Introduction

The recognition of glucagon as a hormone followed Unger's pioneering work reporting on its quantitation by radioimmunoassay.[1] Unger first reported the glycogenolytic, gluconeogenic, and ketogenic effects of glucagon in dogs.[2] Glucagon, manufactured by the alpha cells in the pancreatic islets, stimulates glucose production through glycogenolysis and gluconeogenesis.  Elevated plasma concentrations of glucagon and hyperglucagonemia contribute to the hyperglycemia of diabetes. Hyperglucagonemia also occurs in other clinical conditions such as non-alcoholic fatty liver disease, glucagon-producing tumors, and after gastric bypass surgery.[3] This brief review covers the relevant biochemistry, physiology, measurement, and clinical relevance of glucagon. 

Issues of Concern

Glucagon and insulin work together to maintain euglycemia and glucose transport to tissues. Insulin is a peptide hormone secreted by beta cells of the pancreas and maintains blood glucose by allowing the intracellular transfer of glucose. Insulin also exerts its effect by affecting carbohydrate, lipid, and protein metabolism. Pre-proinsulin becomes proinsulin while it is being transferred through the endoplasmic reticulum. Proinsulin is converted to insulin and C-peptide by an enzyme complex on the outside membrane of the Golgi complex. The ratio of insulin to glucagon affects the degree of phosphorylation or dephosphorylation of the relevant enzymes like glycogen synthase and glycogen phosphorylase.[4] 

The blood glucose levels determine whether glucagon or insulin is activated or inhibited. Low plasma glucose stimulates glucagon secretion, which in turn promotes hepatic gluconeogenesis and glycogenolysis to normalize plasma glucose levels. Excessive glucagon might result from a tumor in the tail or the body of the pancreas, potentially leading to the glucagonoma syndrome, comprising weight loss, necrolytic migratory erythema (NME), diabetes, and mucosal abnormalities including stomatitis, cheilitis, and glossitis.[5] Necrolytic migratory erythema (NME) is a characteristic skin rash most often associated with glucagonoma, an alpha-cell tumor of the pancreatic islets. Erythematous regions with superficial epidermal necrosis spreading in a centrifugal pattern are commonly seen with NME.[6]

Cellular Level

Glucagon plays a role in hepatic glucose production after blocking endogenous insulin production.[7] The proglucagon expressing alpha cells in the pancreas are inhibited by insulin.[8] Proglucagon is cleaved by prohormone convertase 2 (PC2) to form fully processed bioactive glucagon and is secreted from the pancreatic alpha cells in response to hypoglycemia or increasing concentration of amino acids.  Glucagon secretion is affected by blood glucose levels and enhanced by oxyntomodulin and glucose-dependent insulinotropic polypeptide (GIP) while inhibited by glucagon-like peptide-1 (GLP-1).[9] The autonomic nervous system may also play an important role in glucagon secretion. Paracrine factors were shown to regulate glucagon production from pancreatic alpha cells. In the islets, GABA, somatostatin, and insulin inhibit glucagon secretion.[10][11][12] Insulin is produced by beta cells, while somatostatin is produced by delta cells. [13] 

Acinar cells of the pancreas are responsible for the exocrine function. [14] Increasing glucose levels also inhibit glucagon secretion while hypoglycemia stimulates glucagon and other counter-regulatory hormones, such as epinephrine and cortisol, to correct the hypoglycemia and avoid its serious sequelae. Hypoglycemia occurs when the fasting blood sugar levels fall below 70 mg/dl or when you have symptoms of hypoglycemia. Signs of hypoglycemia can either be adrenergic, which includes tremor, palpitations, anxiety, or cholinergic, which is hunger, sweating, paresthesia. Other possible symptoms could include behavioral changes, confusion, lethargy, seizure, coma, and death if the hypoglycemia is not rectified. Hypoglycemia is more likely in type I DM than type II DM. In terms of medications, sulfonylurea, meglitinides, and insulin are the most common culprits. When hypoglycemia is noted, questions about medications taken, history of alcohol or drug use, history of kidney injury, unintentional weight changes, or history of psychiatric disorders must be asked. Hypoglycemia is treated with IV dextrose or glucose in unconscious patients, while oral glucose (fruit juice preferred) can be attempted in conscious patients. If patients are not responsive or are unable to take oral medications, a 1-mg intramuscular (IM) injection of glucagon is an option.[15] 

The incretin hormone GLP-1 also inhibits glucagon secretion. Gluconeogenic amino acids such as alanine stimulate glucagon secretion. Although it remains unknown whether glucagon feedback to alpha cells is in an autocrine manner, according to recent studies, alpha cells do not express glucagon receptor (GCGR), and it may thus regulate its own secretion by activating insulin.[16]  

Development

The proglucagon gene encodes it, and the proglucagon peptide is processed by prohormone convertase-2 (PC2) to produce the 29-amino acid mature peptide. Enhancement of glucagon transcription is by amino acids and cyclic AMP in the pancreas and intestinal cells, and Wnt signaling in the intestine.[17]

Organ Systems Involved

Similar to insulin, glucagon secretion is tightly regulated depending on blood glucose levels. In the pancreas, the prohormone proprotein convertase 2 processes proglucagon (160 amino acids (aa)) to the glicentin-related pancreatic polypeptide (GRPP, 1–30 aa), glucagon (33–61 aa), intervening peptide-1 (IP-1), and major proglucagon fragment (72–158 aa). In the intestine, proglucagon is processed by PC1/3 activity to glicentin (1–69 aa), GLP-1 (78–107 aa), IP-2, and GLP-2 (152–158 aa). Glicentin is further cleaved into GRPP (1–30 aa) and oxyntomodulin (33–69 aa). In the intestine, glucagon with N-terminally elongated form (1–61 aa) also forms. Hence, the entire amino acid sequence of proglucagon is also present in oxyntomodulin and glicentin, which the intestinal L-cells secrete in response to food intake. Reports exist of extra-pancreatic glucagon secretion after pancreatectomy in humans and dogs.[18][19] Also, a molecule with a molecular weight similar to that of pancreatic glucagon has been reported to be produced in the gastrointestinal tract of several species.[20] 

Function

The primary function of glucagon is to increase the hepatic glucose output, thereby restoring euglycemia. Administration of glucagon increases glucose levels via gluconeogenesis and glycogenolysis in fasted or fed animals and in humans.[17] Incubation of glucagon-induced glucose output from primary hepatocytes in culture was observed. Inhibition of glucagon signaling leads to decreased plasma glucose concentrations.[21]

Mechanism

Glucagon binds to its membrane-bound receptor, a seven-pass transmembrane G-protein-coupled receptor (G-stimulatory protein). [8] Glucagon receptor gene (GCGR) encodes the receptor and has an abundant expression in the liver and kidney and less expression in the heart, adipocytes, lymphoblasts, spleen, pancreas, brain, retina, adrenal gland, and the gastrointestinal tract.[22] Upon glucagon binding, the receptor stimulates adenylate cyclase, which induces cAMP levels and activates the protein kinase A (PKA) pathway, the most common pathway activated. Other studies also have shown that glucagon could activate other pathways involving 5’-AMP-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). 

Glucagon signaling, while promoting glycogenolysis, it inhibits glycogen synthesis in the liver. Upon glucagon stimulation, activated PKA phosphorylates glycogen phosphorylase kinase, which phosphorylates serine-14 residue of glycogen phosphorylase. This activated glycogen phosphorylase phosphorylates glycogen, resulting in increased glycogenolysis and the production of glucose-6-phosphate. Glucose-6-phosphate is converted to glucose by glucose-6-phosphatase, resulting in increased plasma glucose levels. Glucagon modulates glucose-6-phosphatase activity via the PKA-dependent mechanism. 

In addition to promoting glycogenolysis, glucagon also inhibits glycogenesis in the liver at the same time. Glycogen synthase is a significant regulatory enzyme in the glycogenesis pathway. This enzyme catalyzes the transfer of a glucosyl residue from UDP-glucose to a nonreducing end of the branched glycogen molecule. Glucagon induces phosphorylation of glycogen synthase and thereby inhibits glycogen synthase activity in the liver. Phosphorylation of glycogen synthase by multiple kinases, including PKA and other serine/threonine kinases leading to a graded inactivation, which leads to decreased glycogen synthesis and therefore increases the glucose output to the blood.[8][23] Glucagon-induced phosphoenolpyruvate carboxykinase (PEPCK) is the enzyme in a rate-limiting step in hepatic gluconeogenesis. PKA activation leads to the activation of CREB, leading to the increased synthesis of PGC-1 protein. Both PGC-1 and HNF-4 are coactivators that increase the transcription of the PEPCK gene. In addition to the above functions, glucagon inhibits glycolysis by inhibiting phosphofructokinase-1. Glucagon also stimulates lipolysis and ketogenesis, and the ratio of glucagon to insulin perceived by the liver is a critical signal for the liver to convert fatty acids to ketone bodies as in diabetic hyperglycemic coma.[24] 

Related Testing

Glucagon has a half-life of 3-6 minutes. Hence, the sample collection should be in chilled tubes with a proteolytic enzyme inhibitor. In adults and children, the normal plasma levels are less than 120 pg/ml, while in cold blood, the levels can go as high as 215 pg/ml. The sample collection should be in chilled ethylenediaminetetraacetate (EDTA) or dipotassium EDTA plus aprotinin tubes after overnight fasting. Storage pending the taking of measurements should be at -70 degrees celsius after separation from the cells.

In patients with kidney failure, there may be increased levels of  N-terminally elongated proglucagon (1-61 aa); thus, glucagon measurements in subjects with impaired kidney function may lead to an overestimation of endogenous glucagon secretion. The stability of the glucagon molecule and the sensitivity of the assay are considerations when measuring glucagon levels.

Pathophysiology

In patients with type II diabetes mellitus (TIIDM), elevated plasma glucagon levels are observed in the fasting state and defective suppression in the postprandial state, resulting in high glucagon levels in the blood.[25] This defective state has led to the bi-hormonal hypothesis and the importance of glucagon in the pathogenesis of diabetes. Hyperglucagonemia has been observed in patients with pancreatic neuroendocrine tumors.[26] Hyperglucagonemia due to N-terminally elongated forms of the glucagon molecule may be found in patients with renal dysfunction.[27]

Clinical Significance

Although the measurement of plasma glucagon levels is not recommended in the diagnosis and management of diabetes, the role played by glucagon in the pathogenesis of diabetes, particularly TIIDM, has been confirmed by drugs that modulate the incretin axis such as GLP-1 receptor agonists that inhibit glucagon and decrease hyperglycemia and the DPP4 inhibitors that increase endogenous GLP-1 and hence inhibit glucagon and hyperglycemia.