Islet isolation and cell culture and treatment

Mouse islets were isolated as previously described [34]. Briefly, islets were isolated by 2 mg/ml liberase (Roche, Mannheim, Germany) injection into the pancreas and digested for 10 minutes at 37°C. Islets were purified by a density gradient of Histopaque (1:1; 1077 and 1119, Sigma) and by subsequent hand-picking. Human islets were isolated from six pancreases of healthy organ donors and from ten with T2D at the University of Illinois at Chicago, Lille University or at ProdoLabs. Informed consent was obtained from all subjects or their relatives and ethical approval given to the respective institutions. Research with human islets from brain dead donors applies to NIH regulations PHS 398, exemption 4. The use of human islets in the experiments have been approved by the "University of Bremen ethical committee". Islet purity was greater than 95% as judged by dithizone staining (if this degree of purity was not achieved by routine isolation, islets were handpicked). Mouse and human islets were cultured on extracellular matrix (ECM-) coated dishes (Novamed Ltd., Jerusalem, Israel, [35]) for 48h. For Ang-2 overexpression, islets from RIP-rtTA;Tet-O-Ang-2 and RIP-rtTA control mice were maintained in 10 μg/ml doxycycline prior to and during treatments. Islets were treated with diabetogenic conditions of glucolipotoxicity (22.2 mM glucose+ 0.5 mM palmitate) or a cytokine milieu (2 ng/ml IL-1β, 1000U/ml IFN-ɣ and TNF-α) for 3 days. MS-1 cells (mouse islet endothelial cell line ATCC CRL-2279) were cultured in DMEM 5.5 mM glucose/ 5% FCS and treated with glucolipotoxic (22.2 mM glucose+ 0.25 mM palmitate) or a cytokine milieu (2 ng/ml IL-1β, 1000U/ml IFN-ɣ and TNF-α) for 24h. Recombinant human Ang-2 (Peprotech) and 100 nM Tie-2 kinase inhibitor 4-(6-Methoxy-2-naphthyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (CAS 948557-43-5; Calbiochem, Merck Millipore # 612085), a potent and selective Tie-2 tyrosine kinase inhibitor [36] was added in parallel to treatments. The highly selective Tie2 kinase inhibitor was developed by Semones et al. [36] by library screenings and in silico design and its in vitro and in vivo activity was shown [36, 37].