No abstract

Although the peroxisome proliferator-activated receptor (PPAR␣) binds and is activated by a variety of synthetic xenobiotics, the identity of the high affinity endogenous ligand(s) is incompletely resolved. Likewise, it is not known how putative endogenous ligands alter PPAR␣ conformation in order to affect transcriptional regulation. Direct fluorescence binding and fluorescence displacement assays showed for the first time that PPAR␣ exhibits high affinity (1-14 nM K d values) for unsaturated long chain fatty acyl-CoAs as well as unsaturated long chain fatty acids commonly found in mammalian cells. Fluorescence resonance energy transfer between PPAR␣ aromatic amino acids and bound corresponding naturally occurring fluorescent ligands (i.e. cis-parinaroyl-CoA, trans-parinaric acid) yielded intermolecular distances of 25-29 Å, confirming close molecular interaction. Interestingly, although PPAR␣ also exhibited high affinity for saturated long chain fatty acylCoAs, regardless of chain length (1-13 nM K d values), saturated long chain fatty acids were not significantly bound. In contrast to the similar affinities of PPAR␣ for fatty acyl-CoAs and unsaturated fatty acids, CoA thioesters of peroxisome proliferator drugs were bound with 5-6-fold higher affinities than their free acid forms. Circular dichroism demonstrated that high affinity ligands (long chain fatty acyl-CoAs, unsaturated fatty acids), but not weak affinity ligands (saturated fatty acids), elicited conformational changes in PPAR␣ structure, a hallmark of ligand-activated nuclear receptors. Finally, these ligand specificities and induced conformational changes correlated functionally with co-activator binding. In summary, since nuclear concentrations of these ligands are in the nanomolar range, long chain fatty acyl-CoAs and unsaturated fatty acids may both represent endogenous PPAR␣ ligands. Furthermore, the finding that saturated fatty acylCoAs, rather than saturated fatty acids, are high affinity PPAR␣ ligands provides a mechanism accounting for saturated fatty acid transactivation in cell-based assays.

Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that affect lipid and glucose catabolism and storage.

Fluorescent sterols, dehydroergosterol and NBD-cholesterol, were used to examine high density lipoproteinmediated cholesterol uptake and intracellular targeting in L-cell fibroblasts. The uptake, but not esterification or targeting to lipid droplets, of these sterols differed >100-fold, suggesting significant differences in uptake pathways. NBD-cholesterol uptake kinetics and lipoprotein specificity reflected high density lipoprotein-mediated sterol uptake via the scavenger receptor B1. Fluorescence energy transfer showed an average intermolecular distance of 26 Å between the two fluorescent sterols in L-cells. Indirect immunofluorescence revealed that both fluorescent sterols localized to L-cell lipid droplets, the surface of which contained adipose differentiation-related protein. This lipid droplet-specific protein specifically bound NBD-cholesterol with high affinity (K d ‫؍‬ 2 nM) at a single site. Thus, NBDcholesterol and dehydroergosterol were useful fluorescent probes of sterol uptake and intracellular sterol targeting. NBD-cholesterol more selectively probed high density lipoprotein-mediated uptake and rapid intracellular targeting of sterol to lipid droplets. Targeting of sterol to lipid droplets was correlated with the presence of adipose differentiation related protein, a lipid droplet-specific protein shown for the first time to bind unesterified sterol with high affinity.Because of cholesterol's dual role in both normal cell function and the pathobiology of atherosclerosis, it is essential to resolve the mechanisms whereby exogenous cholesterol is taken up and distributed within the cell (reviewed in Refs. 1-3). Unesterified cholesterol uptake shares some, but not all, aspects of cholesterol ester uptake. Unesterified cholesterol enters the cell either by the slower LDL 1 receptor mediated pathway wherein it leaves the LDL endocytosed within clathrin-coated vesicles prior to vesicle fusion at the lysosome (for review, see Ref.3) or by the rapid "alternate" HDL receptor pathway (3, 5). However, most attention has focused on the HDL-mediated cholesterol efflux from rather than uptake of cholesterol into the cell (for review, see Ref. 4). Almost nothing is known regarding the uptake and intracellular targeting of unesterified cholesterol via the HDL receptor pathway nor has the process been directly visualized. Fluorescent cholesterol analogs represent an opportunity for real-time monitoring of the rapid, HDL-mediated uptake of unesterified sterol uptake and movement in living cells. The main requirement for choice of an appropriate fluorescent cholesterol analogue is that it should mimic the behavior of cholesterol. Unfortunately, the functional properties of many fluorescent sterol analogues do not closely resemble those of cholesterol (for review, see Refs. 6 -9). The advent of dehydroergosterol (DHE), whose structure closely resembles that of cholesterol, represents a major advance for examining the structure of lipoproteins (10, 11) and membranes (12). DHE is a naturally occurring sterol where it...

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) ...