1. 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 receptor (IR) is a transmembrane receptor that is activated by insulin, IGF-I, IGF-

II and belongs to the large class of tyrosine kinase receptors.[1] Metabolically, the insulin receptor

plays a key role in the regulation of glucose homeostasis, a functional process that under

degenerate conditions may result in a range of clinical manifestations

including diabetes and cancer.[2][3] Biochemically, the insulin receptor is encoded by a

single gene INSR, from which alternate splicing during transcription results in either IR-A or IR-

B isoforms.[4] Downstream post-translational events of either isoform result in the formation of a

proteolytically cleaved α and β subunit, which upon combination are ultimately capable of homo

or hetero-dimerisation to produce the ≈320 kDa disulfide-linked transmembrane insulin receptor.[4]

Solubility: Insulin is not soluble at neutral pH. It can be solubilized (1 - 10 mg/ml) in dilute acetic

acid or dilute hydrochloric acid, pH 2-3. A stock solution can be aliquoted and stored at -20°C.

Multiple freeze-thaw cycles should be avoided. Alternatively, it can be stored for up to 6 months

at 2-8°C if it is sterile filtered through a low protein binding membrane or if it contains a suitable

bacteriostat, such as 0.1% thimerosal or sodium azide. Insulin solutions cannot be autoclaved.

Insulin can also be solubilized in 125 mM NaHCO3.23 However, alkaline stock solutions are not

recommended since high pH increases the rate of deamidation and aggregation.16 Brange, et

al.4 have reviewed insulin structure and stability.

Description: Insulin is the primary polypeptide hormone responsible for controlling the cellular

uptake, utilization and storae of glucose, amino acids and fatty acids while inhibiting the

breakdown of glycogen, protein and fat. It is produced by pancreatic beta cells. The precursor

protein (preproinsulin) contains a 23-30 amino acid signal peptide attached to the amino terminal

of proinsulin. Proinsulin is composed of the insulin B-chain followed by a connecting peptide (C-

Peptide) and the A-chain. The signal peptide assists in translocating preproinsulin into the lumen

of the endoplasmic reticulum, after which it is rapidly cleaved. Proinsulin is then transported to

the Golgi complex where it is packaged into granules and converted to insulin. On secretion,

equimolar amounts of insulin and C-peptide are released into the blood.12,26,28

The insulin receptor is a tyrosine kinase that phosphorylates 185 kDa insulin receptor substrate

(IRS-1) found in most cell types. IRS-1 activates phosphatidylinositol 3 kinase (IP3

Kinase).8,10,21The Kd for insulin at its receptor is approximately 0.5 nM (approximately 2.9

ng/ml).21

The comparison of solution structural flexibility and zinc binding domains for insulin, proinsulin

and miniproinsulin has been reviewed by Kaarsholm, et al.20

Biosynthesis

Insulin is produced in beta cells which constitute 75% of the islets of Langerhans of the pancreas. Alpha cells secrete glucagon, delta cells somatostatin.

Insulin is synthesized in the form of a single polypeptide chain, preproinsulin which is transformed into proinsulin which, itself, catalyzed by proteases called furines, gives insulin and C peptide (C for connecting, because connecting the two chains A and B). Bound to two zinc atoms, insulin is stored in granules as a polymer, probably a hexamer.

Secretion

Insulin, as well as C peptide, are released by exocytosis into the portal venous system which leads it directly to the liver, which takes up nearly 50%. The remainder of insulin is distributed throughout the body.

With a basal secretion of approximately 40 microgram/h under fasting conditions, there are increases of secretion linked to meals. To these slow variations are superimposed peaks of pulsatile secretion. The aim of the treatments by exogenous insulin is to approach the physiological curve of secretion.

The principal stimulant of insulin secretion is glucose; it elicits a biphasic release: an immediate effect of short duration and a sustained effect. The cells of the islets are connected by tight junctions, which allow the transfer of ions, of metabolites, secondary messengers from one cell to another, and thus play an important part in synchronizing the secretions.

The stimulation of insulin secretion by glucose requires several steps:

penetration of glucose into beta cells, by Glut2 carriers, independently of the presence of insulin.

phosphorylation of glucose by glucokinase, then its metabolisation with synthesis of ATP whose intracellular concentration increases. This increase in ATP induces the closing of ATP-dependant potassium channels and the cessation of potassium exit, with as a consequence depolarization and opening of the voltage-dependant calcium channels. The entry of calcium elicits the activation of A2 and C phospholipases and the secretion of insulin.

The other stimulants of insulin secretion are amino acids (arginine, lysine), fatty acids and ketonic bodies.

Hormones, others that insulin, and transmitters modulate insulin secretion.

Some increase it:

catecholamines by ß2-mimetic effect. The ß-blockers could theoretically worsen hyperglycemia by decreasing insulin secretion but, in fact, they inhibit especially the hyperglycemic effect of catecholamines and worsen hypoglycemia.

acetylcholine and various hormones of intestinal origin: gastrin, secretin, cholecystokinin, glucagon.

Others decrease it:

alpha-2 sympathomimetics, of which antihypertensive drugs with central effect which can thus worsen hyperglycemia.

somatostatin which inhibits glucagon and insulin secretion and could take part in the regulation of their secretion. Somatostatin is present in delta cells of the pancreas. In large doses, somatostatin inhibits also the release of other hormones such as growth hormone and TSH. It also inhibits intestinal absorption and

motility. Somatostatin secretion is stimulated by glucose, various amino acids and various intestinal hormones.

leptin, by activating leptin receptors of the pancreas.

Hormones that work against the action of insulin, raising blood glucose levels in response

to hypoglycemia (low blood sugar). The main counterregulatory hormones are glucagon,

epinephrine (also known as adrenaline), cortisol, and growth hormone.

People who don’t have diabetes have a number of defense mechanisms against

hypoglycemia. First, the pancreas decreases its insulin output, allowing blood glucose to

rise. Second, the alpha cells of the pancreas secrete the counterregulatory

hormone glucagon, which signals the liver to release more glucose. Third, the adrenal glands

secrete epinephrine, which signals the liver and kidneys to produce more glucose; in addition,

epinephrine keeps certain body tissues, such as muscle, from using as much glucose from

the bloodstream, and it acts to reduce insulin secretion. Epinephrine is the same “fight or

flight” hormone that revs the body up in response to danger, and it produces the symptoms

that normally herald an episode of hypoglycemia, such as hunger, sweating, trembling,

“butterflies,” and heart palpitations. In some cases, especially when glucagon and

epinephrine fail to adequately raise blood glucose levels, the body releases cortisol and

growth hormone, which can also increase blood glucose levels.

2.