Proteomic Analysis of Disease Stratified Human Pancreas Tissue Indicates Unique Signature of Type 1 Diabetes

<div><p>Type 1 diabetes (T1D) and type 2 diabetes (T2D) are associated with functional beta cell loss due to ongoing inflammation. Despite shared similarities, T1D is an autoimmune disease with evidence of autoantibody production, as well as a role for exocrine pancreas involvement. Our hypothesis is that differential protein expression occurs in disease stratified pancreas tissues and regulated proteins from endocrine and exocrine tissues are potential markers of disease and potential therapeutic targets. The study objective was to identify novel proteins that distinguish the pancreas from donors with T1D from the pancreas from patients with T2D, or autoantibody positive non-diabetic donors. Detailed quantitative comprehensive proteomic analysis was applied to snap frozen human pancreatic tissue lysates from organ donors without diabetes, with T1D-associated autoantibodies in the absence of diabetes, with T1D, or with T2D. These disease-stratified human pancreas tissues contain exocrine and endocrine tissues (with dysfunctional islets) in the same microenvironment. The expression profiles of several of the proteins were further verified by western blot. We identified protein panels that are significantly and uniquely upregulated in the three disease-stratified pancreas tissues compared to non-disease control tissues. These proteins are involved in inflammation, metabolic regulation, and autoimmunity, all of which are pathways linked to, and likely involved in, T1 and T2 diabetes pathogenesis. Several new proteins were differentially upregulated in prediabetic, T1D, and T2D pancreas. The results identify proteins that could serve as novel prognostic, diagnostic, and therapeutic tools to preserve functional islet mass in Type 1 Diabetes.</p></div

Validation of differentially upregulated of specific proteins in pancreas tissue lysates.

<p>Normalized total protein lysates from pooled normal, Aab+, T1D and T2D samples were subjected Western blot analysis to detect for REGIIIα/γ (Panel A), Olfactomedin 4 (Panel B), and ENPP1 (Panel C),, . GAPDH detection was included as a loading control. ImageJ analysis was used to confirm the expression of the three proteins after normalization using GAPDH values. The bar graphs represent data from triplicate analyses.</p

Functional categories and biological processes of all the proteins that are identified in ND, AAb+, T1D and T2D cases.

<p>Functional categories and biological processes of all the proteins that are identified in ND, AAb+, T1D and T2D cases.</p

Comparative IPA analysis of proteins that are uniquely upregulated in AAb+, T1D and T2D cases.

<p>There is a unique upregulation of specific pathways, diseases and biofunctions in the different sample categories.</p

(A) Ingenuity pathway analysis showing top interaction network for differentially regulated proteins in AAb+ cases.

<p>Those highlighted with red color are upregulated genes and those with green are downregulated genes. The names of these genes are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135663#pone.0135663.s017" target="_blank">S7 Table</a>. (<b>B).</b> Ingenuity pathway analysis depicting the activated transcription factor KDM5B) in AAb+ cases when compared to controls. This leads to the inhibition of FHL1, IARS2, ARL6IP5, PSIP1 and PRPS1.</p

(A) Venn diagram comparison of uniquely upregulated proteins in AAb+, T1D and T2D cases compared to ND.

<p>The cut-off for the threshold fold change differences were ≥ 2.0 for upregulation with p- values < 0.05. <b>(B).</b> Ingenuity pathway analysis showing top interaction network for uniquely upregulated proteins in AAb+ cases. Those highlighted with red color are upregulated genes and those with green are downregulated genes. The names of these genes are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135663#pone.0135663.s018" target="_blank">S8 Table</a>.</p

Loss of intra-islet heparan sulfate is a highly sensitive marker of type 1 diabetes progression in humans

<div><p>Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.</p></div

HS optimizes human beta cell survival <i>in vitro</i>.

<p>Flow cytometry analyses of the viability of freshly isolated human islet cells (Con) on day 0 and after culture for 2 days with or without heparin, PI-88 or BT548 at 50 μg/ml. (<b>A-C</b>) Islet cells were stained with Newport Green (NG) to identify beta cells and with 7AAD (<b>B</b> and <b>C</b>) to label non-viable cells; NG+ve, 7AAD-ve staining identified viable beta cells (<b>C</b>). Con, control; Hep, Heparin; BT548, chemically modified LMWH. Data (% islet cells) shows mean ± SEM; n = 8–10 independent experiments and significance was measured by non-parametric ANOVA (Kruskal-Wallis test) with Dunn’s Multiple Comparisons test. * P<0.001, **P<0.01, ***P<0.05.</p

Expression of HS and Col18 in freshly isolated human beta cells and after culture in the absence or presence of heparin.

<p>Expression of HS and Col18 in freshly isolated human beta cells and after culture in the absence or presence of heparin.</p

HS mimetics protect human beta cells from oxidative damage.

<p>Flow cytometric analyses of isolated human islet cells cultured with or without HS mimetics for 2 days and then treated acutely with hydrogen peroxide. Islet cell death/damage was measured by Sytox green fluorescence. Con, control; Hep, Heparin; BT548, chemically modified LMWH. Data shows mean ± SEM; n = 11–12 independent experiments. Con d0 versus Con d0 + hydrogen peroxide, Unpaired t-test, *P<0.0001; Con d2 + hydrogen peroxide versus PI-88 d2 + hydrogen peroxide, non-parametric ANOVA with Dunn’s Multiple Comparisons test, ** P<0.05.</p