Sirolimus

Sirolimus was purchased from LC Laboratories (Boston, MA, USA) and soy bean phosphatidylcholine (S100PC) from Avanti Polar Lipids (Alabaster, AL, USA). The liposomes were prepared using the thin lipid film hydration method followed by extrusion, as described previously [33 (link)–35 (link)]. The mixture of Sirolimus (20 mg) and phosphatidylcholine (120 mg) was dissolved in chloroform and dried with nitrogen gas to form a thin lipid film on a round bottom glass tube. The tube was maintained under vacuum for 12 h to ensure complete removal of chloroform. The resulting film was hydrated with 4 mL of TES buffer containing 6% trehalose (pH 7.0) for self-assembly of the lipids into multilamellar liposomes. The liposome dispersion was then passed through 0.1 and 0.2 μm pore size polycarbonate membranes in an extruder (Northern Lipids, Inc., Burnaby, Canada) under nitrogen pressure, and was freeze–dried and stored at 4 °C.

The dual IGF1R/IR inhibitor linsitinib and the mTOR inhibitors sirolimus and everolimus were purchased from LC Laboratories (Inc. Woburn, MA, USA) and prepared as a 10⁻³M stock solution in dimethylsulfoxide (DMSO). Compounds were stored at −20 °C and further diluted in 40% DMSO before the use. Final DMSO concentration, also added as vehicle to controls, was 0.4%.

Kinase inhibitors of ATM (KU-60019, AZD1390, and AZD0156) and ATR (VE-821) were obtained from Selleck Chemicals (Houston, TX, USA). Sirolimus was obtained from LC Laboratories (Woburn, MA, USA). The concentrations and timepoints of the pharmacological inhibitors used in all experiments were based on previous studies [26 (link),27 (link),28 (link),29 (link),30 (link),31 (link),32 (link),33 (link),34 (link)].

Sirolimus (#R-5000) and rosuvastatin calcium (#RHR1928) obtained from LC Laboratories (Woburn, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively, were dissolved in dimethyl sulfoxide (#D0457; Samchun Chemical, Seoul, Korea). Heparin sodium was obtained from Hanlim (Seoul, Korea). Zoletil® 50 was obtained from VIRBAC (Carros, France), and aspirin was obtained from Bayer Korea (Seoul, Korea). Rhodamine B and agarose were obtained from Sigma-Aldrich.

Human islets with >90% purity and viability were obtained from non-diabetic de-identified cadaveric donors through the Integrated Islet Distribution Program (IIDP). The characteristics of the donors are reported in Supplementary Table 1. Upon receipt, the islets were cultured as described^{70 (link)}. Murine islets of Langerhans were isolated as previously described^{31 (link)}. Procedures on rodents have been performed according to guidelines and regulations approved by the Einstein Animal Care and Use Committee. INS-1 cells were maintained in monolayer culture in RPMI-1640 medium, as previously described by our group^{71 (link)}. Insulin levels were determined as described and validated^{31 (link),71 (link)–73 (link)}. In some experiments the cells were treated with glucose (5.5 and 16.7 mM, Bio-Techne, Abingdon, UK), sirolimus (LC Laboratories, Woburn, MA, dissolved in dymethylsulfoxide), or L-leucine (10 mM, MyBioSource, San Diego, CA, USA) and glutamine (2 mM, MyBioSource). Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT colorimetric assay, spectophotometrically (570 nm) measuring the ability of metabolically active cells to reduce MTT.