Peptides 67 (2015) 74–81

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Peptides journal homepage: www.elsevier.com/locate/peptides

Review

Glucagon – Early breakthroughs and recent discoveries Bo Ahrén ∗ Department of Clinical Sciences Lund, Lund University, Lund, Sweden

a r t i c l e

i n f o

Article history: Received 28 January 2015 Received in revised form 14 March 2015 Accepted 16 March 2015 Available online 23 March 2015 Keywords: Glucagon Glucose Diabetes History

a b s t r a c t Glucagon was discovered in 1922 as a hyperglycemic factor in the pancreas. During its early history up to 1970, glucagon was shown to increase circulating glucose through stimulating glycogenolysis in the liver. It was also shown to be a constituent of islet non-␤ cells and to signal through G protein coupled receptors and cyclic AMP. Furthermore, its chemical characteristics, including amino acid sequence, and its processing from the preproglucagon gene had been established. During the modern research during the last 40 years, glucagon has been established as a key hormone in the regulation of glucose homeostasis, including a key role for the glucose counterregulation to hypoglycemia and for development of type 2 diabetes, and today glucagon is a potential target for treatment of the disease. Glucagon has also been shown to be a key factor beyond glucose control and involved in many processes. For the coming, future research, studies will be focused on ␣-cell biology beyond glucagon, hyperglucagonemia in other conditions than diabetes, its involvement in the regulation of body weight and energy expenditure and the potential of glucagon as a target for other diseases than type 2 diabetes, such as type 1 diabetes and obesity. This review summarizes the more than 90 years history of this important hormone as well as discusses potential future research regarding glucagon. © 2015 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early breakthroughs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identification of glucagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of glucagon effect and cellular origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of glucagon structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Processing from the preproglucagon gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon signaling mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technological discoveries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Somatostatin infusion technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon receptor knockout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other technological breakthroughs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modern history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon is a key actor in glucose regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon contributes to counterregulation in hypoglycaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon is involved in the paracrine regulation of the neighboring ␤-cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon is an important player in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon is a target for treatment of diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon has multiple effects beyond glucose control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . On the threshold to the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ␣-Cell biology is more than glucagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hyperglucagonemia is seen in other conditions than T2D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Correspondence to: B11 BMC, Sölvegatan 19, 22457 Lund, Sweden. Tel.: +46 462220758; fax: +46 462220757. E-mail address: [email protected] http://dx.doi.org/10.1016/j.peptides.2015.03.011 0196-9781/© 2015 Elsevier Inc. All rights reserved.

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Glucagon is involved in the regulation of body weight and, energy expenditure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon may have a role in central regulation of glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon may be targeted also for other reasons than reducing glucose in type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon – a long history with a prosperous future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements and duality declaration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Glucagon has emerged as a key hormone for the regulation of glucose homeostasis and for development of type 2 diabetes, and glucagon is a potential target for treatment of the disease. Glucagon is also a key factor beyond glucose control and involved in many processes. Here the more than 90 years history of this important hormone is summarized along with the results of the modern glucagon science as well as outlook for the future research. Early breakthroughs Identification of glucagon Following the discovery by von Mering and Minkowski that pancreatectomy results in hyperglycemia and diabetes in dogs [1] several researchers worked to isolate the putative antidiabetic factor in the pancreas. Murlin was one of those pioneers. He had started working in 1912 on aqueous extracts of the pancreas and showed, together with Kramer when they were working at the Cornell University Medical College, that when alkali was administered together with the pancreatic extracts to pancreatectomized dogs, urine glucose completely disappeared. After the move to University of Rochester, Murlin intensified his work in October 1921 [2]. He then found that, besides the glucose lowering ability of pancreatic extracts, pancreas contains also a factor which raises glucose. This discovery was performed in December 1922 when an extract was injected in two pancreatectomized dogs and found to raise blood sugar after three hours (from 21.1 to 32.8 mmol/l and from 7.8 to 21.7 mmol/l, respectively). The results were repeated four days later when the extract was injected in normal rabbits with similar results. It was therefore concluded that a hyperglycemic substance exists in the pancreas and this putative substance was named glucagon [3]. This name, however, was not used in general until the 1950s; in the meantime “the hyperglycemic factor of the pancreas” was more commonly used.

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hormone produced in non ␤-cells, using modern language, which is the basis of our present-day understanding of the islet topography with the glucagon producing ␣-cells in close apposition to the insulin-producing ␤-cells (Fig. 1). Both these researchers received the Nobel price; Sutherland in 1971 for the discovery of cyclic AMP and de Duve in 1974 for the discovery of lysosomes and other cell organelles. Characterization of glucagon structure During the 1950s attempts to isolate and characterize the structure of the hyperglycemic factor of the pancreas was intensified in the Lilly Research Laboratories in Indianapolis by a research group led by Staub. They successfully crystallized a pancreatic material for which they applied the name glucagon [6]; hereafter the name glucagon was used for a specific and identified factor. In later work, the purification and crystallization of glucagon was described in detail [7] and in 1957 the complete amino acid sequence of the hormone was reported [8]. Processing from the preproglucagon gene In 1983, Bell and coworkers at the University of Chicago established the structure of the human preproglucagon gene [9]. The gene was found to encode proglucagon which consists of 160 amino acids and is expressed mainly in pancreatic ␣ cells, in the gut L cells and in the brain. Later studies have established that these cells exhibit differential posttranslational processing of this prohormone. In the pancreatic ␣-cells, through the action of proconvertase 2, the main products are glucagon together with glucagon related

Characterization of glucagon effect and cellular origin Studies in the 1930s and 1940s aimed at further characterizing the hyperglycemic effect of pancreatic extracts and it was shown that following intravenous injection, circulating glucose rose within a few minutes to reach a peak after 5–10 min. Then the glucose-lowering effect of insulin remaining in the extracts reduced glucose. Main effort in characterization of the glucagon effect was performed by Bürger and Brandt at Bonn University who also aimed at isolating glucagon from insulin in the preparations [4]. Although producing a partially purified extract with hyperglycemic property, Bürger and Brandt never succeeded to completely dissociate this factor from insulin and, in fact, concluded that insulin and glucagon must be closely related. Further studies by Sutherland and de Duve showed that the hyperglycemic factor of the pancreas stimulated glycogenolysis from liver slices in vitro and characterized the source of this factor [5]. They then found that its occurrence in the pancreas followed the distribution of islets and that it remained in pancreatic extracts from alloxan-treated animals. They thus presented evidence that glucagon is an islet

Fig. 1. Schematic view of pancreatic islet anatomy with the glucagon producing ␣-cells in close apposition to insulin-producing ␤-cells, somatostatin-producing ␦-cells, pancreatic polypeptide producing F cells and ghrelin-producing ␧-cells as based on comparative and developmental morphological studies [85].

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polypeptide and the major proglucagon fragment, all of which are secreted [10]. In contrast, in the L cells and in the brain processing is different due to expression of proconvertase 1/3 and main products are glicentin, glucagon-like peptide-1 and glucagon-like peptide-2 [11]. Glucagon signaling mechanism Sutherland, who had described the presence of glucagon in islet non-␤-cells and who was the discoverer of cyclic AMP, showed early that glucagon increased cyclic AMP levels in isolated liver slices and suggested that the hormone works through this signal molecule [12]. This idea was later explored in detail by Exton and collaborators at Vanderbilt University, who characterized the raising of cyclic AMP by the hormone in detail [13]. Furthermore, Rodbell, who received the Nobel Prize in 1974, together with Gilman, for the discovery of G-protein coupled receptors (GPCR), showed that glucagon is signaling through such a receptor [14]. These discoveries bring us to the end of the early history of glucagon and to the footstep of the modern era. The knowledge by then was that glucagon is an islet hormone which is distinct from insulin both in structure and cellular origin. Its structure had been clarified down to detailed amino acid sequence, its processing from the preproglucagon gene and its glycogenolytic and hyperglycemic effects had been characterized both in vitro and in vivo. Furthermore, its signaling pathway through a GPCR and cyclic AMP had been established. Technological discoveries As often is the case in the history of science, leaps in discoveries are taken by technological steps in combination with researchers understanding the research history and opportunities with novel techniques. Several important novel techniques paved the way for the modern era of glucagon research and were fundamental for the knowledge of the hormone which we have today. Glucagon assays A problem in the early days of glucagon research was that it was not possible to measure glucagon with non-bioassay techniques, i.e., techniques not using the hyperglycemic property or the glycogenolytic activity as read-outs. The discovery of radioimmunoassay for measuring glucagon by Unger in Dallas in 1961 was therefore of huge importance for the whole field [15]. This discovery opened up for an entirely new field in the history, allowing conclusions on the physiology and also pathophysiology of glucagon, a field which is still very active more than 50 years later. The analytical techniques themselves have also improved. It has, in fact, been problematic that many antibodies used in radioimmunoassays are not specific for glucagon. This was highlighted in a study in mouse plasma from 1982, showing that more than 50% of what was measured as glucagon in fact determined immunoglobulins [16]. There has therefore been a very active development for improvement of the glucagon assay, and today reliable and specific assays exist [17]. Somatostatin infusion technique In 1973, somatostatin was discovered [18] and it was early shown to be a powerful inhibitor of glucagon secretion [19]. This allowed the development of the somatostatin infusion technique (with or without insulin replacement) as an interventional tool [20]. This was a breakthrough since it opened up the possibility

to explore the detailed role of glucagon in physiology and pathophysiology. Glucagon receptor knockout In 2003, the mouse with genetic deletion of glucagon receptors was described and characterized [21]. It opened up the science for using genetic tools for further characterizing function of glucagon. Other technological breakthroughs Besides these three techniques which specifically involved technological development for glucagon research, a few other technological breakthroughs have been instrumental for building the present day knowledge of glucagon. The two most important are the technique of isolating pancreatic islets [22,23] and using liver perfusion in vivo [24], which have been essential for discoveries on glucagon synthesis and secretion and for detailed studies on glucagon action. Modern history The modern history of glucagon started in the early 1970s and has allowed many groundbreaking discoveries which have resulted in the today’s impressive knowledge on glucagon. It is not possible to cover all aspects of this development, but six major themes have been particularly instrumental to create not only the knowledge but also the foundation for the future of glucagon. Glucagon is a key actor in glucose regulation Although early discoveries allowed the conclusion that the pancreatic islets contain a hormone with hyperglycemic properties and radioimmunoassays allowed the measurement of this hormone under a variety of conditions, it was not until the development of the somatostatin infusion technique that reliable conclusions that glucagon is of importance for glucose regulation could be drawn. A first study showed that after 2 h infusion of somatostatin in subjects with type 1 diabetes, plasma glucagon levels were reduced from 150 ± 15 to 77 ± 10 pg/ml and this was associated with a reduction in plasma glucose from 14.4 ± 1.1 to 10.6 ± 1.1 mmol/l, i.e., by 25% [20]. Since the radioimmunoassay used at that time was not entirely specific for glucagon, the reduction in glucagon in this study was probably underestimated; however, the conclusion that glucagon is of major importance for normal glucose concentrations is nevertheless valid. This study was followed by a similar study using the somatostatin infusion technique in healthy subjects for measurements not only of circulating glucose but also of hepatic glucose production using tracer technique [25]. It was then demonstrated that already after 15 min of somatostatin infusion, hepatic glucose production was reduced by more than 75%, showing the profound importance of glucagon in this respect. Furthermore, the detailed studies performed by Cherrington and coworkers at Vanderbilt University have established the close relation between portal (and hepatic sinusoidal) glucagon levels and hepatic glucose production, showing that within physiological levels, the relation is linear [26]. In the year 2000, it was also shown that glucagon is important for postprandial glucose; the somatostatin infusion technique was used by Shah and collaborators during an oral glucose tolerance test in subjects with type 2 diabetes and it was found that when reducing glucagon levels the raise of glucose during the OGTT was diminished [27]. Also the genetically manipulated mice with deletion of glucagon receptors show the same: baseline glucose and glucose levels during an OGTT are reduced in these mice [21]. These studies thus together show that glucagon is a major player in

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Fig. 2. Potential factors responsible for the release of glucagon from the ␣-cells during hypoglycemia, which is a key process to stimulate glucose release from the liver.

the regulation of circulating glucose both under fasting and postprandial conditions through its action to stimulate hepatic glucose production. Glucagon contributes to counterregulation in hypoglycaemia Glucagon had since the 1950s been used to combat hypoglycaemia since its injection stimulates hepatic glucose production also during hypoglycaemia. In the 1970s it was also demonstrated that glucagon levels increased during hypoglycaemia [28–30]. In fact, the glucagon counter-regulation to hypoglycaemia is a key mechanism for preventing and defending hypoglycaemia as demonstrated in glucagon receptor knock-out mice, who have a defective counterregulation as evident from hypoglycemic clamp studies [31]. Several mechanisms may explain this, as is illustrated in Fig. 2. These effects include a direct effect of low glucose to stimulate glucagon secretion from the ␣-cells, inhibition of islet ␤-cell secretion of insulin during hypoglycaemia which leads to stimulation of glucagon secretion since insulin inhibits glucagon secretion, and extrapancreatic mechanism of adrenaline released from the adrenals which stimulates glucagon secretion [32]. However, there may also be other mechanisms, and we have presented evidence that also the autonomic nerves are important. Thus, autonomic nerves, both the sympathetic and parasympathetic nerves, are known to stimulate glucagon secretion [33–35] and when interrupting the autonomic input by ganglionic blockade we have shown both in experimental animals and in humans that the glucagon response to hypoglycaemia is impaired [36,37]. Recently, however, an additional mechanism has been proposed based on results from Christensen and collaborators that the incretin hormone glucosedependent insulinotropic polypeptide (GIP) stimulates glucagon secretion during hypoglycaemia [38]. This would open the possibility that GIP through stimulation of glucagon secretion at low glucose prevents hypoglycemia and this was recently demonstrated in studies in mice with genetic deletion of the GIP receptor [31]. Glucagon is involved in the paracrine regulation of the neighboring ˇ-cells Since ␣-cells are located in close proximity to the ␤-cells (Fig. 1), it is possible that there is a functional crosstalk between these two major islet cell types. Studies in the 1980s and 1990s by Samols and Stagner used anterograde and retrograde infusion of antibody directed against insulin and glucagon into the isolated rat pancreas and showed that immunoneutralization of insulin increased glucagon secretion whereas immunoneutralization of glucagon did not affect insulin secretion [39]. They then concluded that the circulation passed from ␤-cells to ␣-cells, and not the reverse, which

suggested that circulating glucagon was not a key player for regulation of insulin secretion. Nevertheless, glucagon could be of importance for insulin secretion through a paracrine mechanism and this has been explored by three different techniques. One technique used specific ␣-cell ablation by administering diphtheria toxin to mice with ␣-cell overexpression of dipheteria toxin receptor [40]. It was then found that insulin secretion is reduced by more than 70% implying that ␣-cells are indeed of importance for insulin secretion. Moreover, Pipeleers and collaborators demonstrated that insulin secretion from isolated ␤-cells was lower than from ␤-cells in the presence of ␣-cells [41]. However, whether these effects are due to glucagon remains to be established, since ␣-cells produce also other factors of key relevance for ␤-cell function, such as GLP-1 [42] or GIP [43]. A more specific technique to analyze the relevance of glucagon for ␤-cell function is to examine isolated islets from mice with genetic deletion of glucagon receptors. In such a study, it has been shown, indeed, that glucose-stimulated insulin secretion is impaired [44]. Furthermore, using mice with ␤-cell overexpression of the glucagon receptor it has also been demonstrated that glucose-stimulated insulin secretion is stimulated [45]. Hence, these studies show that glucagon secreted from the ␣cells is of importance for insulin secretion from ␤-cells through a paracrine mechanism.

Glucagon is an important player in diabetes That glucagon is a pathophysiological factor in type 2 diabetes was suggested in the 1970s by Unger and Orci who coined the “bihormonal hypothesis” of the disease [46]. An important evidence for this was the demonstration that subjects with type 2 diabetes had higher baseline glucagon levels than non-diabetic subjects and these levels were not suppressed by a carbohydrate meal, as was the case in the non-diabetics [47]. The hyperglucagonemia in type 2 diabetes which is less suppressible by raising glucose has been confirmed in many studies [48] and is clearly evident by the correlation in improvement in glycemia and reduction in glucagon by dipeptidyl peptidase-4 (DPP-4) inhibition [49] and GLP-1 receptor agonism [50]. We have also in our long-term population study in Malmö evaluated the contribution of perturbations in glucagon secretion for changes in glucose tolerance. The study was a 12 year evaluation of glucagon secretion (performed with glucose-dependent arginine-stimulation clamp test), insulin sensitivity (using euglycemic, hyperinsulinemic clamp) and glucose tolerance (using an OGTT). Fig. 3 shows the main design of the project. From the start, all subjects had normal glucose tolerance, and already at the initial analyses of glucagon secretion in relation to glucose tolerance, it was evident that subjects with impaired glucose tolerance (IGT) had a higher, i.e., non-suppressed, glucagon secretion than those having normal glucose tolerance (NGT) [51].

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Fig. 3. The design of the Malmö study. Subjects with normal glucose tolerance (NGT) was recruited and examined at baseline and after 3, 5, 8 and 12 years in regard to insulin and glucagon secretion, glucose tolerance and insulin sensitivity. Some subjects developed impaired glucose tolerance (IGT) after three years, whereas others developed IGT after 8 years; other subjects remained NGT throughout the 12 year study period. Results from this study are discussed [51–53].

Furthermore, at the first follow-up after 3 years, some subjects had developed impaired glucose tolerance (IGT). This allowed an evaluation of the predictive potential of glucagon secretion for changes in glucose tolerance and it was found that a rise of glucagon secretion during the 3 year period was a key predictive factor for worsening of glucose tolerance [52]. Finally, in the study, some subjects had NGT until follow-up after 8 years but then developed IGT during the following 4 years until follow-up after 12 years. By now comparing glucagon secretion in those subjects with those remaining NGT throughout the 12 year study period, it was found that those who developed IGT had an elevated glucagon secretion already at the 3 year test, i.e., at least five years before the onset of IGT [53]. Therefore, this study verified that hyperglucagonemia is a predictive factor for development of IGT and also a key factor for the condition itself. The reason for the hyperglucagonemia in type 2 diabetes is not clearly established. It may be due to defective suppression of glucagon secretion by high glucose and/or by insulin resistance in the ␣-cells making them less responsive to the inhibitory action of insulin. However, recently an alternative hypothesis was suggested from studies showing that the defective suppression of glucagon secretion is more evident after oral than after intravenous glucose administration, which points to a mechanism related to the glucoincretin hormones released after oral glucose, such as GLP-1 and GIP [48]. However, although hyperglucagonemia per se is established and important in type 2 diabetes, its mechanism remains to be established. In contrast to the elevated levels of glucagon in type 2 diabetes, glucagon action and ␣-cell number seem not to be affected. Thus, as demonstrated by Matsuda and coworkers, intravenous infusion of glucagon at various dose levels increases glucose levels to the same extent and with no difference in subjects with type 2 diabetes compared to non-diabetic subjects [54]. Furthermore, a detailed study on ␣-cell mass showed that this was not significantly different in subjects with versus without type 2 diabetes, which was in contrast the ␤-cell mass which was shown to be reduced in subjects with type 2 diabetes [55]. An important finding in 2011 by Lee and collaborators has also suggested that glucagon is important, and even essential, for development of type 1 diabetes [56]. Thus, it was shown that diabetes did not develop in glucagon receptor knockout mice after administration of the ␤-cell toxin streptozotocin. This suggested that if glucagon signaling is blocked not even complete insulin deficiency

Fig. 4. Results from the first study demonstrating that infusion of GLP-1 reduces postprandial glucagon in both healthy controls and in subjects with type 2 diabetes (T2D) and type 1 diabetes (T1D). Adapted from Ref. [59].

can induce diabetes. Glucagon is, however, not the only contributor to this effect, since it was later shown that immunoneutralization of FGF-21 could reduce glucose even further in glucagon-receptor knockout mice given streptozotocin, and that administration of a GLP-1 receptor antagonist increased glycemia [57]. Similar results were recently presented also in mice with double genetic deletion of the glucagon receptor and the GLP-1 receptor [58]. This suggests that glucagon is very important for the development of diabetes in this model, but that also FGF-21 contributes and that the elevated GLP-1 levels associated with this model masks a hyperglycemia and therefore overexaggerates the influence of glucagon. Nevertheless, glucagon is important also for this condition, suggesting that it is important also for the development of type 1 diabetes. Glucagon is a target for treatment of diabetes The importance of glucagon for circulating glucose both under fasting and postprandial conditions and the hyperglucagonemia in type 2 diabetes have made glucagon an attractive target for glucose-lowering therapy in the disease. This has been enforced by the successful improvement of hyperglycemia by DPP-4 inhibition and GLP-1 receptor agonism in association with reduction in glucagon [49,50]. The first demonstration that GLP-1 reduces glucagon diabetes was presented by Gutniak et al., who showed that GLP-1 infusion in subjects with type 2 diabetes reduced glucagon levels after meal ingestion (Fig. 4; [59]). A later study by Nauck and collaborators showed that 4 h infusion of GLP-1 in fasted subjects with type 2 diabetes reduced glucagon levels through a glucose-dependent effect [60]. Also attempts to prevent glucagon action have been undertaken as potential treatments, using immunoneutralization of glucagon, glucagon receptor antisense and glucagon receptor antagonists [61]. However, although successful in experimental settings, these have not yet reached the clinic. Gutniak et al. showed also in 1992 that GLP-1 reduces postprandial glucagon levels in type 1 diabetes (Fig. 4; [59]) which suggests that incretin therapy may also be feasible in type 1 diabetes. This has repeatedly been confirmed in studies using GLP-1 infusion in subjects with type 1 diabetes with or without residual ␤-cell function [62] and using the DPP-4 inhibitor vildagliptin during a four week treatment period in type 1 diabetes [63]. These promising results are now followed by clinical trials in regard to the potential of using incretin therapy in type 1 diabetes.

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Fig. 5. The three phases of glucocentric glucagon research history with bullet points referring to paragraphs in the text.

Glucagon has multiple effects beyond glucose control Most research on glucagon has been glucocentric which is explained by the history of glucagon, its clear association with glucose control both in physiology and during development of diabetes and its potential as a target for treatment of diabetes. However, several studies have demonstrated that glucagon is of importance also for other processes. This was recently reviewed [64] and involves stimulation of lipolysis, fatty acid oxidation and ketogenesis in the liver, increased satiety and energy expenditure, and increased bile acid synthesis. These effects have not been characterized in such detail as the glucocentric effects, but may allow glucagon to be targets also in the treatment of lipid disorders, eating disorders and obesity.

a small amount of GLP-1 is expressed [42,66]. However, under some conditions, such as hyperglycemia and cytokine stimulation, GLP-1 production is increased [67]. It has also, interestingly, been demonstrated that in some species, also DPP-4 is expressed in the ␣-cells [68] which makes it possible that local islet GLP-1 may be of relevance during DPP-4 inhibition [69]. The contribution of these non-glucagon products is important to establish to completely understand ␣-cell biology. Recent studies on ␣-cell biology has also demonstrated that under some conditions, these cells may be differentiated into ␤cells [70–72]. This intraislet cell plasticity and transdifferentiation may be of relevance to the defective islet function in type 2 diabetes and is a strong candidate for being a target to be explored in detail in the future.

On the threshold to the future

Hyperglucagonemia is seen in other conditions than T2D

Glucagon has a long history and an impressive amount of knowledge has accumulated regarding various aspects of glucagon and its function. Sometimes, however, glucagon has been thought to be “forgotten” or “neglected”. One reason for this has been the difficulties in handling the compound for research due to its instability, and another reason might be difficulties in measurements of glucagon due to non-specific assays. Furthermore, glucagon has often been analyzed in the context of studies aiming at understanding insulin physiology and therefore studies have seldom been optimized for studies on glucagon and, finally, islet-oriented diabetologists have been focused to present evidence that it is insulin secretion rather than insulin resistance which is the factor behind type 2 diabetes and in that context the arguments in relation to glucagon have been suppressed. Nevertheless, glucagon is now in the center stage considering its main relevance for normal physiology and pathophysiology in diabetes [65]. Therefore, we are now on the footstep for the future research, leaving the present day modern research with glucagon now in the center. What research will the future hold? In regard to the glucocentric aspects of glucagon, five important areas can be anticipated.

Hyperglycemia has been demonstrated also in conditions other than basal pathophysiology of type 2 diabetes. Thus, it was shown in 2002 that phloridzin, which is an inhibitor of glucose reabsorption in the proximal nephrones by targeting sodium glucose-transporters, increases glucagon levels [73]. This, which was recently confirmed in clinical trials using SGLT2-inhibitors as glucose-lowering agents in type 2 diabetes [74,75], suggests a reno-islet axis. The mechanism of this is not known, nor is the consequence and is a novel area of research. Furthermore, it has been shown that also other gut hormones than GLP-1 and GIP affect glucagon levels. Of particular interest is the demonstration by Meier and collaborators that GLP-2 increases glucagon [76]. This raises questions on not only the physiological relevance of this finding but also whether it exists and, if so, the consequences for this effect during treatment with GLP-2 receptor agonists, such as teduglutide, for short bowel syndrome [77]. Finally, there is also evidence that postprandial glucagon levels are elevated after gastric bypass surgery, the importance of which needs to be examined further [78].

˛-Cell biology is more than glucagon

Glucagon is involved in the regulation of body weight and, energy expenditure

Glucagon is processed from proglucagon, but this prohormone can also be processed to GLP-1. Normally, the majority of proglucagon is processed to glucagon in the islet ␣-cells, and also

Glucagon administration has been shown to reduce body weight and to increase energy expenditure [79]. These effects, which are of

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tremendous potential clinical interest, need to be explored in more detail. Glucagon may have a role in central regulation of glucose Glucagon and glucagon receptors have been shown to be expressed in the brain [80,81] and central administration of glucagon has been shown to inhibit rather than stimulate hepatic glucose production [82]. These effects suggest a central regulation of hepatic glucose production with involvement of central glucagon, which is of high relevance to penetrate further. Glucagon may be targeted also for other reasons than reducing glucose in type 2 diabetes As discussed above, antagonism of glucagon in both type 2 and type 1 diabetes has been discussed and for incretin therapy a reality in type 2 diabetes and examined in clinical trials in type 1 diabetes. However, it has also been discussed whether glucagon may be coadministered with GLP-1 in obesity for reducing body weight [83] and with insulin in type 1 diabetes to prevent hypoglycemia [84]. Glucagon – a long history with a prosperous future This article has summarized the development for the early breakthroughs in glucagon research over the modern research demonstrating a role of glucagon in physiology and pathophysiology to the future research with diverse and advanced basic and clinical research on glucagon. The three phases of glucagon research can be summarized as in Fig. 5. We are today in the transition between modern and future research where glucagon is occupying the center court [65] and we can expect exciting and novel results explaining in further all aspects of this now more than 90 years old hormone. Acknowledgements and duality declaration The study reported by the author has been funded by the Swedish Research Council, Region Skåne, Faculty of Medicine, Lund University, Novartis A/S and Sanofi. The authors has consulted for Novartis, Glaxo-SmithKline, Merck, Sanofi, Novo Nordisk, Boehringer Ingelheim and Takeda, and has received lecture fees from Novartis, Merck, Novo Nordisk, Sanofi, Bristol Myers Squibb, AstraZeneca and GlaxoSmithKline which all are companies producing DPP-4 inhibitors or GLP-1 receptor agonists. References [1] von Mering J, Minkowski O. Diabetes mellitus nach Pankreasextirpation. Arch Exp Pathol Pharmakol 1890;26:371–8. [2] Murlin JR, Clough HD, Gibbs CBF, Stokes AM. Aqueous extracts of the pancreas. I. Influence on the carbohydrate metabolism of depancreatized animals. J Biol Chem 1923;56:253–96. [3] Kimball CP, Murlin JR. Aqueous extracts of pancreas. III. Some precipitation reactions of insulin. J Biol Chem 1923;58:337–46. [4] Bürger M, Brandt W. Über das Glukagon (die Hyperglykamisierende Substanz des Pancreas). Z Ges Exp Med 1935;96:375. [5] Sutherland EW, de Duve C. Origin and distribution of the hyperglycemicglycogenolytic factor of the pancreas. J Biol Chem 1948;175:663–74. [6] Staub A, Sinn L, Behrens OK. Purification and crystallization of hyperglycemic glycogenolytic factor (HGF). Science 1953;117:628–9. [7] Staub A, Sinn L, Behrens OK. Purification and crystallization of glucagon. J Biol Chem 1955;214:619–32. [8] Bromer WW, Sinn LG, Staub A, Behrens OK. The amino acid sequence of glucagon. Diabetes 1957;6:234–8. [9] Bell GI, Sanchez-Pescador R, Laybourn PJ, Najarian RC. Exon duplication and divergence in the human preproglucagon gene. Nature 1983;304:368–714. [10] Holst JJ, Bersani M, Johnsen AH, Kofod H, Hartmann B, Orskov C. Proglucagon processing in porcine and human pancreas. J Biol Chem 1994;269:18827–33. [11] Holst JJ. Thye physiology of glucagon-like peptide 1. Physiol Rev 2007;87: 1409–39.

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