Monomeric insulins

Fig. 3. Refolding model of insulin protofilaments, from Jimenez et al. (2002). (A) Ribbon diagram of the crystal structure of porcine insulin (PDB ID code 3INS), generated with Pymol (DeLano, 2002). The two chains are shown as dark and light gray with N- and C-termini indicated. The dotted lines represent the three disulfide bonds 1 is the intrachain and 2 and 3 are the interchain bonds. (B) Cartoon representation of the structure of monomeric insulin in the fibril, as proposed by Jimenez et al. (2002). The thick, arrowed lines represent /1-strands, and thinner lines show the remaining sequence. The disulfide bonds are as represented in panel A, and N- and C-termini are indicated. (Components of this panel are not to scale.) (C) Cartoon representation of an insulin protofilament, showing a monomer inside. The monomers are proposed to stack with a slight twist to form two continuous /(-sheets. (Components of this panel, including the protofilament twist, are not to scale.) In the fibril cross sections presented byjimenez et al. (2002), two, four, or six protofilaments are proposed to associate to form the amyloid-like fibrils.

Fig. 15 Schematic drawing of the formation of amyloid fibrils, (a) Monomeric insulin having an a-helical conformation, (b) [i-sheet (arrows) rich oligomers are being formed, (c) Amyloid fibrils having a diameter around 10 nm are being formed, (d) Higher magnification of the intrinsic repetitive (S-pIcatcd sheet structure of the amyloid fibril. The pictures were taken by transmission electron microscopy (TEM)...

The opposite page presents models of insulin, a small protein. The biosynthesis and function of this important hormone are discussed elsewhere in this book (pp.l60,388). Monomeric insulin consists of 51 amino acids, and with a molecular mass of 5.5 kDa it is only half the size of the smallest enzymes. Nevertheless, it has the typical properties of a globular protein. 

The van der Waals model of monomeric insulin (1) once again shows the wedge-shaped tertiary structure formed by the two chains together. In the second model (3, bottom), the side chains of polar amino acids are shown in blue, while apolar residues are yellow or pink. This model emphasizes the importance of the hydrophobic effect for protein folding (see p. 74). In insulin as well, most hydrophobic side chains are located on the inside of the molecule, while the hydrophilic residues are located on the surface. Apparently in contradiction to this rule, several apolar side chains (pink) are found on the surface. However, all of these residues are involved in hydrophobic interactions that stabilize the dimeric and hexameric forms of insulin. 

Brange, J., Ribel, U., Hansen, J.F., Dodson, G., Hansen, M.T., Havelund, S. et al. (1988) Monomeric insulins obtained by protein engineering and their medical imphcations. 

It may be that the exposure time to monomeric insulin plays a role in allergic effects. Pumps with extra short acting insulin give a short contact of monomeric insulin with the body. The same may be true for zinc insulin the released monomers are only for a short period in the circulation. 

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. 

Langkjaer, L. Brange, J. Grodsky, G.M. Guy, R.H. Iontophoresis of monomeric insulin analogues in vitro effects of insulin charge and skin pretreatment. J. Control Release... 

Figure 9.19 Suggested scheme of events after the s.c. administration of soluble human insulin the concentration of hexa-meric zinc-insulin, which is the predominant form of insulin in soluble insulin (40 Unit or 100 Unit - i.e. 0.6 mmol dm ), decreases as diffusion of insulin occurs and os a result the hexamer dissociates into smaller units to achieve monomeric insulin requires a 1000-fold dilution. The importance of the association state is that the larger species have more difficulty dispersing and passing through the capillary membrane.

J. Brange, D. R. Owens, S. Kang and A. Volund. Monomeric insulins and their experimental and clinical implications. Diabetes Care, 13, 923-54 (1990)... 

Recent development of insulin analogues has altered the rates of absorption. Insulin with aspartate and glutamate substituted at positions B9 and B27 respectively crystallizes poorly and has been termed "monomeric insulin (Vora et al., 1988). This insulin is absorbed more rapidly from subcutaneous depots and thus may be useful in meeting postprandial demands. In contrast, other insulin analogues tend to crystallize at the site of injection and are absorbed more slowly (Markussen et al., 1988). Insulins with enhanced biological potency have been produced by substitution of aspartate for histidine at position BIO and by modification of the C-terminal residues of the B-chain (Schwartz et al., 1989). 

Page 1084 is adapted from crystallographic coordinates deposited with the Protein Data Barrk. PDB ID IPID. Brange, J., Dodson, G. G., Edwards, D. J., Holden, P. H., Whittingham, J. L., A Model of Insulin Fibrils Derived from the X-Ray Crystal Structure of a Monomeric Insulin (Despentapeptide Insulin). To be published. 

The polymerization of insuHn and proinsulin to dimers and hexamers is a very important process which takes place in the pancreas and impacts upon the pharmaceutical application of insuHn, because the dissociation of the polymers is the rate-limiting processes in the absorption and action in the tissues of the biological active monomeric insulin [45]. The polymerization diminishes osmotic pressure and hy-drophobicity and improves solubility. A positive correlation between expression yield and the degree of polymerization was found [46] that supports yeast in vivo polymerization of insulin precursors and accentuates its importance. However, polymerization can also account for a drawback by retention of product in the vacuole [47]. Intracellular retention of a substantial quantity of the synthesized insulin precursor indicated that the insulin precursor followed two different intracellular routes in the late secretory pathway [12, 47]. Constitutive secretion to the culture supernatant may reflect saturation of a sorting mechanism in the late Golgi due to overexpression. The kexin cleaves the leader-insulin precursor peptide in a late Golgi compartment to yield free insulin precursor, and... 

By the end of the 1980s, to accommodate postprandial hyperglycemia, monomeric insulin formulations, insulin lispro (the Lys-Pro analog) and insulin aspart (the Asp-Pro analog) had been developed for clinical use [19, 20] (Figure 6.3-1). 

Vola J P, Owens D R, Dolben J, et al. (1988). Recombinant DNAderived monomeric insulin analogue Comparison with soluble human insulin in normal subjects. Br. Med. J. 297 1236-1239. 

The authors then tried cathodal iontophoresis of a monomeric form of insulin, sulfated insulin, in pigs in the belief that the smaller molecular size of the monomeric insulin (MW 5800) versus hexameric regular insulin (MW 35,000) would iontophorese better. In one of the animals studied, a large rise in serum insulin which correlated with a large fall in serum glucose was observed. However, in each of the other animals studied, no similar result was obtained. The authors concluded that insulin iontophoresis is possible and that a smaller monomeric insulin is important. The reasons for failure in all the animals but one remain unknown. 

Figure 8. Transdermal flux of monomeric insulins during in vitro iontophoresis (cathodal...

Brange, J., Owens, D. R., Kang, S., and Volund, A., 1990, Monomeric insulins, and their experimental and clinical imphcations, Diabetes Care 13 923-954. 

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