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

Somatostatin receptors.

AbstractIn 1972, Brazeau et al. isolated somatostatin (somatotropin release-inhibiting factor, SRIF), a cyclic polypeptide with two biologically active isoforms (SRIF-14 and SRIF-28). This event prompted the successful quest for SRIF receptors. Then, nearly a quarter of a century later, it was announced that a neuropeptide, to be named cortistatin (CST), had been cloned, bearing strong resemblance to SRIF. Evidence of special CST receptors never emerged, however. CST rather competed with both SRIF isoforms for specific receptor binding. And binding to the known subtypes with affinities in the nanomolar range, it has therefore been acknowledged to be a third endogenous ligand at SRIF receptors.This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing the characteristic seven-transmembrane-segment (STMS) topography. Years of intensive research have resulted in cloning of five receptor subtypes (sst1-sst5), one of which is represented by two splice variants (sst2A and sst2B). The individual subtypes, functionally coupled to the effectors of signal transduction, are differentially expressed throughout the mammalian organism, with corresponding differences in physiological impact. It is evident that receptor function, from a physiological point of view, cannot simply be reduced to the accumulated operations of individual receptors. Far from being isolated functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst2 and sst5 receptors) in characteristic proportions. In other words, levels of individual receptor subtypes are highly cell-specific and vary with the co-expression of different-ligand receptors. However, the question is how to quantify the relative contributions of individual receptor subtypes to the integration of transduced signals, ultimately the result of collective receptor activity. The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype-selective analogues. Several have become available

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

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

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 ¹²⁵I-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

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

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

The specific antibody binding capacity (SABC) for human mammary epithelial cells (HMEC) and L6-hIR cells.

<p>The SABC (average number of antibodies capable of binding to each cell) was measured using either the murine monoclonal antibody 83–7 recognising the human IR or 24–31 recognising the human IGF-1R. Data represent mean (±SD) of at least three independent experiments. No antibody is available that recognises the extracellular domains of the rat IGF-1R, therefore it is not possible to determine the relative number of rat IGF-1R on L6-hIR cells.*unpublished results obtains by Western blot.</p><p>IGF-1R, insulin-like growth factor 1 receptor; N/A, not applicable.</p