5 REVIEW

Physiology of the pancreatic a-cell and glucagon secretion: role in glucose homeostasis and diabetes

Ivan Quesada, Eva Tudurı´, Cristina Ripoll and A´ ngel Nadal CIBER de Diabetes y Enfermedades Metabo´licas Asociadas (CIBERDEM) and Instituto de Bioingenierı´a, Universidad Miguel Herna´ndez, Avenida de la Universidad s/n, 03202 Elche, Spain (Correspondence should be addressed to I Quesada; Email: [email protected])

Abstract The secretion of glucagon by pancreatic a-cells plays a critical glucagon secretion from glucose and nutrients to paracrine role in the regulation of glycaemia. This hormone counteracts and neural inputs. Additionally, we have described the hypoglycaemia and opposes insulin actions by stimulating glucagon actions on glycaemia and energy metabolism, and hepatic glucose synthesis and mobilization, thereby increasing discussed their involvement in the pathophysiology of blood glucose concentrations. During the last decade, diabetes. Finally, some of the present approaches for diabetes knowledge of a-cell physiology has greatly improved, therapy related to a-cell function are also discussed in this especially concerning molecular and cellular mechanisms. review. A better understanding of the a-cell physiology is In this review, we have addressed recent findings on a-cell necessary for an integral comprehension of the regulation of physiology and the regulation of ion channels, electrical glucose homeostasis and the development of diabetes. activity, calcium signals and glucagon release. Our focus in Journal of Endocrinology (2008) 199, 5–19 this review has been the multiple control levels that modulate

Introduction to those of insulin can be determinant in the higher rate of hepatic glucose output, which seems to be critical The principal level of control on glycaemia by the islet in maintaining hyperglycaemia in diabetic patients of Langerhans depends largely on the coordinated (Dunning et al.2005). secretion of glucagon and insulin by a-andb-cells Despite the importance of the a-cell and glucagon respectively. Both cell types respond oppositely to changes secretion in the regulation of glycaemia and nutrient in blood glucose concentration: while hypoglycaemic homeostasis, little is known about the physiology of these conditions induce a-cell secretion, b-cells release insulin cells compared with the overwhelming information about when glucose levels increase (Nadal et al. 1999, Quesada b-cells. Several factors may explain this lack of information et al. 2006a). Insulin and glucagon have opposite effects about glucagon secretion. First, the scarcity of this cell on glycaemia as well as on the metabolism of nutrients. population in islets of animal models such as mice and rats Insulin acts mainly on muscle, liver and adipose tissue along with several technical limitations of conventional with an anabolic effect, inducing the incorporation methods have made it more difficult to study a-cells than of glucose into these tissues and its accumulation as b-cells (Quoix et al. 2007). Second, the lack of functional glycogen and fat. By contrast, glucagon induces a catabolic effect, mainly by activating liver glycogenolysis identification patterns has also been an important limitation in and gluconeogenesis, which results in the release of a-cell research. However, in recent years notable progress has glucose to the bloodstream. An abnormal function been made in the study of a-cell function at the cellular and of these cells can generate failures in the control of molecular levels. This review attempts to describe recent glycaemia, which can lead to the development of diabetes advances in a-cell physiology and the regulation of glucagon (Dunning et al.2005). Actually, diabetes is associated with secretion. Additionally, it focuses on the pathophysiology of disorders in the normal levels of both insulin and these cells, their role in diabetes, as well as potential glucagon.Anexcessofglucagonplasmalevelsrelative therapeutic strategies.

Journal of Endocrinology (2008) 199, 5–19 DOI: 10.1677/JOE-08-0290 0022–0795/08/0199–005 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org

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Islet of Langerhans: cell architecture and function deeply into the islet (Ahren 2000). Thus, multiple regulation levels determine hormone release from pancreatic islets. Glucagon-secreting a-cells are one of the main endocrine cell populations that coexist in the islet of Langerhans along with insulin-secreting b-cells. The islet is further composed a by other scarce secretory populations such as d- and poly- Glucagon secretion by pancreatic -cells peptide releasing (PP)-cells, which release somatostatin and Stimulus-secretion coupling in a-cells: from ion channel activity to pancreatic polypeptide respectively. This multicellular exocytosis structure constitutes the endocrine unit of the pancreas and is responsible for the regulation of blood glucose homeostasis. Pancreatic a-cells are equipped with a specific set of channels C 2C Approximately one million islets are distributed throughout a that generate action potentials of Na and Ca in the healthy adult human pancreas, representing 1 and 2% of the absence or at low levels of glucose (Gromada et al. 1997). This 2C total mass of the organ. Each islet, with sizes varying from electrical activity triggers Ca signals and glucagon secretion. Elevated glucose concentrations inhibit all these 100 to 500 mm, is made up of 1000–3000 cells. In mouse C and rat islets, b-cells are the main population accounting for events. ATP-dependent K (KATP) channels play a funda- 60–80% of the total number of cells, while 15–20% are mental role in a-cells, such as they do in b-cells, since they a-cells, !10% are d-cells and less than 1% correspond to the couple variations in extracellular glucose concentrations to PP-cell population (Brelje et al.1989, Brissova et al.2005). changes in membrane potential and electrical activity. In The architecture of rodent islets is characterized by the intact rat a-cells, KATP channels have a lower ATP sensitivity Z . location of b-cells in the core and the non-b cells distributed (Ki 0 94 mM) than the one observed in excised patches Z in a mantle around the insulin-secreting cell population. This (Ki 16 mM; Bokvist et al. 1999, Gromada et al. 2007), but a cellular distribution along with several studies on micro- very similar one to the values recorded in mouse and rat intact b circulation within the islet suggests that the order of -cells. However, KATP channels exhibit a higher ATP a Z . paracrine interactions is from b-toa-andd-cells sensitivity in intact mouse -cells (Ki 0 16 mM; Leung et al. 2005). Consequently, lower ATP concentrations are required (Bonner-Weir 1991). The rich vascularization within the to obtain the maximal inhibition of K conductance islet ensures a rapid sensing of plasma glucose levels by these ATP compared with mouse b-cells. Recent evidence has indicated endocrine cells, allowing an appropriate secretory response. that the densities of these channels are similar in mouse a- and In human islets, however, there are important differences b-cells (Leung et al. 2005). The repolarization of action in composition and spatial organization compared with C potentials is mediated by voltage-dependent K channels. rodents (Cabrera et al.2006). While the proportion of d-and C While delayed rectifying K channels have been demon- PP-cells are similar in the human islet, b-cells are less strated in rat, mouse and guinea pig a-cells, a tetraethy- a C abundant (48–59%) and the -cell population reaches a lammonium-resistant voltage-dependent K current 33–46%, suggesting that glucagon secretion plays a major (A-current) has only been identified in mice (Barg et al. role in humans (Cabrera et al.2006). These islet cell 2000, Leung et al. 2005). Furthermore, tetrodotoxin-sensitive C populations show a random distribution pattern, where the Na currents are fundamental for the generation of action C majority of b-cells are in contact with non-b-cells, potentials in these cells. Na channels are activated at voltages suggesting that paracrine interactions among different above K30 to K20 mV (Gopel et al. 2000), and their populations may be more active (Cabrera et al.2006). blockade by tetradotoxin leads to the inhibition of glucagon Another divergence between human and rodent islets is the secretion (Gromada et al. 2004, Olsen et al. 2005, MacDonald intercellular communication among the different populations. et al. 2007). Additionally, a-cells have a heterogeneous C In mice, b-cells work as a syncytium in terms of electrical presence of Ca2 channel subtypes with different roles. 2C activity and Ca signalling due to the high level of coupling While L and N channels have been reported in rat a-cells mediated by gap junctions of connexin36 (Gopel et al.1999, (Gromada et al. 1997), mouse a-cells express L-, T-, N- and C Nadal et al.1999, Quesada et al.2003). This coupling favours probably R-type Ca2 channels (Gopel et al. 2000, Gromada a more vigorous insulin secretion (Vozzi et al.1995). By et al. 2004, Pereverzev et al. 2005, MacDonald et al. 2007). contrast, coupling can be found between several human The low voltage-activated T-type channels work as pace- b-cells in clusters within the same islet but not in the whole makers in the initiation of action potentials in mice (Gopel b-cell population (Quesada et al.2006b). This kind of et al. 2000). They open around K60 mV, the action potential intercellular communication is probably the result of the initiation threshold in a-cells. The high voltage-activated L human islet cytoarchitecture and its functional meaning is and N channels open during action potentials when the still unknown (Cabrera et al.2006). Unlike b-cells, a-and membrane potential exceeds K40 to K30 mV. Although C d-cells from rodents and humans are not functionally coupled most of the Ca2 current goes through L-type channels in C and work as independent units. In addition to nutrients a-cells, the Ca2 required for exocytosis at low-glucose levels and paracrine signals, islet function is further regulated by is mediated by N-type channels, and their blockade by sympathetic, parasympathetic and sensory nerves that go u-conotoxin-GVIA inhibits glucagon secretion in these

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Downloaded from Bioscientifica.com at 09/28/2021 04:17:15AM via free access Regulation of a-cell and glucagon secretion . I QUESADA and others 7 conditions (Gromada et al. 1997, 2004, Olsen et al. 2005, MacDonald et al. 2007). However, in the presence of cAMP- C elevating agents, L channels are the major conduit for Ca2 (Gromada et al. 1997). A model to explain the glucose regulation of electrical activity in mouse a-cells has been postulated in the light of recent studies (Fig. 1). At low-glucose levels, the activity of KATP channels renders a membrane potential of about K60 mV. At this voltage, T-type channels open, which C depolarize the membrane potential to levels where Na and C N-type Ca2 channels are activated, leading to regenerative action potentials (Gromada et al. 2004, MacDonald et al. C 2007). Ca2 entry through N-type channels induces glucagon secretion. The repolarization of action potentials C is mediated by the flowing of K A-currents. At low-glucose concentrations, this electrical activity triggers oscillatory C Ca2 signals in both human and mouse a-cells in intact islets (Nadal et al. 1999, Quesada et al. 1999, 2006b; Fig. 2).

C Figure 2 Opposite Ca2 signalling patterns in a- and b-cells in response to glucose. At low-glucose concentrations (0.5 mM), C electrical activity triggers oscillatory Ca2 signals in a-cells that lead to glucagon release. Elevation of glucose levels (11 mM) inhibits all these events. By contrast, 11 mM glucose stimulate C Ca2 signalling and insulin secretion in b-cells. Both fluorescence records were obtained by confocal microscopy from two cells within an intact mouse islet. Inset shows a thin optical section C (w6 mm) of a mouse islet loaded with the Ca2 -sensitive fluorescent probe Fluo-3.

However, the increase in extracellular glucose levels rises the cytosolic ATP/ADP ratio which blocks KATP channels, depolarizing a-cells to a membrane potential range where the channels involved in action potentials become inactivated (Gromada et al. 2004, MacDonald et al.2007). As a C consequence, electrical activity, Ca2 signals and glucagon secretion are inhibited (Figs 1 and 2). Thus, glucagon release from a-cells is mainly supported by an intermediate KATP channel activity that maintains a membrane potential range able to sustain regenerative electrical activity (MacDonald et al. 2007). A similar model has been also proposed for human a-cells (MacDonald et al. 2007). Nevertheless, this scheme has been argued by some reports indicating that glucose may be hyperpolarizing rather than depolarizing (Liu et al. 2004, Figure 1 Schematic model for glucose-dependent regulation of Manning Fox et al. 2006). It has also been proposed that glucagon secretion in the mouse a-cell. Glucose is incorporated glucose would inhibit glucagon secretion by suppressing into the a-cell by the transporter SLC2A1. At low-glucose C a depolarizing Ca2 store-operated current independent concentrations, the moderate activity of KATP channels situates the a-cell membrane potential in a range that allows the opening of of KATP channels (Liu et al. 2004, Vieira et al. 2007). C voltage-dependent T- and N-type Ca2 channels and voltage- a C In rat -cells, the activity of KATP channels at low-glucose dependent Na channels. Their activation triggers action 2C concentrations also keeps the membrane potential at about potentials, Ca influx and exocytosis of glucagon granules. The C 2C C K60 mV, where spontaneous Na and Ca action opening of A-type K channels is necessary for action potential repolarization. However, high-glucose concentrations elevate the potentials are produced (Gromada et al. 1997). However, in intracellular ATP/ADP ratio, blocking KATP channels and depolar- contrast to the situation in mice, the stimulus-secretion izing the membrane potential to a range where the inactivation of coupling in rat a-cells is similar to that of b-cells. That is, voltage-dependent channels takes place. This results in the C elevations of extracellular glucose levels increase the inhibition of electrical activity, Ca2 influx and glucagon secretion. The function of L-type channels predominates when cAMP levels intracellular ATP/ADP ratio, blocking KATP channels and 2C are elevated. See text for further details. depolarizing the membrane potential, which stimulates Ca www.endocrinology-journals.org Journal of Endocrinology (2008) 199, 5–19

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influx through N channels and glucagon secretion (Franklin ratio is higher in non-b cells (Sekine et al. 1994). Additionally, et al. 2005, Olsen et al. 2005). Accordingly, the pharma- a-cells may express higher levels of the lactate/monocarbox- cological inhibition of glucose metabolism increases KATP ylate transporter than b-cells but lower ones of pyruvate channel activity in rat a-cells (Olsen et al. 2005). This model carboxylase (Sekine et al. 1994, Zhao et al. 2001). These indicating a b-cell-like stimulus-secretion coupling is based biochemical differences indicate that b-cells are more efficient on recent studies that have used isolated rat a-cells. However, in the mitochondrial oxidation of glucose, while a-cells rely these results contrast with the observations showing that more on anaerobic glycolysis (Schuit et al. 1997, Quesada et al. glucose inhibits a-cell electrical activity and glucagon 2006a). This lower coupling between glycolytic events in the secretion in intact rat islets (Franklin et al. 2005, Manning cytosol and ATP synthesis in mitochondrial respiration of Fox et al. 2006). Therefore, the blocking effect observed in rat a-cells would explain the fact that, in response to glucose, islets at high-glucose concentrations is most likely the result of cytosolic ATP increases are small in these cells (Ishihara et al. paracrine signalling by b-cell activation (Wendt et al. 2004). 2003, Ravier & Rutter 2005) and that ATP/ADP changes are almost invariable (Detimary et al. 1998). Therefore, some aspects at the above-mentioned models for a-cell stimulus- Regulation of a-cell function by glucose: direct or paracrine effect? secretion coupling deserve more attention, especially those Whether glucose inhibits a-cells directly or by paracrine concerning the modulation of KATP channel activity by mechanisms has been a matter of debate, and, probably, the glucose metabolism and ATP production. Other mechanisms predominant level of control may depend on the physiologi- regulating KATP channels may also have an important role. cal situation. Part of this controversy is also due to the divergences found in the stimulus-secretion coupling of Regulation of glucagon secretion by fatty acids and amino acids different animal models. Although paracrine signalling may be critical for the glucose inhibition of glucagon secretion in Although the lipotoxicity theory and its role in obesity- rats (Wendt et al. 2004, Franklin et al. 2005, Olsen et al. 2005), induced diabetes have increased the interest in the interactions a direct effect has been observed in mice and humans (Asplin between fatty acids and islet functions, little is known about et al. 1983, MacDonald et al. 2007). In mice and humans, a their effect on the regulation of the a-cell compared with those glucose direct action on a-cells has been proven in isolated on b-cells. While initial studies suggested an inhibitory effect cells under conditions where paracrine effects are negligible, on glucagon secretion (Andrews et al. 1975), more recent and in intact islets incubated with different paracrine investigations have indicated that short-term exposure to fatty signalling inhibitors (Gromada et al. 2004, Ravier & Rutter acids stimulates the release of this hormone (Bollheimer et al. 2005, Shiota et al. 2005, MacDonald et al. 2007, Vieira et al. 2004, Olofsson et al. 2004, Hong et al. 2005). The short-term 2007). Moreover, secretion studies prove that glucose inhibits stimulatory action depends on the chain length, spatial glucagon release at concentrations below the threshold for configuration and degree of saturation of the fatty acid b-cell activation and insulin release (MacDonald et al. 2007, (Hong et al. 2005). The action of palmitate has been studied in Vieira et al. 2007). mice at the cell level. This fatty acid increases a-cell exocytosis C C Several reports on experiments using genetic mouse models by enhancing Ca2 entry through L-type Ca2 channels and support the role of glucose-modulated KATP channels in a-cell also, by relief of the inhibitory paracrine action of the function. The regulation of glucagon secretion by glucose is somatostatin secreted from d-cells (Olofsson et al. 2004). A impaired in ABCC8-deficient mice lacking functional KATP study using clonal a-cells on the long-term effect of palmitate channels (Gromada et al. 2004, Munoz et al. 2005). A similar and oleate concluded that they also enhance glucagon situation occurs in KCNJ11Y12X mouse with a KCNJ11 secretion and triglyceride accumulation in a time- and dose- mutation in the KATP channel (MacDonald et al. 2007). In dependent manner but inhibit cell proliferation (Hong et al. humans, the Glu23Lys polymorphism in the KCNJ11 subunit 2007). In agreement with this, the long-term exposure of rat of these channels is associated with diminished suppression of islets to fatty acids induces a marked increase in glucagon glucagon release in response to hyperglycaemia (Tschritter release, a decrease in glucagon content and no changes in et al. 2002). Nevertheless, since KATP channels seem to be glucagon gene expression (Gremlich et al. 1997, Dumonteil essential for the a-cell regulation in the proposed models, some et al. 2000). In addition to fatty acids, amino acids are also considerations on glucose metabolism should be taken into relevant in the modulation of the a-cell function. Amino acids account. Although a-cells possess the high-affinity, low- such as arginine, alanine and glutamine are potent stimulators capacity glucose transporter SLC2A1, instead of the high- of glucagon secretion (Pipeleers et al. 1985, Kuhara et al. 1991, capacity SLC2A2 present in the b-cell, it has been Dumonteil et al. 2000). However, a few amino acids such as demonstrated that glucose transport is not a limiting factor isoleucine can also inhibit a-cell secretion while leucine has a in a-cell glucose metabolism (Gorus et al. 1984, Heimberg dual effect: it is a positive stimulus at physiological et al. 1995, 1996). However, several studies indicate that concentrations but becomes a negative one at elevated levels important biochemical differences exist between both cell (15 mM; Leclercq-Meyer et al. 1985). In any case, the function types. While the ratio of lactate dehydrogenase/mitochondrial of amino acids and fatty acids in the a-cell requires further glycerol phosphate dehydrogenase is low in the b-cell, this investigation at the cellular and molecular levels.

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Autocrine, paracrine, endocrine and neural regulation of glucagon Insulin and zinc. One of the most important paracrine secretion mechanisms responsible for inhibiting glucagon release is Autocrine, paracrine and endocrine signalling The conducted by insulin, acting via several pathways. An a spatial distribution of a-cells and the vascular organization appropriate expression of the insulin receptor in mouse -cells within the islet sustain an important intercellular communi- seems to be essential for glucose-regulated glucagon secretion (Diao et al.2005). In INR1-G9 clonal a-cells, insulin has been cation through autocrine and paracrine mechanisms (Fig. 3). found to inhibit glucagon release through the activation of In addition to insulin, glucagon or somatostatin, secretory phosphatidylinositol 3-kinase (PIK3; Kaneko et al.1999). The granules from islet cells contain other molecules with insulin receptor–PIK3 signalling pathway is also involved in the biological activity, which are released to the extracellular modificationof thesensitivityof KATP channelstoATP in mouse space by exocytosis, activating surface receptors in the same a-cells, which may affect the secretory response (Leung et al. cell, in neighbouring islet cells, or in distant cells within the 2006). Furthermore, insulin increases KATP channel activity in islet via the vascular system. Several paracrine mechanisms are isolated rat a-cells, inducing an inhibitory effect on glucagon activated at high-glucose concentrations as a result of b- and release via membrane hyperpolarization (Franklin et al. 2005). d-cell stimulations, and thus, they may participate in the In addition to the effects on KATP channels, insulin can glucose-induced inhibition of glucagon release. translocate A-type GABA receptors to the cell membrane,

Figure 3 Paracrine signalling in the a-cell. See text for details. ADCY, adenylate cyclase; AMPA-R, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; GABA, g-aminobutyric acid; GLP1, glucagon-like peptide-1; GRM, metabotrophic glutamate receptor; PKA, protein kinase A; SSTR2, somatostatin receptor-2. www.endocrinology-journals.org Journal of Endocrinology (2008) 199, 5–19

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which increases the response to GABA secreted by b-cells, deficiency as well as hyperglucagonaemia (Dunning et al.2005). favouring membrane hyperpolarization and suppression of The observed suppressing effect of GLP1 on glucagon secretion C glucagon secretion (Xu et al.2006). Since a-cell Ca2 signals in vivo and in perfused pancreas contrasts with those effects found C rely on electrical activity, insulin also inhibits Ca2 signals in single a-cells (Dunning et al.2005). In isolated rat a-cells, induced by low-glucose concentrations (Ravier & Rutter 2005). GLP1 stimulates glucagon secretion by interacting with specific Therefore, several pieces of evidence indicatethat insulin inhibits receptors coupled to G-proteins that activate adenylate cyclase, glucagon release mainly by altering a-cell membrane potential. which increases cAMP levels (Ding et al.1997, Ma et al.2005). Insulin is stored within the secretory granule forming stable Thus, it seems that paracrine mechanisms may be responsible for C hexamers around two atoms of Zn2 . After exocytosis, these the GLP1 suppressing action (Dunning et al.2005). This hexameric crystals are exposed to a change in pH from 5.5to possibility has been underscored by the findings in experiments C 7.4, become dissociated and release both atoms of Zn2 . using b-cell-specific knock-out mice for the transcription factor Recent studies have claimed that zinc atoms can also work as Pdx1. In these mice, the lack of effect of GLP1 on b-cells is also modulators of the a-cell function (Gyulkhandanyan et al. accompanied by its inability to induce an inhibitory action on 2008), although their role remains controversial. On one glucagon plasma levels (Li et al.2005). Moreover, GLP1 may also 2C hand, it has been found that Zn activates KATP channels affect the a-cell function by interacting with the autonomic and decreases glucagon release in isolated rat a-cells (Ishihara nervous system (Balkan & Li 2000). C et al. 2003, Franklin et al. 2005). Zn2 seems to be the switch- off signal to initiate glucagon secretion during hypoglycaemia Other extracellular messengers The neurotransmitter in streptozotocin-induced diabetic rats (Zhou et al. 2007a). g-aminobutyric acid (GABA) is another a-cell modulator. C However, these results contrast with the absence of effects on GABA accumulates in b-cell vesicles and is released by Ca2 - mouse a-cells (Ravier & Rutter 2005). dependent exocytosis, stimulating A-type GABA receptors in neighbouring a-cells. Activation of these receptors is coupled to K Somatostatin and glucagon Somatostatin is produced and inward Cl currents that hyperpolarize the a-cell plasma secreted by several tissues in addition to the d-cell population membrane, decreasing glucagon release in rats and guinea pigs of the islet and works as an inhibitor of both glucagon and (Rorsman et al.1989, Wendt et al.2004). Similar conclusions insulin release (Fehmann et al. 1995). Immunocytochemical were obtained in mouse islets and clonal aTC1–9 cells (Xu et al. studies in human islets have demonstrated that, among the 2006, Bailey et al.2007). The neurotransmitter L-glutamate also five identified somatostatin receptor (SSTR) subtypes, accumulates in the a-cell secretory granules because of vesicular SSTR2 is highly expressed in a-cells while SSTR1 and glutamate transporters 1 and 2 found in these cells (Hayashi et al. SSTR5 are expressed in b-cells (Kumar et al. 1999). In mice 2003a). In low-glucose conditions, L-glutamate is cosecreted and rats, SSTR2 also predominates in the a-cell and SSTR5 with glucagon, triggering GABA release from neighbouring in the b-cell population (Hunyady et al. 1997, Strowski et al. b-cells and, subsequently, inhibiting the a-cell function as 2000). These receptors are coupled to G-proteins and induce previously described (Hayashi et al. 2003b). Additionally, multiple intracellular effects. Electrophysiological studies have glutamate can activate autocrine signalling pathways in a-cells C shown that somatostatin activates K channels in a-cells, through the multiple glutamate receptors expressed in these inducing membrane hyperpolarization and suppressing cells, which include ionotrophic AMPA and kainate subtypes C electrical activity, which affects Ca2 -dependent exocytosis and metabotrophic receptors (Inagaki et al.1995, Uehara et al. (Yoshimoto et al. 1999, Gromada et al. 2001). Capacitance 2004, Cabrera et al.2008). Although activation of ionotrophic measurements have further elucidated that somatostatin receptors may stimulate glucagon release (Bertrand et al.1993), directly decreases exocytosis by depriming secretory granules metabotrophic glutamate receptors inhibit rat glucagon through the activation of the serine/threonine protein secretion at low-glucose concentrations through a down- phosphatase calcineurin pathway (Gromada et al. 2001). regulation of cAMP levels (Uehara et al.2004). Another a-cell Also, a negative interaction of somatostatin with adenylate regulator is amylin or islet amyloid pancreatic polypeptide cyclase and cAMP levels has been reported in rat a-cells (Iapp). This polypeptide is a 37 amino acid hormone mainly (Schuit et al. 1989). In addition to the effects of insulin and synthesized in b-cells, although it can be produced in d-cells as somatostatin on a-cells, glucagon itself works as an well. This peptide is cosecreted with insulin by exocytosis and extracellular messenger. It exerts an autocrine positive has an inhibitory effect on glucagon basal concentrations as well feedback that stimulates secretion in both isolated rat and as on those levels observed after arginine stimulation (Akesson mouse a-cells by an increase in exocytosis associated to a rise et al.2003, Gedulin et al.2006). This glucagonostatic effect has in cAMP levels (Ma et al. 2005). been reported in the plasma levels of mice and rats as well as in perfused pancreas or intact islets. Since amylin also reduces GLP1 The incretin hormone glucagon-like peptide 1 (GLP1) somatostatin and insulin release, some authors have proposed is released from the L-cells of the small intestine after food intake, that endogen amylin within the islet may establish a negative stimulating insulin production and inhibiting glucagon release. feedback to avoid excessive secretion from a-, b-andd-cells Because of this dual effect, GLP1 is a potential therapeutic agent (Wa ng et al.1999). Also, the purinergic messenger ATP is highly in the treatment of diabetic patients that manifest insulin accumulated in b-cell secretory granules and in nerve terminals.

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C It has recently been reported that ATP inhibits Ca2 signalling in each tissue mediates the production of each different and glucagon secretion in mouse a-cells, indicating that peptide. In a-cells, the predominance of PCSK2 leads to a purinergic receptors are involved in a-cell function (Tu du rı´ major production of glucagon together with the products et al.2008). Purinergic regulation of glucagon release has also glicentin, glicentin-related pancreatic polypeptide, interven- been described in rat islets (Grapengiesser et al.2006). ing peptide 1 and the major proglucagon fragment (Dey et al. 2005). The absence of PCSK2 in knock-out mice leads to a Neural regulation As previously stated, the islet of lack of mature glucagon (Furuta et al. 2001). In enteroendo- Langerhans is highly innervated by parasympathetic and crine cells, PCSK1/3 enzymes cleave the preproglucagon sympathetic nerves that ensure a rapid response to hypoglycae- hormone to generate GLP1 and GLP2 along with glicentin, mia and protection from potential brain damage (Ahren 2000). intervening peptide 2 and oxyntomodulin (Mojsov et al. Some terminals of these nerves store and release classical 1986). neurotransmitters, such as acetylcholine and noradrenaline, as The regulation of glucagon gene expression has not been well as several neuropeptides, which stimulate or inhibit studied as extensively as the insulin gene. The inhibitory effect glucagon secretion depending on the neural messenger released. of insulin on glucagon secretion has also been confirmed in Cholinergic stimulation involving muscarinic receptors and gene expression and it occurs at the transcriptional level C intracellular Ca2 mobilization enhances a-cell function both (Philippe et al. 1995). In diabetic rats, glucagon gene in vivo and in isolated cells (Ahren & Lundquist 1982, Berts et al. expression is augmented and is accompanied by hypergluca- 1997). Noradrenaline increases glucagon secretion as well gonaemia in conditions of hyperglycaemia and insulin (Ahren et al.1987). Sympathetic activation can also induce deficiency. Insulin treatment normalized glucagon expression adrenaline release from the adrenal medulla, which potently and plasma levels in these rats, an effect that was not attributed C stimulates glucagon secretion by enhancing Ca2 influx to the restoration of normal glucose levels (Dumonteil et al. C through L-type Ca2 channels and accelerating granule 1998). It was concluded that insulin, unlike glucose, mobilization (Gromada et al.1997). In addition to classical modulates glucagon expression. The lack of response to neurotransmitters, several neuropeptides such as vasoactive glucose was further confirmed in isolated rat islets (Gremlich intestinal polypeptide, pituitary adenylate cyclase-activating et al. 1997, Dumonteil et al. 2000), contrasting with the polypeptide and gastrin-releasing peptide, which may stimulate up-regulation of glucagon expression observed in clonal glucagon release from pancreas, can be accumulated in a-cells (Dumonteil et al. 1999, McGirr et al. 2005). The effect parasympathetic nerves, while galanin and neuropeptide Y of amino acids on glucagon gene regulation has also been can be stored in sympathetic nerve terminals (Ahren 2000). studied. While arginine increases glucagon expression in Multiple actions have been reported for the latter neuro- isolated rat islets: a process that is mediated by protein kinase peptides. The effects and mechanisms involved in neural C (PKA; Yamato et al. 1990, Dumonteil et al. 2000), histidine regulation of a-cells have yet to be established at the cellular plays a fundamental role in clonal aTC1–6 cells (Paul et al. and molecular levels. These systems are mainly regulated by 1998). Other nutrients, such as the fatty acid palmitate, glucose-sensing neurons of the ventromedial hypothalamus, produces a down-regulated glucagon expression at short term which respond to plasma glucose levels with mechanisms very in rat islets in a dose-dependent manner (Bollheimer et al. similar to those of the b-cell, including the activity of glucose- 2004). By contrast, no effect with palmitate has been observed regulated KATP channels (Borg et al.1994, Miki et al.2001, Song in other long-term studies (Gremlich et al. 1997, Dumonteil et al.2001). Actually, it has been observed that the a-cell et al. 2000). Like insulin, somatostatin also inhibits glucagon response to hypoglycaemia is also impaired in KCNJ11- expression. It has been reported that somatostatin down- deficient mice whose neurons of the ventromedial regulates glucagon expression basal levels as well as those hypothalamus lack functional KATP channels and glucose produced by forskolin stimulation in clonal INR1G9 cells responsiveness (Miki et al.2001). (Fehmann et al. 1995, Kendall et al. 1995).

Glucagon receptor Glucagon physiological and pathophysiological The rat and mouse glucagon receptor is a 485 amino acid actions and its role in diabetes protein, belonging to the secretin–glucagon receptor II class family of G protein-coupled receptors (Mayo et al. 2003). Glucagon synthesis Glucagon binding to this receptor is coupled to GTP-binding The preproglucagon-derived peptides glucagon, GLP1 and heterotrimeric G proteins of the Gas type that leads to the GLP2, are encoded by the preproglucagon gene, which is activation of adenylate cyclase, cAMP production and PKA. expressed in the central nervous system, intestinal L-cells and This receptor can also activate the phospholipase C/inositol 2C pancreatic a-cells. A post-translational cleavage by prohor- phosphate pathway via Gq proteins, resulting in Ca release mone convertases (PC) is responsible for the maturation of from intracellular stores (Fig. 4; Wakelam et al. 1986, Mayo the preproglucagon hormone that generates all these peptides et al. 2003). The glucagon receptor is present in multiple (Mojsov et al. 1986). The different expression of PC subtypes tissues including the liver, pancreas, heart, kidney, brain and www.endocrinology-journals.org Journal of Endocrinology (2008) 199, 5–19

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Figure 4 The role of glucagon and the glucagon receptor in the liver. Glucagon signalling regulates positively (C)or negatively (K) the multiple steps of the hepatic glucose metabolism. This modulation affects the expression and/or the activity of several enzymes of glucose metabolism. See text for details. ADCY, Adenylate cyclase; CREB, cAMP response element binding; F(1,6)P2, fructose-1,6-bisphosphate; F(2,6)P2, fructose-2,6-bisphosphate; F-6-P, fructose 6-phosphate; FBP1, fructose-1,6-bisphosphatase; FBP2, fructose-2,6-bisphosphatase; G-1-P, glucose 1-phosphate; G-6-P, glucose 6-phosphate; G6PC, glucose-6-phosphatase; GP, glycogen phosphorylase; GS, glycogen synthase; IP3, inositol 1,4,5-trisphosphate; OAA, oxaloacetate; PC, pyruvate carboxylase; PEP, phosphoenolpyruvate; PCK2, phosphoenol- pyruvate carboxykinase; PFKM, phosphofructokinase-1; PPARGC1A, peroxisome proliferators-activated receptor-g coactivator-1; PIP2, phosphatidylinositol 4,5-bisphosphate; PKLR, pyruvate kinase; PLC, phospholipase C; Pyr, pyruvate. Dashed lines: red, inhibition; blue, stimulation.

smooth muscle. Thus, it modulates multiple responses in glycaemia changes, as previously mentioned. Among all these tissues, including effects on ion transport and glomerular these regulatory systems, glucagon plays a central role in the filtration rate in kidney among others (Ahloulay et al. 1992). response to hypoglycaemia and also opposes to insulin effects. In any case, the regulation of glucose homeostasis is the major The main action of glucagon occurs in the liver where the function of glucagon and its receptor. This role will be insulin/glucagon ratio controls multiple steps of hepatic described in the next paragraph. metabolism. Glucagon stimulates gluconeogenesis and glyco- genolysis, which increases hepatic glucose output, ensuring an appropriate supply of glucose to body and brain, and at the Glucagon control of glucose homeostasis and metabolism same time, it decreases glycogenesis and glycolysis. The Several lines of defence protect the organism against glucagon receptor in the liver is highly selective for glucagon, hypoglycaemia and its potential damaging effects, especially but it exhibits a modest affinity for glucagon-like peptides in the brain, which depends on a continuous supply of (Hjorth et al. 1994). Its main action on the liver is mediated by glucose, its principal metabolic fuel. These defences include the activation of adenylyl cyclase and the PKA pathway. decreased insulin release and increased secretion of adrenaline Glucagon regulates gluconeogenesis mainly by the up-regula- and glucagon. Additionally, glucose-sensing neurons of the tion of key enzymes such as glucose-6-phosphatase (G6PC) ventromedial hypothalamus further control responses to and phosphoenolpyruvate carboxykinase (PCK2) through the

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Downloaded from Bioscientifica.com at 09/28/2021 04:17:15AM via free access Regulation of a-cell and glucagon secretion . I QUESADA and others 13 activation of the cAMP response element-binding protein higher rate of hepatic glucose production than glucose (CREB) and peroxisome proliferator-activated receptor utilization, favouring hyperglycaemia. At present, there exists g-coactivator-1 (PPARGC1A; Herzig et al. 2001, Yoon multiple clinical and experimental evidence that support this et al. 2001; Fig. 4). PCK2 and G6PC, along with fructose- hypothesis. The rate of hepatic glucose output has been 1,6-biphosphatase (FBP1) have a key role in the rate of correlated with the hyperglycaemia found in animal models gluconeogenesis (Fig. 4). PCK2 mediates the conversion of of diabetes as well as in human diabetes, and the maintenance oxalacetate into phosphoenolpyruvate while G6PC regulates of this abnormality has also been associated with hyperglu- glucose production from glucose-6-phosphate. FBP1 is cagonaemia (Baron et al. 1987, Consoli et al. 1989, Gastaldelli responsible for the conversion of fructose-1,6-biphosphate et al. 2000, Dunning & Gerich 2007, Li et al. 2008). In type 2 (F(1,6)P2) into fructose-6-phosphate (F6P). Its activity is diabetes, the impairment of insulin release and development regulated by glucagon since this hormone decreases the of insulin resistance is often accompanied by absolute or intracellular levels of fructose-2,6-biphosphate (F(2,6)P2), an relative increased levels of glucagon in the fasting and allosteric inhibitor of FBP1 (Kurland & Pilkis 1995). postprandial states (Reaven et al. 1987, Larsson & Ahren Additionally, this decrease in F(2,6)P2 also reduces the 2000). In this situation, insulin is not effective as a negative activity of phosphofructokinase-1 (PFKM), down-regulating feedback for hepatic glucose output while glucagon glycolysis. The glycolytic pathway is further inhibited by potentiates glucose mobilization from the liver, thus glucagon at the pyruvate kinase (PKLR) level (Slavin et al. contributing to hyperglycaemia. Another malfunction 1994). Glycogen metabolism is mainly determined by the reported in diabetic patients is the lack of suppression of activity of glycogen synthase (GS) and glycogen phos- glucagon release in hyperglycaemic conditions, which would phorylase (GP). While glucagon is important for GP contribute further to postprandial hyperglycaemia in both phosphorylation and activation, it inhibits GS function by type 1 and type 2 diabetes (Dinneen et al. 1995, Shah et al. phosphorylation and its conversion into an inactive form of 2000). However, this irregular a-cell behaviour does not the enzyme (Band & Jones 1980, Ciudad et al. 1984, occur when insulin levels are adequate, suggesting that Andersen et al. 1999). abnormalities in glucagon release are relevant for hypergly- Glucagon can also stimulate the uptake of amino acids for caemia in the context of diabetes or impairment of insulin gluconeogenesis in the liver. Indeed, subjects with hyperglu- secretion or action (Shah et al. 1999). Hyperglucagonaemia is cagonaemia can develop plasma hypoaminoacidaemia, also responsible for the development of hyperglycaemia and especially of amino acids involved in gluconeogenesis, such diabetes in patients with the glucagonoma syndrome, a as alanine, glycine and proline (Cynober 2002). Glucagon is paraneoplastic phenomenon characterized by an islet a-cell also involved in the regulation of fatty acids in adipocytes. pancreatic tumour (Chastain 2001). Hormone-sensitive lipase mediates the lipolysis of triacylgly- Another defect in normal glucagon secretion has important cerol into the non-esterified fatty acids and glycerol, which are consequences in the management of hypoglycaemia. The released from adipocytes. It has been reported that although secretory response of a-cells to low-glucose concentrations is glucagon does not modify the transcriptional levels of this impaired in type 1 and long-lasting type 2 diabetes, increasing enzyme, it increases the release of glycerol from adipocytes the risk of episodes of severe hypoglycaemia, especially in (Slavin et al. 1994). This mobilization of glycerol from adipose patients treated with insulin (Cryer 2002). In this regard, tissue can further be used in the liver during gluconeogenesis. iatrogenic hypoglycaemia is a situation that implies insulin However, the existence of a lipolytic action of glucagon excess and compromised glucose counter-regulation, and it is observed in several animal models is still controversial in responsible for a major complication in diabetes treatment, humans. While a positive effect of glucagon on lipolysis has increasing the morbidity and mortality of this disease (Cryer been reported in human subjects (Carlson et al. 1993), several 2002). This lack of glucagon response to hypoglycaemia has recent studies have indicated that it lacks a role in a been associated with multiple failures in a-cell regulation; yet, physiological context (Gravholt et al. 2001). An elevated the mechanisms are still under study (Bolli et al. 1984, Cryer glucagon to insulin ratio accelerates gluconeogenesis as well as 2002, Zhou et al. 2007b). Even though islet allotransplanta- fatty acid b-oxidation and ketone bodies formation (Vons et al. tion can provide prolonged insulin independence in patients 1991). Thus, glucagon may also be involved in diabetic with type 1 diabetes, the lack of a-cell response to ketoacidosis, a medical complication in diabetes derived from hypoglycaemia usually persists after transplantation, indicating the overproduction of ketone bodies (Eledrisi et al. 2006). that this procedure does not restore the physiological behaviour of a-cells (Paty et al. 2002). All these problems in the glucagon secretory response The role of a-cell function in diabetes observed in diabetes have been attributed to several defects in More than 30 years ago, Unger & Orci (1975) proposed the a-cell regulation including defective glucose sensing, loss of bihormonal hypothesis to explain the pathophysiology of b-cell function, insulin resistance or autonomic malfunction. diabetes. According to this hypothesis, this metabolic disease However, the mechanisms involved in a-cell pathophysiology is the result of an insulin deficiency or resistance along with an still remain largely unknown and deserve more investigation absolute or relative excess of glucagon, which can cause a for better design of therapeutic strategies. In this regard, www.endocrinology-journals.org Journal of Endocrinology (2008) 199, 5–19

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although direct therapeutic approaches to correct the lack of concentrations up to 1 mM, but higher doses are inhibitory a-cell response to hypoglycaemia are missing, several (MacDonald et al. 2007). This biphasic effect is due to the proposals have been developed to amend glucagon excess, mouse a-cell electrical behaviour (Fig. 1): glucagon release as we will see in the next section. takes place within a narrow window of intermediate KATP channel activity (and membrane potential), and thus it is inhibited when the cell is hyperpolarized or depolarized Molecular pharmacology of glucagon release and beyond this membrane potential range (MacDonald et al. action: therapeutic potential in diabetes treatment 2007). Accordingly, with this scheme, the KATP channel opener diazoxide can also have a biphasic effect on glucagon Given that absolute or relative glucagon excess seems to be secretion. These effects will change depending on the critical in the development and/or maintenance of hypergly- extracellular glucose concentrations that necessarily influence caemia in diabetes by increasing hepatic glucose output, the KATP channel activity (MacDonald et al. 2007). This biphasic strategies targeted to correct this malfunction are suitable for behaviour may explain the disparity of effects found for the improvement of glucose levels. In this respect, several sulphonylureas (Loubatieres et al. 1974, Ostenson et al. 1986). experimental and therapeutic approaches have been In humans, sulphonylureas are associated to a glucagon developed (for a further review, see Dunning & Gerich secretion decrease in healthy and type 2 diabetic subjects 2007). The specific control of glucagon secretion by (Landstedt-Hallin et al. 1999), while they stimulate glucagon pharmacological modulation is complex since several levels in type 1 diabetic patients (Bohannon et al. 1982). Since components of the a-cell stimulus-secretion coupling are sulphonylureas also induce insulin and somatostatin secretion, also present in b- and d-cells. Thus, the manipulation of which affect a-cells, these drugs offer a poor specific control glucagon action by modulating the glucagon receptor of glucagon secretion. signalling seems to be an effective alternative (Li et al. 2008). This strategy has been supported by several studies. GLP1 mimetics and DPP4 inhibitors In addition to Glucagon receptor knock-out mice have hyperglucagonae- stimulating insulin release, GLP1 can suppress glucagon mia and a-cell hyperplasia, but their glucose tolerance is secretion in humans, perfused rat pancreas and isolated rat improved and they develop only a mild fasting hypoglycaemia islets in a glucose-dependent manner (Guenifi et al. 2001, (Gelling et al. 2003). These mice have a normal body weight, Nauck et al. 2002). Because GLP1 is rapidly cleaved and food intake and energy expenditure although less adiposity inactivated by the enzyme dipeptidyl peptidase-IV (DPP4), a and lower leptin levels. These results are consistent with the good alternative would be to design either GLP1 derivatives experiments with anti-sense oligonucleotides for the gluca- with higher resistance to DPP4 or agents that increase GLP1 gon receptor. Diabetic db/db mice treated with these endogenous levels. Among the GLP1 mimetics, exenatide is a oligonucleotides had lower glucose, triglyceride and free synthetic polypeptide with high resistance to DPP4 cleavage fatty acids blood levels, as well as improved glucose tolerance, that decreases glucagon levels in normal and diabetic subjects and they developed hyperglucagonaemia without apparent (Degn et al. 2004). Liraglutide, another GLP1 derivative with effects on a-cell size or number (Liang et al. 2004). This long-lasting actions, can reduce glucagon release after a meal approach is also accompanied by an increase in GLP1 and in patients with type 2 diabetes (Juhl et al. 2002). Alternatively, insulin levels in Zucker diabetic fatty rats and db/db and ob/ DPP4 inhibitors like sitagliptin and vildagliptin increase the ob mice (Sloop et al. 2004). Furthermore, the use of high endogen effects of GLP1, reducing glucagon plasma affinity, neutralizing glucagon monoclonal antibodies concentrations in diabetic individuals (Rosenstock et al. improved glycaemic control and reduced hepatic glucose 2007). Since all these alternatives produce opposing actions production in diabetic ob/ob mice (Sorensen et al. 2006). on insulin and glucagon, they generate promising expec- Therefore, these experimental results are a further support tations for diabetes treatment. that glucagon antagonism may be beneficial for diabetes treatment. Imidazolines The insulinotrophic effects of imidazoline compounds are mediated by KATP channel blockade, which C leads to depolarization, Ca2 influx and secretion, and by Modulation of glucagon secretion direct interactions with the exocytotic machinery (Zaitsev Sulphonylureas Sulphonylureas are efficient KATP channel et al.1996). Remarkably, imidazoline compounds such as blockers that have been extensively used for the clinical phentolamine also suppress glucagon secretion in both rat treatment of diabetes. In rat a-cells, sulphonylureas stimulate and mouse islets, an action mediated by a direct effect on C electrical activity, Ca2 signalling and glucagon release a-cell exocytosis via activation of phosphatase calcineurin 2C (Franklin et al. 2005). In mice, however, tolbutamide proteins, and independent of KATP channels or Ca C produces membrane depolarization, but a decrease in Ca2 currents (Hoy et al. 2001). Given that imidazoline signalling and glucagon release (Gromada et al. 2004). Recent compounds stimulate insulin release while inhibiting experiments in mouse a-cells have shown that, in the absence glucagon secretion, these drugs are potentially valuable in of glucose, this drug increases glucagon secretion at diabetes.

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Somatostatin analogues Because of the different Despite the success of several approaches to modulate expression of SSTR in the islet (Kumar et al. 1999), several glucagon secretion or action and improve glucose control in studies have explored the modulation of glucagon secretion animal models or in humans, more information is still required. by subtype-specific somatostatin analogues (Strowski et al. Long-standing studies should address whether the utilization of 2006). It has been shown that SSTR2 is the subtype receptor these agents could lead to undesired hypoglycaemia in humans, predominantly expressed in rodent a-cells, and that SSTR2- accumulation of lipids or compensatory mechanisms that deficient mice develop hyperglycaemia and non-fasting decrease the benefits of these therapies in the long term. In hyperglucagonaemia (Singh et al. 2007). In mice, the use of this aspect, the results obtained in animal models are positive: a highly SSTR2-selective non-peptide agonist inhibited although the glucagon receptor knock-out mouse develops glucagon release without affecting insulin release (Strowski hyperglucagonaemia, it is not hypoglycaemic and does not have et al. 2006). However, there is some overlapping in human an abnormal accumulation of lipids (Gelling et al.2003). islets between the different SSTR subtypes in a- and b-cells Additionally,recent long-term studies in mice further prove the that limit, at present, the use of subtype-specific somatostatin viability of glucagon antagonism (Winzell et al.2007). Thus, analogues (Singh et al. 2007). present data are promising and indicate that several therapeutic agents targeted to glucagon signalling and a-cell secretion may Amylin and pramlintide Amylin, which is cosecreted be useful for the management of diabetes. with insulin from b-cells, inhibits glucagon secretion stimulated by amino acids but does not affect hypoglycae- mia-induced glucagon release (Young 2005). Since a-cell Conclusions response to amino acids is often exaggerated in diabetic patients, amylin or amylinomimetic compounds such as Pancreatic a-cells and glucagon secretion are fundamental pramlintide are used as an effective alternative for the components of the regulatory mechanisms that control glucose treatment of postprandial and amino acid-induced excess of homeostasis. However, a-cell physiology has remained elusive glucagon secretion (Dunning et al. 2005, Young 2005). compared with the overwhelming information about insulin secretion and the b-cell. In recent years, however, several groups have initiated intensive efforts to understand a-cell physiology Modulation of glucagon action and glucagon receptor signalling and identified essential pieces of its stimulus-secretion coupling. Peptide-based glucagon receptor antagonists Several Additionally, important aspects of the regulation of a-cell linear and cyclic glucagon analogues have been developed to metabolism and the control of glucagon expression are being work as glucagon receptor antagonists. Essentially, they impair elucidated. All of this information will favour an overall the ability of glucagon to stimulate adenylate cyclase activity comprehension of the a-cell function and its role in glucose in liver, thus reducing hepatic glucose output and improving homeostasis. Nevertheless, more research is required to under- plasma glucose levels. This is the case of [des-His1, des-Phe6, stand the a-cell behaviour, not only in healthy subjects but in 9 pathological conditions as well. In conclusion, since the Glu ] glucagon-NH2, which reduces glucose levels in streptozotocin-induced diabetic rats (Van Tine et al. 1996). malfunction of the glucagon secretory response is involved in diabetes and its complications, a complete understanding of the Recent investigations have demonstrated that the antagonist a-cell will allow for a better design of therapeutic approaches for des-His-glucagon binds preferentially to the hepatic glucagon the treatment of this disease. receptor in vivo, and this correlates with the glucose lowering effects (Dallas-Yang et al. 2004). Declaration of interest Non-peptide glucagon receptor antagonists Multiple, competitive and non-competitive, non-peptide antagonists The authors declare that there is no conflict of interest that could be perceived have been reported to act on glucagon binding and/or as prejudicing the impartiality of the research reported. function. For instance, a novel competitive antagonist (N-[3-cyano-6-(1, 1-dimethylpropyl)-4, 5, 6, 7-tetrahydro- Funding 1-benzothien-2-yl]-2-ethylbutanamide) was recently shown to inhibit glucagon-mediated glycogenolysis in primary This work was supported by grants from the Ministerio de Educacio´ny human hepatocytes and to block the increase in glucose Ciencia (BFU2007-67607 and PCI2005-A7-0131 to I Q; BFU2005-01052 levels after the administration of exogenous glucagon in mice to A N). CIBERDEM is an initiative of the Instituto de Salud Carlos III. (Qureshi et al. 2004). The information about the effect of these antagonists on humans is, however, scarce. In this respect, Bay 27-9955 is an oral glucagon receptor antagonist References that has been tested in humans, demonstrating its efficacy in Ahloulay M, Bouby N, Machet F, Kubrusly M, Coutaud C & Bankir L 1992 reducing glucose levels induced by exogenous glucagon Effects of glucagon on glomerular filtration rate and urea and water (Petersen & Sullivan 2001). excretion. American Journal of Physiology. Renal Physiology 263 F24–F36. www.endocrinology-journals.org Journal of Endocrinology (2008) 199, 5–19

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