Differential impact of re-stimulation on pathways downstream of the IR.

<p>(<b>A</b>) pERK, (<b>B</b>) total ERK, (<b>C</b>) pAKT, (<b>D</b>) total AKT (<b>E</b>) pGSKβ and (<b>F</b>) total GSKβ proteins quantified by western blot in response to repeated insulin stimulation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g002" target="_blank">Fig. 2</a> for measurement details. Data are represented as mean ± SD from three (170 nM) and four (17 nM) independent studies. Data normalization and symbols are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">figure 1</a>.</p

Chronic insulin exposure down-regulates IR activity.

<p>Timecourse and insulin dose response of IR phosphorylation at (<b>A</b>) pY IR⁹⁷² and (<b>B</b>) pY IR^1162,1163 detected by western blot with phospho-specific rabbit polyclonal antibodies. Detection and quantification was by infrared immunofluorescence linked to the anti-rabbit secondary antibody. Representative westerns shown for a dose response (right panels; only shown for the shortest insulin incubation period). (<b>C</b>) Total IR protein quantified on the same western blots with an anti-IR mouse monoclonal antibody conjugated with an anti-mouse secondary antibody linked to a distinct infrared fluorophore. Total (<b>D</b>) tyrosine and (<b>E</b>) serine phosphorylation, over all sites on the IR determined by ELISA, in response to insulin stimulation. Data are presented as mean ± sd from 3, 10 and 9 independent studies for the 5 nm, 17 nM and 170 nM doses. The intermediate 45 mins time point was included in a subset of those studies (3, 4 and 4 studies for the 5 nm, 17 nM and 170 nM doses). Since the absolute ‘light unit’ values quantified differ from experiment to experiment, the data were normalized to a treatment condition common amongst all experimental conditions. The earliest time point of induction (2–10′) with 17 nM insulin was set as 1.0 and all other data points within each study were normalized to that. <b>*</b>, increased IR phosphorylation (p<0.05) at the 2–10′ time point relative to the no insulin control. #, diminished IR phosphorylation at longer insulin incubation periods (p<0.05) relative to the 2–10′ time point. The data normalization and statistical symbols are applied similarly to the data presented in the remaining figures.</p

Insulin refractory IR structure in HTC cells.

<p>Energy transfer of IR-CFP-YFP probe transiently expressed in HTC cells subjected to acute (10′) and chronic (120′) insulin exposure. Gray bars, minimal energy transfer of CFP and YFP attached to the amino and carboxy termini of IRS1 (too far apart for energy transfer) demonstrates FRET measurement accuracy. Data represent the mean ± sd of the average FRET measurements of an average of 44 cells per point in each of three independent studies.</p

Chronically stimulated IR is refractory to subsequent re-stimulation following removal of insulin.

<p>Insulin receptor tyrosine phosphorylation at (<b>A</b>) pY IR⁹⁷² (170 nM: n = 4 independent studies; 17 nM: n = 5) and (<b>B</b>) pY IR^1162,1163 (170 nM: n = 4; 17 nM: n = 3) quantified by western blot in response to repeated insulin stimulation. (<b>C</b>) total IR protein quantified on the same blots. (<b>D</b>) Total tyrosine (n = 5) and (<b>E</b>) total serine (n = 5) phosphorylation, measured by ELISA (17 nM treatments). Data normalization and symbols are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">figure 1</a>.</p

The IR remains membrane localized following chronic insulin stimulation of HTC-IR-YFP cells.

<p>(<b>A</b>) YFP fused to the IR fluoresces green and co-localizes with the membrane-specific stain (orange) and not with nuclei (blue). (<b>B</b>) Total IR tyrosine phosphorylation in HTC-IR-YFP cells, detected by ELISA, mirrors that of the unfused similar to HTC-IR in response to insulin stimulation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">Fig. 1D</a>). The fluorescence intensity of (<b>C</b>) YFP (IR) and (<b>D</b>) the membrane marker were quantified at the membrane and in the cytosol of HTC-IR-YFP cells.</p

Differential down-regulation of downstream signaling pathways following chronic insulin stimulation.

<p>(<b>A</b>) phospho-ERK, (<b>B</b>) total ERK (<b>C</b>) phospho-AKT, (<b>D</b>) total AKT, (<b>E</b>) phospho-GSKβ, and (<b>F</b>) total GSKβ proteins quantified by western blot using the same extracts analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">figure 1</a>. The fluorescence intensities of pERK and ERK, or pGSK and GSK, were quantified on the same blot with the phospho-specific and total protein antibodies (of rabbit and mouse origin) conjugated to specific-specific secondary antibodies with distinguishable fluorophores. The pAKT and total AKT antibodies both originated from rabbits and were probed in separate blots. Data normalization, number of studies, symbols and representative westerns are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">figure 1</a>.</p

IR-CFP-YFP structure induced by chronic insulin exposure is similar to that induced by expression of PC-1.

<p>FRET levels of the IR-CFP-YFP probe transiently expressed in CHO cells or in CHO cells stably overexpressing PC-1. Measurements are conducted prior to insulin treatment or after chronic (120′) insulin exposure (n = 3 independent studies). Data represent the mean ± sd of the average FRET measurements of an average of 27 cells per point in each of three independent studies.</p

FRET measurement of altered IR structure or environment associated with insulin resistance in CHO cells.

<p>(<b>A</b>) Schematic representation of the IR-CFP-YFP probe and alterations in (<b>B</b>) total IR tyrosine phosphorylation or (<b>C</b>) energy transfer in that probe under acute or chronic insulin stimulation and after re-stimulation following insulin removal. The probe was transiently expressed in CHO cells. Symbols are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g001" target="_blank">figure 1</a>. Gray bars (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108693#pone-0108693-g006" target="_blank">Fig. 6B</a>) represent IR phosphorylation levels normalized to IR expression levels determined by IR-CFP-YFP fluorescence.</p