Gahk20

Glucose assay was performed as described [25 (link)] with minor modifications. Log phase cells were harvested and re-suspended at a density of 8 × 10⁶ cells ml⁻¹ and kept under shaken conditions for 6 h at 22°C. Cells were harvested and lysed by the freeze–thaw method. Glucose assay was performed as per instructions given by the manufacturer (GAHK20; Sigma-Aldrich). Protein levels were measured using Bradford reagent (BIO-RAD).

Quantification of trehalose and glucose levels in whole fly extracts were performed as described previously (Meunier et al., 2007 (link)). Briefly, 10 males were weighed and homogenized in 250 µL of 0.25 M Na₂CO₃ buffer and incubated in a water bath at 95°C for 5 min to inactivate all enzymes. Then 150 µL 1 M acetic acid and 600 µL 0.25 M sodium acetate (pH 5.2) were added, and the solution was centrifuged (10 min,12,500 g force, 24°C); 200 µL of each supernatant was incubated overnight at 37°C with 2 µL porcine kidney trehalose (Sigma: T8778 UN) to convert trehalose into glucose, and 100 µL of this solution was added to 1 mL glucose hexokinase solution (Sigma: GAHK-20) and incubated for 20 min at 37°C. Glucose levels were quantified at 340 nm. We determined glucose concentrations using a glucose standard curve.

Calculation of the minimal Feret’s diameter, the closest possible distance between the two parallel tangents of an object, was determined as previously described^{49 (link)}. Images were obtained using an Olympus IX series microscope and analyzed using the CellP Software. The fiber type composition was assessed by determining the expression of different myosin heavy chain isoforms by high resolution gel electrophoresis followed by Coomassie Brilliant blue gel staining as described^{50 (link),51 (link)}.
Glycogen content was assessed enzymatically on snap frozen EDL and soleus muscles using a hexokinase-dependent, kit according to the manufacturer’s instructions (GAHK-20, Sigma-Aldrich; USA). A control to measure free glucose levels in the muscle (not related to glycogen stores), was also performed.

Glycogen content was assessed in frozen heart, gastrocnemius, and liver tissues by determining the amount of glucose released from glycogen with a commercially available kit (Sigma Aldrich, GAHK-20, St. Louis, MO, USA). Glycogen was separated from exogenous glucose in the tissue using an alkaline extraction procedure [69 (link)]. Triglycerides were extracted and measured from frozen heart, gastrocnemius, and liver tissue using a triglyceride colorimetric assay kit (Cayman Chemicals, #10010303, Ann Arbor, MI, USA).

Periodic acid Schiff’s (PAS) staining for glycogen was done on fresh frozen 10-μm-thick sections from quadriceps muscle as previously described [30 (link)]. In brief, sections were fixed in Carnoy’s fixative (60% ethanol, 30% chloroform, 10% acetic acid) for 5 min, followed by rinsing three times with water. Sections were immersed in 0.5% periodic acid (Sigma P7875) for 5 min, followed by rinsing with water four times and then transferred to Schiff’s solution (Sigma 3952016) for 10 min, followed by rinsing in running tap water for 10 min. Sections were mounted using Cytoseal 60 mounting medium (Richard-Allan Scientific 8310-16).
Total glycogen content in muscle was quantified using an adaptation of a previously described method [31 ]. In brief, 30 mg of frozen gastrocnemius muscle was dissolved in 1 ml 5 N KOH in a boiling water bath, then 0.2 ml of saturated sodium sulfate and 1.5 ml ethanol were added. Samples were spun at 2,000 × g for 10 min at 4°C and the pelleted material was resuspended in 0.5 ml 2 N HCl and incubated in a boiling water bath for 2 to 2.5 h. Samples were cooled and neutralized to pH 6 to 8 with 4 N KOH in 0.1 M triethanolamine. Glycogen content was determined by measuring the glucose released from glycogen using a glucose assay kit as recommended by the manufacturer (Sigma GAHK20).

Glucose concentrations were assayed using a specific enzymatic oxidation method coupled to NAD reduction (GAHK20; Sigma Aldrich, St. Louis, MO) according to the manufacturer’s instructions. Briefly, filter weighing paper (#531; VWR) was cut into 0.75 mm-radius discs, and single disks were placed between the agar gel and membrane before inoculation. After 1 week of growth, the filters were picked up, soaked in 20–180 μL dosing solution, and the absorbance value of the supernatants at 340 nm was read after 1 h incubation and compared to a calibration curve created using filters placed far from the colony or in colony-free gels.