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Insulin is an essential peptide hormone produced by beta cells in pancreas for regulating glucose homeostasis. The production of insulin occurs throughout the day and is stimulated after meals when large amount of glucose is generated from food digestion in a short time. The normal insulin profile found in blood would have a basal level throughout the day and spikes that are seen after meals. Insulin binds to insulin receptor located on insulin-responsive cells, primarily muscle and adipose cell, to activate an internal cellular mechanism that involves phosphorylation of cellular proteins and ultimately leads to an increase in glucose transporters (GLUT4) on the cell surface. An influx of glucose results and it is converted to glycogen, lipid for storage, or pyruvate for glycolysis. Insulin binding also activates other cascades that lead to decrease in protein, lipid and glucose synthesis. Consequently, the blood glucose level is reduced.

=Insulin and diabetes= Diabetes is the disorder in glucose regulation and affects over 300 million people worldwide in 2013. Diabetes patients are characterized by high glucose concentration in their blood circulation which can lead to symptoms such as frequent urination, increased hunger and thirst, and complications such as heart disease, kidney failure. Insulin therapy is the predominant form of treatment for type 1 diabetes and in some cases, type 2 diabetes. Type 1 diabetes, also known as insulin-dependent diabetes, is caused by autoimmune destruction of beta-producing cells. Genetic or environmental factors such as virus infection, application of certain drugs can cause the disease. Type 2 diabetes, on the other hand, is caused by insulin resistance or insensitivity of human cells to insulin. Obesity is believed to be the major cause of it, and changing lifestyle is the usual treatment. In some cases, insulin therapy is also prescribed for the treatment.

=Insulin structure= Insulin is the first protein to be sequenced, by Frederick Sanger, in 1949. The three dimensional structure of insulin was determined by X-ray crystallography by Dorothy Hodgkin in 1969 (1INS). . Insulin is composed of two polypeptide chains, A and B chain. The A chain contains 21 amino acids in the form of two α-helices. The first helical segment (A1-A7) is followed by a turn and the second helical segment (A14-A20) is antiparallel to the first one. The B chain contains 30 amino acids in the form of an α-helix (B9-B19) and a β-strand (B22-B29). Two interchain disulfide bonds (A7-B7 & A20-B19) connect the two chains, and another intrachain disulfide bond (A6-A11) is also present. Hydrophobic residues including A16 Leu, B11 Leu, B15 Leu, A2 Ile and B24 Phe stabilize the insulin structure by exerting hydrophobic interactions within the protein core. The charged residues, such as A15 Glu, B29 Lys, A21 Asn, are predominately on the surface of the protein to interact with water molecules.



=Insulin monomer, dimer and hexamer= The active form of insulin is its monomer form containing 51 amino acids. Two binding sites with its receptor were proposed: binding site 1 (B12, B24, B25, A21) and 2 (A13, B17). Residues B24, B25 & B26 are also involved in dimerization (1MSO). Dimerization of insulin occurs spontaneously between two monomers by hydrogen bonding and van der Waals interaction along the β-strand in chain B. This implies that the dimer has to be dissociated into monomers before insulin can bind to its receptor. Both monomer (~5.8kDa) and dimer (~11.6kDa) are small enough to diffuse freely in blood and thus are the major forms of insulin found in the blood circulation. When metal ions such as zinc are present, three dimers would associate to form a hexamer (1AI0). The structure is stabilized by interactions between two zinc ions in the core and three histidine residues on each side pointing inwards. The hexamer structure of insulin is stable and mainly found in pancreas where insulin is stored. In fact, the precursor form of insulin, proinsulin, is synthesized and stored in hexameric form as well. After the insulin hexamer is released from pancreas, the lower zinc concentration environment will promote the dissociation of hexamer into dimer or monomer. It is also noted that negatively charged residues glutamic acid are located around the core and the charge repulsion is believed to facilitate the dissociation process once zinc ions are removed.

=Insulin analog= Insulin analog is an altered form of insulin which differs in amino acid sequence from human insulin yet produces similar functions as the human insulin in terms of glycemic control. The insulin analogs that exist in nature are those animal insulins that perform similar functions as the human ones. The development of recombinant DNA technology has fostered the creation of functional insulin with different characteristics in terms of adsorption, distribution, metabolism, and excretion. The U.S. Food and Drug Administration (FDA) call them "insulin receptor ligands", although they are more commonly referred to as insulin analogs.



Animal insulin
The amino acid sequences of insulin in different mammals are similar to human insulin while those across different species of vertebrate are much different. For instance, porcine insulin differs from human insulin by one residue only (Ala in B30 instead of Thr) (3FHP). Bovine insulin differs by three residues (Ala in A8 instead of Thr, Val in A10 instead of Ile, Ala in B30 instead of Thr) (2A3G). Structurally these residues are located at neither protein core nor regions involved with dimerization or receptor binding, hence they display similar properties and functions as human insulin and could be used clinically at the time when biosynthetic human insulin was not available.

Examples of biosynthetic insulin with modified amino acid sequences
Most people who require insulin therapy deliver insulin via syringe or pen subcutaneously. . Those insulin are usually stored in hexameric form and have to dissociate into dimer or monomer and diffuse into blood to be transported to the target cells. Therefore, the timeframe of action of delivered insulin always lag behind the native insulin and is not very effective at controlling blood glucose level after meals when a bolus of insulin is required. The effect of those delivered insulin lasts for only around 3-6 hours so they also cannot be used to supplement the basal level of insulin required throughout the day. By modifying the amino acid sequences of insulin, it is possible to produce insulin that acts faster or maintain their level throughout the day, yet eliciting the same cellular response after binding to its receptor.



Rapid-acting insulin
Rapid-acting insulin begins to work around 15 minutes after injection. The effect would peak in 1 hour and lasts for 2 to 4 hours. This closely mimicks the insulin concentration profile found in blood of normal people after meal. One strategy employed to achieve this is to alter the amino acid sequence at the end of the β strand such that dimerization is prevented. Once the hexameric form of insulin dissociates, most of them will be available as the active monomer form, reducing the time required for further dissociation from dimer to monomer. The key is to replace the proline at B28 which has been shown to take part in intermolecular contact with B21 and B23 of the other monomer. Replacing proline at B28 with other residues has been shown to reduce dimerization.

Insulin Aspart
Insulin aspart marketed by Novo Nordisk as "NovoLog/NovoRapid" was approved by FDA in June 2000 to be used as rapid-acting insulin analog (4GBC). The proline at B28 has been replaced with aspartic acid, which removes the intermolecular contact between proline and the other monomer. Consequently the end of β strand becomes more flexible conformationally and less likely bonds with other monomer to form dimer. The dimer association constant of insulin aspart is around 200 times lower than the normal human insulin.

Insulin Lispro
Insulin lispro marketed by Eli Lilly and Company as "Humalog" is another rapid-acting insulin analog approved by FDA in 1996 (1LPH). The proline at B28 is switched in position with lysine at B29. Same effect of removing the intermolecular contact results that leads to reduction in dimerization. The dimer association constant of insulin lispro is around 300 times lower than the normal human insulin.

Long-acting insulin
Long-acting insulin would reach the blood stream several hours after injection and maintain their level in blood for a 24 hour period. The strategies used for developing this kind of analogs include altering the isoelectric constant of insulin by modifying its amino acid sequence so that it will precipitate in neutral pH and be absorbed slowly. Attaching molecules such as fatty acid to the native insulin can slow down its release due to the binding with large proteins or formation of multihexamers.



Insulin Detemir
In insulin detemir marketed by Novo Nordisk as "Levemir", the B30 residue is removed and the lysine at B29 is conjugated with 14-carbon myristic acid (1XDA). The conjugated myristic acid would not interfere with binding to receptor or dimerization. The delayed action of insulin detemir is achieved primarily through slow absorption into the blood due to two mechanisms: self-association and albumin binding. After injection under the skin, dihexamer that is slowly adsorbed in blood is generated via the interaction between two myristic acid side chains at each pole of the hexamer complex. Subsequent albumin binding to the myristic acid side chains is then thought to further prolong the depot residence time of insulin detemir. After the insulin determir is adsorbed in blood, rapid dilution will cause immediate dissociation into monomers. Plasma albumin binding of insulin detemir would slow its distribution and reduce the rate of transendothelial transport from the bloodstream into the interstitial fluid. Compared with plasma albuming binding, self-association and albumin binding at the injection site produces the dominant effect of protracted action.

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