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PubMed2003

Molecular and cellular physiology of apolipoprotein A-I lipidation by the ATP-binding cassette transporter A1 (ABCA1).

The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Specific binding parameters of (125)I-apoA-I to ABCA1 at 37 degrees C were determined: K(d) = 0.65 microg/ml, B(max) = 0.10 ng/microg cell protein. Lipid-free apoA-I inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than pre-beta(1)-LpA-I, reconstituted HDL particles r(LpA-I), or HDL(3) (IC(50) = 0.35 +/- 1.14, apoA-I; 1.69 +/- 1.07, pre-beta(1)-LpA-I; 17.91 +/- 1.39, r(LpA-I); and 48.15 +/- 1.72 microg/ml, HDL(3)). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither (125)I-apoA-I binding nor (125)I-apoA-I/ABCA1 cross-linking. We next investigated the dynamics of apoA-I lipidation by monitoring the kinetic of apoA-I dissociation from ABCA1. The dissociation of (125)I-apoA-I from normal cells at 37 degrees C was rapid (t((1/2)) = 1.4 +/- 0.66 h; n = 3) but almost completely inhibited at either 15 or 4 degrees C. A time course analysis of apoA-I-containing particles released during the dissociation period showed nascent apoA-I-phospholipid complexes that exhibited alpha-electrophoretic mobility with a particle size ranging from 9 to 20 nm (designated alpha-LpA-I-like particles), whereas lipid-free apoA-I incubated with ABCA1 mutant (Q597R) cells was unable to form such particles. These results demonstrate that: 1) the physical interaction of apoA-I with ABCA1 does not depend on membrane phosphatidylcholine or sphingomyelin; 2) the association of apoA-I with lipids reduces its ability to interact with ABCA1; and 3) the lipid translocase activity of ABCA1 generates alpha-LpA-I-like particles. This process plays in vivo a key role in HDL biogenesis.

ATP Binding Cassette Transporter 1↗
PubMed2003

Apomine, a novel hypocholesterolemic agent, accelerates degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and stimulates low density lipoprotein receptor activity.

Apomine, a novel 1,1-bisphosphonate ester, has been shown to lower plasma cholesterol concentration in several species. Here we show that Apomine reduced the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme in the mevalonate pathway, both in rat liver and in cultured cells. Apomine resembles sterols such as 25-hydroxycholesterol in its ability to potently accelerate the rate of HMGR degradation by the ubiquitin-proteasome pathway, a process that depends on the transmembrane domain of the enzyme. The similarity between Apomine and sterols in promoting rapid HMGR degradation extends to its acute requirements for ongoing protein synthesis and mevalonate-derived non-sterol product(s) as a co-regulator. Yet, at suboptimal concentrations, sterols potentiated the effect of Apomine in stimulating HMGR degradation, indicating that these agents act via distinct modes. Furthermore, unlike sterols, Apomine inhibited the activity of acyl-CoA:cholesterol acyltransferase in intact cells but not in cell-free extracts. Apomine stimulated the cleavage of the precursor of sterol-regulatory element-binding protein-2 and increased the activity of low density lipoprotein receptor pathway. This Apomine-enhanced activation of sterol-regulatory element-binding protein-2 was prevented by sterols or mevalonate. Taken together, our results provide a molecular mechanism for the hypocholesterolemic activity of Apomine.

Animals↗
PubMed2003

Apolipoprotein A-I activates cellular cAMP signaling through the ABCA1 transporter.

It has been suggested that the signal transduction pathway initiated by apoA-I activates key proteins involved in cellular lipid efflux. We investigated apoA-I-mediated cAMP signaling in cultured human fibroblasts induced with (22R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Treatment of stimulated fibroblasts with apoA-I for short periods of time (<or=45 min) increased ATP binding cassette A1 (ABCA1) phosphorylation in a concentration-dependent manner. Concomitantly, apoA-I increased the intracellular level of cAMP in a concentration- and time-dependent manner. The maximal cAMP level was reached within 10 min at 10 microg/ml apoA-I representing a 1-fold increase. The ability of apoA-I to mediate cAMP production was only observed in stimulated fibroblasts. Furthermore, overexpression of ABCA1 in Chinese hamster ovary cells resulted in a 1.5-fold increase in apoA-I-mediated cAMP accumulation as compared with untransfected cells. In contrast, forskolin increased cAMP production significantly in unstimulated fibroblasts as well as in untransfected Chinese hamster ovary cells. Pharmacological inhibition of protein kinase A (H89) completely blocked apoA-I-mediated ABCA1 phosphorylation. Naturally occurring mutations of ABCA1 associated with Tangier disease (C1477R, 2203X, and 2145X) severely reduced apoA-I-mediated cAMP production, ABCA1 phosphorylation, (125)I-apoA-I binding, and lipid efflux, without affecting forskolin-mediated cAMP elevation. In contrast, the protein kinase A catalytic subunit was able to phosphorylate ABCA1 similarly from mutant and normal cell lines in vitro. Together, our results indicate that apoA-I activates ABCA1 phosphorylation through the cAMP/protein kinase A-dependent pathway, apoA-I-mediated cAMP production required high level expression of functional ABCA1, and Tangier disease mutants have defective apoA-I-mediated cAMP signaling. These findings suggest that apoA-I may activate cAMP signaling through G protein-coupled ABCA1 transporter.

ATP Binding Cassette Transporter 1↗
PubMed2004

Adipocytic differentiation and liver x receptor pathways regulate the accumulation of triacylglycerols in human vascular smooth muscle cells.

Lipid accumulation by vascular smooth muscle cells (VSMC) is a feature of atherosclerotic plaques. In this study we describe two mechanisms whereby human VSMC foam cell formation is driven by de novo synthesis of fatty acids leading to triacylglycerol accumulation in intracellular vacuoles, a process distinct from serum lipoprotein uptake. VSMC cultured in adipogenic differentiation medium accumulated lipids and were induced to express the adipocyte marker genes adipsin, adipocyte fatty acid-binding protein, C/EBPalpha, PPARgamma, and leptin. However, complete adipocyte differentiation was not observed as numerous genes present in mature adipocytes were not detected, and the phenotype was reversible. The rate of lipid accumulation was not affected by PPARgamma agonists, but screening for the effects of other nuclear receptor agonists showed that activation of the liver X receptors (LXR) dramatically promoted lipid accumulation in VSMC. Both LXRalpha and LXRbeta were present in VSMC, and their activation with TO901317 resulted in induction of the lipogenic genes fatty acid synthetase, sterol regulatory element binding protein (SREBP1c), and stearoyl-CoA desaturase. 27-Hydroxycholesterol, an abundant oxysterol synthesized by VSMC acted as an LXR antagonist and, therefore, may have a protective role in preventing foam cell formation. Immunohistochemistry showed that VSMC within atherosclerotic plaques express adipogenic and lipogenic markers, suggesting these pathways are present in vivo. Moreover, the development of an adipogenic phenotype in VSMC is consistent with their known phenotypic plasticity and may contribute to their dysfunction in atherosclerotic plaques and, thus, impinge on plaque growth and stability.

ATP-Binding Cassette Transporters↗
PubMed2005

Isolation of sterol-resistant Chinese hamster ovary cells with genetic deficiencies in both Insig-1 and Insig-2.

Insig-1 and Insig-2, a pair of endoplasmic reticulum (ER) membrane proteins, mediate feedback control of cholesterol synthesis through their sterol-dependent binding to the following two polytopic ER membrane proteins: sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Sterol-induced binding of Insigs to SCAP prevents the proteolytic processing of SREBPs, membrane-bound transcription factors that enhance the synthesis of cholesterol, by retaining complexes between SCAP and SREBP in the ER. Sterol-induced binding of Insigs to reductase leads to the ubiquitination and ER-associated degradation of the enzyme, thereby slowing a rate-controlling step in cholesterol synthesis. Here we report the isolation of a new line of mutant Chinese hamster ovary cells, designated SRD-15, deficient in both Insig-1 and Insig-2. The SRD-15 cells were produced by gamma-irradiation of Insig-1-deficient SRD-14 cells, followed by selection in high levels of the oxysterol, 25-hydroxycholesterol. Sterols neither inhibit SREBP processing nor promote reductase ubiquitination/degradation in SRD-15 cells. Sterol regulation of SREBP processing and reductase ubiquitination/degradation is fully restored in SRD-15 cells when they are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results demonstrate an absolute requirement for Insig proteins in the regulatory system that mediates lipid homeostasis in animal cells.

Animals↗
PubMed2005

A novel principle for partial agonism of liver X receptor ligands. Competitive recruitment of activators and repressors.

Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoR-dissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs.

Benzoates↗
PubMed2006

Regulation of human cytochrome P450 4F2 expression by sterol regulatory element-binding protein and lovastatin.

This report provides the first evidence that human P450 4F2 (CYP4F2) is induced by statins, which are widely used to treat hypercholesterolemia. Real time PCR and immunoblots indicate that lovastatin treatment increases expression of the endogenous CYP4F2 gene in human primary hepatocytes and HepG2 cells. The effects of lovastatin on gene expression are often mediated through sterol regulatory element-binding proteins (SREBPs). Immunoblots indicate that lovastatin-treated human hepatocytes display increased proteolytic processing of SREBP-2. In HepG2 cells, co-administration of a potent suppressor of SREBP-2 activation, 25-hydroxycholesterol, inhibits CYP4F2 mRNA induction by lovastatin. HepG2 cells transfected with an expression vector for the active nuclear form of SREBP-1a (nSREBP-1a) also display elevated endogenous CYP4F2 expression. Luciferase reporters containing the CYP4F2 proximal promoter are transactivated by nSREBPs (-1a, -1c, and -2) or a dominant positive form of the SREBP cleavage-activating protein (SCAP), which facilitates activation of endogenous SREBPs. Lovastatin-induced reporter expression is inhibited by overexpressed Insig-1, which prevents proteolytic activation of endogenous SREBPs. Electrophoretic mobility shift assays with in vitro translated nSREBP-1a identified two SREBP binding sites at -169/-152 and -109/-92, relative to the CYP4F2 transcription start site. Mutations in each site abolish SREBP binding. Chromatin immunoprecipitation experiments indicate that more SREBP-1 is associated with the CYP4F2 promoter after overexpression of nSREBP-1a. Transfection studies and mutagenesis indicate that the -109/-92 region is the primary site responsible for the effects of statins. Collectively, these results demonstrate that SREBPs transactivate CYP4F2 transcription and that CYP4F2 induction by statins is mediated by SREBP-2.

Anticholesteremic Agents↗
PubMed2000

The regulatory effects of thyroid hormone on the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The effects of 3, 3', 5-triiodothyronine (T3) on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity were evaluated in the C100 baby hamster kidney cell line. Cells cultured in Minimal Essential Medium (MEM) were supplemented with 10% thyroid hormone-depleted fetal bovine serum (THDS-MEM) and had a 70.1% lower level of HMG-CoA reductase activity than the cells grown in a medium supplemented with fetal bovine serum (FBS). When T3 was added to THDS-MEM, the reduction of the reductase activity was blocked in a dose-dependent manner. In the cells grown in THDS-MEM for 48 hours, T3 (10(-6) M) treatment rapidly increased HMG-CoA reductase activity, achieving the control level six hours after treatment. Such effects of T3 were blocked by actinomycin D (5 microg/ml) or cycloheximide (10 microg/ml). The transcriptional rate of the HMG-CoA reductase gene did not change significantly regardless of the presence of T3, while T3 inhibited the 25-hydroxycholesterol-mediated decay of the reductase mRNA significantly. Our results show that T3-dependent regulation of HMG-CoA reductase activity, via the de novo synthesis of the reductase enzyme, seems to be mediated at least partially by the stabilization of HMG-CoA reductase mRNA.

Animals↗
PubMed2002

Effect of chronic ethanol feeding on oxysterols in rat liver.

It was our hypothesis that, as a consequence of increased oxidative stress, cholesterol-derived hydroperoxides and oxysterols are increased in livers of rats exposed to ethanol. To test this we dosed Wistar rats (approximately 0.1 kg initial body weight) with ethanol chronically (rats fed a nutritionally complete liquid diet containing ethanol as 35% of total calories; sampled liver at approximately 6-7 weeks). We measured concentrations of 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH) as well as 7 alpha- and 7 beta-hydroxycholesterol (7 alpha-OH and 7 beta-OH), and 3 beta-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto). In response to chronic alcohol feeding, there were significant elevations in the concentrations of 7 alpha-OOH (+169%, P = 0.005) and 7 beta-OOH (+199%, P = 0.011). Increases in the concentrations of hepatic 7-keto (+74%, P = 0.01) and decreases in cholesterol (-43%; P = 0.03) also occurred. In contrast, the concentrations of both 7 alpha-OH and 7 beta-OH were not significant (NS). However, when oxysterols in chronic ethanol-fed rats were expressed relative to cholesterol there were significant increases in 7-keto/cholesterol (P = 0.0006), 7 alpha-OH/cholesterol (P = 0.0018) and 7 beta-OH/cholesterol (P = 0.0047). In conclusion, this is the first report of increased 7 alpha-OOH, 7 beta-OOH, and 7-keto in liver of rats and their elevation in chronic experimental alcoholism represent evidence of increased oxidative stress.

Alcoholism↗
PubMed2003

Oral alpha-tocopherol supplementation inhibits lipid oxidation in established human atherosclerotic lesions.

BACKGROUND: Much experimental evidence suggests that lipid oxidation is important in atherogenesis and in epidemiological studies dietary antioxidants appear protective against cardiovascular events. However, most large clinical trials failed to demonstrate benefit of oral antioxidant vitamin supplementation in high-risk subjects. This paradox questions whether ingestion of antioxidant vitamins significantly affects lipid oxidation within established atherosclerotic lesions. METHODS AND RESULTS: This placebo-controlled, double blind study of 104 carotid endarterectomy patients determined the effects of short-term alpha-tocopherol supplementation (500 IU/day) on lipid oxidation in plasma and advanced atherosclerotic lesions. In the 53 patients who received alpha-tocopherol there was a significant increase in plasma alpha-tocopherol concentrations (from 32.66 +/- 13.11 at baseline to 38.31 +/- 13.87 (mean +/- SD) micromol/l, p < 0.01), a 40% increase (compared with placebo patients) in circulating LDL-associated alpha-tocopherol (p < 0.0001), and their LDL was less susceptible to ex vivo oxidation than that of the placebo group (lag phase 115.3 +/- 28.2 and 104.4 +/- 15.7 min respectively, p < 0.02). Although the mean cholesterol-standardised alpha-tocopherol concentration within lesions did not increase, alpha-tocopherol concentrations in lesions correlated significantly with those in plasma, suggesting that plasma alpha-tocopherol levels can influence lesion levels. There was a significant inverse correlation in lesions between cholesterol-standardised levels of alpha-tocopherol and 7beta-hydroxycholesterol, a free radical oxidation product of cholesterol. CONCLUSIONS: These results suggest that within plasma and lesions alpha-tocopherol can act as an antioxidant. They may also explain why studies using < 500 IU alpha-tocopherol/day failed to demonstrate benefit of antioxidant therapy. Better understanding of the pharmacodynamics of oral antioxidants is required to guide future clinical trials.

Administration, Oral↗
PubMed2004

Application of water-soluble radical initiator, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, to a study of oxidative stress.

It is essential to generate free radicals at a controled and constant rate for specific duration and at specific site to study the dynamics of oxidation and also antioxidation. Both hydrophilic and lipophilic azo compounds have been used for such purpose. In the present work, the action of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) was examined and compared with those of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)-propionamide] (AMHP). The rate constant of free radical formation (ek(d)) for AIPH was 2.6 x 10(-6)/s at 37 degrees C in PBS (pH 7.4) solution, indicating that AIPH gives 3.8 times more free radicals than AAPH under the same conditions. It was found that the dynamics of oxidation and antioxidation induced by AIPH can be studied satisfactorily in the oxidation in micelles, LDL and erythrocyte suspensions, plasma, and cultured cells. The extent of cell death induced by AIPH and AAPH was directly proportional to the total free radicals formed. Interestingly, it was found that rats would not drink water containing AAPH, but they drank water containing AIPH. The levels of 8-iso-prostaglandin F2alpha (8-isoPs), 7-hydroxycholesterol (FCOH), lysophosphatidylcholine in the plasma of rats given water containing 50 mM AIPH for 1 month increased compared with those of control rats which drank water without AIPH. It may be concluded that AIPH is useful for kinetic and mechanistic studies on oxidative stress to membranes, lipoproteins, cells, and even animal models.

Adult↗
PubMed1999

Antioxidant BO-653 and human macrophage-mediated LDL oxidation.

Oxidation of LDL is now widely accepted to be involved in atherogenesis. The aim of this study was to examine the effect of BO-653, a strong radical scavenger and antioxidant, on oxidation of LDL by human macrophages in vitro. Fifty microg/ml LDL protein was incubated with macrophages in Ham's F10 medium, supplemented with additional Fe2+, for up to 48 h. Then the medium was analysed by LDL agarose gel electrophoresis, the thiobarbituric acid assay and gas chromatography. In the absence of added exogenous antioxidants, after 24h LDL oxidation produced 30.48 nmoles MDA equivalents/mg LDL protein and a relative electrophoretic mobility of 4.74. Linoleic acid (18:2), arachidonic acid (20:4) and cholesterol were depleted and 7beta-hydroxycholesterol was generated. BO-653 completely inhibited this cell-mediated oxidation of LDL in concentrations as low as 5 microM, being more effective than either alpha-tocopherol or probucol, which completely inhibited oxidation at 200 and 80 microM and only partially at 80 and 8 microM, respectively. This inhibition of cell-mediated LDL oxidation was not due to toxicity, as alpha-tocopherol, probucol and BO-653 were not toxic for the macrophages at the concentrations tested. Eighty microM alpha-tocopherol, 8 microM probucol and 5 microM BO-653 significantly reduced the toxicity to the oxidising culture caused by LDL oxidation. The results show that in this system BO-653 is a more effective antioxidant than alpha-tocopherol or probucol.

Antioxidants↗
PubMed1980

Cholesterol availability modulates myoblast fusion.

The requirement of cholesterol for myoblast fusion has been linked to the primary step in the fusion process, calcium-dependent aggregation (recognition). Inhibition of cholesterol synthesis with 25-hydroxycholesterol or compactin in the absence of exogenous lipid dramatically inhibits calcium-mediated aggregation and concomitant fusion within several hours. Restimulating cholesterol synthesis or supplying exogenous cholesterol rapidly restores aggregation activity. Over this time period, however, the sterol:phospholipid ratio is unaltered, suggesting a local rather than a general membrane cholesterol requirement for the expression of aggregation activity. The aggregation response to a change in sterol availability occurs on a shorter time scale than that required to inhibit the synthesis of the protein(s) with aggregation activity; thus, the cholesterol-requiring step is posttranslational. We suggest that the assembly or maintenance of the aggregation activity depends on a continued local supply of cholesterol.

Animals↗
PubMed2004

Conjugated linoleic acid upregulates LDL receptor gene expression in HepG2 cells.

Conjugated linoleic acid (CLA) exerts anticarcinogenic and antiatherosclerotic effects in animals. The present study was conducted to examine the effects of CLA on LDL receptor (LDLr) expression in HepG2 cells, and to evaluate whether the sterol response element binding protein 1 (SREBP-1) and acyl CoA:cholesterol acyltransferase (ACAT) were involved in the regulation of LDLr expression by CLA. When HepG2 cells were cultured with serum-free DMEM for 48 h, there was a three- to fivefold (P<0.05) increase in LDLr protein and mRNA levels. Incubation of HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH, 5 mg/L) for 24 h decreased LDLr protein and mRNA by 50-70% (P<0.05) and mature SREBP-1 by 20-40% (P<0.05). CLA, but not linoleic acid, antagonized the depressive effects of 25OH and increased both LDLr protein and mRNA abundance twofold (P<0.05). LDLr protein and mRNA abundance were not different when HepG2 cells were cultured with CLA (0.4 mmol/L) plus 25OH in the presence or absence of an ACAT inhibitor (58-035, 1 mg/L). Furthermore, CLA had no effect on SREBP-1 abundance. These results suggest that CLA upregulates LDLr expression via a mechanism that is independent of ACAT and SREBP-1.

Blotting, Western↗
PubMed2005

Long-chain polyunsaturated fatty acids upregulate LDL receptor protein expression in fibroblasts and HepG2 cells.

The objective of this study was to investigate the effect of individual PUFAs on LDL receptor (LDLr) expression in human fibroblasts and HepG2 cells, and to evaluate whether acyl CoA:cholesterol acyltransferase (ACAT) and sterol regulatory element-binding protein 1 (SREBP-1) were involved in the regulation of LDLr expression by fatty acids. When fibroblasts and HepG2 cells were cultured with serum-free defined medium for 48 h, there was a 3- to 5-fold (P < 0.05) increase in LDLr protein and mRNA levels. Incubation of fibroblasts and HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH-cholesterol, 5 mg/L) for 24 h decreased LDLr protein and mRNA levels by 50-90% (P < 0.05). Arachidonic acid [AA, 20:4(n-6)], EPA [20:5(n-3)], and DHA [22:6(n-3)] antagonized the depression of LDLr gene expression by 25OH-cholesterol and increased LDLr protein abundance 1- to 3-fold (P < 0.05), but had no significant effects on LDLr mRNA levels. Oleic (18:1), linoleic (18:2), and alpha-linolenic acids [18:3(n-3)] did not significantly affect LDLr expression. ACAT inhibitor (58-035, 1 mg/L) attenuated the regulatory effect of AA on LDLr protein abundance by approximately 40% (P < 0.05), but did not modify the regulatory effects of other unsaturated fatty acids in HepG2 cells. The present results suggest that AA, EPA, and DHA increase LDLr protein levels, and that ACAT plays a role in modulating the effects of AA on LDLr protein levels. Furthermore, the effects of the fatty acids appeared to be independent of any change in SREBP-1 protein.

Arachidonic Acid↗
PubMed2001

Increased oxysterols associated with iron accumulation in the brains and visceral organs of acaeruloplasminaemia patients.

Acaeruloplasminaemia is characterized by excessive neurovisceral iron accumulation due to mutation of the caeruloplasmin gene. Excess iron functions as a potent catalyst of biological oxidation, and increased iron concentration is associated with the products of lipid peroxidation in the serum and cerebrospinal fluid. We investigated whether the amount of iron accumulated paralleled lipid peroxidation levels in acaeruloplasminaemia tissues, examining brains and visceral organs of two affected patients at autopsy for iron and copper content, and oxysterols, including 7-hydroxycholesterol and 7-ketocholesterol, which are directly produced from cholesterol by active oxygen species. The amount of iron accumulated in various tissues was correlated with the levels of the oxysterols. These findings suggest that lipid peroxidation produced by the intracellular accumulation of iron is involved in the pathogenesis of acaeruloplasminaemia.

Aged↗
PubMed1989

Physiologic characterization of transformed and cloned rat granulosa cells.

Properties of a clonal line of SV40-transformed rat granulosa cells (DC3 cells) were elucidated. DC3 cells were maintained in vitro in Iscove Modified Dulbecco Medium that contained 20% fetal bovine serum. The cells had a logarithmic growth phase doubling time of approximately 18 h and produced detectable quantities of estrone, estradiol, and progesterone. Steroidogenesis was increased by supplementation with either steroidogenic substrates or agents that stimulated activity of adenylate cyclase. Production of progesterone and estrogens was enhanced when medium was supplemented with 25-hydroxycholesterol, and production of estradiol was enhanced by medium supplementation with androstenedione. Treatments with forskolin and cholera toxin resulted in marked increases of cyclic adenosine 3',5'-monophosphate (cAMP) in medium and cells and enhanced steroidogenesis. Isoproterenol and vasoactive intestinal peptide, but not follicle-stimulating hormone (FSH), luteinizing hormone (LH), insulin or prolactin, stimulated cAMP secretion by suspended cells. DC3 cells had small but detectable levels of binding to FSH, but binding of LH and epidermal growth factor could not be detected. DC3 cells possess characteristics expected of granulosa cells arrested in an early stage of differentiation and may provide a useful model for studies of "immature" granulosa cell functions.

Animals↗
PubMed1991

Effect of activin A on dehydroepiandrosterone and testosterone secretion by primary immature porcine Leydig cells.

In the present study, we evaluated the effect of the homodimer activin A on immature porcine Leydig cell functions in primary culture. Activin A (0.5-100 ng/ml) reduced hCG-stimulated dehydroepiandrosterone (DHEA) accumulation in a dose- and time-dependent manner, with a maximal inhibitory effect (58% decrease) at 20 ng/ml (8 x 10(-10) M). Activin A was found not to control steroidogenesis, either through a modulation of the gonadotropin LH/hCG binding or low-density lipoprotein cholesterol binding and internalization. However, activin A significantly decreased pregnenolone (p less than 0.002) and DHEA (p less than 0.001) formation (evaluated in the presence of 10(-5) M of WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase [3 beta-HSDI]activity) in Leydig cells maximally stimulated with hCG (3 ng/ml, 3 h) or incubated in the presence of 22R-hydroxycholesterol (5 micrograms/ml, 2 h). These findings indicate that activin A probably exerts a partial inhibitory effect on cholesterol side-chain cleavage cytochrome P450 (P450scc) activity. On the other hand, activin A significantly (p less than 0.001) enhanced the conversion of exogenous pregnenolone and DHEA (500 ng/ml) but not of progesterone and androstenedione (500 ng/ml) into testosterone, suggesting that activin A potentially enhances 3 beta-HSDI activity in Leydig cells. Activin A action on 3 beta-HSDI activity was found to be closely related to that of transforming growth factor-beta 1 (TGF beta 1), since both activin A (20 ng/ml) and TGF beta 1 (2 ng/ml) induced a comparable and non-additive increase in 3 beta-HSDI activity.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗