A possible cellular explanation for the NMR-visible mobile lipid (ML) changes in cultured C6 glioma cells with growth

The NMR-visible mobile lipid (ML) signals of C6 glioma cells have been monitored at 9.4 and 11.7 T (single pulse and 136 ms echo time) from cell pellets by 1H NMR spectroscopy. A reproducible behavior with growth has been found. ML signals increase from log phase (4 days of culture) to postconfluence (7 days of culture). This ML behavior is paralleled by the percentage of cells containing epifluorescence detectable Nile Red stained cytosolic droplets (range 23%–60% of cells). The number of positive cells increases after seeding (days 0–1), decreases at log phase (days 2–4), increases again at confluence (day 5) and even further at post-confluence (day 7). C6 cells proliferation arrest induced by growth factors deprivation induces an even higher accumulation of cytosolic droplets (up to 100% of cells) and a large ML increase (up to 21-fold with respect to 4-day log phase cells). When neutral lipid content is quantified by thin-layer chromatography (TLC) on total lipid extracts of C6 cells, no statistically significant change can be detected (in μg/108 cells) with growth or growth arrest in major neutral lipid containing species (triacylglycerol, TAG, diacylglycerol, DAG, cholesteryl esters, ChoEst) except for DAG, which decreased in post-confluent, 7-day cells. The apparent discrepancy between NMR, optical microscopy and TLC results can be reconciled if possible biophysical changes in the neutral lipid pool with growth are taken into account. A cellular explanation for the observed results is proposed: the TAG-droplet-size-change hypothesis.