Glucose

One of the main stimuli that modulate glucagon secretion is plasma glucose. The ingestion, injection or infusion of glucose in animal models and human subjects has been proven to suppress glucagon secretion, while decreased plasma glucose levels are correlated with enhanced glucagon release⁽ Reference Unger, Aguilar-Parada and Muller ^{10 ,} Reference Gerich, Lorenzi and Schneider ^{16 ,} Reference Ohneda, Aguilar-Parada and Eisentraut ^{17 )}. Similarly, in vitro experiments have shown that glucagon release is stimulated in hypoglycaemic conditions, whereas high glucose levels down-regulate α-cell secretory activity⁽ Reference Salehi and Vieira ^{18 ,} Reference Vieira and Salehi ^{19 )}. Pancreatic α-cells are excitable cells able to respond to changes in extracellular glucose concentrations. At low glucose levels, these cells develop action potentials that trigger intracellular Ca signals and glucagon secretion in rodent and human islets⁽ Reference Nadal, Quesada and Soria ^{3 ,} Reference Quesada, Todorova and Soria ^{4 ,} Reference Gopel, Zhang and Eliasson ^{20 –} Reference Tuduri, Marroqui and Soriano ^{22 )}. In contrast, hyperglycaemic conditions inhibit α-cell functional activity, lowering glucagon release⁽ Reference Quesada, Tuduri and Ripoll ^{9 )}. This glucose modulation may rely on different control levels: the neural sensing of glucose and the subsequent neural regulation of α-cells⁽ Reference Taborsky, Ahren and Havel ^{23 ,} Reference Thorens ^{24 )}, the sensing of glucose by neighbouring β- and δ-cells within the islet and the inhibition of pancreatic α-cells by paracrine regulation (see the following sections) and, lastly, the glucose sensing and intracellular metabolism directly by pancreatic α-cells⁽ Reference Rorsman, Salehi and Abdulkader ^{25 )}. The latter possibility has been the subject of intense debate. While some studies indicate that ATP-dependent K⁺ (K_ATP) channels play a central role in coupling glucose-modulated metabolic changes with pancreatic α-cell electrical activity and secretion⁽ Reference Rorsman, Salehi and Abdulkader ^{25 ,} Reference MacDonald, De Marinis and Ramracheya ^{26 )}, other reports support a K_ATP-independent glucose effect⁽ Reference Gylfe ^{27 –} Reference Quoix, Cheng-Xue and Mattart ^{29 )}. Some of these controversies have emerged from studies of α-cell metabolism. The analysis of mitochondrial membrane potential, NADH autofluorescence or direct measurements of cytosolic ATP levels have shown both measurable and negligible glucose-induced changes in α-cell metabolism⁽ Reference Quesada, Todorova and Soria ^{4 ,} Reference Quoix, Cheng-Xue and Mattart ^{29 –} Reference Ravier and Rutter ^{31 )}. In this regard, it has been described that glucose-induced ATP/ADP changes in α-cells are very low compared with the ones in β-cells, probably because of the biochemical differences existing between both islet cell types⁽ Reference Detimary, Dejonghe and Ling ^{32 )}. In experiments with either samples of non-β-cells or purified α-cells, it has been determined that GLUT-1 is the high-affinity, low-capacity GLUT present in α-cells, in contrast to GLUT-2, which is present in β-cells. Despite this difference, this metabolic step is not a limiting factor in α-cell glucose utilisation⁽ Reference Gorus, Malaisse and Pipeleers ^{33 ,} Reference Heimberg, De Vos and Pipeleers ^{34 )}. Other biochemical differences between both cell types also suggest that α-cells are less competent in the use they make of mitochondrial glucose oxidation⁽ Reference Quesada, Todorova and Soria ^{4 ,} Reference Schuit, De Vos and Farfari ^{35 )}. In fact, compared with pancreatic β-cells, non-β-cells exhibit a higher expression of the lactate/monocarboxylate transporter and a lower expression of pyruvate carboxylase⁽ Reference Sekine, Ito and Hashimoto ^{36 ,} Reference Zhao, Wilson and Schuit ^{37 )}. Additionally, non-β-cells present higher rates of lactate dehydrogenase activity, while mitochondrial glycerol phosphate dehydrogenase activity is low⁽ Reference Sekine, Ito and Hashimoto ^{36 )}. These characteristics may explain why the coupling between glycolysis and mitochondrial glucose oxidation is not high in α-cells. Recent reports, however, have shown that in pancreatic α-cells labelled with yellow fluorescent protein, glucose hyperpolarises the mitochondrial membrane potential, further indicating that it directly affects mitochondrial metabolism and pointing to a K_ATP-dependent modulation of glucagon secretion by glucose⁽ Reference Allister, Robson-Doucette and Prentice ^{38 )}. In contrast, another recent study demonstrates that glucose acts directly on α-cells to control glucagon release but through K_ATP-independent mechanisms⁽ Reference Cheng-Xue, Gomez-Ruiz and Antoine ^{39 )}. Additionally, the authors suggest that glucagon output may depend on the balance between the direct and indirect (paracrine) effects of glucose. In recent years, several molecules have been identified that may play a key role in the glucose-sensing capacity of α-cells and its transduction into glucagon secretion. These include the per-arnt-sim domain-containing protein kinase (PASK) and AMP-activated protein kinase (AMPK), which are well-known energy sensors, as well as Rev-erb α, a clock gene involved in metabolism⁽ Reference da Silva Xavier, Farhan and Kim ^{40 –} Reference Vieira, Marroquí and Figueroa ^{42 )}. Thus, a larger consensus is required regarding the role of α-cell K_ATP channels and glucose metabolism in glucagon release. In addition to the role of glucose in glucagon secretion, it may also be involved in the regulation of α-cell survival, although few studies have been made regarding this. It has been documented that high levels of glucose (33.3 mm) combined with palmitate to simulate in vitro glucolipotoxicity conditions induce apoptosis in pancreatic α-cells⁽ Reference Ellingsgaard, Ehses and Hammar ^{43 )}.

Amino acids

Although amino acids are important modulators of α-cell secretion, little is known about this process at the molecular level and the mechanisms involved. Amino acids such as arginine, alanine and glutamine are potent stimulators of glucagon release, while leucine or lysine contribute to a lesser extent to α-cell secretion⁽ Reference Gerich, Lorenzi and Schneider ^{16 ,} Reference Dumonteil, Magnan and Ritz-Laser ^{44 –} Reference Pipeleers, Schuit and Van Schravendijk ^{46 )}. It is postulated that the function of this increase in glucagon release is to physiologically prevent hypoglycaemia after protein intake, since amino acids also stimulate insulin secretion. An increased glucagon secretion is observed if α-cells are incubated in the presence of a mixture of different amino acids but not when exposed to each one of the nutrients separately⁽ Reference Quoix, Cheng-Xue and Mattart ^{29 ,} Reference Cheng-Xue, Gomez-Ruiz and Antoine ^{39 ,} Reference Pipeleers, Schuit and Van Schravendijk ^{46 )}. In fact, the effect of glucose on intracellular Ca signalling and glucagon release is further detected in the presence of mediums containing a mixture of amino acids⁽ Reference Cheng-Xue, Gomez-Ruiz and Antoine ^{39 )}, indicating that these nutrients are critical for α-cell function. Ostenson & Grebing⁽ Reference Ostenson ^{47 )} showed that glutamine is a positive modulator of glucagon release. This effect involves the oxidation of this amino acid and is likely to be related to actions on glucose metabolism and ATP levels in the pancreatic α-cell⁽ Reference Ostenson ^{47 )}. Moreover, a recent study performed in human islets from healthy and type 2 diabetic individuals showed that glycine, acting via an α-cell-specific glycine receptor, was the predominant amino acid stimulating glucagon release via an intracellular Ca influx⁽ Reference Li, Liao and Zhuo ^{48 )}. Similarly, ingested glycine increases plasma glucagon in human subjects⁽ Reference Gannon, Nuttall and Nuttall ^{49 )}. Amino acids like arginine may be involved in the release of glucagon by direct plasma membrane depolarisation and Ca influx in the pancreatic α-cell. However, other amino acids act as negative modulators. For instance, isoleucine can inhibit α-cell secretion while leucine has a dual effect: it is a positive stimulus at physiological concentrations but becomes inhibitory at elevated levels⁽ Reference Leclercq-Meyer, Marchand and Woussen-Colle ^{50 )}. In In-R1-G9 glucagonoma cells, concentrations of alanine and glutamine in the micromolar range contribute to the triggering of intracellular Ca oscillations, whereas these signals are inhibited with millimolar levels of these amino acids⁽ Reference Bode, Weber and Fehmann ^{51 )}. This effect has been attributed to amino acid-induced hyperpolarisation of the plasma membrane by the modulation of the Na–K pump, and involves the intracellular metabolism of these nutrients. Therefore, divergent effects may take place depending on the type and concentration of amino acids.

Fatty acids

Even though lipotoxicity in pancreatic β-cells is one of the main factors related to obesity-induced diabetes, less is known about the role and potential toxic effect of fatty acids in the α-cell. It has been shown that the modulation of glucagon release by fatty acids depends on the chain length, spatial configuration and degree of saturation of the fatty acid as well as the incubation time, which may lead to different acute or chronic effects⁽ Reference Collins, Luan and Thompson ^{52 –} Reference Olofsson, Salehi and Gopel ^{54 )}. The majority of studies show that short-term exposure to fatty acids stimulates glucagon release in isolated islets and clonal α-cell lines⁽ Reference Olofsson, Salehi and Gopel ^{54 ,} Reference Bollheimer, Landauer and Troll ^{55 )}. Palmitate increases glucagon secretion by enhancing α-cell intracellular Ca entry and also, by relieving the inhibitory paracrine action of somatostatin, which is secreted from δ-cells⁽ Reference Olofsson, Salehi and Gopel ^{54 )}. Linoleic acid increases glucagon secretion in isolated mouse and rat islets as well as in In-R1-G9 glucagonoma cells⁽ Reference Flodgren, Olde and Meidute-Abaraviciene ^{56 ,} Reference Wang, Zhao and Gui ^{57 )}. This effect depends on the activation of the NEFA receptor G-protein coupled receptor 40 (GPR40) and the intracellular release of InsP3. Similar findings have been reported in rat islets with oleic acid⁽ Reference Fujiwara, Maekawa and Dezaki ^{58 )}. The long-term effects of fatty acids have been examined alone or in the presence of high concentrations of glucose. When αTC1-6 clonal α-cells were cultured with palmitate and oleate for up to 3 d, glucagon secretion was enhanced by means of fatty acid oxidation and TAG accumulation in a time- and dose-dependent manner but cell proliferation was decreased⁽ Reference Hong, Jeppesen and Nordentoft ^{59 )}. Accordingly, the long-term culture of this clonal α-cell line with palmitate augmented glucagon release and enhanced glucagon expression and protein content, probably by activating the mitogenic mitogen-activated protein kinase (MAPK) pathway⁽ Reference Piro, Maniscalchi and Monello ^{60 )}. In contrast, it was observed that the inhibitory action of insulin on glucagon release was impaired in a long-term incubation with fatty acids. This was attributed to palmitate-induced insulin resistance due to defects in the insulin receptor substrate-1 (IRS-1)/phosphatidylinositol kinase (PI3K)/serine-threonine protein kinase (Akt) pathway⁽ Reference Piro, Maniscalchi and Monello ^{60 )}. In mouse isolated islets, long-term exposure to oleate and palmitate results in an over-secretion of glucagon at both low and high glucose concentrations but diminished glucagon protein content⁽ Reference Collins, Luan and Thompson ^{52 )}. Similar results were observed in rat islets: the chronic effects of fatty acids produced a marked increase in glucagon release, but decreased glucagon content and did not change glucagon gene expression⁽ Reference Dumonteil, Magnan and Ritz-Laser ^{44 ,} Reference Gremlich, Bonny and Waeber ^{61 )}. It has been recently reported that the survival of α-cells, like of β-cells, is also sensitive to glucolipotoxic conditions: palmitate (0.5 mm) combined with high glucose levels are able to induce apoptosis in rodent α-cells⁽ Reference Ellingsgaard, Ehses and Hammar ^{43 )}. In general, all these data support the hypothesis that chronic elevation of fatty acids might contribute to α-cell deregulation in type 2 diabetes.

Other nutrients

α-Cell function is also sensitive to vitamins. For instance, plasma glucagon levels and glucagon release have been found to be augmented in rats with a diet deficiency of vitamin D₃ ⁽ Reference Bourlon, Faure-Dussert and Billaudel ^{62 )} while no major effects have been reported in non-insulin-dependent diabetic patients supplemented with vitamin D⁽ Reference Orwoll and Riddle ^{63 )}. Glucagon secretion is markedly impaired in rats subjected to vitamin A dietary deficiencies⁽ Reference Chertow, Driscoll and Blaner ^{64 )}. Additionally, retinol- and retinoic acid-binding proteins have been detected in glucagon-secreting α-cell lines while retinol and retinoic acid have been demonstrated to inhibit glucagon secretion in cultured rat islets⁽ Reference Chertow, Driscoll and Blaner ^{64 ,} Reference Chertow, Driscoll and Primerano ^{65 )}. Important increases in plasma glucagon levels are also related to biotin deficiencies due to fasting⁽ Reference Klandorf and Clarke ^{66 )}.

Other levels of regulation

In addition to nutrients, pancreatic α-cells are subjected to other control levels. These include neural regulation as well as autocrine, paracrine and endocrine signalling pathways that interact with the different steps involved in glucagon release or synthesis⁽ Reference Quesada, Tuduri and Ripoll ^{9 )}. Although numerous neurotransmitters released by nerve endings situated within the islet are associated with a stimulatory effect on glucagon secretion, the molecular and cellular mechanisms are still not well understood⁽ Reference Ahren ^{7 )}. Parasympathetic neurotransmitters such as vasoactive intestinal polypeptide, gastrin-releasing peptide, pituitary adenylate cyclase-activating polypeptide and acetylcholine have a stimulatory effect on pancreatic α-cells⁽ Reference Wilkes, Bailey and Thompson ^{67 ,} Reference Winzell ^{68 )}. Similarly, sympathetic nerves within the islet have a glucagonotropic effect via neurotransmitters such as noradrenaline, galanin and neuropeptide Y⁽ Reference Ahren ^{7 ,} Reference Miralles, Peiro and Degano ^{69 )}. Sympathetic activation can also induce adrenaline release, which is a potent stimulator of pancreatic α-cell exocytosis⁽ Reference De Marinis, Salehi and Ward ^{70 )}. Sensory nerves containing calcitonin gene-related protein and substance P may also trigger glucagon release from the pancreatic islet⁽ Reference Ahren ^{7 )}. It has been also shown that α-cells can secrete neural factors such as acetylcholine or glutamate⁽ Reference Cabrera, Jacques-Silva and Speier ^{71 ,} Reference Rodriguez-Diaz, Dando and Jacques-Silva ^{72 )}. Neural regulation of islet function has been related to the control of the pulsatile release of islet hormones and the synchronisation of individual cell responses within the islet⁽ Reference Quesada, Tuduri and Ripoll ^{9 )}. Additionally, neural regulation allows for a further control of the glucagon secretory response to hypoglycaemia as well as the suppression of glucagon secretion in conditions of hyperglycaemia⁽ Reference Quesada, Tuduri and Ripoll ^{9 ,} Reference Cryer ^{15 )}. This neural control is mediated by glucose-sensing neurons located in the ventromedial hypothalamus⁽ Reference Miki, Liss and Minami ^{73 )}.

Additionally, islet hormones such as insulin, glucagon and somatostatin or β-cell-secretory products such as ATP, γ-aminobutyric acid, amylin or Zn also act as paracrine and autocrine signals that affect α-cell physiology⁽ Reference Quesada, Tuduri and Ripoll ^{9 ,} Reference Leibiger, Moede and Muhandiramlage ^{74 ,} Reference Tuduri, Filiputti and Carneiro ^{75 )}. Extrapancreatic hormones such as leptin and gastrointestinal incretins can also modulate α-cell function⁽ Reference Tuduri, Marroqui and Soriano ^{22 ,} Reference Ma, Zhang and Gromada ^{76 )}. In this latter group of hormones, glucagon-like peptide-1 (GLP-1) is one of the most important bioactive peptides with numerous islet effects. GLP-1 is produced by intestinal ileum L-cells by post-translational processing of proglucagon and is secreted after a meal in response to nutrients⁽ Reference Tuduri, Marroqui and Soriano ^{22 ,} Reference Ma, Zhang and Gromada ^{76 )}. Given that GLP-1 has a potent glucose-induced insulinotropic action but reduces glucagon release, some pharmacological approaches in diabetes have led to the design of GLP-1 derivatives of improved duration and resistance to degradation compared with the natural hormone⁽ Reference Tuduri, Marroqui and Soriano ^{22 ,} Reference Ma, Zhang and Gromada ^{76 )}. In contrast to the suppressive effect on glucagon release observed in animal models and human subjects⁽ Reference Dunning and Foley ^{77 )}, initial experiments at the cellular level reported an enhanced protein kinase A (PKA)-dependent exocytosis in rat pancreatic α-cells, which was associated with increased glucagon secretion⁽ Reference Ding, Renstrom and Rorsman ^{78 )}. In light of these findings, it was proposed that the GLP-1 inhibitory effect on pancreatic α-cells may rely on paracrine mechanisms⁽ Reference Ding, Renstrom and Rorsman ^{78 ,} Reference de Heer, Rasmussen and Coy ^{79 )}. However, recent studies in mice have shown that GLP-1 can indeed reduce glucagon secretion in α-cells by PKA-dependent inhibition of a specific subset of Ca channels via a small increase in intracellular cyclic AMP (cAMP) concentrations⁽ Reference De Marinis, Salehi and Ward ^{70 )}. These last observations support a molecular basis for the studies showing an inhibitory GLP-1 action on glucagon release. Unlike GLP-1, a stimulatory effect has been reported for the gastrointestinal hormone glucose-dependent insulinotropic polypeptide (GIP), which is produced by intestinal K-cells. Studies with perifused mouse pancreatic islets showed that GIP was able to reverse the suppressive effect of high glucose levels on glucagon release⁽ Reference Opara, Burch and Taylor ^{80 )}. In primary rat α-cells and perfused rat pancreas, GIP stimulated glucagon release at low glucose concentrations⁽ Reference de Heer, Rasmussen and Coy ^{79 )}.

Glucagon synthesis

The preproglucagon gene is mainly expressed in pancreatic α-cells, in intestinal L-cells and in the central nervous system and is closely regulated by nutritional status. The preproglucagon promoter contains up to six regulatory sequences called G1–5 and CRE (cAMP response element), which confers responsiveness to cAMP. The promoter consists of a region comprising the regulatory sequences G1 and G4 while the remaining sequences are part of the enhancer regions. Numerous transcription factors are involved in the control of glucagon expression such as LIM-homeobox transcription factor islet-1 (Isl1), paired box gene 6 (Pax6), musculoaponeurotic fibrosarcoma oncogene homolog (Mafs), forkhead box protein A1 (Foxa1) and forkhead box protein A2 (Foxa2) among others⁽ Reference Gosmain and Cheyssac ^{81 ,} Reference Jin ^{82 )}. The transcription of the preproglucagon gene results in a peptide of 160 amino acids. The processing of this peptide into glucagon, GLP-1 and GLP-2 hormones depends on the post-translational cleavage mediated by different prohormone convertase (PC) subtypes. The differential cell-specific expression of PC generates different peptides in each tissue⁽ Reference Mojsov, Heinrich and Wilson ^{83 )}. PC2 is responsible in α-cells for the production of glucagon in addition to other products such as glicentin, glicentin-related pancreatic polypeptide, intervening peptide 1 and the major proglucagon fragment⁽ Reference Dey, Lipkind and Rouille ^{84 –} Reference Patzelt, Tager and Carroll ^{86 )}. The importance of PC2 for the correct processing of glucagon has been proven in experiments using PC2 knockout (KO) mice⁽ Reference Furuta, Zhou and Webb ^{87 )}. In contrast to the results in some clonal α-cell lines⁽ Reference Dumonteil, Ritz-Laser and Magnan ^{88 ,} Reference McGirr, Ejbick and Carter ^{89 )}, studies in isolated rat islets indicate that the effect of glucose on glucagon gene expression is not direct but occurs via paracrine mechanisms that stimulate the inhibitory insulin signal⁽ Reference Dumonteil, Magnan and Ritz-Laser ^{44 )}. This hormone decreases glucagon gene expression in pancreatic α-cells by activating phosphatidylinositol kinase (PI3K) and PKB pathways⁽ Reference Grzeskowiak, Amin and Oetjen ^{90 –} Reference Schinner, Barthel and Dellas ^{92 )}. In line with these findings, the enhanced glucagon expression found in rats with insulinopenic diabetes was corrected by administrating insulin⁽ Reference Dumonteil, Ritz-Laser and Magnan ^{88 )}. As mentioned earlier, it has been reported that lipids are able to modulate glucagon gene expression both in a paracrine way through their effects on β-cells⁽ Reference Piro, Maniscalchi and Monello ^{60 )} and by direct mechanisms⁽ Reference Bollheimer, Landauer and Troll ^{55 )}. In the latter case, while short-term experiments indicate a down-regulation of the glucagon gene after being treated with palmitate⁽ Reference Bollheimer, Landauer and Troll ^{55 )}, no effect has been observed in long-term studies⁽ Reference Dumonteil, Magnan and Ritz-Laser ^{44 ,} Reference Gremlich, Bonny and Waeber ^{61 )}. Amino acids may also play a role in glucagon gene regulation, although much further investigation is still necessary: while arginine has been shown in some studies to increase glucagon expression and protein synthesis via protein kinase C activation⁽ Reference Yamato, Noma and Tahara ^{93 )}, other researchers have failed to observe these changes⁽ Reference Dumonteil, Magnan and Ritz-Laser ^{44 )}. In clonal αTC1-6 cells, the removal of histidine from the culture leads to a decrease in preproglucagon gene expression similar to what is observed in an amino acid-free medium⁽ Reference Paul, Waegner and Gaskins ^{94 )}.

Glucagon receptor and intracellular signalling

The glucagon receptor is a protein belonging to the secretin–glucagon receptor II class family of the G protein-coupled receptors and consists of 477 amino acids in humans and 485 amino acids in rodents with a primary sequence homology of more than 80 % between them⁽ Reference Mayo, Miller and Bataille ^{95 )}. This receptor is characterised by seven transmembrane-spanning domains connected by three intracellular and extracellular loops. Its activation is coupled to GTP-binding heterotrimeric G proteins of the Gαs type that stimulate adenylate cyclase and PKA⁽ Reference Weinstein, Yu and Warner ^{96 )}. Although glucagon-induced PKA signalling is the most important biochemical cascade, this hormone can also activate the phospholipase C/inositol phosphate pathway via G_q proteins, resulting in the release of Ca²⁺ from intracellular stores⁽ Reference Mayo, Miller and Bataille ^{95 )}. In addition, glucagon has also been implicated in signalling via 5′-AMPK⁽ Reference Longuet, Sinclair and Maida ^{13 ,} Reference Kimball, Siegfried and Jefferson ^{97 )}, p38 MAPK and c-Jun N-terminal kinase (JNK)⁽ Reference Longuet, Sinclair and Maida ^{13 ,} Reference Chen, Ishac and Dent ^{98 )} (Fig. 2). Although the glucagon receptor is mainly expressed in the liver, its expression is also observed in multiple tissues including the pancreas, heart, kidney, brain, smooth muscle, adipocytes, lymphoblast, spleen, retina, adrenal gland and gastrointestinal tract⁽ Reference Svoboda, Tastenoy and Vertongen ^{99 )}. Although a preferential expression of the glucagon receptor is found in pancreatic β-cells, it is also expressed, to a lesser degree, in non-β-cells, including glucagon-secreting α-cells⁽ Reference Kedees, Grigoryan and Guz ^{100 ,} Reference Kieffer, Heller and Unson ^{101 )}. Mice lacking the glucagon receptor exhibit several phenotypic alterations such as pancreatic α-cell hyperplasia, hyperglucagonaemia, mild hypoglycaemia, increased number of islets per pancreas and increased GLP-1 plasma levels⁽ Reference Gelling, Du and Dichmann ^{102 )}. Additionally, these mice show resistance to high-fat diet-induced hyperinsulinaemia and streptozotocin-induced diabetes, while exhibit improved insulin sensitivity compared with wild-type controls⁽ Reference Conarello, Jiang and Mu ^{103 –} Reference Vuguin, Kedees and Cui ^{106 )}. However, a specific over-expression of the glucagon receptor in pancreatic β-cells also improves glucose tolerance in mice exposed to high-fat diets, suggesting an improvement in β-cell function⁽ Reference Gelling, Vuguin and Du ^{107 )}.

Fig. 2 Glucagon intracellular signalling and actions in different tissues. Intracellular signalling and biochemical pathways activated by glucagon are illustrated in the top of the figure. The different target tissues of glucagon as well as its effects are summarised in the lower part of the figure. For further information, see the text. PLC, phospholipase c; PIP2, phosphatidylinositol 4,5-bisphosphate; AC, adenylate cyclase; Pi, inorganic phosphate; IP3, inositol 1,4,5-trisphosphate; AMPK, AMP-activated protein kinase; JNK, c-Jun N-terminal kinase; cAMP, cyclic AMP; PKA, protein kinase A; MAPK, mitogen-activated protein kinase.

Effects of glucagon on hepatic nutrient metabolism

The primary target organ of glucagon is the liver where the insulin:glucagon ratio controls multiple key steps of hepatic metabolism (Fig. 2). The action of glucagon on the liver plays a key role in glucose homeostasis, particularly in adaptive and counter-regulatory responses to hypoglycaemic conditions, fasting and starvation⁽ Reference Longuet, Sinclair and Maida ^{13 )} as well as in situations of increased fuel demand such as vigorous exercise⁽ Reference Berglund, Lustig and Baheza ^{108 )} or states of metabolic stress such as trauma, inflammation or sepsis⁽ Reference Jones, Tan and Bloom ^{109 )}. Given that the brain relies on a continuous glucose supply, several lines of defence against hypoglycaemia take place to counterbalance this situation. These include glucose-sensitive neural responses, an increased release of hyperglycaemic hormones such as adrenaline and an enhanced glucagon secretion. Additionally, glucagon opposes numerous insulin actions. Glucagon has a dual effect on the liver: on the one hand, it stimulates gluconeogenesis and glycogenolysis increasing hepatic glucose output and, on the other hand, it decreases glycogenesis and glycolysis⁽ Reference Quesada, Tuduri and Ripoll ^{9 ,} Reference Yoon, Puigserver and Chen ^{110 )}. These effects along with decreased plasma insulin levels allow for the restoration of normoglycaemia by favouring hepatic glucose release. Although the glucagon receptor is highly selective for glucagon, it can also bind to other glucagon-related peptides⁽ Reference Hjorth, Adelhorst and Pedersen ^{111 )}. One of the main effects of glucagon to modulate gluconeogenesis is the up-regulation of key enzymes involved in this process such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase through the activation of the cAMP response element-binding protein and PPARγ-coactivator-1⁽ Reference Yoon, Puigserver and Chen ^{110 ,} Reference Herzig, Long and Jhala ^{112 )}. The modulatory effect of glucagon on pyruvate kinase accounts for the down-regulation of glycolysis⁽ Reference Quesada, Tuduri and Ripoll ^{9 )}. Glucagon also regulates glycogen synthesis and lysis by modulating the activity of glycogen synthase and glycogen phosphorylase via the phosphorylation of these enzymes⁽ Reference Band and Jones ^{113 )}.

Glucagon is not only implicated in glucose homeostasis but in hepatic lipid metabolism as well. Several reports indicate that TAG production is decreased in perfused livers treated with glucagon⁽ Reference De Oya, Prigge and Grande ^{114 ,} Reference Penhos, Wu and Daunas ^{115 )}. The potential hypolipidaemic actions of glucagon after its administration also include a reduction in plasma and hepatic TAG, decreased VLDL and cholesterol levels, as well as increased fatty acid oxidation⁽ Reference Longuet, Sinclair and Maida ^{13 ,} Reference Xiao, Pavlic and Szeto ^{14 ,} Reference Aubry, Marcel and Davignon ^{116 ,} Reference Eaton ^{117 )}. Moreover, reduced glucagon signalling has been related to the development of fatty liver⁽ Reference Charbonneau, Couturier and Gauthier ^{118 )}. Experiments using KO and wild-type mice for the glucagon receptor showed that administering glucagon not only reduces the synthesis and secretion of TAG and their plasma levels but also stimulates fatty acid oxidation⁽ Reference Longuet, Sinclair and Maida ^{13 )}. These effects depend on the activation of PPARα and are necessary for the adaptive metabolic response to fasting. In line with these results, these KO mice rapidly develop hepatosteatosis compared with wild-type controls when exposed to a high-fat diet⁽ Reference Longuet, Sinclair and Maida ^{13 )}. This finding in glucagon receptor KO mice contrasts with the resistance to hepatosteatosis found in this model by others⁽ Reference Conarello, Jiang and Mu ^{103 )}. In diet-induced hepatic steatosis, glucagon signalling may be impaired as a consequence of a reduced number of hepatic plasma membrane glucagon receptors⁽ Reference Charbonneau, Melancon and Lavoie ^{119 )}. Recently, it has been demonstrated in db/db mice that the inhibition of hepatic glucagon signalling leads to an increase in LDL due to an over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis⁽ Reference Han, Akiyama and Previs ^{120 )}. In addition to lipid metabolism, glucagon can also stimulate the hepatic uptake of amino acids. Accordingly, hyperglucagonaemia may be related to plasma hypoaminoacidaemia, particularly with those amino acids involved in gluconeogenesis, such as alanine, glycine and proline⁽ Reference Cynober ^{121 )}.

Effects of glucagon on food intake, body weight and body energy

Glucagon is able to exert several actions on food intake behaviour as well as on the regulation of weight and body energy (Fig. 2). However, the precise signalling mechanisms that allow this hormone to perform these actions are still unclear. It has been shown that glucagon receptors are present in the rat brain, including the hypothalamus⁽ Reference Hoosein and Gurd ^{122 )}, and that glucagon is able to bind to mouse astrocyte suspensions and induce cAMP production⁽ Reference Cockram, Kum and Ho ^{123 )}. Additionally, this hormone is able to suppress the electrical activity of glucose-sensitive hypothalamic neurons⁽ Reference Inokuchi, Oomura and Nishimura ^{124 )}. Intraventricular infusion of glucagon leads to hyperglycaemia⁽ Reference Amir ^{125 )} but it also decreases food intake, an effect that is less potent when glucagon is administered peripherally⁽ Reference Inokuchi, Oomura and Nishimura ^{124 )}. In addition to a potential direct action on central regions, it has been reported that glucagon may be sensed by peripheral vagal nerves that communicate the signal to satiety control areas of the hypothalamus to exert an anorexigenic action. This is supported by experiments in which a glucagon-induced decrease in food intake was suppressed when animals were subjected to hepatic vagotomy⁽ Reference Geary and Smith ^{126 )}. This reduced food intake when glucagon is infused has been observed not only in rodents but in human subjects as well⁽ Reference Geary and Smith ^{127 –} Reference Penick and Hinkle ^{129 )}. The anorectic action of glucagon has been related to its ability to reduce meal size⁽ Reference Woods, Lutz and Geary ^{130 )}, an effect that can be abolished with the infusion of neutralising glucagon antibodies⁽ Reference Langhans, Zeiger and Scharrer ^{131 )}. Severe anorexia is also found in rats when transplanted with glucagonomas, further supporting the satiating role of this hormone⁽ Reference Jensen, Blume and Mikkelsen ^{132 )}. In contrast with these findings, glucagon receptor KO mice fed with high-fat diets exhibit lower food intake and body-weight increase than wild-type controls⁽ Reference Conarello, Jiang and Mu ^{103 )}. In addition to the central actions mentioned above, it has been suggested that the satiety effect of glucagon may involve the suppression of the orexigenic hormone ghrelin via the hypothalamic–pituitary axis⁽ Reference Arafat, Perschel and Otto ^{133 )}. It has recently been reported that hypothalamic glucagon signalling inhibits hepatic glucose production and that the central resistance to this hormone might be involved in the hyperglycaemia that characterises diabetes⁽ Reference Mighiu, Yue and Filippi ^{134 )}.

Considerable evidence supports the role of glucagon as a thermogenic agent, thereby favouring the body's energy expenditure. In rats, an energy-increasing effect of glucagon was reported some time ago⁽ Reference Davidson, Salter and Best ^{135 )}. In healthy volunteers, RMR increases after glucagon infusion in conditions of insulin deficiency⁽ Reference Nair ^{136 )}. The presence of insulin seems to counterbalance this glucagon action⁽ Reference Calles-Escandon ^{137 )}. Recently, indirect calorimetry measurements in obese, non-diabetic individuals showed that glucagon infusion alone or in combination with GLP-1 is able to increase resting energy expenditure⁽ Reference Tan, Field and McCullough ^{138 )}. These thermogenic effects have been associated with direct actions on brown adipose tissue due to increased oxygen consumption, metabolism and heat production in both in vitro and in vivo experiments⁽ Reference Billington, Briggs and Link ^{139 –} Reference Yahata and Kuroshima ^{141 )}. Additionally, chronic glucagon administration in rats increases the mass of brown adipose tissue as well as its thermogenic capacity and fatty acid uptake⁽ Reference Billington, Bartness and Briggs ^{142 )}. Several reports suggest that these effects are mediated by the sympathetic nervous system via catecholamines⁽ Reference Filali-Zegzouti, Abdelmelek and Rouanet ^{143 )}. This is supported by experiments in which the glucagon effect is prevented or reduced in conditions of brown adipose tissue denervation or pharmacological β-adrenergic inhibition with propranolol⁽ Reference Billington, Bartness and Briggs ^{142 ,} Reference Dicker, Zhao and Cannon ^{144 )}. It has also been shown that glucagon reduces body weight in both humans and normal rats⁽ Reference Billington, Briggs and Link ^{139 ,} Reference Schulman, Carleton and Whitney ^{145 )} as well as in obese Zucker rats⁽ Reference Chan, Mackey and Snover ^{146 )}, a model of genetic obesity. These effects may probably be due to the above-mentioned glucagon action on food intake and energy expenditure. Recent results in mice confirm that glucagon not only induces a loss of body weight and fat mass but also lowers plasma cholesterol levels⁽ Reference Habegger, Stemmer and Cheng ^{147 )}. All these processes are attributed to the enhanced secretion of fibroblast growth factor 21 (FGF21) in plasma, whose levels are increased when glucagon is infused in mice and human subjects. Thus, glucagon may have potential therapeutic implications not only for diabetes but also for obesity.

Other effects of glucagon

Although the effect of glucagon on the liver is the best known, this hormone also affects other extra-hepatic organs and tissues (Fig. 2). Glucagon-receptor KO mice exhibit α-cell hyperplasia⁽ Reference Gelling, Du and Dichmann ^{102 )}, which is consistent with in vitro experiments showing that glucagon down-regulates α-cell proliferation in an autocrine manner⁽ Reference Liu, Kim and Chen ^{148 )}. The secretion of this hormone also increases in mouse and rat pancreatic α-cells via cAMP production⁽ Reference Ma, Zhang and Gromada ^{76 )}. The over-expression of the glucagon receptor in pancreatic β-cells improves glucagon- and glucose-stimulated insulin secretion, and enhances β-cell mass as well as glucose tolerance⁽ Reference Gelling, Vuguin and Du ^{107 )}. The direct effect of glucagon on β-cells is further supported by experiments showing that this hormone increases insulin release in perfused pancreas and isolated β-cells⁽ Reference Kieffer, Heller and Unson ^{101 ,} Reference Moens, Heimberg and Flamez ^{149 )}. Additionally, glucagon produces cardiac ionotropic and chronotropic actions, partly by stimulating Ca currents via cAMP production and the inhibition of phosphodiestarases⁽ Reference Mery, Brechler and Pavoine ^{150 )}, thus leading to increased contractility⁽ Reference Gonzalez-Munoz, Nieto-Ceron and Cabezas-Herrera ^{151 )}. It has also been indicated that this hormone regulates cardiac metabolism at the level of glucose utilisation⁽ Reference Harney and Rodgers ^{152 )}. The role of glucagon in adipose tissue has been controversial for a long time. Glucagon has been reported to augment lipolysis in isolated rodent and human adipocytes⁽ Reference Heckemeyer, Barker and Duckworth ^{153 ,} Reference Perea, Clemente and Martinell ^{154 )}. The physiological meaning of these results has been disputed based on findings that show no effect of glucagon on the abdominal fat tissue of healthy individuals submitted to microdialysis⁽ Reference Gravholt, Moller and Jensen ^{155 )}. However, new studies have reported glucagon-induced lipolysis in healthy volunteers, diabetic patients, diabetic animal models and isolated adipocytes from these animals⁽ Reference Arafat, Kaczmarek and Skrzypski ^{11 )}. All these effects were attributed to a glucagon-induced release of FGF21, which has lipolytic activity. Actually, recent reports demonstrate that FGF21 also mediates other different glucagon actions, as we previously commented⁽ Reference Habegger, Stemmer and Cheng ^{147 )}. Given that the lipolytic action of glucagon is blunted in denervated rats, its effect on adipose tissue has also been associated with sympathetic signals⁽ Reference Lefebvre, Luyckx and Bacq ^{156 )}. Kidneys are also a target site for glucagon, since its intravenous infusion is related to changes produced in urea synthesis and excretion as well as in water conservation⁽ Reference Ahloulay, Bouby and Machet ^{157 )}. In in vitro perfused inner medullary collecting ducts, glucagon induces cAMP production and changes in aquaporin 2 expression that affect water excretion⁽ Reference Yano, Cesar and Araujo ^{158 )}. Glucagon is also used as an anti-peristaltic agent because of its effects on small intestine motility⁽ Reference Gutzeit, Binkert and Koh ^{159 ,} Reference Mochiki, Suzuki and Takenoshita ^{160 )}. Moreover, it has recently been reported that glucagon also enhances sweet taste responses by acting directly on the mouse gustatory epithelium⁽ Reference Amanda, Manuela and Antonia ^{161 )}.

Role of glucagon in the control of glycaemia during diabetes mellitus

Type 1 diabetes is characterised by an autoimmune specific β-cell loss that results in insulin deficiency, leading to hyperglycaemia. These high plasma glucose levels are filtered by kidneys, which may result in glycosuria and, eventually, osmolar alterations that can produce a non-ketotic hyperosmolar coma. Since glucose utilisation as an energy substrate is limited as a consequence of insulin deficiency, increased levels of fatty acids are oxidised to acetyl CoA, increasing the production of ketonic bodies and ketonuria with the concomitant risk of ketoacidosis. Other symptoms that develop in poorly controlled diabetes are polyuria, polydipsia and polyphagia. Since the basic treatment for diabetes is insulin administration, another major complication may be hypoglycaemia (iatrogenic hypoglycaemia), which results from an excess of insulin in a condition of impaired homeostatic responses against the decline in plasma glucose levels⁽ Reference Cryer ^{15 )}. In the case of type 2 diabetes, hyperglycaemia is due to a combination of increased peripheral resistance to the action of insulin along with inappropriate insulin secretion. This situation can eventually progress to β-cell death⁽ Reference Quesada, Tuduri and Ripoll ^{9 )}, thus leading to a greater deterioration of glucose homeostasis and a worsening of hyperglycaemia. This type of diabetes is often associated with obesity. In both types of diabetes, poorly controlled glycaemia can lead to micro- and macrovascular, neural, retinal and renal complications as well as skin ulcers and, eventually, amputation.

In addition to the central role of insulin and pancreatic β-cells in the pathophysiology of diabetes, abundant evidence demonstrates that glucagon is also involved in this metabolic disorder. Absolute or relative (to insulin) hyperglucagonaemia is frequently found in diabetes during fasting and postprandial periods⁽ Reference Larsson ^{162 –} Reference Sherwin, Wahren and Felig ^{164 )}. Hyperglucagonaemia in the context of insufficient insulin secretion and/or insulin resistance is associated with increased hepatic glucose output, which contributes to hyperglycaemia⁽ Reference Li, Liao and Zhuo ^{48 ,} Reference Gastaldelli, Baldi and Pettiti ^{165 )}. Additionally, α-cell function in response to changes in glucose is impaired in diabetes⁽ Reference Siafarikas, Johnston and Bulsara ^{166 )}. For instance, hyperglycaemia fails to suppress glucagon secretion in diabetic patients, which further exacerbates their high glucose levels during postprandial periods⁽ Reference Dinneen, Alzaid and Turk ^{167 ,} Reference Shah, Vella and Basu ^{168 )}. Although the mechanism for this failure is not clearly known, it has been suggested that the α-cell might be refractory to the insulin paracrine signal or insensitive to high glucose levels⁽ Reference Quesada, Tuduri and Ripoll ^{9 ,} Reference Cryer ^{15 )}. Patients with a glucagonoma develop hyperglucagonaemia and the characteristic hyperglycaemia of diabetes, which disappears when the tumour is removed, further suggesting that high levels of glucagon are involved in this disease⁽ Reference Eldor, Glaser and Fraenkel ^{169 )}. Another problem of diabetes is that pancreatic α-cells do not respond adequately to hypoglycaemia, which is particularly important in insulin-treated patients, and leads to increased morbidity and mortality rates in this disease⁽ Reference Taborsky and Mundinger ^{8 ,} Reference Taborsky, Ahren and Havel ^{23 )}. This life-threatening situation seems to be the result of several altered processes: impaired α-cell sensing of falling glucose levels, an inefficient effect of the withdrawal of the insulin inhibitory signal during hypoglycaemia as well as a defective function of the autonomous nervous system and of the different defence mechanisms against hypoglycaemia⁽ Reference Taborsky and Mundinger ^{8 ,} Reference Bolli, De Feo and Perriello ^{170 –} Reference Zhou, Zhang and Oseid ^{172 )}. In addition to an altered α-cell function, diabetes has been also associated with increases in either absolute or relative (to β-cells) α-cell mass in animal models and humans⁽ Reference Deng, Vatamaniuk and Huang ^{173 –} Reference Yoon, Ko and Cho ^{175 )}, which may explain the higher plasma glucagon levels in these patients.

Glucagon is also involved in protein and fat metabolic alterations in diabetes. This hormone plays a catabolic role during a protein load⁽ Reference Charlton, Adey and Nair ^{176 )} and hyperglucagonaemia induces phenylalanine oxidation in healthy humans⁽ Reference Tessari, Inchiostro and Barazzoni ^{177 )}. Individuals with a glucagonoma exhibit higher levels of free amino acids in muscles and the liver⁽ Reference Roth, Muhlbacher and Karner ^{178 )}. In type 1 diabetic patients, hyperglucagonaemia has been related to different catabolic effects including an increased RMR and increased leucine oxidation, effects that were independent of insulin deficiency⁽ Reference Charlton and Nair ^{179 )}. Because of the effect of this hormone on amino acid hepatic uptake and the stimulation of gluconeogenesis, hyperglucagonaemia can lead to marked hypoaminoacidaemia, particularly from those amino acids that participate in gluconeogenesis⁽ Reference Cynober ^{121 ,} Reference Boden, Rezvani and Owen ^{180 )}. Glucagon has also been involved in lipid metabolism. Although the role of glucagon in adipose tissue lipolysis has remained controversial for a long time, new evidence in animal models and human subjects, including diabetic patients, demonstrates this effect⁽ Reference Arafat, Kaczmarek and Skrzypski ^{11 )}. These actions may be the result of the activation of hormone-sensitive lipase⁽ Reference Slavin, Ong and Kern ^{181 )} and probably involve the participation of FGF21⁽ Reference Arafat, Kaczmarek and Skrzypski ^{11 )}. In animal models, chronic administration of glucagon induces hypolipidaemic effects⁽ Reference Guettet, Rostaqui and Mathe ^{182 ,} Reference Guettet, Rostaqui and Navarro ^{183 )}. Furthermore, it has been recently shown that hyperglucagonaemia modulates hepatic lipoprotein particle metabolism in humans both by decreasing hepatic lipoprotein particle production and by inhibiting particle clearance⁽ Reference Xiao, Pavlic and Szeto ^{14 )}. In the context of insulin deficiency and high levels of fatty acids, glucagon can accelerate the formation of ketonic bodies from the liver⁽ Reference Beylot, Picard and Chambrier ^{184 –} Reference Okuda, Kawai and Yamashita ^{186 )}. Consistent with this, it has been observed that diabetic ketoacidosis is associated with hyperglucagonaemia combined with hypoinsulinaemia⁽ Reference Taborsky ^{187 ,} Reference Wahid, Naveed and Hussain ^{188 )}. Thus, in view of the role that glucagon plays in the pathophysiology of diabetes, it would be advisable to develop therapeutic strategies to limit either glucagon secretion or action.

Therapeutic potential of modulating glucagon secretion and action in diabetes

Numerous studies have shown that restrained glucagon action is beneficial for the control of hyperglycaemia and/or diabetes management. For instance, glucagon-receptor KO mice exhibit reduced fasting hypoglycaemia and improved glucose tolerance⁽ Reference Gelling, Du and Dichmann ^{102 )}. Additionally, these mice are resistant to diet-induced obesity, as well as streptozotocin-induced β-cell loss and hyperglycaemia⁽ Reference Conarello, Jiang and Mu ^{103 )}. Similarly, lower hepatic glucose production and improved glucose tolerance are observed in mice that are deficient in the α-cell transcription factor ARX (aristaless-related homeobox), leading to the loss of glucagon-producing α-cells⁽ Reference Hancock, Du and Liu ^{189 )}. More recently, it has been shown that the metabolic and clinical alterations caused by type 1 diabetes are absent in glucagon receptor KO mice treated with streptozotocin and that the restoration of the hepatic glucagon receptor in this model leads to the reappearance of hyperglycaemia⁽ Reference Lee, Wang and Du ^{104 ,} Reference Lee, Berglund and Wang ^{190 )}. However, a limited glucagon action could interfere with the role of glucagon in hepatic lipid metabolism and lead to an increased susceptibility to hepatosteatosis after a high-fat diet⁽ Reference Longuet, Sinclair and Maida ^{13 )}, or it could also interfere with the hepatic survival function of glucagon⁽ Reference Sinclair, Yusta and Streutker ^{191 )}. Additionally, in this kind of KO mice the exocrine and endocrine cell masses are augmented, increasing the risk of developing tumours⁽ Reference Gelling, Du and Dichmann ^{102 )}. Thus, all these aspects require further examination in human subjects. Consistent with these findings in KO mice, other strategies such as glucagon antibodies, glucagon receptor antisense oligonucleotides and glucagon receptor antagonists revealed similar findings in hepatic glucose production and showed an antihyperglycaemic effect in diabetic animal models as well as in a few human studies⁽ Reference Quesada, Tuduri and Ripoll ^{9 ,} Reference Parker, McPherson and Andrews ^{192 –} Reference Winzell, Brand and Wierup ^{195 )}. The antagonism of glucagon action on the liver also partly mediates the glucose-lowering effect of biguanides⁽ Reference Miller, Chu and Xie ^{196 )}. It has been recently demonstrated that the design of peptides with dual agonism for both the glucagon and the GLP-1 receptors is useful to fight against obesity in mice without any apparent adverse effects⁽ Reference Day, Ottaway and Patterson ^{197 ,} Reference Pocai, Carrington and Adams ^{198 )}. This is mainly the result of the antihyperglycaemic, lipolytic, satiating and energy expenditure actions of this combined treatment. In human subjects, the co-administration of GLP-1 during glucagon infusion leads to increased resting energy expenditure and lower hyperglycaemia⁽ Reference Tan, Field and McCullough ^{138 )}. In the present study, the augmented energy expenditure was attributed to glucagon alone. Similarly, chronic glucagon receptor agonism in diet-induced obese mice lowered food intake, body weight, fat mass and cholesterolaemia while increasing energy expenditure⁽ Reference Habegger, Stemmer and Cheng ^{147 )}. The actions of glucagon were mediated by FGF21. This growth factor has also been involved in the lipolytic action of glucagon in diabetic and non-diabetic animals and humans⁽ Reference Arafat, Kaczmarek and Skrzypski ^{11 )}. Additionally, peripherally administered glucagon inhibits food intake, presumably by the neural activation of appetite-regulating brain centres⁽ Reference Parker, McCullough and Field ^{199 )}. Thus, the combined activation of glucagon and GLP-1 receptors has been proved to be useful for the design of strategies against obesity and diabetes.

The modulation of glucagon secretion with a therapeutic potential has also been explored. Exogenous GLP-1 administration is known to stimulate insulin release while inhibiting glucagon secretion in isolated rodent islets, in perfused rat pancreas and in human subjects, which allows for the improvement of hyperglycaemia⁽ Reference Quesada, Tuduri and Ripoll ^{9 ,} Reference Nauck, Heimesaat and Behle ^{200 )}. The effect on glucagon is mediated by the direct action on pancreatic α-cells⁽ Reference De Marinis, Salehi and Ward ^{70 )}. Accordingly, GLP-1 agonists widely used in diabetes treatment such as exenatide or liraglutide also exert hypoglycaemic effects by lowering plasma glucagon levels⁽ Reference Dupre, Behme and McDonald ^{201 )}. Similar actions have been reported for inhibitors of the GLP-1-degrading enzyme dipeptidyl peptidase 4 (DPP-4)⁽ Reference Edgerton, Johnson and Cherrington ^{202 )}. DPP-4 inhibitors commonly used in diabetes such as vildagliptin or sitagliptin suppress hepatic glucose production by augmenting insulin release and lowering glucagon secretion⁽ Reference Balas, Baig and Watson ^{203 )}. Other drugs such as pramlintide, a synthetic analogue of islet amyloid polypeptide, also decreases glucagon secretion after a meal in diabetic individuals and may act on reducing hepatic glucose output to improve hyperglycaemia⁽ Reference Edgerton, Johnson and Cherrington ^{202 ,} Reference Lebovitz ^{204 )}.