A critical role for cAMP response element-binding protein (CREB) as a Co-activator in sterol-regulated transcription of 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter. (1/1766)

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a key regulatory enzyme in the pathway for endogenous cholesterol synthesis, is a target for negative feedback regulation by cholesterol. When cellular sterol levels are low, the sterol regulatory element-binding proteins (SREBPs) are released from the endoplasmic reticulum membrane, allowing them to translocate to the nucleus and activate SREBP target genes. However, in all SREBP-regulated promoters studied to date, additional co-regulatory transcription factors are required for sterol-regulated activation of transcription. We have previously shown that, in addition to SREBPs, NF-Y/CBF is required for sterol-regulated transcription of HMG-CoA synthase. This heterotrimeric transcription factor has recently been shown to function as a co-regulator in several other SREBP-regulated promoters, as well. In addition to cis-acting sites for both SREBP and NF-Y/CBF, the sterol regulatory region of the synthase promoter also contains a consensus cAMP response element (CRE), an element that binds members of the CREB/ATF family of transcription factors. Here, we show that this consensus CRE is essential for sterol-regulated transcription of the synthase promoter. Using in vitro binding assays, we also demonstrate that CREB binds to this CRE, and mutations within the CRE that result in a loss of CREB binding also result in a loss of sterol-regulated transcription. We further show that efficient activation of the synthase promoter in Drosophila SL2 cells requires the simultaneous expression of all three factors: SREBPs, NF-Y/CBF, and CREB. To date this is the first promoter shown to require CREB for efficient sterol-regulated transcription, and to require two different co-regulatory factors in addition to SREBPs for maximal activation.  (+info)

Comparison of synthetic saponin cholesterol absorption inhibitors in rabbits: evidence for a non-stoichiometric, intestinal mechanism of action. (2/1766)

The hypocholesterolemic activities of pamaqueside and tiqueside, two structurally similar saponins, were evaluated in cholesterol-fed rabbits. The pharmacological profiles of the saponins were virtually identical: both dose-dependently decreased the intestinal absorption of labeled cholesterol 25-75%, increased fecal neutral sterol excretion up to 2.5-fold, and decreased hepatic cholesterol content 10-55%. High doses of pamaqueside (>5 mg/kg) or tiqueside (>125 mg/kg) completely prevented hypercholesterolemia. Decreases in plasma and hepatic cholesterol levels were strongly correlated with increased neutral sterol excretion. Ratios of neutral sterol excreted to pamaqueside administered were greater than 1:1 at all doses, in opposition to the formation of a stoichiometric complex previously suggested for tiqueside and other saponins. Ratios in tiqueside-treated rabbits were less than unity, a reflection of its lower potency. Pamaqueside-treated rabbits exhibited a more rapid decline in plasma cholesterol concentrations than control animals fed a cholesterol-free diet, indicating that the compound also inhibited the absorption of biliary cholesterol. Intravenous administration of pamaqueside had no effect on plasma cholesterol levels despite plasma levels twice those observed in rabbits given pamaqueside orally. These data indicate that pamaqueside and tiqueside induce hypocholesterolemia by blocking lumenal cholesterol absorption via a mechanism that apparently differs from the stoichiometric complexation of cholesterol hypothesized for other saponins.  (+info)

High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2). (3/1766)

Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to beta-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12-beta-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the beta-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible.  (+info)

Serum sterols during stanol ester feeding in a mildly hypercholesterolemic population. (4/1766)

We investigated the changes of cholesterol and non-cholesterol sterol metabolism during plant stanol ester margarine feeding in 153 hypercholesterolemic subjects. Rapeseed oil (canola oil) margarine without (n = 51) and with (n = 102) stanol (2 or 3 g/day) ester was used for 1 year. Serum sterols were analyzed with gas-liquid chromatography. The latter showed a small increase in sitostanol peak during stanol ester margarine eating. Cholestanol, campesterol, and sitosterol proportions to cholesterol were significantly reduced by 5-39% (P < 0.05 or less for all) by stanol esters; the higher their baseline proportions the higher were their reductions. The precursor sterol proportions were significantly increased by 10- 46%, and their high baseline levels predicted low reduction of serum cholesterol. The decrease of the scheduled stanol dose from 3 to 2 g/day after 6-month feeding increased serum cholesterol by 5% (P < 0. 001) and serum plant sterol proportions by 8-13% (P < 0.001), but had no consistent effect on precursor sterols. In twelve subjects, the 12-month level of LDL cholesterol exceeded that of baseline; the non-cholesterol sterol proportions suggested that stimulated synthesis with relatively weak absorption inhibition contributed to the non-responsiveness of these subjects. In conclusion, plant stanol ester feeding lowers serum cholesterol in about 88% of subjects, decreases the non-cholesterol sterols that reflect cholesterol absorption, increases the sterols that reflect cholesterol synthesis, but also slightly increases serum plant stanols. Low synthesis and high absorption efficiency of cholesterol results in the greatest benefit from stanol ester consumption.  (+info)

Characterization of phosphomevalonate kinase: chromosomal localization, regulation, and subcellular targeting. (5/1766)

Phosphomevalonate kinase catalyzes the conversion of mevalonate-5-phosphate to mevalonate-5-diphosphate and was originally believed to be a cytosolic enzyme. In this study we have localized the phosphomevalonate kinase gene to chromosome 1p13-1q22-23 and present a genomic map indicating that the gene spans more than 8.4 kb in the human genome. Furthermore, we show that message levels and enzyme activity of rat liver phosphomevalonate kinase are regulated in response to dietary sterol levels and that this regulation is coordinate with 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of cholesterol biosynthesis. In addition, we demonstrate that phosphomevalonate kinase is a peroxisomal protein which requires the C-terminal peroxisomal targeting signal, Ser-Arg-Leu, for localization to the organelle.  (+info)

Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains. (6/1766)

In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction. To establish a system in which raft-dependent processes could be studied genetically, we have analyzed the protein and lipid composition of these microdomains in Drosophila melanogaster. Using mass spectrometry, we identified the phospholipids, sphingolipids, and sterols present in Drosophila membranes. Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved. Consistent with this, we have identified a detergent-insoluble fraction of Drosophila membranes that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins. We show that the sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction. Our findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures. They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association.  (+info)

Non-insulin-dependent diabetes mellitus and hypertriglyceridemia impair lipoprotein metabolism in chronic hemodialysis patients. (7/1766)

Patients with diabetes mellitus undergoing chronic hemodialysis treatment have the worst outcome on dialysis due to an increased rate of cardiovascular complications. Nearly all patients present with dyslipidemia, a prominent vascular risk factor, probably responsible for the high rate of vascular injury. Since both uremia and diabetes predispose to hypertriglyceridemia, the present study was conducted to investigate the influence of diabetes mellitus and/or hypertriglyceridemia on lipoprotein metabolism in hemodialysis patients. LDL was isolated and characterized from hyper- and normotriglyceridemic diabetic and nondiabetic hemodialysis patients (n = 40; 10 in each group); also, LDL-receptor-dependent uptake and intracellular cholesterol metabolism were studied in HepG2 cells. In addition, scavenger-receptor-mediated uptake was examined in mouse peritoneal macrophages. LDL isolated from nondiabetic normotriglyceridemic hemodialysis patients exhibited impaired cellular uptake via the LDL receptor. Additionally, intracellular sterol synthesis was less inhibited and cholesterol esterification was reduced compared with LDL from healthy control subjects. Reduction of catabolic capacities was more marked in hemodialysis patients who were either diabetic or hypertriglyceridemic and even more pronounced in patients presenting with a combination of both diabetes and hypertriglyceridemia. Hypertriglyceridemic and diabetic patients showed reduced lipase activity and increased LDL oxidation. Furthermore, they accumulated a fraction of small, dense LDL, and LDL was predominantly taken up via the scavenger-receptor pathway in peritoneal macrophages. This study elucidates the distinct influence of diabetes and/or hypertriglyceridemia in hemodialysis patients on cellular LDL metabolism via specific and nonspecific metabolic pathways. Furthermore, it underscores the cumulative impact of these pathologic entities on impairment of lipoprotein metabolism and increase of cardiovascular risk.  (+info)

The interaction of lipodepsipeptide toxins from Pseudomonas syringae pv. syringae with biological and model membranes: a comparison of syringotoxin, syringomycin, and two syringopeptins. (8/1766)

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.  (+info)