Type 2 diabetic multiorgan donors.

<p>The details of the diabetic organ donors. Data are expressed as means ± SEM.</p

Reduced transcript levels of insulin signaling and cell-cycle proteins and protein levels of insulin signaling components in islets isolated from patients with T2DM.

<p>(a) Expression of insulin receptor isoforms A and B; (b) transcript levels of insulin signaling proteins, and (c) transcript levels of cell-cycle proteins. Data are expressed after normalization for TATA binding protein (TBP) (<i>n</i> = 5–7); *, p<0.05; ***, p<0.001; controls vs T2DM. (d) protein expression of total insulin receptors, (e), total insulin receptor substrate-1, (f) total insulin receptor substrate-2, (g), tyrosine-612 IRS-1, (h) tyrosine-612 IRS-2, (i) or total phosphatidyl inositol 3-kinase. *,p<0.05; **,p<0.001 Control vs T2D. Data in 2d–i are immunoprecipitates. (j) β-actin was used to normalize the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028050#pone-0028050-g002" target="_blank">Figures 2d–i</a>.</p

Reduced β-cell mass, enhanced PCNA+ β- and α-cells and altered expression of insulin signaling and cell adhesion proteins in pancreas from patients with T2DM.

<p>Representative confocal mages of pancreas sections from type 2 diabetic and control pancreas showing (a) quantification of islet cell number with immunostaining for insulin (red), glucagon (blue) and somatostatin (red); PCNA+ cells co-staining with (b) insulin or glucagons (red) and PCNA (green) and (c) CK19 (green), PCNA (red) and insulin (blue); (d) alterations in expression of β-catenin with immunstaining for PCNA (blue), β-catenin (green), insulin (red), light blue in the merged image indicates co-localization between β-catenin and PCNA; (e) alterations in E-cadherin with immunostaining for PCNA (blue), E-cadherin (green) and insulin (red) and the merged image in yellow; Immunostaining of pancreas sections for (f) insulin receptor α-subunit (green), insulin (red) and glucagon (blue); (g) BAD (green) and insulin (red) and merged image in yellow; (h) phospho-BAD (green), insulin (red) and merged image in yellow, and (i) the cell-cycle inhibitor p27-kip1 (green), insulin (red) and glucagon (blue), (<i>n</i> = 7). Yellow in merged images indicates co-localization between insulin staining and insulin signalling proteins. Immunostaining was performed as described in Methods. Bars = 50 µm. *, p<0.05 controls vs T2D.</p

Schematic linking the growth factor pathway to defects in cell cycle progression and apoptosis in diabetic β-cells.

<p>Schematic linking the growth factor pathway to defects in cell cycle progression and apoptosis in diabetic β-cells.</p

Insulin receptor-deficient β-cells exhibit slow growth and block in G1 to S-phase transition.

<p>(a) Defects in growth of βIRKO cells is evident from total number of cells 4 and 12 days after the same number of cells are plated at day 0; the panel on the right shows poor spread and clumping of βIRKO cells (b) β-cell size in control and βIRKO cell lines and islets determined by FACS analyses with forward scatter; (c) significantly lower G1 and G2 in βIRKO cells, and (d) block in G1 to S-phase transition in βIRKO cells after synchronization with hydroxyurea for 18h. Analyses were performed by FACS. In each case, representative data from 3 clones each derived from 3 RIP-<i>Cre</i> controls and 3 βIRKO mice are shown.</p

Insulin receptor-deficient β-cells show reduced expression of cyclins and cyclin dependent kinases and nuclear restriction of FoxO1.

<p>(a) Reduced expression of cdk2 and cdk4; (b) reduced expression of cyclin D2, cyclin D3 and cyclin E; (c) increased expression and nuclear restriction of FoxO1; (d) re-expression of the insulin receptor in βIRKO β-cells (βIRKO+hIRB) restores the expression of CDK2, CDK4 and cyclin E. Actin is used as a loading control; (e) reduced p-IRS2 and p-S473-Akt in βIRKO and restoration of total IRS2, pIRS2 and p-S473-Akt in insulin receptor re-expressing cells (βIRKO+hIRB); (f) Re-expression of insulin receptors in βIRKO cells restores FoxO1 to nucleus; (g) proliferation analyzed by the deuterated water technique <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028050#pone.0028050-Neese1" target="_blank">[57]</a>. Representative data from at least 3 independent Western blotting experiments from at least 2 independent clones are shown. The plots below the blots are shown in arbitrary units. The loading control for experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028050#pone-0028050-g004" target="_blank">Fig. 4a–c</a> is presented under the blot for cyclin D3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028050#pone-0028050-g004" target="_blank">Fig. 4b</a>. *, p<0.05 βIRKO vs Control or βIRKO+hIRB.</p

Supplementary Material for: The Clinicopathologic Heterogeneity of Grade 3 Gastroenteropancreatic Neuroendocrine Neoplasms: Morphological Differentiation and Proliferation Identify Different Prognostic Categories

<b><i>Background/Aims:</i></b> Gastroenteropancreatic (GEP) neuroendocrine carcinomas (NECs) are defined as neuroendocrine neoplasms (NENs) with a Ki-67 index >20% according to the 2010 WHO classification. Some reports suggest that this category is heterogeneous. We retrospectively studied a series of 136 patients affected by grade 3 GEP-NECs with the aim to clarify the prognostic role of tumor morphological differentiation, proliferation, defect in mismatch repair proteins (MMRd), CD117 expression, and site of origin. The primary endpoint was the correlation between these parameters and the overall survival (OS). <b><i>Methods:</i></b> Univariate and multivariable Cox proportional hazards regression analyses were used to assess the prognostic significance of various clinical and histopathologic features. <b><i>Results:</i></b> With a median follow-up of 81 months, the median OS was 12.9 months. At multivariate analysis, morphological differentiation, Ki-67 index, MMRd, stage, and CD117 expression were independent prognostic markers in NECs. Three different prognostic categories of NECs were identified according to the degree of morphologic differentiation (well vs. poorly differentiated) and Ki-67 index (<55% vs. ≥55%). On this basis, median OS was 43.6 months in well-differentiated neoplasms with a Ki-67 index 20-55% (named type A), 24.5 months in poorly differentiated neoplasms with a Ki-67 index 20-55% (type B), and 5.3 months (p < 0.0001) in poorly differentiated neoplasms with a Ki-67 index ≥55% (type C). <b><i>Conclusions:</i></b> The present study suggests that GEP-NECs represent a heterogeneous group of neoplasms which can be better classified in different prognostic categories using both tumor morphology and Ki-67 index