Plant sterol and stanol substrate specificity of pancreatic cholesterol esterase

Consumption of plant sterols or stanols (collectively referred to as phytosterols) and their esters results in decreased low-density lipoprotein cholesterol, which is associated with decreased atherosclerotic risk. The mechanisms by which phytosterols impart their effects, however, are incompletely characterized. The objective of the present study is to determine if pancreatic cholesterol esterase (PCE; EC 3.1.1.13), the enzyme primarily responsible for cholesterol ester hydrolysis in the digestive tract, is capable of hydrolyzing various phytosterol esters and to compare the rates of sterol ester hydrolysis in vitro. We found that PCE hydrolyzes palmitate, oleate and stearate esters of cholesterol, stigmasterol, stigmastanol and sitosterol. Furthermore, we found that the rate of hydrolysis was dependent on both the sterol and the fatty acid moieties in the following order of rates of hydrolysis: cholesterol>(sitosterol=stigmastanol)>stigmasterol; oleate>(palmitate=stearate). The addition of free phytosterols to the system did not change hydrolytic activity of PCE, while addition of palmitate, oleate or stearate increased activity. Thus, PCE may play an important but discriminatory role in vivo in the liberation of free phytosterols to compete with cholesterol for micellar solubilization and absorption.     

Short-term LDL cholesterol-lowering efficacy of plant stanol esters

Background  

The short-term cholesterol-lowering efficacy of plant stanol esters has been open to debate, and the data from different clinical studies with hypercholesterolemic subjects are variable, partly due to lack of systematic studies. Therefore, we investigated the time in days needed to obtain the full cholesterol-lowering effect of stanol esters in hypercholesterolemic subjects.     

Modulation of plant mitochondrial VDAC by phytosterols

We have investigated the effect of cholesterol and two abundant phytosterols (sitosterol and stigmasterol) on the voltage-dependent anion-selective channel (VDAC) purified from mitochondria of bean seeds (Phaseolus coccineus). These sterols differ by the degree of freedom of their lateral chain. We show that VDAC displays sensitivity to the lipid-sterol ratio and to the type of sterol found in the membrane. The main findings of this study are: 1), cholesterol and phytosterols modulate the selectivity but only stigmasterol alters the voltage-dependence of the plant VDAC in the range of sterol fraction found in the plant mitochondrial membrane; 2), VDAC unitary conductance is not affected by the addition of sterols; 3), the effect of sterols on the VDAC is reversible upon sterol depletion with 10 μM methyl-β-cyclodextrins; and 4), phytosterols are essential for the channel gating at salt concentration prevailing in vivo. A quantitative analysis of the voltage-dependence indicates that stigmasterol inhibits the transition of the VDAC in the lowest subconductance states.     

Free phytosterols facilitate excretion of endogenous cholesterol in gerbils

To determine whether phytosterols (PST) facilitate excretion of whole body cholesterol and whether dietary fat or enhancing gallbladder contraction with curcumin might influence this process, four experiments were conducted in gerbils. In Experiment 1, naive gerbils received cholesterol-free purified diets with 30% energy from fat and 0% or 0.75% free PST from tall oil for 4 weeks. In Experiment 2, body cholesterol pools were expanded by feeding a diet containing 0.3% cholesterol for 3 weeks. Subsequently, PST was provided in either fat-free or normal-fat diets without cholesterol for only 2 h each morning, followed by a low-fat diet for the rest of the day and food restriction overnight. In Experiment 3, gerbils were preloaded with cholesterol, followed by either PST alone or PST+curcumin to enhance gallbladder contraction. In Experiment 4, curcumin or curcumin+PST were fed with 30% as fat and 0.15% cholesterol throughout the study. Because of the small whole body cholesterol pool in Experiment 1, the impact of PST was limited. When whole body cholesterol was expanded in Experiments 2 and 3, subsequent reductions of liver esterified cholesterol by PST were significant. In the presence of dietary fat, PST caused a greater reduction (23%) than in a fat-free diet (8%) compared to respective controls. Curcumin (Experiments 3 and 4) proved ineffective in reducing liver or plasma cholesterol pools, and the 3:1 ratio between PST/diet cholesterol was less effective at blocking cholesterol absorption than a 5:1 ratio previously employed. Thus, free PST removed whole body cholesterol, which was enhanced by concomitant fat intake, but was unaffected by a gallbladder contracting agent.     

A moderate intake of phytosterols from habitual diet affects cholesterol metabolism

Cholesterol metabolism homeostasis is the result of a balance between synthesis, degradation and intestinal absorption. It is well established that intestinal cholesterol absorption efficiency can be modified by the intake of phytosterol-enriched food and, therefore, have a serum cholesterol-lowering effect. Recent epidemiological and clinical studies have shown that presence of phytosterols at normal diet levels could also be effective on lowering total and LDL serum cholesterol since they affect whole-body cholesterol metabolism even at those moderate doses. The aim of this study was to analyze the effect of the levels of the naturally-occurring phytosterols in the diet on cholesterol metabolism parameters. In order to do that a group of 99 healthy volunteers was studied for their dietary habits and surrogate markers of cholesterol synthesis and absorption. The mean daily dietary intake of phytosterols, measured by a food semiquantitative frequency questionnaire, was found to be 494 mg being β-sitosterol the major contributor to it. Subjects were classified into tertiles according to their total phytosterol intake and comparisons were done between subgroups. No statistical differences were observed for surrogate markers of intestinal cholesterol absorption, but a significant increase in the cholesterol synthesis surrogate marker lathosterol-to-cholesterol ratio associated to highest dietary phytosterol intake was observed. Regardless of this, only a non significant trend toward a less atherogenic lipid profile was observed in the upper tertile. In conclusion, the intake of moderate amounts of phytosterols naturally present in habitual diet may affect cholesterol metabolism and specially the rate of cholesterol synthesis as estimated by the surrogate marker lathosterol-to-cholesterol ratio in serum.     

The role of phytosterols in plant adaptation to temperature

Membranes of composition approaching those found in “rafts” of plants, fungi and mammals were investigated by means of solidstate ²H-NMR, using deuterated dipalmitoyl-phosphatidylcholine (²H-DPPC) as a reporter. The dynamics of such membranes was determined through measuring of membrane ordering or disordering properties. The presence of the liquid-ordered, lo, phase, as an indicator of rigid sterol-sphingolipid domains, was detected in all cases. Of great interest, the dynamics of mixtures mimicking rafts in plants showed the lesser temperature sensitivity to thermal shocks. The presence of an additional ethyl group branched on the alkyl chain of major plant sterols (sitosterol and stigmasterol) is proposed as reinforcing the membrane cohesion. The fine tuning of the sterol structure thus appears to be the evolution response for plant adaptation to large temperature variations.Key words: sitosterol, stigmasterol and glucosylcerebrosides, regulation of membrane dynamics, membrane rafts, deuterium NMRIt is widely recognized that lipids play multiple roles that either individually or collectively influence cell processes. Glycerolipids and sphingolipids through charge and structure are involved in DNA replication, protein translocation, cell recognition, signalling pathways, energetic, signal transduction, and cell trafficking. Together with diacylglycerols their collective properties modulate lipid polymorphism, through phase transitions (lamellar, hexagonal, cubic, micelles), which are involved in enzyme conformational changes, cell division, cell fusion, and apoptosis.¹Sterols, the third lipid class, also regulate biological processes and sustain the domain structure of cell membranes where they are considered as membrane reinforcers.^2,3 While cholesterol (CHO) is the major sterol of vertebrates, ergosterol plays a key role in fungi. Plants usually possess more complex sterol compositions. Stigmasterol (STI) and sitosterol (SIT), two 24-ethyl sterols, are major constituents of the sterol profiles of plant species. They are involved in the embryonic growth of plants.^4,5 Sterols are critical for the formation of liquid-ordered (lo) lipid domains (lipid rafts) that are supposed to play an important role in fundamental biological processes like signal transduction, cellular sorting, cytoskeleton reorganization and infectious diseases.^6,7 In plants, specialized lipid domains are involved in the polarized growth of pollen tube and root hair⁸ and the asymmetric growth of plant cells is in general due to the asymmetric distribution of membrane components.We recently documented the effect of sitosterol and stigmasterol, two major plant sterols, on the structure and dynamics of membranes whose composition is representative of domains (rafts) in plants.⁹ Liposomes of phytosterols associated with glucosylcerebroside (GC) and with deuterium-labelled dipalmitoylphosphatidylcholine (²H-DPPC) were analysed with deuterium solid state nuclear magnetic resonance (²H-NMR). For comparison, membrane systems representative of raft composition in fungi and mammals were also investigated. ²H-NMR is known to be the best non-invasive technique to analyse membrane dynamics¹⁰ because it is non-destructive and because replacement of DPPC protons with their deuterium isotope brings very little membrane perturbation.^11,12 Acyl chain deuteration affords analysis of both structure and dynamics of the hydrophobic membrane interior. Spectra such as that shown in Figure 1 insert, allow detection of the lo phase, characteristic of a membrane state half-way between solid-ordered (so) and liquid-disordered (ld) states. The so state, also called “gel”, is found at low temperatures (below 35°C), when membranes are essentially composed of sphingomyelins (SM)¹³ or GC (Fig. 1). This membrane state allows little biological function because in forbids membrane trafficking due to its very rigid state (order parameter close to 1). In turn, the ld or “fluid” state is found at high temperatures, in the absence of SM, GC and sterols (low order parameter). At the opposite such a high membrane dynamics may lead to excessive membrane passages. Following with ²H-NMR the temperature behaviour of membrane systems containing GC and plant sterols, we found that the so-ld, order-disorder, transition was totally abolished: SIT and STI fluidized the so state and ordered the ld state to produce the lo state where membrane fluctuations vary smoothly with temperature (Fig. 1). This effect was already documented with CHO in mammals^14–16 but on a much narrower temperature range. The case of the fungus system was found in between that of plants and mammals.Open in a separate windowFigure 1Regulation of temperature-driven membrane dynamics by plant sterols. Central panel: first spectral moment (left y-axis) or order parameter (right y-axis) as a function of temperature; solid line: ²H-DPPC with glucosylcerebroside; open circles: plus stigmasterol; filled circles: plus sitosterol. Insert: ²H-NMR spectrum typical of a liquid-ordered, lo, state. Left panel: schematics of solid-ordered, so (gel), and liquid-disordered, ld (fluid), membrane states. Right panel: schematics of the lo (raft) membrane state together with the structures of cholesterol and sitosterol. Adapted from reference ⁹.Summarizing, it appears that plant membranes of “raft” composition are less sensitive to temperature variations than those of animals. This suggests that cell membrane components like sitosterol, stigmasterol and glucosylcere-brosides, which are typical of plants, are produced in order to extend the temperature range in which membrane-associated biological processes can take place. This observation is well in accordance with the fact that plants have to face higher temperature variations than animals, which usually can either regulate their body temperature or change their location in order to avoid extreme heat or coldness.Compared to cholesterol, the two phytosterols possess additional ethyl groups branched on C-24 (Fig. 1). We proposed that the presence of an additional ethyl group may reinforce the attractive van der Waals interactions leading to more membrane cohesion and therefore less temperature sensitivity. Our results also suggest that domains of smaller size would be promoted in the presence of phytosterols and especially with sitosterol. Such domains may be viewed as dynamic, with sterols laterally exchanging at the microsecond time scale.¹⁴ In plant cells, enzymes transfer alkyl groups to the C-24 of sterols. If we suppose that the relative activities of the different branches of the plant sterol biosynthesis are regulated, the concentrations of major sterols in plants, like sitosterol, stigmasterol, and cholesterol could be controlled.^4,17 This shows the importance of equilibrated sterol concentrations for plant growth and development. Sterols have been historically considered as membrane reinforcers because they bring order to membranes.^2,3Our works^{9,15,16,18,19} show that they could better be named as “membrane dynamics regulators”, by maintaining the membrane in a state of microfluidity suitable for cell function on large temperature scales. It thus appears that a fine tuning of the sterol structure, i.e., the presence of branched ethyl groups in plant sterols increasing membrane cohesion through formation of smaller membrane domains, may be the evolution response for plant adaptation to large temperature variations.     

Competitive solubilization of cholesterol and six species of sterol/stanol in bile salt micelles

Slight differences in the molecular structures of a category of sterol/stanol species affect the solubility of cholesterol in a bile salt solution. We systematically studied the preferential solubilization of cholesterol and sterol/stanol in sodium taurodeoxycholate solutions using relatively minor plant species of sterol/stanol (brassicasterol and stigmasterol) and a non-plant sterol (cholestanol). As relatively major sterol/stanol species (β-sitosterol, β-sitostanol, and campesterol) have already been examined using nearly identical procedures to that used in our system, we were able to sufficiently discuss the cholesterol-lowering effects resulting from the molecular structures of six sterol/stanol species. The results of competitive solubilization revealed that cholestanol has the largest cholesterol-lowering effect, decreasing cholesterol solubility to 33% of that in a single solubilizate system. The molecular structure of cholestanol is also most similar to that of cholesterol. In contrast, brassicasterol and stigmasterol have little ability to decrease cholesterol solubility in a mixed binary system. Both have an unsaturated double bond at the side chain of the steroid ring. By applying thermodynamic analyses to these results, we found that the Gibbs energy changes (ΔG°) of solubilization for sterol/stanol species with cholesterol-lowering effects show larger negative values than that for cholesterol.     

Control of ovarian cholesterol ester biosynthesis

1. Experimental evidence is presented for a role of progesterone and 20alpha-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [(14)C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3':5'-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [(14)C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20alpha-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.     

Xylanase treatment of plant cells induces glycosylation and fatty acylation of phytosterols

Treatment of tobacco suspension cells ( Nicotiana tabacum cv. KY 14) with a purified β -1,4-endoxylanase from Trichoderma viride [1 μg enzyme (ml cells)⁻¹] caused a 13-fold increase in the levels of acylated sterol glycosides and elicited the synthesis of phytoalexins. A commercial preparation of xylanase from Trichoderma viride caused an identical shift in sterols. In contrast, a commerical xylanase from Aureobasidium pullaulans had no effect on the levels of acylated sterol glycosides, but did elevate the levels of sterol esters. Treatment of the cells with Cu²⁺ or Ag⁺ also evoked a severalfold increase in the levels of acylated sterol glycosides. Analysis of the various sterol lipid classes revealed that the large xylanase-induced increase in acylated sterol glycosides occurred at the expense of sterol esters, free sterols and sterol glycosides. Further analyses revealed that the most abundant phytosterol in each of the four classes of sterol lipids was β -sitosterol. Linoleic acid was the most abundant fatty acid in the sterol esters, and palmitic and linoleic acids were the most abundant fatty acids in the acylated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acvlated sterol glycosides. Glucose was the only sugar moiety in the sterol glycoside and acylated sterol glycoside fractions. The results of the present study demonstrate that xylanase from Trichoderma viride induces a dramatic shift in the level of acylated sterol glycosides, indicating that endoxylanase was probably the active component in the cellulase enzyme preparations used in our previous study.     

Selectivity and contribution of lecithin: cholesterol acyltransferase to plasma cholesterol ester formation

Selectivity factors (Vm/Km) for human and rat lecithin: cholesterol acyltransferases (LCAT) for the transfer of various acyl groups from the 2-position of phosphatidylcholine were determined. By multiplying these values by the proportions of acyl groups at the 2-position of phosphatidylcholine, one can predict the proportions of molecular species of cholesterol ester which will be synthesized by LCAT. In human subjects fasted overnight, the molecular composition of plasma cholesterol ester was found to reflect the LCAT selectivity relatively accurately. This result supports the concepts that hepatic acyl-CoA:cholesterol acyltransferase (ACAT) does not contribute significantly to the synthesis of plasma cholesterol ester and that removal of cholesterol ester from plasma is not selective with respect to molecular species under these conditions. In contrast to the results with humans, the molecular composition of plasma cholesterol ester formed in spontaneously hypertensive rats fed a high-cholesterol diet and then fasted overnight differs from that which is predicted from LCAT selectivity and the proportion of various fatty acids at the 2-position of phosphatidylcholine: these results suggest that cholesterol ester is formed mainly via the ACAT reaction.     

HDL and reverse cholesterol transport. Role of cholesterol ester transfer protein]

As most of peripheral cells are not able to catabolize cholesterol, the transport of cholesterol excess from peripheral tissues back to the liver, namely "reverse cholesterol transport", is the only way by which cholesterol homeostasis is maintained in vivo. Reverse cholesterol transport pathway can be divided in three major steps: 1) uptake of cellular cholesterol by the high density lipoproteins (HDL), 2) esterification of HDL cholesterol by the lecithin: cholesterol acyltransferase and 3) captation of HDL cholesteryl esters by the liver where cholesterol can be metabolized and excreted in the bile. In several species, including man, cholesteryl esters in HDL can also follow an alternative pathway which consists in their transfer from HDL to very low density (VLDL) and low density (LDL) lipoproteins. The transfer of cholesteryl esters to LDL, catalyzed by the Cholesteryl Ester Transfer Protein (CETP), might affect either favorably or unfavorably the reverse cholesterol transport pathway, depending on whether LDL are finally taken up by the liver or by peripheral tissues, respectively. In order to understand precisely the implication of CETP in reverse cholesterol transport, it is essential to determine its role in HDL metabolism, to know the potential regulation of its activity and to identify the mechanism by which it interacts with lipoprotein substrates. Results from recent studies have demonstrated that CETP can promote the size redistribution of HDL particles. This may be an important process in the reverse cholesterol transport pathway as HDL particles with various sizes have been shown to differ in their ability to promote cholesterol efflux from peripheral cells and to interact with lecithin: cholesterol acyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of thermodynamic parameters for solubilization of plant sterol and stanol in bile salt micelles

We investigated the difference between the molecular structures of plant sterols and stanols that affect the solubilization of cholesterol in bile salt micelles (in vitro study). First, the aqueous solubility of beta-sitosterol, beta-sitostanol, and campesterol was determined by considering the specific radioactivity by using a fairly small quantity of each radiolabeled compound. The order of their aqueous solubilities was as follows: cholesterol > campesterol > beta-sitostanol > beta-sitosterol. The maximum solubility of cholesterol and the above mentioned sterol/stanol in sodium taurodeoxycholate and sodium taurocholate solutions (single solubilizate system) was measured. Moreover, the preferential solubilization of cholesterol in bile salt solutions was systematically studied by using different types of plant sterols/stanols. The solubilization results showed that the cholesterol-lowering effect was similar for sterols and stanol. Thermodynamic analysis was applied to these experimental results. The Gibbs energy change (Delta G degrees ) for the solubilization of plant sterols/stanols showed a negative value larger than that for cholesterol.     

Intracellular cholesterol transporter StarD4 binds free cholesterol and increases cholesteryl ester formation

StarD4 protein is a member of the StarD4 subfamily of steroidogenic acute regulatory-related lipid transfer (START) domain proteins that includes StarD5 and StarD6, proteins whose functions remain poorly defined. The objective of this study was to isolate and characterize StarD4's sterol binding and to determine in a hepatocyte culture model its sterol transport capabilities. Utilizing purified full-length StarD4, in vitro binding assays demonstrated a concentration-dependent binding of [(14)C]cholesterol by StarD4 similar to that of the cholesterol binding START domain proteins StarD1 and StarD5. Other tested sterols showed no detectable binding to StarD4, except for 7alpha-hydroxycholesterol, for which StarD4 demonstrated weak binding on lipid protein overlay assays. Subsequently, an isolated mouse hepatocyte model was used to study the ability of StarD4 to bind/mobilize/distribute cellular cholesterol. Increased expression of StarD4 in primary mouse hepatocytes led to a marked increase in the intracellular cholesteryl ester concentration and in the rates of bile acid synthesis. The ability and specificity of StarD4 to bind cholesterol and, as a function of its level of expression, to direct endogenous cellular cholesterol suggest that StarD4 plays an important role as a directional cholesterol transporter in the maintenance of cellular cholesterol homeostasis.     

Concept of sequential analysis of free and conjugated phytosterols in different plant matrices

A unique concept and method for the determination of the total plant sterol content as sum of free sterols (FS), steryl esters (SE), steryl glycosides (SG) and acylated steryl glycosides (ASG) in different plant materials (pumpkin seeds, lecithins) and phytopharmaceuticals derived thereof, was developed. For this purpose, a multidimensional sample clean-up protocol based on efficient solid-phase extraction materials was elaborated and the SG were isolated employing a novel phenyl boronic acid modified silica gel material. Along this line also a set of steryl glucosides was synthesised and employed as internal standard and for calibration in the course of quantitative analysis. Final quantification of SG was carried out with reversed-phase HPLC in combination with evaporative light scattering detection (ELSD); the ASG were determined after conversion to SG by mild alkaline hydrolysis. In order to determine the total plant sterol profile the sum of FS and SE was additionally analysed from the unsaponifiable lipid fraction by GC-FID. The yields obtained from recovery tests for the determination of SG using soya lecithin as matrix to which 2, 20 and 40 mg/g of cholesterol-beta-D-glucoside was added were 99.10, 98.07 and 90.00%, and the RSDs were 4.11, 2.62 and 4.50%, respectively. Application related to the qualitative and quantitative analysis of total phytosterol profiles in different plant matrices and extracts demonstrate the validity of the method.