Angiii

Rat primary neurons were seeded in 96-well plates and maintained as previously described (Mateos et al., 2009 (link)). However, rat primary neurons were substituted for SH-SY5Y cells, as similar results were found in both cell types (Figs. 3 A and 5 C). This is in accordance with the replacement requirement of the guiding principles of ethical use of animals 3Rs (replacement, reduction, and refinement). SH-SY5Y cells were grown in 24-well plates with MEM supplemented with 10% FBS until 80% confluence. Treatments were with 1 µM 27-OH (24 h) and 10 µM bestatin (24 h); however, when 1 µM AngIV and 1 µM AngIII (Bachem) were added, this was done in the last 3 h of the 27-OH treatment. Insulin was used at 100 nM. For the combination of 27-OH and insulin, 1 µM 27-OH was applied for 24 h, followed by insulin for 3 h. A fluorescent glucose analogue, 2-NBDG (Invitrogen), was used at 100 µM diluted in HBSS (Sigma-Aldrich). After incubation at 37°C for 1 h with the analogue in addition to the treatments, culture medium was removed and fluorescence was measured (after repeated washing and addition of PBS) in a fluorescence microplate reader, set at an excitation wavelength of 466 nm and an emission wavelength of 540 nm. Experimental outcomes are indicated as a percent reduction of fluorimetric response with respect to controls. Results are expressed as means ± SEM.

Primary cortical and hippocampal neuronal cultures from embryonic day (E) 18 Sprague Dawley rat embryos were established as described previously (Mateos et al., 2009 (link)). 27-OH was obtained from Steraloids, and treatments were done at 1 µM for 24 h. Blocking LXR was done by preincubating cells for 3 h with 10 µM 22(S)-OH (Sigma-Aldrich). AngIV and AngIII (Bachem) treatments were added at 1 µM in the last 3 h of 27- OH treatment. Furthermore, 10 µM bestatin (Sigma-Aldrich) was added for 24 h or co-incubated with 27-OH. Insulin signaling experiments were done by incubating 1 µM 27-OH for 24 h, and 100 nM insulin was added in the last 3 h. Experiments with primary cultures were conducted following approval from the regional ethical committee of Karolinska Institutet, Stockholm.