diabetes research and clinical practice 106 (2014) 1–10
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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Invited review
The forgotten members of the glucagon family Dominique Bataille a,*, Ste´phane Dalle b a
INSERM, 269, Rue Adrien Proby, 34090 Montpellier, France INSERM, Research—Pathophysiology of the Pancreatic b Cell, Institute of Functional Genomic, INSERM U 661, CNRS UMR 5203, Universities Montpellier 1 & 2, Montpellier, France b
article info
abstract
Article history:
From proglucagon, at least six final biologically active peptides are produced by tissue-
Received 13 June 2014
specific post-translational processing. While glucagon and GLP-1 are the subject of perma-
Accepted 26 June 2014
nent studies, the four others are usually left in the shadow, in spite of their large biological
Available online 3 July 2014
interest. The present review is devoted to oxyntomodulin and miniglucagon, not forgetting glicentin, although much less is known about it. Oxyntomodulin (OXM) and glicentin are
Keywords:
regulators of gastric acid and hydromineral intestinal secretions. OXM is also deeply
Proglucagon
involved in the control of food intake and energy expenditure, properties that make this
Glucagon
peptide a credible treatment of obesity if the question of administration is solved, as for any
Oxyntomodulin
peptide. Miniglucagon, the C-terminal undecapeptide of glucagon which results from a
Glicentin
secondary processing of original nature, displays properties antagonistic to that of the
Miniglucagon
mother-hormone glucagon: (a) it inhibits glucose-, glucagon- and GLP-1-stimulated insulin
Post-translational processing
release at sub-picomolar concentrations, (b) it reduces the in vivo insulin response to glucose with no change in glycemia, (c) it displays insulin-like properties at the cellular level using only a part of the pathway used by insulin, making it a good basis for developing a pharmacological workaround of insulin resistance. # 2014 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon-containing C-terminally elongated peptides . . . . . . . . . . . 2.1. Structures of oxyntomodulin and glicentin . . . . . . . . . . . . . . 2.2. Compared activities of OXM and glucagon . . . . . . . . . . . . . . . 2.2.1. Gastric acid secretion . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Other sites of action in the GI tract . . . . . . . . . . . . . . 2.2.3. Sites of action in the CNS. . . . . . . . . . . . . . . . . . . . . . 2.3. Questions about the mode(s) of action of OXM and glicentin
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* Corresponding author. Tel.: +33 467541697; fax: +33 467541697. E-mail addresses: [email protected] (D. Bataille), [email protected] (S. Dalle). http://dx.doi.org/10.1016/j.diabres.2014.06.010 0168-8227/# 2014 Elsevier Ireland Ltd. All rights reserved.
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diabetes research and clinical practice 106 (2014) 1–10
3.
4.
1.
Glucagon–insulin relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Glucagon–insulin relationship in the islets of Langerhans . 3.1.1. The observations . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. The molecular mechanisms . . . . . . . . . . . . . . . . . . 3.2. Glucagon and insulin secretion. . . . . . . . . . . . . . . . . . . . . . 3.3. Miniglucagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Miniglucagon in the islet of Langerhans . . . . . . . . 3.3.2. Miniglucagon in the whole organism . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction
Proglucagon (Fig. 1) is, besides the proopiomelanocortin (POMC), the archetype of a prohormone the post-translational processing of which provides the organism with a series of peptides with different, yet complementary biological activities. All activities of the proglucagon-derived peptides are related to the control of nutrition and metabolism: Glucagon, by its hyperglycaemic effect and GLP-1 via its incretin action, control the carbohydrate metabolism. GLP-2 maintains a healthy morphology of the gut epithelium and its functions, while the glucagon-extended molecules oxyntomodulin and glicentin regulate gastric acid secretion, hydromineral transport across the intestine, gut motility and food intake. Miniglucagon, which derives from a secondary processing of glucagon, plays a strange score in this orchestra, blocking possible unwanted effects of its mother-hormone on insulin secretion, while having insulin-like properties. Since many excellent reviews exist on glucagon itself (see, in particular, the three famous books edited by Lefe`bvre [1–3])
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4 4 4 5 5 6 6 8 8 9
and on glucagon-like peptide-1 ([4] for a recent review), the present review will focus on the three peptides that contain at least a part of the glucagon sequence, namely glicentin, oxyntomodulin and miniglucagon that have been partially ‘forgotten’ by the researchers in the last years, in spite of their importance in the metabolic regulation. The glucagon-containing peptides derive from tissue specific post-translational processing (Fig. 2). Expression of the prohormone convertase PC-1/3 in intestinal L cells leads to the production of the C-terminally extended peptides glicentin and oxyntomodulin. Expression of the prohormone convertase PC-2 in the a-cells of the islets of Langerhans leads to the production of glucagon. A secondary processing due to the presence of NRD convertase and aminopeptidaseB allows the production of a small, yet biologically significant, proportion of the C-terminal undecapeptide miniglucagon. Every peptide of the family is a true hormone, reaching its remote target(s) through the blood flow, with the exception of miniglucagon, a local regulator acting at the close vicinity of its site of production and quickly degraded.
Fig. 1 – The glucagon-containing peptides and its derivatives. Besides the four peptides with proved natural existence, are added the two oxyntomodulin fragments with demonstrated biological activities.
diabetes research and clinical practice 106 (2014) 1–10
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Fig. 2 – Tissue-specific post-translational processing of proglucagon into the glucagon-containing peptides. GRPP: glicentinrelated pancreatic polypeptide; SP-1 and SP-2: spacer peptides 1- & -2; GLP-1 et GLP-2: glucagon-like peptide-1 & -2; PC1/3: prohormone convertase-1/3; PC2: prohormone convertase-2; CPE: carboxypeptidase-E; NDRc: N-arginine dibasic convertase; APB: aminopeptidase-B; KR: lysine–arginine cleavage site: RR: arginine–arginine cleavage site; RK: arginine– lysine cleavage site; R: arginine cleavage site.
2. Glucagon-containing C-terminally elongated peptides 2.1.
Structures of oxyntomodulin and glicentin
Oxyntomodulin (OXM) differs from glucagon (see Fig. 1) by an additional C-terminal octapeptide [5,6], while glicentin is Nterminally elongated, as compared to OXM, by the 32-amino acid (32-AA) glicentin-related polypeptide (GRPP). Although much less is known about glicentin as compared to OXM, both peptides share most, if not all, of their biological activities. The two peptides are produced from proglucagon in cells, such as the intestinal L cells or some specialized neurones in the central nervous system (C.N.S.), which express the prohormone convertase PC1/3 rather than PC2, the expression of which in pancreatic a-cells leads to the glucagon production. During the same process, GLP-1 and GLP-2 are produced and co-secreted with OXM and glicentin from intestinal L cells.
2.2.
Compared activities of OXM and glucagon
Since the single difference between OXM and glucagon is the C-terminal octapeptide, it was of considerable interest to compare the biological activities of the two peptides.
2.2.1.
Gastric acid secretion
The inhibitory effect of OXM on pentagastrin-stimulated gastric acid secretion in the rat was contemporary with determination of its structure [7]. OXM displayed in that model a potency ca 15-fold higher than that of glucagon, showing the
importance of the octapeptide in the biological specificity of OXM. It was shown later that OXM also inhibited histaminestimulated gastric acid secretion [8,9]. Interestingly, the Cterminal (19–37) fragment displayed a roughly two-fold lower potency as compared to the entire molecule. When corrected for comparative clearance rate, both the 37-AA OXM and its Cterminal 19-AA fragment have the same potency, indicating that, on that parameter, the N-terminal fragment of glucagon does not enter into the OXM mode of action on gastric acid secretion. Although much less potent, mostly due to its very short half-life, the C-terminal octapeptide on its own, contains the inhibitor message in both the rat [10] and in the human species [11]. The minimal fully active structure was the Nterminally acetylated hexapeptide (octapeptide lacking the last two aminoacids) [12].
2.2.2.
Other sites of action in the GI tract
Besides its gastric target, OXM is able to modulate the hydromineral transport through the rat small intestine [13]. Its mode of action seems to be the same as, or at least similar to that present in the stomach, since the C-terminal [19–37] fragment mimics the effects of the whole molecule, at least in the jejunum. The same holds true in gastric smooth muscles [14]. The receptor(s) implicated in that model was shown to be linked, via a Gi or Go protein, to phospholipase C (production of inositol triphosphate -IP3-) and inhibition of adenylate cyclase (decreased cyclic AMP production). OXM and glicentin were also shown to inhibit the motility pattern of the small intestine [15], while it was shown that glicentin (OXM was not tested in that study) is able to induce contraction of smooth muscle cells from the human colon and that this effect is abolished by
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the GLP-1 receptor antagonist exendin (9–39) [16]. See the chapter on the mode of action of OXM and glicentin for more details.
2.2.3.
Sites of action in the CNS
The major effect of OXM observed on the central nervous system (CNS) is a negative control of food intake (suppression of hunger -anorexigenic action-). Indeed, OXM inhibits food intake in the rat, either applied centrally [17] or via a peripheral administration [18]. Similar findings were obtained in humans [19], with a reduction of body weight in overweight and obese patients using the subcutaneous route [20]. In addition to its action on energy intake, OXM increases energy expenditure [21]. The effect of peripherally administered OXM on food intake is due to the fact that, like other gastro-intestinal hormones, the peptide is able to cross the blood–brain barrier at the level of the median eminence and reaches the arcuate nucleus of hypothalamus which contains both stimulatory and inhibitory neurons. OXM also inhibits the orexigenic signal carried by ghrelin [23]. Altogether, these features make OXM a good basis for the development of an anti-obesity drug [22]. For recent reviews on appetite regulation see [23,24].
2.3. Questions about the mode(s) of action of OXM and glicentin It is clear that the receptor(s), the precise nature of which is still unknown, which transduce(s) the OXM actions on the GI tract displays a strong preference towards the C-terminal end (including the octapeptide itself) of the molecule, with little or no importance of the N-terminal half common with glucagon and which resembles that of GLP-1. This part of the molecules (glucagon or GLP-1) is crucial for triggering the cyclic AMP pathway associated with classical Gs protein-linked glucagon and GLP-1 receptors. In sharp contrast, the recognition system responsible for its CNS actions seems to be, at least in part, transduced by the GLP-1 receptor. Indeed, OXM has no longer effect on food intake in mice lacking the GLP-1 receptor [25] and the (not entirely specific) GLP-1 antagonist exendin (9–39) blocks the actions of both OXM and GLP-1 [25].
However, the figure is much more complex, since (a) OXM and GLP-1 are equipotent in their central effects despite a large difference between their respective affinities for the GLP-1 receptor; (b) OXM and GLP-1 behave differently in vivo and seem to act at different places of the CNS, namely primarily arcuate nucleus and brain stem, respectively [25,18]. Thus, whether these confusing observations are related to the existence of a distinct, still unrecognized, receptor or that of sub-classes of GLP-1 receptors with different specificities remains an open question. More recently, however, it appears that the glucagon receptor is also involved in the body-lowering effect of OXM [26]. A likely conclusion is thus that OXM acts on the CNS via two pathways using both GLP-1 and glucagon receptors.
3.
Glucagon–insulin relationship
Although the focus of the present review is not glucagon itself, it is still necessary to briefly recall basic informations on the impact of the relationship of this peptide with insulin, inasmuch as these data are necessary to understand the biological role of miniglucagon. Glucagon represents, in the regulation of metabolism and particularly that of the carbohydrates, the opposite counterpart of insulin. Both peptides are produced in the islets of Langerhans and display complex local relationships. While insulin is secreted after a meal and allows to store fuels for future usage, glucagon is secreted during the interprandial state or when a particular need for energy-producing molecules (mostly glucose and lipids) occurs (e.g. physical exercise). By its effects on the liver and adipose tissue, it liberates the stored fuels and makes them available to the whole body. This catabolic/anabolic hormonal balance is of major importance for the metabolic regulation. If the details on this balance in the whole organism has been the subject of a huge amount of studies since the discovery of glucagon and, accordingly, not much is probably still to be discovered, the relationship between the two antagonistic peptides was still unclear inside the islet of Langerhans, the place of synthesis and secretion of both peptides.
3.1. Glucagon–insulin relationship in the islets of Langerhans 3.1.1.
Fig. 3 – Architecture of the islets of Langerhans. After Orci et al. [27,28].
The observations
In contrast to the clearly opposite nature of glucagon towards insulin recalled above, the relationship between the two hormones inside their production site, the islets of Langerhans, are much more complex. This is possible thanks to the particular architecture of the islets in which many direct contacts between the a- and the b-cells exist. Indeed, 40–50% of the b-cells have direct contacts with a-cells, according to the precise schematic drawings made by Lelio Orci and colleagues (Fig. 3). In the same line, the human islets have particular structure ‘‘allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between a- and bcells . . .’’ [29]. See also [30] for a pathological approach of paracrinology inside the islets.
diabetes research and clinical practice 106 (2014) 1–10
The close vicinity between the two types of cells allows mutual interactions. If the direct inhibition of glucagon secretion by insulin is well documented [31], many data indicate that glucagon displays important effects on the b-cell. This is possible thanks to the presence of authentic glucagon receptors at the surface of the insulin-secreting cells [32–34]. These receptors are coupled to adenylate cyclase and allow glucagon to control beta cells functions as follows: (a) Glucagon is required for early differentiation of insulinsecreting cells during the mouse development [35]: Addition of a proglucagon antisense to pancreas from 11-week mouse embryo not only blocks differentiation of a-cells but also that of b-cells and that this differentiation may be rescued by addition of glucagon. Also, ablation of the glucagon receptor gene produces alteration in islet development and differentiation [36]. This fits well with the fact that a-cell is the first islet cell to appear during development [37] and thus precedes the b-cell. (b) Rat islets from dorsal glucagon-rich pancreas have a better insulin response than islets taken from the ventral lobe [38]. (c) Purified b-cells separated from a cells have a poor secretory response to glucose [39]. (d) Glucose competence may be restored by adding nM concentrations of glucagon [40]. (e) Addition of a glucagon receptor antagonist in human islets not only blunts the glucagon-induced but also the glucoseinduced insulin release [34]. The conclusion [34] is that ‘‘the exquisite responsiveness of intact islets is not only dependent on the process of glucose recognition, but also on the presence of a certain threshold of intra-islet glucagon concentration which results in activation of glucagon receptors on b cells’’. It is interesting to note that, if GLP-1 regulates insulin release via its incretin effect, it does not seem to share with glucagon its action on glucose competence, since this parameter is preserved in islets with disrupted GLP-1 receptors [41].
3.1.2.
The molecular mechanisms
The control by glucagon of both b-cell differentiation and the ability of the same cells to respond to glucose (concept of glucose competence) relies on intracellular pathways that have been analyzed in details. A key role in those pathways is played by the extracellularsignal regulated kinases 1 and 2 (ERK1/2) also known as Mitogen-Activated Protein Kinases 1 and 2 (P44/42 MAPKinases). These kinases are activated by double phosphorylation (on a tyrosine and a threonine residues) by the MAPkinase–kinases MEK1/2. The cascade may be triggered either by activation of tyrosine-kinase receptors (such as those for insulin, IGF or EGF) or by activation of G protein-coupled receptors (such as the one for glucagon) via production of cyclic AMP which may act at two different steps of the pathway, namely the small GTPases Rap and P21-Ras or the kinase–kinase–kinases Raf which exists under three different forms (Raf-1, A-Raf, B-raf). A sophistication (which may turn into pathology) of this system is that an excess of PKA
5
activation by cyclic AMP may phosphorylate Raf-1 isoform in an inadequate manner decreasing the activity of the pathway instead of increasing it. The ERK pathway exists in the b-cell as shown by the effect of low glucose in the absence and in the presence of glucagon on ERK activation by double phosphorylation in the MIN6 bcell line [42]. At a low glucose concentration, which corresponds to the physiological situation of glucagon secretion, glucagon stimulates ERK phosphorylation within 10 min by a 10-fold factor. Interestingly, glucagon is able to stimulate ERK even in the absence of added glucose during the experiment [42]. A major target for ERK is the transcription factor CREB (cyclic AMP response element binding protein) which controls expression of genes implicated in many cell functions. Indeed, CREB phosphorylation by glucagon is suppressed by addition of a MEK (MAP kinase–kinase) inhibitor. Unexpectedly, GLP-1, which, similarly to glucagon, stimulates cyclic AMP production, stimulates PKA and phosphorylates ERK, appears to skip the ERK step: Acute GLP-1 stimulation phosphorylates CREB in a non ERK-dependent manner, since the MEK inhibitor has no effect on CREB phosphorylation by GLP-1, while prolonged GLP-1 stimulation phosphorylates CREB in a b-arrestin-1-ERK-dependent manner. The pathways used by glucagon, glucose and GLP-1 in phosphorylating CREB in the b-cell are different. Only glucagon uses a straight pathway (glucagon ! cAMP ! PKA ! ERK ! CREB), while glucose uses increased intracellular calcium concentrations and GLP-1 uses a b-arrestin-1-ERK-dependent manner. This might be related to the different timing of actions of the three regulators, glucagon having plenty of time (the interprandial state) to produce in-depth regulation of CREB-dependent mechanisms, and, thus ‘preparing’ the ß-cell to respond to glucose, as compared to GLP-1 which acts, as an incretin, shortly before glucose reaches the islets. Fig. 4 outlines the central role of ERK in the b-cell biology, particularly in transmission of the glucagon actions: at the level of the nucleus, ERK is directly implicated in activation by phosphorylation of CREB, which controls expression of major genes such as that of proinsulin and that of the anti-apoptotic molecule Bcl-2. Another role of ERK in the b-cell is to phosphorylate proteins participating in insulin secretory granule exocytosis, favouring the secretory action of glucose [43]. In the cytoplasm, glucose-activated ERK phosphorylates proteins which have been proposed to be implicated in insulin granule exocytosis, such as Fak, paxillin, and synapsin-1 [43– 45]. ERK activated by glucose accumulates in the cytoplasm at actin filament tips adjacent to the plasma membrane, indicating that these are the main sites of action for these kinases during insulin secretion [46].
3.2.
Glucagon and insulin secretion
The beneficial effect of glucagon on the b-cell asks a question about its potential direct role in regulating insulin secretion. Indeed, while the maintenance of glucose competence of the insulin cells by glucagon is obviously beneficial, the eventuality of a direct effect of the peptide on insulin exocytosis would ruin the necessary hyperglycaemic action of glucagon. Fortunately, if exogenous glucagon is known for long to
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diabetes research and clinical practice 106 (2014) 1–10
Fig. 4 – Central role of ERK in b-cell regulation by glucagon and insulin. Synthesis from [42–46].
trigger insulin secretion [47,32], endogenous (interstitial) glucagon is unable by itself to do so [48]. Indeed, acute addition of a glucagon receptor antagonist in the rat perfused pancreas does not modify the ability of glucose to stimulate insulin secretion. Furthermore, under hypoglycaemic conditions where glucagon is physiologically secreted, endogenous secretion of glucagon by isoproterenol does not stimulate insulin secretion [48]. Thus, while glucagon acts on the b-cell via its own receptors to maintain the capacity of those cells to fully respond to glucose, glucagon secreted from the a-cells under hypoglycaemic conditions does not trigger insulin secretion from the neighbouring b-cells. The explanation for this discrepancy is the presence together with glucagon in the secretory granules of a-cells of a powerful inhibitor of insulin secretion.
3.3.
Miniglucagon
3.3.1.
Miniglucagon in the islet of Langerhans
Miniglucagon is the product of a secondary processing of glucagon, due to an original enzymatic system, different from the classical duo [49–51] prohormone-convertase (PC) + carboxypeptidase-E (CPE). This system [52,53], which may be considered as the opposite (cleavage at the N-terminus of basic residues followed by elimination of the remaining basic aminoacids by aminopeptidase) of the PC + CPE system (cleavage at the C-terminus of basic doublet by PC followed by elimination of the remaining basic aminoacids by carboxypeptidase), is made of N-arginine dibasic convertase (NRDc) + aminopeptidase-B. It was shown to catalyze, at least in some tissues, the cut of somatostatin-28 into somatostatin14 and is responsible for the production of miniglucagon from glucagon [54]. About 5% of the glucagon stores present in the a granules are transformed into miniglucagon [55]. This relatively small proportion is more than compensated by the huge (ca 104) difference in the respective potencies of the peptides.
Miniglucagon was shown to inhibit, at sub-picomolar concentrations, glucose-, sulfonylurea-, glucagon- and GLP1-stimulated insulin secretion from the MIN6 b-cell line [56] and from the rat isolated perfused pancreas [55]. The inhibitory effect of miniglucagon is due to blockage of calcium entry, necessary for insulin secretion stimulated by these agents [56]. Whole cell patch-clamp experiments [54] indicated that the inhibitory action of miniglucagon on calcium entry was linked to repolarization of the membrane (see Fig. 5) which forces the voltage-dependent calcium channels (VDCC) to close, and not to a direct action of the peptide on the VDCC (see Fig. 6), in contrast to another peptide, a-endosulfine [57]. Indeed, 10 mM glucose induces a depolarization from the resting potential (ca 70 mV) up to the potential (ca 40 mV) of activation of the VDCC (Fig. 5 upper panel) and addition of miniglucagon to a depolarized cell induces a repolarization up to the resting potential, suppressing the calcium entry and, consecutively, insulin secretion (Fig. 5 lower panel). As shown
Fig. 5 – Repolarization by miniglucagon of a MIN6 cell depolarized by 10 mM glucose. Data from [56].
diabetes research and clinical practice 106 (2014) 1–10
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Fig. 6 – Calcium currents at different degrees of depolarization: S40, S30, S20, S10, 0 mV (upper traces), +10, +20, +30, +40 mV, lower traces, in the absence (control) and in the presence of miniglucagon. In contrast to a-endosulfine, miniglucagon does not act directly on the voltage-dependent calcium channels. Data from [56,57] for the inset.
in Fig. 6, when the polarity of the plasma membrane is set to various values from 40 to +40 mV, the calcium currents are not modified by miniglucagon, in contrast to a-endosulfine. Altogether, the data indicate that miniglucagon closes indirectly the VDCC by modifying the membrane polarity, most probably by opening a pertussis toxin-sensitive Gi or Go protein-linked potassium channel (see [58] for a review on those channels), since the miniglucagon effect is suppressed by a treatment with pertussis toxin [56].
The fact that miniglucagon suppresses any insulinosecretagogue effect of glucagon raises the possibility that it also suppresses the beneficial effect of glucagon on the glucose competence of the b-cell. Fortunately, this is not the case [59]. Indeed, since Miniglucagon does not interfere with the ability of glucagon to stimulate cyclic AMP production, it does not interfere either with activation by glucagon of ERK and CREB which rely on the cyclic AMP/PKA pathway. It is interesting to note that somatostatin, which also suppresses the ability of
Fig. 7 – a-Cell–b-cell relationship inside the islet of Langerhans.
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Fig. 8 – Pathways used by insulin and miniglucagon in triggering Glut4 translocation in the adipose tissue. From [64].
glucagon to stimulate insulin secretion, does so by inhibiting adenylate cyclase via a Gi protein. This is an indirect indication that the miniglucagon receptor, the nature of which is unknown, is probably linked to the potassium channel via a Go rather than a Gi protein. In conclusion (Fig. 7), miniglucagon co-released with glucagon suppresses any prejudicial effect of its motherhormone on the b-cell, leaving untouched its beneficial effect: Glucagon, secreted during the interprandial state, maintains the b-cell in a glucose-competent condition via the cyclic AMP/ PKA/ERK/CREB pathway. Co-secreted miniglucagon does not modify this glucagon action, since it is disconnected from that pathway, but, via opening of a G protein-regulated potassium channel, drives the membrane via repolarization to the resting potential, preventing opening of the VDCC and thus insulin secretion. The single situation in which insulin raises in blood together with a significant glucagon (and thus miniglucagon) secretion is shortly after the beginning of a meal. The explanation for such an apparent contradiction is that the early insulin secretion is triggered essentially by vagal stimulation (cephalic phase) [60] which liberates Ca2+ from intracellular stores. Since the miniglucagon inhibitory action is solely mediated by closure of VDCC, it does not interfere with intracellular Ca2+ release and, thus, not with this early phase of insulin secretion.
3.3.2.
Miniglucagon in the whole organism
The data about the relationship between a- and b-cells prompted us to study a possible effect of miniglucagon in vivo, in spite of its extremely short half-life. The data obtained [61–63] indicated that, in the conscious rat, injection of miniglucagon together with a bolus of glucose reduced the insulin by about 40% at 3 min with no change in variations of glycaemia. This suggests either that miniglucagon facilitates the insulin action (potentiation) or that miniglucagon displays,
by itself, an insulin-like effect. Experiments conducted on the 3T3-L1 adipocytes showed that miniglucagon displays effects on Glut4 translocation similarly to insulin and displays insulin-like activity on the same parameter when used in the absence of added insulin. Analysis of its mode of action (Fig. 8) indicates that the miniglucagon receptor forms a scaffold with the insulin receptor and IRS-2 and, when activated, phosphorylates a 50-kDa protein which transmits the message to PI3-kinase. The next steps in the pathway are common with that of insulin. The major interest of such an architecture is that, in case of insulin resistance which implicates mostly the first steps (e.g. Ser/Thr phosphorylation which hampers the Tyr phosphorylation necessary for activation), the use of alternative steps by miniglucagon allows a workaround of insulin resistance. The physiological, pathological and pharmacological implications of these discoveries have still to be analyzed in details.
4.
Conclusion
The variety of peptides with different, yet complementary biological activities that derive from a single original molecular motif (glucagon plus its N-terminal and C-terminal extensions present in the gene and consequently in the prohormone), is a particularly rich example of the pivotal role of post-translational processing in the biological regulation. If we add the duplication of this motif during animal evolution (leading to the series of glucagon-like peptides) and the fact that this processing differs from a tissue to another, this is, altogether, not less than six peptides with demonstrated biological activity(ies) that are produced, four of them containing at least a part of glucagon, the original molecular motif. This importance of post-translational processing related to the presence in different tissues of various pro-hormone
diabetes research and clinical practice 106 (2014) 1–10
convertases [65,66] which allows the production of various peptides/proteins with specific roles has also, in some way, been ‘forgotten’, when the focus in biological research was made on genetics, while, although of major importance, the genes are the beginning but not the end of the story.
Conflict of interest The authors declare no conflict of interest.
references
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Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Invited review
The forgotten members of the glucagon family Dominique Bataille a,*, Ste´phane Dalle b a
INSERM, 269, Rue Adrien Proby, 34090 Montpellier, France INSERM, Research—Pathophysiology of the Pancreatic b Cell, Institute of Functional Genomic, INSERM U 661, CNRS UMR 5203, Universities Montpellier 1 & 2, Montpellier, France b
article info
abstract
Article history:
From proglucagon, at least six final biologically active peptides are produced by tissue-
Received 13 June 2014
specific post-translational processing. While glucagon and GLP-1 are the subject of perma-
Accepted 26 June 2014
nent studies, the four others are usually left in the shadow, in spite of their large biological
Available online 3 July 2014
interest. The present review is devoted to oxyntomodulin and miniglucagon, not forgetting glicentin, although much less is known about it. Oxyntomodulin (OXM) and glicentin are
Keywords:
regulators of gastric acid and hydromineral intestinal secretions. OXM is also deeply
Proglucagon
involved in the control of food intake and energy expenditure, properties that make this
Glucagon
peptide a credible treatment of obesity if the question of administration is solved, as for any
Oxyntomodulin
peptide. Miniglucagon, the C-terminal undecapeptide of glucagon which results from a
Glicentin
secondary processing of original nature, displays properties antagonistic to that of the
Miniglucagon
mother-hormone glucagon: (a) it inhibits glucose-, glucagon- and GLP-1-stimulated insulin
Post-translational processing
release at sub-picomolar concentrations, (b) it reduces the in vivo insulin response to glucose with no change in glycemia, (c) it displays insulin-like properties at the cellular level using only a part of the pathway used by insulin, making it a good basis for developing a pharmacological workaround of insulin resistance. # 2014 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucagon-containing C-terminally elongated peptides . . . . . . . . . . . 2.1. Structures of oxyntomodulin and glicentin . . . . . . . . . . . . . . 2.2. Compared activities of OXM and glucagon . . . . . . . . . . . . . . . 2.2.1. Gastric acid secretion . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Other sites of action in the GI tract . . . . . . . . . . . . . . 2.2.3. Sites of action in the CNS. . . . . . . . . . . . . . . . . . . . . . 2.3. Questions about the mode(s) of action of OXM and glicentin
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* Corresponding author. Tel.: +33 467541697; fax: +33 467541697. E-mail addresses: [email protected] (D. Bataille), [email protected] (S. Dalle). http://dx.doi.org/10.1016/j.diabres.2014.06.010 0168-8227/# 2014 Elsevier Ireland Ltd. All rights reserved.
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3.
4.
1.
Glucagon–insulin relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Glucagon–insulin relationship in the islets of Langerhans . 3.1.1. The observations . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. The molecular mechanisms . . . . . . . . . . . . . . . . . . 3.2. Glucagon and insulin secretion. . . . . . . . . . . . . . . . . . . . . . 3.3. Miniglucagon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Miniglucagon in the islet of Langerhans . . . . . . . . 3.3.2. Miniglucagon in the whole organism . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction
Proglucagon (Fig. 1) is, besides the proopiomelanocortin (POMC), the archetype of a prohormone the post-translational processing of which provides the organism with a series of peptides with different, yet complementary biological activities. All activities of the proglucagon-derived peptides are related to the control of nutrition and metabolism: Glucagon, by its hyperglycaemic effect and GLP-1 via its incretin action, control the carbohydrate metabolism. GLP-2 maintains a healthy morphology of the gut epithelium and its functions, while the glucagon-extended molecules oxyntomodulin and glicentin regulate gastric acid secretion, hydromineral transport across the intestine, gut motility and food intake. Miniglucagon, which derives from a secondary processing of glucagon, plays a strange score in this orchestra, blocking possible unwanted effects of its mother-hormone on insulin secretion, while having insulin-like properties. Since many excellent reviews exist on glucagon itself (see, in particular, the three famous books edited by Lefe`bvre [1–3])
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and on glucagon-like peptide-1 ([4] for a recent review), the present review will focus on the three peptides that contain at least a part of the glucagon sequence, namely glicentin, oxyntomodulin and miniglucagon that have been partially ‘forgotten’ by the researchers in the last years, in spite of their importance in the metabolic regulation. The glucagon-containing peptides derive from tissue specific post-translational processing (Fig. 2). Expression of the prohormone convertase PC-1/3 in intestinal L cells leads to the production of the C-terminally extended peptides glicentin and oxyntomodulin. Expression of the prohormone convertase PC-2 in the a-cells of the islets of Langerhans leads to the production of glucagon. A secondary processing due to the presence of NRD convertase and aminopeptidaseB allows the production of a small, yet biologically significant, proportion of the C-terminal undecapeptide miniglucagon. Every peptide of the family is a true hormone, reaching its remote target(s) through the blood flow, with the exception of miniglucagon, a local regulator acting at the close vicinity of its site of production and quickly degraded.
Fig. 1 – The glucagon-containing peptides and its derivatives. Besides the four peptides with proved natural existence, are added the two oxyntomodulin fragments with demonstrated biological activities.
diabetes research and clinical practice 106 (2014) 1–10
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Fig. 2 – Tissue-specific post-translational processing of proglucagon into the glucagon-containing peptides. GRPP: glicentinrelated pancreatic polypeptide; SP-1 and SP-2: spacer peptides 1- & -2; GLP-1 et GLP-2: glucagon-like peptide-1 & -2; PC1/3: prohormone convertase-1/3; PC2: prohormone convertase-2; CPE: carboxypeptidase-E; NDRc: N-arginine dibasic convertase; APB: aminopeptidase-B; KR: lysine–arginine cleavage site: RR: arginine–arginine cleavage site; RK: arginine– lysine cleavage site; R: arginine cleavage site.
2. Glucagon-containing C-terminally elongated peptides 2.1.
Structures of oxyntomodulin and glicentin
Oxyntomodulin (OXM) differs from glucagon (see Fig. 1) by an additional C-terminal octapeptide [5,6], while glicentin is Nterminally elongated, as compared to OXM, by the 32-amino acid (32-AA) glicentin-related polypeptide (GRPP). Although much less is known about glicentin as compared to OXM, both peptides share most, if not all, of their biological activities. The two peptides are produced from proglucagon in cells, such as the intestinal L cells or some specialized neurones in the central nervous system (C.N.S.), which express the prohormone convertase PC1/3 rather than PC2, the expression of which in pancreatic a-cells leads to the glucagon production. During the same process, GLP-1 and GLP-2 are produced and co-secreted with OXM and glicentin from intestinal L cells.
2.2.
Compared activities of OXM and glucagon
Since the single difference between OXM and glucagon is the C-terminal octapeptide, it was of considerable interest to compare the biological activities of the two peptides.
2.2.1.
Gastric acid secretion
The inhibitory effect of OXM on pentagastrin-stimulated gastric acid secretion in the rat was contemporary with determination of its structure [7]. OXM displayed in that model a potency ca 15-fold higher than that of glucagon, showing the
importance of the octapeptide in the biological specificity of OXM. It was shown later that OXM also inhibited histaminestimulated gastric acid secretion [8,9]. Interestingly, the Cterminal (19–37) fragment displayed a roughly two-fold lower potency as compared to the entire molecule. When corrected for comparative clearance rate, both the 37-AA OXM and its Cterminal 19-AA fragment have the same potency, indicating that, on that parameter, the N-terminal fragment of glucagon does not enter into the OXM mode of action on gastric acid secretion. Although much less potent, mostly due to its very short half-life, the C-terminal octapeptide on its own, contains the inhibitor message in both the rat [10] and in the human species [11]. The minimal fully active structure was the Nterminally acetylated hexapeptide (octapeptide lacking the last two aminoacids) [12].
2.2.2.
Other sites of action in the GI tract
Besides its gastric target, OXM is able to modulate the hydromineral transport through the rat small intestine [13]. Its mode of action seems to be the same as, or at least similar to that present in the stomach, since the C-terminal [19–37] fragment mimics the effects of the whole molecule, at least in the jejunum. The same holds true in gastric smooth muscles [14]. The receptor(s) implicated in that model was shown to be linked, via a Gi or Go protein, to phospholipase C (production of inositol triphosphate -IP3-) and inhibition of adenylate cyclase (decreased cyclic AMP production). OXM and glicentin were also shown to inhibit the motility pattern of the small intestine [15], while it was shown that glicentin (OXM was not tested in that study) is able to induce contraction of smooth muscle cells from the human colon and that this effect is abolished by
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the GLP-1 receptor antagonist exendin (9–39) [16]. See the chapter on the mode of action of OXM and glicentin for more details.
2.2.3.
Sites of action in the CNS
The major effect of OXM observed on the central nervous system (CNS) is a negative control of food intake (suppression of hunger -anorexigenic action-). Indeed, OXM inhibits food intake in the rat, either applied centrally [17] or via a peripheral administration [18]. Similar findings were obtained in humans [19], with a reduction of body weight in overweight and obese patients using the subcutaneous route [20]. In addition to its action on energy intake, OXM increases energy expenditure [21]. The effect of peripherally administered OXM on food intake is due to the fact that, like other gastro-intestinal hormones, the peptide is able to cross the blood–brain barrier at the level of the median eminence and reaches the arcuate nucleus of hypothalamus which contains both stimulatory and inhibitory neurons. OXM also inhibits the orexigenic signal carried by ghrelin [23]. Altogether, these features make OXM a good basis for the development of an anti-obesity drug [22]. For recent reviews on appetite regulation see [23,24].
2.3. Questions about the mode(s) of action of OXM and glicentin It is clear that the receptor(s), the precise nature of which is still unknown, which transduce(s) the OXM actions on the GI tract displays a strong preference towards the C-terminal end (including the octapeptide itself) of the molecule, with little or no importance of the N-terminal half common with glucagon and which resembles that of GLP-1. This part of the molecules (glucagon or GLP-1) is crucial for triggering the cyclic AMP pathway associated with classical Gs protein-linked glucagon and GLP-1 receptors. In sharp contrast, the recognition system responsible for its CNS actions seems to be, at least in part, transduced by the GLP-1 receptor. Indeed, OXM has no longer effect on food intake in mice lacking the GLP-1 receptor [25] and the (not entirely specific) GLP-1 antagonist exendin (9–39) blocks the actions of both OXM and GLP-1 [25].
However, the figure is much more complex, since (a) OXM and GLP-1 are equipotent in their central effects despite a large difference between their respective affinities for the GLP-1 receptor; (b) OXM and GLP-1 behave differently in vivo and seem to act at different places of the CNS, namely primarily arcuate nucleus and brain stem, respectively [25,18]. Thus, whether these confusing observations are related to the existence of a distinct, still unrecognized, receptor or that of sub-classes of GLP-1 receptors with different specificities remains an open question. More recently, however, it appears that the glucagon receptor is also involved in the body-lowering effect of OXM [26]. A likely conclusion is thus that OXM acts on the CNS via two pathways using both GLP-1 and glucagon receptors.
3.
Glucagon–insulin relationship
Although the focus of the present review is not glucagon itself, it is still necessary to briefly recall basic informations on the impact of the relationship of this peptide with insulin, inasmuch as these data are necessary to understand the biological role of miniglucagon. Glucagon represents, in the regulation of metabolism and particularly that of the carbohydrates, the opposite counterpart of insulin. Both peptides are produced in the islets of Langerhans and display complex local relationships. While insulin is secreted after a meal and allows to store fuels for future usage, glucagon is secreted during the interprandial state or when a particular need for energy-producing molecules (mostly glucose and lipids) occurs (e.g. physical exercise). By its effects on the liver and adipose tissue, it liberates the stored fuels and makes them available to the whole body. This catabolic/anabolic hormonal balance is of major importance for the metabolic regulation. If the details on this balance in the whole organism has been the subject of a huge amount of studies since the discovery of glucagon and, accordingly, not much is probably still to be discovered, the relationship between the two antagonistic peptides was still unclear inside the islet of Langerhans, the place of synthesis and secretion of both peptides.
3.1. Glucagon–insulin relationship in the islets of Langerhans 3.1.1.
Fig. 3 – Architecture of the islets of Langerhans. After Orci et al. [27,28].
The observations
In contrast to the clearly opposite nature of glucagon towards insulin recalled above, the relationship between the two hormones inside their production site, the islets of Langerhans, are much more complex. This is possible thanks to the particular architecture of the islets in which many direct contacts between the a- and the b-cells exist. Indeed, 40–50% of the b-cells have direct contacts with a-cells, according to the precise schematic drawings made by Lelio Orci and colleagues (Fig. 3). In the same line, the human islets have particular structure ‘‘allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between a- and bcells . . .’’ [29]. See also [30] for a pathological approach of paracrinology inside the islets.
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The close vicinity between the two types of cells allows mutual interactions. If the direct inhibition of glucagon secretion by insulin is well documented [31], many data indicate that glucagon displays important effects on the b-cell. This is possible thanks to the presence of authentic glucagon receptors at the surface of the insulin-secreting cells [32–34]. These receptors are coupled to adenylate cyclase and allow glucagon to control beta cells functions as follows: (a) Glucagon is required for early differentiation of insulinsecreting cells during the mouse development [35]: Addition of a proglucagon antisense to pancreas from 11-week mouse embryo not only blocks differentiation of a-cells but also that of b-cells and that this differentiation may be rescued by addition of glucagon. Also, ablation of the glucagon receptor gene produces alteration in islet development and differentiation [36]. This fits well with the fact that a-cell is the first islet cell to appear during development [37] and thus precedes the b-cell. (b) Rat islets from dorsal glucagon-rich pancreas have a better insulin response than islets taken from the ventral lobe [38]. (c) Purified b-cells separated from a cells have a poor secretory response to glucose [39]. (d) Glucose competence may be restored by adding nM concentrations of glucagon [40]. (e) Addition of a glucagon receptor antagonist in human islets not only blunts the glucagon-induced but also the glucoseinduced insulin release [34]. The conclusion [34] is that ‘‘the exquisite responsiveness of intact islets is not only dependent on the process of glucose recognition, but also on the presence of a certain threshold of intra-islet glucagon concentration which results in activation of glucagon receptors on b cells’’. It is interesting to note that, if GLP-1 regulates insulin release via its incretin effect, it does not seem to share with glucagon its action on glucose competence, since this parameter is preserved in islets with disrupted GLP-1 receptors [41].
3.1.2.
The molecular mechanisms
The control by glucagon of both b-cell differentiation and the ability of the same cells to respond to glucose (concept of glucose competence) relies on intracellular pathways that have been analyzed in details. A key role in those pathways is played by the extracellularsignal regulated kinases 1 and 2 (ERK1/2) also known as Mitogen-Activated Protein Kinases 1 and 2 (P44/42 MAPKinases). These kinases are activated by double phosphorylation (on a tyrosine and a threonine residues) by the MAPkinase–kinases MEK1/2. The cascade may be triggered either by activation of tyrosine-kinase receptors (such as those for insulin, IGF or EGF) or by activation of G protein-coupled receptors (such as the one for glucagon) via production of cyclic AMP which may act at two different steps of the pathway, namely the small GTPases Rap and P21-Ras or the kinase–kinase–kinases Raf which exists under three different forms (Raf-1, A-Raf, B-raf). A sophistication (which may turn into pathology) of this system is that an excess of PKA
5
activation by cyclic AMP may phosphorylate Raf-1 isoform in an inadequate manner decreasing the activity of the pathway instead of increasing it. The ERK pathway exists in the b-cell as shown by the effect of low glucose in the absence and in the presence of glucagon on ERK activation by double phosphorylation in the MIN6 bcell line [42]. At a low glucose concentration, which corresponds to the physiological situation of glucagon secretion, glucagon stimulates ERK phosphorylation within 10 min by a 10-fold factor. Interestingly, glucagon is able to stimulate ERK even in the absence of added glucose during the experiment [42]. A major target for ERK is the transcription factor CREB (cyclic AMP response element binding protein) which controls expression of genes implicated in many cell functions. Indeed, CREB phosphorylation by glucagon is suppressed by addition of a MEK (MAP kinase–kinase) inhibitor. Unexpectedly, GLP-1, which, similarly to glucagon, stimulates cyclic AMP production, stimulates PKA and phosphorylates ERK, appears to skip the ERK step: Acute GLP-1 stimulation phosphorylates CREB in a non ERK-dependent manner, since the MEK inhibitor has no effect on CREB phosphorylation by GLP-1, while prolonged GLP-1 stimulation phosphorylates CREB in a b-arrestin-1-ERK-dependent manner. The pathways used by glucagon, glucose and GLP-1 in phosphorylating CREB in the b-cell are different. Only glucagon uses a straight pathway (glucagon ! cAMP ! PKA ! ERK ! CREB), while glucose uses increased intracellular calcium concentrations and GLP-1 uses a b-arrestin-1-ERK-dependent manner. This might be related to the different timing of actions of the three regulators, glucagon having plenty of time (the interprandial state) to produce in-depth regulation of CREB-dependent mechanisms, and, thus ‘preparing’ the ß-cell to respond to glucose, as compared to GLP-1 which acts, as an incretin, shortly before glucose reaches the islets. Fig. 4 outlines the central role of ERK in the b-cell biology, particularly in transmission of the glucagon actions: at the level of the nucleus, ERK is directly implicated in activation by phosphorylation of CREB, which controls expression of major genes such as that of proinsulin and that of the anti-apoptotic molecule Bcl-2. Another role of ERK in the b-cell is to phosphorylate proteins participating in insulin secretory granule exocytosis, favouring the secretory action of glucose [43]. In the cytoplasm, glucose-activated ERK phosphorylates proteins which have been proposed to be implicated in insulin granule exocytosis, such as Fak, paxillin, and synapsin-1 [43– 45]. ERK activated by glucose accumulates in the cytoplasm at actin filament tips adjacent to the plasma membrane, indicating that these are the main sites of action for these kinases during insulin secretion [46].
3.2.
Glucagon and insulin secretion
The beneficial effect of glucagon on the b-cell asks a question about its potential direct role in regulating insulin secretion. Indeed, while the maintenance of glucose competence of the insulin cells by glucagon is obviously beneficial, the eventuality of a direct effect of the peptide on insulin exocytosis would ruin the necessary hyperglycaemic action of glucagon. Fortunately, if exogenous glucagon is known for long to
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Fig. 4 – Central role of ERK in b-cell regulation by glucagon and insulin. Synthesis from [42–46].
trigger insulin secretion [47,32], endogenous (interstitial) glucagon is unable by itself to do so [48]. Indeed, acute addition of a glucagon receptor antagonist in the rat perfused pancreas does not modify the ability of glucose to stimulate insulin secretion. Furthermore, under hypoglycaemic conditions where glucagon is physiologically secreted, endogenous secretion of glucagon by isoproterenol does not stimulate insulin secretion [48]. Thus, while glucagon acts on the b-cell via its own receptors to maintain the capacity of those cells to fully respond to glucose, glucagon secreted from the a-cells under hypoglycaemic conditions does not trigger insulin secretion from the neighbouring b-cells. The explanation for this discrepancy is the presence together with glucagon in the secretory granules of a-cells of a powerful inhibitor of insulin secretion.
3.3.
Miniglucagon
3.3.1.
Miniglucagon in the islet of Langerhans
Miniglucagon is the product of a secondary processing of glucagon, due to an original enzymatic system, different from the classical duo [49–51] prohormone-convertase (PC) + carboxypeptidase-E (CPE). This system [52,53], which may be considered as the opposite (cleavage at the N-terminus of basic residues followed by elimination of the remaining basic aminoacids by aminopeptidase) of the PC + CPE system (cleavage at the C-terminus of basic doublet by PC followed by elimination of the remaining basic aminoacids by carboxypeptidase), is made of N-arginine dibasic convertase (NRDc) + aminopeptidase-B. It was shown to catalyze, at least in some tissues, the cut of somatostatin-28 into somatostatin14 and is responsible for the production of miniglucagon from glucagon [54]. About 5% of the glucagon stores present in the a granules are transformed into miniglucagon [55]. This relatively small proportion is more than compensated by the huge (ca 104) difference in the respective potencies of the peptides.
Miniglucagon was shown to inhibit, at sub-picomolar concentrations, glucose-, sulfonylurea-, glucagon- and GLP1-stimulated insulin secretion from the MIN6 b-cell line [56] and from the rat isolated perfused pancreas [55]. The inhibitory effect of miniglucagon is due to blockage of calcium entry, necessary for insulin secretion stimulated by these agents [56]. Whole cell patch-clamp experiments [54] indicated that the inhibitory action of miniglucagon on calcium entry was linked to repolarization of the membrane (see Fig. 5) which forces the voltage-dependent calcium channels (VDCC) to close, and not to a direct action of the peptide on the VDCC (see Fig. 6), in contrast to another peptide, a-endosulfine [57]. Indeed, 10 mM glucose induces a depolarization from the resting potential (ca 70 mV) up to the potential (ca 40 mV) of activation of the VDCC (Fig. 5 upper panel) and addition of miniglucagon to a depolarized cell induces a repolarization up to the resting potential, suppressing the calcium entry and, consecutively, insulin secretion (Fig. 5 lower panel). As shown
Fig. 5 – Repolarization by miniglucagon of a MIN6 cell depolarized by 10 mM glucose. Data from [56].
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Fig. 6 – Calcium currents at different degrees of depolarization: S40, S30, S20, S10, 0 mV (upper traces), +10, +20, +30, +40 mV, lower traces, in the absence (control) and in the presence of miniglucagon. In contrast to a-endosulfine, miniglucagon does not act directly on the voltage-dependent calcium channels. Data from [56,57] for the inset.
in Fig. 6, when the polarity of the plasma membrane is set to various values from 40 to +40 mV, the calcium currents are not modified by miniglucagon, in contrast to a-endosulfine. Altogether, the data indicate that miniglucagon closes indirectly the VDCC by modifying the membrane polarity, most probably by opening a pertussis toxin-sensitive Gi or Go protein-linked potassium channel (see [58] for a review on those channels), since the miniglucagon effect is suppressed by a treatment with pertussis toxin [56].
The fact that miniglucagon suppresses any insulinosecretagogue effect of glucagon raises the possibility that it also suppresses the beneficial effect of glucagon on the glucose competence of the b-cell. Fortunately, this is not the case [59]. Indeed, since Miniglucagon does not interfere with the ability of glucagon to stimulate cyclic AMP production, it does not interfere either with activation by glucagon of ERK and CREB which rely on the cyclic AMP/PKA pathway. It is interesting to note that somatostatin, which also suppresses the ability of
Fig. 7 – a-Cell–b-cell relationship inside the islet of Langerhans.
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Fig. 8 – Pathways used by insulin and miniglucagon in triggering Glut4 translocation in the adipose tissue. From [64].
glucagon to stimulate insulin secretion, does so by inhibiting adenylate cyclase via a Gi protein. This is an indirect indication that the miniglucagon receptor, the nature of which is unknown, is probably linked to the potassium channel via a Go rather than a Gi protein. In conclusion (Fig. 7), miniglucagon co-released with glucagon suppresses any prejudicial effect of its motherhormone on the b-cell, leaving untouched its beneficial effect: Glucagon, secreted during the interprandial state, maintains the b-cell in a glucose-competent condition via the cyclic AMP/ PKA/ERK/CREB pathway. Co-secreted miniglucagon does not modify this glucagon action, since it is disconnected from that pathway, but, via opening of a G protein-regulated potassium channel, drives the membrane via repolarization to the resting potential, preventing opening of the VDCC and thus insulin secretion. The single situation in which insulin raises in blood together with a significant glucagon (and thus miniglucagon) secretion is shortly after the beginning of a meal. The explanation for such an apparent contradiction is that the early insulin secretion is triggered essentially by vagal stimulation (cephalic phase) [60] which liberates Ca2+ from intracellular stores. Since the miniglucagon inhibitory action is solely mediated by closure of VDCC, it does not interfere with intracellular Ca2+ release and, thus, not with this early phase of insulin secretion.
3.3.2.
Miniglucagon in the whole organism
The data about the relationship between a- and b-cells prompted us to study a possible effect of miniglucagon in vivo, in spite of its extremely short half-life. The data obtained [61–63] indicated that, in the conscious rat, injection of miniglucagon together with a bolus of glucose reduced the insulin by about 40% at 3 min with no change in variations of glycaemia. This suggests either that miniglucagon facilitates the insulin action (potentiation) or that miniglucagon displays,
by itself, an insulin-like effect. Experiments conducted on the 3T3-L1 adipocytes showed that miniglucagon displays effects on Glut4 translocation similarly to insulin and displays insulin-like activity on the same parameter when used in the absence of added insulin. Analysis of its mode of action (Fig. 8) indicates that the miniglucagon receptor forms a scaffold with the insulin receptor and IRS-2 and, when activated, phosphorylates a 50-kDa protein which transmits the message to PI3-kinase. The next steps in the pathway are common with that of insulin. The major interest of such an architecture is that, in case of insulin resistance which implicates mostly the first steps (e.g. Ser/Thr phosphorylation which hampers the Tyr phosphorylation necessary for activation), the use of alternative steps by miniglucagon allows a workaround of insulin resistance. The physiological, pathological and pharmacological implications of these discoveries have still to be analyzed in details.
4.
Conclusion
The variety of peptides with different, yet complementary biological activities that derive from a single original molecular motif (glucagon plus its N-terminal and C-terminal extensions present in the gene and consequently in the prohormone), is a particularly rich example of the pivotal role of post-translational processing in the biological regulation. If we add the duplication of this motif during animal evolution (leading to the series of glucagon-like peptides) and the fact that this processing differs from a tissue to another, this is, altogether, not less than six peptides with demonstrated biological activity(ies) that are produced, four of them containing at least a part of glucagon, the original molecular motif. This importance of post-translational processing related to the presence in different tissues of various pro-hormone
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convertases [65,66] which allows the production of various peptides/proteins with specific roles has also, in some way, been ‘forgotten’, when the focus in biological research was made on genetics, while, although of major importance, the genes are the beginning but not the end of the story.
Conflict of interest The authors declare no conflict of interest.
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