Seiji Ohgaku

Supernormal insulin: [D-PheB24]-insulin with increased affinity for insulin receptors

Abstract [D-Phe B24 ]- and [D-Phe B25 ]-human insulin were semisynthesized from porcine insulin by enzyme assisted coupling method. Receptor binding ability of [D-Phe B24 ]- and [D-Phe B25 ]-insulin was 180% and 4%, respectively, of that of human insulin. Increased affinity of [D-Phe B24 ]-insulin was ascribed to markedly decreased dissociation rate in binding to human cultured lymphocytes. Negative cooperative effect of [D-Phe B24 ]insulin was also increased to twice of that of human insulin. Biological activity of these analogues was assessed by 2-deoxy-glucose uptake studies in isolated adipocytes and the ability of [D-Phe B24 ]- and [D-Phe B25 ]-insulin was 140% and 4%, respectively, of that of human insulin. These findings suggest that B25 L-Phe is more crucial for receptor binding and that [D-Phe B24 ]-insulin is the first semisynthetic insulin to show increased affinity for insulin receptors.

A new potentiator of insulin action: Post-receptor activation in vitro

A new potentiator of insulin action, 5‐[4‐(1‐methylcyclohexylmethoxy)‐benzyl]thiazolidine‐2,4‐dione, was tested for activation of insulin action in vitro. The agent (50 mg.kg−1.day−1) was orally administered to rats for 14 days and adipocytes from treated rats were used to assess insulin‐binding, glucose uptake and glucose oxidation. In obese rats, the agent increased glucose uptake and oxidation without any change in insulin binding, whereas in lean or streptozotocin‐treated rats it failed to increase glucose metabolism. Fat tissues were cultured with the agent for 24 h and were tested for insulin action. In the presence of insulin (10 ng/ml) in the culture media, the agent increased glucose oxidation in these cells without any change in insulin binding. However, without insulin in the culture media the agent did not increase glucose oxidation. Thus, the agent appeared to potentiate insulin action at the post receptor process.

Immunoreactivity and biologic activity of semisynthetic [LeuB-30]-insulin: potential value in the treatment of insulin antibody-mediated insulin resistance

Insulin analogues with different amino acids, including threonine, alanine, L-leucine, D-leucine, L-leucine amide, phenylalanine, tri-alanine, or desalanine, at the B-30 position were semisynthesized from pork insulin by the new enzymatic method. The order of ability of the insulin analogues to bind to anti-insulin sera was [AlaB−30] > desalanine > [ThrB−30] > [Ala-Ala-AlaB−30] > [D-LeuB−30], [Leu-NH2B−30], [PheB−30] > desoctapep-tide > [LeuB−30]. The ability of insulin analogues with different amino acids at B-30 to bind to receptors, as well as their biologic potency tested with glucose uptake in isolated rat adipocytes, was comparable among the analogues. These results suggest that [LeuB−30]-insulin demonstrated the least immunoreactivity and has full activity in receptor binding and biologic effect, and that it may be useful for treatment of anti-insulin antibody-mediated insulin resistance.

Receptor-binding kinetics of A-14 and A-19 125I-labelled insulin

Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 125I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.