Glucose hk

Glycogen measurement was adapted from the protocol described by Monin and Sellier^{48 (link)}. The YSM (100 mg) was homogenised in 500 µL of H₂O and 500 µL of cold perchloric acid (PCA, 1 M) using TissueLyser II for 4 min. A volume of 500 µL was centrifuged (20 min, 15,000g, 4 °C) to determine the free-glucose concentration (Glucose HK, ref 981779, ThermoFisher Scientific). The remaining volume was suspended with potassium hydroxide (KOH, 5.4 M) and sodium acetate buffer (0.2 M) added with amyloglucosidase (2 mg/mL, Sigma). The samples were incubated for 3 h at 38 °C with stirring (1400 rpm.). Cold PCA (3 M) was added and, after 10 min at 4 °C, samples were centrifuged (15 min, 15,000g, 4 °C). Supernatants were collected for total glucose determination (Glucose HK, ref 981779, ThermoFisher Scientific). Scal (ref 981831, ThermoFisher Scientific) and Abtrol (ref 981044, ThermoFisher Scientific) were used for calibration and quality control, respectively.
A range of glycogen was used to determine the regression line y = ax + b. $$\text{Adjusted}\left[\text{Glu HK hydrolysis}\right]\text{in mg/L}=2\ast \left[\text{Glu HK hydrolysis}\right]\ast 3.3\left(\text{dilution factor}\right)$$ $$\left[\text{Glucose derived from glycogen}\right]\text{in mg/L}=\text{Adjusted}\left[\text{Glu HK hydrolysis}\right]-\left[\text{Glu HK free}\right]$$ $$\text{Amount of glycogen in mg}=(\left[\text{Glucose derived from glycogen}\right]-\text{b})/\text{a}$$ $$\left[\text{Glycogen}\right]\text{in mg/g of tissue}=\frac{(\left[\text{Glucose derived from glycogen}\right]-\text{b})/\text{a}}{\text{Weight}\left(\text{g}\right)}$$

The serum glucose (Glucose GOD-POD, ref 981,780, Thermo Fisher Scientific, Dardilly, France), uric acid (Uric Acid AOX, ref 981,788, Thermo Fisher Scientific, Dardilly, France), triglyceride (ref 981,786, Thermo Fisher Scientific, Dardilly, France), non-esterified fatty acid (NEFA-HR(2), FUJIFILM, Sobioda, Montbonnot Saint Martin) and hydroxybutyrate (RANBUT, RB 1007/MD, RANDOX, Roissy) concentrations; the serum total antioxidant status (TAS, NX2332, RANDOX, Roissy); the creatine kinase activity (CK-NAC, ref 92,307, BIOLABO, Abliance, Compiègne, France); and the liver glycolytic potential (Glucose HK, ref 981,779 and L-lactic acid, ref 984,308, Thermo Fisher Scientific, Dardilly, France) were measured spectrophotometrically using commercial kits.