The insulin molecule

Insulin was first identified as an anti-diabetic factor in 1921, and was introduced clinically the following year. Its complete amino acid sequence was determined in 1951. Although mature insulin is a dimeric structure, it is synthesized as a single polypeptide precursor, i.e. preproinsulin. This 108 amino acid polypeptide contains a 23 amino acid signal sequence at its amino terminal end. This guides it through the endoplasmic reticulum membrane, where the signal sequence is removed by a specific peptidase. 

Mature insulin consists of two polypeptide chains connected by two interchain disulfide linkages. The A-chain contains 21 amino acids, whereas the larger B-chain is composed of 30 residues. Insulins from various species conform to this basic structure, while varying slightly in their amino acid sequence. Porcine insulin (5777 Da) varies from the human form (5807 Da) by a single amino acid, whereas bovine insulin (5733 Da) differs by three residues. 

Although a high degree of homology is evident between insulins from various species, the same is not true for proinsulins, as the C peptide sequence can vary considerably. This has therapeutic implications, as the presence of proinsulin in animal-derived insulin preparations can potentially elicit an immune response in humans. 

The S5Tnptoms of diabetes have been recognized and recorded for thousands of years. They generally consist of excessive thirst and urination, loss of weight and, until recently, certain death. 

Diabetes meUitus, the most common form of diabetes, is caused by the partial or complete absence of insulin-triggered biological responses. Two forms of diabetes melUtus exist insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes melUtus (NIDDM). 

also known as t5 pe I diabetes, normally occurs during childhood or teens. It is caused by an autoimmune-mediated destruction of the insulin-producing pancreatic B cells. It is thus characterized by the absence or near-absence of insulin in the blood, even at elevated blood glucose levels. 

In the case of NIDDM (maturity onset or type II diabetes mellitus), insulin is present in the blood at normal (or even elevated) levels, but fails to promote any of its characteristic effects. A number of factors can contribute to such insulin resistance, including  

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Insulin and Amylin. Insulin is a member of a family of related peptides, the insulin-like growth factors (IGFs), including IGF-I and IGF-II (60) and amylin (75), a 37-amino acid peptide that mimics the secretory pattern of insulin. Amylin is deficient ia type 1 diabetes meUitus but is elevated ia hyperinsulinemic states such as insulin resistance, mild glucose iatolerance, and hypertension (33). Insulin is synthesized ia pancreatic P cells from proinsulin, giving rise to the two peptide chains, 4. and B, of the insulin molecule. IGF-I and IGF-II have stmctures that are homologous to that of proinsulin (see INSULIN AND OTHER ANTIDIABETIC DRUGS). 

Insulin is composed of two peptide chains covalently linked by disulfide bonds (Figures 5.17 and 6.35). This monomer of insulin is the active form that binds to receptors in target cells. However, in solution, insulin spontaneously forms dimers, which themselves aggregate to form hexamers. The surface of the insulin molecule that self-associates to form hexamers is also the surface that binds to insulin receptors in target cells. Thus, hexamers of insulin are inactive. 

However, if a triplet genetic code system really did exist around 3.5 billion years ago, an RNA strand containing about 100 nucleotides would only have been able to code for a maximum of 33 amino acids. With 33 amino acids, the polypeptide formed would have been only two thirds as long as the insulin molecule, and it is doubtful whether such a chain length would have sufficed for an active replication system. 

The three-dimensional structure of insulin remained recalcitrant in spite of the knowledge of its primary sequence. The early crystals had been found by Scott (1936) to contain zinc which could be replaced by other divalent metals. The zinc atom is not heavy enough to be unambiguously distinguishable. Eventually it proved possible to introduce uranyl acetate and uranyl fluoride into the insulin molecule and to obtain the three-dimensional structure, first at 2.8 A resolution and then at 1.9 A (see Blundell, Dodson, Hodgkin, and Mercola, 1972). 

Physical properties of the protein structure should be considered in designing strategies to achieve stable formulations because they can often yield clues about which solution environment would be appropriate for stabilization. For example, the insulin molecule is known to self-associate via a nonspecific hydrophobic mechanism66 Stabilizers tested include phenol derivatives, nonionic and ionic surfactants, polypropylene glycol, glycerol, and carbohydrates. The choice of using stabilizers that are amphiphilic in nature to minimize interactions where protein hydrophobic surfaces instigate the instability is founded upon the hydro-phobic effect.19 It has already been mentioned that hydrophobic surfaces prefer... 

The significance of Sanger s work is immense. It proved for the first time that the structure of a protein is unique that is, aU molecules of bovine insulin, for example, possess the same sequence of amino acids along the polypeptide chains. This sequence has no obvious order, but it is unique. This singular finding requires that there is a genetic code information encoded in a molecule which specifies the sequence of amino acids in the insulin molecule and, for that matter, in all protein molecules. 

An apparently more effective method for prolonging the half-life of insulin in the blood is to add substituents at the end of the A- or B-chain (or both) that alter the chemical properties of the molecule and delay its breakdown in the body. A product known as HOE 901 (insulin glargine) has two glycine residues added to one end of the B-chain and the A21 asparagine residue replaced with another glycine residue. These changes modify the acidity of the insulin molecule, reducing the rate at which it is absorbed and metabolized in the body. 

The higher molecular mass contaminants in conventional insulin preparations include various proteases. Such preparations are generally maintained in solution at acidic pH values (often as low as pH 2.5-3.5). This minimizes the risk of proteolytic degradation of the insulin molecules, as contaminant proteases are inactive at such pH values. 

The short-acting insulins are dispensed with the insulin molecules in solution, thereby enabling a rapid onset of action. The intermediate- and long-acting insulins are dispensed as turbid suspensions such that mobilization of the insulin molecule from the... 

Insulin is composed of 51 amino acids arranged in two polypeptide chains, designated A and B, which are linked together by two di- sulfide bridges (Figure 23.3A). The insulin molecule also contains an j intramolecular disulfide bridge between amino acid residues of the A chain. Beef insulin differs from human insulin at three amino add positions, whereas pork insulin varies at only one position. 

Figure 7-17 The structure of insulin. (A) The amino acid sequence of the A and B chains linked by disulfide bridges. (B) Sketch showing the backbone structure of the insulin molecule as revealed by X-ray analysis. The A and B chains have been labeled. Positions and orientations of aromatic side chains are also shown. (C) View of the paired N-terminal ends of the B chains in the insulin dimer. View is approximately down the pseudo-twofold axis toward the center of the hexamer. (D) Schematic drawing showing packing of six insulin molecules in the zinc-stabilized hexamer.

If peptide chains can be oriented in a regular fashion, it may be useful to measure infrared linear dichroism.24 25 Absorption spectra are recorded by passing plane polarized light through the protein in two mutually perpendicular directions, with the electric vector either parallel to the peptide chains or perpendicular to the chains. Such a pair of spectra is shown in Fig. 23-3A for oriented fibrils of insulin. In this instance, the insulin molecules are thought to assume a P conformation and to be stacked in such a way that they extend transverse to the fibril axis (a cross-P structure). When the electric vector is parallel to the fibril axis, it is perpendicular to the peptide chains. Since the amide I band is dominated by a carbonyl stretching motion that is perpendicular to the... 

Insulin lispro induces more rapid and constant release of insulin from the injection site, since it consists of monomeric insulin. The change of one or more amino acids in the insulin molecule prevents insulin from forming dimers or hexamers. More rapid absorption, rapid availability, and rapid inactivation make the action better than that of endogenously secreted insulin. When the interval between meals is long, the premeal blood glucose concentration increases rapidly. 

Insulin, a large polypeptide, is not suitable for oral administration. Even if the insulin molecule survived digestion by proteases in the stomach and small intestine, this compound is much too large to be absorbed through the gastrointestinal wall. Consequently, insulin is usually administered through subcutaneous injection. Insulin may also be administered by the intravenous route in emergency situations (e.g., diabetic coma). 

Alternative routes for administering insulin are also being considered.51 In particular, a form of insulin (Exubera) has been developed that can be administered by inhalation or nasal spray, thus precluding the need for subcutaneous injection.88,104 Other modifications of the insulin molecule or use of chemical enhancers can increase the permeability of this hormone so that insulin can be administered through the skin (transcu-taneously) or even via oral or buccal routes.2,35 Technologic and practical advancements in insulin delivery continue to be explored, and methods for administering insulin may be safer and more convenient in the future. 

Insulin preparations that are commercially available differ in their relative onset of action, maximal activity, and duration of action. Conjugation of the insulin molecule with either zinc or protamine, or both, will convert the normally rapidly absorbed parenterally administered insulin to a preparation with a more prolonged duration of action. The various formulations of insulin are usually classified as short acting (0.5 to 14 h), intermediate acting (1 to 28 h), and long acting (4 to 36 h). The duration of action can vary, however, depending on injection volume, injection site, and blood flow at the site of administration. 

If the injected volume is large, diffusion of insulin in the tissue has little significance for the size of the depot and, hence, for the concentration of insulin. However, at the smallest injection volumes, the insulin molecules can rapidly... 

The insulin molecule contains no post-Uanslational modifications such as glycosylation. A and B chains expressed independently, or as proinsulin molecule, were obtained in a stable form at high yields by... 

Pal G, Fong SY, Kossiakoff AA, Sidhu SS. Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis. Protein Sci. 2005 14 2405-2413. Conlon JM. Evolution of the insulin molecule insights into structure-activity and phylogenetic relationships. Peptides 2001 22 1183-1193. 

With oxidized insulin as substrate, a progressive drop in activity of oxidized trypsin samples was observed. In all cases, the extent of cleavage was restricted to the two known sites for tryptic cleavage in the insulin molecule. 

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