6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoglucose

A fluorescent glucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2-NBDG) (Invitrogen #N13195), was used to measure glucose uptake in INS-1 cells according to the manufactory's instructions. Briefly, a 100 µM of 2-NBDG (dissolved in ethanol) was added over the transfected cells (as described above) (100 µM/1 ml medium/well) and incubated for one hour at 37 °C. Next, trypsin harvested cells were washed twice with cold-PBS, then centrifuged for 5 min at 1500 rpm at 4 °C. The supernatant was aspirated and cell suspended in 200 µl of cold-PBS in each tube. Cells were immediately analyzed and quantified by flow cytometry (BD FACS Aria™ III) at excitation 465 nm and emission at 540 nm .

A fluorescently labeled deoxyglucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]- 2-deoxy-glucose (2-NBDG) (Invitrogen #N13195, Waltham, MA, USA), was utilized as a probe for the detection of glucose uptake in cultured cells, as previously described [28 (link)]. Briefly, after 48 h transfection, cells were incubated with the 2-NBDG reagent (1 h). Cells were then trypsinized and analyzed by flow cytometry (BD FACS Aria III flow cytometer, San Jose, CA, USA) with excitation at 465 nm and emission at 540 nm.

To evaluate the impact of avarone (2) on the insulin signaling pathway, C2C12 cells were plated on P35 dishes and grown until 70% confluence. Then, cells were washed with phosphate-buffered saline (PBS) and incubated for 24 h in the presence of a starvation medium (DMEM high glucose containing 2 mM glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin). After this time, cells were stimulated for 30 min with 10 nM insulin (Humulin R, Ely Lilly, Italy) with avarone (2) or the insulin-avarone combination. After this time, cells were washed with cold PBS and lysed using 100 µL of 1X Laemmli sample buffer solution (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue). Cell proteins were withdrawn, transferred in an Eppendorf tube and boiled for 5 min. All samples were stored at −20 °C before the analysis. Analysis of samples was carried out using SDS-PAGE; after electrophoresis, proteins were transferred on a PVDF membrane by Western blot. Phosphorylation levels of the insulin receptor β chain and of the kinase Akt were evaluated using specific antibodies: IR β subunit, clone CT-3 (MABS65) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); pIR β subunit, Y1162/1163 (sc-25103-R) was from Merck-Millipore (Burlington, MA, USA). Akt (9272S) and p-Akt (9271S) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Finally, β-actin and clone C-4 (sc-47778) were from Merck-Millipore (Burlington, MA, USA). Anti-rabbit and anti-mouse secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Detection was performed using Clarity western ECL substrates (BioRad Laboratories, Inc. 1000 Alfred Nobel DriveHercules, CA, USA). Quantitation of the images obtained was carried out using iBright Analysis Software (Thermo Fisher Scientific 168 Third Avenue Waltham, MA, USA, 02451). Glucose uptake was evaluated as previously described [26 (link)]. Briefly, 70% confluent C2C12 cells were starved and then stimulated for 30 min with 10 nM insulin, avarone, or the insulin-avarone combination. After this time, cells were washed with PBS and incubated for 3 h with starvation medium containing 40 μM 2-NBDG (Invitrogen). Then, cells were washed with PBS, trypsinized, pelleted by centrifugation (1000× g for 5 min), and resuspended in 500 μL of PBS before the analysis. Intrinsic and 2-NBDG-derived cell fluorescences were determined using a flow cytometer (FACSCanto II, BD Biosciences, San Jose, CA, USA). For each sample, 1 × 104 events were acquired. Quantitative analysis was carried out using FlowJo software (FlowJo^TM v10. BD FlowJo™ Software for Windows Version 10. Ashland, OR: Becton, Dickinson and Company; 2021).