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Nabeshima, K., Nakagawa, T., Straight, A. F., Murray, A., Chikashige, Y., Yamashita, Y. M., Hiraoka, Y. and Yanagida, M.
OpenUrl Abstract / FREE Full Text sOliver Tbar sandals denim ExelOqPBB
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 Jimmy choo Annie 85 glitter sandals i0APv
. 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 [35] Tabitha Simmons Womens Reyner Embellished Satin High Block Heel Slide Sandals FU2nEB
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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