Cxcl5

Whole blood was collected via cardiac puncture at euthanasia, serum was isolated, and stored at −80°C. IL17A (Quantikine, R&D Systems), TNF (Quantikine, R&D Systems), P1NP (Immunodiagnostics Systems Inc, Gaithersburg, MD, USA), and TRAP5b (Immunodiagnostics Systems Inc) were assessed in serum isolates. CXCL1 (Quantikine, R&D Systems), CCL2 (Quantikine, R&D Systems), and CXCL5 (Quantikine, R&D Systems) was evaluated in osteoblast culture supernatants. ELISA kits were used following manufacturer’s protocols. All reactions were performed in triplicate.

Pancreatic islets were isolated by collagenase-P digestion (Roche Diagnostics, Indianapolis, IN) followed by centrifugation with Histopaque 1100 (Sigma-Aldrich, St. Louis, MO) as previously described (Carter, et al. 2009 (link)). Islets were incubated overnight in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin to allow recovery from collagenase digestion before further treatment. Various islet stressors were prepared as follows: Rotenone and thapsigargin were purchased from Sigma-Aldrich and prepared in stocks of DMSO to final concentrations less than 0.1%. Glucose-free RPMI medium (10% FBS and 1% penicillin/streptomycin) was supplemented with 1M glucose stock to produce the 28G condition. Stocks of the murine forms of IL-1B, IL-6, CXCL1, and CXCL5 (purchased from R&D Systems, Inc., Minneapolis, MN) were prepared in PBS with 0.1% BSA. Oleate, linOleate, and palmitate were purchased from Sigma-Aldrich and were prepared in 100mM stock concentrations in methanol and stored in -80. To treat the islets, the required amount of each fatty acid was taken in a glass tube and dried under a stream of nitrogen to remove methanol. The dried mixture of fatty acids thus obtained was resuspended in the RPMI medium with 0.1% BSA followed by vortexing and sonication. All experiments were completed within 48-72 hours post isolation.