Characterization of [LeuB-24]- and [LeuB-25]-insulin analogues. Receptor binding and biological activity

Abstract

Human [LeuB-24]- and [LeuB-25]-insulins were semi-synthesized from porcine insulin by an enzyme-assisted coupling method. The receptor-binding ability of [LeuB-24]- and [LeuB-25]-insulins was 30--48% and 2--5% respectively of that of human insulin. There was no significant difference in degradation between human insulin and these analogues on incubation with isolated adipocytes. The decreased affinity of these analogues was due to an increased dissociation rate rather than a change in the association rate of their binding to human cultured lymphocytes. The negative co-operative effect of [LeuB-24]- and [LeuB-25]-insulin was decreased to 50 and 1% respectively of that of human insulin at a concentration of 100 ng/ml. The ability of [LeuB-24]- and [LeuB-25]-insulin to stimulate 2-deoxyglucose uptake in isolated rat adipocytes was 35 and 4% respectively of that of human insulin. These analogues did not have an antagonistic effect on the biological activity of human insulin. The immunoreactivity of [LeuB-25]insulin was similar to that of porcine or human insulin, whereas [LeuB-24]insulin demonstrated decreased binding to anti-(porcine insulin) antibodies. These findings suggest that B-chain phenylalanine-25 residue is more crucial for receptor binding and negative co-operativity, whereas the B-chain phenylalanine-24 residue may play a more important role in binding to anti-insulin antibody.

Human insulin B24 (Phe----Ser). Secretion and metabolic clearance of the abnormal insulin in man and in a dog model.

“…1 (18) and that immunometric equivalency must be separately assessed for each new or uncommon insulin analogue under investigation.…”

Contribution of TyrB26 to the Function and Stability of Insulin

“…Vertebrate insulin sequences exhibit broad conservation (17), including several invariant residues recently shown to pack at the primary hormone-receptor interface (4) (13,(55)(56)(57). Co-evolution of the hormone-receptor interface has presumably led to the strict conservation of such contact sites.…”

Ultrastructural basis for chloroquine-induced increase in intracellular insulin in adipocytes: alteration of lysosomal function.

“…Adipocytes internalize membrane-associated insulin and this internalization results in the degradation of the hormone (2)(3)(4)(5)(6)(7)(8) or the receptor (25,26). The degradation occurs at a chloroquine-sensitive location (6-8, 25, 26) and therefore, it has been presumed that adipocyte lysosomes accumulate and degrade This study showed that chloroquine had no effects on the initial binding of insulin to its receptor, on the distribution of insulin receptors on the cell surface, or on the initial phases of the uptake process.…”

“…An increased accumulation of intact insulin in adipocytes has been reported when lysosome function was inhibited by chloroquine (6-8); quantitative biochemical analyses of the insulin uptake process in adipocytes have estimated that about 15-30% of the receptorbound hormone was internalized (6-8) and approximately 25-50% ofthe internalized hormone was degraded at a chloroquinesensitive site (5, 9). In a number ofstudies chloroquine-sensitive insulin degradation was found to be a small percentage of the total insulin-degradative activity of adipocytes (6)(7)(8)10).Several laboratories have used morphological techniques to study the binding and uptake of insulin (for a review, see ref.11). We have found that monomeric ferritin-labeled insulin initially occupied receptor sites on the adipocyte plasma membrane and was endocytosed in pinocytotic invaginations and transported to cytoplasmic vesicles (unpublished data).…”

“…An increased accumulation of intact insulin in adipocytes has been reported when lysosome function was inhibited by chloroquine (6-8); quantitative biochemical analyses of the insulin uptake process in adipocytes have estimated that about 15-30% of the receptorbound hormone was internalized (6-8) and approximately 25-50% ofthe internalized hormone was degraded at a chloroquinesensitive site (5, 9). In a number ofstudies chloroquine-sensitive insulin degradation was found to be a small percentage of the total insulin-degradative activity of adipocytes (6)(7)(8)10).…”