Movement Of Cholesterol Research Articles

The effect of non-receptor-mediated uptake of cholesterol on intracellular cholesterol metabolism in human skin fibroblasts.

Unilamellar lipid vesicles of various cholesterol:phosphatidylcholine molar ratios were used to alter, via passive exchange at the plasma membrane, the cellular free cholesterol content of cultured human skin fibroblasts which had been preincubated in lipoprotein-deficient serum. The effects of these net surface transfers of cholesterol on cellular cholesterol biosynthesis, cholesterol esterification and low density lipoprotein (LDL) binding were determined and were compared with the effects of cholesterol delivered to the cell interior via the receptor-mediated endocytosis of LDL. Both LDL and cholesterol-rich lipid vesicles increased cell cholesterol within 6 h. Cells exposed to LDL also showed, within 6 h, decreased cholesterol synthesis, decreased LDL binding and increased cholesterol esterification. Cells incubated with the cholesterol-rich vesicles showed similar changes but these were delayed and did not occur until 24 h. Fibroblasts incubated with cholesterol-free phosphatidylcholine vesicles had decreased cell cholesterol, increased cholesterol synthesis, increased LDL binding, and decreased esterification, but only after 24 h of incubation. These results suggest that passive net transfers of cholesterol occurring at the cell surface can with time modulate intracellular cholesterol metabolism. These findings are consistent with the idea that the movement of cholesterol from the cell surface to the cell interior is a limited and relatively slow process.

Regulation of mitochondrial compartment volumes in rat adrenal cortex by ether stress

In vivo ether stress of rats causes release of pituitary adrenocorticotropin (ACTH) leading to activation of steroidogenesis in adrenal cortex mitochondria. The present studies show that this treatment also induces a decrease in the volume of the intermembrane space in isolated adrenal mitochondria. This decrease is accompanied by an increase in the volume of the matrix, thus leaving the total mitochondrial volume approximately constant. These effects are prevented by the protein synthesis inhibitor, cycloheximide, and are specific to the adrenal gland. The decrease in the intermembrane space (or increase in the matrix volume) is correlated with activation of the cholesterol side chain cleavage reaction (the regulated step in steroidogenesis). We propose as a working hypothesis that these changes reflect a hormonally regulated alteration in the relationship between the outer and inner mitochondrial membranes, which may facilitate the rate-limiting movement of cholesterol from the outer to the inner membrane where the side chain cleavage enzyme is located.

Movement of cholesterol between vesicles prepared with different phospholipids or sizes.

The rates of exchange of [4-14C]cholesterol between lipid vesicles prepared with different phospholipids and with different sizes have been measured. The first-order rate constants were higher using vesicles prepared from phosphatidylcholines with highly branched or polyunsaturated fatty acyl chains than with saturated diacyl or di-O-alkyl chains. The rate measurements indicate that the affinity of cholesterol for phospholipid does not vary significantly on change of the type of linkage (ether or ester) in phosphatidylcholine (PC) or of the positions of the fatty acyl chains in 1,2-diacyl-PC bearing one saturated and one unsaturated chain; furthermore, egg phosphatidylglycerol and egg phosphatidylethanolamine appear to have comparable affinities for cholesterol. However, the molecular packing in the bilayer and nearest-neighbor interactions involving cholesterol appear tightened more by N-palmitoylsphingomyelin than by dipalmitoyl-PC; on incorporation of 44 mol % of these phospholipids (which have the same fatty acyl chain composition) into either small or large unilamellar vesicles prepared with egg phosphatidylglycerol, the exchange rates were strikingly slower when the donor species contained sphingomyelin compared with PC. The rate of cholesterol exchange was 100% faster with small unilamellar vesicles than with large unilamellar vesicles as donors, suggesting that the looser packing in the highly curved small vesicles facilitates cholesterol desorption. The cholesterol exchange rate did not vary with the size of the acceptor vesicles, which indicates that desorption is the rate-limiting step in the exchange process in the presence of excess acceptors.

The effects of taxol, a microtubule-stabilizing drug, on steroidogenic cells.

The effects of taxol on steroid production and microtubule polymerization were examined using Y-1 adrenocortical tumor cells, MLTC-1 Leydig tumor cells, and primary cultures of bovine adrenocortical cells. Taxol inhibited the following steroidogenic processes within the Y-1 and MLTC-1 cells: (1) hormonal increase of steroid production, (2) dibutyryl cyclic AMP-increased steroid production, and (3) hormone-stimulated pregnenolone production. The inhibitory action of taxol was concentration dependent and also resulted in an increase in cytoplasmic microtubules. In addition, the inhibitory action of taxol on hormone-stimulated steroid production was reversible. Taxol appeared to inhibit cholesterol movement to the mitochondrial site of cholesterol side-chain cleavage enzyme but did not affect overall protein synthesis. Interestingly, taxol did not affect hormone-stimulated steroid production in bovine adrenocortical cells. This lack of inhibition may correspond to the ultrastructural observation that microtubule bundling after taxol treatment was observed in the tumor cells but not in similarly treated bovine adrenal cells. With this conflicting information between cell types, a direct relationship between taxol treatment and inhibition of steroid production has not been established. However, these results suggest that taxol alters the rate of transport of cholesterol to the cholesterol side-chain cleavage enzyme within the steroidogenic tumor cells.

Kinetics of cholesterol and phospholipid exchange from membranes containing cross-linked proteins or cross-linked phosphatidylethanolamines.

Mono- and dipalmitoylphosphatidylethanolamine derivatives have been synthesized and used to evaluate the role of cross-links between the amino groups of two phospholipid molecules in the rate of cholesterol movement between membranes. Incorporation of the cross-linked phospholipids into small unilamellar vesicles (the donor species) decreased the rate of spontaneous cholesterol exchange with acceptor membranes (small unilamellar vesicles or Mycoplasma gallisepticum cells). These results suggest that the cross-linking of aminophospholipids by reactive intermediates, which may be one of the degenerative transformations associated with peroxidation of unsaturated lipids and cellular aging, can inhibit cholesterol exchangeability in biological membranes. The rates of spontaneous [14C]cholesterol and protein-mediated 14C-labeled phospholipid exchange from diamide-treated mycoplasma and erythrocyte membranes have also been measured. The formation of extensive disulfide bonds in the membrane proteins of M. gallisepticum enhanced the 14C-labeled phospholipid exchange rate but did not affect the rate of [14C]cholesterol exchange. The rates of radiolabeled cholesterol and phospholipid exchange between erythrocyte ghosts and vesicles were both enhanced (but to different extents) when ghosts were treated with diamide. These observations suggest that diamide-induced oxidative cross-linking of sulfhydryl groups in membrane proteins does not lead to random defects in the lipid domain.

The role of the cytoskeleton in hormone action.

The structure and functions of microfilaments and microtubules is briefly reviewed. Based on evidence from a variety of cells with various experimental approaches, it is proposed that the cytoskeleton is important in the functions of cells as follows: the cytoskeleton appears to be involved in attachment (to other cells and to substrata used in cell culture) and in movement; the cytoskeleton appears to be involved in the complex relationship between the surface and the interior of the cell. This is a two-way relationship in which the cell uses the cytoskeleton to influence the distribution of proteins within the plasma membrane and perhaps as a go-between to transmit information from the surface of the cell to the interior. Within the cell, the cytoskeleton appears to coordinate intracellular activities (e.g., between nucleus and cytoplasm) and to provide a compartmentation which restricts the movements of intracellular structures (from molecules to organelles), thereby facilitating productive encounters between such structures with an efficiency that is presumed to exceed that of random motion. The cytoskeleton may arrange the various components of the cell in space; it may direct intracellular traffic and promote intracellular movement. This loose picture of cytoskeletal activity may be more emphatic than the available data warrant. It should be regarded as informed extrapolation based upon fragments of information. When these ideas are applied to endocrine function, we can see that the secretion of hormones (exemplified by thyroid hormones and insulin) requires cytoskeletal activity to provide direction and perhaps to promote movement. In steroidogenic cells the movement of cholesterol from cytoplasm to mitochondria requires microfilaments. The antidiuretic hormone requires microfilaments and microtubules to promote transport of water through cells in response to osmotic gradients. Microfilaments adjust the compliance of the cell to provide a path for water and microtubules influence the permeability of the cell membrane to water.

Intramitochondrial movement of adrenal sterol carrier protein with cholesterol in response to corticotropin.

Sterol carrier protein (SCP) is a highly abundant, ubiquitous, low molecular weight protein that has a rapid turnover and multifunctional roles in lipid metabolism and transport. It is also known as the fatty acid-binding protein. These and other characteristics led to studies on the possible role of SCP in mediation of steroidogenic responses of the adrenal gland to corticotropin. To quantitate the level of SCP in adrenal tissue and subcellular fractions, a specific immunochemical assay was developed using an antibody to homogeneous liver SCP. SCP is a major protein in adrenal cells (greater than 100 micrograms/mg of total protein). The bulk of it is present in the soluble (60%) and mitochondrial (35%) fractions. Nearly all mitochondrial SCP is present in the inner membrane. Adrenal SCP undergoes a dramatic diurnal variation, varying from 2% to 12% of total protein. When corticotropin was administered in vivo in the presence of aminoglutethimide, an inhibitor of steroidogenesis, there was a parallel movement of SCP with cholesterol to the inner mitochondrial membrane. Other work showed mitochondrial SCP levels increase only in situations where there is an increase in cholesterol levels. These findings strongly suggest that one of the functions of adrenal SCP is to participate in teh corticotropin-stimulated movement of cholesterol to the inner mitochondrial membrane for steroidogenesis.

Cytochrome P-450scc: enzymology, and the regulation of intramitochondrial cholesterol delivery to the enzyme.

The mechanism and properties of the adrenal cortex enzyme system which catalyzes the side chain cleavage of cholesterol to form pregnenolone are summarized. Cytochrome P-450scc, an integral inner mitochondrial membrane protein, interacts with its electron donor adrenodoxin via an aqueous-exposed (matrix side) site, and with its substrate cholesterol via an active site in communication with the hydrophobic phospholipid milieu. In a purified, phospholipid vesicle-reconstituted system, membrane-dissolved cholesterol interacts rapidly with and can be readily metabolized by the membrane-associated cytochrome, and thus represents a readily accessible cholesterol pool. Evidence for a rapidly metabolizable mitochondrial substrate pool (presumably that in the inner mitochondrial membrane) and the regulation by ACTH of cholesterol movement from other site(s) (presumably the outer mitochondrial membrane) into the reactive pool is reviewed; additional evidence is provided which supports the idea that the outer mitochondrial membrane/intermembrane space provides the rate-limiting block to cholesterol utilization. Possible mechanisms by which ACTH might regulate intramitochondrial cholesterol movement are discussed. ACTH has been found to regulate intramitochondrial aqueous volumes (both the matrix and the intermembrane space) in a cycloheximide-inhibitable manner, and it is proposed that these volume changes reflect an altered relationship of outer and inner membranes which may promote movement of cholesterol.

Regulation of Lipid Metabolism by a Lipid-Carrying Protein

The overall conclusion to be made from the information presented here is that for many reasons SCP is a highly unusual protein. Some of these reasons are, first, SCP serves as cofactor for a number of different membrane-bound enzymes catalyzing specific steps in lipid metabolism. Second, SCP is involved in intracellular transport or movement of both cholesterol and fatty acids. Third, SCP is remarkably abundant and ubiquitous; its structure is conserved throughout nature. Fourth, SCP is exported to the blood stream from its site of synthesis by some, perhaps unique, mechanism and then rapidly taken up by specific tissues, e.g., the adrenal. Fifth, SCP is free in the cytosol and can also move to the inner mitochondrial membrane, where it is tightly bound. Sixth, SCP undergoes a dramatic diurnal variation in amount, reflecting changes in synthetic rate. Its half-life is less than an hour. Seventh, the diurnal variation in amount is triggered by feeding and influenced by several hormones. The diurnal variation is lost but a high level of SCP is maintained in the face of debilitating conditions, i.e., starvation, diabetes. Eighth, malignant cells exhibit defects in the uptake, synthesis, or turnover of SCP. Ninth, the synthesis of SCP is regulated by the efficiency of translation of its ever abundant mRNA. Tenth, there is much more to be learned about the functions and regulation of SCP.

The movement of cholesterol within cells.

The movement of cholesterol within cells.

Cholesterol metabolism in the adrenal cortex

Adrenal cortical mitochondria contain a mixed function oxidase capable of converting cholesterol to pregnenolone; this enzyme requires NADPH, oxygen and cholesterol. This cholesterol side chain cleavage enzyme system contains a Flavoprotein, an iron sulphur protein and a specific cytochrome P450 termed cytochrome P450 scc. ACTH stimulates the adrenal cortex by activating adenyl cyclase producing an elevated intracellular concentration of cAMP. This in turn increases the activity of a cytosolic cAMP dependent protein kinase. Adrenal cortical cytosol contains a cholesterol ester hydrolase which is activated by ATP and a protein kinase. This enzyme may be deactivated by a phosphoprotein phosphatase. The adrenal cortex contains lipid droplets that are rich in esterified cholesterol. Cholesterol ester hydrolase can release free cholesterol from the lipid droplets. The free cholesterol released may be used to supplement the mitochondrial cholesterol as a pregnenolone precursor. Steroid hormone production by the adrenal cortex exhibits a diurnal rhythm and correlates with the activity of the cytosolic cholesterol ester hydrolase. The acute steroidogenic response to ACTH may be in part attributed to the availability of free cholesterol to the mitochondrial cholesterol side chain cleavage enzyme complex. The intracellular movement of free cholesterol from lipid droplets to mitochondrial inner membranes may be impeded by protein synthesis inhibitors such as cycloheximide. The precise mechanism of this block in steroidogenesis remains to be elucidated. Various drugs and oestrogenic hormones suppress the plasma and adrenal cholesterol concentrations. If adrenal cells are deficient in cholesterol, these cells exhibit a diminished response to ACTH. The response to this hormone can be corrected by supplying cholesterol via exogenous plasma lipoproteins. The route that free cholesterol follows within the adrenal cortical cell and the physiological factors influencing free cholesterol movement in such cells are important issues to be explored in future.

Mechanism of ACTH action : Intracellular flux of cholesterol during ACTH-induced corticoidogenesis

Corticoidogenic response to ACTH in the adrenal cortex is strictly controlled by the rate of cholesterol bound to cyt.P450scc which is located in the inner mitochondrial membrane. Thus, the rate of the intracellular movement of cholesterol induced by ACTH was investigated in bovine adrenocortical cells. Cholesterol and pregnenolone were measured by enzymatic assay and Cortisol by the fluorometrical assay. (1) In the presence of 1 nM ACTH, the amount of the intracellular free cholesterol was not affected, whereas about 0.3 nmoles of the cholesterol esters per 10^ cells decreased in 60 min incubation. Most of the diminished cholesterol esters in the cells were thought to be utilized as a substrate of steroidogenesis. (2) The rate_of pregnenolone formation induced by 1 nM ACTH was about 0.3 nmoles/105 cells/hr, which was just the same as that of Cortisol production for 60 min incubation. There was a stoichiometrical correlation between the decrement of the intracellular cholesterol esters and the increment of either pregnenolone or Cortisol formation in the cells. It is therefore concluded that the intracellular cholesterol esters might be the sole substrate in the corticoidogenic response to ACTH in the adrenal cortex. Thus, ACTH could activate cholesterol ester hydrolase in adrenocortical cells to facilitate the transport of the free cholesterol to the inner mitochondrial membrane, and the translocated cholesterol in the inner membrane rapidly interacts with cyt.P450scc to convert to pregnenolone and other corticoids.

The role of cholesteryl ester hydrolysis and synthesis in cholesterol transport across rat intestinal mucosal membrane a new concept

The rate of accumulation of cholesteryl ester in rat intestinal sacs is enhanced up to 3-fold by the addition of porcine cholesterol esterase to the uptake medium. For this the enzyme must be catalytically active and does not exert its effect via its activity in the medium. A mechanism for this rate enhancement is proposed in which the action of cholesterol esterases on both sides of a bilayer membrane can catalyze the net transmembrane movement of cholesterol.

Phospholipid methylation of kidney cortex brush border membranes. Effect on fluidity and transport

Exposure of intact brush border membrane vesicles of hog kidney cortex to cholesterol oxidase resulted in 24% oxidation of membrane cholesterol compared with more than 95% oxidation of cholesterol in lipids isolated from membranes, showing that cholesterol is asymmetrically distributed in membranes. Phospholipase C, hydrolyzed 76% of phosphatidylcholine and 10–12% phosphatidylethanolamine while phosphatidylserine was not hydrolyzed, thus indicating that majority of phosphatidylcholine is present on the outer surface of these vesicles while phosphatidylethanolamine and phosphatidylserine are present on the inner surface. Methylation of phospholipids in brush border membrane with S- adenosyl-[methyl- 3 H] methionine resulted in the formation of phosphatidyl-N- monomethylethanolamine , phosphatidyl-N,N- dimethylethanolamine and phosphatidylcholine from endogenous phosphatidylethanolamine. The K m for S- adenosylmethionine was 1·10 −4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg 2+ was without any effect between pH 5 to 10. Addition of exogenous mono- and dimethylphosphatidylethanolamine derivatives enhanced methyl group incorporation by 4–5-fold as compared to the addition of phosphatidylethanolamine. The conversion of endogenous phosphatidylethanolamine to phosphatidyl-N- monomethylethanolamine or addition of exogenous phosphatidylmonomethylethanolamine to brush border membrane did not result in a change in bulk membrane fluidity as determined by fluorescence polarization of diphenylhexatriene. Methylation of phosphatidylethanolamine in brush border membrane did not affect the Na +-dependent uptake of either d-glucose or phosphate, although the accessibility of cholesterol in membrane to cholesterol oxidase was diminished by 21%, presumably due to altered flip-flop movement of cholesterol in the membrane.

Cholesterol movement between human skin fibroblasts and phosphatidylcholine vesicles

Cholesterol readily exchanges between human skin fibroblasts and unilamellar phospholipid vesicles. Only a fraction of the exchangeable cholesterol and only 10–15% of the total cellular free cholesterol is available for net movement or depletion to cholesterol-free phosphatidylcholine vesicles. [ 14C]Cholesterol introduced into the fibroblast plasma membrane by exchange from lipid vesicles does not readily equilibrate with fibroblast cholesterol labelled endogenously from [ 3H]mevalonic acid. While endogenously-synthesized [ 3H]cholesterol readily becomes incorporated into a pool of esterified cholesterol, little, if any, of the [ 14C]cholesterol introduced into the fibroblast plasma membrane by exchange from lipid vesicles becomes available for esterification. We interpret these findings as suggesting that: (1) net cholesterol movement from fibroblasts to an acceptor membrane is limited to a small percentage of the plasma membrane cholesterol, and (2) separate pools of cholesterol exist in human skin fibroblasts, one associated with the plasma membrane and the second associated with intracellular membranes, and equilibration of cholesterol between the two pools is a very limited process.

The rate of transmembrane movement of cholesterol in the human erythrocyte.

Cholesterol appears to be abundant on both sides of the human erythrocyte membrane, but its precise distribution and rate of transmembrane movement are undetermined. Although the cholesterol in the intact cell normally was resistant to cholesterol oxidase, enrichment of cells with exogenous cholesterol or preincubation at very low ionic strength rendered the entire cholesterol pool susceptibility to attack. Under these experimental conditions, all of the membrane cholesterol was oxidized in a strictly first order fashion with a half-time as short as 10 s at 37 degrees C. Since the enzyme had access only to the outer membrane surface, these data suggest that the transmembrane movement (flip-flop) of cholesterol is extremely rapid. From an error analysis, we estimate an upper bound on the half-time of the transmembrane movement of cholesterol of 3 s at 37 degrees C. A physiological function for rapid sterol flip-flop is suggested.

Effect of LH factors regulating ovarian cholesterol metabolism and progesterone synthesis in PMSG-primed immature rats.

Administration of an ovulatory dose of LH (10 micrograms, i.v.) to PMSG (4 i.u., s.c.)-primed immature rats increased ovarian pregnenolone levels 5-fold and ovarian progesterone levels 40-fold within 6 h and the levels, although fluctuating, remained elevated for 72 h. Serum progesterone levels mimicked those of the luteal phase in the normal cycle. The sustained increase in steroidogenesis was accompanied by a decrease in both basal and cAMP-stimulatable ovarian cholesterol ester hydrolase activity and a net increase in ovarian cholesterol ester content. Ovarian free cholesterol levels were essentially unchanged during the 72 h study. LH does not, therefore, chronically stimulate steroidogenesis by providing additional substrate for the steroidogenic enzymes through activating cholesterol ester hydrolase to bring about hydrolysis of cholesterol esters. Moreover, ovarian cholesterol esters are unlikely to be the primary source of cholesterol utilized to support luteal steroidogenesis. Studies with isolated mitochondria suggested that the mechanisms by which LH stimulated steroidogenesis were by (a) stimulating mitochondrial pregnenolone production probably by facilitating the intramitochondrial movement of cholesterol of the site of side-chain cleavage, and (b) promoting the metabolism of pregnenolone to progesterone.

Transmembrane movement of cholesterol in small unilamellar vesicles detected by cholesterol oxidase.

Greater than 90% of the cholesterol in small unilamellar vesicles composed of egg lecithin and cholesterol (molar ratio 1:0.7) was oxidized by a cholesterol oxidase from Brevibacterium sp., with a single time constant and a half-time of 1 min at 37 degrees C. The enzyme preparation used was at least 95% pure and possessed no detectable phospholipase C activity. Since cholesterol is present in both halves of the bilayer, it was concluded that transmembrane movement of cholesterol in these vesicles occurs with a half-time of 1 min or less at 37 degrees C.

Intracellular movement of cholesterol in rat adrenal cells. Kinetics and effects of inhibitors.

Intracellular movement of cholesterol in rat adrenal cells. Kinetics and effects of inhibitors.

Mechanism of cholesterol efflux from cells

Experiments have been conducted to gain information on the mechanisms underlying the surface transfer of free cholesterol between cells and extracellular cholesterol acceptors. The movement of radiolabeled cholesterol has been monitored using hepatoma cells, fibroblasts, and primary hepatocytes as cholesterol donors and phospholipid vesicles as acceptors. The data obtained from these studies are compared to those obtained from studies using two populations of phospholipid vesicles; one contained labeled cholesterol, which served as a donor, while the second population acted as an acceptor. In the presence of phospholipid vesicles, the loss of cholesterol from the cells follows first order kinetics if the data are corrected for the changing specific activity produced by the initiation of cholesterol synthesis. In hepatoma cells, at least two-thirds of the cellular cholesterol is in one kinetic pool. The activation energy for transfer of cholesterol from hepatoma cells to unilamellar vesicles was determined to be 12 kcal/mole. A similar value of 16 kcal/mole was obtained in the vesicle/vesicle system. The transfer of cholesterol between vesicles was shown to be independent of acceptor concentration, whereas the half-time for the movement of cholesterol between cells and vesicles was shown to be a function of the extracellular vesicle concentration. Analysis and comparison of the parameters describing the kinetics of cholesterol transfer in these systems suggests that the mechanism involves diffusion of cholesterol molecules through the aqueous phase, with the overall rate being influenced by the rate of desorption from the donor phospholipid-cholesterol bilayer membrane. The differences observed between the two experimental systems, particularly with regard to the influence of acceptor concentration, is attributed to the effects of the presence of a large unstirred water layer around cells which is not present around vesicles.