Sterol utilization in honey bees fed a synthetic diet: Analysis of prepupal sterols

The sterols of prepupal honey bees, Apis mellifera L., from brood reared by workers fed chemically-defined synthetic diets containing cholesterol, campesterol, sitosterol, stigmasterol, 24-methylenecholesterol, or no sterol over a 12-week period were isolated, identified, and quantified. The major sterol present in each prepupal sample was 24-methylenecholesterol, but significant levels of sitosterol and isofucosterol were also present in every case, as was a very small percentage of desmosterol (usually < 1%). This is the first report of isofucosterol being identified in the sterols of the honey bee. A considerably larger percentage of each dietary sterol was found in prepupae reared by workers fed that particular sterol in the diet. This was most dramatic in the case of the cholesterol diet in which case cholesterol content increased to as much as 17.2% of the prepupal sterols, whereas cholesterol had not exceeded 2.2% in samples from other diet regimens. However, stigmasterol comprised no more than 6.3% of the total sterols in any sample from prepupae fed the stigmasterol diet. The preponderance of 24-methylenecholesterol in all prepupae, regardless of the dietary sterol provided to the workers, as well as the lesser quantities of sitosterol and isofucosterol present in all samples, suggest a unique system of utilization and metabolism of these dietary sterols by the worker bees. Apparently they make available to the brood varying amounts of unchanged dietary sterol plus considerable and fairly constant portions of 24-methylenecholesterol, sitosterol, and isofucosterol drawn from their own sterol pools.     

Effects of dietary sterols on development and reproduction of southwestern corn borer Diatraea grandiosella Dyar

Southwestern corn borer larvae, Diatraea grandiosella Dyar, were reared on artificial diets containing individual sterols (cholesterol, sitosterol, or stigmasterol) in concentrations ranging from 0.05 to 0.2%. Female larvae developed to pupae more rapidly as sitosterol and stigmasterol were increased in the diets. Increased cholesterol concentrations did not affect the larval period significantly, and development was not as rapid as with the phytosterols. Female larvae developed at significantly slower rates in all diets than did males, except at the highest concentrations of sitosterol and stigmasterol. Female pupae and adults were significantly heavier than the males, and pupal and adult weight increased as sterol concentrations increased. Number of eggs laid per fertilized female and egg hatchability were significantly increased as concentrations of the three sterols were increased in the larval diets. Sitosterol-reared females produced more eggs than did females reared on other sterols but egg hatchability was not significantly different among sterols.     

Die quantitative verteilung der sterine und sterinderivate in organen von Solanum dulcamara

The following sterols were found in the roots, stems, leaves, unripe and ripe fruits of Solanum dulcamara: cholesterol, sitosterol, stigmasterol, campesterol, brassicasterol, isofucosterol and 24-methylenecholesterol. The most abundant components are cholesterol, sitosterol and stigmasterol (77–84%). In all parts of the plant the sterols are present in the free form and as esters, glycosides and acylated glycosides. The total sterol content and the content of combined forms were determined photometrically. In the leaves 58% of the sterols were found in the form of glycoside (26%), acylated glycoside (29%) and ester (2%). In the roots only 25% of the sterol were found in combined form. In the other organs the ratio of free and combined sterols was intermediate. In all cases, the ester fraction was the least.     

Effects of the growth retardant tetcyclacis on the sterol composition of oat (Avena sativa)

The norbornenodiazetine plant growth regulator tetcyclacis, when applied to roots of Avena sativa, caused a substantial increase in the cholesterol content of the shoots. Amounts of the C-24 alkylated sterols campesterol, stigmasterol and sitosterol all declined. A similar alteration in the sterol profile was observed for a plasma membrane preparation from the shoots. Changes in the sterol composition of root tissue were much less pronounced.     

Differential modulation of membrane structure and fluctuations by plant sterols and cholesterol

We have studied the concentration and temperature dependent influence of cholesterol, stigmasterol, and sitosterol on the global structure and the bending fluctuations of fluid dimyristoyl phosphatidylcholine and palmitoyl oleoyl phosphatidylcholine bilayers applying small-angle x-ray scattering, as well as dilatometry and ultrasound velocimetry. Independent of the lipid matrix, cholesterol was found to be most efficient in modulating bilayer thickness and elasticity, followed by sitosterol and stigmasterol. This can be attributed to the additional ethyl groups and double bond at the C₁₇ alkyl side-chain of the two plant sterols. Hence, it seems that some flexibility of the sterol hydrocarbon chain is needed to accommodate within the lipid bilayer. In addition, we did not observe two populations of membranes within the putative liquid-ordered/liquid-disordered phase coexistence regime of binary sterol/lipid mixtures. Instead, the diffraction patterns could be interpreted in terms of a uniform phase. This lends further support to the idea of compositional fluctuations of unstable sterol rich domains recently brought up by fluorescence microscopy experiments, which contrasts the formation of stable domains within the miscibility gap of binary lipid/sterol mixtures.     

Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.     

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.     

Overexpression of CYP710A1 and CYP710A4 in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants increases the level of stigmasterol at the expense of sitosterol

Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008–1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008–1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.     

Comparative study of in vivo stigmasterol biosynthesis in Nicotiana tabacum and Hordeum vulgare

Six-day-old tobacco (Nicotiana tabacum) and barley (Hordeum vulgare) seedlings rapidly incorporated and metabolized exogenously supplied [4-¹⁴C]sitosterol but neither plant was able to convert it into stigmasterol. However, a sterol metabolite was isolated from both species and the acetate derivative was slightly more polar, on AgNO₃—silica gel TLC, than stigmasteryl acetate. A similar metabolite was also obtained with [4-¹⁴C]cholesterol, indicating a general metabolic reaction of plants to exogenous sterols. Both species incorporated [2-¹⁴C]mevalonic acid into sitosterol and stigmasterol. We suggest that in vascular plants, whether monocotyledons or dicotyledons, the pathway of stigmasterol biosynthesis is not via sitosterol but through a common precursor which is derived from mevalonic acid.     

α-Onocerin and sterol content of twelve species of Ononis

The sterols and triterpenoids of 12 species of the genus Ononis were analysed by GLC. α-Onocerin was found in all but one of these species, although in some others its concentration was low. In all species examined, sitosterol was the major sterol; stigmasterol, campesterol, cholesterol and the triterpenoids cycloartenol and 24-methylene cycloartanol also occurred. The patterns of α-onocerin and sterols found seem to be consistent with the accepted classification of species within the genus.     

Characterization of Soybean Plasma Membrane during Development: FREE STEROL COMPOSITION AND CONCANAVALIN A BINDING STUDIES

Plasma membrane preparations from soybean root and hypocotyl contained the following free sterols: cholesterol, campesterol, stigmasterol, and sitosterol. The cholesterol level was relatively low in root plasma membrane (less than 0.5%) but was 1.4 to 2.4% in hypocotyl membrane. The relative levels of the three other sterols fluctuated with cellular development and tissue source. Campesterol level decreased with the development of both root and hypocotyl membrane. With development, stigmasterol increased greatly in root membrane but remained constant in hypocotyl membrane, and sitosterol, the major free sterol component of all membrane preparations, decreased in root membrane but increased slightly in hypocotyl membrane.     

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.     

NaCl effects on 4-desmethylsterol composition of plasma-membrane-enriched preparations from citrus roots

Abstract The free 4-desmethylsterol composition of plasma-membrane-enriched preparations from white fibrous roots of Rangpur lime (Citrus reticulata var. austera hybrid?), Kharna khatta (C. kharna Raf.) and Etrog citron (C. medica L.) seedlings grown in the presence of 0, 50, or 100 mol m^?3 NaCl for 28 d was quantitated by gas chromatography (GC) on analytical capillary (SE-54 fused silica) columns and the sterols were identified by combined gas chromatography-mass spectrometry (GC-MS). Only three 4-desmethylsterols were positively identified by GC-MS, viz. campesterol, stigmasterol and sitosterol. Cholesterol could not be positively identified in any of the membrane preparations. Campesterol levels were generally similar for all treatments and for all three genotypes, approximating 30% of the total free 4-desmethylsterol content of the plasma membranes. At all levels of salinity (0, 50 or 100 mol m^?3 NaCl) sitosterol levels decreased in the order Rangpur lime > Kharna khatta > Etrog citron and stigmasterol levels decreased in the reverse order. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron at each level of salinity and was reduced by salt treatment in all three genotypes. Salt-induced reductions in the ratio of ‘more planar’ to ‘less planar’ sterols correlated inversely with the accumulation of Cl^? in the leaves of the three genotypes suggesting a role for plasma membrane sterols in the Cl^? exclusion mechanism. A model relating sterol structure, membrane sterol composition and membrane permeability to Cl^? exclusion ability in citrus is presented.     

STEROL PROFILES IN 18 MACROALGAE OF THE PORTUGUESE COAST1

The sterol profiles of dominant macroalgae occurring in the western Portuguese coast were evaluated. An analytical procedure, involving alkaline hydrolysis and extraction followed by separation by reversed‐phase HPLC–diode array detection (HPLC–DAD), was optimized for the study of their sterols composition. The validated methodology is short in analysis time (as the compounds are determined in <20 min), sensitive, reproducible, and accurate. It was then successfully applied to the determination of campesterol, cholesterol, desmosterol, ergosterol, fucosterol, stigmasterol, and β‐sitosterol in 18 species (three Chlorophyta, five Rhodophyta, and 10 Phaeophyta). The profiles obtained for the several macroalgal species were considerably different. C29 sterols were predominant in Phaeophyta and Chlorophyta (71%–95% of total sterol content), while in Rhodophyta cholesterol content is significantly higher (34%–87%). Among the studied species, Asparagopsis armata Harv. contained the lowest sterol amount (555 mg · kg^?1 dry weight), and Cystoseira tamariscifolia (Huds.) Papenf. the highest one (6,502 mg · kg^?1 dry weight). Data obtained may be helpful in identifying suitable marine sources of sterols, with potential applications in the food and pharmaceutical industries.     

Complex formation between solanaceous steroidal glycoalkaloids and free sterols in vitro

Both the steroidal glycoalkaloid mixture from potato (α-solanine and α-chaconine) and pure α-tomatine are able to complex with the sterols cholesterol, sitosterol, stigmasterol, campesterol and ergosterol in vitro. The sterol-complexing ability of tomatine was greater than that of the potato alkaloids and more akin to that of the steroidal saponin, digitonin. With all three compounds, cholesterol was the least-readily bound sterol while binding to other sterols was of a similar order. Complex formation with tomatine was not markedly influenced by temperature, and with the aglycone tomatidine did not appear to occur at all.     

A dynamic role for sterols in embryogenesis of Pisum sativum

Molecular roles of sterols in plant development remain to be elucidated. To investigate sterol composition during embryogenesis, the occurrence of 25 steroid compounds in stages of developing seeds and pods of Pisum sativum was examined by GC-MS analysis. Immature seeds containing very young embryos exhibited the greatest concentrations of sterols. Regression models indicated that the natural log of seed or pod fr. wt was a consistent predictor of declining sterol content during embryonic development. Although total sterol levels were reduced in mature embryos, the composition of major sterols sitosterol and campesterol remained relatively constant in all 12 seed stages examined. In mature seeds, a significant decrease in isofucosterol was observed, as well as minor changes such as increases in cycloartenol branch sterols and campesterol derivatives. In comparison to seeds and pods, striking differences in composition were observed in sterol profiles of stems, shoots, leaves, flowers and flower buds, as well as cotyledons versus radicles. The highest levels of isofucosterol, a precursor to sitosterol, occurred in young seeds and flower buds, tissues that contain rapidly dividing cells and cells undergoing differentiation. Conversely, the highest levels of stigmasterol, a derivative of sitosterol, were found in fully-differentiated leaves while all seed stages exhibited low levels of stigmasterol. The observed differences in sterol content were correlated to mRNA expression data for sterol biosynthesis genes from Arabidopsis. These findings implicate the coordinated expression of sterol biosynthesis enzymes in gene regulatory networks underlying the embryonic development of flowering plants.     

Microbial Transformation of Sterols

Cholesterol decomposing ability of 1589 microbial strains was examined. Two hundreds and thirty six strains from actinomycetes, bacteria, molds, and yeasts were found capable of oxidizing cholesterol into cholestenone. Cholesta-1,4-dien-3-one was produced by 5 strains of Streptomyces. The complete decomposition of cholesterol molecule was observed in the genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. α,α′-Dipyridyl and arsenite inhibited decomposing enzymes giving rise to cholestenone, cholesta-1,4-dien-3-one, and an intermediate probably devoid of the sterol side chain.

Selective cleavage of the side chains of various sterols at C-17, giving rise to androsta-1,4-diene-3,17-dione (ADD), occurred in the presence of α,α′-dipyridyl by microorganisms of the following genera: Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Mycobacterium, Nocardia, Protaminobacter, Serratia, and Streptomyces. The degradation pathway of cholesterol, for example, was shown as follows:

Other sterols such as campesterol, β-sitosterol, stigmasterol and 7-dehydrocholesterol were degraded by the same sequence. The pathway exemplified in cholesterol is considered to be the general degradation pathway of sterols by their decomposing microorganisms.

It was further demonstrated that ADD thus formed from sterols was converted into 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione.     

亚洲玉米螟对常见植物甾醇代谢利用研究

亚洲玉米螟Ostrinia furndcalis G.在缺少甾醇的饲料上不能正常生长发育,能通过脱烷基化作用将△⁵-植物甾醇,如谷甾醇和豆甾醇脱去支链上的烷基变成胆甾醇加以利用,而对△⁷-烯甾烷醇的代谢则有困难。     

Dietary sterols/steroids and the generalist caterpillar Helicoverpa zea: Physiology,biochemistry and midgut gene expression

Sterols are essential nutrients for insects because, in contrast to mammals, no insect (or arthropod for that matter) can synthesize sterols de novo. Plant-feeding insects typically generate their sterols, commonly cholesterol, by metabolizing phytosterols. However, not all phytosterols are readily converted to cholesterol. In this study we examined, using artificial diets containing single sterols/steroids, how typical (cholesterol and stigmasterol) and atypical (cholestanol and cholestanone) sterols/steroids affect the performance of a generalist caterpillar (Helicoverpa zea). We also performed sterols/steroids analyses, using GC/MS techniques, to explore the metabolic fate of these different dietary sterols/steroids. Finally, we used a microarray approach to measure, and compare, midgut gene expression patterns that arise as a function of dietary sterols/steroids. In general, H. zea performed best on the cholesterol and stigmasterol diets, with cholesterol as the dominant tissue sterol on these two treatments. Compared to the cholesterol and stigmasterol diets, performance was reduced on the cholestanol and cholestanone diets; on these latter treatments stanols were the dominant tissue sterol. Finally, midgut gene expression patterns differed as a function of dietary sterol/steroid; using the cholesterol treatment as a reference, gene expression differences were smallest on stigmasterol, intermediate on cholestanol, and greatest on cholestanone. Inspection of our data revealed two broad insights. First, they identify a number of genes potentially involved in sterol/steroid metabolism and absorption. Second, they provide unique mechanistic insights into how variation in dietary sterol/steroid structure can affect insect herbivores.