Insulin synthesis, secretion and degradation
Insulin is produced by the beta-cells in the pancreatic islets. Its synthesis involves sequential cleavage of its two precursor molecules preproinsulin and proinsulin. The gene encoding preproinsulin is located on the short arm of chromosome 11. Following synthesis the preproinsulin molecule undergoes rapid enzymatic cleavage to proinsulin, which contains the insulin A and B chains linked by connecting or C-peptide. Proinsulin is packaged into small granules within the Golgi complex, which then migrate towards the cell surface. As the granules mature, proteases split proinsulin into equal amounts of insulin and C-peptide, allowing the insulin molecule, consisting of A and B chains linked by two disulfide bridges, to assume its active configuration. Insulin forms microcrystals around zinc ions within the secretory granules, producing hexamers which separate rapidly following release. Rising intracellular glucose triggers insulin secretion by activation of glucokinase followed by an increase in intracellular ATP, resulting in closure of the ATP-sensitive potassium channel. This causes depolarisation of the beta-cell membrane and the influx of calcium ions, leading to fusion of the insulin granules with the cell membrane and the release of insulin, C-peptide and other molecules into the circulation by exocytosis.
The insulin molecule
The primary structure of the insulin molecule was elucidated by Frederick Sanger in 1951, and its tertiary structure by Dorothy Hodgkin in 1969. Human insulin is a protein consisting of a A-chain with 21 amino acids, and a B-chain with 30 amino acids. The chains are linked by two disulfide bridges between the cystein residues at positions A7 and B7, and A20 and C19. An additional disulfide-bridge connects the cystein residues at A6 and A11, which is important for determining the tertiary structure and receptor binding of the molecule. Insulin has a molecular weight of 5808. Its iso-electric point (point of least ionisation/ water solubility) is at an pH of 5.4. Human insulin aggregates to dimers, hexamers and more complex crystalline structures in the presence of zinc ions and low pH, as found in the secretory granule.
The insulin gene
The insulin gene is evolutionary remarkably conserved across species, and diverged from its sister molecule insulin-like growth factor-1 (IGF-1) early in the course of chordate evolution. The human gene lies on the short arm of chromosome 11. The regulation of insulin gene expression is of course influenced by glucose but other factors such as Glucagon-Like Peptide-1 (GLP-1) and Growth Hormone play a part. The product of the insulin gene is the elongated, single-chain preproinsulin (98 amino acids) which is processed in the rough endoplasmatic reticulum to proinsulin, by removal of the so-called signal peptide (12 amino-acids). In the endoplasmatic reticulum, the single chain proinsulin folds back onto itself, aligning the future A- and B-chain and creating the disulfide bonds in this process. The A-chain and B-chain are still connected by the Connecting Peptide (C-peptide). In the Golgi-complex, the proinsulin is stored in so-called beta-granules. These contain the proteolytic enzymes that will cleave and remove the C-peptide from proinsulin, resulting in equimolar amounts of insulin and C-peptide in the mature beta-granule.
The mature beta-granules form a large storage pool for insulin, well in excess of the daily requirement. Insulin is released into the circulation by fusion of the granules with the beta-cell membrane and exocytosis. A series of events triggers insulin secretion. Physiologically, glucose enters the beta-cell through an insulin independent process (probably involving the glucose transporter 1, GLUT-1). There it is phosphorylated by the enzyme glucokinase and metabolized through glycolysis and entry into the mitochondrial TCA cycle. This results in the generation of ATP which is transferred back to the cytosol and increases the ATP/ADP ratio. This increased ATP/ADP ratio leads to closure of the ATP-dependent potassium channel (KATP channel) which leads to depolarisation of the beta-cell membrane. The depolarisation of the cell membrane activates voltage-sensitive Ca2+ channels, leading to an influx of Ca2+ into the cell. This forms the final trigger for insulin exocytosis. The granule membrane is recycled to the Golgi apparatus following release of insulin.
Insulin has a short half-life in the circulation following release, estimated at 4-6 minutes, allowing minute-to-minute regulation of metabolism. Circulating insulin is cleared by the liver as it passes through the portal circulation, which means that portal levels of insulin are higher than those in the systemic circulation. The kidney is largely responsible for insulin clearance in the systemic circulation, and delayed insulin clearance may cause problems with control in those with kidney disease. Some degradation occurs within the insulin granule, and insulin is degraded in other tissues after binding to the insulin receptor. In this receptor-mediated degradation, the insulin-insulin receptor complexes come together on the plasma membrane of the target cell, forming groups that are sequestered in so-called coated-pits. These invaginate to fuse with intracellular lysosomes, in which the insulin is enzymatically degraded.