Sterols regulate development and gene expression in Arabidopsis

Sterols are important not only for structural components of eukaryotic cell membranes but also for biosynthetic precursors of steroid hormones. In plants, the diverse functions of sterol-derived brassinosteroids (BRs) in growth and development have been investigated rigorously, yet little is known about the regulatory roles of other phytosterols. Recent analysis of Arabidopsis fackel (fk) mutants and cloning of the FK gene that encodes a sterol C-14 reductase have indicated that sterols play a crucial role in plant cell division, embryogenesis, and development. Nevertheless, the molecular mechanism underlying the regulatory role of sterols in plant development has not been revealed. In this report, we demonstrate that both sterols and BR are active regulators of plant development and gene expression. Similar to BR, both typical (sitosterol and stigmasterol) and atypical (8, 14-diene sterols accumulated in fk mutants) sterols affect the expression of genes involved in cell expansion and cell division. The regulatory function of sterols in plant development is further supported by a phenocopy of the fk mutant using a sterol C-14 reductase inhibitor, fenpropimorph. Although fenpropimorph impairs cell expansion and affects gene expression in a dose-dependent manner, neither effect can be corrected by applying exogenous BR. These results provide strong evidence that sterols are essential for normal plant growth and development and that there is likely a BR-independent sterol response pathway in plants. On the basis of the expression of endogenous FK and a reporter gene FK::beta-glucuronidase, we have found that FK is up-regulated by several growth-promoting hormones including brassinolide and auxin, implicating a possible hormone crosstalk between sterol and other hormone-signaling pathways.     

Plant sterols: Diversity,biosynthesis, and physiological functions

Sterols, which are isoprenoid derivatives, are structural components of biological membranes. Special attention is now being given not only to their structure and function, but also to their regulatory roles in plants. Plant sterols have diverse composition; they exist as free sterols, sterol esters with higher fatty acids, sterol glycosides, and acylsterol glycosides, which are absent in animal cells. This diversity of types of phytosterols determines a wide spectrum of functions they play in plant life. Sterols are precursors of a group of plant hormones, the brassinosteroids, which regulate plant growth and development. Furthermore, sterols participate in transmembrane signal transduction by forming lipid microdomains. The predominant sterols in plants are β-sitosterol, campesterol, and stigmasterol. These sterols differ in the presence of a methyl or an ethyl group in the side chain at the 24th carbon atom and are named methylsterols or ethylsterols, respectively. The balance between 24-methylsterols and 24-ethylsterols is specific for individual plant species. The present review focuses on the key stages of plant sterol biosynthesis that determine the ratios between the different types of sterols, and the crosstalk between the sterol and sphingolipid pathways. The main enzymes involved in plant sterol biosynthesis are 3-hydroxy-3methylglutaryl-CoA reductase, C24-sterol methyltransferase, and C22-sterol desaturase. These enzymes are responsible for maintaining the optimal balance between sterols. Regulation of the ratios between the different types of sterols and sterols/sphingolipids can be of crucial importance in the responses of plants to stresses.     

Interactions between sterol biosynthesis genes in embryonic development of Arabidopsis

The sterol biosynthesis pathway of Arabidopsis produces a large set of structurally related phytosterols including sitosterol and campesterol, the latter being the precursor of the brassinosteroids (BRs). While BRs are implicated as phytohormones in post-embryonic growth, the functions of other types of steroid molecules are not clear. Characterization of the fackel (fk) mutants provided the first hint that sterols play a role in plant embryogenesis. FK encodes a sterol C-14 reductase that acts upstream of all known enzymatic steps corresponding to BR biosynthesis mutants. Here we report that genetic screens for fk-like seedling and embryonic phenotypes have identified two additional genes coding for sterol biosynthesis enzymes: CEPHALOPOD (CPH), a C-24 sterol methyl transferase, and HYDRA1 (HYD1), a sterol C-8,7 isomerase. We describe genetic interactions between cph, hyd1 and fk, and studies with 15-azasterol, an inhibitor of sterol C-14 reductase. Our experiments reveal that FK and HYD1 act sequentially, whereas CPH acts independently of these genes to produce essential sterols. Similar experiments indicate that the BR biosynthesis gene DWF1 acts independently of FK, whereas BR receptor gene BRI1 acts downstream of FK to promote post-embryonic growth. We found embryonic patterning defects in cph mutants and describe a GC-MS analysis of cph tissues which suggests that steroid molecules in addition to BRs play critical roles during plant embryogenesis. Taken together, our results imply that the sterol biosynthesis pathway is not a simple linear pathway but a complex network of enzymes that produce essential steroid molecules for plant growth and development.     

Boosting crop yields with plant steroids

Plant sterols and steroid hormones, the brassinosteroids (BRs), are compounds that exert a wide range of biological activities. They are essential for plant growth, reproduction, and responses to various abiotic and biotic stresses. Given the importance of sterols and BRs in these processes, engineering their biosynthetic and signaling pathways offers exciting potentials for enhancing crop yield. In this review, we focus on how alterations in components of sterol and BR metabolism and signaling or application of exogenous steroids and steroid inhibitors affect traits of agronomic importance. We also discuss areas for future research and identify the fine-tuning modulation of endogenous BR content as a promising strategy for crop improvement.     

Overexpression of 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases causes growth defects possibly due to abnormal auxin transport in arabidopsis

Sterols play crucial roles as membrane components and precursors of steroid hormones (e.g., brassinosteroids, BR). Within membranes, sterols regulate membrane permeability and fluidity by interacting with other lipids and proteins. Sterols are frequently enriched in detergent-insoluble membranes (DIMs), which organize molecules involved in specialized signaling processes, including auxin transporters. To be fully functional, the two methyl groups at the C-4 position of cycloartenol, a precursor of plant sterols, must be removed by bifunctional 3β-hydroxysteroid dehydrogenases/C-4 decarboxylases (3βHSD/D). To understand the role of 3βHSD/D in Arabidopsis development, we analyzed the phenotypes of knock-out mutants and overexpression lines of two 3βHSD/D genes (At1g47290 and At2g26260). Neither single nor double knock-out mutants displayed a noticeable phenotype; however, overexpression consistently resulted in plants with wrinkled leaves and short inflorescence internodes. Interestingly, the internode growth defects were opportunistic; even within a plant, some stems were more severely affected than others. Endogenous levels of BRs were not altered in the overexpression lines, suggesting that the growth defect is not primarily due to a flaw in BR biosynthesis. To determine if overexpression of the sterol biosynthetic genes affects the functions of membrane-localized auxin transporters, we subjected plants to the auxin efflux carrier inhibitor, 1-N-naphthylphthalamic acid (NPA). Where-as the gravity vectors of wild-type roots became randomly scattered in response to NPA treatment, those of the overexpression lines continued to grow in the direction of gravity. Overexpression of the two Arabidopsis 3βHSD/D genes thus appears to affect auxin transporter activity, possibly by altering sterol composition in the membranes.     

hydra Mutants of Arabidopsis are defective in sterol profiles and auxin and ethylene signaling

The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a Delta8-Delta7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file-specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function.     

Sterol metabolic constraints as a factor contributing to the maintenance of diet mixing in grasshoppers (Orthoptera: Acrididae)

Sterols are essential nutrients for all arthropods, including grasshoppers, but metabolic constraints may limit which sterols can support normal growth and development. In the firsts part of this study, a comparative experiment, which included five different species of grasshoppers (Orthoptera: Acrididae) representing three separate taxonomic groups, was performed to determine how widespread sterol metabolic constraints are within the Acrididae. Grasshoppers were reared on artificial diets containing sterols that differed in the position of double bonds within the sterol structure, and various life history traits were measured. Sterols with double bonds at position 7, within the sterol nucleus, and/or at position 22, on the cholestane side chain, failed to support development to the adult stage for any of the five species. In addition, grasshoppers reared on sterols with these configurations often had extended developmental times and reduced growth rates in the first and second stadium compared with grasshoppers reared on sitosterol or cholesterol diets. In the second half of this study, we examined how mixtures of suitable and unsuitable sterols influenced survival, growth, and development. Artificial foods containing mixtures of suitable and unsuitable sterols were fed to the highly polyphagous grasshopper Schistocerca americana. Results suggest that survival and performance of this grasshopper suffer as the concentration of unsuitable sterols increases and as the ratio of suitable to unsuitable sterols in the diet decreases. We review the literature to document variation in plant sterol profiles and propose that constraints on sterol metabolism may contribute to the maintenance of diet mixing in the Acrididae.     

Survival strategies of a sterol auxotroph

The high sterol concentration in eukaryotic cell membranes is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila cannot synthesize sterols, but do require them for development. Does this simply reflect a requirement for sterols in steroid hormone biosynthesis, or is bulk membrane sterol also essential in Drosophila? If the latter is true, how do they survive fluctuations in sterol availability and maintain membrane homeostasis? Here, we show that Drosophila require both bulk membrane sterol and steroid hormones in order to complete adult development. When sterol availability is restricted, Drosophila larvae modulate their growth to maintain membrane sterol levels within tight limits. When dietary sterol drops below a minimal threshold, larvae arrest growth and development in a reversible manner. Strikingly, membrane sterol levels in arrested larvae are dramatically reduced (dropping sixfold on average) in most tissues except the nervous system. Thus, sterols are dispensable for maintaining the basic membrane biophysical properties required for cell viability; these functions can be performed by non-sterol lipids when sterols are unavailable. However, bulk membrane sterol is likely to have essential functions in specific tissues during development. In tissues in which sterol levels drop, the overall level of sphingolipids increases and the proportion of different sphingolipid variants is altered. These changes allow survival, but not growth, when membrane sterol levels are low. This relationship between sterols and sphingolipids could be an ancient and conserved principle of membrane homeostasis.     

The ratio of campesterol to sitosterol that modulates growth in Arabidopsis is controlled by STEROL METHYLTRANSFERASE 2;1

The Arabidopsis genome contains three distinct genes encoding sterol-C24-methyltransferases (SMTs) involved in sterol biosynthesis. The expression of one of them, STEROL METHYLTRANSFERASE 2;1, was modulated in 35S:SMT2;1 Arabidopsis in order to study its physiological function. Plants overexpressing the transgene accumulate sitosterol, a 24-ethylsterol which is thought to be the typical plant membrane reinforcer, at the expense of campesterol. These plants displayed a reduced stature and growth that could be restored by brassinosteroid treatment. Plants showing co-suppression of SMT2;1 were characterized by a predominant 24-methylsterol biosynthetic pathway leading to a high campesterol content and a depletion in sitosterol. Pleiotropic effects on development such as reduced growth, increased branching, and low fertility of high-campesterol plants were not modified by exogenous brassinosteroids, indicating specific sterol requirements to promote normal development. Thus SMT2;1 has a crucial role in balancing the ratio of campesterol to sitosterol in order to fit both growth requirements and membrane integrity.     

Increased incorporation of dietary plant sterols and cholesterol correlates with decreased expression of hepatic and intestinal Abcg5 and Abcg8 in diabetic BB rats

The aim of this study was to determine the impact of dietary plant sterols and stanols on sterol incorporation and sterol-regulatory gene expression in insulin-treated diabetic rats and nondiabetic control rats. Diabetic BioBreeding (BB) and control BB rats were fed a control diet or a diet supplemented with plant sterols or plant stanols (5 g/kg diet) for 4 weeks. Expression of sterol-regulatory genes in the liver and intestine was assessed by real-time quantitative polymerase chain reaction. Diabetic rats demonstrated increased tissue accumulation of cholesterol and plant sterols and stanols compared to control rats. This increase in cholesterol and plant sterols and stanols was associated with a marked decrease in hepatic and intestinal Abcg5 (ATP-binding cassette transporter G5) and Abcg8 (ATP-binding cassette transporter G8) expressions in diabetic rats, as well as decreased mRNA levels of several other genes involved in sterol regulation. Plant sterol or plant stanol supplementation induced the accumulation of plant sterols and stanols in tissues in both rat strains, but induced a greater accumulation of plant sterols and stanols in diabetic rats than in control rats. Surprisingly, only dietary plant sterols decreased cholesterol levels in diabetic rats, whereas dietary plant stanols caused an increase in cholesterol levels in both diabetic and control rats. Therefore, lower expression levels of Abcg5/Abcg8 in diabetic rats may account for the increased accumulation of plant sterols and cholesterol in these rats.     

Synthesis of hydroxylated sterols in transgenic Arabidopsis plants alters growth and steroid metabolism

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.     

Genetic basis of sitosterolemia

The molecular mechanisms regulating the amount of dietary cholesterol retained by the body, as well as the body's ability to exclude other dietary sterols selectively, are poorly understood. An average Western diet will contain approximately 250-500 mg of dietary cholesterol and approximately 200-400 mg of non-cholesterol sterols, of which plant sterols are the major constituents. Approximately 50-60% of dietary cholesterol is absorbed and retained by the normal human body, but less than 1% of the non-cholesterol sterols are retained. There thus exists a subtle mechanism that allows the body to distinguish between cholesterol and non-cholesterol sterols. In sitosterolemia, a rare autosomal recessive disorder, affected individuals hyperabsorb and retain not only cholesterol but also all other sterols, including plant and shellfish sterols from the intestine. Consequently, patients with this disease have very high levels of plant sterols in the plasma, and develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. The STSL locus has been mapped to human chromosome 2p21. Mutations in two tandem ABC genes, ABCG5 and ABCG8, encoding sterolin-1 and -2, respectively, are now known to be mutant in sitosterolemia. The identification of these genes should now lead to a better understanding of the molecular mechanism(s) governing the highly selective absorption and retention of cholesterol by the body. Indeed, it is the very existence of this disease that has given credence to the hypothesis that there is a molecular pathway that regulates dietary cholesterol absorption and sterol excretion by the body.     

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.     

Importance of plant sterols in pattern formation and hormone signalling

The recent identification of sterol mutants in plants has shown that these molecules play essential roles in development. Although several such mutants are dwarfed, predominantly because of the reduced accumulation of brassinosteroids, others show distinctive phenotypes. We put forward the view that sterols also have roles in mediating brassinosteroid-independent signalling, in the trafficking of membrane vesicles that transport key regulatory proteins and in correct signalling protein conformation and function in membranes.     

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.     

Intracellular sterol transport in eukaryotes,a connection to mitochondrial function?

Eukaryotic cells synthesize sterols in the endoplasmatic reticulum (ER) from where it needs to be efficiently transported to the plasma membrane, which harbors approximately 90% of the free sterol pool of the cell. Sterols that are being taken up from the environment, on the other hand, are transported back from the plasma membrane to the ER, where the free sterols are esterified to steryl esters. The molecular mechanisms that govern this bidirectional movement of sterols between the ER and the plasma membrane of eukaryotic cells are only poorly understood. Proper control of this transport is important for normal cell function and development as indicated by fatal human pathologies such as Niemann Pick type C disease and atherosclerosis, which are characterized by an over-accumulation of free sterols within endosomal membranes and the ER, respectively. Recently, a number of complementary approaches using Saccharomyces cerevisiae as a model organism lead to a more precise characterization of the pathways that control the subcellular transport of sterols and led to the identification of components that directly or indirectly affect sterol uptake at the plasma membrane and its transport back to the ER. A genetic approach that is based on the fact that yeast is a facultative anaerobic organism, which becomes auxotrophic for sterols in the absence of oxygen, resulted in the identification of 17 genes that are required for efficient uptake and/or transport of sterols. Unexpectedly, many of these genes are required for mitochondrial functions. A possible connection between mitochondrial biogenesis and sterol biosynthesis and uptake will be discussed in light of the fact that cholesterol transport into the inner membranes of mitochondria is a well established sterol transport route in vertebrates, where it is required to convert cholesterol into pregnenolone, the precursor of steroids.     

Soybean sterol composition and utilization by Phytophthora sojae

The sterol fraction of Glycine max (soybean) was found to contain a mixture of 13 major sterols which differed dramatically in composition between seeds and shoots. Typical C4-desmethyl Delta(5)-sterols, including sitosterol, predominate the sterol mixture of shoots, whereas C4-methyl sterol intermediates, cycloartenol and 24(28)-methylene cycloartanol, accumulate in seeds. The significance of modified sterol profile of shoot compared to seed was relevant to the physiology of Phytophthora sojae, a phytopathogen of soybean shown to be auxotrophic for sterol. Sterols native to the host plant containing a C4-methyl group, such as cycloartenol, were not utilized by the fungus. Alternatively, all Delta(5)-sterols added to the culture media of P. sojae supported normal growth and promoted viable oospore production. The results demonstrate the importance of sterols in plant-fungal interactions and offer the possibility of bioengineering the phytosterol pathway for resistance to phytopathogens which scavenge specific sterols of the host plant to complete the life cycle.     

Arabidopsis cyp51 mutant shows postembryonic seedling lethality associated with lack of membrane integrity

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14alpha-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14alpha-methyl-delta8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14alpha-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.     

Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols

Statins do not always decrease coronary heart disease mortality, which was speculated based on increased serum plant sterols observed during statin treatment. To evaluate plant sterol atherogenicity, we fed low density lipoprotein-receptor deficient (LDLr(+/-)) mice for 35 weeks with Western diets (control) alone or enriched with atorvastatin or atorvastatin plus plant sterols or stanols. Atorvastatin decreased serum cholesterol by 22% and lesion area by 57%. Adding plant sterols or stanols to atorvastatin decreased serum cholesterol by 39% and 41%. Cholesterol-standardized serum plant sterol concentrations increased by 4- to 11-fold during sterol plus atorvastatin treatment versus stanol plus atorvastatin treatment. However, lesion size decreased similarly in the sterol plus atorvastatin (-99% vs. control) and the stanol plus atorvastatin (-98%) groups, with comparable serum cholesterol levels, suggesting that increased plant sterol concentrations are not atherogenic. Our second study confirms this conclusion. Compared with lesions after a 33 week atherogenic period, lesion size further increased in controls (+97%) during 12 more weeks on the diet, whereas 12 weeks with the addition of plant sterols or stanols decreased lesion size (66% and 64%). These findings indicate that in LDLr(+/-) mice 1) increased cholesterol-standardized serum plant sterol concentrations are not atherogenic, 2) adding plant sterols/stanols to atorvastatin further inhibits lesion formation, and 3) plant sterols/stanols inhibit the progression or even induce the regression of existing lesions.