ISLET TRANSCRIPTION FACTORS AND THE α CELL

The pancreas derives from the upper foregut, with dorsal and ventral pancreatic buds giving rise to pancreatic epithelium that eventually forms the exocrine and endocrine pancreas. This process involves signals from mesenchyme and requires the correct temporal expression of growth factors, transcription factors, and related signaling molecules. Pancreatic islet cells appear to be of endodermal origin and are first detectable by E8.5 in the mouse. The first islet hormone-immunopositive cells detectable in developing islets contain glucagon immunoreactivity. Insulin immunopositive cells are detectable by day E14 in the mouse, followed several days later by the appearance of somatostatin-containing cell types.

Studies of targeted gene disruption in mice have provided new insights into the organization of islet endocrine cells in the pancreas. Neurogenin-3 is critical for pancreatic islet cell development and differentiation and controls the expression of many genes important for α cell differentiation and glucagon gene transcription. Ngn3 interacts in a regulatory network with the zinc-finger protein Myt1 to promote glucagon expression in α cells.² Mice with a null allele in the basic helix-loop-helix transcription factor p48 fail to develop exocrine pancreatic tissue; however, hormone-secreting islet cells, including glucagon-producing α cells, are found within the mesentery during embryonic development, and later in the spleen.³ A key role for cell adhesion molecules in the control of spatial organization of islet cells is illustrated by analysis of the endocrine pancreas in mice expressing a dominant negative E-cadherin receptor in islet β cells. These mice exhibit abnormal clustering of β cells, yet glucagon-producing α cells are still capable of aggregating into islet-like clusters.⁴ In contrast, the normal peripheral distribution of islet α cells is markedly perturbed in neural cell adhesion molecule (NCAM)-/- mice; however, the number of α cells and the glucagon content of the pancreas remain unaffected.⁵ It is intriguing that mice with genetic disruption of the ciliogenic transcription factor regulatory factor X (RFX)-3 exhibit small disorganized islets with markedly reduced insulin and glucagon content, implying a role for cilia in optimal islet organization and cell development.⁶

Considerable insight into the developmental biology of the endocrine pancreas and islet α cells has been derived from studies of islet transcription factors in cell lines and mice. Gene knockout studies demonstrate that the LIM domain protein isl-1 is necessary for the formation of differentiated islet cells, including α cells, in the developing pancreas.⁷ Targeted deletion of the homeobox transcription factor Arx results in mice that exhibit complete failure of α cell development, leading to glucagon deficiency and neonatal hypoglycemia.⁸ In contrast, although mice deficient in the homeobox transcription factor Ipf/Pdx-1 fail to develop a pancreas, a few islet cells immunopositive for insulin or glucagon are observed in Ipf/Pdx-1-/- mice at embryonic day 11.⁹ These observations suggest that Ipf/Pdx-1 is not essential for the formation of islet α cells, whereas loss of Pdx-1 expression appears to be required for acquisition of a differentiated α cell phenotype.

The homeobox transcription factor Nkx2.2 is expressed in adult α, β, and PP islet cells; mice with targeted disruption of NKx2.2 lack β cells, develop diabetes, and exhibit a marked reduction in the numbers of islet α cells.¹⁰ A related phenotype is observed in NeuroD/BETA2-/- mice, which exhibit marked reductions in the numbers of islet β and α cells and develop diabetes shortly after birth,¹¹ a phenotype dependent on the specific genetic background. Similarly, disruption of pax6 function in mice results in poorly formed islets with disorganized islet architecture and markedly reduced but detectable numbers of both β and islet α cells.^12,13 It is intriguing that mice that harbor mutations in islet transcription factors also may exhibit paradoxically increased numbers of islet α cells. For example, targeted deletion of the Pax4 gene results in poorly formed islets, with a marked reduction in islet β cells and comparatively increased numbers of islet α cells.¹⁴ A related phenotype is observed in mice with a homozygous deletion in the glucose transporter 2 (GLUT-2) gene, with a marked increase in the ratio of α to β cells observed in GLUT2-/-islets.¹⁵ Whether these findings are due to a block in the normal islet differentiation program, or to loss of inhibitors that restrain α cell proliferation, is not known. Evidence in support of glucagon itself regulating the numbers of islet α cells derives from analysis of mice with targeted disruption of the genes that encode prohormone convertase-2, or the glucagon receptor. These mice represent models for loss of bioactive glucagon¹⁶ or glucagon action,¹⁷ respectively, and they exhibit marked α cell hyperplasia, which is corrected in the former instance by glucagon replacement therapy.

PROGLUCAGON GENE TRANSCRIPTION FACTORS

A combination of gene transfection experiments using cell lines in vitro and transgenic studies of promoter function in vivo has yielded insight into the molecular control of glucagon gene transcription. Differential gene expression analysis has revealed a remarkably complex pattern of transcription factors preferentially expressed in α versus β cell lines¹⁸ and in single α cells isolated from the developing endocrine pancreas.¹⁹ The rat proglucagon gene promoter directs transcriptional initiation from a single transcription start site that maps to an identical location in brain, pancreas, and intestine.²⁰ Cell transfection experiments utilizing islet cell lines have identified five distinct regions, designated G1 through G5, within the proximal proglucagon promoter that exhibit functional importance for activation of islet cell–specific proglucagon gene transcription. The proximal G1 region is AT rich, interacts with both widely expressed and islet cell–specific proteins, and is functionally important for specifying expression in islet A cells. G1 interacts with the homeobox transcripton factors Isl-1, Cdx-2/3, Brn4, and Pax6.^13,21–24 These transcription factors bind G1 and activate reporter genes containing G1-derived sequences in transfection assays. Pax6 and c-Maf exhibit cooperativity in their ability to increase G1-dependent transcriptional activation,²⁵ whereas Nkx6.1 inhibits Pax6-dependent G1 activation. Reduction of isl-1 expression leads to reduced G1-dependent proglucagon promoter activity and decreased levels of proglucagon mRNA transcripts.²¹ Similarly, increased expression of Cdx-3 in islet cells is associated with induction of both transfected G1-dependent reporter genes and endogenous proglucagon gene expression.^22,26 The G4 element, located just upstream of G1, binds a complex resembling insulin-enhancer factor 1 (IEF1), and recent experiments suggest that IEF1 represents a heterodimer of the helix-loop-helix (HLH) proteins E47 and BETA2.²⁷

The more distal G3 region functions as an islet enhancer–like element and has been divided into two distinct functional domains. Subdomain A contains a sequence element similar to sequences found within the insulin and somatostatin promoters, leading to the designation of this composite sequence as a pancreatic islet cell–specific enhancer sequence, or PISCES element.^28,29 Sequences within domain A of G3 also appear to mediate the inhibition of glucagon gene transcription by insulin.^30–32 The pax6 protein has been identified as a positive activator of proglucagon gene transcription that exerts its function in part through interaction with domain A of the G3 element.¹³ Pax6 also binds the G1 element, either as a monomer or via heterodimer formation with cdx-2/3.³³ Disruption of Pax6 expression in gene-targeted mice results in loss of islet A cells.^12,13 The levels of pancreatic and intestinal proglucagon mRNA transcripts are also markedly reduced in SEY^NEU mice that harbor a dominant negative mutation in the pax6 gene. Thus Pax6, a PISCES binding protein, is functionally important for both islet and enteroendocrine cell development and activation of proglucagon gene transcription via the G3 enhancer and G1 promoter elements.^12,13,33,34

The G2 element mediates the positive and negative actions of HNF-3 (Foxa) proteins, with isoforms of HNF-3β and HNF-3α competing for binding to the G2 element and serving as repressors and activators, respectively, of proglucagon gene transcription.^35,36 HNF-3γ also binds to and transactivates the proglucagon promoter G2 element but does not appear to be essential for islet cell formation or glucagon gene transcription.^37–39 Whether HNF-3β plays an essential role in the development of islet α cell formation or glucagon gene transcription in vivo remains unknown, as targeted inactivation of the HNF-3β gene results in embryonic lethality before formation of the endocrine pancreas.^40,41 Although the numbers of α cells and the morphology of islets appear histologically normal in HNF3α-/- mice, the development of neonatal hypoglycemia in the face of inappropriately reduced levels of circulating glucagon and proglucagon mRNA transcripts demonstrates the essential role of HNF-3α in pancreatic proglucagon gene transcription.³⁷

Activation of the cyclic adenosine monophosphate (cAMP)-dependent pathway leads to transcriptional induction of proglucagon gene expression in both islet and intestinal cells through a cAMP response element (CRE) present upstream of the G3 element in the proximal promoter region of the rat proglucagon gene.^42,43 The CRE also mediates transcriptional activation by ATF3 family members⁴⁴ and is the target for pharmacologic depolarization–dependent induction of rat proglucagon gene transcription.⁴⁵ A second calcium response element has been localized to the G2 element, and calcium responsiveness may be mediated via interaction of nuclear factor of activated T cells (NFAT)-like proteins with members of the HNF-3 family.⁴⁶ The sequence of the CRE element is less well conserved in the human proglucagon promoter, and its functional relevance for proglucagon gene expression in human islets has not yet been elucidated.

Transgenic experiments in mice have identified distinct DNA sequences essential for tissue-specific proglucagon gene transcription in vivo. The first 1253 nucleotides of the rat proglucagon promoter direct heterologous transgene expression to the islets and brain but not the intestine in transgenic mice.⁴⁷ In contrast, targeting of transgene expression to enteroendocrine cells in vivo requires the presence of additional rat proglucagon gene 5′-flanking sequences between −1253 and −2252.⁴⁸ Foxo1 binds to an upstream site at −1798 and appears to be important for both basal and insulin-regulated proglucagon gene transcription in α cells.⁴⁹ DNA sequences constituting the human proglucagon gene promoter exhibit different functional properties than homologous rat sequences, as the first 1600 bp of the human proglucagon gene 5′-flanking region target transgene expression to appropriate cell types in the brain and intestine, but not the pancreatic islets in transgenic mice.⁵⁰

ISLET PROGLUCAGON BIOSYNTHESIS

Consistent with the increased circulating glucagon levels in patients with diabetes, experimental diabetes in rodents is associated with increased levels of pancreatic proglucagon mRNA; correction of the insulin deficiency, but not the hyperglycemia, normalizes the levels of proglucagon mRNA.⁵¹ Paradoxically, insulin may increase proglucagon gene expression in enteroendocrine cells.⁵² Orexin-A also inhibits glucagon secretion and proglucagon gene expression via the phosphatidylinositol-3-kinase–dependent pathway, requiring Foxo1.⁵³ In contrast, few hormones or metabolites have been identified that increase pancreatic proglucagon biosynthesis in vivo. Although experiments using in situ hybridization suggested that 4 days of fasting in the rat leads to a doubling of pancreatic proglucagon mRNA transcripts,⁵⁴ hypoglycemia induced by insulin failed to demonstrate upregulation of proglucagon mRNA.^51,55 Available data suggest that regulation of glucagon secretion, and not biosynthesis, may be a more relevant locus of control in vivo.

Following transcription of proglucagon mRNA and translation of the proglucagon precursor, 29 amino acid mature glucagon is liberated via posttranslational processing by specific prohormone convertases differentially expressed in the islet α cell. In contrast to processing in the enteroendocrine cell (see Fig. 35-1), the amino acid sequences carboxyterminal to glucagon remain unprocessed and are secreted as part of a larger polypeptide designated the major proglucagon fragment (MPGF). PC2 is essential for processing of proglucagon to glucagon in α cells as PC2-/- mice exhibit hypoglycemia, α cell hyperplasia, and glucagon deficiency with an accumulation of incompletely processed PGDPs in the pancreas.¹⁶ Whether PC2 directly liberates glucagon from proglucagon, or cleaves proglucagon to glicentin, which is subsequently processed to glucagon, remains unclear. The enzyme PC1 is responsible for cleavage of proglucagon to yield an intestinal profile of PGDPs,^56,57 and PC1 null mice exhibit impaired processing of proglucagon to the glucagon-like peptides.⁵⁸ Moreover, human subjects with an inactivating mutation in the PC1 gene exhibit incompletely processed proglucagon in gut endocrine cells and deficiency of both GLP-1 and GLP-2.⁵⁹ The convertases important for proglucagon processing in the brain have not been identified definitively.

Glucagon Secretion

The secretion of glucagon is regulated, both positively and negatively, by neuropeptides, hormones, metabolites, and the autonomic nervous system. The islet α cell plays a central role in the defense of blood glucose, with hypoglycemia stimulating and hyperglycemia suppressing glucagon secretion in vivo. A cells express voltage-dependent Na⁺, K⁺, and Ca²⁺ channels that interact in the regulation of membrane potential and, ultimately, depolarization. Amino acids such as glutamine, alanine, pyruvate, and arginine stimulate both insulin and glucagon secretion, and this observation provides the basis for the use of arginine in the assessment of islet and α cell function in rodent and human physiology.⁶⁰ Both α- and β-adrenergic receptors modulate glucagon secretion. Epinephrine stimulates glucagon secretion via protein kinase A (PKA)-dependent enhancement of Ca²⁺ influx through L-type calcium channels, leading to granule exocytosis. Peptidergic activators of glucagon secretion include cholecystokinin (CCK), pituitary adenylate cyclase–activating polypeptide (PACAP), gastrin, urocortin III, vasopressin, and glucose-dependent insulinotropic peptide (GIP).

In contrast, inhibitors of glucagon secretion include glucose, somatostatin-14, and γ-aminobutyric acid (GABA).⁶¹ Somatostatin tonically inhibits glucagon release, and mice with disruption of the somatostatin receptor 2 (SSTR2) gene exhibit mild hyperglycemia and elevated levels of nonfasting glucagon.⁶² Glucose induces GABA release from β cells, providing an indirect mechanism for glucose-mediated suppression of glucagon secretion.^63–65 Glucose also reduces electrical activity and exocytosis via depolarization-induced inactivation of ion channels, specifically KATP, Na⁽⁺⁾ (TTX) and N-type Ca⁽²⁺⁾ channels, as a direct mechanism for inhibition of glucagon secretion. Insulin also may directly inhibit glucagon release, possibly via insulin receptors expressed on α cells. The anatomic arrangement of peripheral islet α cells surrounding a core of β cells, together with the functional results of immunoneutralization studies, provides additional evidence for intra-islet insulin inhibiting downstream of α cells in some species.⁶⁶ Although the mechanisms underlying the paracrine inhibition of α cell activity remain unclear, experimental evidence supports an important role for β cell secretory products such as zinc as negative regulators of glucagon secretion.^65,67

The effects of glucose on glucagon secretion are integrated with the inhibitory effects of insulin on α cell secretion, as hyperglycemia stimulates insulin secretion, whereas a drop in blood glucose suppresses insulin release from the β cell, thereby relieving the α cell from the tonic inhibitory actions of insulin. These actions are exemplified by meal ingestion, which is associated with increased circulating nutrients and insulin secretion, and reduced levels of circulating glucagon. In contrast, blood glucose is maintained in the fasting state by hepatic glucose production due in part to increased levels of glucagon secretion and suppression of insulin release from the β cell.^68,69 Consistent with the effects of individual nutrients on α cell function, infusion of arginine stimulates glucagon secretion in both normal subjects and individuals with diabetes mellitus.⁶⁹ The response to arginine infusion in normal human subjects, namely, stimulation of insulin and glucagon release, is reproducible, and as expected, the insulin response is significantly greater and the glucagon response attenuated as the glucose concentration increases.⁷⁰

In normal subjects, glucagon secretion rises as glucose falls in the fasting state and may be further stimulated by exercise, consistent with the physiologic role for glucagon in regulating glucose production. The levels of both glucagon and plasma catecholamines increase with graded exercise in normal human subjects, with the greatest increments in plasma glucagon observed in subjects undergoing prolonged exhaustive exercise.⁷¹ The exercise-induced increment in plasma glucagon appears dependent on the degree and duration of exercise, with some studies demonstrating no significant changes in plasma glucagon during exercise under normoglycemic conditions.⁷² Trained healthy male subjects exhibit increased hepatic glucose production following glucagon infusion, implying that exercise may be associated with the development of increased glucagon sensitivity.⁷³ Although prevention of exercise-induced rises in plasma glucagon by concomitant somatostatin infusion may result in mild hypoglycemia,⁷⁴ suppression of the rise in plasma glucagon by somatostatin infusion does not always prevent increased hepatic glucose production, likely because of the redundant compensatory mechanisms for maintaining normoglycemia.^74,75

The control of hepatic glucose production (HGP) is highly sensitive to the glucagon/insulin ratio, and secretion of these two hormones generally is regulated in a reciprocal manner. The effect of insulin to suppress HGP actually may be determined in part by the levels of circulating glucagon.⁷⁶ Following meal ingestion, nutrient absorption is associated with energy assimilation and suppression of glucagon secretion. Nevertheless, the integrated response of islet α cells is dependent in part on the nutrient composition of the meal and reflects positive and negative enteric-derived regulators of glucagon secretion. For example, ingestion of carbohydrate, especially glucose, is associated with a decline in plasma glucagon.⁶⁹ Nutrients also promote release of gut-derived peptides such as CCK and GIP, which stimulate glucagon release,^77,78 and GLP-1, which inhibits glucagon release.^79,80

Glucagon secretion is increased during times of stress, and “stress-induced hormones” such as cortisol, vasopressin, and β-endorphin increase glucagon secretion from the α cell. The classic stress hormones epinephrine and norepinephrine also stimulate glucagon secretion, and several mechanisms link the autonomic nervous system to increased secretion from the α cell. Epinephrine secreted from the adrenal medulla increases glucagon secretion in normoglycemic subjects. Furthermore, the pancreas receives innervation from both sympathetic and parasympathetic nerves, and stimulation of these autonomic inputs, all of which are activated by hypoglycemia, increases glucagon secretion.⁸¹ It is intriguing that mice with targeted inactivation of the pro-opiomelanocortin gene (and hence adrenocorticotropic hormone [ACTH]) exhibit a profound defect in the counterregulatory response to insulin-induced hypoglycemia, largely as the result of defective glucagon secretion. Although specific genetic defects in glucagon secretion have not been described, nondiabetic subjects with mutations in the MODY1/HNF-4α gene exhibit decreased arginine-stimulated glucagon secretion and reduced glucose suppression of plasma glucagon,⁸² raising the possibility that the HNF-4α transcription factor influences islet α cell function through a direct or indirect mechanism.

Glucagon Action

The importance of glucagon in the control of hepatic glucose metabolism provides a useful model for analysis of hormone action in metabolic pathways. Glucagon stimulates glucose production via activation of hepatic glycogenolysis and gluconeogenesis, and by inhibition of glycolysis. Following activation of the glucagon receptor, adenylyl cyclase activity is increased, leading to activation of protein kinase A and phosphorylase kinase, and increased rates of glycogenolysis via glycogen phosphorylase and inactivation of glycogen synthase.¹¹⁰ Glucagon action serves to modify the activity of enzymes important for glucose production via effects on specific kinases and phosphatases. Glucagon also modulates the expression of genes encoding enzymes of the glycolytic or gluconeogenic pathways¹¹¹ and regulates fatty acid metabolism via reduction of malonyl CoA and stimulation of fatty acid oxidation. The cAMP-dependent transcription factor is an important downstream mediator for glucagon action, acting in part through activation of the nuclear receptor coactivator PGC-1, and in part through suppression of peroxisome-proliferator–activated receptor-γ (PPAR-γ) agonist activity, which induces a metabolic program that results in the activation of hepatic gluconeogenesis.¹¹²

In adipocytes, glucagon increases cAMP and stimulates lipolysis, thereby providing free fatty acids as substrate for fat-burning tissues. Glucagon inhibits insulin-stimulated glucose transport in adipocytes through effects on insulin binding and via postreceptor mechanisms,¹¹³ and short-term glucagon administration reduces glucose uptake in hepatic and nonhepatic tissues.¹¹⁴ In the peripheral vascular system, glucagon functions as a vasodilator via effects on local vascular tone, and glucagon increases both cardiac output and heart rate, possibly via direct effects on the heart.¹¹⁵ Pharmacologic doses of glucagon increase renal blood flow, glomerular filtration rate, and urinary electrolyte excretion¹¹⁶; the kidney also exhibits significant gluconeogenic capacity and may account for up to 25% of systemic glucose production in humans.¹¹⁷ Although renal glucose output is markedly increased in subjects with diabetes, and hypoglycemia increases renal glucose output in association with increased release of counterregulatory hormones, the available evidence does not support an important role for glucagon in the control of renal glucose output.¹¹⁸

The actions of glucagon are transduced via activation of the glucagon receptor (Gcgr), a seven-transmembrane–spanning G protein–coupled receptor. The cloned receptor responds to glucagon with an increase in both intracellular cAMP and intracellular calcium.¹¹⁹ The human glucagon receptor gene has been localized to chromosome 17q25. Although activating mutations of the Gcgr have been generated by mutagenesis in vitro,¹²⁰ no constitutively active Gcgr mutations have been reported in human subjects. Although several population studies have described a Gly40Ser mutation linked to increased development of diabetes and/or hypertension, whether this mutation itself contributes to a diabetes predisposition or is associated with other genes that increase diabetes susceptibility in certain populations remains unclear.

The tissue distribution of Gcgr expression correlates well with studies localizing high-affinity glucagon-binding sites with Gcgr mRNA transcripts detected in liver, brain, adipocytes, heart, kidney, and islet β cells. Rat Gcgr mRNA transcripts also have been detected in spleen, thymus, adrenal gland, ovary, and testis—nonclassical target tissues where glucagon action remains poorly defined.¹²¹ Although glucagon action has been studied extensively in the liver and adipocytes, the precise biological importance of glucagon in the brain remains unclear. The brain stem is the principal site of central nervous system (CNS) proglucagon gene expression¹²²; however, PGDPs are transported from the brain stem along nerve fibers to multiple brain regions.¹²³ Consistent with these findings, glucagon binding sites are detected in multiple brain regions, and the glucagon receptor is expressed in cortex, cerebellum, hypothalamus, and brain stem; however, the specific biological action of glucagon in each of these CNS regions remains unclear. Although intracerebroventricular injection of glucagon in rodents causes hyperglycemia and increases sympathetic nervous system discharge, whether these findings are relevant to physiologic control of glucose homeostasis requires further analysis.

The β cell expresses receptors for glucagon, GIP, and GLP-1 that all are coupled to cAMP and stimulation of insulin secretion. The threshold for glucagon-stimulated cAMP accumulation in isolated β cells is ≈1 nM glucagon—higher than the concentrations required for cAMP stimulation by GLP-1 or GIP.¹²⁴ The physiologic importance of endogenous glucagon for β cell physiology in vivo remains unclear, given the direction of islet blood flow, the peripheral location of islet α cells in rodents, and the high concentrations of glucagon required to stimulate the β cell in vitro.

Insights into glucagon action derive from characterization of glucagon receptor knockout mice.^17,125 Gcgr-/- mice exhibit marked elevations in levels of plasma glucagon, mild fasting hypoglycemia, increased pancreatic weight, α cell hyperplasia, and increased circulating levels of the PGDPs. The compensatory mechanisms sufficient for maintaining glucose production despite the complete absence of hepatic glucagon receptor signaling remain unknown. Similarly, the glucagon receptor–dependent signals regulating the number and secretory function of islet α cells have not yet been elucidated.

Both the liver and the kidney contribute to glucagon clearance from the circulation; however, these sites account for less than 50% of glucagon clearance, implicating additional tissues as sites for glucagon clearance or degradation.¹²⁶ Glucagon action is terminated via both extracellular and intracellular degradation pathways. Glucagon-degrading activity within hepatic endosomes has been attributed to cathepsins B and D in studies using cathepsin inhibitors,¹²⁷ and both glucagon and GLP-1 are substrates for the widely expressed membrane-bound neutral ectopeptidase (NEP) 24.11.¹²⁸ An endopeptidase activity has been described that cleaves 29 amino acid glucagon to “mini-glucagon,” which is also known as glucagon (19-29) in various tissues. The physiologic importance of mini-glucagon remains uncertain; whether glucagon (19-29) exerts physiologically relevant effects in the heart or β cell via a separate unique receptor remains unclear.

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