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Nabeshima, K., Nakagawa, T., Straight, A. F., Murray, A., Chikashige, Y., Yamashita, Y. M., Hiraoka, Y. and Yanagida, M.
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Nabetani, A., Koujin, T., Tsutsumi, C., Haraguchi, T. and Hiraoka, Y.

First, we analyzed the binding characteristics of insulin glargine and its metabolites M1, M2, and IM to IR-A and IR-B. Using plasma membranes from CHO cells that overexpress the receptor isoforms, the IC 50 value for competition of unlabeled ligand with a constant concentration of radioactively labeled human insulin were determined as a measure of affinity. The results are shown in Table 1 with representative competition curves given in Charlotte Olympia Velvet platform sandals 1Hm6tjV
. Both IR isoforms bound human insulin with high affinity that did not significantly (p = 0.23) differ between IR-A (IC 50  = 0.49 nmol/L) and IR-B (IC 50  = 0.57 nmol/L). In contrast, IR-A displayed a considerably higher affinity for IGF-1 (2.7-fold) and IGF-2 (7.5-fold) than IR-B, but the affinity of IR-A and IR-B for IGF-1 were approximately 130- and 300-fold, respectively, less potent than those for human insulin. Both isoforms bound IGF-2 with higher affinity than IGF-1 but with lower affinity than human insulin. These data are consistent with results published previously Donald Pliner Mens Vinco CrocEmbossed Penny Drivers Mens Shoes HAdxz
[39] and demonstrated the validity of our experimental system.

Figure 2. Binding and signaling of insulin glargine and its metabolites to the human insulin receptor isoform A and B.

Binding of the insulin analogs to the human IR-A ( A ) or IR-B ( B ) was analyzed in a competitive binding assay using SPA technology. The binding of a constant concentration of [ 125 I]insulin to plasma membranes from CHO cells overexpressing either IR-A or IR-B was measured in presence of increasing concentrations of unlabeled competing ligand after incubation at room temperature for 12 h. All data has been corrected for non-specific binding and are expressed as percentage of [ 125 I]insulin in absence of competing ligand. To analyze the insulin-stimulated activation and subsequent autophosphorylation of the insulin receptor CHO cells overexpressing the human IR-A ( C ) or IR-B ( D ) were stimulated for 15 min at 37°C with increasing concentrations of peptides, then the cells were fixed with 3.7% PFA and the amount of phosphotyrosines was analyzed via In-Cell Western. The data represent mean values ± SEM of at least 3 individual experiments measured in quadruplicate.

https://doi.org/10.1371/journal.pone.0009540.g002

Table 1. Summarized data for insulin and glargine metabolites.

https://doi.org/10.1371/journal.pone.0009540.t001

Insulin glargine and its metabolites IM, M1, and M2 showed no difference in their respective binding affinities to the two IR isoforms and were only 40–50% less active than human insulin. In contrast, [Asp B10 ]insulin showed significant isoform selectivity, being 3.5-fold more active towards IR-A (p<0.01), and was 8.2- and 2.7-fold more potent in binding to IR-A and IR-B, respectively, relative to human insulin.

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