Long in the shade, glucagon re‐occupies centre court

Glucagon and its role in the regulation of glucose homeostasis started to be taught in physiology classes in the late 1950s to early 1960s, around the time the first radioimmunoassays for insulin and glucagon were developed (a 'prehistoric' time in the eyes of our current PhD students and postdocs). Currently, which medical student or intern would be unaware that the liver, the prime orchestrator of metabolism, is tightly regulated by the antagonistic couple insulin–glucagon? And yet, so much has been done in the insulin field, while glucagon has languished in backwaters to the limit of oblivion. A Medline search with the words insulin and glucagon yields almost 10 times more hits for insulin than glucagon! Why this neglect? It is mostly because glucagon and the α-cell are difficult to investigate. While the proinsulin gene produces only insulin, the pro-glucagon gene gives rise not only to glucagon but also to several other bioactive peptides. β-Cells are abundant in the islet; thus, studying the biology of the islet leads to data which are mainly representative of β-cells; furthermore, β-cells can be isolated with relative ease, and many β-cell lines are available to allow in-depth studies. This is not so regarding the minority population of α-cells. Not to mention the difficulty in generating specific and sensitive enough assays for glucagon and the other glucagon gene products, while for proinsulin-derived peptides excellent assays have been on the market since decades. Furthermore, no common disease has been assigned to the specific loss of α-cells or of glucagon secretion. Fortunately, of late, glucagon is experiencing a renaissance. This is partly due to the clinical interest in the glucagon gene product GLP-1, but partly also due to technological advances. The 12th Servier-IGIS Symposium, entitled 'Pancreatic α-Cells and Glucagon—Neglected Metabolic Actors' and whose participants have contributed to this proceedings volume, is a timely tribute to this renaissance. Unlike insulin, whose name was coined 1 in referral to its then still hypothetical origin (insula or islet of Langerhans), the name of glucagon referred to the hyperglycaemic properties of a still unpurified substance in pancreatic extracts 2. Indeed, glucagon's physiological function is to increase hepatic glucose production in order to prevent hypoglycaemia during fasting, and to provide glucose to working muscles during exercise. Glucagon also plays a central role in the counter-regulatory response to hypoglycaemia. Its action in hepatocytes is predominantly through enhanced glycogenolysis, with little acute effect on gluconeogenesis, because the latter also requires substrate (lactate, glycerol) delivery under the influence of other hormones such as adrenaline. Glucagon has no effect in skeletal muscle and, at least in humans, barely stimulates lipolysis in adipose tissue. Glucagon is synthesized and secreted by the α-cells of the islet. During embryogenesis, cell differentiation is governed by the precisely timed expression of transcription factors, which, in turn, sequentially induce the expression of specific lineage genes. Loss- and gain-of-function studies have unravelled the mechanisms of specification towards the different islet cell types. After development of the pancreatic lineage under the control of Pdx1, the endocrine differentiation program is initiated by Neurogenin3. These progenitor cells can then give birth to all the endocrine cells of the islets. Separation of cell types is due to distinct expression of Arx and Foxa2 with repression of Pax4 in α- and PP cells, and to the reverse pattern in β- and δ-cells. Later on, these transcription factors also serve to maintain the phenotype of differentiated cells, including expression of the pro-glucagon gene. An important message from a number of novel studies is that islet cells are more plastic than previously believed. They can change their lineage if expression of the above factors is induced or repressed by genetic manipulation or by experimental modification of the environment, such as near total β-cell destruction by diphtheria toxin 3. That may be fine for rodents, but what about human islet cells? There is as yet no answer, particularly because recent data indicate that, like β-cells, α-cells have a long lifespan. This is compatible with various observations indicating that, in contrast to rodents which easily adapt to variations in hormone demand by changing cell mass, humans mainly modulate cell function. There are other differences. Rather than forming a characteristic mantle surrounding the core of β-cells as in rodents, α-cells are dispersed throughout human islets 4-6. These long overlooked interspecies differences in islet architecture have received much attention during recent years, and led to a precise description of the cellular interactions and vascular contacts in human islets 7. It has also become clear that human islets contain a substantially higher proportion of α-cells than rodent islets, normal α-cell mass in the whole pancreas amounting to ∼40% of β-cell mass 8. This improved knowledge of the human islet composition and organization is important for elucidation of islet function, in particular the control of glucagon secretion. Variations in blood glucose level cause changes in glucagon secretion. This could be achieved directly at the α-cell level, via adequate glucose-sensing, and transduction and effector systems that control the rate of exocytosis of glucagon-containing granules. It could also take place at the islet level, through the influence of signals originating from non-α-cells and arriving at the target by a paracrine route or the local microcirculation. Finally, regulation could be systemic, extrinsic to the islet and mediated by neural or hormonal signals. Regulation of insulin secretion by β-cells is also sufficiently complex and multifactorial, but the hierarchy and interplay between the different levels of control are understood well enough to form a coherent picture. In contrast, our knowledge of the α-cell remains fragmentary after four decades of investigation 9, 10. Many discrepancies exist between studies of glucagon secretion. They are at least partly linked to the variability of experimental conditions (for instance, evaluation of the inhibitory effect of high glucose vs. the stimulatory effect of a sudden decrease in glucose, in static vs. dynamic measurements, etc.) and the use of different preparations. Functional studies of primary α-cells are indeed technically quite difficult. Models using single cells meet with the difficulty of recognizing the few α-cells amid the multitude of cells obtained when islets are dispersed. Then comes a classic error, despite plenty of lessons gained from β-cell studies: trusting that the secretory behaviour of single α-cells is normal. Nevertheless, this model is important for identifying the different components of the signalling machinery. We thus know that, like β-cells, α-cells are electrically excitable and use a rise in cytosolic calcium as triggering signal, but they are endowed with a distinct equipment in ionic channels. One model suggests that it is the partial opening of KATP channels at low glucose that promotes Ca2+ influx into α-cells, just the opposite of what occurs in β-cells. This apparent incongruity is explained by the properties of certain Ca2+ channels that, upon mild hyperpolarization, can recover from depolarization-induced inactivation. Not all laboratories agree with this rather sophisticated mode of regulation, and some emphasize the role of intracellular Ca2+ stores and store-operated channels 11. While there is strong evidence that the α-cell itself can adjust the glucagon secretion rate, many data suggest that a paracrine action is also involved. It is worth recalling that such a paracrine action is independent of the direction of blood flow. Products can be released by one cell in the immediate proximity of another cell. In addition to cell proximity, the existence of pseudopods facilitates such interactions. This paracrine interaction might involve somatostatin released from δ-cells and insulin, zinc or GABA released from β-cells. The 'intra-islet insulin' or 'insulin switch-off' model has been proposed to explain the increase in glucagon secretion that occurs when blood glucose levels fall. The model poses that insulin exerts a tonic inhibitory action on α-cells, and that arrest of its secretion, when blood glucose falls, removes the brake on glucagon secretion. Although there is in vivo support for this interpretation 12, it is puzzling that removal of insulin alone is inefficient if blood glucose is not also suppressed, which points to participation of a glucose signal. Another problem is that many experiments have shown that the reverse phenomenon, inhibition of glucagon secretion, already occurred at glucose concentrations that have no effect on insulin secretion 13. Next, assuming that β-cell function indeed suppresses glucagon release, the question is whether it is insulin itself that affects α-cells or zinc that is released with the hormone during exocytosis. That exogenous zinc can inhibit glucagon secretion is widely admitted, but whether zinc released in situ from β-cells indeed exerts the physiological inhibitory action remains debated. Thus, recent data showed that glucose normally inhibits glucagon secretion in islets from mice whose insulin granules contain only low levels of zinc because of suppression of the zinc transporter ZnT8 specifically in β-cells. Some human studies also argue against the role of zinc 12. Is it then insulin itself? Such conclusion is difficult to reconcile with the increase in glucagon levels during a hyperinsulinemic hypoglycaemic clamp in mice lacking insulin receptors selectively in their α-cells. While there is agreement that several neurotransmitters and hormones can profoundly influence glucagon secretion, their relative contribution compared to more direct mechanisms remains undefined. GLP-1 lowers glucagon secretion, but how does it do that? Persistence of its effects in type 1 diabetic subjects and in mice lacking insulin receptor in α-cells argues against an indirect effect mediated by increased insulin secretion. α-Cells barely express the GLP-1 receptor, but is this evidence against a direct action, or appropriate for inhibition mediated by a minor increase in cAMP, as opposed to the stimulation of glucagon secretion by the large increase in cAMP produced by adrenaline acting on the numerous β-adrenergic receptors? The inhibitory action of GLP-1 could also be mediated by a paracrine action of somatostatin. Only a few of the current debates have been alluded to above, but they suffice to illustrate the major difficulty in finding and fitting all the pieces of the puzzle depicting a consensus model of the α-cell. When the picture becomes sharper, a final problem will persist in extrapolating a model from rodent to human α-cells. Several indices suggest that this may be more problematic than for insulin secretion, and not only because of differences in architectural organization of the islets 7. Different abnormalities of glucagon secretion can be encountered in diabetic patients. Insufficient secretion in response to hypoglycaemia underlies the disorders of counter-regulation in long-term type 1 diabetic subjects. The almost complete loss of β-cells profoundly alters islet architecture but is not accompanied by changes in α-cell mass 14. The secretory problem is usually explained by the insulin-switch-off model 12: in the absence of β-cells, intra-islet exogenous insulin does not decrease during hypoglycaemia. Another possibility could be that chronic rather than acute insulin signalling into α-cells is necessary to make them glucose-responsive via changes in regulatory or effector proteins. Much attention has also been focussed on glucagon disorders in the pathogenesis of type 2 diabetes. It is widely recognized that these patients show elevated plasma glucagon levels relative to their high glucose levels 15, 16. Excessive glucagon secretion, in the context of insulin insufficiency and resistance, causes fasting hyperglycaemia by promoting glucose production in the liver. Insufficient and delayed inhibition (or even increase) of glucagon secretion after meals contributes to postprandial hyperglycaemia. Owing to a decrease in β-cell mass, the α/β-cell ratio is increased in many though not all type 2 diabetic subjects, but the α-cell mass is similar to that of non-diabetic subjects 8. The fact that some type 2 diabetics do not show obvious changes in the islet cell composition and architecture does not necessarily acquit the β-cells whose defective insulin secretion might secondarily affect α-cell function 17. Intrinsic or hyperglycaemia-induced perturbation of α-cells is equally a possibility. In this perspective, it is intriguing that in normal rodent islets the greatest inhibition of glucagon secretion occurs within the physiological range rather than at very high glucose concentrations. In vitro studies of islets from patients with type 2 diabetes would be critically important to unravel the underlying defects. A third possibility is that the inappropriate secretion of glucagon after meals in type 2 diabetic subjects is driven by GIP, the glucagon stimulatory action of which overrides the normal inhibitory action of GLP-1. If glucagon is more deleterious than useful when insulin is insufficient, would it be helpful to eliminate it or its effects? Let us first recall that pro-glucagon is processed by pro-hormone convertase 2 (PC2) in α-cells to produce glucagon, and by PC1/3 in intestinal L-cells to produce GLP-1 and GLP-2. Recent genetic models of altered glucagon signalling have yielded only partly overlapping phenotypes, probably because of distinct associated changes: mice with inactivation of the pro-glucagon gene lack both glucagon and GLP-1; PC2-deficient mice lack glucagon but have high levels of GLP-1 and impaired processing of proinsulin; glucagon receptor knockout mice show increased levels of both glucagon and GLP-1. Overall, the impact on glucose homeostasis was mild in the three models, which surprisingly showed a still unexplained marked hyperplasia of α-cells. More spectacular and intriguing was the observation that β-cell destruction with streptozotocin did not cause the expected hyperglycaemic and catabolic disorders in mice protected from glucagon action by elimination of glucagon receptors 18. Pharmacological blockade of these receptors is thus an appealing new avenue for the treatment of diabetes but the approach may be complicated by the importance of glucagon in counter-regulation and exercise, and its numerous effects besides blood glucose control. Other perspectives in this area include production of GLP-1 rather than glucagon in the islets by forced expression of PC1/3 rather than PC2 in α-cells, or even reprogramming α-cells to become β-cells, with the caveat that genetic or epigenetic abnormalities that may follow cell reprogramming could hamper their use in regenerative or cell replacement medicine. Besides these futuristic options, a safe way of decreasing plasma glucagon levels has already been implemented. Thus, GLP-1-based therapies target both insulin and glucagon secretion, inhibition of the latter accounting for no less than 50% of the glucose-lowering effect in type 2 diabetic patients. Whether a similar approach may also be useful in type 1 diabetes is currently under investigation. In short, glucagon promises further surprises in the near future! J.-C. Henquin 1 , D. Accili 2 , B. Ahrén 3 , C. Boitard 4 , S. Seino 5,6 & E. Cerasi 7 We are grateful to Laboratoires Servier, Paris, for the long-term commitment to the IGIS project. We are indebted to Laurence Alliot for her leadership and support of the IGIS Symposia, and we thank Martine Zeitoun and Catriona Donagh (Servier) for their outstanding logistical and editorial support. Alain Ktorza (Servier), as Secretary of the IGIS Board, provided us with invaluable support and counsel; our sincere thanks to him. The authors declare no conflicts of interest.

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