Glucose oxidase method

Serum glucose level was analyzed using glucose oxidase method (Applygen, Beijing, China) (Wang C. et al., 2016 (link)). Serum insulin level was determined by ELISA using an ultra-sensitive mouse insulin kit (Crystal Chem, USA) (Ding et al., 2016 (link)). Serum levels of total cholesterol, triglycerides, LDL-C, HDL-C, and free fatty acids (FFA) levels were assayed using the calorimetric kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) (Jiang et al., 2016 (link); Wang et al., 2019 (link); Liu et al., 2020 (link)). Liver TG was analyzed using Tissue triglyceride assay kit (Applygen, Beijing, China) (Wang C. et al., 2016 (link)).

Before the experiments, the cells were transferred and maintained in 96‐well plates to 80% confluence and then treated with rotenone or amobarbital at various concentrations in FBS‐free DMEM (15 mmol/l D‐glucose) containing 0.25% bovine serum albumin (BSA) for 24 h (n = 4–8/group). The glucose concentration in the medium was determined by the glucose oxidase method (Applygen Technologies Inc., China). The amount of glucose consumption was calculated by subtraction of glucose concentrations between the cell plated wells and the blank wells 7. The cells were collected and lysed. And the total protein amount of each well was measured using a BCA protein assay kit (Beyotime Biotechonology, China) to normalize the glucose consumption.

Glucose uptake was measured using nonradioactive fluorescent glucose 2-NBDG (Sigma, St. Louis, MO, USA), as described previously [45 (link)]. For skeletal muscle experiments, fresh GM from mice after insulin or/and PBMT was incubated with 2-NBDG (50 μM) for 30 min. The specimens were washed and lysed, and 2-NBDG levels in GM were quantified using a microplate fluorimeter (Infinite M200; Tecan, Hillsborough, NC, USA). For cell experiments, IR-L6 myotubes were incubated in KHB containing 50 μM 2-NBDG with or without 8 J/cm² PBMT at 37° C for 30 min. Cells were lysed, then 2-NBDG (Ex/Em, 465/540 nm) was quantified with a microplate fluorimeter. For glucose consumption in DMEM medium, IR-L6 myotubes were serum-starved for 12 hours and irradiated by PBMT. The cells were cultured in fresh free-serum medium in a humidified incubator containing 5% CO₂ at 37° C. After 12 hours, glucose consumption in the DMEM medium was measured using Glucose Oxidase Method (Applygen Technologies Inc., Shanghai, China) following the supplier’s instructions.