REVIEW ARTICLE
review article
Diabetes, Obesity and Metabolism 17: 720–725, 2015. © 2015 John Wiley & Sons Ltd
Inhibiting or antagonizing glucagon: making progress in diabetes care P. J. Lefèbvre1 , N. Paquot1 & A. J. Scheen1,2 1 Division of Diabetes, Nutrition and Metabolic Disorders, Department of Medicine, University of Liège, Liège, Belgium 2 Division of Clinical Pharmacology, Department of Medicine, University of Liège, Liège, Belgium
Absolute or relative hyperglucagonaemia has been recognized for years in all experimental or clinical forms of diabetes. It has been suggested that excess secretion of glucagon by the islet 𝛼 cells is a direct consequence of intra-islet insulin secretory defects. Recent studies have shown that knockout of the glucagon receptor or administration of a monoclonal specific glucagon receptor antibody make insulin-deficient type 1 diabetic rodents thrive without insulin. These observations suggest that glucagon plays an essential role in the pathophysiology of diabetes and that targeting the 𝛼 cell and glucagon are innovative approaches in the management of diabetes. Despite active research and identification of promising compounds, no one selective glucagon antagonist is presently used in the treatment of diabetes. Interestingly, besides insulin, several drugs used today in the management of diabetes appear to exert their effects, in part, by inhibiting glucagon secretion (glucagon-like peptide-1 receptor agonists, dipeptidyl peptidase-4 inhibitors, 𝛼-glucosidase inhibitors and, possibly, sulphonylureas) or glucagon action (metformin). The potential risks associated with total glucagon suppression include 𝛼-cell hyperplasia, increased mass of the pancreas, increased susceptibility to hepatosteatosis and hepatocellular injury and increased risk of hypoglycaemia, and these should be considered in the search and development of new compounds reducing glucagon receptor signalling. More than 40 years after its initial description, hyperglucagonaemia in diabetes can no longer be ignored or minimized, and its correction represents an attractive way to improve diabetes management. Keywords: diabetes, glucagon Date submitted 24 February 2015; date of first decision 28 March 2015; date of final acceptance 27 April 2015
Introduction It has been our privilege to work on glucagon for more than 50 years [1] and to suggest 35 years ago that a search for selective glucagon inhibitors could open a new avenue in diabetes management [2]. In the present concise review, we will recall that hyperglucagonaemia is a constant feature in all forms of diabetes and attempt to explain why. We will briefly review the concept of Unger and Orci [3] that diabetes should be seen as a paracrinopathy of the islets of Langerhans, and recall recent animal studies that have shown that knockout of the glucagon receptor makes insulin-deficient type 1 diabetic rodents thrive without insulin. The effect on glucagon of current antidiabetic drugs will be reviewed, while the search for selective glucagon antagonists will be briefly analysed. Finally, the potential risks associated with total glucagon suppression will be considered. A comprehensive bibliography on glucagon up to 1996 can be found in the three volumes of the Handbook of Experimental Pharmacology that we edited [4,5] and in the proceedings of a more recently held conference published in this journal [6].
Hyperglucagonaemia is a Constant Feature of Diabetes It has been established for years that plasma glucagon levels are increased in all experimental and clinical forms of Correspondence to: Dr Pierre Lefebvre, Department of Medicine, CHU Sart Tilman, B-4000 Liège 1, Belgium. E-mail: [email protected]
diabetes [7]. This disturbance undoubtedly contributes to the hyperglycaemia of the disease and the excessive ketogenesis of diabetic coma [8]. Numerous studies have shown that failure of glucagon suppression contributes to postprandial hyperglycaemia in type 1 [9,10] and type 2 diabetes [11,12]. Impaired glucagon suppression concurs with impaired insulin release to the excessive blood glucose levels in early type 1 diabetes [13], in impaired glucose tolerance [14,15] and in ketosis-prone atypical diabetes [16,17]. Morphological studies have now firmly established that the main abnormality in the islet cell population of human diabetes is a decrease in the 𝛽-cell mass with a relative expansion of the 𝛼-cell mass [18].
Why Hyperglucagonaemia in Diabetes? The mechanisms leading to hyperglucagonaemia in diabetes have long been discussed and include: a direct consequence of intra-islet insulin secretory defects, a resistance of the 𝛼 cell to insulin or a desensitization of the 𝛼 cell to glucose [7]. Unger and Orci have recently proposed that diabetes should be considered as a ‘paracrinopathy’ of the islets of Langerhans [3]. This proposal is based on the concept that the very high concentrations of insulin normally reached inside the stimulated islets exert, directly or by proxy, a major inhibitory effect on glucagon secretion from the neighbouring 𝛽 cells, as previously suggested by us and others [19,20]. Disruption of this mechanism is proposed as a key factor in the pathogenesis of diabetes [21].The concept is supported by recent data on
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A
C
E Frequency (%)
D
B
60 40 20 0 beta wrapping alpha
apha-beta
alpha wrapping beta
Figure 1. Human islet cells were isolated and cultured for 24 h. After a double immunofluorescence for insulin (red) and glucagon (green), islet cells were analysed by confocal microscopy. (A–C) Images showing a cell pair composed of one 𝛼 cell surrounded by one 𝛽 cell, the merged image is shown in (C). The cell pair shown here is representative of cell pairs observed in different human cell preparations from at least 10 different pancreata. (D) One series of consecutive merged images of a cell pair composed of one 𝛼 cell (green) surrounded by a 𝛽 cell (red). Scale bars, 10 μm. All heterologous contacts between 𝛼 and 𝛽 cells were coded according to their type of association: a 𝛽 cell wrapping an 𝛼 cell (beta wrapping alpha), neutral apposition between 𝛼 and 𝛽 cells (alpha-beta) and an 𝛼 cell wrapping a 𝛽 cell (alpha wrapping beta). Results are shown in E as relative frequencies and columns are means ± standard error of the mean (s.e.m.) of five islet cell preparations from five different pancreata. From all heterogenous contacts between 𝛼 and 𝛽 cells, the percentage of 𝛼 cells whose profile was round and perimeter almost completely wrapped by a 𝛽 cell, as in (D), was 38 ± 8 (mean ± s.e.m. of three experiments). Figure from Diabetes 2010; 59: 1202–1210 and reprinted with permission from the American Diabetes Association. Copyright 2010 American Diabetes Association.
the microanatomy of the islets of Langerhans in man [22] and by new data on the intimate relationships between human 𝛽 and 𝛼 cells as shown in Figure 1, reproduced from Bosco et al. [22]. When the association between 𝛼 and 𝛽 cells is assessed in cultured isolated human islet cells, many 𝛼 cells are wrapped by 𝛽 cells and almost never the contrary. This strongly suggests that, in some way, human 𝛼 cells are under the control of 𝛽 cells. As discussed by the authors, this observation reveals a unique plasticity of the 𝛽 cells, which are able to spread around 𝛼 cells, suggesting that this characteristic is intrinsic to 𝛽 cells and not dictated by some islet coercions, such as extracellular matrix or islet vasculature. The molecular mechanism of this extraordinary so-called embracing of 𝛼 cells by 𝛽 cells remains to be elucidated, but the phenomenon in itself must be considered when discussing the paracrinology of the islets and the attractive suggestion that diabetes must be seen as a paracrinopathy in which glucagon cannot be ignored [3,23]. In type 1 diabetes, 𝛼 cells lack constant action of high insulin levels from juxtaposed 𝛽 cells. Replacement with exogenous insulin subcutaneously injected does not approach the paracrine levels of insulin, except with high doses that over-insulinize the peripheral insulin targets, thereby promoting glycaemic volatility [3,21]. In type 2 diabetes, the 𝛼-cell dysfunction may result from the failure of the juxtaposed 𝛽 cells to secrete the first phase of insulin or from the loss of the intra-islet pulsatile secretion of insulin. Observations made in experimental diabetes in minipigs [24], and recently confirmed in human type 2 diabetes [25], are in support of the second mechanism.
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Essential Role for Glucagon in the Pathophysiology of Diabetes? Several recent studies on experimental animal models of diabetes have suggested that glucagon plays an essential role in the pathophysiology of diabetes. Yu et al. [26] have shown that inhibition of glucagon secretion markedly improves experimental diabetes in rodents and that knockout of the glucagon receptor makes insulin-dependent type 1 diabetic rodents thrive without insulin [27]. Interestingly, a recent study found a role for the glucagon-like peptide-1 (GLP-1) receptor in controlling endogenous glucose production in the combined absence of glucagon and insulin in rodents [28]. To determine unambiguously if suppression of glucagon action eliminates manifestations of diabetes, Lee et al. [29] expressed glucagon receptors in livers of glucagon receptor (GcgR)-null (−/−) mice before and after 𝛽-cell destruction by high doses streptozotocin. Wild-type mice developed fatal diabetic ketoacidosis after streptozotocin, whereas GcgR−/− mice remained clinically normal without hyperglycaemia, impaired glucose tolerance or hepatic glycogen depletion. Administration of an adenovirus containing the GcgR cDNA transiently restored hepatic GcgR mRNA expression, while phospho-cAMP response element binding protein (P-CREB) and phosphoenol-pyruvate carboxykinase, markers of glucagon action, rose dramatically and severe hyperglycaemia developed. When GcgR mRNA spontaneously disappeared 7 days later, P-CREB declined and hyperglycaemia disappeared. In this experimental setting, the
doi:10.1111/dom.12480 721
review article metabolic manifestations of diabetes cannot occur without glucagon action, and once present, disappear promptly when glucagon action is abolished. Supporting these data, Wang et al. [30] recently reported that administration of a specific monoclonal antiglucagon receptor antibody completely normalized blood glucose and glycated haemoglobin levels without insulin in murine models of type 1 diabetes. All these experimental observations strongly suggest that targeting the 𝛼 cell and glucagon are innovative approaches in diabetes management.
Current Antidiabetic Agents and Glucagon Secretion or Action Old and new antidiabetic agents may exert part of their therapeutic effects via inhibition of glucagon secretion or inhibition of glucagon action.
Insulin The classic inhibitory effect of insulin on glucagon secretion observed in vitro contrasts with the elevated, or relatively elevated, glucagon circulating levels often reported in people with insulin-treated diabetes. In fact, under intensive insulin therapy either by multiple injections or by portable pumps, hyperglucagonaemia is markedly [31] or fully [32] corrected. Perfect substitution of insulin deficiency, including restoration of normal intra-islet pulsatile insulin secretion, would probably represent the best means to normalize the hypersecretion of glucagon [33]. In practice, and as alluded to above, reproduction of high intra-islet insulin concentrations that allow the achievement of sufficient inhibition of glucagon secretion by subcutaneous insulin administration may induce peripheral tissues over-insulinization and excessively increase the risk of hypoglycaemia [3,21].
Sulphonylureas The effects of sulphonylureas on glucagon secretion appear particularly complex. By stimulating intra-islet insulin release, sulphonylureas potentially reduce glucagon secretion, as elegantly analysed by Cooperberg and Cryer [34]. By contrast, in profound insulin deficiency, as in type 1 diabetes lacking C-peptide, sulphonylureas, such as glibenclamide [35] or glimepiride [34,36], stimulate glucagon secretion. Interestingly, in people with type 2 diabetes, with persistent endogenous insulin secretion, oral glibenclamide inhibits the glucagon response to insulin-induced hypoglycaemia [37]. Recent in vitro studies on the effect of sulphonylureas on pancreatic 𝛼 cells have shown the extreme complexity of the mechanisms involved [38]. Most interestingly, studies using islets of Langerhans from donors with type 2 diabetes have shown that low concentrations of tolbutamide (lower than those required to stimulate insulin secretion) restore normal glucose regulation of glucagon secretion (inhibition at high and stimulation at low glucose concentrations) [39]. Clinical studies are needed to evaluate the role of low-dose sulphonylureas precisely targeting glucagon secretion in both type 1 and type 2 diabetes.
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Biguanides Biguanides, such as metformin [40] or buformin [41], seem to have little or no effect of their own on pancreatic 𝛼-cell function. Interestingly, glucagon signalling in the liver is attenuated by metformin [42].
Glucagon-like Peptide-1 (GLP-1), GLP-1 Receptor Agonists and Dipeptidyl Peptidase-4 Inhibitors As recently reviewed [43], GLP-1 and GLP-1 receptor agonists, such as exenatide, liraglutide, dulaglutide and albiglutide, are now largely used in the treatment of Type 2 diabetes. Regarding GLP-1, Hare et al. [44] elegantly showed that inhibition of glucagon secretion and stimulation of insulin release contribute equally to its glucose-lowering action. Inhibition of glucagon secretion was further demonstrated for exenatide [45,46] and liraglutide [47,48]. This inhibition is most likely explained by a GLP-1 receptor-dependent stimulation of islet somatostatin secretion which, in turn, inhibits glucagon secretion through activation of somatostatin receptor-2 in 𝛼 cells [49–51]. The action of endogenously released GLP-1 is transient because the peptide is rapidly cleared by the kidney and inactivated by cleavage at the N-terminus by dipeptidyl peptidase-4 (DPP-4), a ubiquitous exopeptidase. Numerous DPP-4 inhibitors (vildagliptin, sitagliptin, saxagliptin, linagliptin and alogliptin) have been developed with the aim of prolonging the action of endogenously released GLP-1. It is currently accepted that their efficacy in controlling blood glucose in type 2 diabetes is partly attributable to their inhibitory effect on glucagon secretion [52,53]. Interestingly, despite inhibition of glucagon secretion during hyperglycaemia, gliptins appear not to compromise the glucagon counter-regulatory response to hypoglycaemia [54].
𝛼-Glucosidase Inhibitors The 𝛼-glucosidase inhibitor class of agent includes acarbose, miglitol and voglibose. These cause a significant postprandial increase in active GLP-1 circulating levels, an effect that may contribute to reduced glucagon secretion [55,56].
Thiazolidinediones or Glitazones Thiazolidinediones or glitazones are compounds aimed at reducing insulin resistance in type 2 diabetes, but their use has been limited by cardiovascular side effects and other adverse events [57]. As part of their antidiabetic effect, thiazolidinediones, such as rosiglitazone, have been shown to inhibit glucagon gene transcription in pancreatic 𝛼-cell lines through binding to the nuclear peroxisome proliferator-activated receptor-𝛾 and inhibition of the transcriptional activity of PAX6 that is required for cell-specific activation of the glucagon gene [58]. Clinical studies are needed to assess the importance of this mechanism in man.
Sodium-Glucose Cotransporter Type 2 Inhibitors As recently reviewed [59,60], sodium-glucose cotransporter type 2 inhibitors (canagliflozin, dapagliflozin, empagliflozin and ipragliflozin) are glucose-lowering agents that exert their
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therapeutic activity independently of insulin by facilitating glucose excretion through the kidneys. Quite surprisingly, they appear to increase rather than reduce endogenous glucose production [61]. This appears to be driven by increased glucagon secretion [62].
Search for Selective Glucagon Antagonists ‘A search for selective glucagon inhibitors represents an attractive new way in diabetes management’ was the concluding sentence of a review we published more than 35 years ago [2]. At the time of writing the present review, there is still no selective glucagon inhibitor used in the treatment of diabetes and data concerning compounds in clinical development are not in the public domain. In addition to the effects of eliminating glucagon signalling by knocking out the glucagon receptor mentioned above, animal studies have shown that experimental diabetes is markedly improved by immunoneutralization of glucagon by specific glucagon antibodies [63], by the use of antisense oligonucleotides targeting hepatic glucagon expression [64] or by monoclonal glucagon receptor antibodies [30]. Numerous peptide [65–67] and non-peptide glucagon receptor antagonists have been developed, including substituted ureas [68], benzamidine derivatives [69], alkylidene hydrazides [70–72], 𝛽-alanine derivatives [73] and furan-2-carbohyrazides [74], and have been shown to reduce blood glucose in dogs and monkeys, as well as in non-diabetic and diabetic rodents and, for one compound, in humans [75]. Kelly et al. [76] recently reported that short-term oral administration of the small-molecule glucagon receptor antagonist LY2409021 results in substantial reduction of fasting and postprandial glucose, with minimal hypoglycaemia, but reversible dose-dependent increases in aminotransferases, indicating that longer clinical trials are requested to better evaluate benefits and risks.
A Recent Iconoclastic Proposal: Using a Glucagon Receptor Agonist Rather than an Antagonist? Over the recent years, a new paradigm arose with the development of a GLP-1/glucagon receptor co-agonist [85] and even a GLP-1/glucose-dependent insulinotropic polypeptide (GIP)/glucagon receptor tri-agonist [86]. These newly designed peptides showed remarkable effects in rodent models of diabetes and obesity by improving glucose metabolism and inducing weight loss. In this approach, activation of the glucagon receptor drastically contrasts with previous attempts to inhibit glucagon secretion or action, as described above. The theory is that glucagon could have some beneficial activities by increasing energy expenditure [87] and promoting weight loss, whereas its diabetogenic effects might be counteracted by co-activation of the GLP-1 and GIP receptors [88]. These promising results in rodents have been partly confirmed in humans. Indeed, Tan et al. [89] reported that co-administration of GLP-1 during glucagon infusion results in increased energy expenditure and amelioration of hyperglycaemia, while Cegla et al. [90] reported that co-administration of glucagon and GLP-1, at doses which are individually subanorectic, significantly reduce food intake.
Conclusion Long ignored or neglected, glucagon today appears to be a major factor in the pathophysiology of diabetes. Inhibiting glucagon secretion or antagonizing the action of the hormone are now considered innovative approaches in the treatment of diabetes. Several drugs currently used in the treatment of diabetes may exert part of their effects by reducing glucagon secretion or action. Research is active to identify new compounds reducing glucagon secretion or antagonizing glucagon action. Such compounds should provide a therapeutic benefit without the risk of adverse events.
Risks Associated with Glucagon Suppression Glucagon being recognized to be the first line of defence against hypoglycaemia [77], inhibiting or reducing glucagon action may lead to an increased risk of hypoglycaemia in people with insulin-treated diabetes [78]. It has long been known that glucagon induces hypolipidaemic effects in multiple species [79,80] and, more recently, that GcgR−/− mice exhibit significant defects in lipid synthesis, secretion and oxidation [81]. Consequently, marked attenuation of glucagon signalling potentially increases the risk of hepatosteatosis and hepatocellular injury [81]. Furthermore, glucagon receptor signalling is an important regulator of hepatocyte survival [82], but the minimum level of expression of glucagon for this effect in humans has still to be determined. Last, marked inhibition of glucagon signalling in rodents results in islet hyperplasia, increased endocrine cell proliferation and significant increases in pancreatic weight [83]. The relevance of these observations to humans remains to be established, particularly in view of the fact that rodent islet cells appear to have a greater capacity for replication relative to human [84].
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Conflicts of Interest None declared.
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57. Soccio RE, Chen ER, Lazar MA. Thiazolidinediones and the promise of insulin sensitization in type 2 diabetes. Cell Metab 2014; 20: 573–591. 58. Krätzner R, Fröhlich F, Lepler K et al. A peroxisome proliferator-activated receptor 𝛾-retinoid X receptor heterodimer physically interacts with the transcriptional activator PAX6 to inhibit glucagon gene transcription. Mol Pharmacol 2008; 73: 506–517. 59. Scheen AJ, Paquot N. Metabolic effects of SGLT2 inhibitors beyond increased glycosuria: a review of the clinical experience. Diabetes Metab 2014; 40: S4–11. 60. Scheen AJ. Pharmacodynamics, efficacy and safety of sodium-glucose co-transporter type 2 (SGLT2) inhibitors for the treatment of type 2 diabetes mellitus. Drugs 2015; 75: 33–59. 61. Ferrannini E, Muscelli E, Frascerra S et al. Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic patients. J Clin Invest 2014; 124: 499–508.
77. Cryer PE. The barrier of hypoglycaemia in diabetes. Diabetes 2008; 57: 3169–3176. 78. Ali S, Drucker DJ. Benefits and limitations of reducing glucagon action for the treatment of type 2 diabetes. Am J Physiol Endocrinol Metab 2009; 296: E415–421. 79. Tiengo A, Nosadini R. Glucagon and lipoprotein metabolism. In: Lefèbvre PJ, ed. Glucagon II (Handbook of Experimental Pharmacology). Berlin: Springer, 1983; 441–451. 80. Guettet C, Rostaqui N, Mathe D, Lecuyer B, Navarro N, Jacotot B. Effect of chronic glucagon administration on lipoprotein composition in normally fed, fasted and cholesterol-fed rats. Lipids 1991; 26: 451–458. 81. Longuet C, Sinclair EM, Malda A. The glucagon receptor is required for adaptive metabolic response to fasting. Cell Metab 2008; 8: 359–371.
62. Bonner C, Kerr-Conte J, Gmyr V et al. Glucose transporter SGLT2 inhibition triggers glucagon secretion in alpha cells (abstract). Diabetologia 2014; 57(Suppl. 1): 251.
82. Sinclair EM, Yusta B, Streurker C et al. Glucagon receptor signalling is essential for control of murine hepatocyte survival. Gastroenterology 2008; 135: 2096–2106.
63. Sørensen H, Brand CL, Neschen S et al. Immunoneutralization of endogenous glucagon reduces hepatic glucose output and improves long-term glycemic control in diabetic ob/ob mice. Diabetes 2006; 55: 2843–2848.
83. Drucker DJ. Incretin action in the pancreas: potential promise, possible perils and pathological pitfalls. Diabetes 2013; 62: 3316–3323.
64. Sloop KW, Cao JX, Siesky AM et al. Hepatic and glucagon-like peptide-1mediated reversal of diabetes by glucagon receptor antisense oligonucleotides inhibitors. J Clin Invest 2004; 113: 1571–1581. 65. Bagger JI, Knop FK, Holst JJ, Visbøll T. Glucagon antagonism as a potential therapeutic target in type 2 diabetes. Diabetes Obes Metab 2011; 13: 965–971. 66. McShane LM, Franklin ZJ, O’Harte FP, Irwin N. Ablation of glucagon receptor signalling by peptide-based glucagon antagonists improves glucose tolerance in high fat fed mice. Peptides 2014; 60: 95–101. 67. O’Harte FP, Franklin ZJ, Irwin N. Two novel glucagon receptor antagonists prove effective therapeutic agents in high-fat-fed and obese diabetic mice. Diabetes Obes Metab 2014; 16: 1214–1222.
84. Parnaud G, Bosco D, Berney T et al. Proliferation of sorted human and rat beta cells. Diabetologia 2008; 51: 91–100. 85. Day JW, Ottaway N, Patterson JT et al. A new glucagon and GLP-1 coagonist eliminates obesity in rodents. Nat Chem Biol 2009; 5: 749–757. 86. Finan B, Yang B, Ottaway N et al. A rationally designed monomeric peptide triagonist corrects obesity and diabetes in rodents. Nat Med 2015; 21: 27–36. 87. Arafat AM, Kaczmarek P, Skrzypski M et al. Glucagon regulates orexin A secretion in humans and rodents. Diabetologia 2014; 57: 2108–2116. 88. Scheen AJ, Paquot N. A new paradigm for treating obesity and diabetes mellitus. Nat Rev Endocrinol 2015; 11: 196–198.
68. Shen DM, Zhang F, Brady EJ et al. Discovery of novel, potent, and orally active spiro-urea human glucagon receptor antagonists. Bioorg Med Chem Lett 2005; 15: 4564–4569.
89. Tan TM, Field BC, McCullough KA et al. Coadministration of glucagon-like peptide-1 during glucagon infusion in humans results in increased energy expenditure and amelioration of hyperglycemia. Diabetes 2013; 62: 1131–1138.
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doi:10.1111/dom.12480 725
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Diabetes, Obesity and Metabolism 17: 720–725, 2015. © 2015 John Wiley & Sons Ltd
Inhibiting or antagonizing glucagon: making progress in diabetes care P. J. Lefèbvre1 , N. Paquot1 & A. J. Scheen1,2 1 Division of Diabetes, Nutrition and Metabolic Disorders, Department of Medicine, University of Liège, Liège, Belgium 2 Division of Clinical Pharmacology, Department of Medicine, University of Liège, Liège, Belgium
Absolute or relative hyperglucagonaemia has been recognized for years in all experimental or clinical forms of diabetes. It has been suggested that excess secretion of glucagon by the islet 𝛼 cells is a direct consequence of intra-islet insulin secretory defects. Recent studies have shown that knockout of the glucagon receptor or administration of a monoclonal specific glucagon receptor antibody make insulin-deficient type 1 diabetic rodents thrive without insulin. These observations suggest that glucagon plays an essential role in the pathophysiology of diabetes and that targeting the 𝛼 cell and glucagon are innovative approaches in the management of diabetes. Despite active research and identification of promising compounds, no one selective glucagon antagonist is presently used in the treatment of diabetes. Interestingly, besides insulin, several drugs used today in the management of diabetes appear to exert their effects, in part, by inhibiting glucagon secretion (glucagon-like peptide-1 receptor agonists, dipeptidyl peptidase-4 inhibitors, 𝛼-glucosidase inhibitors and, possibly, sulphonylureas) or glucagon action (metformin). The potential risks associated with total glucagon suppression include 𝛼-cell hyperplasia, increased mass of the pancreas, increased susceptibility to hepatosteatosis and hepatocellular injury and increased risk of hypoglycaemia, and these should be considered in the search and development of new compounds reducing glucagon receptor signalling. More than 40 years after its initial description, hyperglucagonaemia in diabetes can no longer be ignored or minimized, and its correction represents an attractive way to improve diabetes management. Keywords: diabetes, glucagon Date submitted 24 February 2015; date of first decision 28 March 2015; date of final acceptance 27 April 2015
Introduction It has been our privilege to work on glucagon for more than 50 years [1] and to suggest 35 years ago that a search for selective glucagon inhibitors could open a new avenue in diabetes management [2]. In the present concise review, we will recall that hyperglucagonaemia is a constant feature in all forms of diabetes and attempt to explain why. We will briefly review the concept of Unger and Orci [3] that diabetes should be seen as a paracrinopathy of the islets of Langerhans, and recall recent animal studies that have shown that knockout of the glucagon receptor makes insulin-deficient type 1 diabetic rodents thrive without insulin. The effect on glucagon of current antidiabetic drugs will be reviewed, while the search for selective glucagon antagonists will be briefly analysed. Finally, the potential risks associated with total glucagon suppression will be considered. A comprehensive bibliography on glucagon up to 1996 can be found in the three volumes of the Handbook of Experimental Pharmacology that we edited [4,5] and in the proceedings of a more recently held conference published in this journal [6].
Hyperglucagonaemia is a Constant Feature of Diabetes It has been established for years that plasma glucagon levels are increased in all experimental and clinical forms of Correspondence to: Dr Pierre Lefebvre, Department of Medicine, CHU Sart Tilman, B-4000 Liège 1, Belgium. E-mail: [email protected]
diabetes [7]. This disturbance undoubtedly contributes to the hyperglycaemia of the disease and the excessive ketogenesis of diabetic coma [8]. Numerous studies have shown that failure of glucagon suppression contributes to postprandial hyperglycaemia in type 1 [9,10] and type 2 diabetes [11,12]. Impaired glucagon suppression concurs with impaired insulin release to the excessive blood glucose levels in early type 1 diabetes [13], in impaired glucose tolerance [14,15] and in ketosis-prone atypical diabetes [16,17]. Morphological studies have now firmly established that the main abnormality in the islet cell population of human diabetes is a decrease in the 𝛽-cell mass with a relative expansion of the 𝛼-cell mass [18].
Why Hyperglucagonaemia in Diabetes? The mechanisms leading to hyperglucagonaemia in diabetes have long been discussed and include: a direct consequence of intra-islet insulin secretory defects, a resistance of the 𝛼 cell to insulin or a desensitization of the 𝛼 cell to glucose [7]. Unger and Orci have recently proposed that diabetes should be considered as a ‘paracrinopathy’ of the islets of Langerhans [3]. This proposal is based on the concept that the very high concentrations of insulin normally reached inside the stimulated islets exert, directly or by proxy, a major inhibitory effect on glucagon secretion from the neighbouring 𝛽 cells, as previously suggested by us and others [19,20]. Disruption of this mechanism is proposed as a key factor in the pathogenesis of diabetes [21].The concept is supported by recent data on
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A
C
E Frequency (%)
D
B
60 40 20 0 beta wrapping alpha
apha-beta
alpha wrapping beta
Figure 1. Human islet cells were isolated and cultured for 24 h. After a double immunofluorescence for insulin (red) and glucagon (green), islet cells were analysed by confocal microscopy. (A–C) Images showing a cell pair composed of one 𝛼 cell surrounded by one 𝛽 cell, the merged image is shown in (C). The cell pair shown here is representative of cell pairs observed in different human cell preparations from at least 10 different pancreata. (D) One series of consecutive merged images of a cell pair composed of one 𝛼 cell (green) surrounded by a 𝛽 cell (red). Scale bars, 10 μm. All heterologous contacts between 𝛼 and 𝛽 cells were coded according to their type of association: a 𝛽 cell wrapping an 𝛼 cell (beta wrapping alpha), neutral apposition between 𝛼 and 𝛽 cells (alpha-beta) and an 𝛼 cell wrapping a 𝛽 cell (alpha wrapping beta). Results are shown in E as relative frequencies and columns are means ± standard error of the mean (s.e.m.) of five islet cell preparations from five different pancreata. From all heterogenous contacts between 𝛼 and 𝛽 cells, the percentage of 𝛼 cells whose profile was round and perimeter almost completely wrapped by a 𝛽 cell, as in (D), was 38 ± 8 (mean ± s.e.m. of three experiments). Figure from Diabetes 2010; 59: 1202–1210 and reprinted with permission from the American Diabetes Association. Copyright 2010 American Diabetes Association.
the microanatomy of the islets of Langerhans in man [22] and by new data on the intimate relationships between human 𝛽 and 𝛼 cells as shown in Figure 1, reproduced from Bosco et al. [22]. When the association between 𝛼 and 𝛽 cells is assessed in cultured isolated human islet cells, many 𝛼 cells are wrapped by 𝛽 cells and almost never the contrary. This strongly suggests that, in some way, human 𝛼 cells are under the control of 𝛽 cells. As discussed by the authors, this observation reveals a unique plasticity of the 𝛽 cells, which are able to spread around 𝛼 cells, suggesting that this characteristic is intrinsic to 𝛽 cells and not dictated by some islet coercions, such as extracellular matrix or islet vasculature. The molecular mechanism of this extraordinary so-called embracing of 𝛼 cells by 𝛽 cells remains to be elucidated, but the phenomenon in itself must be considered when discussing the paracrinology of the islets and the attractive suggestion that diabetes must be seen as a paracrinopathy in which glucagon cannot be ignored [3,23]. In type 1 diabetes, 𝛼 cells lack constant action of high insulin levels from juxtaposed 𝛽 cells. Replacement with exogenous insulin subcutaneously injected does not approach the paracrine levels of insulin, except with high doses that over-insulinize the peripheral insulin targets, thereby promoting glycaemic volatility [3,21]. In type 2 diabetes, the 𝛼-cell dysfunction may result from the failure of the juxtaposed 𝛽 cells to secrete the first phase of insulin or from the loss of the intra-islet pulsatile secretion of insulin. Observations made in experimental diabetes in minipigs [24], and recently confirmed in human type 2 diabetes [25], are in support of the second mechanism.
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Essential Role for Glucagon in the Pathophysiology of Diabetes? Several recent studies on experimental animal models of diabetes have suggested that glucagon plays an essential role in the pathophysiology of diabetes. Yu et al. [26] have shown that inhibition of glucagon secretion markedly improves experimental diabetes in rodents and that knockout of the glucagon receptor makes insulin-dependent type 1 diabetic rodents thrive without insulin [27]. Interestingly, a recent study found a role for the glucagon-like peptide-1 (GLP-1) receptor in controlling endogenous glucose production in the combined absence of glucagon and insulin in rodents [28]. To determine unambiguously if suppression of glucagon action eliminates manifestations of diabetes, Lee et al. [29] expressed glucagon receptors in livers of glucagon receptor (GcgR)-null (−/−) mice before and after 𝛽-cell destruction by high doses streptozotocin. Wild-type mice developed fatal diabetic ketoacidosis after streptozotocin, whereas GcgR−/− mice remained clinically normal without hyperglycaemia, impaired glucose tolerance or hepatic glycogen depletion. Administration of an adenovirus containing the GcgR cDNA transiently restored hepatic GcgR mRNA expression, while phospho-cAMP response element binding protein (P-CREB) and phosphoenol-pyruvate carboxykinase, markers of glucagon action, rose dramatically and severe hyperglycaemia developed. When GcgR mRNA spontaneously disappeared 7 days later, P-CREB declined and hyperglycaemia disappeared. In this experimental setting, the
doi:10.1111/dom.12480 721
review article metabolic manifestations of diabetes cannot occur without glucagon action, and once present, disappear promptly when glucagon action is abolished. Supporting these data, Wang et al. [30] recently reported that administration of a specific monoclonal antiglucagon receptor antibody completely normalized blood glucose and glycated haemoglobin levels without insulin in murine models of type 1 diabetes. All these experimental observations strongly suggest that targeting the 𝛼 cell and glucagon are innovative approaches in diabetes management.
Current Antidiabetic Agents and Glucagon Secretion or Action Old and new antidiabetic agents may exert part of their therapeutic effects via inhibition of glucagon secretion or inhibition of glucagon action.
Insulin The classic inhibitory effect of insulin on glucagon secretion observed in vitro contrasts with the elevated, or relatively elevated, glucagon circulating levels often reported in people with insulin-treated diabetes. In fact, under intensive insulin therapy either by multiple injections or by portable pumps, hyperglucagonaemia is markedly [31] or fully [32] corrected. Perfect substitution of insulin deficiency, including restoration of normal intra-islet pulsatile insulin secretion, would probably represent the best means to normalize the hypersecretion of glucagon [33]. In practice, and as alluded to above, reproduction of high intra-islet insulin concentrations that allow the achievement of sufficient inhibition of glucagon secretion by subcutaneous insulin administration may induce peripheral tissues over-insulinization and excessively increase the risk of hypoglycaemia [3,21].
Sulphonylureas The effects of sulphonylureas on glucagon secretion appear particularly complex. By stimulating intra-islet insulin release, sulphonylureas potentially reduce glucagon secretion, as elegantly analysed by Cooperberg and Cryer [34]. By contrast, in profound insulin deficiency, as in type 1 diabetes lacking C-peptide, sulphonylureas, such as glibenclamide [35] or glimepiride [34,36], stimulate glucagon secretion. Interestingly, in people with type 2 diabetes, with persistent endogenous insulin secretion, oral glibenclamide inhibits the glucagon response to insulin-induced hypoglycaemia [37]. Recent in vitro studies on the effect of sulphonylureas on pancreatic 𝛼 cells have shown the extreme complexity of the mechanisms involved [38]. Most interestingly, studies using islets of Langerhans from donors with type 2 diabetes have shown that low concentrations of tolbutamide (lower than those required to stimulate insulin secretion) restore normal glucose regulation of glucagon secretion (inhibition at high and stimulation at low glucose concentrations) [39]. Clinical studies are needed to evaluate the role of low-dose sulphonylureas precisely targeting glucagon secretion in both type 1 and type 2 diabetes.
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Biguanides Biguanides, such as metformin [40] or buformin [41], seem to have little or no effect of their own on pancreatic 𝛼-cell function. Interestingly, glucagon signalling in the liver is attenuated by metformin [42].
Glucagon-like Peptide-1 (GLP-1), GLP-1 Receptor Agonists and Dipeptidyl Peptidase-4 Inhibitors As recently reviewed [43], GLP-1 and GLP-1 receptor agonists, such as exenatide, liraglutide, dulaglutide and albiglutide, are now largely used in the treatment of Type 2 diabetes. Regarding GLP-1, Hare et al. [44] elegantly showed that inhibition of glucagon secretion and stimulation of insulin release contribute equally to its glucose-lowering action. Inhibition of glucagon secretion was further demonstrated for exenatide [45,46] and liraglutide [47,48]. This inhibition is most likely explained by a GLP-1 receptor-dependent stimulation of islet somatostatin secretion which, in turn, inhibits glucagon secretion through activation of somatostatin receptor-2 in 𝛼 cells [49–51]. The action of endogenously released GLP-1 is transient because the peptide is rapidly cleared by the kidney and inactivated by cleavage at the N-terminus by dipeptidyl peptidase-4 (DPP-4), a ubiquitous exopeptidase. Numerous DPP-4 inhibitors (vildagliptin, sitagliptin, saxagliptin, linagliptin and alogliptin) have been developed with the aim of prolonging the action of endogenously released GLP-1. It is currently accepted that their efficacy in controlling blood glucose in type 2 diabetes is partly attributable to their inhibitory effect on glucagon secretion [52,53]. Interestingly, despite inhibition of glucagon secretion during hyperglycaemia, gliptins appear not to compromise the glucagon counter-regulatory response to hypoglycaemia [54].
𝛼-Glucosidase Inhibitors The 𝛼-glucosidase inhibitor class of agent includes acarbose, miglitol and voglibose. These cause a significant postprandial increase in active GLP-1 circulating levels, an effect that may contribute to reduced glucagon secretion [55,56].
Thiazolidinediones or Glitazones Thiazolidinediones or glitazones are compounds aimed at reducing insulin resistance in type 2 diabetes, but their use has been limited by cardiovascular side effects and other adverse events [57]. As part of their antidiabetic effect, thiazolidinediones, such as rosiglitazone, have been shown to inhibit glucagon gene transcription in pancreatic 𝛼-cell lines through binding to the nuclear peroxisome proliferator-activated receptor-𝛾 and inhibition of the transcriptional activity of PAX6 that is required for cell-specific activation of the glucagon gene [58]. Clinical studies are needed to assess the importance of this mechanism in man.
Sodium-Glucose Cotransporter Type 2 Inhibitors As recently reviewed [59,60], sodium-glucose cotransporter type 2 inhibitors (canagliflozin, dapagliflozin, empagliflozin and ipragliflozin) are glucose-lowering agents that exert their
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therapeutic activity independently of insulin by facilitating glucose excretion through the kidneys. Quite surprisingly, they appear to increase rather than reduce endogenous glucose production [61]. This appears to be driven by increased glucagon secretion [62].
Search for Selective Glucagon Antagonists ‘A search for selective glucagon inhibitors represents an attractive new way in diabetes management’ was the concluding sentence of a review we published more than 35 years ago [2]. At the time of writing the present review, there is still no selective glucagon inhibitor used in the treatment of diabetes and data concerning compounds in clinical development are not in the public domain. In addition to the effects of eliminating glucagon signalling by knocking out the glucagon receptor mentioned above, animal studies have shown that experimental diabetes is markedly improved by immunoneutralization of glucagon by specific glucagon antibodies [63], by the use of antisense oligonucleotides targeting hepatic glucagon expression [64] or by monoclonal glucagon receptor antibodies [30]. Numerous peptide [65–67] and non-peptide glucagon receptor antagonists have been developed, including substituted ureas [68], benzamidine derivatives [69], alkylidene hydrazides [70–72], 𝛽-alanine derivatives [73] and furan-2-carbohyrazides [74], and have been shown to reduce blood glucose in dogs and monkeys, as well as in non-diabetic and diabetic rodents and, for one compound, in humans [75]. Kelly et al. [76] recently reported that short-term oral administration of the small-molecule glucagon receptor antagonist LY2409021 results in substantial reduction of fasting and postprandial glucose, with minimal hypoglycaemia, but reversible dose-dependent increases in aminotransferases, indicating that longer clinical trials are requested to better evaluate benefits and risks.
A Recent Iconoclastic Proposal: Using a Glucagon Receptor Agonist Rather than an Antagonist? Over the recent years, a new paradigm arose with the development of a GLP-1/glucagon receptor co-agonist [85] and even a GLP-1/glucose-dependent insulinotropic polypeptide (GIP)/glucagon receptor tri-agonist [86]. These newly designed peptides showed remarkable effects in rodent models of diabetes and obesity by improving glucose metabolism and inducing weight loss. In this approach, activation of the glucagon receptor drastically contrasts with previous attempts to inhibit glucagon secretion or action, as described above. The theory is that glucagon could have some beneficial activities by increasing energy expenditure [87] and promoting weight loss, whereas its diabetogenic effects might be counteracted by co-activation of the GLP-1 and GIP receptors [88]. These promising results in rodents have been partly confirmed in humans. Indeed, Tan et al. [89] reported that co-administration of GLP-1 during glucagon infusion results in increased energy expenditure and amelioration of hyperglycaemia, while Cegla et al. [90] reported that co-administration of glucagon and GLP-1, at doses which are individually subanorectic, significantly reduce food intake.
Conclusion Long ignored or neglected, glucagon today appears to be a major factor in the pathophysiology of diabetes. Inhibiting glucagon secretion or antagonizing the action of the hormone are now considered innovative approaches in the treatment of diabetes. Several drugs currently used in the treatment of diabetes may exert part of their effects by reducing glucagon secretion or action. Research is active to identify new compounds reducing glucagon secretion or antagonizing glucagon action. Such compounds should provide a therapeutic benefit without the risk of adverse events.
Risks Associated with Glucagon Suppression Glucagon being recognized to be the first line of defence against hypoglycaemia [77], inhibiting or reducing glucagon action may lead to an increased risk of hypoglycaemia in people with insulin-treated diabetes [78]. It has long been known that glucagon induces hypolipidaemic effects in multiple species [79,80] and, more recently, that GcgR−/− mice exhibit significant defects in lipid synthesis, secretion and oxidation [81]. Consequently, marked attenuation of glucagon signalling potentially increases the risk of hepatosteatosis and hepatocellular injury [81]. Furthermore, glucagon receptor signalling is an important regulator of hepatocyte survival [82], but the minimum level of expression of glucagon for this effect in humans has still to be determined. Last, marked inhibition of glucagon signalling in rodents results in islet hyperplasia, increased endocrine cell proliferation and significant increases in pancreatic weight [83]. The relevance of these observations to humans remains to be established, particularly in view of the fact that rodent islet cells appear to have a greater capacity for replication relative to human [84].
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Conflicts of Interest None declared.
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