Abstract
Insulin (2 mg/ml) was incubated with glucose (36 mg/ml) for 14 days at 37°C in 0.1M sodium phosphate buffer, pH 8.5. Incubation was carried out in the presence and absence of 0.25M sodium cyanoborohydride. D-(14C)-glucose or NaB3H3CN was used as a tracer.
Unreacted glucose was removed by gel filtration on a Bio-Gel P-6, and unreacted insulin was separated from glucosylated insulin by affinity chromatogrophy on phenyl boronate coupled to agarose (glyco-Gel B, Pierce, Illinois). Synthetic 14C-glucosylated insulin reduced with NaBH3CN and 3H-glucosylated insulin reduced with NaB3H3CN were eluted in the same fraction. Moreover, we detected sugar in this fraction by periodate oxidation. Thus, we believe that three glucoses, were taken up per mole of insulin monomer.
The immunological reactivity of glucosylated insulin was 20% lower than native insulin.
The hormonal effects on 14C-glucose oxidation and lipogenesis by rat adipocytes was diminished by 70%.
It was shown that glucose was incorporated into the insulin molecule upon incubation in vitro. The biological and immunological activities of the products were reduced.
Based on these results, it might be suggested that glucosylation of insulin also underwent a modification similar to hemoglobin A. We have considerable interest in the glucosylation of insulin in vivo at the hyperglycemic state.
