Biomedical Engineering Reference
In-Depth Information
Insulin has been available for treatment of diabetes since 1923, and the major form for adminis-
tration is still by subcutaneous injection. Insulin therapy typically involves multiple doses of differ-
ent forms of insulin to maintain near-physiological insulin (and thereby glucose) levels. Long-acting
insulin maintains the basal insulin level over 24 h with a single administration, and fast- and short-
acting insulin analogs, which are instantaneously absorbed, are used to meet the insulin requirements
associated with food intake. Thus, the development of insulin formulations with tailored properties,
e.g., a prolonged effect or a faster onset, has always had a high priority in insulin research.
With the biosynthesis of recombinant human insulin in the 1980s it became possible to optimize
the insulin therapy by designing insulin analogs with optimal pharmacokinetic properties by chang-
ing the amino acid sequence of the insulin molecule. A rational design of insulin analogs was only
possible because a large number of insulin structures were determined experimentally by NMR
spectroscopy and x-ray crystallography. Insulin was one of the i rst proteins whose 3D structure
was determined by x-ray crystallography, and today more than 200 insulin structures are available
in the Protein Data Bank.
Insulin exists in the crystalline phase as hexamers, dimers, and monomers. Actually, several
forms of hexamers, T6, T3R3, and R6, exist depending on the presence of zinc ions and phenol
(Figure 2.13). The presence of several hexameric forms of insulin rel ects that even in the crystal-
line form insulin exerts some kind of conformational l exibility. Both zinc ions and phenol are
stabilizing the hexameric form of insulin and are accordingly added to insulin formulations in order
to improve their stability. After subcutaneous injection the insulin hexamer dissociates to dimeric
insulin and by further dissociation to monomeric insulin, which represents the bioactive form. Thus,
from the beginning it was believed that shifting the equilibrium toward the monomeric form would
lead to faster-acting insulins, whereas stabilization of the hexameric form would lead to longer-
acting insulins.
The insulin molecule consists of two peptide chains, an A-chain of 21 residues and a B-chain of
31 residues. The A- and B-chains are connected by two disuli de bridges linking A7-B7 and A20-B19.
With the introduction of recombinant DNA techniques the residues involved in interaction with the
insulin receptor or involved in the hexamer vs. dimer stabilization were identii ed.
From the x-ray structure of hexamer insulin it was evident that the side chain of the HisB10 resi-
due was involved in zinc binding and thereby in stabilizing the hexamer (Figure 2.13). Mutation of
the B10 residue from His to Asp yielded an insulin analog being absorbed twice as rapidly as normal
insulin. Unfortunately, this analog turned out to be mitogenic and thus not suitable for clinical use.
Based on various structural studies of insulin it could be concluded that the l exibility of the
C-terminus is crucial for the binding of insulin to its receptor (Figure 2.14). It also became evident
that the B24-B26 residues are stabilizing the dimer by making an intermolecular antiparallel
(A)
(B)
FIGURE 2.13
(A) Insulin R6 hexamer (pdb-code 1EV6) showing the threefold symmetry of the three dimers
(red-green, cyan-orange, and magenta-blue). (B) Three HisB10 residues coordinate to a zinc ion.
