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Obesity is frequently associated with excessive accumulation of lipids in ectopic tissue and presents a major risk factor for type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Diacylglycerols (DAGs) and ceramides (CERs) were identified as key players in lipid-induced insulin resistance, typical for such diseases. Recent results suggest that the subcellular distribution of these lipids affects their lipotoxic properties. However, the subcellular dynamics of these lipids and the role of lipid droplets (LDs) as a potential storage site is not understood. Here, we developed a liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS)-method for the rapid and simultaneous quantification of DAG and CER species in tissue sample fractions. The assay is characterized by excellent recovery of analytes, limit of quantification, accuracy and precision. We established a fractionation protocol that allows the separation of subcellular tissue fractions. This method was subsequently tested to measure the concentration of DAGs and CERs in subcellular fractions of human muscle and several mouse tissues. In a mouse model of NAFLD, application of this method revealed a prominent role for LDs as repository for lipotoxic DAG and CER species. In conclusion, the new method proved as a valuable tool to analyse the subcellular dynamics of lipotoxins, related to the pathogenesis of insulin resistance, T2D and NAFLD.

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A New Targeted Lipidomics Approach Reveals Lipid Droplets in Liver, Muscle and Heart as a Repository for Diacylglycerol and Ceramide Species in Non-Alcoholic Fatty Liver