Is reverse cholesterol transport regulated by active cholesterol?

This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. 4 Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed.     

The role of intracellular cholesterol transport in cholesterol homeostasis

How cholesterol is transported among the membranes of the cell is obscure. Similarly, the mechanisms governing the abundance of cell cholesterol are not entirely understood. It may be, however, that a link exists between the intracellular transport of cholesterol and its homeostasis. We propose that cholesterol circulates between the plasma membrane, which contains the bulk of the sterol, and organelle membranes, which contain only traces. A putative sensor translates small fluctuations in plasma membrane cholesterol into relatively large changes in this flux, thereby setting the magnitude of the intracellular pools. The cholesterol concentration in the endoplasmic reticulum and mitochondrial membranes then governs the activities of proteins embedded therein that mediate cholesterol transformations. This arrangement creates a feedback loop through which the intracellular effectors regulate the abundance of plasma membrane cholesterol.     

Modulation of cellular cholesterol transport and homeostasis by Rab11

To analyze the contribution of vesicular trafficking pathways in cellular cholesterol transport we examined the effects of selected endosomal Rab proteins on cholesterol distribution by filipin staining. Transient overexpression of Rab11 resulted in prominent accumulation of free cholesterol in Rab11-positive organelles that sequestered transferrin receptors and internalized transferrin. Sphingolipids were selectively redistributed as pyrene-sphingomyelin and sulfatide cosequestered with Rab11-positive endosomes, whereas globotriaosyl ceramide and GM2 ganglioside did not. Rab11 overexpression did not perturb the transport of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate–labeled low-density lipoprotein (LDL) to late endosomes or the Niemann-Pick type C1 (NPC1)-induced late endosomal cholesterol clearance in NPC patient cells. However, Rab11 overexpression inhibited cellular cholesterol esterification in an LDL-independent manner. This effect could be overcome by introducing cholesterol to the plasma membrane by using cyclodextrin as a carrier. These results suggest that in Rab11-overexpressing cells, deposition of cholesterol in recycling endosomes results in its impaired esterification, presumably due to defective recycling of cholesterol to the plasma membrane. The findings point to the importance of the recycling endosomes in regulating cholesterol and sphingolipid trafficking and cellular cholesterol homeostasis.     

Lipoproteins, cholesterol homeostasis and cardiac health

Cholesterol is an essential substance involved in many functions, such as maintaining cell membranes, manufacturing vitamin D on surface of the skin, producing hormones, and possibly helping cell connections in the brain. When cholesterol levels rise in the blood, they can, however, have dangerous consequences. In particular, cholesterol has generated considerable notoriety for its causative role in atherosclerosis, the leading cause of death in developed countries around the world. Homeostasis of cholesterol is centered on the metabolism of lipoproteins, which mediate transport of the lipid to and from tissues. As a synopsis of the major events and proteins that manage lipoprotein homeostasis, this review contributes to the substantial attention that has recently been directed to this area. Despite intense scrutiny, the majority of phenotypic variation in total cholesterol and related traits eludes explanation by current genetic knowledge. This is somewhat disappointing considering heritability estimates have established these traits as highly genetic. Thus, the continued search for candidate genes, mutations, and mechanisms is vital to our understanding of heart disease at the molecular level. Furthermore, as marker development continues to predict risk of vascular illness, this knowledge has the potential to revolutionize treatment of this leading human disease.     

Release of cellular cholesterol: molecular mechanism for cholesterol homeostasis in cells and in the body

Most mammalian somatic cells are unable to catabolize cholesterol and therefore need to export it in order to maintain sterol homeostasis. This mechanism may also function to reduce excessively accumulated cholesterol, which would thereby contribute to prevention or cure of the initial stage of atherosclerotic vascular lesion. High-density lipoprotein (HDL) has been believed to play a main role in this reaction based on epidemiological evidence and in vitro experimental data. At least two independent mechanisms are identified for this reaction. One is non-specific diffusion-mediated cholesterol 'efflux' from cell surface. Cholesterol molecules desorbed from cells can be trapped by various extracellular acceptors including various lipoproteins and albumin, and extracellular cholesterol esterification mainly on HDL may provide a driving force for the net removal of cell cholesterol by maintaining a cholesterol gradient between lipoprotein surface and cell membrane. The other is apolipoprotein-mediated process to generate new HDL by removing cellular phospholipid and cholesterol. The reaction is initiated by the interaction of lipid-free or lipid-poor helical apolipoproteins with cellular surface resulting in assembly of HDL particles with cellular phospholipid and incorporation of cellular cholesterol into the HDL being formed. Thus, HDL has dual functions as an active cholesterol acceptor in the diffusion-mediated pathway and as an apolipoprotein carrier for the HDL assembly reaction. The impairment of the apolipoprotein-mediated reaction was found in Tangier disease and other familial HDL deficiencies to strongly suggest that this is a main mechanism to produce plasma HDL. The causative mutations for this defect was identified in ATP binding cassette transporter protein A1, as a significant step for further understanding of the reaction and cholesterol homeostasis.     

Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.     

Caveolin-1 and regulation of cellular cholesterol homeostasis

Caveolae are 50- to 100-nm cell surface plasma membrane invaginations present in terminally differentiated cells. They are characterized by the presence of caveolin-1, sphingolipids, and cholesterol. Caveolin-1 is thought to play an important role in the regulation of cellular cholesterol homeostasis, a process that needs to be properly controlled to limit and prevent cholesterol accumulation and eventually atherosclerosis. We have recently generated caveolin-1-deficient [Cav-1(-/-)] mice in which caveolae organelles are completely eliminated from all cell types, except cardiac and skeletal muscle. In the present study, we examined the metabolism of cholesterol in wild-type (WT) and Cav-1(-/-) mouse embryonic fibroblasts (MEFs) and mouse peritoneal macrophages (MPMs). We observed that Cav-1(-/-) MEFs are enriched in esterified cholesterol but depleted of free cholesterol compared with their wild-type counterparts. Similarly, Cav-1(-/-) MPMs also contained less free cholesterol and were enriched in esterified cholesterol on cholesterol loading. In agreement with this finding, caveolin-1 deficiency was associated with reduced free cholesterol synthesis but increased acyl-CoA:cholesterol acyl-transferase (ACAT) activity. In wild-type MPMs, we observed that caveolin-1 was markedly upregulated on cholesterol loading. Despite these differences, cellular cholesterol efflux from MEFs and MPMs to HDL was not affected in the Cav-1-deficient cells. Neither ATP-binding cassette transporter G1 (ABCG1)- nor scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux was affected. Cellular cholesterol efflux to apolipoprotein A-I was not significantly reduced in Cav-1(-/-) MPMs compared with wild-type MPMs. However, ABCA1-mediated cholesterol efflux was clearly more sensitive to the inhibitory effects of glyburide in Cav-1(-/-) MPMs versus WT MPMs. Taken together, these findings suggest that caveolin-1 plays an important role in the regulation of intracellular cholesterol homeostasis and can modulate the activity of other proteins that are involved in the regulation of intracellular cholesterol homeostasis.     

Hormonal regulation of testicular steroid and cholesterol homeostasis

The male sex steroid, testosterone (T), is synthesized from cholesterol in the testicular Leydig cell under control of the pituitary gonadotropin LH. Unlike most cells that use cholesterol primarily for membrane synthesis, steroidogenic cells have additional requirements for cholesterol, because it is the essential precursor for all steroid hormones. Little is known about how Leydig cells satisfy their specialized cholesterol requirements for steroid synthesis. We show that in mice with a unique hypomorphic androgen mutation, which disrupts the feedback loop governing T synthesis, that genes involved in cholesterol biosynthesis/uptake and steroid biosynthesis are up-regulated. We identify LH as the central regulatory molecule that controls both steroidogenesis and Leydig cell cholesterol homeostasis in vivo. In addition to the primary defect caused by high levels of LH, absence of T signaling exacerbates the lipid homeostasis defect in Leydig cells by eliminating a short feedback loop. We show that T signaling can affect the synthesis of steroids and modulates the expression of genes involved in de novo cholesterol synthesis. Surprisingly, accumulation of active sterol response element-binding protein 2 is not required for up-regulation of genes involved in cholesterol biosynthesis and uptake in Leydig cells.     

Activity-independent homeostasis in rhythmically active neurons

The shal gene encodes the transient potassium current (I(A)) in neurons of the lobster stomatogastric ganglion. Overexpression of Shal by RNA injection into neurons produces a large increase in I(A), but surprisingly little change in the neuron's firing properties. Accompanying the increase in I(A) is a dramatic and linearly correlated increase in the hyperpolarization-activated inward current (I(h)). The enhanced I(h) electrophysiologically compensates for the enhanced I(A), since pharmacological blockade of I(h) uncovers the physiological effects of the increased I(A). Expression of a nonfunctional mutant Shal also induces a large increase in I(h), demonstrating a novel activity-independent coupling between the Shal protein and I(h) enhancement. Since I(A) and I(h) influence neuronal activity in opposite directions, our results suggest a selective coregulation of these channels as a mechanism for constraining cell activity within appropriate physiological parameters.     

Intracellular cholesterol homeostasis and amyloid precursor protein processing

Many preclinical and clinical studies have implied a role for cholesterol in the pathogenesis of Alzheimer's disease (AD). In this review we will discuss the movement of intracellular cholesterol and how normal distribution, transport, and export of cholesterol are vital for regulation of the AD related protein, Aβ. We focus on cholesterol distribution in the plasma membrane, transport through the endosomal/lysosomal system, control of cholesterol intracellular signaling at the endoplasmic reticulum and Golgi, the HMG-CoA reductase pathway and finally export of cholesterol from the cell.     

Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL.

Results from several laboratories clearly indicate that expression of scavenger receptor class B type I (SR-BI) enhances the bidirectional flux of cholesterol between cells and lipoproteins. Because the activity of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis, is regulated by cell cholesterol content, we designed experiments to investigate the effect of SR-BI expression on the activity of this enzyme and on net cellular cholesterol mass. In addition, we compared the function of SR-BI with its human homolog, CD36 and LIMPII analogous 1. Our experiments demonstrate that both receptors enhance the flux of unesterified or free cholesterol bidirectionally, down a concentration gradient. Receptor-mediated cholesterol flux can effectively modulate multiple aspects of cellular cholesterol metabolism, including the pool that regulates the activity of HMG-CoA reductase. We also found that constitutive expression of SR-BI alters the steady state level of cellular cholesterol and phospholipid when SR-BI-expressing cells are maintained in medium containing serum lipoproteins. All of these effects are proportional to the level of receptor on the cell surface. These data indicate that the level of SR-BI expression determines both the rate of free cholesterol flux and the steady state level of cellular cholesterol.