Intracellular distribution and mobilization of unesterified cholesterol inadipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures

In addition to their central role in triglyceride storage, fat cells are a primary depot of unesterified cholesterol (FC) in the body In comparison, p eripheral cells contain very little FC, This difference in adipocytes versu s peripheral tissues is inconsistent with the current theory of cholesterol homeostasis. Attempting to resolve this discrepancy, we examined intracell ular storage sites of FC in murine 3T3-F442A adipocytes. Using the choleste rol-binding antibiotic, filipin, in combination with high resolution fluore scence microscopy, intense fluorescent staining characteristically decorate d the periphery of triglyceride droplets (TGD) as well as the plasma membra ne (PM) of fat cells, Filipin-staining was not visible inside the lipid dro plets, Purification of TGD by subcellular fractionation demonstrated that t he rise in total FC content of adipocytes upon differentiation was attribut able to an increase in TGD-FC, which contributed up to one third of the tot al cellular FC, The protein component of purified TGD from cultured adipocy tes as well as from murine adipocytes obtained from fresh tissues contained the lumenal endoplasmic reticulum (ER) immunoglobulin binding protein (BiP ) and the integral ER membrane protein calnexin, Efflux experiments using t he extracellular FC accepters beta-cyclodextrin or apolipoprotein A-I demon strated that TGD-associated FC was releasable from TGD, Whereas FC efflux f rom adipocytes was unaffected in the presence of brefeldin A or monensin, t he secretion of a control protein, lipoprotein lipase, was effectively redu ced, In summary, our findings identify the TGD surface layer as primary intracel lular storage site for FC within adipocytes, We suggest that the structural role of ER-resident proteins in this adipocyte TGD envelope has been previ ously neglected, Our findings support the suggestion that an ER-like struct ure, albeit of modified lipid composition, constitutes the lipid droplets' surface layer, Finally, the efflux process of FC from adipocytes upon extra cellular stimulation with beta-cyclodextrin provides evidence for an energy -dependent intracellular trafficking route between the TGD-FC pool and the PR;I-FC sites which is distinct from the secretory pathway of proteins.