Effect of Insulin Analogues on Insulin/IGF1 Hybrid Receptors: Increased Activation by Glargine but Not by Its Metabolites M1 and M2

<div><h3>Background</h3><p>In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids.</p> <h3>Methodology</h3><p>To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP₃) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP₃ production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells.</p> <h3>Results</h3><p>Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP₃ production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP₃ production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin.</p> <h3>Conclusion</h3><p>Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells.</p> </div

EC50s of insulin, insulin analogues and IGF1 for PIP₃ production in MCF-7 and MDA-MB231 cells.

<p>Results are the means ± S.E.M. of 5 to 6 independent experiments.</p>*, **<p>, p<0.05 or p<0.01 respectively, when compared to insulin.</p

EC50s of insulin, IGF1, glargine and glargine metabolites towards IR homodimers and IR/IGF1R hybrids.

<p>Results are the means ± S.E.M. of 4 to 6 independent experiments.</p>*, **<p>, p<0.05 or p<0.01 respectively, when compared to insulin.</p

Effect of insulin, glargine and IGF1 on PIP₃ production in intact living cells.

<p>Activation of tyrosine kinase receptors by their ligands stimulates the activity of PI-3 kinase, leading to increased phosphorylation of phosphatidyl-inositol 2 phosphate (PIP₂) into phosphatidyl-inositol 3 phosphate (PIP₃) and subsequent recruitment of Akt to the plasma membrane through its pleckstrin homology (PH) domain. To monitor the production of PIP₃ induced by receptor activation, cells were co-transfected with cDNAs coding for the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to the membrane localization sequence of neuromodulin. Cells were pre-incubated for 10 min with coelenterazine and then stimulated with increasing ligand concentrations. (A) Typical experiment showing real-time insulin or IGF1 effects on PIP₃ production in MCF-7 cells. (B) Dose-dependent effect of insulin, glargine and IGF1 on PIP₃ production in MCF-7 and MDA-MB231 cells. Ligand-induced BRET (BRET above basal at the plateau) was determined for each ligand concentration and was used to establish dose-response curves. Results are the means ± S.E.M. of 5 to 6 independent experiments. EC50 for insulin, IGF1, glargine and other insulin analogues are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041992#pone-0041992-t002" target="_blank">Table 2</a>. (C) Left panel: Receptors were partially purified from MDA-MB231 and MCF-7 cells by WGL chromatography. WGL eluates (12 µg of protein) were submitted to electrophoresis and western-blotting. IR and IGF1R expression levels were evaluated by immunoblotting (IB) using anti-IR (C-19) and anti-IGF1R (C-20) antibodies. Right panel: After normalization of the eluates for IR content, immunoprecipitation (IP) was performed using anti-IR antibody (CT1) and hybrid receptors were detected using anti-IGF1R antibody. Blots were then stripped and reprobed with anti-IR antibody. Results are representative of 6 immunoprecipitation experiments performed on three independent batches of receptor preparations (**, p<0.01).</p

Downstream biological effects of insulin, glargine and its metabolites M1 and M2 in MCF-7 cells.

<p>(A) Effect of insulin, IGF1, glargine and its metabolites M1 and M2 on Akt and Erk1/2 phosphorylation in MCF-7 cells. MCF-7 cells were starved overnight and then incubated for 5 min in presence of 10 nM of insulin, glargine, M1, M2 or IGF1. Ligand-induced phosphorylation of Erk1/2 and Akt was evaluated by western blotting. (B) Dose-dependent effect of insulin, IGF1, glargine and its metabolites M1 and M2 on Akt and Erk1/2 phosphorylation in MCF-7 cells. Cells were stimulated for 20 min and ligand-induced phosphorylation of Akt and Erk1/2 was evaluated by in-cell western. Results correspond to mean ± SEM of 4 to 6 independent experiments. (C) MCF-7 cells were incubated for 18 h in serum free medium in the presence or absence of 10 nM of insulin, glargine, M1, M2 or IGF1. mRNA expression level was measured by qRTPCR. Results are normalized to the expression of cyclophilin A mRNA and correspond to the mean ± SEM of 4 to 8 independent experiments (*, **, p<0.05 or p<0.01 respectively, when compared to the control condition). (D) Subconfluent MCF-7 cells cultured in Cytostar-T scintillation microplates were starved for 4 h and then incubated for 19 h with increasing concentrations of IGF-1, insulin or analogues in serum free medium. [¹⁴C]thymidine was added for an additional 6 h and the radioactivity measured in a Wallac 1450 Micro Beta Trilux Scintillation counter. Data are means ± SEM of at least 6 independent experiments. EC50 for insulin, IGF1, glargine and its metabolites on Akt and Erk1/2 phosphorylation and on thymidine incroporation are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041992#pone-0041992-t004" target="_blank">Table 4</a>.</p

Effect of insulin, glargine and IGF1 on IR homodimers and IR/IGF1R hybrid receptors.

<p>HEK-293 cells were co-transfected with IRA-Luc/IRA-YFP, IRB-Luc/IRB-YFP, IRA-Luc/IGF1R-YFP or IRB-Luc/IGF1R-YFP. Receptors were partially purified by WGL chromatography. Ligand binding induces a conformational change that brings the two ß-subunits in close proximity, resulting in an energy transfer between the luciferase and YFP. BRET assays were performed in the presence of increasing ligand concentrations. (A) Typical experiments showing basal and insulin or IGF1 stimulated BRET signals. (B) Dose-response curves showing the effect of insulin, IGF1 and glargine on IR homodimers and IR/IGF1R hybrids. Ligand-induced BRET (BRET above basal) was determined at each ligand concentration and was used to establish dose-response curves. Results are the means ± S.E.M. of 4 to 6 independent experiments. EC50 for insulin, IGF1, glargine and other insulin analogues are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041992#pone-0041992-t001" target="_blank">Table 1</a>.</p

Comparison of the pharmacological profiles of glargine and its metabolites M1 and M2.

<p>(A) Conversion of glargine into M1 and M2 metabolites. (B) Effects of M1 and M2 on IR/IGF1R hybrids and on IR/IR homodimers. Receptors were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041992#pone-0041992-g001" target="_blank">figure 1</a>. BRET assays were performed in the presence of increasing concentrations of insulin, glargine, M1, M2 or IGF1. (C) Dose-dependent effect of insulin, glargine, M1, M2 or IGF1 on PIP₃ production in MCF-7 cells. BRET assays were performed in the presence of increasing concentrations of ligands. Ligand-induced BRET (BRET above basal at the plateau) was determined for each ligand concentration and was used to establish dose-response curves. Results are the means ± S.E.M. of 4 to 6 independent experiments. EC50 for insulin, IGF1, glargine and its metabolites are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041992#pone-0041992-t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041992#pone-0041992-t004" target="_blank">4</a>.</p