A second mutant insulin, identified as [SerB24]-insulin, has a highly hydrophilic character. To determine the biologic activity and the degradation of this mutant insulin, human [SerB24]- and [SerB25]-insulin analogues were semisynthesized from porcine insulin by an enzyme-assisted coupling method. All of the following studies on isolated rat adipocytes were performed at 37°C to directly correlate the binding potency and the biologic activity. The ability of these insulins to displace 125I-porcine insulin bound to adipocytes was 0.5–2% and 1–4%, respectively, of porcine insulin. When the ability of these insulins to stimulate glucose transport and glucose oxidation was measured, both analogues had full activity at high concentrations (250 ng/ml). However, ED50 of the porcine, [SerB24]-, and [SerB25]-insulins tostimulate glucose transport was 0.37 ± 0.05, 46.3 ± 5.4, and 23.3 ± 5.5 ng/ml, respectively. Similarly, for glucose oxidation, ED50 was 0.38 ± 0.06, 33.8 ± 3.6, and 16.6 ± 3.4 ng/ml, respectively. Thus, the biologic activity of [SerB24]- and [SerB25]-insulins was reduced to 0.5–2% and 1–4% of that of porcine insulin, which was compatible with our previous studies under different conditions. No antagonistic effects were observed for either analogue. Degradation of 125I-labeled [SerB24]- and [SerB25]-insulins was also decreased to 62.8% and 55.8%, respectively, of 125I-porcine insulin. These results confirm the importance of the hydrophobic residues at B24 and B25 in the biologic activity of insulin; the patient having this hydrophilic insulin was considered to be in an insulinopenic state despite the hyperinsulinemia due to decreased degradation of the mutant insulin.

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