Glucose standard solution

Uncoated ZnO nanopowder with an advertised 25-nm particle size was purchased from PlasmaChem GmbH, and ZnCl₂ was purchased from Merck Group. Other chemicals used in the present study were of analytical grade unless otherwise stated. Trichloroacetic acid (TCA), Bradford reagent, bovine serum albumin (BSA), glucose standard solution, iodonitrotetrazolium chloride (INT), β-nicotinamide adenine dinucleotide (NADH), β-nicotinamide adenine dinucleotide phosphate (NADPH), polyvinylpyrrolidone (PVP), tris(hydroxymethyl)aminomethane (Tris base), t-octylphenoxypolyethoxyethanol (Triton™ X-100), and glyceryl tripalmitate were purchased from Sigma-Aldrich®. CHCl₃ (chloroform), CH₃OH (methanol), C₆H₆O (phenol), NaOH, HCl, H₂SO₄, MgSO₄, and HNO₃ (69.9–70.0%; Baker Instra-Analyzed) were purchased from Avantor Performance Materials S.A., and H₂O₂ (30.0%; Baker Analyzed) was purchased from WITKO®.

Glucose concentration was analyzed in the supernatant using high-performance liquid chromatography (HPLC). A Waters Acquity UPLC^® H-Class System (Waters, Zellik, Belgium) with an ion-exchange Aminex HPX-87H column 7.8 × 300 mm (Bio-Rad Laboratories N.V., Temse, Belgium) heated up to 50 °C was used for analysis. A metabolite analysis was carried out with an isocratic flow rate of 0.6 mL min⁻¹ for 25 min. The mobile phase was composed of water containing 5 mM H₂SO₄. Elution profiles were monitored through a Waters Acquity^® Refractive Index Detector (RID) (Waters, Zellik, Belgium). A glucose standard solution (Sigma-Aldrich, Overijse, Belgium) was used to determine the retention time and to establish a calibration curve.

Six male flies were collected at ZT8 and homogenized in 250 μL of PBS with 0.2% Triton-X. The homogenates were then incubated at 70 °C for 5 min to inactivate endogenous enzymes. After centrifugation at 13,500 × g for 15 min at 4 °C, 150 μL of supernatant was collected for subsequent measurement. The PGO Enzyme Preparation Solution (Sigma, P7119) was prepared following the manufacturer’s instructions. The PGO Enzyme Reaction Solution was prepared by adding water, glucose standard solution (Sigma, G6918), and the glucose-containing sample according to the manufacturer’s instructions. All tubes were then incubated at 37 °C for 30 min, and the absorbance (A) of the standards and tests was measured at 450 nm using the Blank as the reference. The glucose concentration of the sample was determined using the following formula:
Sample Glucose Concentration (mg/mL) =  $\frac{\mathrm{Absorbance}\left(\mathrm{Test}\right)\times \mathrm{Dilution}\mathrm{of}\mathrm{sample}\times 1\mathrm{mg}/\mathrm{mL}}{\mathrm{Absorbance}\left(\mathrm{Standard}\right)}$