Receptor Binding and Tyrosine Kinase Activation by Insulin Analogues With Extreme Affinities Studied in Human Hepatoma HepG2 Cells

Abstract

The insulin-receptor affinity of five human insulin analogues with one to four amino acid substitutions was measured with human hepatoma cells (HepG2). The binding affinities ranged from 0.05% for AspB25 insulin, 18% for AspB9, GluB27 insulin, 80% for AspB28 insulin, and 327% for AspB10 insulin to 687% for HisA8, HisB4, GluB10, HisB27 insulin relative to human insulin. Binding constants obtained by competition experiments at steady state with [125I]TyrA14-labeled insulin and unlabeled analogues and by kinetic studies with [125I]TyrA14-labeled analogues and insulin gave essentially the same values. The kinetic studies showed that differences in affinity between analogues were due to differences in both dissociation and association rate constants. The affinity for insulinlike growth factor I receptor was low, ranging from less than 0.005% for AspB25 insulin to 0.6% for HisA8, HisB4, GluB10, HisB27 insulin. The potencies of insulin analogues in activation of the tyrosine kinase of solubilized and partially purified insulin receptors from HepG2 cells, measured with the exogenous substrate poly(Glu80-Tyr20), ranked in the same order as the binding affinities, the actual values being somewhat elevated for the high-affinity analogues, however. We conclude that these human insulin analogues are active in insulin-receptor binding and tyrosine kinase stimulation but show wide variation in affinity.

Insulin X10 revisited: a super-mitogenic insulin analogue

“…In the majority of studies the binding affinity of insulin X10 to IR has been found to be increased 200-400% relative to that of human insulin. It was also noted by these early studies that the binding affinity of insulin X10 to IGF-1R was increased compared with that of human insulin [17,18] but with a fairly low affinity compared with IGF-1 itself, an effect that has subsequently been confirmed by a number of authors [19][20][21][22]. In addition to the increased receptor affinities, [12] insulin X10 was shown to have an increased in vitro ability to stimulate cell growth and DNA synthesis, and Bornfeldt and colleagues speculated that this was due to a substitution in the insulin X10 molecule that made this analogue more chemically similar to the IGF-1 molecule [18].…”

“…One of the first molecular characteristics that was observed to differ between insulin X10 and human insulin was increased binding to IR itself [11,17]. In the majority of studies the binding affinity of insulin X10 to IR has been found to be increased 200-400% relative to that of human insulin.…”

Stimulation of cloned human glucagon‐like peptide 1 receptor expressed in HEK 293 cells induces cAMP‐dependent activation of calcium‐induced calcium release

“…[125I]GLP-1 (7-36)amide was prepared by iodinating the peptide with ~25I using HzO2/lactoperoxidase at neutral pH and purified by reverse-phase HPLC as described elsewhere [7], resulting in a specific activity of 1.76/.tCi/pmol. All other chemicals were obtained from Sigma Chemicals (St. Loius, MO, USA).…”

Biosimilar insulins: guidance for data interpretation by clinicians and users

“…A biosimilar insulin should then bind to, and dissociate from, the two main types of insulin receptor, and the insulin‐like growth factor‐1 (IGF‐1) receptor, to the same measurable extent as the original insulin. The dissociation rate is thought to be important because the post‐receptor signalling pathways of insulin include metabolic and growth promotional pathways, and a historical rapid‐acting insulin analogue (B10‐Asp‐human‐insulin) is believed to have manifested its tumorigenic activity as a result of slow dissociation from its receptor 13. Assessment of both receptor binding and post‐receptor activity is further complicated because a range of tissues and cell lines have been used for preclinical studies with different insulins, often with limited clarity as to their pathophysiological relevance.…”