Reviews: current topicsMetabolic effects of plant sterols and stanols (Review)
Introduction
Increased cholesterol concentrations in the low-density lipoproteins (LDLs), a well-known modifiable risk factor for atherosclerosis [1], can be lowered by changing the fatty acid composition of the diet [2]. Recent dietary recommendations, however, also emphasize the possibility of lowering LDL cholesterol levels through consumption of products enriched with plant sterols or stanols [2]. Plant sterols and stanols, which are mainly present in nuts, vegetable oils, seeds, cereals and beans, are structurally related to cholesterol, but are characterized by an extra ethyl (sitosterol) or methyl group (campesterol) in the side chain [3], [4]. They cannot be synthesized by humans, and all plant sterols and stanols in the human body therefore originate from the diet. Sitosterol, campesterol and stigmasterol are the most common plant sterols in nature [5]. Sitostanol and campestanol are saturated plant sterols, which are found in nature in much smaller amounts than plant sterols. Because of their cholesterol-lowering effects, these components are incorporated nowadays into a wide variety of food products, referred to as functional foods.
This review first describes different aspects of cholesterol metabolism, and in the second part describes the effects of plant sterols and stanols on sterol metabolism and atherosclerosis risk. Finally, the effects of plant sterols and stanols on other metabolic processes in the human body are discussed.
In the intestinal tract, esterified cholesterol from the diet is first hydrolyzed. Absorption of free cholesterol now depends on mixed micelles: mixtures of free cholesterol, mono- and di-acylglycerols, fatty acids, phospholipids, and bile salts. The transfer of free cholesterol from the mixed micelles through the apical brush border membrane into the enterocyte is not completely understood [6]. This process was thought to be driven by passive diffusion [3], but recent findings suggest the existence of a specific, saturatable cholesterol transporter in the intestinal mucosa facilitating the transport of cholesterol into the cell [7].
Within the enterocyte, free cholesterol is esterified by acyl-coenzyme A cholesterol acyltransferase (ACAT), incorporated into chylomicrons, which are subsequently excreted into the circulation and converted into a chylomicron-remnant by the action of lipoprotein lipase [8]. The liver then takes up these chylomicron-remnants and forms very-low density lipoproteins (VLDLs). VLDL can subsequently be converted into intermediate lipoproteins (IDL), and IDL into low-density lipoproteins (LDL). LDL particles contain most of the cholesterol and can be - like the IDL - cleared from the circulation by the LDL receptor. Free cholesterol, however, can also be transported back into the intestinal tract through ATP-binding cassette (ABC) transporters, such as ABCA1, ABCG5 and ABCG8 [9].
Cells tightly regulate their intracellular free cholesterol concentrations through ABC transporter mediated cholesterol efflux, LDL-receptor mediated lipoprotein uptake and endogenous cholesterol synthesis [1], [3]. Transcription of ABC transporters depends on intracellular oxysterol concentrations. These oxysterols originate from the oxidation of redundant cellular cholesterol and are potent ligands for the Liver X receptor (LXR). LXR activity may ultimately upregulate transcription of ABC transporters and increase cellular cholesterol efflux.
Gene expression of the LDL-receptor and of HMG CoA reductase - a key enzyme for cholesterol synthesis - is controlled by sterol regulatory element-binding proteins (SREBPs) [10]. When intracellular free cholesterol levels are low, the amount of SREBP increases [11], resulting in increased transcription of the LDL-receptor and HMG CoA reductase genes. In this way, cellular cholesterol influx and synthesis is raised. SREBP transcription and activity is also affected by LXR [12].
Plant sterols are poorly absorbed in the intestine (0.4-3.5%), while absorption of plant stanols (0.02-0.3%) is even lower [13]. For comparison, cholesterol absorption ranges between 35 and 70%. Gender differences may exist as absorption of plant sterols and stanols in female rats appeared to be higher than in male rats [14]. Interestingly, plant stanols may also lower plant sterol absorption and vice versa [15].
A reason for the low absorption of plant sterols and stanols might be that plant sterols and stanols are poorly esterified, possibly due to the low affinity of ACAT for these components [16]. As merely esterified sterols are incorporated into chylomicrons, absorption of the unesterified plant steros and stanols is consequently low. The difference in absorption between plant sterols and stanols is reflected by their serum concentrations. On regular diets as well as on plant sterol- or stanol-enriched diets, serum plant sterol concentrations are 10-30 times higher than plant stanol concentrations [13].
It has been suggested that the extent and rate of plant sterol or stanol absorption depends on the side chain length and the presence of the Δ5 double bond (saturation) [17], [18]. Other factors like mutations and polymorphisms in the ABCG5 or ABCG8 gene may change the intestinal absorption of plant sterols or stanols [19]. For example, mutations in either the ABCG5 or ABCG8 gene may lead to sitosterolemia. This rare autosomal recessive disorder results in accumulation of plant sterols and stanols [19], [20] and can lead to severe atherosclerosis already at a very young age. Furthermore, the use of statins results in higher plasma cholesterol-standardized sterol levels [21], but whether this is due to an increased plant sterol absorption or to a decreased clearance of sterols is not known [22], [23]. In hypercholesterolemic patients, the cholesterol-lowering effects of statins and plant sterols or stanols are additive [23], [24]. This suggests that these patients may need a lower dose of statins, when consuming plant sterol or stanol enriched margarines. In rats, increased amounts of plant sterols were found in adrenal glands, intestinal epithelia and ovaries after intake of a diet enriched with plant sterols [14], [25]. In studies with rabbits, campesterol and sitosterol were also found in trace amounts in the aorta, muscle, skin subcutaneous adipose tissue and liver after intake of a diet enriched with plant sterols [26]. In addition, after consumption of plant stanols, the deposition of these compounds in rats’ tissues is almost negligible compared with plant sterols [27].
Different mechanisms have been suggested to explain the cholesterol-lowering activity of plant sterols and stanols (Figure 1) [28]. Firstly, plant sterols or stanols may displace cholesterol from mixed micelles [29], because they are more hydrophobic than cholesterol. This replacement causes a reduction of micellar cholesterol concentrations and consequently lowers cholesterol absorption. Furthermore, plant sterols or stanols might reduce the esterification rate of cholesterol in the enterocyte [30] and consequently the amount of cholesterol excreted via the chylomicrons. However, in Caco2-cells no effect of sitosterol on ACAT activity was found [31].
The effects of plant stanols on cholesterol absorption continue for at least several hours after ingestion. This was recently demonstrated by Plat et al [32], who showed that the decrease in serum cholesterol in subjects consuming margarines enriched with plant stanol esters once a day was similar to that in subjects who consumed the same amount of plant stanol esters divided over three servings a day. A possible explanation for this effect may be that plant stanols remain present in the intestine for a while. Another explanation may be that the stanols present inside the enterocytes affect intestinal lipoprotein metabolism. In this respect, Plat and Mensink have recently shown in Caco-2 cells that plant stanols upregulate the expression of ABC-transporters in intestinal cells, which may result in an increased excretion of cholesterol by the enterocyte back into the lumen [33]. ABCA1 -/- mice showed increased cholesterol absorption [34], however not all ABCA1 knock out mice show increased cholesterol absorption, probably because of differences in genetic background [35].
An earlier study has suggested that plant stanols are more effective than sterols [18]. However, three independent studies [36], [37] have shown that plant sterol and stanol esters have comparable effects on serum LDL cholesterol.
In response to the decreased cholesterol absorption, cholesterol synthesis increases [38]. Also, LDL receptor mRNA and protein expression increases [39]. This will not only increase clearance of LDL, but also of IDL. As IDL is the precursor for LDL, this will lower LDL production, as indeed has been demonstrated by Gylling et al [15] using radio-labeled LDL. At a daily intake of 2-2.5 g of plant sterols or stanols, the lower cholesterol absorption, the higher LDL receptor expression and the higher endogenous cholesterol synthesis together result in an average reduction of LDL cholesterol of up to 14% [40]. The decrease of total serum cholesterol is completely accounted for by a reduction in LDL. At higher daily intakes, the additional effects on LDL are marginal [36]. Plant sterols and stanols have no effect on triacylglycerol or HDL cholesterol levels [41]. Interestingly, in diabetic patients an increase in HDL cholesterol levels was found, and this affects atherosclerosis in a positive way [38]. The reason for the positive effect remains unclear, but may be related to disturbances in lipoprotein metabolism as found in diabetic subjects, such as increased triacylglycerol concentrations or VLDL production. Effects of plant sterols and stanols in humans on hepatic VLDL production, however, are not known.
No consensus exists about the effects of plant sterols or stanols on bile metabolism [42]. In colectomized patients, biliary secretion of cholesterol, bile salts and phospholipids remained unchanged after consumption of a plant stanol ester-enriched margarine [43]. Consumption of plant sterols also did not change bile salt excretion, measured in ileostomy bags and feeces respectively [4], [44]. In Apolipoprotein E*3-Leiden transgenic mice, however, plant stanol ester consumption reduced cholesterol saturation of bile resulting in less excretion of bile in bile salts [45]. Finally, a study in Wister rats did show a change in bile salt excretion after stigmasterol consumption [42].
Although serum LDL cholesterol concentrations are a well-validated biomarker for atherosclerotic risk, only measurement of lesion development truly reflects the effects of a compound on atherogenesis but this is difficult to measure in humans. In this respect, small animal models are of great benefit to evaluate the potential of a dietary compound to affect lesion formation and to unravel the underlying mechanisms. For this purpose, normal rodents are not very suitable, as their plasma lipoprotein profiles are not human-like, while these rodents are also very resistant to the development of atherosclerosis. However, in transgenic mice with specific defects in lipid and lipoprotein metabolism - such as the LDL receptor-deficient mouse, lipoprotein concentrations are sensitive to dietary changes and drug treatment [46], [47].
Whether the reduction in LDL cholesterol after consumption of plant sterol and stanol esters lowers cardiovascular risk in humans has formerly not been proven. Animal studies, however, are convincing and showed decreased plaque formation after consumption of plant sterols or stanols. In ApoE-deficient mice, a mixture of plant sterols and stanols reduced both atherosclerotic lesion size and complexity [48]. These effects were attributed to the cholesterol-lowering effects of plant sterols and stanols. In a second study, also in apoE-deficient mice [49], the prevention of plaque formation after plant sterol consumption was attributed to a reduction in the concentration of the atherogenic β-VLDL particles, resulting in a decreased foam cell formation. In New Zealand White (NZW) rabbits, pure sitostanol also reduced plaque formation [16]. Finally, in apoE*3-Leiden transgenic mice, plant stanol esters reduced atherosclerotic lesion size and severity mainly by lowering plasma levels of VLDL and IDL [50].
Except for effects on the plasma lipoprotein profile, in vitro studies suggest that plant sterols may affect plaque formation through other mechanisms as well. Awad et al [51] showed decreased growth and proliferation of vascular smooth muscle cells (VSMC), isolated from rats and cultured in the presence of sitosterol and campesterol. Also, prostacyclin release from VSMC in response to plant sterol administration was increased, which may cause vasodilation and decrease platelet aggregation.
Whether plant sterols and stanols induce regression of existing atherosclerotic plaques is less evident. Moghadasian et al [52] were not able to show regression of atherosclerotic plaques after feeding apoE-deficient mice a mixture of plant sterols and plant stanols. It was remarkable, however, that serum cholesterol levels in the sterol-treated animals were not reduced compared with those in the control group.
Section snippets
Other effects of plant sterols and stanols
In addition to the effects on lipid and lipoprotein metabolism, plant sterols and stanols may affect other metabolic processes (Figure 2). Although serum campesterol concentrations are about three times higher than sitosterol concentrations, most studies have focused upon metabolic effects of sitosterol.
Conclusion
Plant sterols and stanols effectively lower serum LDL cholesterol levels and may therefore play an important role in atherosclerotic lesion development. However, future research is necessary to determine the exact effect of plant sterols and stanols on atherosclerotic lesion development and plaque regression. It is clear that plant sterols and stanols are useful for mildly and hypercholesterolemic subjects as an addition to the diet or to cholesterol lowering medication such as statins. The
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