Glucagon and the Glucagon-Like Peptides
Biosynthesis of Pancreatic Glucagon
Proglucagon is encoded by a single gene in mammals that gives rise to a proglucagon mRNA with major sites of expression in pancreatic islets, brain, and enteroendocrine cells of the small and large intestine. Proglucagon also may be expressed in subsets of salivary glands and taste buds.1 The proglucagon gene contains six exons, several of which encode distinct functional peptide domains (Fig. 9-1). Glucagon, a 29 amino acid peptide, is synthesized in the A cells of the pancreatic islets of Langerhans. Islet A cells are distinguishable from insulin-producing β cells in part by the morphology of their respective secretory granules and by their anatomic distribution, predominantly at the periphery of the islet. The peripheral location of islet A cells, together with functional studies demonstrating central (from a core of β cells) to peripheral (A cells) islet blood flow, raises the possibility of a tightly regulated islet microenvironment. Nevertheless, as the distribution of islet α and β cells may vary from species to species, the functional importance of islet cell distribution remains unclear.
FIGURE 9-1 Structure of proglucagon and the proglucagon-derived peptides. The amino acid sequences of glucagon, glucagon-like peptide (GLP)-1, and GLP-2 are shown, as well as the cleavage site for dipeptidyl peptidase-4 (DPP-4). IP-1 and IP-2, Intervening peptides-1 and -2, respectively.
Islet Transcription Factors and the α Cell
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 E11.9 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 SEYNEU 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. 9-1), 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.
Hypoglycemia
Increasing evidence suggests that multiple complementary mechanisms activate α cell secretion during hypoglycemia. Analysis of the glycemic threshold for activation of counterregulatory mechanisms demonstrates that increased epinephrine and glucagon secretion constitute the initial hormonal responses to decreasing blood sugar. Furthermore, the glucose threshold for activation of counterregulatory responses is clearly higher than the threshold for triggering hypoglycemic symptoms in normal subjects.83 Whether hypoglycemia itself directly stimulates glucagon secretion independent of autonomic input remains unclear. Nevertheless, hypoglycemia suppresses insulin (and β cell) secretion, which removes an important inhibitory influence on glucagon secretion. The finding that α cells express the GLUT1 transporter and glucokinase suggests that glucose transport does not appear to be a critical rate-limiting step for glucose metabolism in α cells and provides important insight into how α cells directly sense ambient changes in glucose concentration.84,85 Hypoglycemia stimulates glutamate release from α cells, which, in turn, activates glucagon secretion in an autocrine manner.86 Glutamate release from ventromedial hypothalamic neurons is also a key component of the counterregulatory response, as mice lacking the synaptic vesicular transporters (VGLUT2) exhibit fasting hypoglycemia and a defective glucagon response to hypoglycemia.87 A role for gastrin in the stimulation of glucagon secretion has been proposed, and gastrin-/- mice exhibit defective glucagon secretion in response to insulin-induced hypoglycemia.88
The role of circulating epinephrine in the autonomic response to hypoglycemia is well established. Epinephrine also directly stimulates glucagon secretion in normal human subjects, and stimulation of autonomic sympathetic nervous system innervation to the pancreas elicits an increase in α cell secretion.89,90 Furthermore, parasympathetic nerve stimulation or the neurotransmitter acetylcholine and the neuropeptide VIP all stimulate glucagon secretion. Studies using nerve transection or pharmacologic blockade have illustrated that these pathways exhibit some degree of functional redundancy, providing multiple backup mechanisms to ensure that the α cell responds appropriately to hypoglycemia. Nevertheless, the ganglionic blocker trimethaphan, which impairs autonomic transmission both in ganglia and in the adrenal, markedly attenuated the glucagon response to hypoglycemia in normal subjects,91 emphasizing the important link between autonomic activation and the glucagon counterregulatory response in vivo. In contrast however, pancreas transplantation restores the glucagon response to hypoglycemia, even in patients with severe autonomic neuropathy,92 illustrating the functional redundancy of mechanisms essential for the α cell response to hypoglycemia.
The identity of the specific glucose sensors that trigger the appropriate counterregulatory response to hypoglycemia remains under active investigation. Mice with genetic disruption of the Kir6.2 channel exhibit normal functional α cells, yet display a marked defect in the glucagon response to hypoglycemia.93 GLUT2 expression in glial cells is essential for the glucagon response to hypoglycemia and the central nervous system, principally the ventromedial hypothalamus (VMH),87 and the splanchnic region, specifically consisting of the portal system and liver, contains multiple glucose sensing systems. Selective perfusion of the portal venous system in rats suggests that the portal vein glucose concentration is a key determinant of the sympathoadrenal response to hypoglycemia; however, whether portal glucose sensors are directly or indirectly linked to control of islet glucagon secretion is not clear.94 It is intriguing that injection of glucose into the portal vein activates glucose-sensitive neurons in the lateral hypothalamus and brain stem, suggesting the possibility of a portal-CNS glucoregulatory axis. The detection of a markedly defective counterregulatory response to hypoglycemia, including absent glucagon and catecholamine secretion, in a patient with hypothalamic sarcoidosis further emphasizes the importance of the hypothalamus in sensing glucose and triggering the release of counterregulatory hormones.95 In contrast, analysis of patients after removal of craniopharyngiomas revealed selective impairment of counterregulatory sympathoadrenal activation but normal glucagon responses to hypoglycemia. Furthermore, human subjects with liver transplants and denervated livers exhibit increased levels of circulating glucagon and defective insulin suppression of glucagon, and human islet transplantation is associated with a defective glucagon response to hypoglycemia. These findings emphasize the importance of the autonomic nervous system and the CNS for the regulation of basal and hypoglycemia-stimulated glucagon secretion.
Glucagon Secretion and Diabetes
Glucagon levels are elevated in many patients with poorly controlled diabetes, and excess glucagon secretion in response to meal ingestion is an early event that is detectable within months of the onset of type 1 diabetes.96 The correction of hyperglucagonemia, using somatostatin or insulin, reverses most metabolic derangements associated with insulin-deficient diabetes.97 Hence there is considerable interest in development of glucagon antagonists for the treatment of diabetes. Moreover, genetic attenuation of glucagon receptor expression in diabetic rodents results in significant amelioration of experimental diabetes.98,99 Despite the central importance of glucagon for control of glucose production, hyperglucagonemia alone, without insulin deficiency, does not significantly increase plasma glucose.100 In the presence of adequate amounts of insulin, glucose production is suppressible despite glucagon excess, emphasizing the insulin:glucagon ratio, and not just the absolute level of glucagon, as a key determinant of glucose homeostasis.101 Nevertheless, hyperglucagonemia is associated with increased leucine oxidation and resting metabolic rate in subjects with type 1 diabetes, reemphasizing the importance of both insulin and glucagon in the catabolism associated with suboptimally treated diabetes.102
The application of intensive insulin therapy to the management of patients with type 1 and type 2 diabetes is associated with an increased incidence of hypoglycemia and heightened awareness of the importance of counterregulatory mechanisms for maintaining normoglycemia. Several factors, including intensive insulin administration, hyperglycemia, and diminished autonomic stimulation of the α cell, inhibit glucagon release in insulin-treated patients with type 1 diabetes.81 Although the glucagon response to hypoglycemia is initially normal in patients with type 1 diabetes, this response frequently becomes impaired, increasing the susceptibility of patients to hypoglycemia.103 Impaired counterregulation and hypoglycemia have been observed in some patients with a very short duration of diabetes, suggesting that frequent episodes of hypoglycemia represent an independent risk for development of an abnormal α cell response. The α cell dysfunction in type 1 diabetes is often selective, as the response to hypoglycemia may be absent, yet glucagon secretion may respond normally to arginine stimulation.104 Further evidence emphasizing the importance of intra-islet hyperinsulinemia or other β cell–derived products in the suppression of glucagon secretion derives from observations that tolbutamide-infused subjects exhibit profound defects in the glucagon response to hypoglycemia.105
It is important to note that restoration of normoglycemia for several months may decrease hypoglycemia unawareness in association with improvement in the glucagon response to hypoglycemia in some studies.106 However, a dissociation between improvement in hypoglycemia unawareness and persistence of defective counterregulatory responses also has been observed.107 Although not completely understood, antecedent hypoglycemia in type 1 diabetes can produce counterregulatory failure during subsequent episodes of prolonged moderate intensity exercise,108 emphasizing the complex interrelationship between insulin-induced and exercise-associated counterregulation.
Multiple defects likely contribute to α cell dysfunction in patients with diabetes. Patients with some residual β cell function, as assessed by C-peptide stimulation, appear to be at decreased risk for hypoglycemia, in part because of preserved counterregulatory glucagon responses. Additional contributing factors may include subtle impairment of autonomic stimulation following repeated hypoglycemic episodes. Nevertheless, impaired epinephrine and glucagon responses to insulin-induced hypoglycemia have been observed after a single antecedent hypoglycemic episode in nondiabetic subjects,109 although increased levels of cortisol alone do not acutely induce defects in hypoglycemic counterregulation. It is intriguing that oral administration of amino acids improves cognitive function and the glucagon response to hypoglycemia in both normal subjects and individuals with type 1 diabetes. Taken together, these observations emphasize the susceptibility of the normal α cell to episodes of hypoglycemia, ultimately leading to sustained defects in the glucagon response to hypoglycemia.
