In Vitro Metabolic and Mitogenic Signaling of Insulin Glargine and Its Metabolites

with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities.The affinity of human insulin, insulin glargine and its metabolites to the IR isoforms A and B or IGF1R was analyzed in a competitive binding assay using SPA technology. Receptor autophosphorylation activities were studied via In-Cell Western in CHO and MEF cells overexpressing human IR-A and IR-B or IGF1R, respectively. The metabolic response of the insulins was studied as stimulation of lipid synthesis using primary rat adipocytes. Thymidine incorporation in Saos-2 cells was used to characterize the mitogenic activity. value for autophosphorylation of the receptor and a more potent stimulation of thymidine incorporation in Saos-2 cells. In contrast, the metabolites M1 and M2 were significantly less active in binding to and activation of the IGF1R and their mitogenicity in Saos-2 cells was equal to human insulin. These findings strongly support the idea that insulin glargine metabolites contribute with the same potency as insulin glargine to blood glucose control but lead to significantly reduced growth-promoting activity

Metabolic activity of insulin glargine and its metabolites in rat adipocytes.

<p>To compare the metabolic activity of glargine and its metabolites insulin-stimulated lipid synthesis in isolated primary rat adipocytes was analyzed by incorporation of [3-³H]glucose into toluene-extractable lipids and subsequent measurement of radioactivity by liquid scintillation counting. The adipocytes were treated with increasing concentrations of insulin, analogs, metabolites or IGF-1 for 90 min at 37°C in the presence of [3-³H]glucose. Each point represents the mean ± SEM of five different adipocyte preparations with activity measurements done in duplicate.</p

Activity of insulin analogs on the human IGF1R and mitogenic potential in Saos-2 cells.

<p>Analysis of binding to the human IGF1R using [¹²⁵I]IGF-1 as radioactive ligand (<b>A</b>) and analysis of the insulin-stimulated autophosphorylation of the human IGF1R overexpressed in MEF cells (<b>B</b>) were done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009540#pone-0009540-g002" target="_blank">figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009540#s2" target="_blank">Materials and Methods</a>. Data are given as means ± SEM. (<b>C</b>) To compare the mitogenic potential of the insulin analogs with that of human insulin, DNA synthesis was determined by [¹⁴C]thymidine incorporation in Saos-2 cells cultured in Cytostar-T scintillation microplates. Confluent cells were starved for 4 h and then incubated for 19 h with increasing concentrations of IGF-1, insulin or analog in serum free medium. [¹⁴C]thymidine was added for further 6 h and the radioactivity measured in a Wallac Micro Scintillation counter. Data are given as means ± SEM from octuplicate sample.</p

Summarized <i>in vitro</i> data for insulin and glargine metabolites.

<p>Data are means ± SEM. All analogs were tested at least three times on different days. Activity was determined within each experiment and then averaged to yield a single reported mean value. IM – intermediate ([Gly^A21,Arg^B31]insulin), M1 – metabolite 1 ([Gly^A21]insulin), M2 – metabolite 2 ([Gly^A21,des-Thr^B30]insulin).</p

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

<p>Binding of the insulin analogs to the human IR-A (<b>A</b>) or IR-B (<b>B</b>) was analyzed in a competitive binding assay using SPA technology. The binding of a constant concentration of [¹²⁵I]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 [¹²⁵I]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 (<b>C</b>) or IR-B (<b>D</b>) 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.</p

Insulin glargine metabolites.

<p>The long-acting insulin glargine (Lantus®, [Gly^A21,Arg^B31,Arg^B32]insulin) is metabolized <i>in vivo</i> in subcutaneous tissue and in bloodstream of healthy humans by sequential cleavage of the C-terminus of the B chain. Two primary degradation products M1 and M2 have been reported, which are both structural similar to human insulin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009540#pone.0009540-Kuerzel2" target="_blank">[10]</a>. Besides M1 and M2 an intermediate IM was also identified but only in minor quantities.</p