13C-labelling studies indicate compartmentalized synthesis of triacylglycerols in C6 rat glioma cells

NMR-visible mobile lipid (ML) signals have been detected in 1H-NMR spectra of tissues in vivo, ex vivo and in vitro, and have been shown to change in apparent intensity in association with pathology (necrosis in brain tumours) and normal processes (cell differentiation, cell growth arrest and apoptosis). Although it is widely accepted that ML signals originate mainly from fatty-acyl chains in triacylglycerols (TAG) contained in cytosolic lipid droplets (LD), the dynamics of TAG in LD is not yet fully understood. In order to better understand the synthesis of cellular TAG and its relationship to ML dynamics we carried out a set of labelling experiments with C6 rat glioma cells in culture. TAG and phospholipid metabolism was monitored by incubating C6 cells with [1-13C]-glucose at two time points during cell growth curve –24 h incubation starting at log-phase; 48 h incubation starting at saturation density– and by acquiring the 2D-HMQC NMR spectra of the respective total lipid extracts. The resulting TAG, diacylglycerol (DAG) and phospholipid labelling patterns can only be explained if TAG synthesis takes place in two different subcellular compartments. One compartment would be the endoplasmic reticulum, which is known to be involved in TAG metabolism, while the other compartment could be the plasma membrane and/or the LD. This possible role of LD is further supported by the recent description of diacylglycerolacyltranferase-activity associated with LD. Accordingly, we postulate the existence of a carbon-shuttling mechanism between plasma membrane phospholipids and endoplasmic reticulum by way of LD content. The results we have obtained with C6 cells may also apply to other cellular systems and should be taken into account when interpreting ML dynamics detected by NMR in vivo.