Preparation of fatty acid solutions exerts significant impact on experimental outcomes in cell culture models of lipotoxicity

Abstract

Free fatty acids are essentially involved in the pathogenesis of chronic diseases such as diabetes mellitus, non-alcoholic fatty liver disease, and cardiovascular disease. They promote mitochondrial dysfunction, oxidative stress, respiratory chain uncoupling, endoplasmic reticulum-stress and modulate stress-sensitive pathways. These detrimental biological effects summarized as lipotoxicity mainly depend on fatty acid carbon chain length, degree of unsaturation, concentration, and treatment time. Preparation of fatty acid solutions involves dissolving and complexing. Solvent toxicity and concentration, the amount of bovine serum albumin, and the ratio of albumin to fatty acids can vary significantly between equal concentrations, mediating considerable harmful effects and/or interference with certain assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Herein, we studied the impact of commonly used solvents ethanol and dimethyl sulfoxide, varying concentrations of bovine serum albumin, treatment duration, and respectively prepared oleic acid solutions on MTT to formazan conversion, adenosine triphosphate level, and insulin content and secretion of murine β-cell line MIN6. Our data show that experimental outcomes and assay readouts can be significantly affected by mere preparation of fatty acid solutions and should thus be carefully considered and described in detail to ensure comparability and distinct evaluation of data.