Relative receptor affinities, metabolic and mitogenic potencies, and IR off-rates [% of human insulin].

<p>All assays were performed in at least three independent experiments. Data are means ± SD and presented relative to human insulin. For human insulin, IR assay IC₅₀ values were in the picomolar affinity range, IGF-IR assay IC₅₀ values were in the nanomolar affinity range, rFFC assay EC₅₀ values were in the picomolar range, and mitogenic assay EC₅₀ values were in the nanomolar range (HMECs) and low nanomolar range (L6-hIR). The dissociation rate constant for human insulin was (3.7±0.3×10⁻² min⁻¹).</p

Representative dose-response profiles for mitogenic potency determination.

<p>Human insulin (•) or insulin analogue (B10D (♦), B10E (▪), or B10A (▾)) stimulated incorporation of [³H]-thymidine into DNA is shown in (<b>A</b>) L6-hIR cells and (<b>B</b>) HMECs. Data points are means ± SEM (n = 3).</p

Relative receptor binding affinities.

<p>Receptor binding affinities for the solubilised IR-A (light teal), IR-B (dark teal) and IGF-IR (gray) relative to human insulin. The gray dotted line represents 100% binding affinity compared to that of human insulin. Data are means ± SD (n = 3).</p

Representative dissociation curves of [¹²⁵I]-labelled insulin or analogue from BHK-hIR cells.

<p>Dissociation was measured at different time points and the residual binding expressed as a percentage of initial binding. Dissociation of (<b>A</b>) B10D (♦) and B10E (▪); (<b>B</b>) B10W (▴), human insulin (•), and B10R (▾). Data points are means ± SEM (n = 3).</p

Agonistic (mitogenic) effect of S961 in MCF-7 cells.

<p>Cells were stimulated with increasing concentrations of S961 or IGF-I. The graph is representative for three experiments. The increased response for S961 at 1 nM compared to the response at the three lowest concentrations is statistically significant (P<0.001, two-tailed t-test). Data points represent means of triplicate determinations. Error bars show one standard deviation.</p

S961 has antagonist as well as agonist activity on IR-mediated mitogenic effect in L6-hIR cells. A,

<p>"10 nM S961" and "100 nM S961" curves: Cells were pretreated for 2h with 10 nM or 100 nM S961, and stimulated with increasing concentrations of insulin (as indicated on the x-axis) in the continued presence of S961. "HI" curve, insulin stimulation only (without S961). "DMSO" curve, insulin stimulation with equal volume DMSO added instead of S961. <b>B,</b> "10 nM S961" and "100 nM S961" curves: Cells were pretreated for 2h with 10 nM or 100 nM S961, and stimulated with increasing concentrations of IGF-I (as indicated on the x-axis) in the continued presence of S961. "IGF-I" curve, IGF-I stimulation only (without S961). "DMSO" curve, IGF-I stimulation with equal volume DMSO added instead of S961. <b>C,</b> "0.01 nM HI", "0.025 nM HI" and "0.05 nM HI" curves: Cells were pretreated for 2 h with increasing concentrations of S961 (as indicated on the x-axis), and stimulated with 0.01 nM, 0.025 nM or 0.05 nM HI in the continued presence of S961. "S961 alone" curve, insulin was omitted. <b>D</b>, "1 nM HI", "10 nM HI" and "100 nM HI" curves: Cells were pretreated for 2 h with increasing concentrations of S961 (as indicated on the x-axis), and stimulated with 1 nM, 10 nM or 100 nM HI in the continued presence of S961. "S961 alone" curve, insulin was omitted. <b>A</b> and <b>B,</b> Graphs are representative for three independent experiments, each experiment comprising triplicate determinations of each ligand concentration. <b>C,</b> The graph is performed in triplicates once. <b>D,</b> The graph is representative for two independent experiments each performed in triplicates. Error bars indicate one standard deviation.</p

S961 stimulates IR and Akt phosphorylation in CHO-hIR cells.

<p>Cells were stimulated with increasing concentrations of HI or S961. <b>A</b>-<b>E</b>, IR tyrosine phosphorylation. The 6 tyrosine phosphorylation sites which were examined were Y972 in the juxtamembrane domain, Y1158 and Y1162/1163 in the tyrosine kinase domain, and Y1328 and Y1334 in the C-terminal tail end of the IR. <b>F</b>, Akt phosphorylation. Phosphorylation of Ser437 is known to be required for Akt activation. Panels <b>A</b>-<b>E</b>: the increased tyrosine phosphorylation of the IR was significant (compared to 0.0001 nM, 0.001 nM and 0.01 nM S961, P<0.05*, P<0.01**, P<0.001***, two-tailed t-test). Panel <b>F</b>: the increased serine phosphorylation of Akt was significant (compared to 0.0001 nM, 0.001 nM and 0.01 nM S961, P<0.01**, two-tailed t-test). Data points represent average of three independent experiments, each comprising triplicate determinations. Error bars show one standard deviation.</p

S961 did not stimulate lipogenesis in rat adiopocytes.

<p>Primary rat adipocytes were stimulated with increasing concentrations of S961or S661 alone or in combination with 1 nM insulin. Insulin alone was included as a reference. The graph is representative of two independent experiments each comprising duplicate determinations. Error bars show one standard deviation.</p

S961 did not stimulate glycogen synthesis in differentiated adipocytes or in muscle cells. A

<p>, Differentiated 3T3-L1 adipocytes were stimulated with increasing concentrations of HI, IGF-I or S961. The graph is representative of two independent experiments each comprising duplicate determinations. Error bars show one standard deviation. <b>B</b>, L6-hIR muscle cells were stimulated with increasing concentrations of S961/S661 alone or in combination with 3 nM insulin. The graph is representative of two independent experiments each comprising triplicate determinations. Error bars show one standard deviation.</p

Current model of IR activation and proposed binding mechanism for S961. A

<p>. Current model of IR activation. The four blue circles represent the receptor binding sites (sites 1 and 2) seen from a top view. Insulin is depicted as a yellow circle. For a detailed explanation of binding sites 1 and 2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051972#pone.0051972-Whitesell1" target="_blank">[24]</a>. <b>B</b>. Proposed binding mechanism for S961. The four blue circles represent the receptor binding sites (sites 1 and 2) seen from a top view. For a detailed explanation of binding sites 1 and 2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051972#pone.0051972-Whitesell1" target="_blank">[24]</a>. The S961 peptide (Site 1–2 peptide) is shown as two connected yellow circles. At concentrations of 1–10 nM, S961 crosslinks the receptor, leading to agonist activity. At concentrations of above 10 nM, the higher flexibility of S961 in comparison to the insulin molecule allows simultaneous crosslinking of both pairs of binding sites, leading to an inactive conformation and antagonism. The corresponding activation and inactivation sigmoids are also shown. <b>C</b>. Orientation of peptide binding sites. If site 1 is located N-terminally and site 2 C-terminally, a longer distance between the binding sites in S961 in comparison to S661 can be achieved.</p