Fluorescence based enzymatic detection kit

Glucose measurements were performed 3 times on 10 zebrafish larvae per condition using a fluorescence-based enzymatic detection kit (Biovision Inc.) [26 (link)]. The larvae were collected in 1.5 ml microcentrifuge tubes. Excess medium was removed and embryos were frozen on crushed dry ice. After thawing, 200 μl PBS was added and the larvae were homogenized using a hand-held mechanical homogenizer. Reactions were assembled on ice in black, flat bottom 96-well plates (Costar). Standard curves were generated using glucose standard solution (according to instructions) and were included in each assay. To measure glucose in embryo extracts, 15 μl of sample were used. Control reactions without sample lysate were included in each row. Reactions were incubated for 30 minutes at 37°C in the dark. Fluorescence (excitation 535 nm; emission, 590 nm) was measured using a Safire II plate reader equipped with XFLUOR4 software (v 4.51). Fluorescence values were corrected by subtracting measurements from control reactions without sample. Glucose levels were interpolated from standard curves. Each sample was measured in triplicate and each experiment repeated three times.

Measurements of total body glucose and lactate were performed on a pool of 5 larvae per genetic condition using a fluorescence-based enzymatic detection kit (Biovision Inc.) as described before [81 (link)]. Briefly, larvae were collected in 1.5 ml microcentrifuge tubes, the excess of water was removed and samples were frozen stored at -80°C until further analysis. Upon defrosting, 15 μl PBS per larvae was added and tissues were disrupted with a sterile pestle (Axygen), on ice. Samples were spun at 14,000 x g for 10 min, at 4°C. The supernatant was immediately used for metabolite measurements according to manufacturers’ protocol. Fluorescence readings (Ex/Em 535/590) were taken with the FLUOstar Omega instrument (BMG Labtech). Results were read from a linear regression curve based on the dilutions of the standard. Statistical analysis between indicated groups was performed using the unpaired t-test and considered significant at P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).

Glucose measurements were performed 3 times on 10 zebrafish larvae per condition using a fluorescence-based enzymatic detection kit (Biovision Inc.). cAMP content of whole-zebrafish larvae and rat INS-1 cells was analyzed using commercial ELISA kits (Enzo Life Sciences, Inc.). Further details are described in the Supplemental Experimental Procedures.