No abstract

were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.Although the HDL-mediated 1 steps of cholesterol transfer from the cell surface membrane and subsequent fate of cholesterol in the vasculature have been extensively studied, much less is known about intracellular components of cholesterol efflux (reviewed in Refs. 1-5). Plasma membrane cholesterol is distributed into multiple pools or domains (reviewed in Refs. 6 and 7). It is now recognized that there may be a connection between such domains and HDL receptor-mediated reverse cholesterol transport (reviewed in Refs. 7-9). The transbilayer distribution of cholesterol in plasma membranes is asymmetric, with the cholesterol enriched 400% in the cytofacial leaflet versus exofacial leaflet (reviewed in Refs. 10 -14). Transbilayer movement of cholesterol across the plasma membrane appears fast (t1 ⁄2 ϭ 1-6 min; reviewed in Ref. 10). Plasma membrane cholesterol is also distributed into lateral cholesterol-rich and -poor membrane domains (reviewed in Refs. 6 and 10). Most of the cholesterol in the plasma membrane is localized in lateral domains that are, for the most part, relatively inert in terms of transfer kinetics (i.e. t1 ⁄2 ϭ hours to days), and movement between such domains is also slow. However, a small pool of plasma membrane cholesterol appears highly dynamic (reviewed in Ref. 6) and is associated with cholesterol-rich, HDL receptor containing microdomains called caveolae (reviewed in Refs. 8, 9, 15, and 16). Molecular details of cholesterol entry/ exit, cholesterol organization, and mechanism(s) of cholesterol transbilayer movement in caveolae remain to be determined. Likewise, the relationships between caveolae, "rafts," and other cholesterol-rich plasma membrane microdomains is not yet clear (reviewed in Ref. 9).The intracellular steps preceding cellular cholesterol efflux include transfer of cholesterol from the Golgi, endoplasmic reticulum, and lipid droplets to the plasma membrane (reviewed in Refs. 1, 9, and 17). The time frame of bidirectional vesicular transfer of cholesterol between plasma membranes and Golgi has a t1 ⁄2 of 10 -20 min (reviewed in Refs. 8,18,and 19). Alternately, molecular cholesterol transfer, mediated by cholesterol-binding proteins in the cytoplasm, occurs much faster (t1 ⁄2 near 1-2 min) from the lysosome (exogenous cholesterol) ...

Although it is hypothesized that long-chain fatty acyl CoAs (LCFA-CoAs) and long-chain fatty acids (LCFAs) regulate transcription in the nucleus, little is known regarding factors that determine the distribution of these ligands to nuclei of living cells. Immunofluorescence colocalization showed that liver fatty acid-binding protein (L-FABP; binds LCFA-CoA as well as LCFA) significantly colocalized with PPARalpha in nuclei of transfected L-cell fibroblasts. Colocalization with a DNA binding dye (SYTO59) revealed that, within the nucleus of control L-cells, the nonhydrolyzable fluorescent LCFA-CoA (BODIPY-C16-S-S-CoA) was distributed primarily in a punctate pattern throughout the nucleoplasm, while nonmetabolizable fluorescent LCFAs (BODIPY-C16 and BODIPY-C12) were localized primarily near the nuclear envelope membranes. L-FABP overexpression selectively increased the targeting of BODIPY-C16-S-S-CoA by 1.9- and 2.7-fold into the nuclear membrane and nucleoplasm, respectively. L-FABP also increased the targeting of fluorescent LCFAs (especially long-chain-length BODIPY-C16) by 1.7-fold to the nuclear membrane and 7.4-fold into the nucleoplasm. A cis-parinaric acid displacement assay showed that L-FABP bound BODIPY-C12 and BODIPY-C16 with K(i)s of 10.1 +/- 2.5 and 20.7 +/- 1.5 nM, respectively, in the same range as naturally occurring LCFAs. Finally, solid-phase extraction and HPLC analysis revealed that, depending on the fatty acid content of the culture medium, L-FABP expression also increased the cellular LCFA-CoA pool size and altered the LCFA-CoA acyl chain composition. Thus, L-FABP may function as a carrier for selectively enhancing the distribution of LCFA-CoA, as well as LCFA, to nuclei for potential interaction with nuclear receptors.

Cellular cholesterol homeostasis is a balance of influx, catabolism and synthesis, and efflux. Unlike vascular lipoprotein cholesterol transport, intracellular cholesterol trafficking is only beginning to be resolved. Exogenous cholesterol and cholesterol ester enter cells via the low-density lipoprotein (LDL) receptor! lysosomal and less so by nonvesicular, high-density lipoprotein (HDL) receptor!caveolar pathways. However, the mechanism(s) whereby cholesterol enters the lysosomal membrane, translocates, and transfers out of the lysosome to the cell interior are unknown. likewise, the steps whereby cholesterol enters the cytofacial leaflet of the plasma membrane caveolae, rapidly translocates, leaves the exofacial leaflet, and transfers to extracellular HDL are unclear. Increasing evidence obtained with model and isolated cell membranes, transfected cells, genetic mutants, and gene-ablated mice suggests that proteins such as caveolin, sterol carrier proteln-2 (SCP-2), Niemann-Pick C1 protein, steroidogenic acute regulatory protein (StAR), and other intracellular proteins mediate intracellular cholesterol transfer. While these proteins bind cholesterol and/or Interact with cholesterol-rich membrane microdomains (e.g., caveolae, rafts, and annuli), their relative contributions to direct molecular versus vesicular cholesterol transfer remain to be resolved. The formation, regulation, and role of membrane microdomalns in regulating cholesterol uptake/efflux and trafficking are unclear. Some cholesterol-binding proteins exert opposing effects on cellular cholesterol uptake/efflux, transfer of cholesterol out of This manuscript is an update of a previously published minireview entitled, "Recent Advances in Membrane Cholesterol Domain Dynamics and Intracellular Cholesterol Trafficking".