenesis [172]. It can be a downstream target of PKA-induced activation of CREB-mediated gene expression, including the expression of hepatic PEPCK [136, 156]. In isolated hepatocytes, p38 can be phosphorylated and activated by pharmacological concentrations of glucagon (10 100 nM) [156, 158, 172]. Whether lower concentrations might also be effective has apparently not been determined in direct studies. Fasting in mice does increase phosphorylation of hepatic p38 and CREB and enhances PEPCK expression, and all three effects are blocked by prior treatment in vivo with SB 203580, a p38 kinase blocker [158]. However, in glucagon receptor knockout mice, hepatic phospho-CREB levels are not diminished compared to those of the wild type [61]. The last result is inconsistent not only with a role of p38 kinase, but also of AMP/PKA, in regulating CREB and PEPCK expression in vivo. As such, it is difficult to interpret. One experiment that might help to shed some light on this would be to test whether low levels of TH glucagon (a partial glucagon agonist that does not activate AC; see below) can enhance hepatic CREB phosphorylation in isolated hepatocytes. Another might be to determine whether pretreatment with SB 203580 is able to inhibit the ability of either TH glucagon or low glucagon concentrations to phosphorylate CREB and enhance PEPCK expression. Until more definitive data become available, it would seem reasonable to hypothesize for the time being that glucagon can stimulate hepatic PEPCK expression by at least two mechanisms, one by activating PKA at higher concentrations and the other by activating p38 kinase at physiological levels without involvement of PKA, with both pathways converging on CREB. Only the latter would presumably be operative in vivo. Post-translational Regulation of Pyruvate Kinase L (liver)-type pyruvate kinase (PK) is a glycolytic enzyme that is also, like PEPCK, involved in the regulation of gluconeogenesis. Phosphorylation of PK inhibits its activity, simultaneously inhibiting glycolysis and promoting gluconeogenesis by increasing the intracellular ratio of phosphoenolpyruvate (PEP) to pyruvate [92, 173]. Glucagon can phosphorylate PK over a broad concentration range, from physiological to pharmacological [174, 175] (Fig. 7A). Although not always observed [see 176], peak physiological concentrations of glucagon have been reported to produce about 40% maximal phosphorylation of PK [177]. Similar concentrations also inhibit PK activity by 30 – 65% [175, 178]. Correspondingly, increases in gluconeogenesis and glucose output reach approximately 60 – 65% of the maximum attainable by higher, AC-activating hormone concentrations [94-96] (Fig. 7A). This pattern is roughly an inverted image of analogous responses to insulin (Fig. 7B). At its

Robert L. Rodgers

maximum physiological concentration [179], insulin inhibits glucose output by about 70 – 75% [180], while stimulating PK activity to approximately 75 -80% of maximal levels achievable by higher insulin concentrations [180, 181]. Therefore, peak physiological concentrations of either hormone produce approximately 60 – 80% of their respective maximal effects on PK activity and glucose output. With glucagon, however, the apparent physiological maximum may be an underestimate because the ultimate peak response, produced at higher concentrations, appears to be an additive effect resulting from simultaneous activation of both cAMPindependent and cAMP-dependent signals (see below). The stimulation of PK by insulin is predominantly or exclusively mediated by a PI3K/akt-dependent pathway [181]. As we have seen, a similar or identical PI3K-dependent signal is also involved in the stimulation of glycolysis by either insulin or glucagon in heart (Fig. 4). However, a role for that pathway in the anti- insulin effect of glucagon on PK activity in the liver is unlikely [182, but see 183]. The search for the elusive physiological glucagon signal in liver, mediating its rapid effects on glucose metabolism, has been intense but not totally definitive. The most plausible candidate is the well-characterized phospholipase C-inositol triphosphate-diacylglycerol (PLC/IP3/DAG) pathway [94, 110]. Activation of the high-affinity glucagon receptor is associated with robust but submaximal increases in hepatic glucose output [184] and generation of inositol phosphates [109, 185, 186]. Other hormones that activate this pathway, such as alpha agonists, angiotensin II, or vasopressin, elevate hepatic intracellular Ca2+ and inhibit PK, presumably via PK phosphorylation by calcium-calmodulin-dependent protein kinase (CaCAMPK) [117, 187, 188]. High concentrations of angiotensin II (0.1 M) strongly inhibit the activation of AC by glucagon at the maximally effective concentration of 1 M. Nevertheless, the same concentration of angiotensin II actually enhances the activation of gluconeogenesis produced by glucagon at its peak physiological concentration of 0.1 nM [184]. Low concentrations of glucagon can substantially increase intracellular Ca2+ levels sufficient to activate CaCAMPK [189 – 191]. Two sites on the PK enzyme can be phosphorylated in response to glucagon exposure, decreasing the affinity of the enzyme for its substrate phosphoenolpyruvate (PEP) [21, 174, 176, 192 – 197]. Both sites can be phosphorylated by CaCAMPK, and one of them can also be phosphorylated by PKA or high concentrations of glucagon [192 – 194]. Overall, it seems that phosphorylation of PK by CaCAMPK alone is sufficient to account for approximately 65% of the maximal inhibition of the enzyme – and the corresponding 50 - 60% maximal increase in glucose output – produced by CaCAMK activators or peak physiological concentrations of glucagon [177] (Fig. 2C). The remaining 40 – 50% of the PK phosphorylation and further enhancement of glucose output is presumably dependent on PKA activation and further phosphorylation of PK by higher concentrations of the hormone. Although phorbol esters can mimic some of the hepatic actions of insulin [198], insulin itself does not appear to act via this pathway in producing its rapid metabolic effects on this tissue [199, 200]. The collective results thus strongly suggest that activation of the high-affinity glucagon receptor by physiological concentrations substantially



371

Fig. (7). Concentration-effect curves for glucagon (A) and insulin (B) on pyruvate kinase activity in relation to glucose output in rat hepatocytes. Shaded areas represent physiological blood concentration ranges for each hormone (Figs. 2 and 6). Adapted from [94 - 96, 175, 180, 181].

Fig. (8). Concentration-effect curves for glucagon (A) and TH-glucagon (B) on rat hepatocytes. The multiple curves are adapted from references 91, 109, 175, 177, 189, and 191.

inhibits PK, and consequently promotes gluconeogenesis, by activating the PLC/IP3/DAG signal pathway and CaCAMK, without affecting AC or increasing the production of cAMP. This conclusion is further supported by the collective results of various studies comparing hepatic effects of glucagon with those of TH-glucagon, a partial agonist on glucagon receptors (Fig. 8). TH glucagon, like glucagon, stimulates hepatic inositol-phosphate production but unlike glucagon does not activate AC except perhaps at very high concentrations [109, 201]. Glucagon and TH-glucagon are similarly potent on hepatic glucose output. At concentrations up to 0.1 nM, either glucagon (Fig. 8A) or TH-glucagon (Fig. 8B) produces linear increases in inositol-phosphate generation and gluconeogenesis without affecting tissue cAMP levels. As a partial agonist, TH-glucagon elicits a weaker maximal gluconeogenic response at 0.1 nM. With regard to glucagon, these elevations in inositol phosphate generation are associated with parallel increases in intracellular calcium

levels and PK phosphorylation. Whether similar effects can also be produced by TH-glucagon over the same concentration range is apparently not known. At concentrations above 0.1 nM, either glucagon or TH-glucagon produces a surge in inositol-phosphate levels, but only glucagon further enhances gluconeogenesis, presumably by activating AC. In vivo studies support this conclusion. In a comparison of the effects of glucagon and TH-glucagon in the guinea pig [202], both hormones increased hepatic inositol phosphates by approximately the same extent, as predicted in Fig. (8). However, glucagon increased tissue cAMP and elevated plasma glucose by 40%. By contrast, TH glucagon decreased tissue cAMP but still increased plasma glucose by around 20%. Not all of the evidence is consistent with the PLC/IP3/Ca2+ hypothesis, however [21]. For example, Hue et al. [203] reported that the -agonist phenylephrine, an activator of PLC, produced a concentration-dependent increase in


glucose output without altering PK activity in rat hepatocytes. In another study, 1 M glucagon did not change 32P radioactivity in phosphatidylinositol-4-phosphate or phosphatidylinositol-4,5-bisphosphate in hepatocyte membranes. By contrast, 1 M angiotensin, another PLC activator that does not activate AC in liver, decreased the radioactivity of the two phosphoinositides by about 12 and 19%, respectively [204]. Similarly, 10 nM glucagon did not increase IP 3 levels in hepatocytes, but 100 nM vasopressin increased them by about 2 – 4 fold [205, 206]. It should be noted, however, that the stimulation of inositol phosphate production, generated by physiological concentrations, can be suppressed by higher concentrations that are sufficient to strongly activate AC [109 and Fig. 8A] (see below). Collectively, the results summarized in Figures 2C and 6 - 8 are consistent with the view that glucagon is capable of producing substantial increases in hepatic gluconeogenesis and glucose output at physiological concentrations without activating AC. The physiological and pharmacological actions are coincidental but are produced by different mechanisms (Fig. 8). The bulk of the available evidence indicates that the rapid physiological effect on PK activity, manifested below 0.1 nM glucagon, is dependent on activation of PLC (generation of inositol phosphates), mobilization of Ca2+, and activation of CaCAMPK, but is apparently not dependent on attendant activation of protein kinase C (PKC) [88]. Phorbol esters activate PKC without increasing intracellular Ca2+ [207, 208], and do not phosphorylate PK [209] or increase hepatic glucose output [210]. The magnitude of the rise in intracellular Ca2+ in response to glucagon appears to be sufficient to activate CaCAMPK, phosphorylate PK, and correspondingly increase gluconeogenesis to a significant extent. Concentrations of glucagon in excess of 0.1 nM potentiate the stimulation of gluconeogenesis, most likely through a combination of further CaCAMPK- and PKA-dependent phosphorylation of PK. The supplemental rise in intracellular Ca+2 levels at higher concentrations parallels the increases in cAMP, and thus appears to be largely the result of PKA- dependent stimulation of intracellular calcium ATP-ases or membrane calcium channels that are not responsive to CaCAMPK [211 – 213]. The sudden, profound increase in inositol-phosphate generation (Fig. 8A) produced by barely supraphysiological glucagon concentrations (just above 0.1 nM) also seems to be independent of the cAMP/PKA signal pathway, because it is roughly duplicated by TH-glucagon (Fig. 8B). A speculative explanation is that either glucagon or TH-glucagon at those concentrations activates a subpopulation of the high-capacity, low-affinity GPCR that are associated with PLC rather than AC. Finally, the reversal of the rise in inositol-phosphates produced by glucagon at or above 100 nM (Fig. 8B) does appear to be related to the elevations in cAMP, because it is not produced by TH- glucagon. The mechanism is obscure, but the effect may at least partially explain contravening evidence, described above, that pharmacological glucagon concentrations lack substantial effects on inositol-phosphate generation. What About Signal Amplification or Spare Receptors? When the curve for cAMP generation even slightly overlaps the curve for the overt response (e.g. Fig. 2C), questions of signal amplification inevitably arise. For example, Christ

Robert L. Rodgers

and Nath [214] reported that 0.1 nM glucagon slightly elevated tissue cAMP above basal levels - the increase was only 8% of that produced at 1 nM – but robustly increased both expression and activity of pyruvate carboxykinase in hepatocytes. Early on, Sutherland and coworkers were able to detect very small increases in tissue levels of cAMP produced by low concentrations of glucagon, which were associated with relatively larger elevations in glucose output from the perfused rat liver [92]. Birnbaum and Fain [215] and Blair et al. [178] also noted a discrepancy between lower concentrations of the hormone required to activate hepatic glycogen phosphorylase and higher concentrations required to activate AC (see Table 2). They suggested that the activation of phosphorylase by lower concentrations might have been due either to an amplified AC-dependent signal or to a PKAindependent signal. Over the ensuing years, others have hypothesized that an amplified signal might be associated with the activation of a pool of high- affinity spare GPCRs coupled to AC [216, 217]. The available evidence, though, seems to be inconsistent with amplification of the AC/cAMP signal as an explanation for hepatic metabolic effects produced physiological glucagon concentrations. For instance, it would seem reasonable to ask why the phenomenon would apply only to the liver, and not to the heart or adipose tissue. If amplification of the AC signal is responsible for the separation of the curve shown in Fig. (2C) for hepatic glucose output, then the analogous curves for cAMP generation and either lipolysis in adipose or inotropy in heart (Figs. 2A and 2B) might also be expected to be separated rather than congruent. In addition, if hepatic phospho-CREB levels were exclusively dependent on amplification of the PKA/cAMP signal subsequent to GPCR activation, then the extent of CREB phosphorylation should be markedly decreased by interruption of glucagon action in vivo. As discussed above, in glucagon receptor knockout mice, the ratio of hepatic phospho-CREB to CREB values are not diminished [61], but they should be if amplification of the cAMP-dependent signal were operative in vivo [139, 218]. Also, TH-glucagon should have no stimulatory effects on hepatic gluconeogenesis or glucose output, because it presumably stimulates the same receptor as glucagon does but lacks intrinsic activity on AC and the cAMP-dependent signal. But the similarly potent TH derivative does indeed mimic the effects of glucagon on hepatic glucose output at physiological concentrations (Fig. 8). As expected, infusion of the cAMP-independent partial agonist TH-glucagon in vivo is about half as effective as glucagon in elevating blood glucose levels [202]. Finally, as discussed above, the true signal in heart mediating the physiological response (lefthand curve in Fig. 4) was identified as PI3K, with no involvement of cAMP. It seems reasonable to hypothesize that different signals would also explain similarly separated leftand right-hand curves for the liver as well (Fig. 2C). It thus appears very likely that early predictions by Birnbaum and Fain [215] and Blair et al. [178], that activation of AC may not explain the enhancement of hepatic glycogen phosphorylase [219], may be correct and may also be more generally applicable to PK and other metabolic enzymes targeted by the hormone [220] (Table 2). It also seems that there is little direct evidence to support the existence of spare glucagon receptors but substantial evi-



373

Table 3. Comparisons of potency and affinity of glucagon: Lack of evidence for spare glucagon receptors in adipose, heart, or livera. EC50 b (nM)

Kdc (nM)

pD2b (-log M)

Adipose lipolysis or AC activation

4.5

6.4

8.4

8.2

Heart contractility or AC activation

38

52

7.4

7.3

Heart glycolysisd

0.03

-----

10.5

----

Tissue and responses

pKdc (-log M)

Liver glucose output (GR2)

3.5

2.4

8.5

8.6

Liver glucose output (GR1)

0.14

0.14

9.8

9.8

a

Averages of values compiled from references 13, 23-27, 39, 63-67, 69, 71, 72, 75, 88 – 103, 107, 108, 114, 180, 186, and 224. Linear and logarithmic values of the index of glucagon potency. Linear and logarithmic (M) values of the index of glucagon binding affinity. d High-affinity glucagon receptors in heart have not yet been detected in binding studies. b c

dence that refutes it. A key requirement of spare receptor systems is that the EC 50 concentration associated with the response is significantly lower than the Kd concentration describing the agonist’s affinity for the receptor. In the absence of spare receptors, the two values would be similar or identical. For ease of comparison, the EC 50 and Kd values for glucagon in adipose, heart, and liver, discussed above, are presented together here in Table 3. It is evident from the table that the EC 50 and Kd values, describing AC-dependent effects and low-affinity receptor binding, are similar concentrations in all three of the major target tissues. In addition, the EC 50 and Kd values for hepatic glucose output and highaffinity receptor binding are identical. These average values, representing multiple studies, would indicate that there are no spare high- or low-affinity receptors mediating any of glucagon’s effects on adipose, heart, or liver. By the same criteria, however, there is very good evidence of spare insulin receptors in liver [221]. As discussed above, the EC 50 value for the cardiac glycolytic response to glucagon is substantially lower than the analogous value for hepatic glucose output, leaving open the possibility that the inotropic and glycolytic responses in heart might be mediated by receptors belonging to different classes. As stated previously, the predicted high-affinity binding site for glucagon in heart has not yet been revealed. SUMMARY: PROPOSED NEW MODEL OF RAPID METABOLIC ACTIONS OF GLUCAGON IN VIVO The central question addressed by this review is whether cyclic AMP mediates any actions of endogenous glucagon on adipose, heart, or liver in vivo. Evidence cited above strongly suggesting that it does not can be summarized as follows: 1. In adipose tissue, glucagon does not promote lipolysis without activating AC, but the required concentrations are too high to be physiologically relevant. There is no clear evidence that endogenous glucagon, over the normal blood concentration range, can stimulate adipose lipolysis in either experimental animals or humans. 2. In heart, glucagon does not increase contractility without activating AC, but as in adipose the required concen-

trations exceed peak blood levels. A newly-discovered, insulin-like effect of glucagon to promote glucose utilization is physiologically significant because it is produced by physiological concentrations of the hormone, far below the threshold concentration required to activate AC. The effect is mediated by PI3K, the same signal that mediates identical rapid metabolic actions of insulin on the myocardium. 3. In liver, glucagon exerts dual and coincidental metabolic actions depending on the concentration. It can promote glycogenolysis, gluconeogenesis, and glucose output at concentrations exceeding 0.1 nM by activating AC/cAMP. It can also produce many or all of those same effects at physiological concentrations, at or below 0.1 nM, without activating AC. Stimulation of hepatic gluconeogenesis and glucose output by physiological concentrations of the hormone involves delayed increases in hepatic PEPCK expression, possibly mediated by p38 MAPK, and rapid inhibition of pyruvate kinase activity, likely mediated by a PLC/Ca2+/CaCAMPK- dependent signal. 4. None of the physiological actions of glucagon in heart or liver can be explained by amplification of the AC/cAMP signal or by activation of spare glucagon receptors. A proposed new model of glucagon’s metabolic actions in vivo, highlighting rapid effects on hepatic gluconeogenesis and myocardial glucose utilization, is summarized in Fig. (9). Hypoglycemia - induced by prolonged fasting, exercise, or other conditions – simultaneously serves as the predominant stimulator of glucagon secretion from pancreatic islet alpha cells and inhibitor of insulin production from pancreatic beta cells. Elevations in plasma glucagon levels up to a maximum of 0.1 nM have opposite metabolic effects on the liver and extrahepatic tissues. On the liver, glucagon activates a highaffinity receptor coupled to a PLC/IP3/Ca2+-dependent signal pathway. The associated G protein is likely Gq/11 [222]. Among other potential intracellular targets of this signal (Table 2) is the glycolytic/gluconeogenic enzyme pyruvate kinase (PK). An IP3-induced rise in intracellular Ca2+ concentration rapidly activates CaCAMPK, which phosphorylates PK at two sites on the enzyme, inhibiting its activity and consequently promoting gluconeogenesis and glucose output. This rapid effect of glucagon is complemented by


Robert L. Rodgers

Fig. (9). Proposed new model of the rapid actions of glucagon on the liver and heart. In hypoglycemia, glucagon secretion is increased, with blood levels peaking at or around 0.1 nM. On the liver, glucagon stimulates a high-affinity GPCR. The / subunits recruit and activate phospholipase C beta (PLC) to increase the synthesis of inositol triphosphate (IP3). The subsequent increase in intracellular Ca2+ levels activates calcium-calmodulin-dependent protein kinase (CaCAMPK), phosphorylating and inhibiting pyruvate kinase (PK), thus stimulating gluconeogenesis. The G protein mediating the calcium-dependent effect is probably Gq [222]. On the heart, and possibly skeletal muscle, glucagon is depicted as activating a high-affinity GPCR coupled to Gq to recruit and activate phosphoinositide-3-kinase (PI3K), whose regulatory and catalytic subunits are p110 (or ) and p85, respectively. Whether the high-affinity cardiac receptor coupled to PI3K is a GPCR or a receptor belonging to a different class has not been established with certainty. The activation of PI3K leads to increased glucose uptake and glycolysis. Actions of glucagon on glucose metabolism oppose those of insulin on the liver but mimic them on heart. None of these effects of glucagon at physiological concentrations, on heart or liver, requires the activation of adenylate cyclase. Adapted from references [75, 81, 83, 113, 222].

delayed stimulation of PEPCK expression, which adds to the promotion of gluconeogenesis (not shown because the presumed cAMP-independent signal for the delayed effect is much less clear). The hypoglycemic state also lifts insulinimposed inhibition of glucagon’s hepatic metabolic actions. Meanwhile, glucagon exerts a pro-insulin action on the heart. Stimulation of a high-affinity myocardial glucagon receptor activates a PI3K (p110 or ) -dependent signal pathway, promoting glucose uptake, glycolysis, and glucose oxidation. The same or similar signal mediates identical actions of insulin on this organ. Interestingly, an enhancement of glucose utilization has also been implicated in metabolic effects of GLP-1 on the myocardium [223], but the signal or signals that may mediate those effects have not been identified. Thus, as the main source of blood glucose shifts from dietary to hepatic, glucagon assumes a relatively greater role in opposing actions of insulin on the liver while supplanting the actions of insulin on the heart and possibly other extrahepatic tissues such as skeletal muscle, kidney, and intestine [224 – 226]. Overall, glucagon maintains euglycemia and extrahepatic tissue glucose utilization in response to episodes of hypoglycemia, when the levels and influences of insulin recede. IMPLICATIONS FOR THE PATHOPHYSIOLOGY AND TREATMENT OF DIABETES If ultimately confirmed, the new model proposed here could have fundamental implications applicable not only to

normal glucagon physiology but also to the role of glucagon in the pathophysiology and treatment of diabetes. Glucagon levels are often moderately elevated in diabetes [227 – 230]. Increases in glucagon-to-insulin ratios contribute to diabetic hyperglycemia in experimental animals and humans [22, 74, 186, 226, 231-235]. Elimination of glucagon receptor expression or pharmacological antagonism of glucagon receptors confers resistance to hyperglycemic and other adverse effects of diabetes [61, 236-239]. Suppression of endogenous glucagon production or antagonism of glucagon’s metabolic actions on the liver are thus considered to be important therapeutic goals in the treatment of both type I and type II diabetes [231, 233, 240-246]. The amelioration of diabetic hyperglycemia by globally antagonizing the glucagon receptor is most often explained by the inhibition of hepatic glucose output. One implication of the proposed new model, however, is that glucagon may be both an enemy and an ally in the pathogenesis and treatment of diabetes, depending on the affected organ. Its hepatic actions are counterproductive because they exacerbate diabetic hyperglycemia, but its cardiac actions may be beneficial because they would promote glucose utilization by that organ against a background of hypoinsulinemia or insulin resistance. The current standard use of exogenous glucagon itself is to antagonize insulin induced hypoglycemia [246]. The rationale is to promote hepatic glucose output to maintain euglycemia. But an unrecognized


parallel benefit may be the attendant promotion of glucose utilization in muscle. Until now the development of antiglucagon therapeutic agents have focused on global glucagon antagonism, with scant attention paid to the potential advantages of antagonists that are hepatoselective or of glucagon agonists that target the heart or other extrahepatic tissues. As an illustration, administration of anti-human glucagon receptor antibodies improves glucose tolerance in mice and monkeys [247]. In monkeys, this is associated with either no change or a slight decline in blood insulin levels but a significant increase in levels of glucagon. One possible interpretation of the markedly improved glucose tolerance is that the antibody is selective for the hepatic glucagon receptor. As discussed above, the high-affinity receptor on heart, and possibly other extrahepatic tissues, may be a distinct GPCR isoform but might also be a member of a different class, and as such might escape blockade by the antibody. Under those conditions, the improved glucose tolerance may be a combination of inhibited hepatic glucose output and preserved or enhanced glucose utilization in extrahepatic tissues. This review is a call for renewed investigations into the endocrinology of glucagon, with emphasis on cellular actions of the hormone produced by physiological concentrations, at or below 0.1 nM. Eventual confirmation that glucagon regulates systemic glucose metabolism in its target cells and organs without activating AC has the potential to increase our understanding of this important hormone and its role in endocrine metabolic physiology as well as in the pathophysiology and treatment of diabetes and other endocrine disorders.


PKA

=

protein kinase A

PLC

=

phospholipase C

TH-glucagon =

1-N- trinitrophenylhistidine 12 homoarginine - glucagons

TORC2

transducer of regulated CREB activity 2

[1] [2] [3] [4]

[5] [6] [7] [8] [9] [10]

[11]

ACKNOWLEDGEMENTS Declared none.

[12] [13]

ABBREVIATIONS

[14]

AMPK

=

AMP kinase

CaCAMPK

=

calcium-calmodulin-dependent protein kinase

cAMP

=

cyclic adenosine monophosphate

CREB

=

cAMP response element binding protein

DAG

=

diacylglycerol

Fox01

=

forkhead box protein 01/A2 transcription factor

GPCR

=

G protein-coupled receptor

IP3

=

inositide-3-phosphate

PEPCK

=

phosphoenolpyruvate carboxykinase

PGC-1

=

peroxisome proliferator activated receptor--coactovator 1

PI3K

=

phosphoinositide-3 kinase

PK

=

pyruvate kinase

=

REFERENCES

CONFLICT OF INTEREST Declared none.

375

[15]

[16] [17]

[18]

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