Cav-1 and PTRF/Cavin co-immunoprecipitation with IGF-IR.

<p>Serum starved HaCat cells were stimulated with IGF110 nM for the indicated times and then lysed. Equal amount of cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. The graphs represent quantification of co-immunoprecipitation experiments following densitometric analysis of bands and are expressed as fold of increase. Data shown are representative of three independent experiments and are expressed as the mean ± SD.</p

Cav-1 and PTRF/Cavin are required for IGF-IR internalization and plasma membrane recovery.

<p>HaCat cells were transfected with siRNA for Cav-1 (Cav-1-siRNA), for PTRF/Cavin (PTRF/Cavin-siRNA), for Clathrin Heavy Chain (Clathrin HC-siRNA) and with scrambled control siRNA (Ctr-siRNA) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">materials and methods</a>. (A) 72 hours after transfection, serum-starved cells were treated with IGF110 nM for the indicated times, trypsined, washed, blocked and incubated with a mouse PE-conjugated IGF-IR antibody. PE-conjugated IGF-IR labeled cells were analyzed by flow-cytometry to measure plasma membrane IGF-IR expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">Materials and Methods</a>. (B) Ctr-siRNA, Cav-1-siRNA, PTRF/Cavin-siRNA and Clathrin HC-siRNA HaCat cells were serum-starved and subjected to a biotinylation based endocytic assay with NH-SS-biotin at 4°C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">Materials and Methods</a>). The cells were then warmed at 37°C with medium containing IGF110 nM to allow IGF-IR internalization. Glutathione was used to reduce the proteins not internalized from the plasma membrane. IGF-IR was immunoprecipitated with IGF-IR antibody and the internalized IGF-IR was revealed by Western Blot with a Streptavidin-HRP antibody. Data were quantified using NIH-Image and plotted in the graph. The amount of biotinylated internalized IGF-IR was expressed as a percentage of the amount of IGF-IR on the surface at 4°C which we set as 100%. (C) 72 hours from the transfection serum-starved cells were lysed and equal amount of Ctr-siRNA and Cav-1-siRNA or Ctr-siRNA and PTRF/Cavin-siRNA and Clathrin HC-siRNA cell lysates were separated on SDS–PAGE, transferred on nitrocellulose and blotted with an antibody directed against Cav-1, PTRF/Cavin, Clathrin HC, IGF-IR, Flotillin-2 and actin proteins. Data are expressed as the mean ± SD. Statistical analysis was performed using Student's <i>t</i> test. *p<0.05.</p

Expression of CavY14F mutant decreases IGF-IR internalization in IGF1 stimulated cells.

<p>(A) Hacat cells were transiently transfected with pEGFPN1 Cav-1-wt and pEGFN1 Cav-1Y14F plasmids. 48 hours after transfection serum starved HaCat cells were stimulated with IGF1 10 nM for 5 min, fixed in 4% formaldehyde, permeabilized with Methanol at −20°C, labeled with a rabbit anti-PTRF/Cavin (red) and a mouse anti-IGF-IR (blue) imaged by confocal immunofluorescence microscopy. Merged fields show co-localization (white) of Cav-1wt, PTRF/Cavin, IGF-IR (upper pannels) and Cav-1Y14F, PTRF/Cavin and IGF-IR (bottom panels). (B) 48 hours from the transfection, serum-starved cells were treated with IGF110 nM for the indicated times, trypsined, washed, blocked and incubated with a mouse PE-conjugated IGF-IR.antibody. PE-conjugated IGF-IR labeled cells were analyzed by flow-cytometry to measure plasma membrane IGF-IR expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014157#s3" target="_blank">Materials and Methods</a>. Data are expressed as the mean ± SD. Statistical analysis was performed using Student's <i>t</i> test. *p<0.05.</p

IGF1 does not Induces IGF-IR and Clathrin Heavy Chain co-localization.

<p>HaCat cells were treated with IGF1(10 nM) for 5 min, fixed in 4% formaldehyde, permeabilized with Methanol at −20°C, labeled with a rabbit anti-Clathrin Heavy Chain (red) and a mouse anti-IGF-IR (green), antibody and imaged by confocal immunofluorescence microscopy.</p

ABAp increases after an oral glucose load in healthy subjects, but not in T2D patients.

<p>After overnight fasting, a pre-test blood sample was taken from 7 healthy subjects and from 9 T2D patients, all of whom subsequently underwent a standard OGTT. The values of plasma ABA (A), glucose (B) and insulin (C) shown are the mean ± SD from the healthy controls (black rhombi) and from the T2D subjects (grey squares). * p<0.05 relative to time zero values.</p

Fasting ABAp in NGT subjects and T2D patients.

<p>Fasting ABAp was determined by HPLC-MS in 21 male T2D patients (squares) and in 27 sex-, age- and BMI-matched NGT subjects (rhombi). Results are ordered by increasing value. The circled areas indicate the possible existence of two sub-groups within the T2D patients, one with higher-than-normal ABAp levels and one with ABAp values similar to those of the NGT group. Inset: a box-and-whisker plot drawn from the same data sets. * p = 0.013</p

Higher fasting ABAp in T2D patients compared to NGT controls.

<p>Higher fasting ABAp in T2D patients compared to NGT controls.</p

Pre-partum impairment and post-partum restoration of the ABAp increase after oral glucose load in GDM subjects.

<p>The values of plasma ABA (A), glucose (B) and insulin (C) shown are the mean ± SD from seven NGT (black rhombi) and from nine GDM subjects (grey squares), who underwent a standard OGTT at the 24^th-28^th week (pre-partum) and again 2–3 months after childbirth (post-partum). Post-partum restoration of the ABAp increase during OGTT in the GDM subjects was accompanied by restoration of a normal glycemic profile. * p<0.05 compared to time zero values; ^§ p<0.05 compared to NGT.</p

Diminished increase of ABAp after oral glucose load in GDM and reversal to normal after childbirth.

<p>Diminished increase of ABAp after oral glucose load in GDM and reversal to normal after childbirth.</p