Effect of increasing iron supplementation on blood lipids in rats.

The effects of increasing levels of Fe on serum fatty acids, cholesterol, triacylglycerol, liver and heart were examined in male Sprague-Dawley rats fed either Fe-deficient or carbonyl Fe-supplemented diets with 35 (control), 350, 3500 and 20 000 microg Fe/g for 12 weeks. As intake of Fe increased, serum total cholesterol increased from 2.0 mmol/l in controls to 5.2 mmol/l at the highest level of Fe. Also, the total serum phospholipid fatty acids increased from 609 mg/dl in controls to 1292 mg/l at the highest level of Fe. Except for the highest dose of Fe, the ratio of saturated to unsaturated phospholipid fatty acids increased from 1.2 to 1.7. The serum total free fatty acid levels remained constant among all groups with a range from 162 to 228 mg/l, while a ratio of 0.6 to 0.8 for saturated to unsaturated fatty acids was maintained. A dose-related increase in liver non-haem Fe from 18 to 3500 microg/g correlated with increases in lipid peroxidation (r 0.87), measured by the lipid-conjugated diene assay. Oxidative changes in the liver may have resulted in alterations in sterol synthesis, leading to increased serum cholesterol levels with increases in serum phospholipids and changes in the ratios of their saturated to unsaturated fatty acids. Animals with heart damage showed myocardial degeneration and cardiomyopathy with haemosiderin in interstitial macrophages or myocardial fibres and, when these were coupled with the findings of increased non-haem Fe in the heart and lipid peroxidation in the liver, suggested that oxidative stress is involved in the pathogenesis of the lesions.

Effect of sesame oil on serum and liver lipid profiles in the rat.

In our previous study (Satchithanandam, S., Reicks, M., Calvert, R.J., Cassidy, M.M. and Kritchevsky, D. (1993) J. Nutr. 123, 1852-1858), we found that the absorption of lymphatic cholesterol by rats fed diets containing 24% sesame oil was about 50% less than that by rats fed the control diet containing no sesame oil. The effect of sesame oil on serum cholesterol levels was not determined at that time. In the present study, three groups of male Wistar rats (75-100 g) were fed a control diet or a diet containing 12 or 24% sesame oil. To increase serum cholesterol levels, 1% cholesterol and 0.5% cholic acid were added to each diet. After rats were fed for 4 weeks, total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglyceride levels were measured in the serum. Liver weight and cholesterol and triglyceride levels were determined. Liver cholesterol levels were significantly lower in rats fed the 24% sesame oil diet, and the liver lipid level was significantly higher in the 24% sesame oil-fed group, compared with levels in the group fed the control diet. Liver weights and esterified cholesterol and liver triglyceride levels were not significantly different among the groups. Levels of serum total cholesterol and LDL-cholesterol were significantly lower in rats fed the 24% sesame oil diet, compared with levels in the control group. Serum triglyceride and HDL-cholesterol levels did not differ significantly among the groups. The mechanism by which a diet containing 24% sesame oil reduces levels of serum and liver cholesterol, liver LDL cholesterol, and liver lipids is not known. However, the high degree of unsaturation (85%) of sesame oil and the presence of linoleic acid may be important factors.

Effects of alpha-tocopherol and beta-carotene on hepatic lipid peroxidation and blood lipids in rats with dietary iron overload.

The ability of dietary antioxidants to reduce lipid peroxidation induced by iron overload was examined in weanling male Sprague-Dawley rats. Animals were fed ad libitum a modified AIN-76A diet (control) or control diet with 0.5% alpha-tocopherol acid succinate, 0.5% crystalline trans-beta-carotene, or 0.5% alpha-tocopherol acid succinate + 0.5% trans-beta-carotene for four weeks. In the following four-week period, the animals received the above diets with 10,000 micrograms Fe/g; a control group continued to receive 35 micrograms Fe/g, and a high-iron group received 10,000 micrograms Fe/g with no antioxidants. After four weeks of dietary supplementation with alpha-tocopherol. Beta-carotene or alpha-tocopherol + beta-carotene, liver concentrations of alpha-tocopherol and beta-carotene increased significantly (p < 0.001). Liver lipid peroxidation, measured by the lipid-conjugated diene assay, increased significantly from 0.012 mumol/mg of lipid in the controls to 0.021 mumol/mg of lipid in animals receiving the high-iron diet. However, lipid peroxidation was significantly reduced in all animals fed the antioxidants, with the group fed alpha-tocopherol + beta-carotene having a lower level than the high-iron group. Total serum cholesterol was elevated in animals fed a high-iron diet and in animals fed the high-iron diet with alpha-tocopherol. In contrast, total serum cholesterol levels in the two groups of animals receiving the diets containing high iron with beta-carotene alone or high iron with beta-carotene + alpha-tocopherol were significantly reduced to the level of the control group. High-density lipoprotein cholesterol also decreased to baseline in the animals receiving beta-carotene alone. Modulation of lipid peroxidation by alpha-tocopherol or beta-carotene may be an important mechanism for reducing oxidative stress.

Prevention of post-ischemic brain lipid conjugated diene production and neurological injury by hydroxyethyl starch-conjugated deferoxamine.

Hydroxyethyl starch conjugated deferoxamine (DFO) was administered to rats following resuscitation from 6.5 min cardiac arrest (CA) in an attempt to prevent the iron-catalyzed production of oxygen free radicals which may lead to neurologic injury and ultimately death following restoration of spontaneous circulation (ROSC). Brain conjugated dienes were analyzed spectrophotometrically 4 and 24 hr following ROSC, and were found to be significantly elevated when compared to non-ischemic controls. Hydroxyethyl starch-DFO treated rats demonstrated no increased conjugated diene production at either period. Neurologic injury was significantly less in drug treated rats surviving 24 or 72 hours when compared to controls. While mortality was similar in drug treated or control rats for the first 24 hours following ROSC, delayed mortality (days 1-10) was significantly less in drug treated animals, presumably as a result of neurologic protection afforded by post-ischemic drug administration. Administration of DFO conjugated to hydroxyethyl starch appears to modulate the neurologic injury which occurs during brain ischemia and reperfusion.

Elevated cholesterol and decreased sterol carrier protein-2 in peroxisomes from AS-30D hepatoma compared to normal rat liver.

Peroxisomes were isolated from AS-30D hepatoma and compared to normal rat liver cells for the purpose of investigating the cholesterol accumulation in the hepatoma cells. Cholesterol was found to be approximately 10-fold higher relative to protein in AS-30D peroxisomes as compared to peroxisomes from normal liver. The peroxisomes from the hepatoma cells were found to be more stable; catalase was not released from these peroxisomes during isolation or osmotic shock of the peroxisomal fraction. The elevated cholesterol level may stabilize the peroxisomal membrane. Sterol carrier protein-2 (SCP-2) levels were measured using a radioimmunoassay (RIA), which indicated the highest concentration of SCP-2 to be in peroxisomes. Hepatoma peroxisomes had a lower concentration of SCP-2 (2.5 micrograms/mg) than normal liver peroxisomes (8 micrograms/mg). Approximately half of all SCP-2 detected was found to be soluble in both hepatoma and normal rat liver cells. Immunoblots from both rat liver and AS-30D fractions demonstrated the presence of the 14-kDa form of SCP-2. The liver fractions also had a 57-kDa immunoreactive protein, which was barely detectable in the AS-30D fractions. The low abundance of the high molecular weight form of SCP-2 from hepatoma peroxisomes and the lower amounts of SCP-2 detected in the AS-30D peroxisomes may be related to the accumulation of cholesterol in the cells.

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.

Sterol carrier protein2: further evidence for its role in adrenal steroidogenesis.

Homogeneous rat liver sterol carrier protein (SCP2) has been implicated in adrenal steroidogenesis by studies utilizing as a model system various sub-cellular fractions of rat adrenals. Levels of SCP2 were measured in rat adrenal subcellular fractions and various rat tissues using a highly sensitive radioimmunoassay. The levels of SCP2 in various tissues correlate well with the capacity of each tissue to either synthesize or metabolize cholesterol. The high level of SCP2 in adrenal mitochondria (46% of total tissue SCP2) is consistent with its proposed role of enhancing transfer of cholesterol from the outer to the inner mitochondrial membrane. Neither ACTH nor cycloheximide treatment of rats had a significant effect on SCP2 levels or distribution in the adrenal subcellular fractions. Western blot analysis of adrenal subcellular fractions indicates the presence of a protein of identical molecular weight and at least similar antigenicity as homogeneous rat liver SCP2. In the present studies, intact dispersed rat adrenal fasciculata cells fused with liposomal encapsulated anti-SCP2 IgG showed a 40-65% reduction in their ability to produce corticosterone when stimulated with ACTH. The steroidogenic competence of these anti-SCP2 IgG treated cells can be restored by treatment of the cells with liposomal encapsulated SCP2 prior to ACTH stimulation. These findings provide direct evidence for the involvement of SCP2 in ACTH stimulated steroidogenesis in rat adrenocortical cells, and suggests that SCP2 may not be the putative high turnover "labile protein" involved in acute steroidogenesis.

Adrenal cholesterol esters as substrate source for steroidogenesis.

Adrenocortical cells obtained from tissues of unstimulated rats and which contained a high concentration of endogenous esterified cholesterol, were labeled in vitro with unesterified [4-14C]cholesterol, or incubated in the presence of [2-14C]acetate or lipoprotein [4-14C]cholesterol oleate (LP-CE). Incubations were conducted in the absence and presence of ACTH, and changes in the specific radioactivity (SA) of the secreted corticosterone were used to assess the primary sources of cholesterol substrate used for steroidogenesis. Incubations of cells containing [4-14C]cholesterol with ACTH resulted in a marked increase in the output of corticosterone mass, but not of labeled corticosterone. Thus, the SA of corticosterone when cells were incubated with ACTH was only 6.5% of that obtained from cells incubated in the absence of ACTH. During incubations with [2-14C]acetate, the ACTH-induced increase in the output of corticosterone mass was not associated with increased isotope output, and the SA of corticosterone was only 15% of that in control incubations. This dilution was not altered in cells isolated from adrenals of rats treated with 4-aminopyrazalopyrimidine (4-APP), in which increased cholesterogenesis was demonstrable. The uptake, and hydrolysis of LP-CE, and formation of labeled corticosterone was lipoprotein concentration dependent, and was not influenced by ACTH. However, in the presence of ACTH, the SA of the secreted corticosterone was only 4-8% of that in unstimulated cells. The consistent dilution of the SA of corticosterone in ACTH-treated cells in all studies suggest that the large stores of cytoplasmic cholesterol esters in these cells may normally serve as a primary source of the immediate precursor sterol used for steroidogenesis.

Sterol carrier and lipid transfer proteins.

The discovery of the sterol carrier and lipid transfer proteins was largely a result of the findings that cells contained cytosolic factors which were required either for the microsomal synthesis of cholesterol or which could accelerate the transfer or exchange of phospholipids between membrane preparations. There are two sterol carrier proteins present in rat liver cytosol. Sterol carrier protein 1 (SCP1) (Mr 47 000) participates in the microsomal conversion of squalene to lanosterol, and sterol carrier protein 2 (SCP2) (Mr 13 500) participates in the microsomal conversion of lanosterol to cholesterol. In addition SCP2 also markedly stimulates the esterification of cholesterol by rat liver microsomes, as well as the conversion of cholesterol to 7 alpha-hydroxycholesterol - the major regulatory step in bile acid formation. Also, SCP2 is required for the intracellular transfer of cholesterol from adrenal cytoplasmic lipid inclusion droplets to mitochondria for steroid hormone production, as well as cholesterol transfer from the outer to the inner mitochondrial membrane. SCP2 is identical to the non-specific phospholipid exchange protein. While SCP2 is capable of phospholipid exchange between artificial donors/acceptors, e.g. liposomes and microsomes, it does not enhance the release of lipids other than unesterified cholesterol from natural donors/acceptors, e.g. adrenal lipid inclusion droplets, and will not enhance exchange of labeled phosphatidylcholine between lipid droplets and mitochondria. Careful comparison of SCP2 and fatty acid binding protein (FABP) using six different assay procedures demonstrates separate and distinct physiological functions for each protein, with SCP2 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 SCP2 does not specifically bind or transport fatty acid. The results described in the present review support the concept that intracellular lipid transfer is a highly specific process, far more substrate-specific than suggested by the earlier studies conducted using liposomal techniques.

Regulation of bile-acid synthesis. Role of sterol carrier protein 2 in the biosynthesis of 7 alpha-hydroxycholesterol.

Sterol carrier protein2 (SCP2) is known to stimulate utilization of cholesterol in enzymic reactions in which cholesterol is the substrate. Substantial recent experimental evidence indicates that SCP2: activates enzymic conversion of intermediates between lanosterol and cholesterol; stimulates the microsomal conversion of cholesterol into cholesterol ester in rat liver; and enhances mitochondrial utilization of cholesterol for pregnenolone formation in the adrenals. The conversion of cholesterol into 7 alpha-hydroxycholesterol is the rate-limiting step in bile-acid synthesis. We therefore investigated the effect of SCP2 on this physiologically critical reaction by using a gas-chromatography-mass-spectrometry procedure that measures the mass of 7 alpha-hydroxycholesterol formed. The results show that SCP2 enhances 7 alpha-hydroxycholesterol formation by rat liver microsomes (microsomal fractions), utilizing either endogenous membrane cholesterol, cholesterol supplied exogenously in serum or in the form of cholesterol/phospholipid liposomes. Microsomes immunotitrated with anti-SCP2 antibody exhibited considerably less capacity to synthesize 7 alpha-hydroxycholesterol, which was restored to control levels on addition of purified SCP2. These data are consistent with the suggestion that SCP2 may be of physiological significance in the overall metabolism of cholesterol.

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

Rats treated with cycloheximide accumulate cholesterol in the adrenal mitochondria. This cholesterol is largely associated with the mitochondrial outer membrane. The addition of homogeneous sterol carrier protein2 (SCP2) to these mitochondria stimulated pregnenolone production to a greater extent than with mitochondria from untreated rats. Adrenal mitochondria from cycloheximide-treated rats were incubated in the presence of aminoglutethimide and cycloheximide to prevent further utilization of cholesterol for pregnenolone synthesis. Outer and inner membrane fractions of these mitochondria incubated with and without 0.75 microM SCP2, were prepared following hypotonic disruption. Incubations of mitochondria in the presence of SCP2 caused a marked shift in cholesterol from outer to inner membrane fractions. The findings provide direct evidence for a role of SCP2 or a similar peptide in modulating transfer of cholesterol to the inner membrane site of cholesterol side chain cleavage.

Cholesterol ester hydrolase and sterol carrier proteins.

The cholesterol substrate required for sustained adrenal steroidogenesis is largely derived from the endogenous stores of cholesterol esters, which are located in large lipid inclusion droplets in the cytoplasm. In isolated adrenal cells, these esters are hydrolyzed during a variety of stimuli associated with cellular cAMP production. This largely appears to be a response to the action of a neutral cholesterol ester hydrolase, whose activity is modulated by phosphorylation of the enzyme protein, catalyzed by cAMP-dependent protein kinase. Transfer of the resulting unesterified cholesterol to mitochondria can be accomplished in a model system by sterol carrier protein2 (SCP2). This protein is distinct from fatty acid binding protein (FABP), has a Mr of 13,500 and is basic in nature. SCP2 can sequester cholesterol from lipid inclusion droplets in a stoichiometric relationship, and transfer this cholesterol to isolated adrenal mitochondria. SCP2 can also enhance the intermembrane transfer of mitochondrial cholesterol to cytochrome P 450scc, but does not directly affect cholesterol side chain cleavage. The stimulatory effect of adrenal cytosolic preparations on mitochondrial pregnenolone production can be completely abolished by pretreatment with anti SCP2 IgG.

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.

Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization.

Addition of homogeneous rat liver sterol carrier protein2 (SCP2) or an adrenal cytosolic fraction enhanced pregnenolone production by adrenal mitochondria. Pretreatment of SCP2 or adrenal cytosol with anti-SCP2 IgG abolished the stimulatory effect of both preparations on mitochondrial pregnenolone output. Incubation of mitochondria with aminoglutethimide, which blocks interaction of cholesterol with inner membrane cytochrome P-450scc, resulted in decreased pregnenolone production and a decreased level of mitoplast cholesterol. Addition of SCP2 to the incubation media caused an almost 2-fold increase in cholesterol associated with the mitoplast, but did not enhance mitochondrial pregnenolone production. Studies with reconstituted cytochrome P-450scc in phospholipid vesicles also suggested that SCP2 did not affect interaction of cholesterol with the hemoprotein. Treatment of rats with cycloheximide alone or with adrenocorticotropic hormone resulted in a dramatic increase in mitochondrial cholesterol. However, these mitochondria did not exhibit increased levels of pregnenolone output under control incubation conditions. When SCP2 was included in the mitochondrial incubation media, pregnenolone production was significantly increased over that observed with adrenal mitochondria from untreated or adrenocorticotropic hormone-treated rats. The results imply that SCP2 enhances mitochondrial pregnenolone production by improving transfer of mitochondrial cholesterol to cytochrome P-450scc on the inner membrane, but does not directly influence the interaction of substrate with the hemoprotein.

Sterol carrier protein2. Delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis.

The ability of sterol carrier protein2 (SCP2) to mediate transfer of unesterified cholesterol from adrenal lipid inclusion droplets to mitochondria has been tested in an in vitro model system. Unlike mitochondrial utilization of cholesterol added in acetone or dimethyl sulfoxide, the unesterified cholesterol of lipid droplets did not provide a readily available source of substrate for mitochondrial pregnenolone production, without the addition of a transport mediator. Addition of SCP2, but not albumin, stimulated mitochondrial utilization of droplet cholesterol in a concentration-dependent manner. In the absence of mitochondria, SCP2 sequestered lipid droplet cholesterol, and in the presence of mitochondria, which were unable to convert cholesterol to pregnenolone, this cholesterol was quantitatively accumulated by mitochondria. Both processes were concentration-dependent and demonstrated a molar ratio of SCP2 and cholesterol for both binding and transport of 1. SCP2 also enhanced pregnenolone formation by mitochondria which were incubated in the absence of an extramitochondrial source of cholesterol. However, SCP2 had no effect on steroid release from a crude particulate fraction. These studies suggest that the effects of SCP2 are related to delivery of cholesterol from preformed stores to and into mitochondria for initiation of steroid hormone synthesis, and may represent an important modulator of sterol metabolism in adrenal cortical cells.