Evidence that newly synthesized esterified cholesterol is deposited in existing cytoplasmic lipid inclusions

Esterified cholesterol (EC) and triglyceride (TG) can be stored in cells as cytoplasmic inclusions. The physical state of the EC in these lipid droplets varies from liquid to liquid crystalline, depending on a number of factors, including the amount of TG co-deposited in the inclusion. The lipid in these droplets undergoes turnover via hydrolysis and resynthesis. We determined whether newly synthesized lipid is incorporated into existing cytoplasmic droplets, forms a discrete cytoplasmic droplet, or forms a small inclusion that fuses with an existing droplet. This was accomplished by monitoring the physical state of the lipid within the cytoplasmic inclusions following sequential deposition of TG and EC. Fu5AH cells were initially grown in media containing oleic acid to produce TG-rich, isotropic inclusions. The cells were then incubated with medium containing free cholesterol-phospholipid dispersions to promote synthesis and deposition of EC. To inhibit cytoplasmic TG hydrolysis, the lipase inhibitor, diethylumbelliferyl phosphate (UBP), was added at the time of cholesterol enrichment. The phase behavior of lipid droplets isolated from the lipid-rich cells was determined using polarizing light flow cytometry and microscopy. An anisotropic droplet population (EC-rich inclusions) was not detected, although there was an increase in cellular EC mass and no change in cellular TG mass. Therefore, under conditions where there is no turnover of cytoplasmic TG, newly synthesized EC is incorporated into existing TG inclusions.