The participation of sterol carrier protein2 in cholesterol esterification in rat adrenal microsomes

The effect of sterol carrier protein2 (SCP2) purified from rat liver on the formation of cholesterol esters by acyl-CoA: cholesterol acyl-transferase (ACAT: EC 2.3.1.26) in rat adrenal microsomes was studied. The rate of incorporation of [1-14C]oleoyl-CoA into cholesteryl oleate was determined in the presence or absence of exogenously added cholesterol or SCP2, or both. The addition of SCP2 had no effect on the formation of cholesterol esters from endogenous cholesterol by ACAT in rat adrenal microsomes. In contrast, the formation of cholesterol esters from exogenous cholesterol by ACAT was dose-dependently increased by the addition of SCP2. These experiments showed that SCP2 had an enhancing effect on cholesterol esterification by ACAT in rat adrenal microsomes most likely by modulating the availability of exogenous cholesterol and that SCP2 may participate in the formation of cholesterol esters in the rat adrenal gland.     

Phospholipids, sterol carrier protein2 and adrenal steroidogenesis

Rat adrenocortical cells and preparations of plasma membrane and mitochondria have been employed to assess the effects of phospholipids and of sterol carrier protein2 (SCP2) on specific aspects of adrenal steroidogenesis. With intact cells, liposomal dispersions of cardiolipin caused significant stimulation of corticosterone output, while preparations of phosphatidylcholine, phosphatidylinositol, or the 4'-phosphate and the 4',5'-diphosphate derivatives of phosphatidylinositol were without effect. With the adrenal plasma membrane preparation, none of the added phospholipids affected either sodium fluoride or ACTH-responsive adenylate cyclase activity. With intact mitochondria, only cardiolipin, among the various phospholipids, tested, caused a concentration-dependent stimulation of pregnenolone production. However, even at the highest concentration of cardiolipin tested (500 microM), the stimulatory effect was only half that observed with 0.7 microM SCP2, and the two effectors were not synergistic. SCP2 caused a redistribution of cholesterol from mitochondrial outer to inner membranes, while cardiolipin, which is an activator of cytochrome P-450scc, had no effect on distribution of mitochondrial membrane cholesterol.     

Sterol carrier protein2 (SCP2)-mediated transfer of cholesterol to mitochondrial inner membranes

The fluorescence parameters of demetallized alpha-lactalbumin in the range from pH 8 to 2 show an extreme around pH 5-4 (a minimum in quantum yield and wavelength and a maximum in polarization). This extreme is not due to a competition between Ca2+ and protons but rather to a stabilization of the conformation of the protein near the isoelectric pH by the ionic interactions between local positive and negative charges on the protein. The calcium-free protein has similar fluorescence characteristics at pH 2 and 8 but the thermal transition curve is different. The influence of 0.1 M NaCl is also considered.     

Role of sterol carrier protein in cholesterol metabolism

This report summarizes our recent studies on the protein known as sterol carrier protein (SCP) or fatty acid binding protein (FABP). SCP is a highly abundant, ubiquitous protein with multifunctional roles in the regulation of lipid metabolism and transport. SCP in vitro activates membrane-bound enzymes catalyzing cholesterol synthesis and metabolism, as well as those catalyzing long chain fatty acid metabolism. SCP also binds cholesterol and fatty acids with high affinity and rapidly penetrates cholesterol containing model membranes. Studies in vivo showed SCP undergoes a remarkable diurnal cycle in level and synthesis, induced by hormones and regulated in liver by translational events. SCP rapidly responds in vivo to physiological events and manipulations affecting lipid metabolism by changes in level. Thus SCP appears to be an important regulator of lipid metabolism. Preliminary evidence is presented that SCP is secreted by liver and intestine into blood and then taken up by tissues requiring SCP but incapable of adequate SCP synthesis.     

Stimulation of adrenal mitochondrial cholesterol side-chain cleavage by GTP, steroidogenesis activator polypeptide (SAP), and sterol carrier protein2. GTP and SAP act synergistically

Several factors are known to stimulate cholesterol side-chain cleavage in isolated adrenal mitochondria, including steroidogenesis activator polypeptide (SAP), GTP, and sterol carrier protein2 (SCP2). All of these reportedly function at the level of the translocation of cholesterol to the inner membrane wherein side-chain cleavage to form pregnenolone occurs. We have investigated the activating effects of these factors alone and in combination. Under conditions where exogenous cholesterol is provided and multiple turnovers of a transport system are required, GTP stimulated steroidogenesis in isolated mitochondria and in adrenal homogenates, and this effect was enhanced by a GTP regenerating system. SAP alone had little effect under these conditions, but synergized with GTP to stimulate cholesterol metabolism. A truncated SAP analog and a variant from the C terminus of the minor heat-shock protein GRP78 had similar effects, but an unrelated peptide had no effect. GTP stimulated side-chain cleavage with the same EC50 in both resting mitochondria (from dexamethasone-treated rats) and in activated mitochondria (from ether-treated rats), but SAP effects were most apparent in resting mitochondria. In contrast, SCP2 stimulation was additive with other factors, suggesting an independent mechanism of action. While the data are consistent with biological roles for these factors, the relatively small magnitude of the in vitro effects may indicate that cell disruption and mitochondrial isolation disrupt important structural or other features which are necessary for the full expression of the steroidogenic response.     

Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol

Purified sterol carrier protein2 (SCP2) from rat liver stimulated utilization of endogenous cholesterol for pregnenolone synthesis by adrenal mitochondria. Cytosolic preparations of rat liver, adrenal and luteinized ovary were also stimulatory in mitochondrial pregnenolone synthesis to different extents. Treatment of all preparations with rabbit anti-rat SCP2 IgG neutralized the stimulatory effects, and immunoprecipitated proteins gave similar patterns on SDS-gradient polyacrylamide gel electrophoresis. Treatment with rabbit pre-immune IgG had no effect on these parameters. Thus, proteins which are immunochemically compatible with hepatic SCP2 appear to be present in steroidogenic tissues and may play a role in control of mitochondrial cholesterol side chain cleavage activity.     

Rat adrenal corticosteroidogenesis; effect of ACTH, cycloheximide and sterol carrier protein.

Corticosterone formation was determined in the reconstructed rat adrenal system which consisted of the mitochondria and post-mitochondrial supernatant fraction (PM-fraction) supported by l-malate, and effect of ACTH and cycloheximide in vivo and cycloheximide, Ca++ and sterol carrier protein (SCP) in vitro were examined. Mitochondria isolated from adrenals of rats which received ACTH 15 min before sacrifice showed an elevated corticosterone formation. Cycloheximide administration 15 min prior to ACTH injection completely blocked the effect of ACTH but in vitro addition of this drug to the incubation mixture did not modify the rate of corticosterone production even at higher concentrations. Since the PM-fraction isolated from adrenals of rats received ACTH or cycloheximide or both did not change the mitochondrial capacity for corticosterone formation, factor(s) which influenced by ACTH administration seemed to be localized in mitochondria. The SCP-bound cholesterol was utilized for corticosterone formation more efficiently than the free cholesterol when added to the incubation mixture, and this might be due to, at least in part, higher rate of binding to the mitochondrial inner membrane of the SCP-bound cholesterol.     

Sterol carrier protein-2 alters high density lipoprotein-mediated cholesterol efflux

Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on "reverse cholesterol transport" has not been explored. As shown herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of [(3)H]cholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (t(b)(12) near 1 min) and vesicular (t(d)(12) = 10-20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t(b)(12) decreased 36%, while the slower t(d)(12) increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and t(d)(12) 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.     

Sterol carrier protein hypothesis: requirement for three substrate-specific soluble proteins in liver cholesterol biosynthesis.

The 105,000 x g supernatant (S₁₀₅) of liver is required for the conversion of squalene to cholesterol by microsomal membranes. Substantial controversy has existed concerning the properties of what was originally considered to be a single sterol carrier protein present in S₁₀₅ and required for this conversion. We have now resolved this controversy by the discovery that S₁₀₅ contains several sterol carrier proteins. Based upon experiments with three substrates, three substrate-specific soluble proteins (with different properties) have been identified which operate at distinct points in microsomal cholesterol synthesis. These proteins are provisionally designated sterol carrier protein₁ (SCP₁), sterol carrier protein₂ (SCP₂), and sterol carrier protein₃ (SCP₃). SCP₁ is required for the microsomal conversion of squalene to lanosterol, SCP₂ for the microsomal conversion of 4,4-dimethyl-Δ⁸-cholesterol to C₂₇-sterols, and SCP₃ for the microsomal conversion of 7-dehydrocholesterol to cholesterol. Available evidence is consistent with the proposal that a given sterol carrier protein is a soluble constituent of a single microsomal enzyme or enzyme complex, and that it participates both as a carrier for the water-insoluble substrate and as an essential enzyme constituent facilitating catalysis. It may well be that enzymatic transformations of water-insoluble substrates require both microsomal membranes and substrate-specific soluble proteins. This requirement could be a common biological mechanism for water-insoluble substrates.     

Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.     

The role of sterol carrier protein 2 in stimulation of steroidogenesis in rat adrenal mitochondria by adrenal cytosol

Cholesterol side-chain cleavage (CSCC) in isolated rat adrenal mitochondria is enhanced by prior corticotropin (ACTH) stimulation in vivo (8-fold). Part of this stimulation is retained in vitro by addition of cytosol from ACTH-stimulated adrenals to mitochondria from unstimulated rats (2.5- to 6-fold). In vivo cycloheximide (CX) treatment fully inhibits the in vivo response and resolves the in vitro cytosolic stimulation into components: (i) ACTH-sensitive, CX-sensitive; (ii) ACTH-sensitive, CX-insensitive; and (iii) ACTH-insensitive, CX-insensitive. These components contribute approximately equally to stimulation by ACTH cytosol. Components (i) and (iii) most probably correspond to previously identified cytosolic constituents steroidogenesis activator peptide and sterol carrier protein 2 (SCP2). SCP2, as assayed by radioimmunoassay or ability to stimulate 7-dehydrocholesterol reductase, was not elevated in adrenal cytosol or other subcellular fractions by ACTH treatment. Complete removal of SCP2 from cytosol by treatment with anti-SCP2 IgG decreased cytosolic stimulatory activity by an increment that was independent of ACTH or CX treatment. Addition of an amount of SCP2, equivalent to that present in cytosol, restored activity to SCP2-depleted cytosol but had no effect alone or when added with intact cytosol, suggesting the presence of a factor in cytosol that potentiates SCP2 action. Pure hepatic SCP2 stimulated CX mitochondrial CSCC 1.5- to 2-fold (EC50 0.7 microM) but was five times less potent than SCP2 in adrenal cytosol. Two pools of reactive cholesterol were distinguished in these preparations characterized, respectively, by succinate-supported activity and by additional isocitrate-supported activity. ACTH cytosol and SCP2 each stimulated cholesterol availability to a fraction of mitochondrial P450scc that was reduced by succinate but failed to stimulate availability to additional P450scc reduced only by isocitrate.     

Identification of mosquito sterol carrier protein-2 inhibitors

A mosquito sterol carrier protein-2, AeSCP-2, has been shown to aid in the uptake of cholesterol in mosquito cells. The discovery of chemical inhibitors of AeSCP-2 is reported here. AeSCP-2 inhibitors (SCPIs) belong to several chemotypes of hydrophobic compounds. Those inhibitors competed with cholesterol for AeSCP-2, binding with relatively high binding affinities. In cultured insect cells, SCPIs reduced cholesterol uptake by as much as 30% at 1-5 microM concentrations. SCPIs were potent larvicides to the yellow fever mosquito, Aedes aegypti, and to the tobacco hornworm, Manduca sexta, with 50% lethal doses (LD50s) of 5-21 microM and 0.013-15 ng/mg diet, respectively. The results indicate that sterol carrier protein-2 has functional similarity in two different insect species.     

Sterol carrier protein 2 and fatty acid-binding protein. Separate and distinct physiological functions

Sterol carrier protein 2 (SCP-2) participates in the microsomal conversion of lanosterol to cholesterol, in the conversion of cholesterol to cholesterol ester, and in intracellular cholesterol transfers. The stoichiometry of binding between cholesterol and SCP-2 is 1:1. However, reports have appeared attributing sterol carrier protein activity to a protein preparation identical to hepatic fatty acid-binding protein (FABP). Therefore, the present investigation was conducted to compare homogeneous preparations of FABP and SCP-2 with respect to their capacities to participate as carrier proteins in reactions involving sterols or fatty acids. The results show that SCP-2 and FABP have separate and distinct physiological functions, with SCP-2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP-2 does not specifically bind or transport fatty acid. As long as only small quantities of organic solvent (1.6 volume %) were used for substrate addition, the sterol delta 7-reductase liver microsomal assay for SCP-2 correlated well with the physiologically relevant assays employed in the reconstituted adrenal system. The sterol carrier protein activity previously attributed to rat hepatic FABP is explained by the presence of significant quantities of propylene glycol (15 volume %) or Tween 80 in the assay procedure.     

Sterol synthesis. High-pressure liquid chromatography of C27 sterol precursors of cholesterol.

Conditions have been described which permit the rapid (approximately 3.5 hr) column chromatographic separation of the acetate derivatives of a number of C27 sterol precursors of cholesterol differing only in the number and position of double bonds in the sterol nucleus. On columns containing muPorasil with hexane-benzene 9:1 as the eluting solvent, acetate derivatives of the delta5, delta8(14), delta8, delta7, delta8,7, delta 8,14, and delta7,14 sterols were separated.     

Sterol carrier protein2-like activity in rat intestine

A sterol carrier protein2 (SCP2)-like activity has been demonstrated in rat intestinal mucosal homogenates and in isolated intestinal cells from both crypt and villus zones. The results indicate the presence of a protein with similar molecular weight and antigenicity to that of authentic SCP2 purified from rat liver cytosol. Like liver SCP2, mucosal cytosol stimulates pregnenolone production in rat adrenal mitochondria and acyl coenzyme A:cholesterol acyltransferase activity of liver and mucosal microsomes. The distribution of SCP2-like activity as determined by radioimmunoassay indicates high levels in mitochondria and cytosol and relatively lower levels in microsomes and in brush-border membranes. The widespread distribution of SCP2-like protein in the intestine is consistent with potential transfer functions in all phases of cholesterol processing.     

A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria

Mitochondrial cholesterol oxidation rapidly depletes cholesterol from the relatively cholesterol-poor mitochondrial membranes. However, almost nothing is known regarding potential mechanism(s) whereby the mitochondrial cholesterol pool is restored. Since most exogenous cholesterol enters the cell via the lysosomal pathway, this could be a source of mitochondrial cholesterol. In the present study, an in vitro fluorescent sterol transfer assay was used to examine whether the lysosomal membrane could be a putative cholesterol donor to mitochondria. First, it was shown that spontaneous sterol transfer from lysosomal to mitochondrial membranes was very slow (initial rate, 0.316 +/- 0.032 pmol/min). This was due, in part, to the fact that 90% of the lysosomal membrane sterol was not exchangeable, while the remaining 10% also had a relatively long half-time of exchange t(1/2) = 202 +/- 19 min. Second, the intracellular sterol carrier protein-2 (SCP-2) and its precursor (pro-SCP-2) increased the initial rate of sterol transfer from the lysosomal to mitochondrial membrane by 5.2- and 2.0-fold, respectively, but not in the reverse direction. The enhanced sterol transfer was due to a 3.5-fold increase in exchangeable sterol pool size and to induction of a very rapidly (t(1/2) = 4.1 +/- 0.6 min) exchangeable sterol pool. Confocal fluorescence imaging and indirect immunocytochemistry colocalized significant amounts of SCP-2 with the mitochondrial marker enzyme cytochrome oxidase in transfected L-cells overexpressing SCP-2. In summary, SCP-2 and pro-SCP-2 both stimulated molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.