16 research outputs found
Molecular Characterisation of Long-Acting Insulin Analogues in Comparison with Human Insulin, IGF-1 and Insulin X10
AIMS/HYPOTHESIS: There is controversy with respect to molecular characteristics of insulin analogues. We report a series of experiments forming a comprehensive characterisation of the long acting insulin analogues, glargine and detemir, in comparison with human insulin, IGF-1, and the super-mitogenic insulin, X10. METHODS: We measured binding of ligands to membrane-bound and solubilised receptors, receptor activation and mitogenicity in a number of cell types. RESULTS: Detemir and glargine each displayed a balanced affinity for insulin receptor (IR) isoforms A and B. This was also true for X10, whereas IGF-1 had a higher affinity for IR-A than IR-B. X10 and glargine both exhibited a higher relative IGF-1R than IR binding affinity, whereas detemir displayed an IGF-1R:IR binding ratio of ≤ 1. Ligands with high relative IGF-1R affinity also had high affinity for IR/IGF-1R hybrid receptors. In general, the relative binding affinities of the analogues were reflected in their ability to phosphorylate the IR and IGF-1R. Detailed analysis revealed that X10, in contrast to the other ligands, seemed to evoke a preferential phosphorylation of juxtamembrane and kinase domain phosphorylation sites of the IR. Sustained phosphorylation was only observed from the IR after stimulation with X10, and after stimulation with IGF-1 from the IGF-1R. Both X10 and glargine showed an increased mitogenic potency compared to human insulin in cells expressing many IGF-1Rs, whereas only X10 showed increased mitogenicity in cells expressing many IRs. CONCLUSIONS: Detailed analysis of receptor binding, activation and in vitro mitogenicity indicated no molecular safety concern with detemir
Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin
Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts. Because the mitogenic effect of growth factors such as insulin is expected to act in G0/G1, promoting S-phase entry, we developed a combined topoinhibition + serum deprivation strategy to explore the effect of G0/G1 synchronization as an independent parameter in the context of serum deprivation, the latter being routinely used to reduce background in mitogenicity assays. G0/G1 synchronization significantly improved the mitogenic responses of L6-hIR cells to insulin, measured by 3H-thymidine incorporation. Comparison with the parental L6 cells using phospho-mitogen-activated protein kinase, phospho-AKT, as well as 3H-thymidine incorporation end points supported that the majority of the mitogenic effect of insulin in L6-hIR cells was mediated by the overexpressed hIR-A. Using the optimized L6-hIR assay, we found that the X-10 insulin analog was more mitogenic than native human insulin, supporting that X-10 exhibits increased mitogenic signaling through the hIR-A. In summary, this study provides the first demonstration that serum deprivation may not be sufficient, and G0/G1 synchronization may be required to obtain optimal responsiveness of hIR-overexpressing cell lines for preclinical safety testing
Duration of activation of insulin receptor isoform A and isoform B at different phosphorylation sites.
<p>Results are presented as mean ± SE, n = 8. IR, insulin receptor; IGF-1, insulin-like growth factor 1.</p
Potential mechanisms influencing the balance of metabolic and mitogenic actions of insulin-like molecules.
<p>Reprinted with kind permission from Springer Science & Business Media: Hansen <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034274#pone.0034274-Hansen1" target="_blank">[9]</a>, Fig. 2. IGF-1, insulin-like growth factor 1.</p
Relative binding affinities for insulin receptor isoform A and isoform B and IGF-1 receptors.
<p>Affinities were determined by insulin competition binding in a scintillation proximity assay; data are means (± SD) of quadruplicates (solubilised receptors) or duplicates (membrane-bound receptors).</p
Competition curves for displacement of 125I-IGF-1 from Hybrid-A and Hybrid-B with human insulin IGF-1, insulin detemir, insulin glargine, insulin X10 or IGF-1 in SPA binding assay.
<p>The graphs are representatives of three experiments. Each point in the graphs is the mean (±SE) of three measurements. IGF-1, insulin-like growth factor 1; SPA, scintillation proximity assay.</p
Dose-response curves for potencies for activation of insulin receptor isoform A and isoform B at different phosphorylation sites.
<p>Results are presented as absorbance (arbitrary value) mean ± SE, n = 4. IR, insulin receptor; IGF-1, insulin-like growth factor 1.</p
Relative binding affinities for Hybrid-A (IR-A/IGF-1R) and Hybrid-B (IR-B/IGF-1R).
<p>IC50 values were determined in scintillation proximity assays for displacement of <sup>125</sup>I-IGF-1 from receptors with human insulin, IGF-1, insulin X10, insulin detemir or insulin glargine. Relative binding compared to human insulin binding is given in percent. Data represent mean (±SD) from three independent experiments.</p><p>IGF-1, insulin-like growth factor 1.</p
Duration of activation of the IGF-1 receptor.
<p>Data are means (±SE) of three measurements. Results are presented as mean ± SE, n = 3. IGF-1, insulin-like growth factor 1.</p
Relative mitogenic potencies in L6-hIR cells and human mammary epithelial cells (HMEC).
<p>Potencies are presented as mean (±SE) from ≥9 independent experiments.</p