The influence of insulin on hepatocellular glycogenesis in growing rats

Abstract

The influence of insulin on hepatocellular glycogenesis was investigated by isolating hepatocytes obtained from growing rats at several different stages and the following results were observed.1) The isolation of hepatocytes was performed by collagenase perfusion following a slight modification of Seglen's method. Over 80% cell viability was achieved by this method.2) The number of insulin molecules bound to an aliquot of hepatocytes was the highest at 15°C within the three incubating temperatures of 4, 15 and 37°C during a period of 120 minutes. Meanwhile the amount of degradation products from insulin, which was detected by the fall in molecular weight of 125I labelled insulin, was markedly increased at 37°C. This phenomenon suggested an acceleration of the interaction between the ligand and the cells dependent on a rise in temperature.3) The numbers of insulin receptors were 1.41±0.06, 2.48±0.30, and 3.76±0.16×105 site/cell in hepatocytes obtained from rats at 7, 14 and 21 days after birth by a 4°C measurement. However, there was no difference in receptor numbers between weaning and adult rats.4) The patterns of Scatchard analysis were hyperbolic in the three growth stages. There were no significant differences in binding constants of Ke (empty sites) and Kf (filled sites) by De Meyts's negative cooperative method in the three growth stages.5) Incorporation of 14C labelled glucose into hepatocyte glycogen was stimulated significantly by insulin in 7-day-old rat and adult rats, in contrast to the controls which were not administered insulin.6) The levels of insulin which maximally stimulated glycogenesis in hepatocytes were approximately 10ng/ml in the media at 37°C in every stage. When a comparison was made between the saturation phenomenon of insulin receptors and glycogenesis in the cells, only 20% of the insulin conjugated to the receptors was estimated to be a factor contributing to glycogenesis. This phenomenon suggested the concept of "spare receptors" for insulin working other than as glycogenesis on the cell membrane.