Biological Properties of an Initial Degradation Product of Insulin by Insulin-Degrading Enzyme*

Abstract

We previously reported on the degradation of monocomponent porcine insulin by affinity-purified pig skeletal muscle insulin-degrading enzyme (IDE) and on the detection and HPLC separation of the initial degradation product (peak VI). Using relatively high concentration of insulin, peak VI appeared rapidly at 30 sec of incubation, whereas other peaks were not detected within 5 min of incubation. Performate oxidation studies suggested that peak VI is composed of a cleaved A-chain and an intact B-chain. To assess whether the initial degradation product of insulin generated by IDE preserves biological properties, we analyzed several insulin-like activities of peak VI. It had a hypoglycemic effect on rats. In vitro, it bound to the insulin receptors of rat adipocytes and stimulated glucose oxidation there. It also strengthened insulin receptor kinase activity in insulin receptors from rat liver and human placenta. Its biological potency, however, was 1/40th to 1/160th that of insulin itself. This is probably due to reduced affinity for the insulin receptor, since it had 2.5% of insulin's ability to both bind to the receptor and stimulate glucose oxidation. Moreover, peak VI had all of insulin's agonistic effect on glucose oxidation when used at a higher concentration. On the other hand, cross-linking analysis suggested that peak VI preserves almost the same affinity for IDE as does insulin. These results suggest that pig skeletal muscle IDE may cleave peptide bonds within the A-chain early in insulin degradation, generating peak VI; this then serves as the next substrate of IDE while exerting reduced insulin-like activity, and peak VI is converted to several relatively low mol wt products.

Mechanisms Involved in Degradation of Human Insulin by Cytosolic Fractions of Human, Monkey, and Rat Liver

“…However, previous investigations have demonstrated that purified IDE can rapidly degrade HI when used at similar or higher substrate concentrations than used in this study (16,41). These findings, along with the subcellular localization of PDI and IDE reported here, suggest that these enzymes may act in concert, particularly in human and nonrodent species.…”

Short-Term Insulin-Induced Glycogen Formation in Primary Hepatocytes as a Screening Bioassay for Insulin Action

“…Fragment A 14 -21 B 10 -30 is one of the larger known degradation fragments of insulin (16,17), maintaining the region B 24 -B 26 , which is important for the binding of insulin to its receptor (27). Preparations of this fragment isolated from digests of insulin displayed consistent activity between different assays and different preparations (whereas the three other fragments prepared at the same time were always inactive).…”

“…This enzyme may not be specific to insulin since it has been shown to degrade several other peptide hormones, including glucagon (13,14). The fragments produced upon insulin degradation by insulin protease have been well characterized with distinct cleavage sites between residues A [13][14] , A 14 -15 , B 9 -10 , B 10 -11 , B [13][14] , B 16 -17 , B 24 -25 , and B [25][26] (7,(15)(16)(17)(18)(19). Interestingly, partially degraded forms of insulin have been found in the circulation of rats (20), mice (21), and humans (22), where some maintain the capacity to bind to the membrane insulin receptor.…”

“…This suggests that insulin degradation may have distinct biological significance beyond the elimination of excess hormone (7). Some larger fragments that maintain the aromatic region B 24 -B 26 appear to retain insulin-like activity and to potentiate the action of insulin in vitro (23)(24)(25)(26). Importantly, this highly hydrophobic region has been shown to be critical for the high-affinity binding of insulin to its receptor and to insulin protease (27,28).…”

In Vitro Nitrosation of Insulin A- and B-Chains

“…22 Insulin with aminoacid substitutions or deletions and A-or B-chain fragments, obtained either by synthesis or by insulin degrading enzyme (IDE) action, have been described in terms of biological activity. [23][24][25][26] A large number of chemical derivatives have also been reported to retard or increase the activity after administration. 27 Reports in the literature describe insulin molecules that have been submitted to glycation, [28][29][30] phosphorylation, 31 sulfitolysis 32 and peroxinitrite 33 or hypochlorite 34 reaction.…”