Glucagon, discovered in 1923 as a contaminant of early insulin preparations, has long been a neglected hormone.
Glucagon was among the very first polypeptide hormones to be isolated, purified, sequenced, and synthesized. Thanks to the pioneering work of Unger, it was the first polypeptide hormone to become measurable by radioimmunoassay, almost one year before insulin [1,2]. It has served two Nobel Prize winners as a unique tool, which permitted Sutherland and his associates to discover cyclic AMP (cAMP), and Rodbell and his coworkers to discover the role of G-proteins in cell-membrane receptors. Subsequently, the nucleotide sequence of the glucagon gene has been determined and the structure of the human glucagon precursor (preproglucagon) has been deduced. This discovery has been fundamental for clarifying the relationships of glucagon itself with various other peptides derived from the same common precursor, and originating from both the pancreas and the gut. Recent observations suggest that diabetes should be seen as a paracrinopathy of the islets of Langerhans in which intra-islet insulin deficiency results in excessive glucagon release from the neighboring α cells and the resulting hyperglucagonemia being a critical factor in the pathophysiology of diabetes [3,4]. A comprehensive bibliography on glucagon up to 1996 can be found in the three volumes of the Handbook of Experimental Pharmacology edited by Lefèbvre [5,6] and in the proceedings of a recently held conference .
Amino acid composition, extraction, synthesis, and biosynthesis
The amino acid sequence of glucagon isolated from the pancreas of pigs, cattle, and humans is identical. Guinea-pig glucagon is different from all other glucagons that have been isolated, while avian glucagons differ from the predominant mammalian glucagon only by a few conservative replacements. This extreme conservation of primary structure exhibited by glucagon has been considered “at least unusual and at most extraordinary.” In fact, it has now been recognized that glucagon belongs to what has been called the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily that includes nine hormones that are related by structure, distribution (especially the brain and the gut), function (often by activation of cAMP), and receptors (a subset of seven transmembrane receptors) . The nine hormones include glucagon, GLP-1 (glucagon-like peptide 1), GLP-2 (glucagon-like peptide 2), GIP (gastric inhibitory polypeptide), GRF (growth-hormone-releasing factor), PHM (peptide histidine methionine), PACAP, secretin, and VIP (vasoactive intestinal peptide). The origin of the ancestral superfamily members is at least as old as the invertebrates. The most ancient and tightly conserved members are PACAP and glucagon. Evidence to date suggests that the superfamily began with a gene or exon duplication and then continued to diverge with some gene duplications in vertebrates . Mammalian glucagon contains 29 amino acid residues and has a molecular mass of 3485 Da (Figure 10.1). Glucagon can be synthesized in vitro by either classic solution synthesis or solid-phase synthesis; in both cases, two substrategies can be used — either fragment assembly or stepwise assembly. All procedures have given highly purified materials that are homogeneous and indistinguishable from natural glucagon by a range of sensitive analytic methods.
The glucagon gene consists of six exons and five introns spanning 10 kb; the human glucagon gene is located on chromosome 2 . It encodes for a preprohormone of 180 amino acids that contains not only glucagon but several other peptides including two glucagon-like peptides whose amino acid structures are distinct from, but closely resemble, that of glucagon and other members of the glucagon superfamily of peptides. The glucagon gene is expressed in the α cells of the islets of Langerhans, the intestinal L cells, and in some parts of the brain. While the functional relevance of glucagon gene expression in the brain is still poorly understood, a clear image has emerged regarding the processing of proglucagon into several bioactive peptides in the pancreas and in the gut (Figure 10.2).
In the pancreas glucagon is the predominant peptide produced, together with glucagon-related polypeptide (GRPP), while the glucagon-like peptides remain in an incompletely processed prohormone fragment. In the gut two glucagon-like peptides (GLP-1 and GLP-2) are produced, while glucagon remains, in part, as a prohormone fragment, glicentin. However, glicentin can be further processed to oxyntomodulin and GRPP. Of these peptides of the glucagon super-family, oxyntomodulin and possibly glicentin are implicated in the physiologic negative control of gastric acid secretion.
Glucagon-like peptide GLP-1(7-37), a peptide of 31 amino acids, or the equally potent isopeptide GLP-1(7-36), an amide of 30 amino acids, has major insulinotropic action on pancreatic β cells [10,11]. This peptide binds to specific receptors on islet β cells, stimulating cAMP formation, insulin release, proinsulin gene transcription, and proinsulin biosynthesis, all in a glucose-dependent manner. Interestingly, GLP-1 is a strong inhibitor of glucagon secretion. GLP-1 also exerts numerous extrapancreatic actions, including inhibition of food intake, promotion of satiety, cardioprotection, vasodilation, and possibly beneficial effects on endothelial function and inflammation . GLP-1 is rapidly degraded by the enzyme dipeptidyl-peptidase-4 (DPP-4). As reviewed in Chapter 48, GLP-1 analogues, such as exenatide and liraglutide, and DPP-4 inhibitors, such as vildagliptin, sitagliptin, saxagliptin, linagliptin, and alogliptin are now used in the treatment of type 2 diabetes.
GLP-2 is recognized as a major regulator of intestinal growth and function. Its secretion from the intestinal L cells is essen- tially stimulated by nutrient intake. It has specific trophic effects on the gut which appear to be mediated by stimulation of mucosal cell proliferation and inhibition of apoptosis and proteolysis . Additional effects of GLP-2 on the digestive tract include stimulation of enterocyte glucose transport and GLUT-2 expression, increased nutrient absorption, reduction of intestinal permeability, stimulation of intestinal blood flow, relaxation of intestinal smooth muscle, and inhibition of gastric emptying and gastric acid secretion [14,15]. Further effects of GLP-2 include stimulation of glucagon release from the islet α cells and modulation of islet adaptation to metabolic stress. GLP-2 has been reported to reduce bone resorption and to significantly increase bone mineral density in postmenopausal women. Preliminary studies suggest a great therapeutic potential of GLP-2 in total parenteral nutrition, short bowel syndrome following major intestinal resection, nonsteroidal drug-induced enteritis, inflammatory bowel disease, ischemic bowel, and so forth. Recently, teraglutide, a degradation-resistant GLP-2 analogue was approved for the treatment of short-bowel syndrome.
Both glucagon and oxyntomodulin are further processed into N-terminal and C-terminal fragments by cleavage at a dibasic site (Arg17-Arg18) [16,17]. The C-terminal fragments are of particular interest: glucagon-(19-29) modulates the plasma membrane calcium flow in the nanomolar range, whereas oxyntomodulin-(19-37) inhibits gastric acid secretion, as does oxyntomodulin itself. Further studies have shown that glucagon processing to glucagon-(19-29) (mini-glucagon) is probably essential for the positive inotropic effect of glucagon on heart contraction. Thus, the concept has emerged that glucagon and oxyntomodulin are first released into the blood and then processed at the level of their respective targets into the corresponding biologically active C-terminal fragments. Interestingly, mini-glucagon inhibits insulin secretion at picomolar concentrations. It has been proposed that mini-glucagon acts as a local inhibitory regulator of insulin release by turning off the main external calcium source for islet β cells via a specific receptor linked to ion channels that control cell polarity [18,19].
The tissue-specific liberation of proglucagon is controlled by cell-specific expression of enzymes known as prohormone convertases (PC). A major defect in the processing of proglucagon to mature pancreatic glucagon is seen in the PC2 knockout mouse. These animals exhibit mild hypoglycemia and hyper- plasia of the α cells in the islets of Langerhans [20,21]. These abnormalities are corrected by glucagon replacement via a micro-osmotic pump . Numerous studies have recently investigated the transcriptional regulation of α-cell differentiation and that of the proglucagon gene [7,23 – 25]. The differentiation of α cells and the maintenance of α-cell function are influenced at several stages during development and in the maturing islet. Several transcriptional factors such as neurogenin 3 (Ngn 3), pancreatic duodenal homeobox 1 (Pdx-1) and regulatory factor X6 (Rfx6) are crucial in the determination of the endocrine cell fate. Other transcription factors such as aristaless-related homeobox (Arx) and forkhead box A2 (Foxa2) are implicated in the initial or terminal differentiation of α cells. On the other hand, proglucagon transcription, and therefore the maintenance of α-cell function, is regulated by several factors, including forkhead box A1 (Foxa1), paired box 6 (Pax6), brain 4 (Brn4), and islet-1 (isl-1) . Interestingly, recent studies have shown that “endocrine cell reprogramming” is possible with conversion of pancreatic α cells into cells displaying a β-cell phenotype, thus possibly offering a totally new avenue for the treatment (or cure ?) of diabetes [24,25].