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 drople ts varies from liquid to liquid crystalline, depending on a number of facto rs, including the amount of TG co-deposited in the inclusion, The lipid in these droplets undergoes turnover via hydrolysis and resynthesis, We determ ined whether newly synthesized lipid is incorporated into existing cytoplas mic droplets, forms a discrete cytoplasmic droplet, or forms a small inclus ion that fuses with an existing droplet, This was accomplished by monitorin g the physical state of the lipid within the cytoplasmic inclusions followi ng sequential deposition of TG and EC, Fu5AH cells were initially grown in media containing oleic acid to produce TG-rich, isotropic inclusions. The c ells were then incubated with medium containing free cholesterol-phospholip id dispersions to promote synthesis and deposition of EG, To inhibit cytopl asmic TG hydrolysis, the lipase inhibitor, diethylumbelliferyl phosphate (U BP), was added at the time of cholesterol enrichment. The phase behavior of lipid droplets isolated from the lipid-rich cells was determined using pol arizing light flow cytometry and microscopy, An anisotropic droplet populat ion (EC-rich inclusions) was not detected, although there was an increase i n cellular EC mass and no change in cellular TG mass, Therefore, under cond itions where there is no turnover of cytoplasmic TG, newly synthesized EC i s incorporated into existing TG inclusions. - Kellner-Weibel, e., B. McHend ry-Rinde, M. P. Haynes, and S. Adelman. Evidence that newly synthesized est erified cholesterol is deposited in existing cytoplasmic Lipid inclusions.