Islet hypervascularization in T2D and effects of Ang-2 overexpression.

<p>A healthy islet is surrounded by intact capillaries, maintained by the Ang/Tie system and extracellular matrix supporting the function and survival of the islet. An increased insulin demand leads to more islet blood flow, β-cell mass and vascular expansion and consequent compensation. A transient Ang-2 upregulation promotes angiogenesis, leading islet endothelium to a non-quiescent state inhibiting Tie-2 signaling. Towards human T2D progression, β-cell failure and apoptosis occurs together with increased islet and endothelial inflammation and islet hypervascularization. Ang-2 overexpression on the other hand prevents β-cell mass and vascular expansion in response to HFD with persistent islet and endothelial inflammation.</p

Ang/Tie expression in isolated islets correlates with changes in vessel area.

<p>Isolated WT mouse and human islets were cultured for 3 days in control condition (11.1 mM glucose for mouse or 5.5 mM for human) or treated with diabetic conditions of 22.2 mM glucose + 0.5 mM palmitic acid or mixture of cytokines: 2 ng/mL IL-1β, 1000 U/ml IFN-ɣ and TNF-α (cyto). <b>(A,D)</b> GSIS is shown by the stimulatory index assessed by 16.7/2.8 mM glucose stimulation. <b>(B,C,E,F)</b> Graph shows ratio of vessel area to islet area for mouse <b>(B,C)</b> and human <b>(E,F)</b> islets, fixed and immune-labelled for vessel (CD-31,red) and islet (insulin, green). <b>(G-L)</b> qPCR analysis of treated mouse and human islets for mouse CD-31 <b>(G,J)</b>, Ang-1,-2 <b>(H,K)</b>, Tie-1,-2 <b>(I,L)</b>. All genes have been normalized to PPIA or 18s as housekeeping control. *p<0.05, treated vs. control 11.1 mM (mouse) or 5.5 mM (human). <b>(M)</b> Representative western blot from treated human islet lysates (left panel) and densitometric analyses of Ang-2 (right panel). Data are means +/-SE from 3–5 independent experiments from 3–5 different organ donors (human islets) or 3–5 independent mouse islet isolations.</p

Islet vessel area increases in T2DM.

<p><b>(A)</b> Representative images of pancreatic sections from non-diabetic controls and patients with T2D, immune-labelled for CD31 (red) and insulin (green). <b>(B)</b> Graphs show ratio of vessel area to islet area (control: n = 6; T2D: n = 10). <b>(C)</b> Plot shows no correlation of vessel density with BMI. <b>(D-H)</b> qPCR analysis of Ang-2, Tie-1, Tie-2, CD-31 from isolated mouse islets from C57BL/6 WT mice kept on normal diet (ND) or high-fat high-sucrose diet (HFD) for <b>(D)</b> 8 weeks (n = 4/group), <b>(E)</b> 16 weeks (n = 9/group) and <b>(F)</b> 24 weeks (n = 7/group), <b>(G) of</b> eNOS and <b>(H) of</b> ICAM-1. <b>(I)</b> qPCR analysis of Ang-1, Ang-2, Tie-1 and Tie-2 of isolated islets from non-diabetic (control, n = 8) and from patients with T2D (n = 7). *p<0.05, HFD vs ND or T2D vs. control</p

Ang-2 over-expression impairs islet function but protects from cytokine treatment in isolated islets.

<p>Isolated islets from RIP-rtTA;tet-O-Ang-2 (Ang2-rtTA) and RIP-rtTA control (rtTA) mice were cultured for 3 days in presence of 10 μg/ml doxycycline to achieve Ang-2 overexpression. Mouse or human islets were cultured in 11.1 (mouse) or 5.5 mM glucose (human) or treated with diabetic conditions of 22.2 mM glucose + 0.5mM palmitic acid or mixture of cytokines: 2 ng/mL IL-1β, 1000 U/ml IFN-ɣ and TNF-α (cyto). <b>(A)</b> Western blot from treated mouse islets shows Ang-2 overexpression in islets by myc-Ang-2. <b>(B)</b> GSIS is shown by the stimulatory index assessed by 16.7/2.8 mM glucose stimulation and normalized to control. <b>(C,D)</b> Treated mouse islets fixed post-GSIS and apoptotic cells detected by double staining for TUNEL and insulin. Representative images from different treatments. <b>(E,F)</b> qPCR analysis for CD31 <b>(E)</b> and ICAM <b>(F)</b> from mouse islets overexpressing Ang-2. <b>(G,H)</b> Representative western blots (upper panel) and densitometric analyses of proteins (lower panels) showing myc-Ang-2, ICAM-1, cleaved caspase 3 and actin/tubulin as housekeeping control, in human islets overexpressing Ang-2 by Ad-Ang-2 or control Ad-GFP <b>(G;</b> MOI = 50) or treated with 100 nM Tie-2 inhibitor for 72h (<b>H)</b>. Data are means +/-SE from 3–5 independent experiments from 3–5 different organ donors (human islets) or 3–5 independent mouse islet isolations. *p<0.05, treated vs. 11.1 mM glucose control, #p<0.05, Ang2-rtTA vs. rtTA.</p

Decreased β-cell mass in Hq mutant mice.

<p>(A). Representative Western blots (panel 1,3) and PCR analyzes (panel 2,4) of AIF expression in isolated mouse (panel 1,2) and human (panel 3,4) islets. Actin was used as loading control/ house keeping gene. Western blots/ PCRs are representatives of three independent experiments from three mice or from 3 organ donors, respectively. (B) Histological analysis by insulin staining in green and glucagon staining in red show a normal islet cellular composition and smaller islets in 2-week-old <i>Hq</i> mutant mice. (C,D) Analysis of β-cell mass (C) or β-cell mass divided by body weight (D) of WT and <i>Hq</i> mutant mice at 2 and 9 weeks of age. Values are representative of 5 slides spanning the whole pancreas of each mouse and 4 mice for each group at each age (magnification x125). (E) Cell cycle characteristics of β-cells from WT mice and <i>Hq</i> mutant mice as measured by BrdU and pHH3 staining. BrdU⁺insulin⁺ cells are counted as β-cells at S phase (see example in F, upper panel). pHH3⁺ (with punctuated pattern) insulin+ cells are counted as β-cells at G2 phase (see example in F, Hq mice lower right panel). pHH3⁺ (with strong nuclear expression) insulin+ cells were counted as β-cells at M phase (see example in F, WT mice lower left panel). Data are shown as mean±SE. *<i>P</i><0.05 in <i>Hq</i> mutant mice <i>vs</i>. WT mice.</p

AIF depletion leads to increased β-cell apoptosis in human islets without affecting insulin secretion.

<p>(A) Isolated human pancreatic islets were exposed to siRNA to AIF (siAIF) or scrambled control siRNA (siScr) for 3 days. The knockdown efficiency was determined by Western blot analysis. Actin was used as a loading control on the same membrane after stripping. This Western blot is representative of three independent experiments from three different organ donors. The density of expression levels were quantified after scanning and normalised to actin levels. (B–D) 3 days after transfection, islets were exposed to 50 µM H₂O₂ for 2 h. (B) Islet sections were prepared for analysis of β-cell apoptosis by the TUNEL assay. Islets were double-stained for insulin in green and counterstained for DAPI in blue. Results are means±SE of the percentage of TUNEL-positive β-cells. The mean number of β-cells counted was 3400 for each treatment condition. (C,D) GSIS: after the H₂O₂ treatment, islets were washed and basal and stimulated insulin secretion analyzed during successive 1-h incubations at 2.8 mM (basal) and 16.7 mM (stimulated) glucose. Data are normalized to insulin content. (D) Stimulatory index denotes the ratio between stimulated and basal values of insulin secretion. (B–D) All assays were performed in triplicate or quadruplicate in three independent experiments from 3 different organ donors, respectively. *p<0.05 to siScr control, **<i>p</i><0.05 in H₂O₂ treated vs. untreated control.</p

AIF depletion in mice leads to increased β-cell apoptosis.

<p>(A, B) Triple staining for TUNEL in red, insulin in green and 4′,6-diamidino-2-phenylindole (DAPI) in blue was performed on fixed, paraffin-embedded sections from isolated islets from 9-week old mice treated for 2 h with or without 50 µM H₂O₂. (B) Results are expressed as percentage of TUNEL-positive β-cells±SE. The mean number of β-cells counted was 700 for each treatment condition. (C) Glucose tolerance test with 2 mg/g BW glucose: Fasting and glucose stimulated plasma glucose levels are significantly lower in 12–16 week old <i>Hq</i> mutant mice (n = 9) compared to age-matched WT mice. Data are shown as mean + SE. *p<0.05 in <i>Hq</i> mutant mice vs. wt mice. **<i>p</i><0.05 in H₂O₂ treated vs. untreated control.</p