Modified and Efficient Glucose Consumption Assay to Study Insulin Resistance in C2C12 Myotubes

Introduction: Insulin resistance (IR), defined by a diminished cellular response to insulin, leads to reduced glucose uptake and disrupted metabolic homeostasis. IR is a central factor in type-2-diabetes and other major non-communicable diseases, including cardiovascular disease, nonalcoholic fatty liver disease, etc. Although animal and human models exist for studying IR, they are costly and raise ethical concerns. Cell culture models, particularly the colorimetric glucose consumption assay, offer a simpler approach but itself gets affected by number of variations in media composition and assay conditions. This study aims to address these issues and re-standardize the glucose consumption assay by using C2C12 myotubes. Methods: C2C12 myotubes were exposed to lab-modified incubation media (DMEM with 1g/L glucose, 0.25% BSA) with varying insulin concentrations (10, 50, 100, 200 nM) and incubation times (3, 6, 12, 24 hours). Glucose levels were measured using a colorimetric kit, and results were compared with a 2-NBDG fluorescence assay in an oxidative stress-induced IR model. Results: Our findings depict that significant glucose consumption occurs either after 6h of incubation with a 50 nM insulin or after 24h with a 10 nM insulin. Moreover, the data are well comparable with fluorescence assays in an oxidative stress-induced IR model. Discussion: Optimizing the cultured media and insulin concentration reduced the assay time and cost without compromising reliability. Conclusion: By using an optimized glucose consumption assay, future IR cell culture studies can be made more efficient either by reducing incubation time or insulin concentration.