Glycogen assay kit

Glycogen content was identified using the approach proposed by Chen et al. (2019) (link). In brief, 0.5 g of minced LL sample was mixed with 1.5 mL of 2 M NaOH and then kept boiling for 20 min. Subsequently, the cooled mixture was mixed with 8 mL of ultrapure water and subjected to centrifugation at 5,000 × g for 10 min. Finally, the supernate was utilized to identify glycogen level with the assistance of a commercial glycogen assay kit (Sigma‐Aldrich, St. Louis, MO, America).
Lactate content was measured following the method described by Li et al. (2016) (link). In general, 0.5 g of minced LL sample was homogenized in 4.5 mL of ice-cold saline at 12,000 rpm for 30 s. After the centrifugal process at 3,000 × g for 15 min under 4 °C, the supernate was collected and applied to identify lactate level with the assistance of a commercial lactate assay kit (Sigma‐Aldrich).
Glycolytic potential was evaluated based on the formula suggested by Li et al. (2017) (link), where Glycolytic potential = 2 × glycogen + lactate.

To assess liver glycogen levels, ∼50 mg of previously snap-frozen liver was weighed and homogenized in 500 µL of ice-cold PBS. Homogenates were heated to 95 °C and cooled on ice before centrifugation for 10 min at room temperature to remove precipitate. Supernatant was assayed for glycogen content using the Glycogen Assay Kit (Sigma-Aldrich) according to manufacturer’s instructions. Glycogen content was normalized to initial liver weight.

Tissue glycogen levels were measured using a glycogen assay kit (Sigma-Aldrich) following manufacturer’s instructions. In brief, 10 µL tissue homogenate was incubated with hydrolysis enzyme reaction mixture in a total volume of 50 µL at room temperature for 30 min before adding 50 µL development enzyme reaction mixture for 30 min incubation at room temperature. Absorbance at 570 nm was measured using a spectrophotometer (Multiskan FC Microplate Photometer, Thermo Fisher). A standard curve was generated using standard glycogen solution in the reaction. A reaction without hydrolysis enzyme treatment was used for background correction (endogenous glucose) for each sample.

Worms were allowed to develop and grow on agar plates at 20 °C until they reached stage L4 and then transferred to NGM plates with or without glucose. At certain ages, worms were collected in micro-centrifugal tubes, washed quickly two times with distilled water, most of the liquid was removed and the worms were flash frozen in liquid nitrogen and stored at −80 °C until further analysed. For analysis, worms were resuspended in ∼70 μl of dH₂O and immediately boiled for 5 min to inhibit enzymatic degradation of glycogen. Tubes were chilled on ice and the worms were grinded with a disposable pestle and aggregated protein and other insoluble material separated by centrifugation. A Glycogen Assay Kit (Sigma, MAK016-1KT) was used to determine glycogen content in the supernatant. As most of the proteins precipitate after boiling, the pellets were dissolved in M9 with 8 M Guanidine HCl, separated from insoluble material and the amount of protein in the supernatant was determined. The total amount of glycogen reported in the Figures was normalized relative to the total protein in the sample. All experiments were repeated at least three times and the average±s.d. presented in the Figures. The detailed results from individual experiments are presented in Supplementary Table 3.