MTT-Based Normalization of Glycerol Release for Accurate Quantification of Lipolysis in Primary Rat Epididymal Adipocytes

Lipolysis, a critical metabolic pathway, involves the hydrolysis of stored triglycerides into free fatty acids and glycerol. However, accurate quantification of glycerol output in lipid-rich primary adipocytes is often confounded by experimental artifacts, such as variability in cell viability and assay interference. To address these limitations, a straightforward method was established to normalize glycerol release measurements using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell viability assay. This protocol leverages the difference in specific gravity between adipocytes and aqueous solutions, enabling simultaneous quantification of lipolytic output and cell viability. In drug screening experiments, pravastatin and simvastatin significantly enhanced glycerol release from abdominal adipocytes, whereas atorvastatin and lovastatin did not significantly alter glycerol output. These findings suggest that MTT-based normalization offers a robust and convenient approach for accurately quantifying lipolysis, with potential applications in drug discovery targeting obesity and metabolic syndrome. This study underscores the need to re-evaluate conventional lipolysis assays to distinguish genuine metabolic responses from nonspecific membrane leakage.