Amyloid formation reduces protein kinase B phosphorylation in primary islet β-cells which is improved by blocking IL-1β signaling

<div><p>Amyloid formation in the pancreatic islets due to aggregation of human islet amyloid polypeptide (hIAPP) contributes to reduced β-cell mass and function in type 2 diabetes (T2D) and islet transplantation. Protein kinase B (PKB) signaling plays a key role in the regulation of β-cell survival, function and proliferation. In this study, we used human and hIAPP-expressing transgenic mouse islets in culture as two <i>ex vivo</i> models of human islet amyloid formation to: 1. Investigate the effects of amyloid formation on PKB phosphorylation in primary islet β-cells; 2. Test if inhibition of amyloid formation and/or interleukin-1β (IL-1β) signaling in islets can restore the changes in β-cell phospho-PKB levels mediated by amyloid formation. Human and hIAPP-expressing mouse islets were cultured in elevated glucose with an amyloid inhibitor (Congo red) or embedded within collagen matrix to prevent amyloid formation. To block the IL-1β signaling, human islets were treated with an IL-1 receptor antagonist (anakinra) or a glucagon-like peptide-1 agonist (exenatide). β-cell phospho-PKB levels, proliferation, apoptosis, islet IL-1β levels and amyloid formation were assessed. Amyloid formation in both cultured human and hIAPP-expressing mouse islets reduced β-cell phospho-PKB levels and increased islet IL-1β levels, both of which were restored by prevention of amyloid formation either by the amyloid inhibitor or embedding islets in collagen matrix, resulting in improved β-cell survival. Furthermore, inhibition of IL-1β signaling by treatment with anakinra or exenatide increased β-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis in amyloid forming human islets during 7-day culture. These data suggest that amyloid formation leads to reduced PKB phosphorylation in β-cells which is associated with elevated islet IL-1β levels. Inhibitors of amyloid or amyloid-induced IL-1β production may provide a new approach to restore phospho-PKB levels thereby enhance β-cell survival and proliferation in conditions associated with islet amyloid formation such as T2D and clinical islet transplantation.</p></div

Treatment with anakinra or exenatide reduces IL-1β levels in human islets during culture which is associated with increased β-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis.

<p>Islet sections from pre-culture and 7-day cultured human islets with anakinra (An; 10 μg/mL) or exenatide (Ex; 10 nmol/L) were immunolabelled for <b>(A)</b> insulin/IL-1β/Thio S or <b>(B)</b> insulin/p-PKB. <b>(C)</b> The percentage of islet amyloid area to total islet area in each condition. <b>(D)</b> β-cell phospho-PKB immunofluorescence (IF) intensity. The percentage of <b>(E)</b> PCNA-positive (proliferative) β-cells and <b>(F)</b> TUNEL-positive (apoptotic) β-cells. <b>(G)</b> Islet IL-1β release from 4-day cultured exenatide-treated and non-treated human islets. The arrows point to regions corresponding to enlarged areas in each image (original magnification: X400; insert: X1000). Results are expressed as mean +/- SEM of five independent studies (5 donors; 25–30 islets per condition in each study). For β-cell phospho-PKB IF intensity, quantifications were performed on a total of 20 amyloid-positive 7-day cultured human islets and equal number of anakinra- or exenatide-treated human islets (lower amyloid formation).*vs Day 0; <b>#</b>vs corresponding untreated group (<i>P<0</i>.<i>05</i>; one-way ANOVA or Student’s <i>t</i>-test).</p

Progressive amyloid formation in human islets during culture is associated with reduced β-cell phospho-PKB levels and proliferation rate.

<p><b>(A)</b> Paraffin-embedded sections from pre-culture and 7-day cultured human islets with or without amyloid binding dye Congo red (CR; 25 μmol/L) were immunolabelled for insulin, phospho-PKB (p-PKB), and thioflavin S (Thio S). The squares (dashed white lines) correspond to enlarged areas in each image (original magnification: X400; insert: X1000). <b>(B)</b> Immunolabelling for insulin or glucagon and phospho-PKB in amyloid-positive and negative human islets. The percentage of <b>(C)</b> thioflavin S (amyloid)-positive islets and <b>(D)</b> islet amyloid area. <b>(E)</b> Islet phospho-PKB immunofluorescence intensity (IF). The proportion of <b>(F)</b> PCNA-positive (proliferative) β-cells and <b>(G)</b> TUNEL-positive (apoptotic) β-cells in each condition. Results are expressed as mean +/- SEM of five independent studies (25–30 islets per condition in each study). For β-cell phospho-PKB IF intensity, quantifications were performed on a total of 18 amyloid (thio S)-positive 7-day cultured human islets or equal number of CR-treated islets (no or very little amyloid formation). *vs Day 0; <b>#</b>vs corresponding untreated group <i>(P<0</i>.<i>05</i>; one-way ANOVA).</p

Formation of hIAPP aggregates is associated with reduced β-cell phospho-PKB levels and proliferation rate in hIAPP^+/- transgenic mouse islets during culture.

<p><b>(A)</b> Paraffin-embedded islet sections from freshly isolated and 7-day cultured hIAPP^-/- (wild-type) and hIAPP^+/- mice with or without the amyloid binding dye Congo red (CR; 25 μmol/L), were immunolabelled for insulin, phospho-PKB (p-PKB), and thioflavin S (Thio S). The squares (dashed white lines) correspond to enlarged areas in each image (original magnification: X400; insert: X1000). The percentage of <b>(B)</b> thioflavin S (amyloid)-positive islets and <b>(C)</b> islet amyloid area. <b>(D)</b> β-cell phospho-PKB immunofluorescence (IF) intensity. The percentage of <b>(E)</b> PCNA-positive (proliferative) β-cells and <b>(F)</b> TUNEL-positive (apoptotic) β-cells in each condition. Results are expressed as mean +/- SEM of three independent studies (25–30 islets per condition from n = 3 mice per group in each group). For β-cell phospho-PKB IF intensity, quantifications were performed on a total of 20 amyloid (thio S)-positive 7-day cultured hIAPP^+/- islets and equal number of WT islets (amyloid-negative) or CR-treated hIAPP^+/- islets (no or very little amyloid formation). *vs Day 0; <b>#</b>vs corresponding untreated group (<i>P<0</i>.<i>05</i>; one-way ANOVA).</p

Collagen matrix-embedded hIAPP^+/- transgenic mouse islets have lower amyloid formation, β-cell apoptosis, and higher β-cell phospho-PKB levels than free-floating cultured islets.

<p><b>(A)</b> Islet sections from hIAPP^+/- mice were immunolabelled for insulin/A11 (oligomer), insulin/thioflavin S (Thio S), insulin/p-PKB/Thio S or insulin/TUNEL before and after 7-day culture free-floating (FF) or in collagen matrix (CM). The arrows point to regions corresponding to enlarged areas in each image (original magnification: X400; insert: X1000). The percentage of <b>(B)</b> A11 (oligomer)-positive islets, <b>(C)</b> Thioflavin S (amyloid)-positive islets, and <b>(D)</b> islet amyloid area. <b>(E)</b> β-cell phospho-PKB immunofluorescence (IF) intensity. <b>(F)</b> The proportion of apoptotic β-cells. Results are expressed as mean +/- SEM of three independent studies (25–30 islets per condition from n = 3 mice per group in each study). For β-cell phospho-PKB IF intensity, quantifications were performed on a total of 15 amyloid (thio S)-positive 7-day cultured hIAPP^+/- islets and equal number of CM-embedded islets (no or very little amyloid formation). *vs Day 0; <b>#</b>vs FF, one-way ANOVA (<i>P<0</i>.<i>05</i>; one-way ANOVA).</p

Proposed mechanism for amyloid-induced reduction in β-cell phospho-PKB levels and potential strategies to prevent this process.

<p>Amyloid formation leads to reduced β-cell phospho-PKB levels possibly by promoting IL-1β production in islets. Prevention of amyloid formation or blocking IL-1β signaling may provide two strategies to restore amyloid-induced decrease in phospho-PKB levels thereby improve islet β-cell mass. (CM: collagen matrix; CR: Congo red; Ex: Exenatide; An: Anakinra).</p

B7-H4 suppresses activation of ERK, JNK, p38, and AKT.

<p>Western blot analyses of protein extracts from CD3⁺ T cells stimulated with immobilized anti-CD3 (5 µg/mL) and soluble anti-CD28 (2 µg/mL). Fc.Ig or B7-H4.Ig was added after plate-bound anti-CD3 incubation. Representative western blot for the detection of phosphorylated ERK1/2 Thr202/Tyr204, JNK Thr183/Tyr185, p38 Thr180/Tyr182, AKT Ser473, and GSK-3α/β Ser21/9 as indicated in the graph. p-ERK, JNK, p38, AKT, and GSK-3α/β activity were quantitated by using Bio-Rad Quantity One program. The y-axis was normalized for the loading control. Data represent 3 independent experiments and are expressed as means ± SD form 10–16 mice per group.</p

B7-H4 inhibits phosphorylation of AKT on different T cell subsets similarly.

<p>Naïve CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were stimulated with 0.3 µg/ml of plate-bound anti-CD3 and soluble anti-CD28 (1 µg/ml) for 16 h and then rested for 30 h prior to re-activation. Activated CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were stimulated with plate-bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) for 10 min. Representative western blot of protein extracts from CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were detected by phosphorylated AKT Ser473 (A) and quantitated by using Bio-Rad Quantity One program. The y-axis was normalized for the loading control. B7-H4 treatment significantly inhibited AKT phosphorylation at 10 min to a similar degree on different T-cell subsets (B). Data represent 3 independent experiments and are expressed as means ± SD from 3–4 mice per group.</p

Expression of B7-H4 receptor on activated T-cell subsets.

<p>Naïve CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were stimulated with 0.3 µg/ml of plate-bound anti-CD3 and soluble anti-CD28 (1 µg/ml) for 16 h and then rested for 30 h prior to re-activation. Activated CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were stimulated with plate-bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) for 4 d. Expression of putative B7-H4 receptor was detected as follows. Cells were stained with a control human IgG1 (filled) or with a B7-H4.hIgG1 (open), followed by goat anti-human IgG-PE. (A). Representative histograms of expression of B7-H4 receptor on CD3⁺, CD4⁺, or CD8⁺ T-cell subsets. (B). Mean fluorescence intensity (MFI) of B7-H4 receptor was plotted. There is no significant difference in the expression of B7-H4 receptor among three different T-cell subsets. Data represent 3 independent experiments and are expressed as means ± SEM.</p

B7-H4 inhibits T-cell proliferation.

<p>Naïve (A and B) and pre-activated (C,D, and E) CD3⁺ T cells were stimulated with various concentrations of plate-bound anti-CD3 and soluble anti-CD28 (1 µg/mL) for 72 h. 18 h before harvest, cultures were pulsed with 1 µCi of [3H]-thymidine. B7-H4.Ig or Fc.Ig was added at indicated concentrations (B and D). 10 µg/ml and 30 µg/ml of B7-H4.Ig or Fc.Ig was added for naïve and pre-activated T cells, respectively. Pre-activated CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were stimulated with 0.3 µg/ml of plate-bound anti-CD3 and soluble anti-CD28 (1 µg/ml). Triplicate wells were harvested and counted. Data represent three independent experiments and expressed as means ± SEM. One star (*) indicates p<0.05, two stars (**) indicate p<0.01, and three stars (***) indicate p<0.001.”</p